NEISSERIAL ANTIGENS

Abstract

The invention provides proteins from Neisseria meningitidis (strains A & B) and from Neisseria gonorrhoeae, including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data. The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent application Ser. No. 12/653,954, filed, Dec. 18, 2009, which is a Divisional of U.S. patent application Ser. No. 10/864,684, filed Jun. 8, 2004, now U.S. Pat. No. 7,655,245, which is a continuation application of U.S. patent application Ser. No. 09/303,518, filed Apr. 30, 1999, now U.S. Pat. No. 6,914,131, which is a continuation-in-part of International Patent Application PCT/IB1998/001665, filed Oct. 9, 1998, from which applications priority is claimed pursuant to 35 U.S.C. §120. PCT/IB1998/001665 claims priority to Great Britain Patent Applications No. 9723516.2, filed Nov. 6, 1997; No. 9724190.5, filed Nov. 14, 1997; No. 9724386.9, filed Nov. 18, 1997; No. 9725158.1, filed Nov. 27, 1997; No. 9726147.3, filed Dec. 10, 1997; No. 9800759.4, filed Jan. 14, 1998; No. 9819016.8, filed Sep. 1, 1998. All of the above applications are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 223002099611SUBSEQLIST.txt, date recorded: Oct. 1, 2012, size: 2,099 KB).

This invention relates to antigens from Neisseria bacteria.

BACKGROUND ART

Neisseria meningitidis and Neisseria gonorrhoeae are non-motile, gram negative diplococci that are pathogenic in humans. N. meningitidis colonises the pharynx and causes meningitis (and, occasionally, septicaemia in the absence of meningitis); N. gonorrhoeae colonises the genital tract and causes gonorrhea. Although colonising different areas of the body and causing completely different diseases, the two pathogens are closely related, although one feature that clearly differentiates meningococcus from gonococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.

N. gonorrhoeae caused approximately 800,000 cases per year during the period 1983-1990 in the United States alone (chapter by Meitzner & Cohen, “Vaccines Against Gonococcal Infection”, In: New Generation Vaccines, 2nd edition, ed. Levine, Woodrow, Kaper, & Cobon, Marcel Dekker, New York, 1997, pp. 817-842). The disease causes significant morbidity but limited mortality. Vaccination against N. gonorrhoeae would be highly desirable, but repeated attempts have failed. The main candidate antigens for this vaccine are surface-exposed proteins such as pili, porins, opacity-associated proteins (Opas) and other surface-exposed proteins such as the Lip, Laz, IgA1 protease and transferrin-binding proteins. The lipooligosaccharide (LOS) has also been suggested as vaccine (Meitzner & Cohen, supra).

N. meningitidis causes both endemic and epidemic disease. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks (see Lieberman et al. (1996) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275(19):1499-1503; Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. N Engl J Med 337(14):970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries. Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).

Based on the organism's capsular polysaccharide, 12 serogroups of N. meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries. The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Although efficacious in adolescents and adults, it induces a poor immune response and short duration of protection, and cannot be, used in infants [eg. Morbidity and Mortality weekly report, Vol. 46, No. RR-5 (1997)]. This is because polysaccharides are T-cell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influenzae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger W D “New and Improved Vaccines Against Meningococcal Disease” in: New Generation Vaccines, supra, pp. 469-488; Lieberman et al (1996) supra; Costantino et al (1992) Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691-698).

Meningococcus B remains a problem, however. This serotype currently is responsible for approximately 50% of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of α(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if an immune response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N-acetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered (Romero & Outschoorn (1994) Current status of Meningococcal group B vaccine candidates: capsular or non-capsular? Clin Microbiol Rev 7(4):559-575).

Alternative approaches to menB vaccines have used complex mixtures of outer membrane proteins (OMPs), containing either the OMPs alone, or OMPs enriched in porins, or deleted of the class 4 OMPs that are believed to induce antibodies that block bactericidal activity. This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but are not effective at large where there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed (eg. Poolman J T (1992) Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28). Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability (eg. Ala'Aldeen & Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14(1):49-53).

A certain amount of sequence data is available for meningococcal and gonoccocal genes and proteins (eg. EP-A-0467714, WO96/29412), but this is by no means complete. The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that are presumed targets for the immune system and which are not antigenically variable. For instance, some of the identified proteins could be components of efficacious vaccines against meningococcus B, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic Neisseriae.

THE INVENTION

The invention provides proteins comprising the Neisserial amino acid sequences disclosed in the examples. These sequences relate to N. meningitidis or N. gonorrhoeae.

It also provides proteins comprising sequences homologous (ie. having sequence identity) to the Neisserial amino acid sequences disclosed in the examples. Depending on the particular sequence, the degree of identity is preferably greater than 50% (eg. 65%, 80%, 90%, or more). These homologous proteins include mutants and allelic variants of the sequences disclosed in the examples. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between the proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.

The invention further provides proteins comprising fragments of the Neisserial amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.

The proteins of the invention can, of course, be prepared by various means (eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.). They are preferably prepared in substantially pure or isolated form (ie. substantially free from other Neisserial or host cell proteins)

According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means.

According to a further aspect, the invention provides nucleic acid comprising the Neisserial nucleotide sequences disclosed in the examples. In addition, the invention provides nucleic acid comprising sequences homologous (ie. having sequence identity) to the Neisserial nucleotide sequences disclosed in the examples.

Furthermore, the invention provides nucleic acid which can hybridise to the Neisserial nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).

Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the Neisserial sequences and, depending on the particular sequence, n is 10 or more (eg 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).

According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.

It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).

Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (eg. expression vectors) and host cells transformed with such vectors.

According to a further aspect, the invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions.

The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (eg. as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria. Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae, or any strain of N. meningitidis, such as strain A, strain B or strain C).

The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.

A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting said duplexes.

A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.

A summary of standard techniques and procedures which may be employed in order to perform the invention (eg. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.

General

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calm eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification.

All publications, patents, and patent applications cited herein are incorporated in full by reference. In particular, the contents of UK patent applications 9723516.2, 9724190.5, 9724386.9, 9725158.1, 9726147.3, 9800759.4, and 9819016.8 are incorporated herein.

DEFINITIONS

A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” eg. a composition “comprising” X may consist exclusively of X or may include something additional to X, such as X+Y.

The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Neisserial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.

A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg. see U.S. Pat. No. 5,753,235).

Expression Systems

The Neisserial nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].

Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.

Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plagued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alis: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, eg. proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987)

Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.

Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.

The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistakes.” In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21].

Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al., (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34]. The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al: (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria. The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the irp gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage of transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria. For example, expression vectors have been developed for, inter alfa, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl₂ or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial specie to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1).

In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg, ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The repl icon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci. USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.

Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75; 1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Antibodies

As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies.

Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisserial proteins.

Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly): A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the art, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly ³²P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, ¹²⁵I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.

Pharmaceutical Compositions

Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).

Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.

The immunogenic compositions (eg. the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.

The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retroviruses eg. MCF and MCF-M LV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.

Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 5,252,479.

The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEWICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.

Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).

DNA vector systems such as eukarytic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.

Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989)385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Nan Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Nall Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curie] (1992) Hum Gene Titer 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. Ser. No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.

Delivery Methods

Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RH.

B. Hormones, Vitamins, etc.

Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. Iri addition, mono-, di-, or polysaccarides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.

Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 fora description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Ada 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.

The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.

Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phopholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.

Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J. Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in Zuckermann et al. PCT/US97/14465.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, and purtrescine.

The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.

Immunodiagnostic Assays

Neisserial antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisserial antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to Neisserial proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 μg for a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10⁸ cpm/μg, resulting in an exposure time of ˜24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation:

Tm=81+16.6(log₁₀Ci)+0.4[%(G+C)]−0.6(% formamide)−600/n−1.5(% mismatch).

where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.

Nucleic Acid Probe Assays

Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.

The nucleic acid probes will hybridize to the Neisserial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Neisserial sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to the Neisserial sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional Neisserial sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisserial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisserial sequence in order to hybridize therewith and thereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.

Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. [Meth. Enzymol. (1987) 155: 335-350]; U.S. Pat. Nos. 4,683,195 and 4,683,202. Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Neisserial sequence.

A thermostable polymerase creates copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the Neisserial sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-E: For ORF37-1, (A) shows the results of affinity purification of the GST-fusion protein, (B) shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for ELISA (positive result), (C) shows FACS analysis, and (D) shows a bactericidal assay (FIG. 1D), and (E) shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF37-1.

FIG. 2A-B: For ORF5-1, (A) shows the results of affinity purification of the GST-fusion protein, and (B) shows the Western blot analysis of sera from mice immunized with purified GST-fusion protein.

FIG. 3A-D: For ORF2-1, (A) shows the results of affinity purification of the GST-fusion protein, (B) shows the results of expression of the His-fusion in E. coli, (C) shows the Western blot analysis of sera from mice immunized with turified GST-fusion protein, (D) shows the ELISA (positive result), and (D) shows the FACS analysis.

FIG. 4A-C: For ORF15-1, (A) shows the results of affinity purification of the GST-fusion protein, (B) shows the results of expression of the His-fusion in E. coli, and (C) shows the Western blot analysis of sera from mice immunized with purified GST-fusion protein.

FIG. 5A-C: For ORF22-1, (A) shows the results of affinity purification of the GST-fusion protein, (B) shows the results of expression of the His-fusion in E. coli, and (C) shows the FACS analysis using sera from mice immunized with the purified GST-fusion protein.

FIG. 6A-B: For ORF28-1, (A) shows the results of affinity purification of the GST-fusion protein, and (B) shows the results of expression of the His-fusion in E. coli.

FIG. 7A-B: For ORF32-1, (A) shows the results of affinity purification of the His-fusion protein, and (B) shows the results of expression of the GST-fusion in E. coli.

FIG. 8A-F: For ORF4-1, (A) shows the results of affinity purification of the His-fusion, (B) shows the results of affinity purification of the GST-fusion proteins, (C) shows the Western blot analysis of sera from mice immunized with the His-fusion protein, (D) shows the FACS analysis, (E) shows a bactericidal assay, and (F) shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF37-1.

FIG. 9 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF61-1.

FIG. 10A-C: For ORF76-1, (A) shows the results of affinity purification of the His-fusion protein, (B) shows the Western blot analysis of sera from mice immunized with the purified His-fusion protein, and (C) shows the FACS analysis.

FIG. 11 shows the results of affinity purification of the GST-ORF89-1 fusion protein.

FIG. 12A-E: For ORF97-1, (A) show the results of affinity purification of the GST-fusion protein, (B) shows the results of affinity purification of the His-fusion protein, (C) shows the Western blot analysis of sera from mice immunized with purified GST-fusion protein, (D) shows the FACS analysis, and (E) shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF97-1.

FIG. 13A-C: For ORF106-1, (A) shows the results of affinity purification of the His-fusion protein, (B) shows the results of expression of the GST-fusion in E. coli, (C) shows the FACS analysis of sera from mice immunized with the purified His-fusion protein.

FIG. 14A-B: For ORF138-1, (A) shows the results of affinity purification of the GST-fusion protein, and (B) shows the FACS analysis of sera from mice immunized with the purified GST-fusion protein.

FIG. 15A-C: For ORF23-1, (A) shows the results of affinity purification of the His-fusion protein, (B) shows the results of expression of the GST-fusion in E. coli, (C) shows the Western blot analysis of sera from mice immunized with the purified His-fusion protein.

FIG. 16A-E: For ORF25-1, (A) shows the results of affinity purification of the GST-fusion protein, (B) shows the results of expression of the His-fusion in E. coli, (C) shows the Western blot analysis of sera from mice immunized with purified His-fusion protein, (D) shows the FACS analysis, and (E) shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF25-1.

FIG. 17A-B: For ORF27-1, (A) shows the results of affinity purification of the GST-fusion protein, and (B) shows the results of expression of the His-fusion in E. coli.

FIG. 18A-B: For ORF79-1, (A) shows the results of affinity purification of the His-fusion protein, and (B) shows the FACS analysis of sera from mice immunized with purified His-fusion protein.

FIG. 19A-D: For ORF85a, (A) shows the results of affinity purification of the GST-fusion protein, (B) shows Western blot analysis of sera from mice immunized with purified GST-fusion protein, (C) shows FACS analysis, and (D) shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF85a.

FIG. 20A-C: For ORF132-1, (A) shows the results of affinity purification of the His-fusion protein, (B) shows the results of expression of the GST-fusion in E. coli, (C) shows the FACS analysis of sera from mice immunized with purified His-fusion protein.

EXAMPLES

The examples describe nucleic acid sequences which have been identified in N. meningitidis, along with their putative translation products, and also those of N. gonorrhoeae. Not all of the nucleic acid sequences are complete i.e. they encode less than the full-length wild-type protein.

The examples are generally in the following format:

• • a nucleotide sequence which has been identified in N. meningitidis (strain B)
  • the putative translation product of this sequence
  • a computer analysis of the translation product based on database comparisons
  • corresponding gene and protein sequences identified in N. meningitidis (strain A) and in N. gonorrhoeae
  • a description of the characteristics of the proteins which indicates that they might be suitably antigenic
  • results of biochemical analysis (expression, purification, ELISA, FACS etc.)
  • The examples typically include details of sequence identity between species and strains. Proteins that are similar in sequence are generally similar in both structure and function, and the sequence identity often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.

Sequence comparisons were performed at NCBI (http://www.ncbi.nlm.nih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, & tBLASTx [eg. see also Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Searches were performed against the following databases: non-redundant GenBank+EMBL+DDBJ+PDB sequences and non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR sequences.

To compare Meningococcal and Gonococcal sequences, the tBLASTx algorithm was used, as implemented at http://www.genome.ou.edu/gono_blast.html. The FASTA algorithm was also used to compare the ORFs (from GCG Wisconsin Package, version 9.0).

Dots within nucleotide sequences (eg. position 495 in SEQ ID 11) represent nucleotides which have been arbitrarily introduced in order to maintain a reading frame. In the same way, double-underlined nucleotides were removed. Lower case letters (eg. position 496 in SEQ ID 11) represent ambiguities which arose during alignment of independent sequencing reactions (some of the nucleotide sequences in the examples are derived from combining the results of two or more experiments).

Nucleotide sequences were scanned in all six reading frames to predict the presence of hydrophobic domains using an algorithm based on the statistical studies of Esposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) Eur J Biochem 190:207-219]. These domains represent potential transmembrane regions or hydrophobic leader sequences.

Open reading frames were predicted from fragmented nucleotide sequences using the program ORFFINDER (NCBI).

Underlined amino acid sequences indicate possible transmembrane domains or leader sequences in the ORFs, as predicted by the PSORT algorithm (http://www.psort.nibb.ac.jp). Functional domains were also predicted using the MOTIFS program (GCG Wisconsin & PROSITE).

Various tests can be used to assess the in vivo immunogencity of the proteins identified in the examples. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot. A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie. the protein is an immunogen. This method can also be used to identify immunodominant proteins.

The recombinant protein can also be conveniently used to prepare antibodies eg. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (eg. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.

In particular, the following methods (A) to (S) were used to express, purify and biochemically characterise the proteins of the invention:

A) Chromosomal DNA Preparation

N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% Sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension was incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one ChCl₃/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes ethanol, and was collected by centrifugation. The pellet was washed once with 70% ethanol and redissolved in 4 ml buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The DNA concentration was measured by reading the OD at 260 nm.

B) Oligonucleotide Design

Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using (a) the meningococcus B sequence when available, or (b) the gonococcus/meningococcus A sequence, adapted to the codon preference usage of meningococcus as necessary. Any predicted signal peptides were omitted, by deducing the 5′-end amplification primer sequence immediately downstream from the predicted leader sequence.

For most ORFs, the 5′ primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-NheI, or EcoRI-NheI, depending on the gene's own restriction pattern); the 3′ primers included a XhoI restriction site. This procedure was established in order to direct the cloning of each amplification product (corresponding to each ORF) into two different expression systems: pGEX-KG (using either BamHI-XhoI or EcoRI-XhoI), and pET21b+ (using either NdeI-XhoI or NheI-XhoI).

5′-end primer tail:	CGCGGATCCCATATG	(BamHI-NdeI)
	CGCGGATCCGCTAGC	(BamHI-NheI)
	CCGGAATTCTAGCTAGC	(EcoRI-NheI)
3′-end primer tail:	CCCGCTCGAG	(XhoI)

For ORFs 5, 15, 17, 19, 20, 22, 27, 28, 65 & 89, two different amplifications were performed to clone each ORF in the two expression systems. Two different 5′ primers were used for each ORF; the same 3′ XhoI primer was used as before:

5′-end primer tail:	GGAATTCCATATGGCCATGG	(NdeI)
5′-end primer tail:	CGGGATCC	(BamHI)

ORF 76 was cloned in the pTRC expression vector and expressed as an amino-terminus His-tag fusion. In this particular case, the predicted signal peptide was included in the final product. NheI-BamHI restriction sites were incorporated using primers:

5′-end primer tail:	GATCAGCTAGCCATATG	(NheI)
3′-end primer tail:	CGGGATCC	(BamHI)

As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridized to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was determined for each primer using the formulae:

T_m=4(G+C)+2(A+T) (tail excluded)

T_m=64.9+0.41(% GC)−600/N (whole primer)

The average melting temperature of the selected oligos were 65-70° C. for the whole oligo and 50-55° C. for the hybridising region alone.

Table I (page 487) shows the forward and reverse primers used for each amplification. In certain cases, it will be noted that the sequence of the primer does not exactly match the sequence in the ORF. When initial amplifications were performed, the complete 5′ and/or 3′ sequence was not known for some meningococcal ORFs, although the corresponding sequences had been identified in gonococcus. For amplification, the gonococcal sequences could thus be used as the basis for primer design, altered to take account of codon preference. In particular, the following codons were changed: ATA→ATT; TCG→TCT; CAG→CAA; AAG→AAA; GAG→GAA; CGA→CGC; CGG→CGC; GGG→GGC. Italicised nucleotides in Table I indicate such a change. It will be appreciated that, once the complete sequence has been identified, this approach is generally no longer necessary.

Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2 ml NH₄OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were then centrifuged and the pellets resuspended in either 100 μl or 1 ml of water. OD₂₆₀ was determined using a Perkin Elmer Lambda Bio spectophotometer and the concentration was determined and adjusted to 2-10 pmol/μl.

C) Amplification

The standard PCR protocol was as follows: 50-200 ng of genomic DNA were used as a template in the presence of 20-40 μM of each oligo, 400-800 μM dNTPs solution, 1×PCR buffer (including 1.5 mM MgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or Tahara Shuzo Taq polymerase).

In some cases, PCR was optimsed by the addition of 10 μl DMSO or 50 μl 2M betaine.

After a hot start (adding the polymerase during a preliminary 3 minute incubation of the whole mix at 95° C.), each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridization temperature the one of the oligos excluding the restriction enzymes tail, followed by 30 cycles performed according to the hybridization temperature of the whole length oligos. The cycles were followed by a final 10 minute extension step at 72° C.

The standard cycles were as follows:

	Denaturation	Hybridisation	Elongation
First 5 cycles	30 seconds	30 seconds	30-60 seconds
	95° C.	50-55° C.	72° C.
Last 30 cycles	30 seconds	30 seconds	30-60 seconds
	95° C.	65-70° C.	72° C.

The elongation time varied according to the length of the ORF to be amplified.

The amplifications were performed using either a 9600 or a 2400 Perkin Elmer GeneAmp PCR System. To check the results, 1/10 of the amplification volume was loaded onto a 1-1.5% agarose gel and the size of each amplified fragment compared with a DNA molecular weight marker.

The amplified DNA was either loaded directly on a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA fragment corresponding to the right size band was then eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the instructions of the manufacturer. The final volume of the DNA fragment was 30 μl or 50 μl of either water or 10 mM Tris, pH 8.5.

D) Digestion of PCR Fragments

The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with:

• • NdeI/XhoI or NheI/XhoI for cloning into pET-21b+ and further expression of the protein as a C-terminus His-tag fusion
  • BamHI/XhoI or EcoRI/XhoI for cloning into pGEX-KG and further expression of the protein as N-terminus GST fusion.
  • For ORF 76, NheI/BamHI for cloning into pTRC-H isA vector and further expression of the protein as N-terminus His-tag fusion.
  • EcoRI/PstI, EcoRI/SalI, SalI/PstI for cloning into pGex-His and further expression of the protein as N-terminus His-tag fusion

Each purified DNA fragment was incubated (37° C. for 3 hours to overnight) with 20 units of each restriction enzyme (New England Biolabs) in a either 30 or 40 μl final volume in the presence of the appropriate buffer. The digestion product was then purified using the QIAquick PCR purification kit, following the manufacturer's instructions, and eluted in a final volume of 30 or 50 μl of either water or 10 mM Tris-HCl, pH 8.5. The final DNA concentration was determined by 1% agarose gel electrophoresis in the presence of titrated molecular weight marker.

E) Digestion of the Cloning Vectors (pET22B, pGEX-KG, pTRC-His A, and pGex-His)

10 μg plasmid was double-digested with 50 units of each restriction enzyme in 200 μl reaction volume in the presence of appropriate buffer by overnight incubation at 37° C. After loading the whole digestion on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen QIAquick Gel Extraction Kit and the DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. The DNA concentration was evaluated by measuring OD₂₆₀ of the sample, and adjusted to 50 μg/μl. 1 μl of plasmid was used for each cloning procedure.

The vector pGEX-His is a modified pGEX-2T vector carrying a region encoding six histidine residues upstream to the thrombin cleavage site and containing the multiple cloning site of the vector pTRC99 (Pharmacia).

F) Cloning

The fragments corresponding to each ORF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20 μl, a molar ratio of 3:1 fragment/vector was ligated using 0.5 μl of NEB T4 DNA ligase (400 units/μl), in the presence of the buffer supplied by the manufacturer. The reaction was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Boheringer “Rapid Ligation Kit”, following the manufacturer's instructions.

In order to introduce the recombinant plasmid in a suitable strain, 100 μl E. coli DH5 competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37° C. for 3 minutes, then, after adding 800 μl LB broth, again at 37° C. for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorf microfuge and resuspended in approximately 200 μl of the supernatant. The suspension was then plated on LB ampicillin (100 mg/ml).

The screening of the recombinant clones was performed by growing 5 randomly-chosen colonies overnight at 37° C. in either 2 ml (pGEX or pTC clones) or 5 ml (pET clones) LB broth+100 μg/ml ampicillin. The cells were then pelletted and the DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, to a final volume of 30 μl. 5 μl of each individual miniprep (approximately 1 g) were digested with either NdeI/XhoI or BamHI/XhoI and the whole digestion loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 Kb DNA Ladder, GIBCO). The screening of the positive clones was made on the base of the correct insert size.

For the cloning of ORFs 110, 111, 113, 115, 119, 122, 125 & 130, the double-digested PCR product was ligated into double-digested vector using EcoRI-PstI cloning sites or, for ORFs 115 & 127, EcoRI-SalI or, for ORF 122, SalI-PstI. After cloning, the recombinant plasmids were introduced in the E. coli host W3110. Individual clones were grown overnight at 37° C. in L-broth with 50 μl/ml ampicillin.

G) Expression

Each ORF cloned into the expression vector was transformed into the strain suitable for expression of the recombinant protein product. 1 μl of each construct was used to transform 30 μl of E. coli BL21 (pGEX vector), E. coli TOP 10 (pTRC vector) or E. coli BL21-DE3 (pET vector), as described above. In the case of the pGEX-His vector, the same E. coli strain (W3110) was used for initial cloning and expression. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, making sure that the OD₆₀₀ ranged between 0.1 and 0.15. The flasks were incubated at 30° C. into gyratory water bath shakers until OD indicated exponential growth suitable for induction of expression (0.4-0.8 OD for pET and pTRC vectors; 0.8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, the protein expression was induced by addition of 1 mM IPTG, whereas in the case of pGEX system the final concentration of IPTG was 0.2 mM. After 3 hours incubation at 30° C., the final concentration of the sample was checked by OD. In order to check expression, 1 ml of each sample was removed, centrifuged in a microfuge, the pellet resuspended in PBS, and analysed by 12% SDS-PAGE with Coomassie Blue staining. The whole sample was centrifuged at 6000 g and the pellet resuspended in PBS for further use.

H) GST-Fusion Proteins Large-Scale Purification.

A single colony was grown overnight at 37° C. on LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid colture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600 ml of fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀ 0.8-1. Protein expression was induced with 0.2 mM IPTG followed by three hours incubation. The culture was centrifuged at 8000 rpm at 4° C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and centrifuged again. The supernatant was collected and mixed with 150 μl Glutatione-Sepharose 4B resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml cold PBS for 10 minutes, resuspended in 1 ml cold PBS, and loaded on a disposable column. The resin was washed twice with 2 ml cold PBS until the flow-through reached OD₂₈₀ of 0.02-0.06. The GST-fusion protein was eluted by addition of 700 μl cold Glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl) and fractions collected until the OD₂₈₀ was 0.1.21 μl of each fraction were loaded on a 12% SDS gel using either Biorad SDS-PAGE Molecular weight standard broad range (M1) (200, 116.25, 97.4, 66.2, 45, 31, 21.5, 14.4, 6.5 kDa) or Amersham Rainbow Marker (M2) (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26 kDa, this value must be added to the MW of each GST-fusion protein.

I) His-Fusion Solubility Analysis (ORFs 111-129)

To analyse the solubility of the His-fusion expression products, pellets of 3 ml cultures were resuspended in buffer M1 [500 μl PBS pH 7.2]. 25 μl lysozyme (10 mg/ml) was added and the bacteria were incubated for 15 min at 4° C. The pellets were sonicated for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and then separated again into pellet and supernatant by a centrifugation step. The supernatant was collected and the pellet was resuspended in buffer M2 [8M urea, 0.5M NaCl, 20 mM imidazole and 0.1 M NaH₂ PO₄] and incubated for 3 to 4 hours at 4° C. After centrifugation, the supernatant was collected and the pellet was resuspended in buffer M3 [6M guanidinium-HCl, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂PO₄] overnight at 4° C. The supernatants from all steps were analysed by SDS-PAGE.

The proteins expressed from ORFs 113, 119 and 120 were found to be soluble in PBS, whereas ORFs 111, 122, 126 and 129 need urea and ORFs 125 and 127 need guanidium-HCl for their solubilization.

J) His-Fusion Large-Scale Purification.

A single colony was grown overnight at 37° C. on a LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture and incubated overnight in a water bath shaker. Bacteria were diluted 1:30 into 600 ml fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀ 0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000 rpm at 4° C., the supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8) for soluble proteins or (ii) buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8) for insoluble proteins. The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed two times and centrifuged again.

For insoluble proteins, the supernatant was stored at −20° C., while the pellets were resuspended in 2 ml buffer C (6M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5) and treated in a homogenizer for 10 cycles. The product was centrifuged at 13000 rpm for 40 minutes.

Supernatants were collected and mixed with 150 μl Ni²⁺-resin (Pharmacia) (previously washed with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml buffer A or B for 10 minutes, resuspended in 1 ml buffer A or B and loaded on a disposable column. The resin was washed at either (i) 4° C. with 2 ml cold buffer A or (ii) room temperature with 2 ml buffer B, until the flow-through reached OD₂₈₀ of 0.02-0.06.

The resin was washed with either (i) 2 ml cold 20 mM imidazole buffer (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8) or (ii) buffer D (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3) until the flow-through reached the OD₂₈₀ of 0.02-0.06. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8) or (ii) elution buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5) and fractions collected until the OD₂₈₀ was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel.

K) His-Fusion Proteins Renaturation

10% glycerol was added to the denatured proteins. The proteins were then diluted to 20 μg/ml using dialysis buffer I (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4° C. for 12-14 hours. The protein was further dialysed against dialysis buffer II (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was evaluated using the formula:

Protein (mg/ml)=(1.55×OD₂₈₀)−(0.76×OD₂₆₀)

L) His-Fusion Large-Scale Purification (ORFs 111-129)

500 ml of bacterial cultures were induced and the fusion proteins were obtained soluble in buffer M1, M2 or M3 using the procedure described above. The crude extract of the bacteria was loaded onto a Ni-NTA superflow column (Quiagen) equilibrated with buffer M1, M2 or M3 depending on the solubilization buffer of the fusion proteins. Unbound material was eluted by washing the column with the same buffer. The specific protein was eluted with the corresponding buffer containing 500 mM imidazole and dialysed against the corresponding buffer without imidazole. After each run the columns were sanitized by washing with at least two column volumes of 0.5 M sodium hydroxide and reequilibrated before the next use.

M) Mice Immunisations

20 μg of each purified protein were used to immunise mice intraperitoneally. In the case of ORFs 2, 4, 15, 22, 27, 28, 37, 76, 89 and 97, Balb-C mice were immunised with Al(OH)₃ as adjuvant on days 1, 21 and 42, and immune response was monitored in samples taken on day 56. For ORFs 44, 106 and 132, CD1 mice were immunised using the same protocol. For ORFs 25 and 40, CD1 mice were immunised using Freund's adjuvant, rather than AL(OH)₃, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for ORFs 23, 32, 38 and 79, CD1 mice were immunised with Freund's adjuvant, but the immune response was measured on day 49.

N) ELISA Assay (Sera Analysis)

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 7 ml of Mueller-Hinton Broth (Difco) containing 0.25% Glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.3-0.4. The culture was centrifuged for 10 minutes at 10000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN₃ in PBS) were added to each well and the plates incubated for 90 minutes at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H₂O) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl H₂SO₄ was added to each well and OD₄₉₀ was followed. The ELISA was considered positive when OD₄₉₀ was 2.5 times the respective pre-immune sera.

O) FACScan Bacteria Binding Assay Procedure.

The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA, 0.4% NaN₃) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD₆₂₀ of 0.07. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytrin conjugated F(ab)₂ goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H threshold:92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539; compensation values: 0.

P) OMV Preparations

Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes and the bacteria disrupted by sonication for 10 minutes on ice (50% duty cycle, 50% output). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes and the total cell envelope fraction recovered by centrifugation at 50000 g at 4° C. for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in 2% sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, and the supernatant further ultracentrifuged at 50000 g for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in 10 mM Tris-HCl, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.

Q) Whole Extracts Preparation

Bacteria were grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.

R) Western Blotting

Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., in transferring buffer (0.3% Tris base, 1.44% glycine, 20% methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.

S) Bactericidal Assay

MC58 strain was grown overnight at 37° C. on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7 ml Mueller-Hinton broth. The suspension was incubated at 37° C. on a nutator and let to grow until OD₆₂₀ was 0.5-0.8. The culture was aliquoted into sterile 1.5 ml Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microfuge. The pellet was washed once in Gey's buffer (Gibco) and resuspended in the same buffer to an OD₆₂₀ of 0.5, diluted 1:20000 in Gey's buffer and stored at 25° C.

50 μl of Gey's buffer/]% BSA was added to each well of a 96-well tissue culture plate. 25 μl of diluted mice sera (1:100 in Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4° C. 25 μl of the previously described bacterial suspension were added to each well. 25 μl of either heat-inactivated (56° C. waterbath for 30 minutes) or normal baby rabbit complement were added to each well. immediately after the addition of the baby rabbit complement, 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 0). The 96-well plate was incubated for 1 hour at 37° C. with rotation and then 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 1). After overnight incubation the colonies corresponding to time 0 and time 1 hour were counted.

Table II (page 493) gives a summary of the cloning, expression and prurification results.

Example 1

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1>:

1   ATGAAACAGA CAGTCAA.AT GCTTGCCGCC GCCCTGATTG
    CCTTGGGCTT
51  GAACCGACCG GTGTGGNCGG ATGACGTATC GGATTTTCGG
    GAAAACTTGC
101 A.GCGGCAGC ACAGGGAAAT GCAGCAGCCC AATACAATTT
    GGGCGCAATG
151 TAT.TACAAA GGACGCGCGT GCGCCGGGAT GATGCTGAAG
    CGGTCAGATG
201 GTATCGGCAG CCGGCGGAAC AGGGGTTAGC CCAAGCCCAA
    TACAATTTGG
251 GCTGGATGTA TGCCAACGGG CGCGC.GTGC GCCAAGATGA
    TACCGAAGCG
301 GTCAGATGGT ATCGGCAGGC GGCAGCGCAG GGGGTTGTCC
    AAGCCCAATA
351 CAATTTGGGC GTGATATATG CCGAAGGACG TGGAGTGCGC
    CAAGACGATG
401 TCGAAGCGGT CAGATGGTTT CGGCAGGCGG CAGCGCAGGG
    GGTAGCCCAA
451 GCCCAAAACA ATTTGGGCGT GATGTATGCC GAAAGANCGC
    GCGTGCGCCA
501 AGACCG...

This corresponds to the amino acid sequence <SEQ ID 2; ORF37>:

1   MKQTVXMLAA ALIALGLNRP VWXDDVSDFR ENLXAAAQGN
    AAAQYNLGAM
51  YXQRTRVRRD DAEAVRWYRQ PAEQGLAQAQ YNLGWMYANG
    RXVRQDDTEA
101 VRWYRQAAAQ GVVQAQYNLG VIYAEGRGVR QDDVEAVRWF
    RQAAAQGVAQ
151 AQNNLGVMYA ERXRVRQD...

Further work revealed the complete nucleotide sequence <SEQ ID 3>:

1   ATGAAACAGA CAGTCAAATG GCTTGCCGCC GCCCTGATTG
    CCTTGGGCTT
51  GAACCGAGCG GTGTGGGCGG ATGACGTATC GGATTTTCGG
    GAAAACTTGC
101 AGGCGGCAGC ACAGGGAAAT GCAGCAGCCC AATACAATTT
    GGGCGCAATG
151 TATTACAAAG GACGCGGCGT GCGCCGGGAT GATGCTGAAG
    CGGTCAGATG
201 GTATCGGCAG GCGGCGGAAC AGGGGTTAGC CCAAGCCCAA
    TACAATTTGG
251 GCTGGATGTA TGCCAACGGG CGCGGCGTGC GCCAAGATGA
    TACCGAAGCG
301 GTCAGATGGT ATCGGCAGGC GGCAGCGCAG GGGGTTGTCC
    AAGCCCAATA
351 CAATTTGGGC GTGATATATG CCGAAGGACG TGGAGTGCGC
    CAAGACGATG
401 TCGAAGCGGT CAGATGGTTT CGGCAGGCGG CAGCGCAGGG
    GGTAGCCCAA
451 GCCCAAAACA ATTTGGGCGT GATGTATGCC GAAAGACGCG
    GCGTGCGCCA
501 AGACCGCGCC CTTGCACAAG AATGGTTTGG CAAGGCTTGT
    CAAAACGGAG
551 ACCAAGACGG CTGCGACAAT GACCAACGCC TGAAGGCGGG
    TTATTGA

This corresponds to the amino acid sequence <SEQ ID 4; ORF37-1>:

1   MKQTVKWLAA ALIALGLNRA VWADDVSDFR ENLQAAAQGN
    AAAQYNLGAM
51  YYKGRGVRRD DAEAVRWYRQ AAEQGLAQAQ YNLGWMYANG
    RGVRQDDTEA
101 VRWYRQAAAQ GVVQAQYNLG VIYAEGRGVR QDDVEAVRWF
    RQAAAQGVAQ
151 AQNNLGVMYA ERRGVRQDRA LAQEWFGKAC QNGDQDGCDN
    DQRLKAGY*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 5>:

1   ATGAAACAGA CAGTCAAATG GCTTGCCGCC GCCCTGATTG
    CCTTGGGCTT
51  GAACCAAGCG GTGTGGGCGG ATGACGTATC GGATTTTCGG
    GAAAACTTGC
101 AGGCGGCAGC ACAGGGAAAT GCAGCAGCCC AAAACAATTT
    GGGCGTGATG
151 TATGCCGAAA GACGCGGCGT GCGCCAAGAC CGCGCCCTTG
    CACAAGAATG
201 GCTTGGCAAG GCTTGTCAAA ACGGATACCA AGACAGCTGC
    GACAATGACC
251 AACGCCTGAA AGCGGGTTAT TGA

This encodes a protein having amino acid sequence <SEQ ID 6; ORF37a>:

1  MKQTVKWLAA ALIALGLNQA VWADDVSDFR ENLQAAAQGN
   AAAQNNLGVM
51 YAERRGVRQD RALAQEWLGK ACQNGYQDSC DNDQRLKAGY
   *

The originally-identified partial strain B sequence (ORF37) shows 68.0% identity over a 75aa overlap with ORF37a:

Further work identified the corresponding gene in N. gonorrhoeae <SEQ ID 7>:

1   ATGAAACAGA CAGTCAAATG GCTTGCCGCC GCCCTGATTG
    CCTTGGGCTT
51  GAACCAAGCG GTGTGGGCGG GTGACGTATC GGATTTTCGG
    GAAAACTTGC
101 AGgcggcaGA ACaggGAAAT GCAGCAGCCC AATTCAATTT
    GGGCGTGATG
151 TATGAAAATG GACAAGGAGT TCGTCAAGAT TATGTACAGG
    CAGTGCAGTG
201 GTATCGCAAG GCTTCAGAAC AAGGGGATGC CCAAGCCCAA
    TACAATTTGG
251 GCTTGATGTA TTACGATGGA CGCGGCGTGC GCCAAGACCT
    TGCGCTCGCT
301 CAACAATGGC TTGGCAAGGC TTGTCAAAAC GGAGACCAAA
    ACAGCTGCGA
351 CAATGACCAA CGCCTGAAGG CGGGTTATTA A

This encodes a protein having amino acid sequence <SEQ ID 8; ORF37ng>:

1   MKQTVKWLAA ALIALGLNQA VWAGDVSDFR ENLQAAEQGN
    AAAQFNLGVM
51  YENGQGVRQD YVQAVQWYRK ASEQGDAQAQ YNLGLMYYDG
    RGVRQDLALA
101 QQWLGKACQN GDQNSCDNDQ RLKAGY*

The originally-identified partial strain B sequence (ORF37) shows 64.9% identity over a 111aa overlap with ORF37ng:

The complete strain B sequence (ORF37-1) and ORF37ng show 51.5% identity in 198 aa overlap:

Computer analysis of these amino acid sequences indicates a putative leader sequence, and it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF37-1 (11 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 1A shows the results of affinity purification of the GST-fusion protein, and FIG. 1B shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for ELISA (positive result), FACS analysis (FIG. 1C), and a bactericidal assay (FIG. 1D). These experiments confirm that ORF37-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 1E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF37-1.

Example 2

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 9>:

TTCGGCGA CATCGGCGGT TTGAAGGTCA ATGCCCCCGT
CAAATCCGCA GGCGTATTGG TCGGGCGCGT CGGCGCTATC
GGACTTGACC CGAAATCCTA TCAGGCGAGG GTGCGCCTCG
ATTTGGACGG CAAGTATCAG TTCAGCAGCG ACGTTTCCGC
GCAAATCCTG ACTTCsGGAC TTTTGGGCGA GCAGTACATC
GGGCTGCAGC AGGGCGGCGA CACGGAAAAC CTTGCTGCCG
GCGACACCAT CTCCGTAACC AGTTCTGCAA TGGTTCTGGA
AAACCTTATC GGCAAATTCA TGACGAGTTT TGCCGAGAAA
AATGCCGACG GCGGCAATGC GGAAAAAGCC GCCGAATAA

This corresponds to the amino acid sequence <SEQ ID 10>:

1 FGDIGGLKVN APVKSAGVLV GRVGAIGLDP KSYQARVRLD LDGKYQFSSD
51 VSAQILTSGL LGEQYIGLQQ GGDTENLAAG DTISVTSSAM VLENLIGKFM
101 TSFAEKNADG GNAEKAAE*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Hypothetical H. influenzae Protein (ybrd.haein; Accession Number p45029)

SEQ ID 9 and ybrd.haein show 48.4% aa identity in 122 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

SEQ ID 9 shows 99.2% identity over a 118aa overlap with a predicted ORF from N. gonorrhoeae:

The complete yrbd H. influenzae sequence has a leader sequence and it is expected that the full-length homologous N. meningitidis protein will also have one. This suggests that it is either a membrane protein, a secreted protein, or a surface protein and that the protein, or one of its epitopes, could be a useful antigen for vaccines or diagnostics.

Example 3

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 11>:

1   ..ATTTTGATAT ACCTCATCCG CAAGAATCTA GGTTCGCCCG
    TCTTCTTCTT
51  TCAGGAACGC CCCGGAAAGG ACGGAAAACC TTTTAAAATG
    GTCAAATTCC
101 GTTCCATGCG CGACGGCTTG TATTCAGACG GCATTCCGCT
    GCCCGACGGA
151 GAACGCCTGA CACCGTTCGG CAAAAAACTG CGTGCCGcCA
    GTwTGGACGA
201 ACTGCCTGAA TTATGGAATA TCTTAAAAGG CGAGATGAGC
    CTGGTCGGCC
251 CCCGCCCGCT GCTGATGCAA TATCTGCCGC TGTACGACAA
    CTTCCAAAAC
301 CGCCGCCACG AAATGAAACC CGGCATTACC GGCTGGGCGC
    AGGTCAACGG
351 GCGCAACGCg CTTTCGTGGG ACGAAAAATT CGCCTGCGAT
    GTTTGGTATA
401 TCGACCACTT CAGCCTGTGC CTCGACATCA AAATCCTACT
    GCTGACGGTT
451 AAAAAAGTAT TAATCAAGGA AGGGATTTCC GCACAGGGCG
    AACA.aCCAT
501 GCCCCCTTTC ACAGGAAAAC GCAAACTCGC CGTCGTCGGT
    GCGGGCGGAC
551 ACGGAAAAGT CGTTGCCGAC CTTGCCGCCG CACTCGGCCG
    GTACAGGGAA
601 ATCGTTTTTC TGGACGACCG CGCACAAGGC AGCGTCAACG
    GCTTTTCCGT
651 CATCGGCACG ACGCTGCTGC TTGAAAACAG TTTATCGCCC
    GAACAATACG
701 ACGTCGCCGT CGCCGTCGGC AACAACCGCA TCCGCCGCCA
    AATCGCCGAA
751 AAAGCCGCCG CGCTCGGCTT CGCCCTGCCC GTACTGGTTC
    ATCCGGACGC
801 GACCGTCTCG CCTTCTGCAA CAGTCGGACA AGGCAGCGTC
    GTTATGGCGA
851 AAGCGGTCG..

This corresponds to the amino acid sequence <SEQ ID 12; ORF3>:

1   . . . ILIYLIRKNL GSPVFFFQER PGKDGKPFKM VKFRSMRDGL YSDGIPLPDG
51  ERLTPFGKKL RAASXDELPE LWNILKGEMS LVGPRPLLMQ YLPLYDNFQN
101 RRHEMKPGIT GWAQVNGRNA LSWDEKFACD VWYIDHFSLC LDIKILLLTV
151 KKVLIKEGIS AQGEXTMPPF TGKRKLAVVG AGGHGKVVAD LAAALGRYRE
201 IVFLDDRAQG SVNGFSVIGT TLLLENSLSP EQYDVAVAVG NNRIRRQIAE
251 KAAALGFALP VLVHPDATVS PSATVGQGSV VMAKAV . . .

Further sequence analysis revealed the complete nucleotide sequence <SEQ ID 13>:

1    ATGAGTAAAT TCTTCAAACG CCTGTTTGAC ATTGTTGCCT
     CCGCCTCGGG
51   ACTGATTTTC CTCTCGCCAG TATTTTTGAT TTTGATATAC
     CTCATCCGCA
101  AGAATCTAGG TTCGCCCGTC TTCTTCTTTC AGGAACGCCC
     CGGAAAGGAC
151  GGAAAACCTT TTAAAATGGT CAAATTCCGT TCCATGCGCG
     ACGCGCTTGA
201  TTCAGACGGC ATTCCGCTGC CCGACGGAGA ACGCCTGACA
     CCGTTCGGCA
251  AAAAACTGCG TGCCGCCAGT TTGGACGAAC TGCCTGAATT
     ATGGAATATC
301  TTAAAAGGCG AGATGAGCCT GGTCGGCCCC CGCCCGCTGC
     TGATGCAATA
351  TCTGCCGCTG TACGACAACT TCCAAAACCG CCGCCACGAA
     ATGAAACCCG
401  GCATTACCGG CTGGGCGCAG GTCAACGGGC GCAACGCGCT
     TTCGTGGGAC
451  GAAAAATTCG CCTGCGATGT TTGGTATATC GACCACTTCA
     GCCTGTGCCT
501  CGACATCAAA ATCCTACTGC TGACGGTTAA AAAAGTATTA
     ATCAAGGAAG
551  GGATTTCCGC ACAGGGCGAA GCCACCATGC CCCCTTTCAC
     AGGAAAACGC
601  AAACTCGCCG TCGTCGGTGC GGGCGGACAC GGAAAAGTCG
     TTGCCGACCT
651  TGCCGCCGCA CTCGGCCGGT ACAGGGAAAT CGTTTTTCTG
     GACGACCGCG
701  CACAAGGCAG CGTCAACGGC TTTTCCGTCA TCGGCACGAC
     GCTGCTGCTT
751  GAAAACAGTT TATCGCCCGA ACAATACGAC GTCGCCGTCG
     CCGTCGGCAA
801  CAACCGCATC CGCCGCCAAA TCGCCGAAAA AGCCGCCGCG
     CTCGGCTTCG
851  CCCTGCCCGT TCTGGTTCAT CCGGACGCGA CCGTCTCGCC
     TTCTGCAACA
901  GTCGGACAAG GCAGCGTCGT TATGGCGAAA GCCGTCGTAC
     AGGCAGGCAG
951  CGTATTGAAA GACGGCGTGA TTGTGAACAC TGCCGCCACC
     GTCGATCACG
1001 ACTGCCTGCT TAACGCTTTC GTCCACATCA GCCCAGGCGC
     GCACCTGTCG
1051 GGCAACACGC ATATCGGCGA AGAAAGCTGG ATAGGCACGG
     GCGCGTGCAG
1101 CCGCCAGCAG ATCCGTATCG GCAGCCGCGC AACCATTGGA
     GCGGGCGCAG
1151 TCGTCGTACG CGACGTTTCA GACGGCATGA CCGTCGCGGG
     CAATCCGGCA
1201 AAGCCGCTGC CGCGCAAAAA CCCCGAGACC TCGACAGCAT
     AA

This corresponds to the amino acid sequence <SEQ ID 14; ORF3-1>:

1   MSKFFKRLFD IVASASGLIF LSPVFLILIY LIRKNLGSPV
    FFFQERPGKD
51  GKPFKMVKFR SMRDALDSDG IPLPDGERLT PFGKKLRAAS
    LDELPELWNI
101 LKGEMSLVGP RPLLMQYLPL YDNFQNRRHE MKPGITGWAQ
    VNGRNALSWD
151 EKFACDVWYI DHFSLCLDIK ILLLTVKKVL IKEGISAQGE
    ATMPPFTGKR
201 KLAVVGAGGH GKVVADLAAA LGRYREIVFL DDRAQGSVNG
    FSVIGTTLLL
251 ENSLSPEQYD VAVAVGNNRI RRQIAEKAAA LGFALPVLVH
    PDATVSPSAT
301 VGQGSVVMAK AVVQAGSVLK DGVIVNTAAT VDHDCLLNAF
    VHISPGAHLS
351 GNTHIGEESW IGTGACSRQQ IRIGSRATIG AGAVVVRDVS
    DGMTVAGNPA
401 KPLPRKNPET STA*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF3 shows 93.0% identity over a 286aa overlap with an ORF (ORF3a) from strain A of N. meningitidis:

The complete length ORF3a nucleotide sequence <SEQ ID 15> is:

1    ATGAGTAAAT TCTTCAAACG CCTGTTTGAC ATTGTTGCCT
     CCGCCTCGGG
51   ACTGATTTTC CTCTCGCCAG TATTTTTGAT TTTGATATAC
     CTCATCCGCA
101  AGAATCTGGG TTCGCCCGTC TTCTTCTTTC AGGAACGCCC
     CGGAAAGGAC
151  GGAAAACCTT TTAAAATGGT CAAATTCCGT TCCATGCACG
     ACGCGCTTGA
201  TTCAGACGGC ATTCTGCTGC CCGACGGAGA ACGCCTGACA
     CCGTTCGGCA
251  AAAAACTGCG TGCCGCCAGT TTGGACGAAC TGCCCGAACT
     GTGGAACGTC
301  CTCAAAGGCG ACATGAGCCT GGTCGGCCCC CGCCCGCTGC
     TGATGCAATA
351  TCTGCCGCTG TACGACAACT TCCAAAACCG CCGCCACGAA
     ATGAAACCGG
401  GCATTACCGG CTGGGCGCAG GTCAACGGGC GCAACGCGCT
     TTCGTGGGAC
451  GAACGCTTCG CATGCGACAT CTGGTATATC GACCACTTCA
     GCCTGTGCCT
501  CGAGATCAAA ATCCTACTGC TGACGGTTAA AAAAGTATTA
     ATCAAAGAAG
551  GGATTTCCGC ACAGGGCGAA GCCACCATGC CCCCTTTCAC
     AGGAAAACGC
601  AAACTTGCCG TCGTCGGTGC GGGCGGACAC GGCAAAGTCG
     TTGCCGAGCT
651  TGCCGCCGCA CTCGGCACAT ACGGCGAAAT CGTTTTTCTG
     GACGACCGCG
701  TCCAAGGCAG CGTCAACGGC TTCCCCGTCA TCGGCACGAC
     GCTGCTGCTT
751  GAAAACAGTT TATCGCCCGA ACAATTCGAC ATCGCCGTCG
     CCGTCGGCAA
801  CAACCGCATC CGCCGCCAAA TCGCCGAAAA AGCCGCCGCG
     CTCGGCTTCG
851  CCCTGCCCGT CCTGATTCAT CCGGACTCGA CCGTCTCGCC
     TTCTGCAACA
901  GTCGGACAAG GCGGCGTCGT TATGGCGAAA GCCGTCGTAC
     AGGCTGACAG
951  CGTATTGAAA GACGGCGTAA TTGTGAACAC TGCCGCCACC
     GTCGATCACG
1001 ATTGCCTGCT TGATGCTTTC GTCCACATCA GCCCGGGCGC
     GCACCTGTCG
1051 GGCAACACGC GTATCGGCGA AGAAAGCTGG ATAGGCACAG
     GCGCGTGCAG
1101 CCGCCAGCAG ATCCGTATCG GCAGCCGCGC AACCATTGGA
     GCGGGCGCAG
1151 TCGTCGTGCG CGACGTTTCA GACGGCATGA CCGTCGCGGG
     CAACCCGGCA
1201 AAACCATTGG CAGGCAAAAA TACCGAGACC CTGCGGTCGT
     AA

This is predicted to encode a protein having amino acid sequence <SEQ ID 16>:

1   MSKFFKRLFD IVASASGLIF LSPVFLILIY LIRKNLGSPV
    FFFQERPGKD
51  GKPFKMVKFR SMHDALDSDG ILLPDGERLT PFGKKLRAAS
    LDELPELWNV
101 LKGDMSLVGP RPLLMQYLPL YDNFQNRRHE MKPGITGWAQ
    VNGRNALSWD
151 ERFACDIWYI DHFSLCLDIK ILLLTVKKVL IKEGISAQGE
    ATMPPFTGKR
201 KLAVVGAGGH GKVVAELAAA LGTYGEIVFL DDRVQGSVNG
    FPVIGTTLLL
251 ENSLSPEQFD IAVAVGNNRI RRQIAEKAAA LGFALPVLIH
    PDSTVSPSAT
301 VGQGGVVMAK AVVQADSVLK DGVIVNTAAT VDHDCLLDAF
    VHISPGAHLS
351 GNTRIGEESW IGTGACSRQQ IRIGSRATIG AGAVVVRDVS
    DGMTVAGNPA
401 KPLAGKNTET LRS*

Two transmembrane domains are underlined.

ORF3-1 shows 94.6% identity in 410 aa overlap with ORF3a:

Homology with Hypothetical Protein Encoded by yvfc Gene (Accession Z71928) of B. subtilis

ORF3 and YVFC proteins show 55% aa identity in 170 aa overlap (BLASTp):

ORF3	3	IYLIRKNLGSPVFFFQERPGKDGKPFKMVKFRSMRDGLYSDGIPLPDGERLTPFGKKLRA	62
		I ++R  +GSPVFF Q RPG  GKPF + KFR+M D   S G  LPD  RLT  G+ +R
yvfc	27	IAVVRLKIGSPVFFKQVRPGLHGKPFTLYKFRTMTDERDSKGNLLPDEVRLTKTGRLIRK	86
ORF3	63	ASXDELPELWNILKGEMSLVGPRPLLMQYLPLYDNFQNRRHEMKPGITGWAQVNGRNALS	122
		S DELP+L N+LKG++SLVGPRPLLM YLPLY   Q RRHE+KPGITGWAQ+NGRNA+S
yvfc	87	LSIDELPQLLNVLKGDLSLVGPRPLLMDYLPLYTEKQARRHEVKPGITGWAQINGRNAIS	146
ORF3	123	WDEKFACDVWYIDHFSLCLDXXXXXXXXXXXXXXEGISAQGEXTMPPFTG	172
		W++KF  DVWY+D++S  LD              EGI      T   FTG
yvfc	147	WEKKFELDVWYVDNWSFFLDLKILCLTVRKVLVSEGIQQTNHVTAERFTG	196

Homology with a Predicted ORF from N. gonorrhoeae

ORF3 shows 86.3% identity over a 286aa overlap with a predicted ORF (ORF3.ng) from N. gonorrhoeae:

The complete length ORF3ng nucleotide sequence <SEQ ID 17> is:

1    ATGAGTAAAG CCGTCAAACG CCTGTTCGAC ATCATCGCAT
     CCGCATCGGG
51   GCTGATTGTC CTGTCGCCCG TGTTTTTGGT TTTAATATAC
     CTCATCCGCA
101  AAAACTTAGG TTCGCCCGTC TTCTTCattC GGGAACGCCc
     cgGAAAGGAc
151  ggaaaacCTT TTAAAATGGT CAAATTCCGT TCCAtgcgcg
     acgcgcttGA
201  TTCAGACGGC ATTCCGCTGC CCGATAGCGA ACGCCTGACC
     GATTTCGGCA
251  AAAAATTACG CGCCACCAGT TTGGACGAAC TTCCTGAATT
     ATGGAATGTC
301  CTCAAAGGCG AGATGAGCCT GGTCGGCCCC CGCCCGCTTT
     TGATGCAGTA
351  TCTGCCGCTT TACAACAAAT TTCAAAACCG CCGCCACGAA
     ATGAAACCGG
401  GCATTACCGG CTGGGCGCAG GTCAACGGGC GCAACGCGCT
     TTCGTGGGAC
451  GAAAAGTTCT CCTGCGATGT TTGGTACACC GACAATTTCA
     GCTTTTGGCT
501  GGATATGAAA ATCCTGTTTC TGACAGTCAA AAAAGTCTTG
     ATTAAAGAAG
551  GCATTTCGGC GCAAGGGGAA GCCACCATGC CCCCTTTCGC
     GGGGAATCGC
601  AAACTCGCCG TTATCGGCGC GGGCGGACAC GGCAAAGTCG
     TTGCCGAGCT
651  TGCCGCCGCA CTCGGCACAT ACGGCGAAAT CGTTTTTCTG
     GACGACCGCA
701  CCCAAGGCAG CGTCAACGGC TTCCCCGTCA TCGGCACGAC
     GCTGCTGCTT
751  GAAAACAGTT TATCGCCCGA ACAATTCGAC ATCACCGTCG
     CCGTCGGCAA
801  CAACCGCATC CGCCGCCAAA TCACCGAAAA CGCCGCCGCG
     CTCGGCTTCA
851  AACTGCCCGT TCTGATTCAT CCCGACGCGA CCGTCTCGCC
     TTCTGCAATA
901  ATCGGACAAG GCAGCGTCGT AATGGCGAAA GCCGTCGTAC
     AGGCCGGCAG
951  CGTATTGAAA GACGGCGTGA TTGTGAACAC TGCCGCCACC
     GTCGATCACG
1001 ACTGCCTGCT TGACGCTTTC GtccaCATCA GCCCGGGCGC
     GCACCTGTCG
1051 GGCAACACGC GTATCGGCGA AGAAAGCCGG ATAGGCACGG
     GCGCGTGCAG
1101 CCGCCAGCAG ACAACCGTCG GCAGCGGGGT TACCgccgGT
     GCAGGGgcGG
1151 TTATCGTATG CGACATCCCG GACGGCATGA CCGTCGCGGG
     CAACCCGGCA
1201 AAGCCCCTTA CGGGCAAAAA CCCCAAGACC GGGACGGCAT
     AA

This encodes a protein having amino acid sequence <SEQ ID 18>:

1   MSKAVKRLFD IIASASGLIV LSPVFLVLIY LIRKNLGSPV
    FFIRERPGKD
51  GKPFKMVKFR SMRDALDSDG IPLPDSERLT DFGKKLRATS
    LDELPELWNV
101 LKGEMSLVGP RPLLMQYLPL YNKFQNRRHE MKPGITGWAQ
    VNGRNALSWD
151 EKFSCDVWYT DNFSFWLDMK ILFLTVKKVL IKEGISAQGE
    ATMPPFAGNR
201 KLAVIGAGGH GKVVAELAAA LGTYGEIVFL DDRTQGSVNG
    FPVIGTTLLL
251 ENSLSPEQFD ITVAVGNNRI RRQITENAAA LGFKLPVLIH
    PDATVSPSAI
301 IGQGSVVMAK AVVQAGSVLK DGVIVNTAAT VDHDCLLDAF
    VHISPGAHLS
351 GNTRIGEESR IGTGACSRQQ TTVGSGVTAG AGAVIVCDIP
    DGMTVAGNPA
401 KPLTGKNPKT GTA*

This protein shows 86.9% identity in 413 aa overlap with ORF3-1:

In addition, ORF3ng shows significant homology with a hypothetical protein from B. subtilis:

gnl|PID|e238668 (Z71928) hypothetical protein [Bacillus subtilis]
>gi|1945702|gnl|PID|e313004 (Z94043) hypothetical protein
[Bacillus subtilis]
>gi|2635938|gnl|PID|e1186113 (Z99121) similar to capsular polysaccharide
biosynthesis [Bacillus subtilis] Length = 202
Score = 235 bits (594), Expect = 3e−61
Identities = 114/195 (58%), Positives = 142/195 (72%)
Query:	5	VKRLFDIIASASGLIVLSPVFLVLIYLIRKNLGSPVFFIRERPGKDGKPFKMVKFRSMRD	64
		+KRLFD+ A+   L  S + L  I ++R  +GSPVFF + RPG  GKPF + KFR+M D
Sbjct:	3	LKRLFDLTAAIFLLCCTSVIILFTIAVVRLKIGSPVFFKQVRPGLHGKPFTLYKFRTMTD	62
Query:	65	ALDSDGIPLPDSERLTDFGKKLRATSLDELPELWNVLKGEMSLVGPRPLLMQYLPLYNKF	124
		DS G  LPD  RLT  G+ +R  S+DELP+L NVLKG++SLVGPRPLLM YLPLY +
Sbjct:	63	ERDSKGNLLPDEVRLTKTGRLIRKLSIDELPQLLNVLKGDLSLVGPRPLLMDYLPLYTEK	122
Query:	125	QNRRHEMKPGITGWAQVNGRNALSWDEKFSCDVWYTDNFSFWLDMKILFLTVKKVLIKEG	184
		Q RRHE+KPGITGWAQ+NGRNA+SW++KF  DVWY DN+SF+LD+KIL LTV+KVL+ EG
Sbjct:	123	QARRHEVKPGITGWAQINGRNAISWEKKFELDVWYVDNWSFFLDLKILCLTVRKVLVSEG	182
Query:	185	ISAQGEATMPPFAGN	199
		I      T   F G+
Sbjct:	183	IQQTNHVTAERFTGS	197

The hypothetical product of yvfc gene shows similarity to EXOY of R. meliloti, an exopolysaccharide production protein. Based on this and on the two predicted transmembrane regions in the homologous N. gonorrhoeae sequence, it is predicted that these proteins, or their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 4

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 19>:

1   ..AACCATATGG CGATTGTCAT CGACGAATAC GGCGGCACAT
    CCGGCTTGGT
51  CACCTTTGAA GACATCATCG AGCAAATCGT CGGCGAAATC
    GAAGACGAGT
101 TTGACGAAGA CGATAGCGCC GACAATATCC ATGCCGTTTC
    TTCAGACACG
151 TGGCGCATCC ATGCAGCTAC CGAAATCGAA GACATCAACA
    CCTTCTTCGG
201 CACGGAATAC AGCATCGAAG AAGCCGACAC CATT.GGCGG
    CCTGGTCATT
251 CAAGAGTTGG GACATCTGCC CGTGCGCGGC GAAAAAGTCC
    TTATCGGCGG
301 TTTGCAGTTC ACCGTCGCAC GCGCCGACAA CCGCCGCCTG
    CATACGCTGA
351 TGGCGACCCG CGTGAAGTAA GC........ .....ACCGC
    CGTTTCTGCA
401 CAGTTTAG

This corresponds to amino acid sequence <SEQ ID 20; ORF5>:

1   ..NHMAIVIDEY GGTSGLVTFE DIIEQIVGEI EDEFDEDDSA
    DNIHAVSSDT
51  WRIHAATEIE DINTFFGTEY SIEEADTIXR PGHSRVGTSA
    RARRKSPYRR
101 FAVHRRTRRQ PPPAYADGDP REVS....XR RFCTV*

Further sequence analysis revealed the complete DNA sequence to be <SEQ ID 21>:

1   ATGGACGGCG CACAACCGAA AACGAATTTT TTTGAACGCC
    TGATTGCCCG
51  ACTCGCCCGC GAACCCGATT CCGCCGAAGA CGTATTAAAC
    CTGCTTCGGC
101 AGGCGCACGA GCAGGAAGTT TTTGATGCGG ATACGCTTTT
    AAGATTGGAA
151 AAAGTCCTCG ATTTTTCCGA TTTGGAAGTG CGCGACGCGA
    TGATTACGCG
201 CAGCCGTATG AACGTTTTAA AAGAAAACGA CAGCATCGAG
    CGCATCACCG
251 CCTACGTTAT CGATACCGCC CATTCGCGCT TCCCCGTCAT
    CGGCGAAGAC
301 AAAGACGAAG TTTTGGGCAT TTTGCACGCC AAAGACCTGC
    TCAAATATAT
351 GTTTAACCCC GAGCAGTTCC ACCTCAAATC CATTCTCCGC
    CCCGCCGTCT
401 TCGTCCCCGA AGGCAAATCG CTGACCGCCC TTTTAAAAGA
    GTTCCGCGAA
451 CAGCGCAACC ATATGGCGAT TGTCATCGAC GAATACGGCG
    GCACATCCGG
501 CTTGGTCACC TTTGAAGACA TCATCGAGCA AATCGTCGGC
    GAAATCGAAG
551 ACGAGTTTGA CGAAGACGAT AGCGCCGACA ATATCCATGC
    CGTTTCTTCC
601 GAACGCTGGC GCATCCATGC AGCTACCGAA ATCGAAGACA
    TCAACACCTT
651 CTTCGGCACG GAATACAGCA GCGAAGAAGC CGACACCATT
    CGGCCTGGTC
701 ATTCAAGAGT TGGGACATCT GCCCGTGCGC GGCGAAAAAG
    TCCTTATCGG
751 CGGTTTGCAG TTCACCGTCG CACGCGCCGA CAACCGCCGC
    CTGCATACGC
801 TGATGGCGAC CCGCGTGAAG TAAGCACCGC CGTTTCTGCA
    CAGTTTAGGA
851 TGACGGTACG GGCGTTTTCT GTTTCAATCC GCCCCATCCG
    CCAAACATAA

This corresponds to amino acid sequence <SEQ ID 22; ORF5-1>:

1   MDGAQPKTNF FERLIARLAR EPDSAEDVLN LLRQAHEQEV
    FDADTLLRLE
51  KVLDFSDLEV RDAMITRSRM NVLKENDSIE RITAYVIDTA
    HSRFPVIGED
101 KDEVLGILHA KDLLKYMFNP EQFHLKSILR PAVFVPEGKS
    LTALLKEFRE
151 QRNHMAIVID EYGGTSGLVT FEDIIEQIVG EIEDEFDEDD
    SADNIHAVSS
201 ERWRIHAATE IEDINTFFGT EYSSEEADTI RPGHSRVGTS
    ARARRKSPYR
251 RFAVHRRTRR QPPPAYADGD PREVSTAVSA QFRMTVRAFS
    VSIRPIRQT*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 23>:

1   ATGGACGGCG CACAACCGAA AACAAATTTT TTNNAACGCC
    TGATTGCCCG
51  ACTCGCCCGC GAACCCGATT CCGCCGAAGA CGTATTGACC
    CTGTTGCGCC
101 AAGCGCACGA ACAGGAAGTA TTTGATGCGG ATACGCTTTT
    AAGATTGGAA
151 AAAGTCCTCG ATTTTTCTGA TTTGGAAGTG CGCGACGCGA
    TGATTACGCG
201 CAGCCGTATG AACGTTTTAA AAGAAAACGA CAGCATCGAA
    CGCATCACCG
251 CCTACGTTAT CGATACCGCC CATTCGCGCT TCCCCGTCAT
    CGGTGAAGAC
301 AAAGACGAAG TTTTGGGTAT TTTGCACGCC AAAGACCTGC
    TCAAATATAT
351 GTTCAACCCC GAGCAGTTCC ACCTCAAATC GATATTGCGC
    CCTGCCGTCT
401 TCGTCCCCGA AGGCAAATCG CTGACCGCCC TTTTAAAAGA
    GTTCCGCGAA
451 CAGCGCAACC ATATGGCAAT CGTCATCGAC GAATACGGCG
    GCACGTCGGG
501 TTTGGTAACT TTTGAAGACA TCATCGAGCA AATCGTCGGC
    GACATCGAAG
551 ATGAGTTTGA CGAAGACGAA AGCGCGGACA ACATCCACGC
    CGTTTCCGCC
601 GAACGCTGGC GCATCCACGC GGCTACCGAA ATCGAAGACA
    TCAACGCCTT
651 TTTCGGCACG GAATACAGCA GCGAAGAAGC CGACACCATC
    GGCGGCCNTG
701 GTCATTCAGG AATTGGNACA CCTGCCCGTG CGCGGCGAAA
    AAGTCNTTAT
751 CGGCGNNTTG CANTTCACNG TCGCCNGCGC NGACAACCGC
    CGCCTGCATA
801 CGCTGATGGC GACCCGCGTG AAGTAAGCTC CGCCGTTTCT
    GTACAGTTTA
851 GGATGACGGT ACGGGCGTTT TCTGTTTCAA TCCGCCCCAT
    CCGCCANACA
901 TAA

This encodes a protein having amino acid sequence <SEQ ID 24; ORF5a>:

1   MDGAQPKTNF XXRLIARLAR EPDSAEDVLT LLRQAHEQEV
    FDADTLLRLE
51  KVLDFSDLEV RDAMITRSRM NVLKENDSIE RITAYVIDTA
    HSRFPVIGED
101 KDEVLGILHA KDLLKYMFNP EQFHLKSILR PAVFVPEGKS
    LTALLKEFRE
151 QRNHMAIVID EYGGTSGLVT FEDIIEQIVG DIEDEFDEDE
    SADNIHAVSA
201 ERWRIHAATE IEDINAFFGT EYSSEEADTI GGXGHSGIGT
    PARARRKSXY
251 RRXAXHXRXR XQPPPAYADG DPREVSSAVS VQFRMTVRAF
    SVSIRPIRXT
301 *

The originally-identified partial strain B sequence (ORF5) shows 54.7% identity over a 124aa overlap with ORF5a:

The complete strain B sequence (ORF5-1) and ORF5a show 92.7% identity in 300 aa overlap:

Further work identified the a partial DNA sequence in N. gonorrhoeae <SEQ ID 25> which encodes a protein having amino acid sequence <SEQ ID 26; ORF5ng>:

1   MDGAQPKTNF FERLIARLAR EPDSAEDVLN LLRQAHEQEV
    FDADTLTRLE
51  KVLDFAELEV RDAMITRSRM NVLKENDSIE RITAYVIDTA
    HSRFPVIGED
101 KDEVLGILHA KDLLKYMFNP EQFHLKSVLR PAVFVPEGKS
    LTALLKEFRE
151 QRNHMAIVID EYGGTSGLVT FEDIIEQIVG DIEDEFDEDE
    SADDIHSVSA
201 ERWRIHAATE IEDINAFFGT EYGSEEADTI RRLGHSGIGT
    PARARRKSPY
251 RRFAVHRRPR RQPPPAHADG DPREVSRACP HRRFCTV*

Further analysis revealed the complete gonococcal nucleotide sequence <SEQ ID 27> to be:

1   ATGGACGGCG CACAACCGAA AACAAATTTT TTTGAACGCC
    TGATTGCCCG
51  ACTCGCCCGC GAACCCGATT CCGCCGAAGA CGTATTAAAC
    CTGCTTCGGC
101 AGGCGCACGA ACAGGAAGTT TTTGATGCCG ACACACTGAC
    CCGGCTGGAA
151 AAAGTATTGG ACTTTGCCGA GCTGGAAGTG CGCGATGCGA
    TGATTACGCG
201 CAGCCGCATG AACGTATTGA AAGAAAACGA CAGCATCGAA
    CGCATCACCG
251 CCTACGTCAT CGATACCGCC CATTCGCGCT TCCCCGTCAT
    CGGCGAAGAC
301 AAAGACGAAG TTTTGGGCAT TTTGCACGCC AAAGACCTGC
    TCAAATATAT
351 GTTCAACCCC GAGCAGTTCC ACCTGAAATC CGTCTTGCGC
    CCTGCCGTTT
401 TCGTGCCCGA AGGCAAATCT TTGACCGCCC TTTTAAAAGA
    GTTCCGCGAA
451 CAGCGCAACC ATATGGCAAT CGTCATCGAC GAATACGGCG
    GCACGTCGGG
501 TTTGGTCACC TTTGAAGACA TCATCGAGCA AATCGTCGGT
    GACATCGAAG
551 ACGAGTTTGA CGAAGACGAA AGCGccgacg acatCCACTC
    cgTTTccgCC
601 GAACGCTGGC GCATCCacgc ggctaCCGAA ATCGAAGaca
    TCAACGCCTT
651 TTTCGGTACG GAatacggca gcgaagaagc cgacaccatc
    cggcggctTG
701 GTCATTCAGG AATTGGGACA CCTGCCCGTG CGCGGCGAAA
    AAGTCCTTAt
751 cggcgGTTTG Cagttcaccg tCGCCCGCGC CGACAACCGC
    CGCCTGCACA
801 CGCTGATGGC GACCCGCGTG AAGTAAGCAG AGCCTGCCcg
    AccgccgttT
851 CTGCacAGTT TAGGatgACG gtaCGGTCGT TTTCTGTTTC
    AATCCGCCCC
901 ATCCGCCAAA CATAA

This encodes a protein having amino acid sequence <SEQ ID 28; ORF5ng-1>:

1   MDGAQPKTNF FERLIARLAR EPDSAEDVLN LLRQAHEQEV
    FDADTLTRLE
51  KVLDFAELEV RDAMITRSRM NVLKENDSIE RITAYVIDTA
    HSRFPVIGED
101 KDEVLGILHA KDLLKYMFNP EQFHLKSVLR PAVFVPEGKS
    LTALLKEFRE
151 QRNHMAIVID EYGGTSGLVT FEDIIEQIVG DIEDEFDEDE
    SADDIHSVSA
201 ERWRIHAATE IEDINAFFGT EYGSEEADTI RRLGHSGIGT
    PARARRKSPY
251 RRFAVHRRPR RQPPPAHADG DPREVSRACP TAVSAQFRMT
    VRSFSVSIRP
301 IRQT*

The originally-identified partial strain B sequence (ORFS) shows 83.1% identity over a 135aa overlap with the partial gonococcal sequence (ORF5ng):

The complete strain B and gonococcal sequences (ORFS-1 & ORF5ng-1) show 92.4% identity in 304 aa overlap:

Computer analysis of these amino acid sequences indicates a putative leader sequence, and identified the following homologies:

Homology with Hemolysin Homolog TlyC (Accession U32716) of H. influenzae

ORF5 and TlyC proteins show 58% aa identity in 77 aa overlap (BLASTp).

ORF5	2	HMAIVIDEYGGTSGLVTFEDIIEQIVGEIEDEFDEDDSADNIHAVSSDTWRIHAATEIED	61
		HMAIV+DE+G  SGLVT EDI+EQIVG+IEDEFDE++ AD I  +S  T+ + A T+I+D
TlyC	166	HMAIVVDEFGAVSGLVTIEDILEQIVGDIEDEFDEEEIAD-IRQLSRHTYAVRALTDIDD	224
ORF5	62	INTFFGTEYSIEEADTI	78
		N  F T++  EE DTI
TlyC	225	FNAQFNTDFDDEEVDTI	241

ORF5ng-1 also shows significant homology with TlyC:

• • SCORES Init1: 301 Initn: 419 Opt: 668
  • Smith-Waterman score: 668; 45.9% identity in 242 aa overlap

Homology with a Hypothetical Secreted Protein from E. coli:

ORF5a shows homology to a hypothetical secreted protein from E. coli:

sp|P77392|YBEX_ECOLI HYPOTHETICAL 33.3 KD PROTEIN IN CUTE-ASNB INTERGENIC
REGION
>gi|1778577 (U82598) similar to H. influenzae [Escherichia coli]
>gi|1786879 (AE000170) f292; This 292 aa ORF is 23% identical (9 gaps)
to 272 residues of an approx. 440 aa protein YTFL_HAEIN SW: P44717
[Escherichia coli] Length = 292
Score = 212 bits (533), Expect = 3e−54
Identities = 112/230 (48%), Positives = 149/230 (64%), Gaps = 3/230 (1%)
Query:	2	DGAQPKTNFXXRLIARLAR-EPDSAEDVLTLLRQAHEQEVFDADTLLRLEKVLDFSDLEV	60
		D    K  F   L+++L   EP + +++L L+R + + ++ D DT   LE V+D +D  V
Sbjct:	10	DTISNKKGFFSLLLSQLFHGEPKNRDELLALIRDSGQNDLIDEDTRDMLEGVMDIADQRV	69
Query:	61	RDAMITRSRMNVLKENDSIERITAYVIDTAHSRFPVIGEDKDEVLGILHAKDLLKYM-FN	119
		RD MI RS+M  LK N +++     +I++AHSRFPVI EDKD + GIL AKDLL +M  +
Sbjct:	70	RDIMIPRSQMITLKRNQTLDECLDVIIESAHSRFPVISEDKDHIEGILMAKDLLPFMRSD	129
Query:	120	PEQFHLKSILRPAVFVPEGKSLTALLKEFREQRNHMAIVIDEYGGTSGLVTFEDIIEQIV	179
		E F +  +LR AV VPE K +  +LKEFR QR HMAIVIDE+GG SGLVT EDI+E IV
Sbjct:	130	AEAFSMDKVLRQAVVVPESKRVDRMLKEFRSQRYHMAIVIDEFGGVSGLVTIEDILELIV	189
Query:	180	GDIEDEFDEDESADNIHAVSAERWRIHAATEIEDINAFFGTEYSSEEADT	229
		G+IEDE+DE++  D    +S   W + A   IED N  FGT +S EE DT
Sbjct:	190	GEIEDEYDEEDDID-FRQLSRHTWTVRALASIEDFNEAFGTHFSDEEVDT	238

Based on this analysis, including the amino acid homology to the TlyC hemolysin-homologue from H. influenzae (hemolysins are secreted proteins), it was predicted that the proteins from N. meningitidis and N. gonorrhoeae are secreted and could thus be useful antigens for vaccines or diagnostics.

ORF5-1 (30.7 kDa) was cloned in the pGex vector and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 2A shows the results of affinity purification of the GST-fusion protein. Purified GST-fusion protein was used to immunise mice, whose sera were used for Western blot analysis (FIG. 1B). These experiments confirm that ORFS-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 5

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 29>:

1   ATGCGCGGCG GCAGGCCGGA TTCCGTTACC GTGCAGATTA
    TCGAAGGTTC
51  GCGTTTTTCG CATATGAGGA AAGTCATCGA CGCAACGCCC
    GACATCGGAC
101 ACGACACCAA AGGCTGGAGC AATGAAAAAC TGATGGCGGA
    AGTTGCGCCC
151 GATGCCTTCA GCGGCAATCC TGAAgGGCAG TTTTTCCCCG
    ACAGCTACGA
201 AATCGATGCG GGCGGCAGTG ATTTGCAGAT TTACCAAACC
    GCCTACAAgG
251 GCGATGCAAC GCCGCCTGAA TGAgGGCATG GGAAAGCAGG
    CAGGACGGGC
301 TGCCTTATAA AAACCCTTAT GAAATGCTGA TTATGGCGAr
    CCTGGTCGAA
351 AAGGAAACAG GGCATGAAGC CGAsCsCGAC CATGTcGCTT
    CCGTCTTCGT
401 CAACCGCCTG AAAATCGGTA TGCGCCTGCA AACCgAssCG
    TCCGTGATTT
451 ACGGCATGGG TGCGGCATAC AAGGGCAAAA TCCGTAAAGC
    CGACCTGCGC
501 CGCGACACGC CGTACAACAC CTACACGCGC GGCGGTCTGC
    CGCCAACCCC
551 GATTGCGCTG CCC..

This corresponds to the amino acid sequence <SEQ ID 30; ORF7>:

1   MRGGRPDSVT VQIIEGSRFS HMRKVIDATP DIGHDTKGWS
    NEKLMAEVAP
51  DAFSGNPEGQ FFPDSYEIDA GGSDLQIYQT AYKAMQRRLN
    EAWESRQDGL
101 PYKNPYEMLI MAXLVEKETG HEAXXDHVAS VFVNRLKIGM
    RLQTXXSVIY
151 GMGAAYKGKI RKADLRRDTP YNTYTRGGLP PTPIALP..

Further sequence analysis revealed the complete DNA sequence <SEQ ID 31>:

1   ATGTTGAGAA AATTGTTGAA ATGGTCTGCC GTTTTTTTGA
    CCGTGTCGGC
51  AGCCGTTTTC GCCGCGCTGC TTTTTGTTCC TAAGGATAAC
    GGCAGGGCAT
101 ACCGAATCAA AATTGCCAAA AACCAGGGTA TTTCGTCGGT
    CGGCAGGAAA
151 CTTGCCGAAG ACCGCATCGT GTTCAGCAGG CATGTTTTGA
    CGGCGGCGGC
201 CTACGTTTTG GGTGTGCACA ACAGGCTGCA TACGGGGACG
    TACAGATTGC
251 CTTCGGAAGT GTCTGCTTGG GATATCTTGC AGAAAATGCG
    CGGCGGCAGG
301 CCGGATTCCG TTACCGTGCA GATTATCGAA GGTTCGCGTT
    TTTCGCATAT
351 GAGGAAAGTC ATCGACGCAA CGCCCGACAT CGGACACGAC
    ACCAAAGGCT
401 GGAGCAATGA AAAACTGATG GCGGAAGTTG CGCCCGATGC
    CTTCAGCGGC
451 AATCCTGAAG GGCAGTTTTT CCCCGACAGC TACGAAATCG
    ATGCGGGCGG
501 CAGTGATTTG CAGATTTACC AAACCGCCTA CAAGGCGATG
    CAACGCCGCC
551 TGAATGAGGC ATGGGAAAGC AGGCAGGACG GGCTGCCTTA
    TAAAAACCCT
601 TATGAAATGC TGATTATGGC GAGCCTGGTC GAAAAGGAAA
    CAGGGCATGA
651 AGCCGACCGC GACCATGTCG CTTCCGTCTT CGTCAACCGC
    CTGAAAATCG
701 GTATGCGCCT GCAAACCGAC CCGTCCGTGA TTTACGGCAT
    GGGTGCGGCA
751 TACAAGGGCA AAATCCGTAA AGCCGACCTG CGCCGCGACA
    CGCCGTACAA
801 CACCTACACG CGCGGCGGTC TGCCGCCAAC CCCGATTGCG
    CTGCCCGGCA
851 AGGCGGCACT CGATGCCGCC GCCCATCCGT CCGGCGAAAA
    ATACCTGTAT
901 TTCGTGTCCA AAATGGACGG CACGGGCTTG AGCCAGTTCA
    GCCATGATTT
951 GACCGAACAC AATGCCGCCG TCCGCAAATA TATTTTGAAA
    AAATAA

This corresponds to the amino acid sequence <SEQ ID 32; ORF7-1>:

1   MLRKLLKWSA VFLTVSAAVF AALLFVPKDN GRAYRIKIAK
    NQGISSVGRK
51  LAEDRIVFSR HVLTAAAYVL GVHNRLHTGT YRLPSEVSAW
    DILQKMRGGR
101 PDSVTVQIIE GSRFSHMRKV IDATPDIGHD TKGWSNEKLM
    AEVAPDAFSG
151 NPEGQFFPDS YEIDAGGSDL QIYQTAYKAM QRRLNEAWES
    RQDGLPYKNP
201 YEMLIMASLV EKETGHEADR DHVASVFVNR LKIGMRLQTD
    PSVIYGMGAA
251 YKGKIRKADL RRDTPYNTYT RGGLPPTPIA LPGKAALDAA
    AHPSGEKYLY
301 FVSKMDGTGL SQFSHDLTEH NAAVRKYILK K*

Computer analysis of this amino acid sequence gave the following results:

Homology with Hypothetical Protein Encoded by yceg Gene (Accession P44270) of H. influenzae

ORF7 and yceg proteins show 44% aa identity in 192 aa overlap:

ORF7   1 MRGGRPDSVTVQIIEGSRFSHMRKVIDATPDIGHDTKGWSNEKLMA-----EVAPDAFSG  55
+  G+     V+ IEG  F   RK ++  P +    K  SNE++ A      ++  +
yceg 102 LNSGKEVQFNVKWIEGKTFKDWRKDLENAPHLVQTLKDKSNEEIFALLDLPDIGQNLELK 161
ORF7  56 NPEGQFFPDSYEIDAGGSDLQIYQTAYKAMQRRLNEAWESRQDGLPYKNPYEMLIMAXLV 115
N EG  +PD+Y      +DL++ + + + M++ LN+AW  R + LP   NPYEMLI+A +V
yceg 162 NVEGWLYPDTYNYTPKSTDLELLKRSAERMKKALNKAWNERDEDLPLANPYEMLILASIV 221
ORF7 116 EKETGHEAXXDHVASVFVNRLKIGMRLQTXXSVIYGMGAAYKGKIRKADLRRDTPYNTYT 175
EKETG       VASVF+NRLK  M+LQT  +VIYGMG  Y G IRK DL   TPYNTY
yceg 222 EKETGIANERAKVASVFINRLKAKMKLQTDPTVIYGMGENYNGNIRKKDLETKTPYNTYV 281
ORF7 176 RGGLPPTPIALP                                                 187
GLPPTPIA+P
yceg 282 IDGLPPTPIAMP                                                 293

The complete length YCEG protein has sequence:

1   MKKFLIAILL LILILAGVAS FSYYKMTEFV KTPVNVQADE
    LLTIERGTTS
51  SKLATLFEQE KLIADGKLLP YLLKLKPELN KIKAGTYSLE
    NVKTVQDLLD
101 LLNSGKEVQF NVKWIEGKTF KDWRKDLENA PHLVQTLKDK
    SNEEIFALLD
151 LPDIGQNLEL KNVEGWLYPD TYNYTPKSTD LELLKRSAER
    MKKALNKAWN
201 ERDEDLPLAN PYEMLILASI VEKETGIANE RAKVASVFIN
    RLKAKMKLQT
251 DPTVIYGMGE NYNGNIRKKD LETKTPYNTY VIDGLPPTPI
    AMPSESSLQA
301 VANPEKTDFY YFVADGSGGH KFTRNLNEHN KAVQEYLRWY
    RSQKNAK

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF7 shows 95.2% identity over a 187aa overlap with an ORF (ORF7a) from strain A of N. meningitidis:

The complete length ORF7a nucleotide sequence <SEQ ID 33> is:

1   ATGTTGAGAA AATTGTTGAA ATGGTCTGCC GTTTTTTTGA
    CCGTATCGGC
51  AGCCGTTTTC GCCGCGCTGC TTTTCGTCCC TAAAGACAAC
    GGCAGGGCAT
101 ACAGGATTAA AATTGCCAAA AACCAGGGTA TTTCGTCGGT
    CGGCAGGAAA
151 CTTGCCGAAG ACCGCATCGT GTTCAGCAGG CATGTTTTGA
    CGGCGGCGGC
201 CTACGTTTTG GGTGTGCACA ACAGGCTGCA TACGGGGACG
    TACAGACTGC
251 CTTCGGAAGT GTCTGCTTGG GATATCTTGC AGAAAATGCG
    CGGCGGCAGG
301 CCGGATTCCG TTACCGTGCA GATTATCGAA GGTTCGCGTT
    TTTCGCATAT
351 GAGGAAAGTC ATCGACGCAA CGCCCGACAT CGAACACGAC
    ACCAAAGGCT
401 GGAGCAATGA AAAACTGATG GCGGAAGTTG CCCCTGATGC
    CTTCAGCGGC
451 AATCCTGAAG GGCAGTTTTT CCCCGACAGC TACGAAATCG
    ATGCGGGCGG
501 CAGCGATTTA CGGATTTACC AAATCGCCTA CAAGGCGATG
    CAACGCCGAC
551 TGAATGAGGC ATGGGAAAGC AGGCAGGACG GGCTGCCTTA
    TAAAAACCCT
601 TATGAAATGC TGATTATGGC GAGCCTGATC GAAAAGGAAA
    CAGGGCATGA
651 AGCCGACCGC GACCATGTCG CTTCCGTCTT CGTCAACCGC
    CTGAAAATCG
701 GTATGCGCCT GCAAACCGAC CCGTCCGTGA TTTACGGCAT
    GGGTGCGGCA
751 TACAAGGGCA AAATCCGTAA AGCCGACCTG CGCCGCGACA
    CGCCGTACAA
801 CACCTACACG CGCGGCGGTC TGCCGCCAAC CCCGATCGCG
    CTGCCCGGCA
851 AGGCGGCACT CGATGCCGCC GCCCATCCGT CCGGTGAAAA
    ATACCTGTAT
901 TTCGTGTCCA AAATGGACGG TACGGGCTTG AGCCAGTTCA
    GCCATGATTT
951 GACCGAACAC AACGCCGCCG TTCGCAAATA TATTTTGAAA
    AAATAA

This is predicted to encode a protein having amino acid sequence <SEQ ID 34>:

1   MLRKLLKWSA VFLTVSAAVF AALLFVPKDN GRAYRIKIAK
    NQGISSVGRK
51  LAEDRIVFSR HVLTAAAYVL GVHNRLHTGT YRLPSEVSAW
    DILQKMRGGR
101 PDSVTVQIIE GSRFSHMRKV IDATPDIEHD TKGWSNEKLM
    AEVAPDAFSG
151 NPEGQFFPDS YEIDAGGSDL RIYQIAYKAM QRRLNEAWES
    RQDGLPYKNP
201 YEMLIMASLI EKETGHEADR DHVASVFVNR LKIGMRLQTD
    PSVIYGMGAA
251 YKGKIRKADL RRDTPYNTYT RGGLPPTPIA LPGKAALDAA
    AHPSGEKYLY
301 FVSKMDGTGL SQFSHDLTEH NAAVRKYILK K*

A leader peptide is underlined.

ORF7a and ORF7-1 show 98.8% identity in 331 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF7 shows 94.7% identity over a 187aa overlap with a predicted ORF (ORF7.ng) from N. gonorrhoeae:

An ORF7ng nucleotide sequence <SEQ ID 35> is predicted to encode a protein having amino acid sequence <SEQ ID 36>:

1   MRGGRPDSVT VQIIEGSRFS HMRKVIDATP DIGHDTKGWS
    NEKLMAEVAP
51  DAFSGNPEGQ FFPDSYEIDA GGSDLQIYQT AYKAMQRRLN
    EAWAGRQDGL
101 PYKNPYEMLI MASLIEKETG HEADRDHVAS VFVNRLKIGM
    RLQTDPSVIY
151 GMGAAYKGKI RKADLRRDTP YNTYTGGGLP PTRIALPGKA
    AMDAAAHPSG
201 EKYLYFVSKM DGTGLSQFSH DLTEHNAAVR KYILKK*

Further sequence analysis revealed a partial DNA sequence of ORF7ng <SEQ ID 37>:

1   ..taccgaatca AGATTGCCAA AAATCAGGGT ATTTCGTCGG
    TCGGCAGGAA
51  ACTTGCcgaA GACCGCATCG TGTTCAGCAG GCATGTTTTG
    ACAGCGGCGG
101 CCTACGTTTT GGGTGTGCAC AACAGGCTGC ATACGGGGAC
    gTACAGATTG
151 CCTTCGGAAG TGTCTGCTTG GGATATCTTG CAGAAAATGC
    GCGGCGGCAG
201 GCCGGATTCC GTTACCGTGC AGATTATCGA AGGTTCGCGT
    TTTTCGCATA
251 TGAGGAAAGT CATCGACGCA ACGCCCGACA TCGGACACGA
    CACCAAAGGC
301 TGGAGCAATG AAAAACTGAT GGCGGAAGTT GCGCCCGATG
    CCTTCAGCGG
351 CAATCCTGAA GGGCAGTTTT TTCCCGACAG CTACGAAATC
    GATGCGGGCG
401 GCAGCGATTT GCAGATTTAC CAAACCGCCT ACAAGGCGAT
    GCAACGCCGC
451 CTGAACGAGG CATGGGCAGG CAGGCAGGAC GGGCTGCCTT
    ATAAAAACCC
501 TTATGAAATG CTGATTATGG CGAGCCTGAT CGAAAAGGAA
    ACGGGGCATG
551 AGGCCGACCG CGACCATGTC GCTTCCGTCT TCGTCAACCG
    CCTGAAAATC
601 GGTATGCGCC TGCAAACCGA CCCGTCCGTG ATTTACGGCA
    TGGGTGCGGC
651 ATACAAGGGC AAAATCCGTA AAGCCGACCT GCGCCGCGAC
    ACGCCGTACA
701 aCAccTAtac gggcgggggc ttgccgccaa cccggattgc
    gctgcccggC
751 Aaggcggcaa tggatgccgc cgcccacccg tccggcgaAa
    aatacctgTa
801 tttcgtgtcC AAAATGGACG GCACGGGCTT GAGCCAGTTC
    AGCCATGATT
851 TGACCGAACA CAACGCCGCc gTcCGCAAAT ATATTTTGAA
    AAAATAA

This corresponds to the amino acid sequence <SEQ ID 38; ORF7ng-1>:

1   ..YRIKIAKNQG ISSVGRKLAE DRIVFSRHVL TAAAYVLGVH
    NRLHTGTYRL
51  PSEVSAWDIL QKMRGGRPDS VTVQIIEGSR FSHMRKVIDA
    TPDIGHDTKG
101 WSNEKLMAEV APDAFSGNPE GQFFPDSYEI DAGGSDLQIY
    QTAYKAMQRR
151 LNEAWAGRQD GLPYKNPYEM LIMASLIEKE TGHEADRDHV
    ASVFVNALKI
201 GMRLQTDPSV IYGMGAAYKG KIRKADLRRD TPYNTYTGGG
    LPPTRIALPG
251 KAAMDAAAHP SGEKYLYFVS KMDGTGLSQF SHDLTEHNAA
    VRKYILKK*

ORF7ng-1 and ORF7-1 show 98.0% identity in 298 aa overlap:

In addition, ORF7ng-1 shows significant homology with a hypothetical E. coli protein:

sp|P28306|YCEG_ECOLI HYPOTHETICAL 38.2 KD PROTEIN IN PABC-HOLB
INTERGENIC REGION gi|1787339 (AE000210) o340; 100% identical to fragment
YCEG_ECOLI SW: P28306 but has 97 additional C-terminal residues
[Escherichia coli] Length = 340
Score = 79 (36.2 bits), Expect = 5.0e−57, Sum P(2) = 5.0e−57
Identities = 20/87 (22%), Positives = 40/87 (45%)
Query:	10	GISSVGRKLAEDRIVFSRHVLTAAAYVLGVHNRLHTGTYRLPSEVSAWDILQKMRGGRPD	69
		G  ++G +L  D+I+    V      +    +    GTYR   +++  ++L+ +  G+
Sbjct:	49	GRLALGEQLYADKIINRPRVFQWLLRIEPDLSHFKAGTYRFTPQMTVREMLKLLESGKEA	108
Query:	70	SVTVQIIEGSRFSHMRKVIDATPDIGH	96
		++++EG R S   K +   P I H
Sbjct:	109	QFPLRLVEGMRLSDYLKQLREAPYIKH	135
Score = 438 (200.7 bits), Expect = 5.0e−57, Sum P(2) = 5.0e−57
Identities = 84/155 (54%), Positives = 111/155 (71%)
Query:	120	EGQFFPDSYEIDAGGSDLQIYQTAYKAMQRRLNEAWAGRQDGLPYKNPYEMLIMASLIEK	179
		EG F+PD++   A  +D+ + + A+K M + ++ AW GR DGLPYK+  +++ MAS+IEK
Sbjct:	158	EGWFWPDTWMYTANTTDVALLKRAHKKMVKAVDSAWEGRADGLPYKDKNQLVTMASIIEK	217
Query:	180	ETGHEADRDHVASVFVNRLKIGMRLQTDPSVIYGMGAAYKGKIRKADLRRDTPYNTYTGG	239
		ET   ++RD VASVF+NRL+IGMRLQTDP+VIYGMG  Y GK+ +ADL   T YNTYT
Sbjct:	218	ETAVASERDKVASVFINRLRIGMRLQTDPTVIYGMGERYNGKLSRADLETPTAYNTYTIT	277
Query:	240	GLPPTRIALPGKAAMDAAAHPSGEKYLYFVSKMDG	274
		GLPP  IA PG  ++ AAAHP+   YLYFV+   G
Sbjct:	278	GLPPGAIATPGADSLKAAAHPAKTPYLYFVADGKG	312

Based on this analysis, including the fact that the H. influenzae YCEG protein possesses a possible leader sequence, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 6

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 39>:

1   CGTTTCAAAA TGTTAACTGT GTTGACGGCA ACCTTGATTG
    CCGGACAGGT
51  ATCTGCCGCC GGAGGCGGTG CGGGGGATAT GAAACAGCCG
    AAGGAAGTCG
101 GAAAGGTTTT CAGAAAGCAG CAGCGTTACA GCGAGGAAGA
    AATCAAAAAC
151 GAACGCGCAC GGCTTGCGGC AGTGGGCGAG CGGGTTAATC
    AGATATTTAC
201 GTTGCTGGGA GGGGAAACCG CCTTGCAAAA GGGGCAGGCG
    GGAACGGCTC
251 TGGCAACCTA TATGCTGATG TTGGAACGCA CAAAATCCCC
    CGAAGTCGCC
301 GAACGCGCCT TGGAAATGGC CGTGTCGCTG AACGCGTTTG
    AACAGGCGGA
351 AATGATTTAT CAGAAATGGC GGCAGATTGA GCCTATACCG
    GGTAAGGCGC
401 AAAAACGGGC GGGGTGGCTG CGGAACGTGC TGAGGGAAAG
    AGGAAATCAG
451 CATCTGGACG GACGGGAAGA AGTGCTGGCT CAGGCGGACG
    AAGGACAG

This corresponds to the amino acid sequence <SEQ ID 40; ORF9>:

1   ..RFKMLTVLTA TLIAGQVSAA GGGAGDMKQP KEVGKVFRKQ
    QRYSEEEIKN
51  ERARLAAVGE RVNQIFTLLG GETALQKGQA GTALATYMLM
    LERTKSPEVA
101 ERALEMAVSL NAFEQAEMIY QKWRQIEPIP GKAQKRAGWL
    RNVLRERGNQ
151 HLDGREEVLA QADEGQ

Further sequence analysis revealed the complete DNA sequence <SEQ ID 41>:

1    ATGTTACCTA ACCGTTTCAA AATGTTAACT GTGTTGACGG
     CAACCTTGAT
51   TGCCGGACAG GTATCTGCCG CCGGAGGCGG TGCGGGGGAT
     ATGAAACAGC
101  CGAAGGAAGT CGGAAAGGTT TTCAGAAAGC AGCAGCGTTA
     CAGCGAGGAA
151  GAAATCAAAA ACGAACGCGC ACGGCTTGCG GCAGTGGGCG
     AGCGGGTTAA
201  TCAGATATTT ACGTTGCTGG GAGGGGAAAC CGCCTTGCAA
     AAGGGGCAGG
251  CGGGAACGGC TCTGGCAACC TATATGCTGA TGTTGGAACG
     CACAAAATCC
301  CCCGAAGTCG CCGAACGCGC CTTGGAAATG GCCGTGTCGC
     TGAACGCGTT
351  TGAACAGGCG GAAATGATTT ATCAGAAATG GCGGCAGATT
     GAGCCTATAC
101  CGGGTAAGGC GCAAAAACGG GCGGGGTGGC TGCGGAACGT
     GCTGAGGGAA
451  AGAGGAAATC AGCATCTGGA CGGACTGGAA GAAGTGCTGG
     CTCAGGCGGA
501  CGAAGGACAG AACCGCAGGG TGTTTTTATT GTTGGCACAA
     GCCGCCGTGC
551  AACAGGACGG GTTGGCGCAA AAAGCATCGA AAGCGGTTCG
     CCGCGCGGCG
601  TTGAAATATG AACATCTGCC CGAAGCGGCG GTTGCCGATG
     TGGTGTTCAG
651  CGTACAGGGA CGCGAAAAGG AAAAGGCAAT CGGAGCTTTG
     CAGCGTTTGG
701  CGAAGCTCGA TACGGAAATA TTGCCCCCCA CTTTAATGAC
     GTTGCGTCTG
751  ACTGCACGCA AATATCCCGA AATACTCGAC GGCTTTTTCG
     AGCAGACAGA
801  CACCCAAAAC CTTTCGGCCG TCTGGCAGGA AATGGAAATT
     ATGAATCTGG
851  TTTCCCTGCA CAGGCTGGAT GATGCCTATG CGCGTTTGAA
     CGTGCTGTTG
901  GAACGCAATC CGAATGCAGA CCTGTATATT CAGGCAGCGA
     TATTGGCGGC
951  AAACCGAAAA GAAGGTGCTT CCGTTATCGA CGGCTACGCC
     GAAAAGGCAT
1001 ACGGCAGGGG GACGGAGGAA CAGCGGAGCA GGGCGGCGCT
     AACGGCGGCG
1051 ATGATGTATG CCGACCGCAG GGATTACGCC AAAGTCAGGC
     AGTGGCTGAA
1101 AAAAGTATCC GCGCCGGAAT ACCTGTTCGA CAAAGGTGTG
     CTGGCGGCTG
1151 CGGCGGCTGT CGAGTTGGAC GGCGGCAGGG CGGCTTTGCG
     GCAGATCGGC
1201 AGGGTGCGGA AACTTCCCGA ACAGCAGGGG CGGTATTTTA
     CGGCAGACAA
1251 TTTGTCCAAA ATACAGATGC TCGCCCTGTC GAAGCTGCCC
     GATAAACGGG
1301 AGGCTTTGAG GGGGTTGGAC AAGATTATCG AAAAACCGCC
     TGCCGGCAGT
1351 AATACAGAGT TACAGGCAGA GGCATTGGTA CAGCGGTCAG
     TTGTTTACGA
1401 TCGGCTTGGC AAGCGGAAAA AAATGATTTC AGATCTTGAA
     AGGGCGTTCA
1451 GGCTTGCACC CGATAACGCT CAGATTATGA ATAATCTGGG
     CTACAGCCTG
1501 CTGACCGATT CCAAACGTTT GGACGAAGGT TTCGCCCTGC
     TTCAGACGGC
1551 ATACCAAATC AACCCGGACG ATACCGCTGT CAACGACAGC
     ATAGGCTGGG
1601 CGTATTACCT GAAAGGCGAC GCGGAAAGCG CGCTGCCGTA
     TCTGCGGTAT
1651 TCGTTTGAAA ACGACCCCGA GCCCGAAGTT GCCGCCCATT
     TGGGCGAAGT
1701 GTTGTGGGCA TTGGGCGAAC GCGATCAGGC GGTTGACGTA
     TGGACGCAGG
1751 CGGCACACCT TACGGGAGAC AAGAAAATAT GGCGGGAAAC
     GCTCAAACGT
1801 CACGGCATCG CATTGCCCCA ACCTTCCCGA AAACCTCGGA
     AATAA

This corresponds to the amino acid sequence <SEQ ID 42; ORF9-1>:

1   MLPNRFKMLT VLTATLIAGQ VSAAGGGAGD MKQPKEVGKV
    FRKQQRYSEE
51  EIKNERARLA AVGERVNQIF TLLGGETALQ KGQAGTALAT
    YMLMLERTKS
101 PEVAERALEM AVSLNAFEQA EMIYQKWRQI EPIPGKAQKR
    AGWLRNVLRE
151 RGNQHLDGLE EVLAQADEGQ NRRVFLLLAQ AAVQQDGLAQ
    KASKAVRRAA
201 LKYEHLPEAA VADVVFSVQG REKEKAIGAL QRLAKLDTEI
    LPPTLMTLRL
251 TARKYPEILD GFFEQTDTQN LSAVWQEMEI MNLVSLHRLD
    DAYARLNVLL
301 ERNPNADLYI QAAILAANRK EGASVIDGYA EKAYGRGTEE
    QRSRAALTAA
351 MMYADRRDYA KVRQWLKKVS APEYLFDKGV LAAAAAVELD
    GGRAALRQIG
401 RVRKLPEQQG RYFTADNLSK IQMLALSKLP DKREALRGLD
    KIIEKPPAGS
451 NTELQAEALV QRSVVYDRLG KRKKMISDLE RAFRLAPDNA
    QIMNNLGYSL
501 LTDSKRLDEG FALLQTAYQI NPDDTAVNDS IGWAYYLKGD
    AESALPYLRY
551 SFENDPEPEV AAHLGEVLWA LGERDQAVDV WTQAAHLTGD
    KKIWRETLKR
601 HGIALPQPSR KPRK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF9 shows 89.8% identity over a 166aa overlap with an ORF (ORF9a) from strain A of N. meningitidis:

The complete length ORF9a nucleotide sequence <SEQ ID 43> is:

1    ATGTTACCCG CCCGTTTCAC CATTTTATCT GTGCTCGCGG
     CAGCCCTGCT
51   TGCCGGGCAG GCGTATGCCG CCGGCGCGGC GGATGCGAAG
     CCGCCGAAGG
101  AAGTCGGAAA GGTTTTCAGA AAGCAGCAGC GTTACAGCGA
     GGAAGAAATC
151  AAAAACGAAC GCGCACGGCT TGCGGCAGTG GGCGAGCGGG
     TTAATCAGAT
201  ATTTACGTTG CTGGGANGGG AAACCGCCTT GCAAAAGGGG
     CAGGCGGGAA
251  CGGCTCTGGC AACCTATATG CTGATGTTGG AACGCACAAA
     ATCCCCCGAA
301  GTCGCCGAAC GCGCCTTGGA AATGGCCGTG TCNCTGAACG
     CGTTTGAACA
351  GGCGGAAATG ATTTATCAGA AATGGCGGCA GATTGAGCCT
     ATACCGGGTA
401  AGGCGCAAAA ACGGGCGGGG TGGCTGCGGA ACGTGCTGAG
     GGAAAGAGGA
451  AATCAGCATC TAGACGGACT GGAAGAANTG CTGGCTCAGG
     CGGACGAANG
501  ACAGAACCGC AGGGTGTTTT TATTGTTGGC ACAAGCCGCC
     GTGCAACAGG
551  ACGGGTTGGC GCAAAAAGCA TCGAAAGCGG TTCGCCGCGC
     GGCGTTGAGA
601  TATGAACATC TGCCCGAAGC GGCGGTTGCC GATGTGGTGT
     TCAGCGTACA
651  GGNACGCGAA AAGGAAAAGG CAATCGGAGC TTTGCAGCGT
     TTGGCGAAGC
701  TCGATACGGA AATATTGCCC CCCACTTTAA TGACGTTGCG
     TCTGACTGCA
751  CGCAAATATC CCGAAATACT CGACGGCTTT TTCGAGCAGA
     CAGACACCCA
801  AAACCTTTCG GCCGTCTGGC AGGAAATGGA AATTATGAAT
     CTGGTTTCCC
851  TGCACAGGCT GGATGATGCC TATGCGCGTT TGAACGTGCT
     GTTGGAACGC
901  AATCCGAATG CAGACCTGTA TATTCAGGCA GCGATATTGG
     CGGCAAACCG
951  AAAAGAANGT GCTTCCGTTA TCGACGGCTA CGCCGAAAAG
     GCATACGGCA
1001 GGGGGACGGG GGAACAGCGG GGCAGGGCGG CAATGACGGC
     GGCGATGATA
1051 TATGCCGACC GAAGGGATTA CACCAAAGTC AGGCAGTGGT
     TGAAAAAAGT
1101 GTCCGCGCCG GAATACCTGT TCGACAAAGG TGTGCTGGCG
     GCTGCGGCGG
1151 CTGTCGAGTT GGACNGCGGC AGGGCGGCTT TGCGGCAGAT
     CGGCAGGGTG
1201 CGGAAACTTC CCGAACAGCA GGGGCGGTAT TTTACGGCAG
     ACAATTTGTC
1251 CAAAATACAG ATGTTCGCCC TGTCGAAGCT GCCCGACAAA
     CGGGAGGCTT
1301 TGAGGGGGTT GGACAAGATT ATCGAAAAAC CGCCTGCCGG
     CAGTAATACA
1351 GAGTTACAGG CAGAGGCATT GGTACAGCGG TCAGTTGTTT
     ACGATCGGCT
1401 TGGCAAGCGG AAAAAAATGA TTTCAGATCT TGAAAGGGCG
     TTCAGGCTTG
1451 CACCCGATAA CGCTCAGATT ATGAATAATC TGGGCTACAG
     CCTGCTTTCC
1501 GATTCCAAAC GTTTGGACGA AGGCTTCGCC CTGCTTCAGA
     CGGCATACCA
1551 AATCAACCCG GACGATACCG CTGTCAACGA CAGCATAGGC
     TGGGCGTATT
1601 ACCTGAAANG CGACGCGGAA AGCGCGCTGC CGTATCTGCG
     GTATTCGTTT
1651 GAAAACGACC CCGAGCCCGA AGTTGCCGCC CATTTGGGCG
     AAGTGTTGTG
1701 GGCATTGGGC GAACGCGATC AGGCGGTTGA CGTATGGACG
     CAGGCGGCAC
1751 ACCTTACGGG AGACAAGAAA ATATGGCGGG AAACGCTCAA
     ACGTCACGGC
1801 ATCGCATTGC CCCAACCTTC CCGAAAACCT CGGAAATAA

This encodes a protein having amino acid sequence <SEQ ID 44>:

1   MLPARFTILS VLAAALLAGQ AYAAGAADAK PPKEVGKVFR
    KQQRYSEEEI
51  KNERARLAAV GERVNQIFTL LGXETALQKG QAGTALATYM
    LMLERTKSPE
101 VAERALEMAV SLNAFEQAEM IYQKWRQIEP IPGKAQKRAG
    WLRNVLRERG
151 NQHLDGLEEX LAQADEXQNR RVFLLLAQAA VQQDGLAQKA
    SKAVRRAALR
201 YEHLPEAAVA DVVFSVQXRE KEKAIGALQR LAKLDTEILP
    PTLMTLRLTA
251 RKYPEILDGF FEQTDTQNLS AVWQEMEIMN LVSLHRLDDA
    YARLNVLLER
301 NPNADLYIQA AILAANRKEX ASVIDGYAEK AYGRGTGEQR
    GRAAMTAAMI
351 YADRRDYTKV RQWLKKVSAP EYLFDKGVLA AAAAVELDXG
    RAALRQIGRV
401 RKLPEQQGRY FTADNLSKIQ MFALSKLPDK REALRGLDKI
    IEKPPAGSNT
451 ELQAEALVQR SVVYDRLGKR KKMISDLERA FRLAPDNAQI
    MNNLGYSLLS
501 DSKRLDEGFA LLQTAYQINP DDTAVNDSIG WAYYLKXDAE
    SALPYLRYSF
551 ENDPEPEVAA HLGEVLWALG ERDQAVDVWT QAAHLTGDKK
    IWRETLKRHG
601 IALPQPSRKP RK*

ORF9a and ORF9-1 show 95.3% identity in 614 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF9 shows 82.8% identity over a 163aa overlap with a predicted ORF (ORF9.ng) from N. gonorrhoeae:

The ORF9ng nucleotide sequence <SEQ ID 45> was predicted to encode a protein having including acid sequence <SEQ ID 46>:

1   MIMLPARFTI LSVLAAALLA GQAYAAGAAD VELPKEVGKV
    LRKHRRYSEE
51  EIKNERARLA AVGERVNRVF TLLGGETALQ KGQAGTALAT
    YMLMLERTKS
101 PEVAERALEM AVSLNAFEQA EMIYQKWRQI EPIPGEAQKP
    AGWLRNVLKE
151 GGNPHLDRLE EVPAQSDYVH QPMIFLLLVQ AAVQHGGVAQ
    KPSKAVRPAA
201 YNYEVLPETA GADAVFCVQG PQYEKAIQSF PPCGRNPQTE
    NIAPPFNELF
251 RPTARPISPK LLQRFFRTEP NLAKPFRPPG PEMETYQTGF
    PRPLTRNNPT

Amino acids 1-28 are a putative leader sequence, and 173-189 are predicted to be a transmembrane domain.

Further sequence analysis revealed the complete length ORF9ng DNA sequence <SEQ ID 47>:

1    ATGTTACCCG CCCGTTTCAC TATTTTATCT GTCCTCGCAG
     CAGCCCTGCT
51   TGCCGGACAG GCGTATGCTG CCGGCGCGGC GGATGTGGAG
     CTGCCGAAGG
101  AAGTCGGAAA GGTTTTAAGG AAACATCGGC GTTACAGCGA
     GGAAGAAATC
151  AAAAACGAAC GCGCACGGCT TGCGGCAGTG GGCGAACGGG
     TCAACAGGGT
201  GTTTACGCTG TTGGGCGGTG AAACGGCTTT GCAGAAAGGG
     CAGGCGGGAA
251  CGGCTCTGGC AACCTATATG CTGATGTTGG AACGCACAAA
     ATCCCCCGAA
301  GTCGCCGAAC GCGCCTTGGA AATGGCCGTG TCGCTGAACG
     CGTTTGAACA
351  GGCGGAAATG ATTTATCAGA AATGgcggca gatcgagcct
     ataCcgggtg
401  aggcgcaaaa accgGcgggG tggctgcgga acgtattgaa
     ggaagggGGa
451  aaTCAGCATC TGGAcgggtt gaaagaggTG CtggcgcaAT
     cggacgatGT
501  GCAAAAAcgc aggaTATTTT TGCTGCTGGT GCAAGCCGCC
     GTGCagcagg
551  gTGGGGTGGC TCAAAAAGCA TCGAAAGCGG TTCGCcgtgc
     GGcgttgaAG
601  TATGAACATC TGCCcgaagc ggcggTTGCC GATGcggTGT
     TCGGCGTACA
651  GGGACGCGAA AAGGAAAagg caaTCGAAGC TTTGCAGCGT
     TTGGCGAAGC
701  TCGATACGGA AATATTGCCC CCCACTTTAA TGACGTTGCG
     TCTGACTGCA
751  CGCAAATATC CCGAAATACT CGACGGCTTT TTCGAGCAGA
     CAGACACCCA
801  AAACCTTTCG GCCGTCTGGC AGGAAATGGA AATTATGAAT
     CTGGTTTCCC
851  TGCGTAAGCC GGATGATGCC TATGCGCGTT TGAACGTGCT
     GTTGGAACAC
901  AACCCGAATG CAAACCTGTA TATTCAGGCG GCGATATTGG
     CGGCAAACCG
951  AAAAGAAGGT GCGTCCGTTA TCGACGGCTA CGCCGAAAAG
     GCATACGGCA
1001 GGGGGACGGG GGAACAGCGG GGCagggcgg cAATgacggc
     GGCGATGATA
1051 TATGCCGACC GCAGGGATTA CGCCAAAGTC AGGCAGTGGT
     TGAAAAAAGT
1101 GTCCGCGCCG GAATACCTGT TCGACAAAGG CGTGCTGGCG
     GCTGCGGCGG
1151 CTGCCGAATT GGACGGAGGC CGGGCGGCTT TGCGGCAGAT
     CGGCAGGGTG
1201 CGGAAACTTC CCGAACAGCA GGGGCGGTAT TTTACGGCAG
     ACAATTTGTC
1251 CAAAATACAG ATGCTCGCCC TGTCGAAGCT GCCCGACAAA
     CGGGAAGCCC
1301 TGATCGGGCT GAACAACATC ATCGCCAAAC TTTCGGCGGC
     GGGAAGCACG
1351 GAACCTTTGG CGGAAGCATT GGCACAGCGT TCCATTATTT
     ACGaacAGTT
1401 cggCAAACGG GGAAAAATGA TTGCCGACCT tgaAACcgcg
     CTCAAACTTA
1451 CGCCCGATAA TGCACAAATT ATGAATAATC TGGGCTACAG
     CCTGCTTTCC
1501 GATTCCAAAC GTTTGGACGA GGGTTTCGCC CTGCTTCAGA
     CGGCATACCA
1551 AATCAACCCG GACGATACCG CCGTTAACGA CAGCATAGGC
     TGGGCGTATT
1601 ACCTGAAAGG CGACgcggaA AGCGCGCTGC CGTATCTGcg
     gtattcgttt
1651 gAAAACGACC CCGAGCCCGA AGTTGCCGCC CATTTGGGCG
     AAGTGTTGTG
1701 GGCATTGGGC GAACGCGATC AGGCGGTTGA CGTATGGACG
     CAGGCGGCAC
1751 ACCTTAGGGG AGACAAGAAA ATATGGCGGG AGACGCTCAA
     ACGCTACGGA
1801 ATCGCCTTGC CCGAGCCTTC CCGAAAACCC CGGAAATAA

This encodes a protein having amino acid sequence <SEQ ID 48>:

1   MLPARFTILS VLAAALLAGQ AYAAGAADVE LPKEVGKVLR
    KHRRYSEEEI
51  KNERARLAAV GERVNRVFTL LGGETALQKG QAGTALATYM
    LMLERTKSPE
101 VAERALEMAV SLNAFEQAEM IYQKWRQIEP IPGEAQKPAG
    WLRNVLKEGG
151 NQHLDGLKEV LAQSDDVQKR RIFLLLVQAA VQQGGVAQKA
    SKAVRRAALK
201 YEHLPEAAVA DAVFGVQGRE KEKAIEALQR LAKLDTEILP
    PTLMTLRLTA
251 RKYPEILDGF FEQTDTQNLS AVWQEMEIMN LVSLRKPDDA
    YARLNVLLEH
301 NPNANLYIQA AILAANRKEG ASVIDGYAEK AYGRGTGEQR
    GRAAMTAAMI
351 YADRRDYAKV RQWLKKVSAP EYLFDKGVLA AAAAAELDGG
    RAALRQIGRV
401 RKLPEQQGRY FTADNLSKIQ MLALSKLPDK REALIGLNNI
    IAKLSAAGST
451 EPLAEALAQR SIIYEQFGKR GKMIADLETA LKLTPDNAQI
    MNNLGYSLLS
501 DSKRLDEGFA LLQTAYQINP DDTAVNDSIG WAYYLKGDAE
    SALPYLRYSF
551 ENDPEPEVAA HLGEVLWALG ERDQAVDVWT QAAHLRGDKK
    IWRETLKRYG
601 IALPEPSRKP RK*

ORF9ng and ORF9-1 show 88.1% identity in 614 aa overlap:

In addition, ORF9ng shows significant homology with a hypothetical protein from P. aeruginosa:

sp|P42810|YHE3_PSEAE HYPOTHETICAL 64.8 KD PROTEIN IN HEMM-HEMA INTERGENIC
REGION (ORF3)
>gi|1072999|pir||S49376 hypothetical protein 3 - Pseudomonas aeruginosa
>gi|557259 (X82071) orf3 [Pseudomonas aeruginosa] Length = 576
Score = 128 bits (318), Expect = 1e−28
Identities = 138/587 (23%), Positives = 228/587 (38%), Gaps = 125/587 (21%)
Query:	67	VFTLLGGETALQKGQAGTALATYMLMLERTKSPEVAERALEMAVSLNAFEQAEMIYQKWR	126
		+++LL  E A Q+ +   AL+ Y++  ++T+ P V+ERA  +A  L A ++A      W
Sbjct:	53	LYSLLVAELAGQRNRFDIALSNYVVQAQKTRDPGVSERAFRIAEYLGADQEALDTSLLWA	112
Query:	127	QIEPIPGEAQKPAG--------------WLRNVLKEGGNQHLDGLKEVLAQSDDVQKRRI	172
		+  P   +AQ+ A               ++  VL   G+ H D L    A++D   +  +
Sbjct:	113	RSAPDNLDAQRAAAIQLARAGRYEESMVYMEKVLNGQGDTHFDFLALSAAETDPDTRAGL	172
Query:	173	FXXXXXXXXXXXXXXXKASKAVRRAALKYEHLPEAAVADAVFGVQGREKEKAIEALQRLA	232
		++      KY +  +     A+   Q    ++A+  L+  +
Sbjct:	173	L------------------QSFDHLLKKYPNNGQLLFGKALLLQQDGRPDEALTLLEDNS	214
Query:	233	KLDTEILPPTLMTLRLTARK-----YPEILDGFFEQTDTQNLSAVWQEMEIMNLVSLRKP	287
		E+ P  L +  L + K      P +  G  E  D + +   +  +    LV   +
Sbjct:	215	ASRHEVAPLLLRSRLLQSMKRSDEALPLLKAGIKEHPDDKRVRLAYARL----LVEQNRL	270
Query:	288	DDAYARLNVLLEHNPN---------------------ANLYIQAAI--------------	312
		DDA A    L++  P+                     A +Y++  +
Sbjct:	271	DDAKAEFAGLVQQFPDDDDDLRFSLALVCLEAQAWDEARIYLEELVERDSHVDAAHFNLG	330
Query:	313	-LAANRKEGASVIDGYAEKAYGRGTGEQRGRAAMTAAMIYADRRDYAKVRQWLKKVSAPE	371
		LA  +K+ A  +D YA+   G G      +   T  ++ A R D A  R    +   P+
Sbjct:	331	RLAEEQKDTARALDEYAQ--VGPGNDFLPAQLRQTDVLLKAGRVDEAAQRLDKARSEQPD	388
Query:	372	YLFDKXXXXXXXXXXXXXXXXXXRQIGRVRKLPEQQGRYFTADNLSKIQMLALSKLPDKR	431
		Y                                        A  L  I+  ALS    +
Sbjct:	389	Y----------------------------------------AIQLYLIEAEALSNNDQQE	408
Query:	432	EALIGLNNIIAKLSAAGSTEPLAEALAQRSIIYEQFGKRGKMIADLETALKLTPDNAQIM	491
		+A   +   + +       E L   L  RS++ E+     +M  DL   +   PDNA  +
Sbjct:	409	KAWQAIQEGLKQYP-----EDL-NLLYTRSMLAEKRNDLAQMEKDLRFVIAREPDNAMAL	462
Query:	492	NNLGYSLLSDSKRLDEGFALLQTAYQINPDDTAVNDSIGWAYYLKGDAESALPYLRYSFE	551
		N LGY+L   + R  E   L+  A+++NPDD A+ DS+GW  Y +G    A  YLR + +
Sbjct:	463	NALGYTLADRTTRYGEARELILKAHKLNPDDPAILDSMGWINYRQGKLADAERYLRQALQ	522
Query:	552	NDPEPEVAAHLGEVLWALGERDQAVDVWTQAAHLRGDKKIWRETLKR	598
		P+ EVAAHLGEVLWA G +  A  +W +    + D  + R T+KR
Sbjct:	523	RYPDHEVAAHLGEVLWAQGRQGDARAIWREYLDKQPDSDVLRRTIKR	569
gi|2983399 (AE000710) hypothetical protein [Aquifex aeolicus] Length = 545
Score = 81.5 bits (198), Expect = 1e−14
Identities = 61/198 (30%), Positives = 98/198 (48%), Gaps = 19/198 (9%)
Query:	408	GRYFTADNL-SKIQMLALSKLPDKREALIGLNNIIAKLSAAGSTEPLAEALAQ-------	459
		G Y  A  L  K ++LA    PDK+E L    +  +K         + + L +
Sbjct:	335	GNYEDAKRLIEKAKVLA----PDKKEILFLEADYYSKTKQYDKALEILKKLEKDYPNDSR	390
Query:	460	----RSIIYEQFGKRGKMIADLETALKLTPDNAQIMNNLGYSLLS--DSKRLDEGFALLQ	513
		+I+Y+  G        L  A++L P+N    N LGYSLL     +R++E   L++
Sbjct:	391	VYFMEAIVYDNLGDIKNAEKALRKAIELDPENPDYYNYLGYSLLLWYGKERVEEAEELIK	450
Query:	514	TAYQINPDDTAVNDSIGWAYYLKGDAESALPYLRYSF-ENDPEPEVAAHLGEVLWALGER	572
		A + +P++ A  DS+GW YYLKGD E A+ YL  +  E   +P V  H+G+VL  +G +
Sbjct:	451	KALEKDPENPAYIDSMGWVYYLKGDYERAMQYLLKALREAYDDPVVNEHVGDVLLKMGYK	510
Query:	573	DQAVDVWTQAAHLRGDKK	590
		++A + + +A  L  + K
Sbjct:	511	EEARNYYERALKLLEEGK	528

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 7

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 49>:

1   AACCTCTACG CCGGCCCGCA GACCACATCC GTCATCGCAA
    ACATCGCCGA
51  CAACCTGCAA CTGGCCAAAG ACTACGGCAA AGTACACTGG
    TTCGCCTCCC
101 CGCTCTTCTG GCTCCTGAAC CAACTGCACA ACATCATCGG
    CAACTGGGGC
151 TGGGCGATTA TCGTTTTAAC CATCATCGTC AAAGCCGTAC
    TGTATCCATT
201 GACCAACGCC TCTTACCGCT CTATGGCGAA AATGCGTGCC
    GCCGCACCCA
251 AACTGCAAGC CATCAAAGAG AAATACGGCG ACGACCGTAT
    GGCGCAACAA
301 CAGGCGATGA TGCAGCTTTA CACAGACGAG AAAATCAACC
    CGaCTGGGCG
351 GCTGCCTGCC TATGCTGTTG CAAATCCCCG TCTTCATCGG
    ATTGTATTGG
401 GCATTGTTCG CCTCCGTAGA ATTGCGCCAG GCACCTTGGC
    TGGGTTGGAT
451 TACCGACCTC AGCCGCGCCG ACCCCTACTA CATCCTGCCC
    ATCATTATGG
501 CGGCAACGAT GTTCGCCCAA ACTTATCTGA ACCCGCCGCC
    GAcCGACCCG
551 ATGCagGCGA AAATGATGAA AATCATGCCG TTGGTTTTCT
    CsGwCrTGTT
601 CTTCTTCTTC CCTGCCGGks TGGTATTGTA CTGGGTAGTC
    AACAACCTCC
651 TGACCATCGC CCAGCAATGG CACATCAACC GCAGCATCGA
    AAAACAACGC
701 GCCCAAGGCG AAGTCGTTTC CTAA

This corresponds to the amino acid sequence <SEQ ID 50; ORF11>:

1   ..NLYAGPQTTS VIANIADNLQ LAKDYGKVHW FASPLFWLLN
    QLHNIIGNWG
51  WAIIVLTIIV KAVLYPLTNA SYRSMAKMRA AAPKLQAIKE
    KYGDDRMAQQ
101 QAMMQLYTDE KINPLGGCLP MLLQIPVFIG LYWALFASVE
    LRQAPWLGWI
151 TDLSRADPYY ILPIIMAATM FAQTYLNPPP TDPMQAKMMK
    IMPLVFSXXF
201 FFFPAGXVLY WVVNNLLTIA QQWHINRSIE KQRAQGEVVS *

Further sequence analysis revealed the complete DNA sequence <SEQ ID 51>:

1    ATGGATTTTA AAAGACTCAC GGCGTTTTTC GCCATCGCGC
     TGGTGATTAT
51   GATCGGCTGG GAAAAGATGT TCCCCACTCC GAAGCCAGTC
     CCCGCGCCCC
101  AACAGGCAGC ACAACAACAG GCCGTAACCG CTTCCGCCGA
     AGCCGCGCTC
151  GCGCCCGCAA CGCCGATTAC CGTAACGACC GACACGGTTC
     AAGCCGTCAT
201  TGATGAAAAA AGCGGCGACC TGCGCCGGCT GACCCTGCTC
     AAATACAAAG
251  CAACCGGCGA CGAAAATAAA CCGTTCATCC TGTTTGGCGA
     CGGCAAAGAA
301  TACACCTACG TCGCCCAATC CGAACTTTTG GACGCGCAGG
     GCAACAACAT
351  TCTAAAAGGC ATCGGCTTTA GCGCACCGAA AAAACAGTAC
     AGCTTGGAAG
401  GCGACAAAGT TGAAGTCCGC CTGAGCGCGC CTGAAACACG
     CGGTCTGAAA
451  ATCGACAAAG TTTATACTTT CACCAAAGGC AGCTATCTGG
     TCAACGTCCG
501  CTTCGACATC GCCAACGGCA GCGGTCAAAC CGCCAACCTG
     AGCGCGGACT
551  ACCGCATCGT CCGCGACCAC AGCGAACCCG AGGGTCAAGG
     TTACTTTACC
601  CACTCTTACG TCGGCCCTGT TGTTTATACC CCTGAAGGCA
     ACTTCCAAAA
651  AGTCAGCTTT TCCGACTTGG ACGACGATGC CAAATCCGGC
     AAATCCGAGG
701  CCGAATACAT CCGCAAAACC CCGACCGGCT GGCTCGGCAT
     GATTGAACAC
751  CACTTCATGT CCACCTGGAT TCTCCAACCT AAAGGCAGAC
     AAAGCGTTTG
801  CGCCGCAGGC GAGTGCAACA TCGACATCAA ACGCCGCAAC
     GACAAGCTGT
851  ACAGCACCAG CGTCAGCGTG CCTTTAGCCG CCATCCAAAA
     CGGCGCGAAA
901  GCCGAAGCCT CCATCAACCT CTACGCCGGC CCGCAGACCA
     CATCCGTCAT
951  CGCAAACATC GCCGACAACC TGCAACTGGC CAAAGACTAC
     GGCAAAGTAC
1001 ACTGGTTCGC CTCCCCGCTC TTCTGGCTCC TGAACCAACT
     GCACAACATC
1051 ATCGGCAACT GGGGCTGGGC GATTATCGTT TTAACCATCA
     TCGTCAAAGC
1101 CGTACTGTAT CCATTGACCA ACGCCTCTTA CCGCTCTATG
     GCGAAAATGC
1151 GTGCCGCCGC ACCCAAACTG CAAGCCATCA AAGAGAAATA
     CGGCGACGAC
1201 CGTATGGCGC AACAACAGGC GATGATGCAG CTTTACACAG
     ACGAGAAAAT
1251 CAACCCGCTG GGCGGCTGCC TGCCTATGCT GTTGCAAATC
     CCCGTCTTCA
1301 TCGGATTGTA TTGGGCATTG TTCGCCTCCG TAGAATTGCG
     CCAGGCACCT
1351 TGGCTGGGTT GGATTACCGA CCTCAGCCGC GCCGACCCCT
     ACTACATCCT
1401 GCCCATCATT ATGGCGGCAA CGATGTTCGC CCAAACTTAT
     CTGAACCCGC
1451 CGCCGACCGA CCCGATGCAG GCGAAAATGA TGAAAATCAT
     GCCGTTGGTT
1501 TTCTCCGTCA TGTTCTTCTT CTTCCCTGCC GGTCTGGTAT
     TGTACTGGGT
1551 AGTCAACAAC CTCCTGACCA TCGCCCAGCA ATGGCACATC
     AACCGCAGCA
1601 TCGAAAAACA ACGCGCCCAA GGCGAAGTCG TTTCCTAA

This corresponds to the amino acid sequence <SEQ ID 52; ORF11-1>:

1   MDFKRLTAFF AIALVIMIGW EKMFPTPKPV PAPQQAAQQQ
    AVTASAEAAL
51  APATPITVTT DTVQAVIDEK SGDLRRLTLL KYKATGDENK
    PFILFGDGKE
101 YTYVAQSELL DAQGNNILKG IGFSAPKKQY SLEGDKVEVR
    LSAPETRGLK
151 IDKVYTFTKG SYLVNVRFDI ANGSGQTANL SADYRIVRDH
    SEPEGQGYFT
201 HSYVGPVVYT PEGNFQKVSF SDLDDDAKSG KSEAEYIRKT
    PTGWLGMIEH
251 HFMSTWILQP KGRQSVCAAG ECNIDIKRRN DKLYSTSVSV
    PLAAIQNGAK
301 AEASINLYAG PQTTSVIANI ADNLQLAKDY GKVHWFASPL
    FWLLNQLHNI
351 IGNWGWAIIV LTIIVKAVLY PLTNASYRSM AKMRAAAPKL
    QAIKEKYGDD
401 RMAQQQAMMQ LYTDEKINPL GGCLPMLLQI PVFIGLYWAL
    FASVELRQAP
451 WLGWITDLSR ADPYYILPII MAATMFAQTY LNPPPTDPMQ
    AKMMKIMPLV
501 FSVMFFFFPA GLVLYWVVNN LLTIAQQWHI NRSIEKQRAQ
    GEVVS*

Computer analysis of this amino acid sequence gave the following results:

Homology with a 60 kDa Inner-Membrane Protein (Accession P25754) of Pseudomonas putida

ORF11 and the 60 kDa protein show 58% aa identity in 229 aa overlap (BLASTp).

ORF11	2	LYAGPQTTSVIANIADNLQLAKDYGKVHWFASPLFWLLNQLHNIIGNWGWAIIVLTIIVK	61
		LYAGP+  S +  ++  L+L  DYG + + A P+FWLL  +H+++GNWGW+IIVLT+++K
60K	324	LYAGPKIQSKLKELSPGLELTVDYGFLWFIAQPIFWLLQHIHSLLGNWGWSIIVLTMLIK	383
ORF11	62	AVLYPLTNASYRSMAKMRAAAPKLQAIKEKYGDDRXXXXXXXXXLYTDEKINPLGGCLPM	121
		+ +PL+ ASYRSMA+MRA APKL A+KE++GDDR         LY  EKINPLGGCLP+
60K	384	GLFFPLSAASYRSMARMRAVAPKLAALKERFGDDRQKMSQAMMELYKKEKINPLGGCLPI	443
ORF11	122	LLQIPVFIGLYWALFASVELRQAPWLGWITDLSRADPYYILPIIMAATMFAQTYLNPPPT	181
		L+Q+PVF+ LYW L  SVE+RQAPW+ WITDLS  DP++ILPIIM ATMF Q  LNP P
60K	444	LVQMPVFLALYWVLLESVEMRQAPWILWITDLSIKDPFFILPIIMGATMFIQQRLNPTPP	503
ORF11	182	DPMQAKMMKIMPLVXXXXXXXXPAGXVLYWVVNNLLTIAQQWHINRSIE	230
		DPMQAK+MK+MP++        PAG VLYWVVNN L+I+QQW+I R IE
60K	504	DPMQAKVMKMMPIIFTFFFLWFPAGLVLYWVVNNCLSISQQWYITRRIE	552

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF 11 shows 97.9% identity over a 240aa overlap with an ORF (ORF11a) from strain A of N. meningitidis:

The complete length ORF11a nucleotide sequence <SEQ ID 53> is:

     ANGGATTTTA AAAGACTCAC NGNGTTTTTC GCCATCGCAC
     TGGTGATTAT
51   GATCGGATNG NAAANGATGT TCCCCACTCC GAAGCCCGTC
     CCCGCGCCCC
101  AACAGACGGC ACAACAACAG GCCGTAANCG CTTCCGCCGA
     AGCCGCGCTC
151  GCGCCCGNAN CGCCGATTAC CGTAACGACC GACACGGTTC
     AAGCCGTCAT
201  TGATGAAAAA AGCGGCGACC TGCGCCGGCT GACCCTGCTC
     AAATACAAAG
251  CAACCGGCGA CNAAAATAAA CCGTTCATCC TGTTTGGCGA
     CGGCAAANAA
301  TACACCTACN TCGCCCANTC CGAACTTTTG GACGCGCAGG
     GCAACAACAT
351  TCTAAAAGGC ATCGGCTTTA GCGCACCGAA AAAACAGTAC
     AGCTTGGAAG
401  GCGACAAAGT TGAAGTCCGC CTGAGCGCAC CTGAAACACG
     CGGTCTGAAA
451  ATCGACAAAG TTTATACTTT CACCAAAGGC AGCTATCTGG
     TCAACGTCCG
501  CTTCGACATC GCCAACGGCA GCGGTCAAAC CGCCAACCTG
     AGCGCGGACT
551  ACCGCATCGT CCGCGACCAC AGCGAACCCG AGGGTCAAGG
     CTACTTTACC
601  CACTCTTACG TCGGCCCTGT TGTTTATACC CCTGAAGGCA
     ACTTCCAAAA
651  AGTCAGCTTC TCCGACTTGG ACGACGATGC CAANTCCGGN
     AAATCCGAGG
701  CCGAATACAT CCGCAAAACC CNGACCGGCT GGCTCGGCAT
     GATTGAACAC
751  CACTTCATGT CCACCTGGAT CCTCCAACCC AAAGGCGGAC
     AAAGCGTTTG
801  CGCCGCTGGC GACTGCNGTA TNGACATCAA ACGCCGCAAC
     GACAAGCTGT
851  ACAGCACCAG CGTCAGCGTG CCTTTAGCCG CTATCCAAAA
     CGGTGCGAAA
901  TCCNAAGCCT CCATCAACCT CTACGCCGGC CCACAGACCA
     CATCNGTTAT
951  CGCAAACATC GCCGACAACC TGCAACTGGN CAAAGACTAC
     GGCAAAGTAC
1001 ACTGGTTCGC CTCCCCCCTC TTTTGGCTTT TGAACCAACT
     GCACAACATC
1051 ATCGGCAACT GGGGCTGGGC GATTATCGTT TTAACCATCA
     TCGTCAAAGC
1101 CGTACTGTAT CCATTGACCA ACGCCTCTTA CCGTTCGATG
     GCGAAAATGC
1151 GTGCCGCCGC GCCCAAACTG CAAGCCATCA AAGAGAAATA
     CGGCGACGAC
1201 CGTATGGCGC AGCAACAAGC CATGATGCAG CTTTACACAG
     ACGAGAAAAT
1251 CAACCCGCTG GGCGGCTGCC TGCCTATGCT GTTGCAAATC
     CCCGTCTTCA
1301 TCGGATTGTA TTGGGCATTG TTCGCCTCCG TAGAATTGCG
     CCAGGCACCT
1351 TGGCTGGGTT GGATTACCGA CCTCAGCCGC GCCGACCCNT
     ACTACATCCT
1401 GCCCATCATT ATGGCGGCAA CGATGTTCGC CCAAACCTAT
     CTGAACCCGC
1451 CGCCGACCGA CCCGATGCAG GCGAAAATGA TGAAAATCAT
     GCCTTTGGTT
1501 NTNTCNNNNA NGTTCTTCNN CTTCCCTGCC GGTCTGGTAT
     TGTACTGGGT
1551 GATCAACAAC CTCCTGACCA TCGCCCAGCA ATGGCACATC
     AACCGCAGCA
1601 TCGAAAAACA ACGCGCCCAA GGCGAAGTCG TTTCCTAA

This encodes a protein having amino acid sequence <SEQ ID 54>:

1   XDFKRLTXFF AIALVIMIGX XXMFPTPKPV PAPQQTAQQQ
    AVXASAEAAL
51  APXXPITVTT DTVQAVIDEK SGDLRRLTLL KYKATGDXNK
    PFILFGDGKX
101 YTYXAXSELL DAQGNNILKG IGFSAPKKQY SLEGDKVEVR
    LSAPETRGLK
151 IDKVYTFTKG SYLVNVRFDI ANGSGQTANL SADYRIVRDH
    SEPEGQGYFT
201 HSYVGPVVYT PEGNFQKVSF SDLDDDAXSG KSEAEYIRKT
    XTGWLGMIEH
251 HFMSTWILQP KGGQSVCAAG DCXXDIKRRN DKLYSTSVSV
    PLAAIQNGAK
301 SXASINLYAG PQTTSVIANI ADNLQLXKDY GKVHWFASPL
    FWLLNQLHNI
351 IGNWGWAIIV LTIIVKAVLY PLTNASYRSM AKMRAAAPKL
    QAIKEKYGDD
401 RMAQQQAMMQ LYTDEKINPL GGCLPMLLQI PVFIGLYWAL
    FASVELRQAP
451 WLGWITDLSR ADPYYILPII MAATMFAQTY LNPPPTDPMQ
    AKMMKIMPLV
501 XSXXFFXFPA GLVLYWVINN LLTIAQQWHI NRSIEKQRAQ
    GEVVS*

ORF11a and ORF11-1 show 95.2% identity in 544 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF11 shows 96.3% identity over a 240aa overlap with a predicted ORF (ORF11.ng) from N. gonorrhoeae:

An ORF11ng nucleotide sequence <SEQ ID 55> was predicted to encode a protein having amino acid sequence <SEQ ID 56>:

1   MAVNLYAGPQ TTSVIANIAD NLQLAKDYGK VHWFASPLFW
    LLNQLHNIIG
51  NWGWAIVVLT IIVKAVLYPL TNASYRSMAK MRAAAPELQT
    IKEKYGDDRM
101 AQQQAMMQLF EDEEINPLGG CLPMLLQIPV FIGLYWALFA
    SVELRQAPWL
151 GWITDLSRAD PYYILPIIMA ATMFAQTYLN PPPTDPMQAK
    MMKIMPLVFS
201 VMFFFFPAGL VLYWVVNNLL TIAQQWHINR SIEKQRAQGE
    VVS*

Further sequence analysis revealed the complete gonococcal DNA sequence <SEQ ID 57> to be:

1    ATGGATTTTA AAAGACTCAC GGCGTTTTTC GCCATCGCGC
     TGGTGATTAT
51   GATCGGCTGG GAAAAAATGT TCCCCACCCC GAAACCCGTC
     CCCGCGCCCC
101  AACAGGCGGC ACAAAAACAG GCAGCAACCG CTTCCGCCGA
     AGCCGCGCTC
151  GCGCCCGCAA CGCCGATTAC CGTAACGACC GACACGGTTC
     AAGCCGTTAT
201  TGATGAAAAA AGTGGCGACC TGCGCCGGCT GACCCTGCTC
     AAATACAAAG
251  CAACCGGCGA CGAAAACAAA CCGTTCGTCC TGTTTGGCGA
     CGGCAAAGAA
301  TACACCTACG TCGCCCAATC CGAACTTTTG GACGCGCAGG
     GCAACAACAT
351  TCTGAAAGGC ATCGGCTTTA GCGCACCGAA AAAACAGTAC
     ACCCTCAACG
401  GCGACACAGT CGAAGTCCGC CTGAGCGCGC CCGAAACCAA
     CGGACTGAAA
451  ATCGACAAAG TCTATACCTT TACCAAAGAC AGCTATCTGG
     TCAACGTCCG
501  CTTCGACATC GCCAACGGCA GCGGTCAAAC CGCCAACCTG
     AGCGCGGACT
551  ACCGCATCGT CCGCGACCAC AGCGAACCCG AGGGTCAAGG
     CTACTTTACC
601  CACTCTTACG TCGGCCCTGT TGTTTATACC CCTGAAGGCA
     ACTTCCAAAA
651  AGTCAGCTTC TCCgacTTgg acgACGATGC gaaaTccggc
     aaATccgagg
701  ccgaatacaT CCGCAAAACC ccgaccggtt ggctcggcat
     gattgaacac
751  cacttcatgt ccacctggat cctccAAcct aaaggcggcc
     aaaacgtttg
801  cgcccaggga gactgccgta tcgacattaa aCgccgcaac
     gacaagctgt
851  acagcgcaag cgtcagcgtg cctttaaccg ctatcccaac
     ccgggggcca
901  aaaccgaaaa tggcggTCAA CCTGTATGCC GGTCCGCAAA
     CCACATCCGT
951  TATCGCAAAC ATCGCcgacA ACCTGCAACT GGCAAAAGAC
     TACGGTAAAG
1001 TACACTGGTT CGCATCGCCG CTCTTCTGGC TCCTGAACCA
     ACTGCACAAC
1051 ATTATCGGCA ACTGGGGCTG GGCAATCGTC GTTTTGACCA
     TCATCGTCAA
1101 AGCCGTACTG TATCCATTGA CCAACGcctc ctACCGTTCG
     ATGGCGAAAA
1151 TGCGTGccgc cgcacCcaaA CTGCAGACCA TCAAAGAAAA
     ATAcgGCGAC
1201 GACCGTATGG CGCAACAGCA AGCGATGATG CAGCTTTACA
     AAgacgAGAA
1251 AATCAACCCG CTGGGCGGCT GTctgcctat gctgttgCAA
     ATCCCCGTCT
1301 TCATCGGCTT GTACTGGGCA TTGTTCGCCT CCGTAGAATT
     GCGCCAGGCA
1351 CCTTGGCTGG GCTGGATTAC CGACCTCAGC CGCGCCGACC
     CCTACTACAT
1401 CCTGCCCATC ATTATGGCGG CAACGATGTT CGCCCAAACC
     TATCTGAACC
1451 CGCCGCCGAC CGACCCGATG CAGGCGAAAA TGATGAAAAT
     CATGCCGTTG
1501 GTTTTCTCCG TCATGTTCTT CTTCTTCCCT GCCGGTTTGG
     TTCTCTACTG
1551 GGTGGTCAAC AACCTCCTGA CCATCGCCCA GCAGTGGCAC
     ATCAACCGCA
1601 GCATCGAAAA ACAACGCGCC CAAGGCGAAG TCGTTTCCTA
     A

This encodes a protein having amino acid sequence <SEQ ID 58; ORF11ng-1>:

1   MDFKRLTAFF AIALVIMIGW EKMFPTPKPV PAPQQAAQKQ
    AATASAEAAL
51  APATPITVTT DTVQAVIDEK SGDLRRLTLL KYKATGDENK
    PFVLFGDGKE
101 YTYVAQSELL DAQGNNILKG IGFSAPKKQY TLNGDTVEVR
    LSAPETNGLK
151 IDKVYTFTKD SYLVNVRFDI ANGSGQTANL SADYRIVRDH
    SEPEGQGYFT
201 HSYVGPVVYT PEGNFQKVSF SDLDDDAKSG KSEAEYIRKT
    PTGWLGMIEH
251 HFMSTWILQP KGGQNVCAQG DCRIDIKRRN DKLYSASVSV
    PLTAIPTRGP
301 KPKMAVNLYA GPQTTSVIAN IADNLQLAKD YGKVHWFASP
    LFWLLNQLHN
351 IIGNWGWAIV VLTIIVKAVL YPLTNASYRS MAKMRAAAPK
    LQTIKEKYGD
401 DRMAQQQAMM QLYKDEKINP LGGCLPMLLQ IPVFIGLYWA
    LFASVELRQA
451 PWLGWITDLS RADPYYILPI IMAATMFAQT YLNPPPTDPM
    QAKMMKIMPL
501 VFSVMFFFFP AGLVLYWVVN NLLTIAQQWH INRSIEKQRA
    QGEVVS*

ORF11ng-1 and ORF11-1 shown 95.1% identity in 546 aa overlap:

In addition, ORF11ng-1 shows significant homology with an inner-membrane protein from the database (accession number p25754):

Based on this analysis, including the homology to an inner-membrane protein from P. putida and the predicted transmembrane domains (seen in both the meningococcal and gonoccal proteins), it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 8

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 59>:

1   ..GCCGTCTTAA TCATCGAATT ATTGACGGGA ACGGTTTATC
    TTTTGGTTGT
51  NAGCGCGGCT TTGGCGGGTT CGGGCATTGC TTACGGGCTG
    ACCGGCAGTA
101 CGCCTGCCGC CGTCTTGACC GNCGCTCTGC TTTCCGCGCT
    GGGTATTTNG
151 TTCGTACACG CCAAAACCGC CGTTAGAAAA GTTGAAACGG
    ATTCATATCA
201 GGATTTGGAT GCCGGACAAT ATGTCGAAAT CCTCCGNCAC
    ACAGGCGGCA
251 ACCGTTACGA AGTT.TTTAT CGCGGTACG. ACTGGCAGGC
    TCAAAATACG
301 GGGCAAGAAG AGCTTGAACC AGGAACTCGC GCCCTCATTG
    TCCGCAAGGA
351 AGGCAACCTT CTTATTATCA CACACCCTTA A

This corresponds to the amino acid sequence <SEQ ID 60; ORF13>:

1   ..AVLIIELLTG TVYLLVVSAA LAGSGIAYGL TGSTPAAVLT
    XALLSALGIX
51  FVHAKTAVRK VETDSYQDLD AGQYVEILRH TGGNRYEVXY
    RGTXWQAQNT
101 GQEELEPGTR ALIVRKEGNL LIITHP*

Further sequence analysis elaborated the DNA sequence slightly <SEQ ID 61>:

1   ..GCCGTCTTAA TCATCGAATT ATTGACGGGA ACGGTTTATC
    TTTTGGTTGT
51  nAGCGCGGCT TTGGCGGGTT CGGGCATTGC TTACGGGCTG
    ACCGGCAGTA
101 CGCCTGCCGC CGTCTTGACC GnCGCTCTGC TTTCCGCGCT
    GGGTATTTnG
151 TTCGTACACG CCAAAACCGC CGTTAGAAAA GTTGAAACGG
    ATTCATATCA
201 GGATTTGGAT GCCGGACAAT ATGTCGAAAT CCTCCGACAC
    ACAGGCGGCA
251 ACCGTTACGA AGTTTTtTAT CGCGGTACGc ACTGGCAGGC
    TCAAAATACG
301 GGGCAAGAAG AGCTTGAACC AGGAACTCGC GCCCTCATTG
    TCCGCAAGGA
351 AGGCAACCTT CTTATTATCA CACACCCTTA A

This corresponds to the amino acid sequence <SEQ ID 62; ORF13-1>:

1   ..AVLIIELLTG TVYLLVVSAA LAGSGIAYGL TGSTPAAVLT
    XALLSALGIX
51  FVHAKTAVRK VETDSYQDLD AGQYVEILRH TGGNRYEVFY
    RGTHWQAQNT
101 GQEELEPGTR ALIVRKEGNL LIITHP*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF13 shows 92.9% identity over a 126aa overlap with an ORF (ORF13a) from strain A of N. meningitidis:

The complete length ORF13a nucleotide sequence <SEQ ID 63> is:

1   ATGACTGTAT GGTTTGTTGC CGCTGTTGCC GTCTTAATCA
    TCGAATTATT
51  GACGGGAACG GTTTATCTTT TGGTTGTCAG CGCGGCTTTG
    GCGGGTTCGG
101 GCATTGCTTA CGGGCTGACC GGCAGCACGC CTGCCGCCGT
    CTTGACCGCC
151 GCTCTGCTTT CCGCGCTGGG TATTTGGTTC GTACACGCCA
    AAACCGCCGT
201 GGGAAAAGTT GAAACGGATT CATATCAGGA TTTGGATGCC
    GGGCAATATG
251 CCGAAATCCT CCGGCACGCA GGCGGCAACC GTTACGAAGT
    TTTTTATCGC
301 GGTACGCACT GGCAGGCTCA AAATACGGGG CAAGAAGAGC
    TTGAACCAGG
351 AACGCGCGCC CTAATCGTCC GCAAGGAAGG CAACCTTCTT
    ATCATCGCAA
401 AACCTTAA

This encodes a protein having amino acid sequence <SEQ ID 64>:

1   MTVWFVAAVA VLIIELLTGT VYLLVVSAAL AGSGIAYGLT
    GSTPAAVLTA
51  ALLSALGIWF VHAKTAVGKV ETDSYQDLDA GQYAEILRHA
    GGNRYEVFYR
101 GTHWQAQNTG QEELEPGTRA LIVRKEGNLL IIAKP*

ORF13a and ORF13-1 show 94.4% identity in 126 aa overlap

Homology with a Predicted ORF from N. gonorrhoeae

ORF13 shows 89.7% identity over a 126aa overlap with a predicted ORF (ORF13.ng) from N. gonorrhoeae:

The complete length ORF13ng nucleotide sequence <SEQ ID 65> is:

1   ATGACTGTAT GGTTTGTTGC CGCTGTTGCC GTCTTAATCA
    TCGAATTATT
51  GACGGGAACG GTTTATCTTT TGGTTGTCAG CGCGGCTTTG
    GCGGGTTCGG
101 GCATTGCCTA CGGGCTGACT GGCAGCACGC CTGCCGCCGT
    CTTGACCGCC
151 GCACTGCTTT CCGCGCTGGG CATTTGGTTC GTACATGCCA
    AAACCGCCGT
201 GGGAAAAGTT GAAACGGATT CATATCAGGA TTTGGATACC
    GGAAAATATG
251 CCGAAATCCT CCGATACACA GGCGGCAACC GTTACGAAGT
    TTTTTATCGC
301 GGTACGCACT GGCAGGCGCA AAATACGGGG CAGGAAGTGT
    TTGAACCGGG
351 AACGCGCGCC CTCATCGTCC GCAAAGAAGG TAACCTTCTT
    ATCATCGCAA
401 ACCCTTAA

This encodes a protein having amino acid sequence <SEQ ID 66>:

1   MTVWFVAAVA VLIIELLTGT VYLLVVSAAL AGSGIAYGLT
    GSTPAAVLTA
51  ALLSALGIWF VHAKTAVGKV ETDSYQDLDT GKYAEILRYT
    GGNRYEVFYR
101 GTHWQAQNTG QEVFEPGTRA LIVRKEGNLL IIANP*

ORF13ng shows 91.3% identity in 126 aa overlap with ORF13-1:

Based on this analysis, including the extensive leader sequence in this protein, it is predicted that ORF13 and ORF13ng are likely to be outer membrane proteins. It is thus predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 9

The following DNA sequence was identified in N. meningitidis <SEQ ID 67>:

1   ATGTwTGATT TCGGTTTrGG CGArCTGGTT TTTGTCGGCA
    TTATCGCCCT
51  GATwGtCCTC GGCCCCGAAC GCsTGCCCGA GGCCGCCCGC
    AyCGCCGGAC
101 GGcTCATCGG CAGGCTGCAA CGCTTTGTCG GcAGCGTCAA
    ACAGGAATTT
151 GACACTCAAA TCGAACTGGA AGAACTGAGG AAGGCAAAGC
    AGGAATTTGA
201 AGCTGCCGcC GCTCAGGTTC GAGACAGCCT CAAAGAAACC
    GGTACGGATA
251 TGGAAGGCAA TCTGCACGAC ATTTCCGACG GTCTGAAGCC
    TTGGGAAAAA
301 CTGCCCGAAC AGCGGACACC TGCCGATTTC GGTGTCGATG
    AAAACGGCAA
351 TCCGCT.TCC CGATGCGGCA AACACCCTAT CAGACGGCAT
    TTCCGACGTT
401 ATGCCGTC..

This corresponds to the amino acid sequence <SEQ ID 68; ORF2>:

1   MXDFGLGELV FVGIIALIVL GPERXPEAAR XAGRLIGRLQ
    RFVGSVKQEF
51  DTQIELEELR KAKQEFEAAA AQVRDSLKET GTDMEGNLHD
    ISDGLKPWEK
101 LPEQRTPADF GVDENGNPXS RCGKHPIRRH FRRYAV..

Further work revealed the complete nucleotide sequence <SEQ ID 69>:

1   ATGTTTGATT TCGGTTTGGG CGAGCTGGTT TTTGTCGGCA
    TTATCGCCCT
51  GATTGTCCTC GGCCCCGAAC GCCTGCCCGA GGCCGCCCGC
    ACCGCCGGAC
101 GGCTCATCGG CAGGCTGCAA CGCTTTGTCG GCAGCGTCAA
    ACAGGAATTT
151 GACACTCAAA TCGAACTGGA AGAACTGAGG AAGGCAAAGC
    AGGAATTTGA
201 AGCTGCCGCC GCTCAGGTTC GAGACAGCCT CAAAGAAACC
    GGTACGGATA
251 TGGAAGGCAA TCTGCACGAC ATTTCCGACG GTCTGAAGCC
    TTGGGAAAAA
301 CTGCCCGAAC AGCGGACACC TGCCGATTTC GGTGTCGATG
    AAAACGGCAA
351 TCCGCTTCCC GATGCGGCAA ACACCCTATC AGACGGCATT
    TCCGACGTTA
401 TGCCGTCCGA ACGTTCCTAC GCTTCCGCCG AAACCCTTGG
    GGACAGCGGG
451 CAAACCGGCA GTACAGCCGA ACCCGCGGAA ACCGACCAAG
    ACCGCGCATG
501 GCGGGAATAC CTGACTGCTT CTGCCGCCGC ACCCGTCGTA
    CAGACCGTCG
551 AAGTCAGCTA TATCGATACT GCTGTTGAAA CGCCTGTTCC
    GCACACCACT
601 TCCCTGCGCA AACAGGCAAT AAGCCGCAAA CGCGATTTTC
    GTCCGAAACA
651 CCGCGCCAAA CCTAAATTGC GCGTCCGTAA ATCATAA

This corresponds to the amino acid sequence <SEQ ID 70; ORF2-1>:

1   MFDFGLGELV FVGIIALIVL GPERLPEAAR TAGRLIGRLQ
    RFVGSVKQEF
51  DTQIELEELR KAKQEFEAAA AQVRDSLKET GTDMEGNLHD
    ISDGLKPWEK
101 LPEQRTPADF GVDENGNPLP DAANTLSDGI SDVMPSERSY
    ASAETLGDSG
151 QTGSTAEPAE TDQDRAWREY LTASAAAPVV QTVEVSYIDT
    AVETPVPHTT
201 SLRKQAISRK RDFRPKHRAK PKLRVRKS*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 71>:

1   ATGTTTGATT TCGGTTTGGG CGAGCTGGTT TTTGTCGGCA
    TTATCGCCCT
51  GATTGTCCTC GGCCCCGAAC GCCTGCCCGA GGCCGCCCGC
    ACCGCCGGAC
101 GGCTCATCGG CAGGCTGCAA CGCTTTGTCG GCAGCGTCAA
    ACAGGAATTT
151 GACACGCAAA TCGAACTGGA AGAACTAAGG AAGGCAAAGC
    AGGAATTTGA
201 AGCTGCCGCT GCTCAGGTTC GAGACAGCCT CAAAGAAACC
    GGTACGGATA
251 TGGAGGGTAA TCTGCACGAC ATTTCCGACG GTCTGAAGCC
    TTGGGAAAAA
301 CTGCCCGAAC AGCGCACGCC TGCTGATTTC GGTGTCGATG
    AAAACGGCAA
351 TCCCTTTCCC GATGCGGCAA ACACCCTATT AGACGGCATT
    TCCGACGTTA
401 TGCCGTCCGA ACGTTCCTAC GCTTCCGCCG AAACCCTTGG
    GGACAGCGGG
451 CAAACCGGCA GTACAGCCGA ACCCGCGGAA ACCGACCAAG
    ACCGTGCATG
501 GCGGGAATAC CTGACTGCTT CTGCCGCCGC ACCCGTCGTA
    CAGACCGTCG
551 AAGTCAGCTA TATCGATACC GCTGTTGAAA CCCCTGTTCC
    GCATACCACT
601 TCGCTGCGTA AACAGGCAAT AAGCCGCAAA CGCGATTTGC
    GTCCTAAATC
651 CCGCGCCAAA CCTAAATTGC GCGTCCGTAA ATCATAA

This encodes a protein having amino acid sequence <SEQ ID 72; ORF2a>:

1   MFDFGLGELV FVGIIALIVL GPERLPEAAR TAGRLIGRLQ
    RFVGSVKQEF
51  DTQIELEELR KAKQEFEAAA AQVRDSLKET GTDMEGNLHD
    ISDGLKPWEK
101 LPEQRTPADF GVDENGNPFP DAANTLLDGI SDVMPSERSY
    ASAETLGDSG
151 QTGSTAEPAE TDQDRAWREY LTASAAAPVV QTVEVSYIDT
    AVETPVPHTT
201 SLRKQAISRK RDLRPKSRAK PKLRVRKS*

The originally-identified partial strain B sequence (ORF2) shows 97.5% identity over a 118aa overlap with ORF2a:

The complete strain B sequence (ORF2-1) and ORF2a show 98.2% identity in 228 aa overlap:

Further work identified a partial DNA sequence <SEQ ID 73> in N. gonorrhoeae encoding the following amino acid sequence <SEQ ID 74; ORF2ng>:

1   MFDFGLGELI FVGIIALIVL GPERLPEAAR TAGRLIGRLQ
    RFVGSVKQEL
51  DTQIELEELR KVKQAFEAAA AQVRDSLKET DTDMQNSLHD
    ISDGLKPWEK
101 LPEQRTPADF GVDEKGNSLS RYGKHRIRRH FRRYAV*

Further work identified the complete gonococcal gene sequence <SEQ ID 75>:

1   ATGTTTGATT TCGGTTTGGG CGAGCTGATT TTTGTCGGCA
    TTATCGCCCT
51  GATTGTCCTT GGTCCAGAAC GCCTGCCCGA AGCCGCCCGC
    ACTGCCGGAC
101 GGCTTATCGG CAGGCTGCAA CGCTTTGTAG GAAGCGTCAA
    ACAAGAACTT
151 GACACTCAAA TCGAACTGGA AGAGCTGAGG AAGGTCAAGC
    AGGCATTCGA
201 AGCTGCCGCC GCTCAGGTTC GAGACAGCCT CAAAGAAACC
    GATACGGATA
251 TGCAGAACAG TCTGCACGAC ATTTCCGACG GTCTGAAGCC
    TTGGGAAAAA
301 CTGCCCGAAC AGCGCACGCc tgccgatttc gGTGTCGATg
    AAAacggcaa
351 tccccttccc gATACGGCAA ACACCGTATC AGACGGCATT
    TCCGACGTTA
401 TGCCGTCTGA ACGTTCCGAT ACTtccgcCG AAACCCTTGG
    GGACGACAGG
451 CAAACCGGCA GTACAGCCGA ACCTGCGGAA ACCGACAAAG
    ACCGCGCATG
501 GCGGGAATAC CTGactgctt ctgccgccgc acctgtcgta
    Cagagggccg
551 tcgaagtcag ctaTATCGAT ACTGCTGTTG AAacgcctgT
    tccgcaCacc
601 acttccctgc gcaAACAGGC AATAAACCGC AAACGCGATT
    TttgtccgaA
651 ACACCGCGCc aAACCGAAat tgcgcgtcCG TAAATCATAA

This encodes a protein having the amino acid sequence <SEQ ID 76; ORF2ng-1>:

1   MFDFGLGELI FVGIIALIVL GPERLPEAAR TAGRLIGRLQ
    RFVGSVKQEL
51  DTQIELEELR KVKQAFEAAA AQVRDSLKET DTDMQNSLHD
    ISDGLKPWEK
101 LPEQRTPADF GVDENGNPLP DTANTVSDGI SDVMPSERSD
    TSAETLGDDR
151 QTGSTAEPAE TDKDRAWREY LTASAAAPVV QRAVEVSYID
    TAVETPVPHT
201 TSLRKQAINR KRDFCPKHRA KPKLRVRKS*

The originally-identified partial strain B sequence (ORF2) shows 87.5% identity over a 136aa overlap with ORF2ng:

The complete strain B and gonococcal sequences (ORF2-1 & ORF2ng-1) show 91.7% identity in 229 aa overlap:

Computer analysis of these amino acid sequences indicates a transmembrane region (underlined), and also revealed homology (59% identity) between the gonococcal sequence and the TatB protein of E. coli:

gnl|PID|e1292181 (AJ005830) TatB protein [Escherichia coli] Length = 171
Score = 56.6 bits (134), Expect = 1e−07
Identities = 30/88 (34%), Positives = 52/88 (59%), Gaps = 1/88 (1%)
Query:  1 MFDFGLGELIFVGIIALIVLGPERLPEAARTAGRLIGRLQRFVGSVKQELDTQIELEELR 60
MFD G  EL+ V II L+VLGP+RLP A +T    I  L+     +V+ EL +++L+E  +
Sbjct:  1 MFDIGFSELLLVFIIGLVVLGPQRLPVAVKTVAGWIRALRSLATTVQNELTQELKLQEFQ 60
Query: 61 -KVKQAFEAAAAQVRDSLKETDTDMQNS 87
+K+  +A+   +   LK +  +++ +
Sbjct: 61 DSLKKVEKASLTNLTPELKASMDELRQA 88

Based on this analysis, it was predicted that ORF2, ORF2a and ORF2ng are likely to be membrane proteins and so the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF2-1 (16 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 3A shows the results of affinity purification of the GST-fusion protein, and FIG. 3B shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for Western blots (FIG. 3C), ELISA (positive result), and FACS analysis (FIG. 3D). These experiments confirm that ORF37-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 10

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 77>:

1   ATGCAAGCAC GGCTGCTGAT ACCTATTCTT TTTTCAGTTT
    TTATTTTATC
51  CGC.TGCGGG ACACTGACAG GTATTCCATC GCATGGCGgA
    GkTAAACgCT
101 TTgCGGTCGA ACAAGAACTT GTGGCCGCTT CTGCCAGAGC
    TGCCGTTAAA
151 GACATGGATT TACAGGCATT ACACGGACGA AAAGTTGCAT
    TGTACATTGC
201 CACTATGGGC GACCAAGGTT CAGGcAGTTT GACAGGGGGG
    TCGCTACTCC
251 ATTGATGCAC kGrTwCsTGG CGAATACATA AACAGCCCTG
    CCGTCCGTAC
301 CGATTACACC TATCCACGTT ACGAAACCAC CGCTGAAACA
    ACATCAGGCG
351 GTTTGACAGG TTTAACCACT TCTTTATCTA CACTTAATGC
    CCCTGCACTC
401 TCTCGCACCC AATCAGACGG TAGCGGAAGT AAAAGCAGTC
    TGGGCTTAAA
451 TATTGGCGGG ATGGGGGATT ATCGAAATGA AACCTTGACG
    ACTAACCCGC
501 GCGACACTGC CTTTCTTTCC CACTTGGTAC AGACCGTATT
    TTTCCTGCGC
551 GGCATAGACG TTGTTTCTCC TGCCAATGCC GATACAGATG
    TGTTTATTAA
601 CATCGACGTA TTCGGAACGA TACGCAACAG AACCGAAATG..

This corresponds to the amino acid sequence <SEQ ID 78; ORF15>:

1   MQARLLIPIL FSVFILSACG TLTGIPSHGG XKRFAVEQEL
    VAASARAAVK
51  DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDAXXXG
    EYINSPAVRT
101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTQSDG
    SGSKSSLGLN
151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP
    ANADTDVFIN
201 IDVFGTIRNR TEM..

Further work revealed the complete nucleotide sequence <SEQ ID 79>:

1   ATGCAAGCAC GGCTGCTGAT ACCTATTCTT TTTTCAGTTT
    TTATTTTATC
51  CGCCTGCGGG ACACTGACAG GTATTCCATC GCATGGCGGA
    GGTAAACGCT
101 TTGCGGTCGA ACAAGAACTT GTGGCCGCTT CTGCCAGAGC
    TGCCGTTAAA
151 GACATGGATT TACAGGCATT ACACGGACGA AAAGTTGCAT
    TGTACATTGC
201 CACTATGGGC GACCAAGGTT CAGGCAGTTT GACAGGGGGT
    CGCTACTCCA
251 TTGATGCACT GATTCGTGGC GAATACATAA ACAGCCCTGC
    CGTCCGTACC
301 GATTACACCT ATCCACGTTA CGAAACCACC GCTGAAACAA
    CATCAGGCGG
351 TTTGACAGGT TTAACCACTT CTTTATCTAC ACTTAATGCC
    CCTGCACTCT
401 CTCGCACCCA ATCAGACGGT AGCGGAAGTA AAAGCAGTCT
    GGGCTTAAAT
451 ATTGGCGGGA TGGGGGATTA TCGAAATGAA ACCTTGACGA
    CTAACCCGCG
501 CGACACTGCC TTTCTTTCCC ACTTGGTACA GACCGTATTT
    TTCCTGCGCG
551 GCATAGACGT TGTTTCTCCT GCCAATGCCG ATACAGATGT
    GTTTATTAAC
601 ATCGACGTAT TCGGAACGAT ACGCAACAGA ACCGAAATGC
    ACCTATACAA
651 TGCCGAAACA CTGAAAGCCC AAACAAAACT GGAATATTTC
    GCAGTAGACA
701 GAACCAATAA AAAATTGCTC ATCAAACCAA AAACCAATGC
    GTTTGAAGCT
751 GCCTATAAAG AAAATTACGC ATTGTGGATG GGGCCGTATA
    AAGTAAGCAA
801 AGGAATTAAA CCGACGGAAG GATTAATGGT CGATTTCTCC
    GATATCCGAC
851 CATACGGCAA TCATACGGGT AACTCCGCCC CATCCGTAGA
    GGCTGATAAC
901 AGTCATGAGG GGTATGGATA CAGCGATGAA GTAGTGCGAC
    AACATAGACA
951 AGGACAACCT TGA

This corresponds to the amino acid sequence <SEQ ID 80; ORF15-1>:

1   MQARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL
    VAASARAAVK
51  DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDALIRG
    EYINSPAVRT
101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTQSDG
    SGSKSSLGLN
151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP
    ANADTDVFIN
201 IDVFGTIRNR TEMHLYNAET LKAQTKLEYF AVDRTNKKLL
    IKPKTNAFEA
251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIRPYGNHTG
    NSAPSVEADN
301 SHEGYGYSDE VVRQHRQGQP *

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 81>:

1   ATGCAAGCAC GGCTGCTGAT ACCTATTCTT TTTTCAGTTT
    TTATTTTATC
51  CGCCTGCGGG ACACTGACAG GTATTCCATC GCATGGCGGA
    GGTAAACGCT
101 TTGCGGTCGA ACAAGAACTT GTGGCCGCTT CTGCCAGAGC
    TGCCGTTAAA
151 GACATGGATT TACAGGCATT ACACGGACGA AAAGTTGCAT
    TGTACATTGC
201 AACTATGGGC GACCAAGGTT CAGGCAGTTT GACAGGGGGT
    CGCTACTCCA
251 TTGATGCACT GATTCGTGGC GAATACATAA ACAGCCCTGC
    CGTCCGTACC
301 GATTACACCT ATCCACGTTA CGAAACCACC GCTGAAACAA
    CATCAGGCGG
351 TTTGACAGGT TTAACCACTT CTTTATCTAC ACTTAATGCC
    CCTGCACTCT
401 CGCGCACCCA ATCAGACGGT AGCGGAAGTA AAAGCAGTCT
    GGGCTTAAAT
451 ATTGGCGGGA TGGGGGATTA TCGAAATGAA ACCTTGACGA
    CTAACCCGCG
501 CGACACTGCC TTTCTTTCCC ACTTGGTACA GACCGTATTT
    TTCCTGCGCG
551 GCATAGACGT TGTTTCTCCT GCCAATGCCG ATACGGATGT
    GTTTATTAAC
601 ATCGACGTAT TCGGAACGAT ACGCAACAGA ACCGAAATGC
    ACCTATACAA
651 TGCCGAAACA CTGAAAGCCC AAACAAAACT GGAATATTTC
    GCAGTAGACA
701 GAACCAATAA AAAATTGCTC ATCAAACCAA AAACCAATGC
    GTTTGAAGCT
751 GCCTATAAAG AAAATTACGC ATTGTGGATG GGACCGTATA
    AAGTAAGCAA
801 AGGAATTAAA CCGACAGAAG GATTAATGGT CGATTTCTCC
    GATATCCAAC
851 CATACGGCAA TCATATGGGT AACTCTGCCC CATCCGTAGA
    GGCTGATAAC
901 AGTCATGAGG GGTATGGATA CAGCGATGAA GCAGTGCGAC
    GACATAGACA
951 AGGGCAACCT TGA

This encodes a protein having amino acid sequence <SEQ ID 82; ORF15a>:

1   MQARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL
    VAASARAAVK
51  DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDALIRG
    EYINSPAVRT
101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTQSDG
    SGSKSSLGLN
151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP
    ANADTDVFIN
201 IDVFGTIRNR TEMHLYNAET LKAQTKLEYF AVDRTNKKLL
    IKPKTNAFEA
251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIQPYGNHMG
    NSAPSVEADN
301 SHEGYGYSDE AVRRHRQGQP *

The originally-identified partial strain B sequence (ORF15) shows 98.1% identity over a 213aa overlap with ORF15a:

The complete strain B sequence (ORF15-1) and ORF15a show 98.8% identity in 320 aa overlap:

Further work identified the corresponding gene in N. gonorrhoeae <SEQ ID 83>:

1   ATGCGGGCAC GGCTGCTGAT ACCTATTCTT TTTTCAGTTT
    TTATTTTATC
51  CGCCTGCGGG ACACTGACAG GTATTCCATC GCATGGCGGA
    GGCAAACGCT
101 TCGCGGTCGA ACAAGAACTT GTGGCCGCTT CTGCCAGAGC
    TGCCGTTAAA
151 GACATGGATT TACAGGCATT ACACGGACGA AAAGTTGCAT
    TGTACATTGC
201 AACTATGGGC GACCAAGGTT CAGGCAGTTT GACAGGGGGT
    CGCTACTCCA
251 TTGATGCACT GATTCGCGGC GAATACATAA ACAGCCCTGC
    CGTCCGCACC
301 GATTACACCT ATCCGCGTTA CGAAACCACC GCTGAAACAA
    CATCAGGCGG
351 TTTGACGGGT TTAACCACTT CTTTATCTAC ACTTAATGCC
    CCTGCACTCT
401 CGCGCACCCA ATCAGACGGT AGCGGAAGTA GGAGCAGTCT
    GGGCTTAAAT
451 ATTGGCGGGA TGGGGGATTA TCGAAATGAA ACCTTGACGA
    CCAACCCGCG
501 CGACACTGCC TTTCTTTCCC ACTTGGTGCA GACCGTATTT
    TTCCTGCGCG
551 GCATAGACGT TGTTTCTCCT GCCAATGCCG ATACAGATGT
    GTTTATTAAC
601 ATCGACGTAT TCGGAACGAT ACGCAACAGA ACCGAAATGC
    ACCTATACAA
651 TGCCGAAACA CTGAAAGCCC AAACAAAACT GGAATATTTC
    GCAGTAGACA
701 GAACCAATAA AAAATTGCTC ATCAAACCCA AAACCAATGC
    GTTTGAAGCT
751 GCCTATAAAG AAAATTACGC ATTGTGGATG GGGCCGTATA
    AAGTAAGCAA
801 AGGAATCAAA CCGACGGAAG GATTGATGGT CGATTTCTCC
    GATATCCAAC
851 CATACGGCAA TCATACGGGT AACTCCGCCC CATCCGTAGA
    GGCTGATAAC
901 AGTCATGAGG GGTATGGATA CAGCGATGAA GCAGTGCGAC
    AACATAGACA
951 AGGGCAACCT TGA

This encodes a protein having amino acid sequence <SEQ ID 84; ORF15ng>:

1   MRARLLIPIL FSVFILSACG TLTGIPSHGG GKRFAVEQEL
    VAASARAAVK
51  DMDLQALHGR KVALYIATMG DQGSGSLTGG RYSIDALIRG
    EYINSPAVRT
101 DYTYPRYETT AETTSGGLTG LTTSLSTLNA PALSRTQSDG
    SGSRSSLGLN
151 IGGMGDYRNE TLTTNPRDTA FLSHLVQTVF FLRGIDVVSP
    ANADTDVFIN
201 IDVFGTIRNR TEMHLYNAET LKAQTKLEYF AVDRTNKKLL
    IKPKTNAFEA
251 AYKENYALWM GPYKVSKGIK PTEGLMVDFS DIQPYGNHTG
    NSAPSVEADN
301 SHEGYGYSDE AVRQHRQGQP *

The originally-identified partial strain B sequence (ORF15) shows 97.2% identity over a 213aa overlap with ORF15ng:

The complete strain B sequence (ORF15-1) and ORF15ng show 98.8% identity in 320 aa overlap:

Computer analysis of these amino acid sequences reveals an ILSAC motif (putative membrane lipoprotein lipid attachment site, as predicted by the MOTIFS program).

Indicates a putative leader sequence, and it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF15-1 (31.7 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 4A shows the results of affinity purification of the GST-fusion protein, and FIG. 4B shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for Western blot (FIG. 4C) and ELISA (positive result). These experiments confirm that ORFX-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 11

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 85>:

1   ..GG.CAGCACA AAAAACAGGC GGTTGAACGG AAAAACCGTA
    TTTACGATGA
51  TGCCGGGTAT GATATTCGGC GTATTCACGG GCGCATTCTC
    CGCAAAATAT
101 ATCCCCGCGT TCGGGCTTCA AATTTTCTTC ATCCTGTTTT
    TAACCGCCGT
151 CGCATTCAAA ACACTGCATA CCGACCCTCA GACGGCATCC
    CGCCCGCTGC
201 CCGGACTGCC CrGACTGACT GCGGTTTCCA CACTGTTCGG
    CACAATGTCG
251 AGCTGGGTCG GCATAGGCGG CGGTTCACTT TCCGTCCCCT
    TCTTAATCCA
301 CTGCGGCTTC CCCGCCCATA AAGCCATCGG CACATCATCC
    GGCCTTGCCT
351 GGCCGATTGC ACTCTCCGGC GCAATATCGT ATCTGCTCAA
    CGGCCTGAAT
401 ATTGCAGGAT TGCCCGAAGG GTCACTGGGC TTCCTTTACC
    TGCCCGCCGT
451 CGCCGTCCTC AGCGCGGCAA CCATTGCCTT TGCCCCGCTC
    GGTGTCAAAA
501 CCGCCCACAA ACTTTCTTCT GCCAAACTCA AAAAATC.TT
    CGGCATTATG
551 TTGCTTTTGA TTGCCGGAAA AATGCTGTAC AACCTGCTTT
    AA

This corresponds to the amino acid sequence <SEQ ID 86; ORF17>:

1   ..GQHKKQAVNG KTVFTMMPGM IFGVFTGAFS AKYIPAFGLQ
    IFFILFLTAV
51  AFKTLHTDPQ TASRPLPGLP XLTAVSTLFG TMSSWVGIGG
    GSLSVPFLIH
101 CGFPAHKAIG TSSGLAWPIA LSGAISYLLN GLNIAGLPEG
    SLGFLYLPAV
151 AVLSAATIAF APLGVKTAHK LSSAKLKKSF GIMLLLIAGK
    MLYNLL*

Further work revealed the complete nucleotide sequence <SEQ ID 87>:

1   ATGTGGCATT GGGACATTAT CTTAATCCTG CTTGCCGTAG
    GCAGTGCGGC
51  AGGTTTTATT GCCGGCCTGT TCGGCGTAGG CGGCGGCACG
    CTGATTGTCC
101 CTGTCGTTTT ATGGGTGCTT GATTTGCAGG GTTTGGCACA
    ACATCCTTAC
151 GCGCAACACC TCGCCGTCGG CACATCCTTC GCCGTCATGG
    TCTTCACCGC
201 CTTTTCCAGT ATGCTGGGGC AGCACAAAAA ACAGGCGGTC
    GACTGGAAAA
251 CCGTATTTAC GATGATGCCG GGTATGATAT TCGGCGTATT
    CACGGGCGCA
301 CTCTCCGCAA AATATATCCC CGCGTTCGGG CTTCAAATTT
    TCTTCATCCT
351 GTTTTTAACC GCCGTCGCAT TCAAAACACT GCATACCGAC
    CCTCAGACGG
401 CATCCCGCCC GCTGCCCGGA CTGCCCGGAC TGACTGCGGT
    TTCCACACTG
451 TTCGGCACAA TGTCGAGCTG GGTCGGCATA GGCGGCGGTT
    CACTTTCCGT
501 CCCCTTCTTA ATCCACTGCG GCTTCCCCGC CCATAAAGCC
    ATCGGCACAT
551 CATCCGGCCT TGCCTGGCCG ATTGCACTCT CCGGCGCAAT
    ATCGTATCTG
601 CTCAACGGCC TGAATATTGC AGGATTGCCC GAAGGGTCAC
    TGGGCTTCCT
651 TTACCTGCCC GCCGTCGCCG TCCTCAGCGC GGCAACCATT
    GCCTTTGCCC
701 CGCTCGGTGT CAAAACCGCC CACAAACTTT CTTCTGCCAA
    ACTCAAAAAA
751 Tc.TTCGGCA TTATGTTGCT TTTGATTGCC GGAAAAATGC
    TGTACAACCT
801 GCTTTAA

This corresponds to the amino acid sequence <SEQ ID 88; ORF17-1>:

1   MWHWDIILIL LAVGSAAGFI AGLFGVGGGT LIVPVVLWVL
    DLQGLAQHPY
51  AQHLAVGTSF AVMVFTAFSS MLGQHKKQAV DWKTVFTMMP
    GMIFGVFTGA
101 LSAKYIPAFG LQIFFILFLT AVAFKTLHTD PQTASRPLPG
    LPGLTAVSTL
151 FGTMSSWVGI GGGSLSVPFL IHCGFPAHKA IGTSSGLAWP
    IALSGAISYL
201 LNGLNIAGLP EGSLGFLYLP AVAVLSAATI AFAPLGVKTA
    HKLSSAKLKK
251 XFGIMLLLIA GKMLYNLL*

Computer analysis of this amino acid sequence gave the following results:

Homology with Hypothetical H. influenzae Transmembrane Protein H10902 (Accession number P44070)

ORF17 and HI0902 proteins show 28% aa identity in 192 aa overlap:

ORF17	3	HKKQAVNGKTVFTMMPGMIFGVFT-GAFSAKYIPAFGLQIF--FILFLTAVAFKTLHTDP	59
		HK   +  + V  + P ++  VF  G F  +       +IF   +++L      ++  D
HI0902	72	HKLGNIVWQAVRILAPVIMLSVFICGLFIGRLDREISAKIFACLVVYLATKMVLSIKKD-	130
ORF17	60	QTASRPLPGLPXLTAVSTLFGTMSSWVGIGGGSLSVPFLIHCGFPAHKAIGTSSGLAWPI	119
		Q  ++ L  L  +     L G  SS  GIGGG   VPFL   G    +AIG+S+     +
HI0902	131	QVTTKSLTPLSSVIG-GILIGMASSAAGIGGGGFIVPFLTARGINIKQAIGSSAFCGMLL	189
ORF17	120	ALSGAISYLLNGLNIAGLPEGSLGFLYLPAVAVLSAATIAFAPLGVXXXXXXXXXXXXXX	179
		+SG  S++++G     +PE SLG++YLPAV  ++A +   + LG
HI0902	190	GISGMFSFIVSGWGNPLMPEYSLGYIYLPAVLGITATSFFTSKLGASATAKLPVSTLKKG	249
ORF17	180	FGIMLLLIAGKM	191
		F + L+++A  M
HI0902	250	FALFLIVVAINM	261

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF17 shows 96.9% identity over a 196aa overlap with an ORF (ORF17a) from strain A of N. meningitidis:

The complete length ORF17a nucleotide sequence <SEQ ID 89> is:

1   ATGTGGCATT GGGACATTAT CTTAATCCTG CTTGCCGTAG
    GCAGTGCGGC
51  AGGTTTTATT GCCGGCCTGT TCGGCGTAGG CGGCGGCACG
    CTGATTGTCC
101 CTGTCGTTTT ATGGGTGCTT GATTTGCAGG GTTTGGCACA
    ACATCCTTAC
151 GCGCAACACC TCGCCGTCGG CACATCCTTC GCCGTCATGG
    TCTTCACCGC
201 CTTTTCCAGT ATGCTGGGGC AGCACAAAAA ACAGGCGGTC
    GACTGGAAAA
251 CCGTATTTAC GATGATGCCG GGTATGGTAT TCGGCGTATT
    CGCTGGCGCA
301 CTCTCCGCAA AATATATCCC AGCGTTCGGG CTTCAAATTT
    TCTTCATCCT
351 GTTTTTAACC GCCGTCGCAT TCAAAACACT GCATACCGAC
    CCTCAGACGG
401 CATCCCGCCC GCTGCCCGGA CTGCCCGGAC TGACTGCGGT
    TTCCACACTG
451 TTCGGCACAA TGTCGAGCTG GGTCGGCATA GGCGGCGGTT
    CACTTTCCGT
501 CCCCTTCTTA ATCCACTGCG GCTTCCCCGC CCATAAAGCC
    ATCGGCACAT
551 CATCCGGCCT TGCCTGGCCG ATTGCACTCT CCGGCGCAAT
    ATCGTATCTG
601 CTCAACGGCC TGAATATTGC AGGATTGCCC GAAGGGTCAC
    TGGGCTTCCT
651 TTACCTGCCC GCCGTCGCCG TCCTCAGCGC GGCAACCATT
    GCCTTTGCCC
701 CGCTCGGTGT CAAAACCGCC CACAAACTTT CTTCTGCCAA
    ACTCAAAAAA
751 TCCTTCGGCA TTATGTTGCT TTTGATTGCC GGAAAAATGC
    TGTACAACCT
801 GCTTTAA

This encodes a protein having amino acid sequence <SEQ ID 90>:

1   MWHWDIILIL LAVGSAAGFI AGLFGVGGGT LIVPVVLWVL
    DLQGLAQHPY
51  AQHLAVGTSF AVMVFTAFSS MLGQHKKQAV DWKTVFTMMP
    GMVFGVFAGA
101 LSAKYIPAFG LQIFFILFLT AVAFKTLHTD PQTASRPLPG
    LPGLTAVSTL
151 FGTMSSWVGI GGGSLSVPFL IHCGFPAHKA IGTSSGLAWP
    IALSGAISYL
201 LNGLNIAGLP EGSLGFLYLP AVAVLSAATI AFAPLGVKTA
    HKLSSAKLKK
251 SFGIMLLLIA GKMLYNLL*

ORF17a and ORF17-1 show 98.9% identity in 268 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF17 shows 93.9% identity over a 196aa overlap with a predicted ORF (ORF17.ng) from N. gonorrhoeae:

An ORF17ng nucleotide sequence <SEQ ID 91> is predicted to encode a protein having amino acid sequence <SEQ ID 92>:

1   MWHWDIILIL LAVGSAAGFI AGLFGVGGGT LIVPVVLWVL
    DLQGLAQHPY
51  AQHLAVGTSF AVMVFTAFSS MLGQHKKQAV DWKTIFAMMP
    GMIFGVFAGA
101 LSAKYIPAFG LQIFFILFLT AVAFKTLHTG RQTASRPLPG
    LPGLTAVSTL
151 FGAMSSWVGI GGGSLSVPFL IHCGFPAHKA IGTSSGLAWP
    IALSGAISYL
201 VNGLNIAGLP EGSLGFLYLP AVAVLSAATI AFAPLGVKTA
    HKLSSAKLKE
251 SFGIMLLLIA GKMLYNLL*

Further work revealed the complete gonococcal DNA sequence <SEQ ID 93>:

1   ATGTGGCATT GGGACATTAT CTTAATCCTG CTTGCcgtag
    gcAGTGCGGC
51  AGGTTTTATT GCCGGCCTGT Tcggtgtagg cggcgGTACG
    CTGATTGTCC
101 CTGTCGTTTT ATGGGTGCTT GATTTGCAGG GTTTGGCACA
    ACATCCTTAC
151 GCGCAACACC TCGCCGTCGG CAcaTccttc gcCGTCATGG
    TCTTCACCGC
201 CTTTTCCAGT ATGTTGGGGC AGCACAAAAA ACAGGCGGTC
    GACTGGAAAA
251 CCATATTTGC GATGATGCCG GGTATGATAT TCGGCGTATT
    CGCTGGCGCA
301 CTCTCCGCAA AATATATCCC CGCGTTCGGG CTTCAAATTT
    TCTTCATCCT
351 GTTTTTAACC GCCGTCGCAT TCAAAACACT GCATACCGGT
    CGTCAGACGG
401 CATCCCGCCC GCTGCCCGGG CTGCCCGGAC TGACTGCGGT
    TTCCACACTG
451 TTCGGCGCAA TGTCGAGCTG GGTCGGCATA GGCGGCGGTT
    CACTTTCCGT
501 CCCCTTCTTA ATCCACTGCG GCTTCCCCGC CCATAAAGCC
    ATCGGCACAT
551 CATCCGGCCT TGCCTGGCCG ATTGCACTCT CCGGCGCAAT
    ATCGTATCTG
601 GTCAACGGTC TGAATATTGC AGGATTGCCC GAAGGGTCGC
    TGGGCTTCCT
651 TTACCTGCCC GCCGTCGCCG TCCTCAGCGC GGCAACCATT
    GCCTTTGCCC
701 CGCTCGGTGT CAAAACCGCC CACAAACTTT CTTCTGCCAA
    ACTCAAAGAA
751 TCCTTCGGCA TTATGTTGCT TTTGATTGCC GGAAAAATGC
    TGTACAACCT
801 GCTTTAA

This corresponds to the amino acid sequence <SEQ ID 94; ORF17ng-1>:

1   MWHWDIILIL LAVGSAAGFI AGLFGVGGGT LIVPVVLWVL
    DLQGLAQHPY
51  AQHLAVGTSF AVMVFTAFSS MLGQHKKQAV DWKTIFAMMP
    GMIFGVFAGA
101 LSAKYIPAFG LQIFFILFLT AVAFKTLHTG RQTASRPLPG
    LPGLTAVSTL
151 FGAMSSWVGI GGGSLSVPFL IHCGFPAHKA IGTSSGLAWP
    IALSGAISYL
201 VNGLNIAGLP EGSLGFLYLP AVAVLSAATI AFAPLGVKTA
    HKLSSAKLKE
251 SFGIMLLLIA GKMLYNLL*

ORF17ng-1 and ORF17-1 show 96.6% identity in 268 aa overlap:

In addition, ORF17ng-1 shows significant homology with a hypothetical H. influenzae protein:

sp|P44070|Y902_HAEIN HYPOTHETICAL PROTEIN HI0902 pir||G64015
hypothetical protein HI0902 - Haemophilus influenzae (strain Rd KW20)
gi|1573922 (U32772) H. influenzae
predicted coding region HI0902 [Haemophilus influenzae]Length = 264
Score = 74 (34.9 bits), Expect = 1.6e−23, Sum P(2) = 1.6e−23
Identities = 15/43 (34%), Positives = 23/43 (53%)
Query:	55	AVGTSFAVMVFTAFSSMLGQHKKQAVDWKTIFAMMPGMIFGVF	97
		A+GTSFA +V T   S    HK   + W+ +  + P ++  VF
Sbjct:	52	ALGTSFATIVITGIGSAQRHHKLGNIVWQAVRILAPVIMLSVF	94
Score = 195 (91.9 bits), Expect = 1.6e−23, Sum P(2) = 1.6e−23
Identities = 44/114 (38%), Positives = 65/114 (57%)
Query:	150	LFGAMSSWVGIGGGSLSVPFLIHCGFPAHKAIGTSSGLAWPIALSGAISYLVNGLNIAGL	209
		L G  SS  GIGGG   VPFL   G    +AIG+S+     + +SG  S++V+G     +
Sbjct:	148	LIGMASSAAGIGGGGFIVPFLTARGINIKQAIGSSAFCGMLLGISGMFSFIVSGWGNPLM	207
Query:	210	PEGSLGFLYLPAVAVLSAATIAFAPLGVKTAFIKLSSAKLKESFGIMLLLIAGKM	263
		PE SLG++YLPAV  ++A +   + LG     KL  + LK+ F + L+++A  M
Sbjct:	208	PEYSLGYIYLPAVLGITATSFFTSKLGASATAKLPVSTLKKGFALFLIVVAINM	261

This analysis, including the homology with the hypothetical H. influenzae transmembrane protein, suggests that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 12

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 95>:

1   ..GGAAACGGAT GGCAGGCAGA CCCCGAACAT CCGCTGCTCG
    GGCTTTTTGC
51  CGTCAGTAAT GTATCGATGA CGCTTGCTTT TGTCGGAATA
    TGTGCGTTGG
101 TGCATTATTG CTTTTCGGGA ACGGTTCAAG TGTTTGTGTT
    TGCGGCACTG
151 CTCAAACTTT ATGCGCTGAA GCCGGTTTAT TGGTTCGTGT
    TGCAGTTTGT
201 GCTGATGGCG GTTGCCTATG TCCACCGCTG CGGTATAGAC
    CGGCAGCCGC
251 CGTCAACGTT CGGCGGCTCG CAGCTGCGAC TCGGCGGGTT
    GACGGCAGCG
301 TTGATGCAGG TCTCGGTACT GGTGCTGCTG CTTTCAGAAA
    TTGGAAGATA
351 A

This corresponds to the amino acid sequence <SEQ ID 96; ORF18>:

1   ..GNGWQADPEH PLLGLFAVSN VSMTLAFVGI CALVHYCFSG
    TVQVFVFAAL
51  LKLYALKPVY WFVLQFVLMA VAYVHRCGID RQPPSTFGGS
    QLRLGGLTAA
101 LMQVSVLVLL LSEIGR*

Further work revealed the complete nucleotide sequence <SEQ ID 97>:

1   ATGATTTTGC TGCATTTGGA TTTTTTGTCT GCCTTACTGT
    ATGCGGCGGT
51  TTTTCTGTTT CTGATATTCC GCGCAGGAAT GTTGCAATGG
    TTTTGGGCGA
101 GTATTATGCT GTGGCTGGGC ATATCGGTTT TGGGGGCAAA
    GCTGATGCCC
151 GGCATATGGG GAATGACCCG CGCCGCGCCC TTGTTCATCC
    CCCATTTTTA
201 CCTGACTTTG GGCAGCATAT TTTTTTTCAT CGGGCATTGG
    AACCGGAAAA
251 CAGATGGAAA CGGATGGCAG GCAGACCCCG AACATCCGCT
    GCTCGGGCTT
301 TTTGCCGTCA GTAATGTATC GATGACGCTT GCTTTTGTCG
    GAATATGTGC
351 GTTGGTGCAT TATTGCTTTT CGGGAACGGT TCAAGTGTTT
    GTGTTTGCGG
401 CACTGCTCAA ACTTTATGCG CTGAAGCCGG TTTATTGGTT
    CGTGTTGCAG
451 TTTGTGCTGA TGGCGGTTGC CTATGTCCAC CGCTGCGGTA
    TAGACCGGCA
501 GCCGCCGTCA ACGTTCGGCG GCTCGCAGCT GCGACTCGGC
    GGGTTGACGG
551 CAGCGTTGAT GCAGGTCTCG GTACTGGTGC TGCTGCTTTC
    AGAAATTGGA
601 AGATAA

This corresponds to the amino acid sequence <SEQ ID 98; ORF18-1>:

1   MILLHLDFLS ALLYAAVFLF LIFRAGMLQW FWASIMLWLG
    ISVLGAKLMP
51  GIWGMTRAAP LFIPHFYLTL GSIFFFIGHW NRKTDGNGWQ
    ADPEHPLLGL
101 FAVSNVSMTL AFVGICALVH YCFSGTVQVF VFAALLKLYA
    LKPVYWFVLQ
151 FVLMAVAYVH RCGIDRQPPS TFGGSQLRLG GLTAALMQVS
    VLVLLLSEIG
201 R*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF18 shows 98.3% identity over a 116aa overlap with an ORF (ORF18a) from strain A of N. meningitidis:

The complete length ORF18a nucleotide sequence <SEQ ID 99> is:

1   ATGATTTTGC TGCATTTGGA TTTTTTGTCT GCCTTACTGT
    ATGCGGCGGT
51  TTTTCTGTTT CTGATATTCC GCGCAGGAAT GTTGCAATGG
    TTTTGGGCGA
101 GTATTATGCT GTGGCTGGGC ATATCGGTTT TGGGGGCAAA
    GCTGATGCCC
151 GGCATATGGG GAATGACCCG CGCCGCGCCC TTGTTCATCC
    CCCATTTTTA
201 CCTGACTTTG GGCAGCATAT TTTTTTTCAT CGGGCATTGG
    AACCGGAAAA
251 CGGATGGAAA CGGATGGCAG GCAGACCCCG AACATCCTCT
    GCTCGGGCTG
301 TTTGCCGTCA GTAATGTATC GATGACGCTT GCTTTTGTCG
    GAATATGTGC
351 GTTGGTGCAT TATTGCTTTT CGNGAACGGT TCAAGTGTTT
    GTGTTTGCGG
401 CACTGCTCAA ACTTTATGCG CTGAAGCCGG TTTATTGGTT
    CGTGTTGCAG
451 TTTGTGCTGA TGGCGGTTGC CTATGTCCAC CGCTGCGGTA
    TAGACCGGCA
501 GCCGCCGTCA ACGTTCGGCG GNTCGCAGCT GCGACTCGGC
    GGGTTGACGG
551 CAGCGTTGAT GCAGNTCTCG GTACTGGTGC TGCTGCTTTC
    AGAAATTGGA
601 AGATAA

This encodes a protein having amino acid sequence <SEQ ID 100>:

1   MILLHLDFLS ALLYAAVFLF LIFRAGMLQW FWASIMLWLG
    ISVLGAKLMP
51  GIWGMTRAAP LFIPHFYLTL GSIFFFIGHW NRKTDGNGWQ
    ADPEHPLLGL
101 FAVSNVSMTL AFVGICALVH YCFSXTVQVF VFAALLKLYA
    LKPVYWFVLQ
151 FVLMAVAYVH RCGIDRQPPS TFGGSQLRLG GLTAALMQXS
    VLVLLLSEIG
201 R*

ORF18a and ORF18-1 show 99.0% identity in 201 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF18 shows 93.1% identity over a 116aa overlap with a predicted ORF (ORF18.ng) from N. gonorrhoeae:

The complete length ORF18ng nucleotide sequence is <SEQ ID 101>:

1   ATGATTTTGC TGCATTTGGA TTTTTTGTCT GCCTTACTGt
    aTGCGGcggt
51  tttTctgTTT CTGATATTCC GCGCAGGAAT GTTGCAATGG
    TTTTGGGCGA
101 GTATTGCGTT GTGGCTCGGC ATCTCGGTTT TAGGGGTAAA
    GCTGATGCCG
151 GGGATGTGGG GAATGACCCG CGCCGCGCCT TTGTTCATCC
    CCCATTTTTA
201 CCTGACTTTG GGCAGCATAT TTTTTTTCAT CGGGTATTGG
    AACCGGAAAA
251 CAGATGGAAA CGGATGGCAG GCAGACCCCG AACATCCGCT
    GCTCGGGCTT
301 TTTGCCGTCA GTAATGTATC GATGACGCTT GCTTTTGTCG
    GAATATGTGC
351 GTTGGTGCAT TATTGCTTTT CGGGAACGGT TCAAGTGTTT
    GTGTTTGCGG
401 CATTGCTCAA ACTTTATGCG CTGAAGCCGG TTTATTGGTT
    CGTGTTGCAG
451 TTTGTATTGA TGGCGGttgC CTATGTCCAC CGCTGCGGTA
    TAGACCGGCA
501 GCCGCCGTCA ACGTTCGGCG GTTCGCAGCT GCGACTCGGC
    GTGTTGGCGG
551 CGATGTTGAT GCAGGTTGCG GTAACGGCGA TGCTGCTTGC
    CGAAATCGGC
601 AGATGA

This encodes a protein having amino acid sequence <SEQ ID 102>:

1   MILLHLDFLS ALLYAAVFLF LIFRAGMLQW FWASIALWLG
    ISVLGVKLMP
51  GMWGMTRAAP LFIPHFYLTL GSIFFFIGYW NRKTDGNGWQ
    ADPEHPLLGL
101 FAVSNVSMTL AFVGICALVH YCFSGTVQVF VFAALLKLYA
    LKPVYWFVLQ
151 FVLMAVAYVH RCGIDRQPPS TFGGSQLRLG VLAAMLMQVA
    VTAMLLAEIG
201 R*

This ORF18ng protein sequence shows 94.0% identity in 201 aa overlap with ORF18-1:

Based on this analysis, including the presence of several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 13

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 103>:

1   ATGAAAACCC CACTCCTCAA GCCTCTGCTN ATTACCTCGC
    TTCCCGTTTT
51  CGCCAGTGTT TTTACCGCCG CCTCCATCGT CTGGCAGCTA
    GGCGAACCCA
101 AGCTCGCCAT GCCCTTCGTA CTCGGCATCA TCGCCGGCGG
    CCTTGTCGAT
151 TTGGACAACC NCNTGACCGG ACGGCTNAAA AACATCATCA
    CCACCGTCGC
201 CCTGTTCACC CTCTCCTCGC TCACGGCACA AAGCACCCTC
    GGCACAGGGC
251 TGCCCTTCAT CCTCGCCATG ACCCTGATGA CTT.CG.CTT
    CACCATTTTA
301 GGCGCGGNCG ...

This corresponds to the amino acid sequence <SEQ ID 104; ORF 19>:

1   MKTPLLKPLL ITSLPVFASV FTAASIVWQL GEPKLAMPFV
    LGIIAGGLVD
51  LDNXXTGRLK NIITTVALFT LSSLTAQSTL GTGLPFILAM
    TLMTXXFTIL
101 GAX...

Further work revealed the complete nucleotide sequence <SEQ ID 105>:

1    ATGAAAACCC CACTCCTCAA GCCTCTGCTC ATTACCTCGC
     TTCCCGTTTT
51   CGCCAGTGTT TTTACCGCCG CCTCCATCGT CTGGCAGCTA
     GGCGAACCCA
101  AGCTCGCCAT GCCCTTCGTA CTCGGCATCA TCGCCGGCGG
     CCTTGTCGAT
151  TTGGACAACC GCCTGACCGG ACGGCTGAAA AACATCATCA
     CCACCGTCGC
201  CCTGTTCACC CTCTCCTCGC TCACGGCACA AAGCACCCTC
     GGCACAGGGC
251  TGCCCTTCAT CCTCGCCATG ACCCTGATGA CCTTCGGCTT
     CACCATTTTA
301  GGCGCGGTCG GGCTCAAATA CCGCACCTTC GCCTTCGGTG
     CACTCGCCGT
351  CGCCACCTAC ACCACACTTA CCTACACCCC CGAAACCTAC
     TGGCTGACCA
401  ACCCCTTCAT GATTTTATGC GGCACCGTAC TGTACAGCAC
     CGCCATCCTC
451  CTGTTCCAAA TCGTCCTGCC CCACCGCCCC GTCCAAGAAA
     GCGTCGCCAA
501  CGCCTACGAC GCACTCGGCG GCTACCTCGA AGCCAAAGCC
     GACTTCTTCG
551  ACCCCGATGA GGCAGCCTGG ATAGGCAACC GCCACATCGA
     CCTCGCCATG
601  AGCAACACCG GCGTCATCAC CGCCTTCAAC CAATGCCGTT
     CCGCCCTGTT
651  TTACCGCCTT CGCGGCAAAC ACCGCCACCC GCGCACCGCC
     AAAATGCTGC
701  GTTACTACTT TGCCGCCCAA GACATACACG AACGCATCAG
     CTCCGCCCAC
751  GTCGATTATC AGGAAATGTC CGAAAAATTC AAAAACACCG
     ACATCATCTT
801  CCGCATCCAC CGCCTGCTCG AAATGCAGGG ACAAGCCTGC
     CGCAACACCG
851  CCCAAGCCCT GCGCGCAAGC AAAGACTACG TTTACAGCAA
     ACGCCTCGGC
901  CGCGCCATCG AAGGCTGCCG CCAATCGCTG CGCCTCCTTT
     CAGACAGCAA
951  CGACAGTCCC GACATCCGCC ACCTGCGCCG CCTTCTCGAC
     AACCTCGGCA
1001 GCGTCGACCA GCAGTTCCGC CAACTCCAGC ACAACGGCCT
     GCAGGCAGAA
1051 AACGACCGCA TGGGCGACAC CCGCATCGCC GCCCTCGAAA
     CCAGCAGCCT
1101 CAAAAACACC TGGCAGGCAA TCCGTCCGCA GCTAAACCTC
     GAATCAGGCG
1151 TATTCCGCCA TGCCGTCCGC CTGTCCCTCG TCGTTGCCGC
     CGCCTGCACC
1201 ATCGTCGAAG CCCTCAACCT CAACCTCGGC TACTGGATAC
     TACTGACCGC
1251 CCTTTTCGTC TGCCAACCCA ACTACACCGC CACCAAAAGC
     CGCGTCCGCC
1301 AGCGCATCGC CGGCACCGTA CTCGGCGTAA TCGTCGGCTC
     GCTCGTCCCC
1351 TACTTCACCC CGTCTGTCGA AACCAAACTC TGGATTGTCA
     TCGCCAGTAC
1401 CACCCTCTTT TTCATGACCC GCACCTACAA ATACAGTTTC
     TCCACCTTCT
1451 TCATTACCAT TCAAGCCCTG ACCAGCCTCT CCCTCGCAGG
     TTTGGACGTA
1501 TACGCCGCCA TGCCCGTACG CATCATCGAC ACCATTATCG
     GCGCATCCCT
1551 TGCCTGGGCG GCAGTCAGCT ACCTGTGGCC AGACTGGAAA
     TACCTCACGC
1601 TCGAACGCAC CGCCGCCCTT GCCGTATGCA GCAACGGTGC
     CTATCTCGAA
1651 AAAATCACCG AACGCCTCAA AAGCGGCGAA ACCGGCGACG
     ACGTCGAATA
1701 CCGCGCCACC CGCCGCCGCG CCCACGAACA CACCGCCGCC
     CTCAGCAGCA
1751 CCCTTTCCGA CATGAGCAGC GAACCCGCAA AATTCGCCGA
     CAGCCTGCAA
1801 CCCGGCTTTA CCCTGCTCAA AACCGGCTAC GCCCTGACCG
     GCTACATCTC
1851 CGCCCTCGGC GCATACCGCA GCGAAATGCA CGAAGAATGC
     AGCCCCGACT
1901 TTACCGCACA GTTCCACCTC GCCGCCGAAC ACACCGCCCA
     CATCTTCCAA
1951 CACCTGCCCG AAACCGAACC CGACGACTTT CAGACAGCAC
     TGGATACACT
2001 GCGCGGCGAA CTCGACACCC TCCGCACCCA CAGCAGCGGA
     ACACAAAGCC
2051 ACATCCTCCT CCAACAGCTC CAACTCATCG CCCGACAGCT
     CGAACCCTAC
2101 TACCGCGCCT ACCGCCAAAT TCCGCACAGG CAGCCCCAAA
     ATGCAGCCTG
2151 A

This corresponds to the amino acid sequence <SEQ ID 106; ORF19-1>:

1   MKTPLLKPLL ITSLPVFASV FTAASIVWQL GEPKLAMPFV
    LGIIAGGLVD
51  LDNRLTGRLK NIITTVALFT LSSLTAQSTL GTGLPFILAM
    TLMTFGFTIL
101 GAVGLKYRTF AFGALAVATY TTLTYTPETY WLTNPFMILC
    GTVLYSTAIL
151 LFQIVLPHRP VQESVANAYD ALGGYLEAKA DFFDPDEAAW
    IGNRHIDLAM
201 SNTGVITAFN QCRSALFYRL RGKHRHPRTA KMLRYYFAAQ
    DIHERISSAH
251 VDYQEMSEKF KNTDIIFRIH RLLEMQGQAC RNTAQALRAS
    KDYVYSKRLG
301 RAIEGCRQSL RLLSDSNDSP DIRHLRRLLD NLGSVDQQFR
    QLQHNGLQAE
351 NDRMGDTRIA ALETSSLKNT WQAIRPQLNL ESGVFRHAVR
    LSLVVAAACT
401 IVEALNLNLG YWILLTALFV CQPNYTATKS RVRQRIAGTV
    LGVIVGSLVP
451 YFTPSVETKL WIVIASTTLF FMTRTYKYSF STFFITIQAL
    TSLSLAGLDV
501 YAAMPVRIID TIIGASLAWA AVSYLWPDWK YLTLERTAAL
    AVCSNGAYLE
551 KITERLKSGE TGDDVEYRAT RRRAHEHTAA LSSTLSDMSS
    EPAKFADSLQ
601 PGFTLLKTGY ALTGYISALG AYRSEMHEEC SPDFTAQFHL
    AAEHTAHIFQ
651 HLPETEPDDF QTALDTLRGE LDTLRTHSSG TQSHILLQQL
    QLIARQLEPY
701 YRAYRQIPHR QPQNAA*

Computer analysis of this amino acid sequence gave the following results:

Homology with Predicted Transmenbrane protein YHFK of H. influenzae (Accession Number P44289)

ORF19 and YHFK proteins show 45% aa identity in 97 aa overlap:

orf19	6	LKPLLITSLPVFASVFTAASIVWQLGEPKLAMPFVLGIIAGGLVDLDNXXTGRLKNIITT	65
		L   +I+++PVF +V  AA  +W       +MP +LGIIAGGLVDLDN  TGRLKN+  T
YHFK	5	LNAKVISTIPVFIAVNIAAVGIWFFDISSQSMPLILGIIAGGLVDLDNRLTGRLKNVFFT	64
orf19	66	VALFTLSSLTAQSTLGTGLPFILAMTLMTXXFTILGA	102
		+  F++SS   Q  +G  + +I+ MT++T  FT++GA
YHFK	65	LIAFSISSFIVQLHIGKPIQYIVLMTVLTFIFTMIGA	101

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF19 shows 92.2% identity over a 102aa overlap with an ORF (ORF19a) from strain A of N. meningitidis:

The complete length ORF19a nucleotide sequence <SEQ ID 107> is:

1    ATGAAAACCC CACCCCTCAA GCCTCTGCTC ATTACCTCGC
     TTCCCGTTTT
51   CGCCAGTGTC TTTACCGCCG CCTCCATCGT CTGGCAGCTG
     GGCGAACCCA
101  AGCTCGCCAT GCCCTTCGTA CTCGGCATCA TCGCTGGCGG
     CCTGGTCGAT
151  TTGGACAACC GCCTGACCGG ACGGCTGAAA AACATCATCG
     CCACCGTCGC
201  CCTGTTCACC CTCTCCTCAC TTGTCGCGCA AAGCACCCTC
     GGCACAGGTT
251  TGCCATTCAT CCTCGCCATG ACCCTGATGA CTTTCGGCTT
     TACCATCATG
301  GGCGCGGTCG GGCTGAAATA CCGCACCTTC GCCTTCGGCG
     CACTCGCCGT
351  CGCCACCTAC ACCACACTTA CCTACACCCC CGAAACCTAC
     TGGCTGACCA
401  ACCCCTTTAT GATTCTGTGC GGAACCGTAC TGTACAGCAC
     CGCCATCATC
451  CTGTTCCAAA TCATCCTGCC CCACCGCCCC GTTCAAGAAA
     ACGTCGCCAA
501  CGCCTACGAA GCACTCGGCA GCTACCTCGA AGCCAAAGCC
     GACTTTTTCG
551  ATCCCGACGA AGCCGAATGG ATAGGCAACC GCCACATCGA
     CCTCGCCATG
601  AGCAACACCG GCGTCATCAC CGCCTTCAAC CAATGCCGTT
     CCGCCCTGTT
651  TTACCGCCTT CGCGGCAAAC ACCGCCACCC GCGCACCGCC
     AAAATGCTGC
701  GCTACTACTT CGCCGCCCAA GACATACACG AACGCATCAG
     CTCCGCCCAC
751  GTCGACTACC AAGAGATGTC CGAAAAATTC AAAAACACCG
     ACATCATCTT
801  CCGCATCCAC CGCCTGCTCG AAATGCAGGG ACAAGCCTGC
     CGCAACACCG
851  CCCAAGCCCT GCGCGCAAGC AAAGACTACG TTTACAGCAA
     ACGCCTCGGC
901  CGCGCCATCG AAGGCTGCCG CCAATCGCTG CGCCTCCTTT
     CAGACAGCAA
951  CGACAATCCC GACATCCGCC ACCTGCGCCG CCTTCTCGAC
     AACCTCGGCA
1001 GCGTCGACCA GCAGTTCCGC CAACTCCAGC ACAACGGCCT
     GCAGGCAGAA
1051 AACGACCGCA TGGGCGACAC CCGCATCGCC GCCCTCGAAA
     CCGGCAGCCT
1101 CAAAAACACC TGGCAGGCAA TCCGTCCGCA GCTAAACCTC
     GAATCAGGCG
1151 TATTCCGCCA TGCCGTCCGC CTGTCCCTTG TCGTTGCCGC
     CGCCTGCACC
1201 ATCGTCGAAG CCCTCAACCT CAACCTCGGC TACTGGATAC
     TACTGACCGC
1251 CCTTTTCGTC TGCCAACCCA ACTACACCGC CACCAAAAGC
     CGCGTCCGCC
1301 AGCGCATCGC CGGCACCGTA CTCGGCGTAA TCGTCGGCTC
     GCTCGTCCCC
1351 TACTTTACCC CCTCCGTCGA AACCAAACTC TGGATCGTCA
     TCGCCAGTAC
1401 CACCCTCTTT TTCATGACCC GCACCTACAA ATACAGCTTC
     TCGACATTTT
1451 TCATCACCAT TCAAGCCCTG ACCAGCCTCT CCCTCGCAGG
     GTTGGACGTA
1501 TACGCCGCCA TGCCCGTACG CATCATCGAC ACCATTATCG
     GCGCATCCCT
1551 TGCCTGGGCG GCAGTCAGCT ACCTGTGGCC AGACTGGAAA
     TACCTCACGC
1601 TCGAACGCAC CGCCGCCCTT GCCGTATGCA GCAACGGCGC
     CTATCTCGAA
1651 AAAATCACCG AACGCCTCAA AAGCGGCGAA ACCGGCGACG
     ACGTCGAATA
1701 CCGCGCCACC CGCCGCCGCG CCCACGAACA CACCGCCGCC
     CTCAGCAGCA
1751 CCCTTTCCGA CATGAGCAGC GAACCCGCAA AATTCGCCGA
     CAGCCTGCAA
1801 CCCGGCTTTA CCCTGCTCAA AACCGGCTAC GCCCTGACCG
     GCTACATCTC
1851 CGCCCTCGGC GCATACCGCA GCGAAATGCA CGAAGAATGC
     AGCCCCGACT
1901 TTACCGCACA GTTCCACCTC GCCGCCGAAC ACACCGCCCA
     CATCTTCCAA
1951 CACCTGCCCG AAACCGAACC CGACGACTTT CAGACAGCAC
     TGGATACACT
2001 GCGCGGCGAA CTCGACACCC TCCGCACCCA CAGCAGCGGA
     ACACAAAGCC
2051 ACATCCTCCT CCAACAGCTC CAACTCATCG CCCGGCAGCT
     CGAACCCTAC
2101 TACCGCGCCT ACCGACAAAT TCCGCACAGG CAGCCCCAAA
     ACGCAGCCTG
2151 A

This encodes a protein having amino acid sequence <SEQ ID 108>:

1   MKTPPLKPLL ITSLPVFASV FTAASIVWQL GEPKLAMPFV
    LGIIAGGLVD
51  LDNRLTGRLK NIIATVALFT LSSLVAQSTL GTGLPFILAM
    TLMTFGFTIM
101 GAVGLKYRTF AFGALAVATY TTLTYTPETY WLTNPFMILC
    GTVLYSTAII
151 LFQIILPHRP VQENVANAYE ALGSYLEAKA DFFDPDEAEW
    IGNRHIDLAM
201 SNTGVITAFN QCRSALFYRL RGKHRHPRTA KMLRYYFAAQ
    DIHERISSAH
251 VDYQEMSEKF KNTDIIFRIH RLLEMQGQAC RNTAQALRAS
    KDYVYSKRLG
301 RAIEGCRQSL RLLSDSNDNP DIRHLRRLLD NLGSVDQQFR
    QLQHNGLQAE
351 NDRMGDTRIA ALETGSLKNT WQAIRPQLNL ESGVFRHAVR
    LSLVVAAACT
401 IVEALNLNLG YWILLTALFV CQPNYTATKS RVRQRIAGTV
    LGVIVGSLVP
451 YFTPSVETKL WIVIASTTLF FMTRTYKYSF STFFITIQAL
    TSLSLAGLDV
501 YAAMPVRIID TIIGASLAWA AVSYLWPDWK YLTLERTAAL
    AVCSNGAYLE
551 KITERLKSGE TGDDVEYRAT RRRAHEHTAA LSSTLSDMSS
    EPAKFADSLQ
601 PGFTLLKTGY ALTGYISALG AYRSEMHEEC SPDFTAQFHL
    AAEHTAHIFQ
651 HLPETEPDDF QTALDTLRGE LDTLRTHSSG TQSHILLQQL
    QLIARQLEPY
701 YRAYRQIPHR QPQNAA*

ORF19a and ORF19-1 show 98.3% identity in 716 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF19 shows 95.1% identity over a 102aa overlap with a predicted ORF (ORF19.ng) from N. gonorrhoeae:

An ORF19ng nucleotide sequence <SEQ ID 109> is predicted to encode a protein having amino acid sequence <SEQ ID 110>:

1   MKTPLLKPLL ITSLPVFASV FTAASIVWQL GEPKLAMPFV
    LGIIAGGLVD
51  LDNRLTGRLK NIIATVALFT LSSLTAQSTL GTGLPFILAM
    TLMTFGFTIL
101 GAVGLKYRTF AFGALAVATY TTLTYTPETY WLTNPFMILC
    GTVLYSTAII
151 LFQIILPHRP VQESVANAYE ALGGYLEAKA DFFDPDEAAW
    IGNRHIDLAM
201 SNTGVITAFN QCRSALFYRL RGKHRHPRTA KMLRYYFAAQ
    DIHERISSAH
251 VDYQEMSEKF KNTDIIFRIR RLLEMQGQAC RNTAQAIRSG
    KDYVYSKRLG
301 RAIEGCRQSL RLLSDGNDSP DIRHLSRLLD NLGSVDQQFR
    QLRHSDSPAE
351 NDRMGDTRIA ALETGSFKNT *

Further work revealed the complete nucleotide sequence <SEQ ID 111>:

1    ATGAAAACCC CACTCCTCAA GCCTCTGCTC ATTACCTCGC
     TTCCCGTTTT
51   CGCCAGTGTC TTTACCGCCG CCTCCATCGT CTGGCAGCTA
     GGCGAACCCA
101  AGCTCGCCAT GCCCTTCGTA CTCGGCATCA TCGCCGGCGG
     CCTGGTCGAT
151  TTGGACAACC GCCTGACCGG ACGGCTGAAA AACATCATCG
     CCACCGTCGC
201  CCTGTTTACC CTCTCCTCGC TCACGGCGCA AAGCACCCTC
     GGCACAGGGC
251  TGCCCTTCAT CCTCGCCATG ACCCTGATGA CCTTCGGCTT
     TACCATTTTA
301  GGCGCGGTCG GGCTGAAATA CCGCACCTTC GCCTTCGGCG
     CACTCGCCGT
351  CGCCACCTAC ACCACGCTTA CCTACACCCC CGAAACCTAC
     TGGCTGACCA
401  ACCCCTTCAT GATTTTATGC GGCACCGTAC TGTACAGCAC
     CGCCATCATC
451  CTGTTCCAAA TCATCCTGCC CCACCGCCCC GTCCAAGAAA
     GCGTCGCCAA
501  TGCCTACGAA GCACTCGGCG GCTACCTCGA AGCCAAAGCC
     GACTTCTTCG
551  ACCCCGATGA GGCAGCCTGG ATAGGCAACC GCCACATCGA
     CCTCGCCATG
601  AGCAACACCG GCGTCATCAC CGCCTTCAAC CAATGCCGTT
     CCGCCCTGTT
651  TTACCGTTTG CGCGGCAAAC ACCGCCACCC GCGCACCGCC
     AAAATGCTGC
701  GCTACTACTT CGCCGCCCAA GACATCCACG AACGCATCAG
     CTCCGCCCAC
751  GTCGACTACC AAGAGATGTC CGAAAAATTC AAAAACACCG
     ACATCATCTT
801  CCGCATCCGC CGCCTGCTCG AAATGCAGGG GCAGGCGTGC
     CGCAACACCG
851  CCCAAGCCAT CCGGTCGGGC AAAGACTAcg tTTACAGCAA
     ACGCCTCGGA
901  CGCGCCATcg aaggctgCCG CCAGTCGCtg cgcctCCTTt
     cagacggcaA
951  CGACAGTCCC GACATCCGCC ACCTGAGccg CCTTCTCGAC
     AACCTCGgca
1001 GCGTcgacca gcagtTCcgc caactCCGAC ACAgcgactC
     CCCCGCcgaa
1051 Aacgaccgca tgggcgacaC CCGCATCGCC GCCCtcgaaa
     ccggcagctT
1101 caaaaaCAcc tggcaggCAA TCCGTCCGCa gctgaaCCTC
     GAATCatgCG
1151 TATTCCGCCA TGCCGTCCGC CTGTCCCTCG TCGTTGCCGC
     CGCCTGCACC
1201 ATCGTCgaag cCCTCAACCT CAACCTCGGC TACTGGATAC
     TGCTGACCGC
1251 CCTTTTCGTC TGCCAACCCA ACTACACCGC CACCAAAAGC
     CGCGTGTACC
1301 AACGCATCGC CGGCACCGTA CTCGGCGTAA TCGTCGGCTC
     GCTCGTCCCC
1351 TACTTCACCC CCTCCGTCGA AACCAAACTC TGGATTGTCA
     TCGCCGGTAC
1401 CACCCTGTTC TTCATGACCC GCACCTACAA ATACAGTTTC
     TCCACCTTCT
1451 TCATCACCAT TCAGGCACTG ACCAGCCTCT CCCTCGCAGG
     TTTGGACGTA
1501 TACGCCGCCA TGCCCGTGCG CATCATcgaC ACCATTATCG
     GCGCATCCCT
1551 TGCCTGGGCG GCGGTCAGCT ACCTGTGGCC AGACTGGAAA
     TACCTCACGC
1601 TCGAACGCAC CGCCGCCCTT GCCGTATGCA GCAGCGGCAC
     ATACCTCCAA
1651 AAAATTGCCG AACGCCTCAA AACCGGCGAA ACCGGCGACG
     ACATAGAATA
1701 CCGCATCACC CGCCGCCGCG CCCACGAACA CACCGCCGCC
     CTCAGCAGCA
1751 CCCTTTCCGA CATGAGCAGC GAACCCGCAA AATTCGCCGA
     CAGCCTGCAA
1801 CCCGGCTTTA CCCTGCTCAA AACCGGCTAC GCCCTGACCG
     GCTACATCTC
1851 CGCCCTCGGC GCATACCGCA GCGAAATGCA CGAAGAATGC
     AGCCCCGACT
1901 TTACCGCACA GTTCCACCTT GCCGCCGAAC ACACCGCCCA
     CATCTTCCAA
1951 CACCTGCCCG ACATGGGACC CGACGACTTT CAGACGGCAT
     TGGATACACT
2001 GCGCGGCGAA CTCGGCACCC TCCGCACCCG CAGCAGCGGA
     ACACAAAGCC
2051 ACATCCTCCT CCAACAGCTC CAACTCATCG CccgGCAACT
     CGAACCCTAC
2101 TACCGCGCCT ACCGACAAAT TCCGCACAGG CAGCCCCAAA
     ACGCAGCCTG
2151 A

This corresponds to the amino acid sequence <SEQ ID 112; ORF19ng-1>:

1   MKTPLLKPLL ITSLPVFASV FTAASIVWQL GEPKLAMPFV
    LGIIAGGLVD
51  LDNRLTGRLK NIIATVALFT LSSLTAQSTL GTGLPFILAM
    TLMTFGFTIL
101 GAVGLKYRTF AFGALAVATY TTLTYTPETY WLTNPFMILC
    GTVLYSTAII
151 LFQIILPHRP VQESVANAYE ALGGYLEAKA DFFDPDEAAW
    IGNRHIDLAM
201 SNTGVITAFN QCRSALFYRL RGKHRHPRTA KMLRYYFAAQ
    DIHERISSAH
251 VDYQEMSEKF KNTDIIFRIR RLLEMQGQAC RNTAQAIRSG
    KDYVYSKRLG
301 RAIEGCRQSL RLLSDGNDSP DIRHLSRLLD NLGSVDQQFR
    QLRHSDSPAE
351 NDRMGDTRIA ALETGSFKNT WQAIRPQLNL ESCVFRHAVR
    LSLVVAAACT
401 IVEALNLNLG YWILLTALFV CQPNYTATKS RVYQRIAGTV
    LGVIVGSLVP
451 YFTPSVETKL WIVIAGTTLF FMTRTYKYSF STFFITIQAL
    TSLSLAGLDV
501 YAAMPVRIID TIIGASLAWA AVSYLWPDWK YLTLERTAAL
    AVCSSGTYLQ
551 KIAERLKTGE TGDDIEYRIT RRRAHEHTAA LSSTLSDMSS
    EPAKFADSLQ
601 PGFTLLKTGY ALTGYISALG AYRSEMHEEC SPDFTAQFHL
    AAEHTAHIFQ
651 HLPDMGPDDF QTALDTLRGE LGTLRTRSSG TQSHILLQQL
    QLIARQLEPY
701 YRAYRQIPHR QPQNAA*

ORF19ng-1 and ORF19-1 show 95.5% identity in 716 aa overlap:

In addition, ORF19ng-1 shows significant homology to a hypothetical gonococcal protein previously entered in the databases:

sp|O33369|YOR2_NEIGO HYPOTHETICAL 45.5 KD PROTEIN (ORF2) gnl|PID|e1154438
(AJ002423) hypothetical protein [Neisseria gonorrh] Length = 417
Score = 1512 (705.6 bits), Expect = 5.3e−203, P = 5.3e−203
Identities = 301/326 (92%), Positives = 306/326 (93%)
Query:	307	RQSLRLLSDGNDSPDIRHLSRLLDNLGSVDQQFRQLRHSDSPAENDRMGDTRIAALETGS	366
		RQSLRLLSDGNDS DIRHLSRLLDNLGSVDQQFRQLRHSDSPAENDRMGDTRIAALETGS
Sbjct:	1	RQSLRLLSDGNDSXDIRHLSRLLDNLGSVDQQFRQLRHSDSPAENDRMGDTRIAALETGS	60
Query:	367	FKNTWQAIRPQLNLESCVFRHAVRLSLVVAAACTIVEALNLNLGYWILLTALFVCQPNYT	426
		FKNTWQAIRPQLNLES VFRHAVRLSLVVAAACTIVEALNLNLGYWILLT LFVCQPNYT
Sbjct:	61	FKNTWQAIRPQLNLESGVFRHAVRLSLVVAAACTIVEALNLNLGYWILLTRLFVCQPNYT	120
Query:	427	ATKSRVYQRIAGTVLGVIVGSLVPYFTPSVETKLWIVIAGTTLFFMTRTYKYSFSTFFIT	486
		ATKSRVYQRIAGTVLGVIVGSLVPYFTPSVETKLWIVIAGTTLFFMTRTYKYSFSTFFIT
Sbjct:	121	ATKSRVYQRIAGTVLGVIVGSLVPYFTPSVETKLWIVIAGTTLFFMTRTYKYSFSTFFIT	180
Query:	487	IQALTSLSLAGLDVYAAMPVRIIDTIIGASLAWAAVSYLWPDWKYLTLERTAALAVCSSG	546
		IQALTSLSLAGLDVYAAMPVRIIDTIIGASLAWAAVSYLWPDWKYLTLERTAALAVCSSG
Sbjct:	181	IQALTSLSLAGLDVYAAMPVRIIDTIIGASLAWAAVSYLWPDWKYLTLERTAALAVCSSG	240
Query:	547	TYLQKIAERLKTGETGDDIEYRITRRRAHEHTAALSSTLSDMSSEPAKFADSLQPGFTLL	606
		TYLQKIAERLKTGETGDDIEYRITRRRAHEHTAALSSTLSDMSSEPAKFAD+  P
Sbjct:	241	TYLQKIAERLKTGETGDDIEYRITRRRAHEHTAALSSTLSDMSSEPAKFADTCNPALPCS	300
Query:	607	KTGYALTGYISALGAYRSEMHEECSP	632
		K   ALTGYISALG   ++  +  +P
Sbjct:	301	KPATALTGYISALGHTAAKCTKNAAP	326

Based on this analysis, including the presence of several putative transmembrane domains in the gonococcal protein (the first of which is also seen in the meningococcal protein), and on homology with the YHFK protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 14

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 113>:

1    ATGAATATGC TGGGAGCTTT GGCAAAAGTC GGCAGCCTGA
     CGATGGTGTC
51   GCGCGTTTTG GGATTTGTGC GCGATACGGT CATTGCGCGG
     GCATTCGGCG
101  CGGGTATGGC GACGGATGCG TTTTTTGTCG CGTTCAAACT
     GCCCAACCTG
151  CTTCGCCGCG TGTTTGCGGA GGGGGCGTTT GCCCAAGCGT
     TTGTGCCGAT
201  TTTGGCGGAA TACAAGGAAA CGCGTTCAAA AGAGGCGG.C
     GAAGCCTTTA
251  TCCGCCATGT GGCGGGGATG CTGTCGTTTG TACTGGTTAT
     CGTTACCGCG
301  CTGGGCATAC TTGCCGCGCC TTGGGTGATT TATGTTTCCG
     CACCCGAGTT
351  TTGCCCAAGA TGCCGACAAA TTTCAGCTCT CCATCGATTT
     GCTGCGGATT
401  ACGTTTCCTT ATATATTATT GATTTCCCTG TCTTCATTTG
     TCGGCTCGGT
451  ACTCAATTCT TATCATAAGT TCGGCATTCC GGCGTTTACG
     CCAC.GTTTC
501  TGAACGTGTC GTTTATCGTA TTCGCGCTGT TTTTCGTGCC
     GTATTTCGAT
551  CCGCCCGTTA CCGCGCyGGC GTGGGCGGTC TTTGTCGGCG
     GCATTTTGCA
601  ACTCGrmTTC CAACTGCCCT GGCTGGCGAA ACTGGGCTTT
     TTGAAACTGC
651  CCAAACtGAG TTTCAAAGAT GCGGCGGTCA ACCGCGTGAT
     GAAACAGATG
701  GCGCCTGCgA TTTTgGGCGT GAgCGTGGCG CAGGTTTCTT
     TGGTGATCAA
751  CACGATTTTc GCGTCTTATC TGCAATCGGG CAGCGTTTCA
     TGGATGTATT
801  ACGCCGACCG CATGATGGAG CTGCCCAGCG GCGTGCTGGG
     GGCGGCACTC
851  GGTACGATTT TGCTGCCGAC TTTGTCCAAA CACTCGGCAA
     ACCaAGATAC
901  GGaACAGTTT TCCGCCCTGC TCGACTGGGG TTTGCGCCTG
     TGCATGCtgc
951  TGACGCTGCC GGCGgcGGTC GGACTGGCGG TGTTGTCGTT
     cCCgCtGGTG
1001 GCGACGCTGT TTATGTACCG CGwATTTACG CTGTTTGACG
     CGCAGATGAC
1051 GCAACACGCG CTGATTGCCT ATTCTTTCGG TTTAATCGGC
     TTAATCATGA
1101 TTAAAGTGTT GGCACCCGGC TTCTATGCGC GGCAAAACAT
     CAAwAmGCCC
1151 GTCAAAATCG CCATCTTCAC GCTCATCTGC mCGCAGTTGA
     TGAACCTTGs
1201 CTTTAyCGGC CCACTrrAAC rCasTCGGAC TTTCGCTTGC
     CATCGGTCTG
1251 GGCGCGTGTA TCAATGCCGG ATTGTTGTTT TACCTGTTGC
     GCAGACACGG
1301 TATTTACCAA CCTGG.CAAG GGTTGGGCAG CGTTCTT.AG
     CAAAAATGCT
1351 GcTCTCGCTC GCCGTGA

This corresponds to the amino acid sequence <SEQ ID 114; ORF20>:

1   MNMLGALAKV GSLTMVSRVL GFVRDTVIAR AFGAGMATDA
    FFVAFKLPNL
51  LRRVFAEGAF AQAFVPILAE YKETRSKEAX EAFIRHVAGM
    LSFVLVIVTA
101 LGILAAPWVI YVSAPSFAQD ADKFQLSIDL LRITFPYILL
    ISLSSFVGSV
151 LNSYHKFGIP AFTPXFLNVS FIVFALFFVP YFDPPVTAXA
    WAVFVGGILQ
201 LXFQLPWLAK LGFLKLPKLS FKDAAVNRVM KQMAPAILGV
    SVAQVSLVIN
251 TIFASYLQSG SVSWMYYADR MMELPSGVLG AALGTILLPT
    LSKHSANQDT
301 EQFSALLDWG LRLCMLLTLP AAVGLAVLSF PLVATLFMYR
    XFTLFDAQMT
351 QHALIAYSFG LIGLIMIKVL APGFYARQNI XXPVKIAIFT
    LICXQLMNLX
401 FXGPLXXIGL SLAIGLGACI NAGLLFYLLR RHGIYQPXQG
    LGSVLXQKCC
451 SRSP*

These sequences were elaborated, and the complete DNA sequence <SEQ ID 115> is:

1    ATGAATATGC TGGGAGCTTT GGCAAAAGTC GGCAGCCTGA
     CGATGGTGTC
51   GCGCGTTTTG GGATTTGTGC GCGATACGGT CATTGCGCGG
     GCATTCGGCG
101  CGGGTATGGC GACGGATGCG TTTTTTGTCG CGTTCAAACT
     GCCCAACCTG
151  CTTCGCCGCG TGTTTGCGGA GGGGGCGTTT GCCCAAGCGT
     TTGTGCCGAT
201  TTTGGCGGAA TACAAGGAAA CGCGTTCAAA AGAGGCGGCG
     GAGGCTTTTA
251  TCCGCCATGT GGCGGGGATG CTGTCGTTTG TACTGGTTAT
     CGTTACCGCG
301  CTGGGCATAC TTGCCGCGCC TTGGGTGATT TATGTTTCCG
     CACCCGGTTT
351  TGCCCAAGAT GCCGACAAAT TTCAGCTCTC CATCGATTTG
     CTGCGGATTA
401  CGTTTCCTTA TATATTATTG ATTTCCCTGT CTTCATTTGT
     CGGCTCGGTA
451  CTCAATTCTT ATCATAAGTT CGGCATTCCG GCGTTTACGC
     CCACGTTTCT
501  GAACGTGTCG TTTATCGTAT TCGCGCTGTT TTTCGTGCCG
     TATTTCGATC
551  CGCCCGTTAC CGCGCTGGCG TGGGCGGTCT TTGTCGGCGG
     CATTTTGCAA
601  CTCGGCTTCC AACTGCCCTG GCTGGCGAAA CTGGGCTTTT
     TGAAACTGCC
651  CAAACTGAGT TTCAAAGATG CGGCGGTCAA CCGCGTGATG
     AAACAGATGG
701  CGCCTGCGAT TTTGGGCGTG AGCGTGGCGC AGGTTTCTTT
     GGTGATCAAC
751  ACGATTTTCG CGTCTTATCT GCAATCGGGC AGCGTTTCAT
     GGATGTATTA
801  CGCCGACCGC ATGATGGAGC TGCCCAGCGG CGTGCTGGGG
     GCGGCACTCG
851  GTACGATTTT GCTGCCGACT TTGTCCAAAC ACTCGGCAAA
     CCAAGATACG
901  GAACAGTTTT CCGCCCTGCT CGACTGGGGT TTGCGCCTGT
     GCATGCTGCT
951  GACGCTGCCG GCGGCGGTCG GACTGGCGGT GTTGTCGTTC
     CCGCTGGTGG
1001 CGACGCTGTT TATGTACCGC GAATTTACGC TGTTTGACGC
     GCAGATGACG
1051 CAACACGCGC TGATTGCCTA TTCTTTCGGT TTAATCGGCT
     TAATCATGAT
1101 TAAAGTGTTG GCACCCGGCT TCTATGCGCG GCAAAACATC
     AAAACGCCCG
1151 TCAAAATCGC CATCTTCACG CTCATCTGCA CGCAGTTGAT
     GAACCTTGCC
1201 TTTATCGGCC CACTGAAACA CGTCGGACTT TCGCTTGCCA
     TCGGTCTGGG
1251 CGCGTGTATC AATGCCGGAT TGTTGTTTTA CCTGTTGCGC
     AGACACGGTA
1301 TTTACCAACC TGGCAAGGGT TGGGCAGCGT TCTTAGCAAA
     AATGCTGCTC
1351 TCGCTCGCCG TGATGTGCGG CGGACTGTGG GCAGCGCAGG
     CTTACCTGCC
1401 GTTTGAATGG GCGCACGCCG GCGGAATGCG GAAAGCGGGG
     CAGCTCTGCA
1451 TCCTGATTGC CGTCGGCGGC GGACTGTATT TCGCATCACT
     GGCGGCTTTG
1501 GGCTTCCGTC CGCGCCATTT CAAACGCGTG GAAAACTGA

This corresponds to the amino acid sequence <SEQ ID 116; ORF20-1>:

1   MNMLGALAKV GSLTMVSRVL GFVRDTVIAR AFGAGMATDA
    FFVAFKLPNL
51  LRRVFAEGAF AQAFVPILAE YKETRSKEAA EAFIRHVAGM
    LSFVLVIVTA
101 LGILAAPWVI YVSAPGFAQD ADKFQLSIDL LRITFPYILL
    ISLSSFVGSV
151 LNSYHKFGIP AFTPTFLNVS FIVFALFFVP YFDPPVTALA
    WAVFVGGILQ
201 LGFQLPWLAK LGFLKLPKLS FKDAAVNRVM KQMAPAILGV
    SVAQVSLVIN
251 TIFASYLQSG SVSWMYYADR MMELPSGVLG AALGTILLPT
    LSKHSANQDT
301 EQFSALLDWG LRLCMLLTLP AAVGLAVLSF PLVATLFMYR
    EFTLFDAQMT
351 QHALIAYSFG LIGLIMIKVL APGFYARQNI KTPVKIAIFT
    LICTQLMNLA
401 FIGPLKHVGL SLAIGLGACI NAGLLFYLLR RHGIYQPGKG
    WAAFLAKMLL
451 SLAVMCGGLW AAQAYLPFEW AHAGGMRKAG QLCILIAVGG
    GLYFASLAAL
501 GFRPRHFKRV EN*

Computer analysis of this amino acid sequence gave the following results:

Homology with the MviN Virulence Factor of S. typhimurium (Accession Number P37169)

ORF20 and MviN proteins show 63% aa identity in 440aa overlap:

Orf20	1	MNMLGALAKVGSLTMVSRVLGFVRDTVIARAFGAGMATDAFFVAFKLPNLLRRVFAEGAF	60
		MN+L +LA V S+TM SRVLGF RD ++AR FGAGMATDAFFVAFKLPNLLRR+FAEGAF
MviN	14	MNLLKSLAAVSSMTMFSRVLGFARDAIVARIFGAGMATDAFFVAFKLPNLLRRIFAEGAF	73
Orf20	61	AQAFVPILAEYKETRSKEAXEAFIRHVAGMLSFVLVIVTALGILAAPWVIYVSAPSFAQD	120
		+QAFVPILAEYK  + +EA   F+ +V+G+L+  L +VT  G+LAAPWVI V+AP FA
MviN	74	SQAFVPILAEYKSKQGEEATRIFVAYVSGLLTLALAVVTVAGMLAAPWVIMVTAPGFADT	133
Orf20	121	ADKFQLSIDLLRITFPYILLISLSSFVGSVLNSYHKFGIPAFTPXFLNVSFIVFALFFVP	180
		ADKF L+  LLRITFPYILLISL+S VG++LN++++F IPAF P FLN+S I FALF  P
MviN	134	ADKFALTTQLLRITFPYILLISLASLVGAILNTWNRFSIPAFAPTFLNISMIGFALFAAP	193
Orf20	181	YFDPPVTAXAWAVFVGGILQLXFQLPWLAKLGFLKLPKLSFKDAAVNRVMKQMAPAILGV	240
		YF+PPV A AWAV VGG+LQL +QLP+L K+G L LP+++F+D    RV+KQM PAILGV
MviN	194	YFNPPVLALAWAVTVGGVLQLVYQLPYLKKIGMLVLPRINFRDTGAMRVVKQMGPAILGV	253
Orf20	241	SVAQVSLVINTIFASYLQSGSVSWMYYADRMMELPSGVLGAALGTILLPTLSKHSANQDT	300
		SV+Q+SL+INTIFAS+L SGSVSWMYYADR+ME PSGVLG ALGTILLP+LSK  A+ +
MviN	254	SVSQISLIINTIFASFLASGSVSWMYYADRLMEFPSGVLGVALGTILLPSLSKSFASGNH	313
Orf20	301	EQFSALLDWGLRLCMLLTLPAAVGLAVLSFPLVATLFMYRXFTLFDAQMTQHALIAYSFG	360
		+++  L+DWGLRLC LL LP+AV L +L+ PL  +LF Y  FT FDA MTQ ALIAYS G
MviN	314	DEYCRLMDWGLRLCFLLALPSAVALGILAKPLTVSLFQYGKFTAFDAAMTQRALIAYSVG	373
Orf20	361	LIGLIMIKVLAPGFYARQNIXXPVKIAIFTLICXQLMNLXFXXXXXXXXXXXXXXXXXCI	420
		LIGLI++KVLAPGFY+RQ+I  PVKIAI TLI  QLMNL F                 C+
MviN	374	LIGLIVVKVLAPGFYSRQDIKTPVKIAIVTLIMTQLMNLAFIGPLKHAGLSLSIGLAACL	433
Orf20	421	NAGLLFYLLRRHGIYQPXQG	440
		NA LL++ LR+  I+ P  G
MviN	434	NASLLYWQLRKQNIFTPQPG	453

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF20 shows 93.5% identity over a 447aa overlap with an ORF (ORF20a) from strain A of N. meningitidis:

The complete length ORF20a nucleotide sequence <SEQ ID 117> is:

1    ATGAATATGC TGGGAGCTTT GGTAAAAGTC GGCAGCCTGA
     CGATGGTGTC
51   GCGCGTTTTG GGATTTGTGC GCGATACGGT CATTGCGCGC
     GCATTCGGCG
101  CAGGCATGGC GACGGATGCG TTCTTTGTCG CGTTCAAACT
     GCCCAACCTG
151  CTTCGCCGCG TGTTTGCGGA GGGGGCGTTT GCCCAAGCGT
     TTGTGCCGAT
201  TTTGGCGGAA TATAAGGAAA CGCGTTCTAA AGAGGCGACG
     GAGGCTTTTA
251  TCCGCCATGT GGCGGGGATG CTGTCGTTTG TACTGGTCAT
     CGTTACCGCG
301  CTGGGCATAC TTGCCGCGCC TTGGGTGATT TATGTTTCCG
     CACCCGGTTT
351  TGCCAAAGAT GCCGACAAAT TTCAGCTCTC TATCGATTTG
     CTGCGGATTA
401  CGTTTCCTTA TATCTTATTG ATTTCACTTT CCTCTTTTGT
     CGGCTCGGTA
451  CTCAATTCCT ATCATAAATT CAGCATTCCT GCGTTTACGC
     CCACGTTCCT
501  GAACGTGTCG TTTATCGTAT TCGCGCTGTT TTTCGTGCCG
     TATTTCGATC
551  CTCCCGTTAC CGCGCTGGCT TGGGCGGTTT TTGTCGGCGG
     CATTTTGCAA
601  CTCGGCTTCC AACTGCCCTG GCTGGCGAAA CTGGGTTTTT
     TGAAACTGCC
651  CAAACTGAGT TTCAAAGATG CGGCGGTCAA CCGCGTGATG
     AAACAGATGG
701  CGCCTGCGAT TTTGGGCGTG AGCGTGGCGC AGATTTCTTT
     GGTGATCAAC
751  ACGATTTTCG CGTCTTATCT GCAATCGGGC AGCGTTTCAT
     GGATGTATTA
801  CGCCGACCGC ATGATGGAAC TGCCCGGCGG CGTGCTGGGG
     GCGGCACTCG
851  GTACGATTTT GCTGCCGACT TTGTCCAAAC ACTCGGCAAA
     CCAAGATACG
901  GAACAGTTTT CCGCCCTGCT CGACTGGGGT TTGCGCNTGT
     GCATGCTGCT
951  GACGCTGCCG GCGGCGGTCG GAATGGCGGT GTTGTCGTTC
     CCGCTGGTGG
1001 CAACCTTGTT TATGTACCGA GAATTCACGC TGTTTGACGC
     GCAGATGACG
1051 CAACACGCGC TGATTGCCTA TTCTTTCGGT TTAATCGGTT
     TAATCATGAT
1101 TAAAGTGTTG GCGCCCGGCT TTTATGCGCG GCAAAACATC
     AAAACGCCCG
1151 TCAAAATCGC CATCTTCACG CTCATTTGCA CGCAGTTGAT
     GAACCTTGCC
1201 TTTATCGGCC CACTGAAACA CGTCGGACTT TCGCTTGCCA
     TCGGTCTGGG
1251 CGCGTGTATC AATGCCGGAT TGTTGTTTTA CCTGTTGCGC
     AGACACGGTA
1301 TTTACCAACC TGGCAAGGGT TGGGCAGCGT TCTTGGCAAA
     AATGCTGCTC
1351 TCGCTCGCCG TGATGGGAGG CGGCCTGTAT GCCGCCCAAA
     TCTGGCTGCC
1401 GTTCGACTGG GCACACGCCG GCGGAATGCA AAAGGCCGCC
     CGGCTCTTCA
1451 TCCTGATTGC CGTCGGCGGC GGACTGTATT TCGCATCACT
     GGCGGCTTTG
1501 GGCTTCCGTC CGCGCCATTT CAAACGCGTG GAAAGCTGA

This encodes a protein having amino acid sequence <SEQ ID 118>:

1   MNMLGALVKV GSLTMVSRVL GFVRDTVIAR AFGAGMATDA
    FFVAFKLPNL
51  LRRVFAEGAF AQAFVPILAE YKETRSKEAT EAFIRHVAGM
    LSFVLVIVTA
101 LGILAAPWVI YVSAPGFAKD ADKFQLSIDL LRITFPYILL
    ISLSSFVGSV
151 LNSYHKFSIP AFTPTFLNVS FIVFALFFVP YFDPPVTALA
    WAVFVGGILQ
201 LGFQLPWLAK LGFLKLPKLS FKDAAVNRVM KQMAPAILGV
    SVAQISLVIN
251 TIFASYLQSG SVSWMYYADR MMELPGGVLG AALGTILLPT
    LSKHSANQDT
301 EQFSALLDWG LRXCMLLTLP AAVGMAVLSF PLVATLFMYR
    EFTLFDAQMT
351 QHALIAYSFG LIGLIMIKVL APGFYARQNI KTPVKIAIFT
    LICTQLMNLA
401 FIGPLKHVGL SLAIGLGACI NAGLLFYLLR RHGIYQPGKG
    WAAFLAKMLL
451 SLAVMGGGLY AAQIWLPFDW AHAGGMQKAA RLFILIAVGG
    GLYFASLAAL
501 GFRPRHFKRV ES*

ORF20a and ORF20-1 show 96.5% identity in 512 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF20 shows 92.1% identity over a 454aa overlap with a predicted ORF (ORF20ng) from N. gonorrhoeae:

An ORF20ng nucleotide sequence <SEQ ID 119> was predicted to encode a protein having amino acid sequence <SEQ ID 120>:

1   MNMLGALAKV GSLTMVSRVL GFVRDTVIAR AFGAGMATDA
    FFVAFKLPNL
51  LRRVFAEGAF AQAFVPILAE YKETRSKEAT EAFIRHVAGM
    LSFVLIVVTA
101 LGILAAPWVI YVSAPGFTKD ADKFQLSISL LRITFPYILL
    ISLSSFVGSI
151 LNSYHKFGIP AFTPTFLNIS FIVFALFFVP YFDPPVTALA
    WAVFVGGILQ
201 LGFQLPWLAK LGFLKLPKLN FKDAAVNRVM KQMAPAILGV
    SVAQISLVIN
251 TIFASYLQSG SVSWMYYADR MMELPGGVLG AALGTILLPT
    LSKHSANQDT
301 EQFSALLDWG LRLCMLLTLP AAAGLAVLSF PLVATLFMYR
    EFTLFDAQMT
351 QHALIAYSFG LIGLIMIKVL ASGFYARQNI KTPVKIAIFT
    LICTQLMNLA
401 FIGPLKHAGL SLAIGLGACI NAGLLFFLFR KHGIYRPGQG
    LGQPSWRKCC
451 SRSP*

Further DNA sequence analysis revealed the following DNA sequence <SEQ ID 121>:

1    ATGAATATGC TTGGAGCTTT GGCAAAAGTC GGCAGCCTGA
     CGATGGTGTC
51   GCGCGTTTTG GGATTTGTGC GCGATACGGT CATTGCGCGG
     GCATTCGGCG
101  CGGGTATGGC GACGGATGCG TTTTTTGTCG CGTTCAAACT
     GCCCAACCTG
151  CTTCGCCGCG TGTTTGCGGA GGGGGCGTTT GCCCAAGCGT
     TTGTGCCGAT
201  TTTGGCGGAA TATAAGGAAA CGCGTTCTAA AGAGGCGAcg
     gAGGCTTTTA
251  TCCGCCACGt tgcgggAatg CTGTCGTTTG TGCTGATcgt
     cGttacCGCG
301  CTGGGCATAC TTGCCGCgcc tTGGGTGATT TATGTTtccg
     CgcccGGCTT
351  TACCAAAGAC GCGGACAAGT TCCAACTTTC CATCAGCCTG
     CTGCGGATTA
401  CGTTTCCTTA TATATTATTG ATTTCTTTGT CTTCTTTTGT
     CGGCTCGATA
451  CTCAATTCCT ACCATAAGTT CGGCATTCCC GCGTTTACGC
     CCACGTTTTT
501  AAACATCTCT TTTATCGTAT TCGCACTGTT TTTCGTGCCG
     TATTTCGATC
551  CGCCCGTTAC CGCGCTGGCG TGGGCGGTTT TTGTCGGCGG
     TATTTTGCAG
601  CTCGGTTTCC AACTGCCGTG GCTGGCGAAA CTGGGCTTTT
     TGAAACTGCC
651  CAAACTGAAT TTCAAAGATG CGGCGGTCAA CCGCGTCATG
     AAACAGATGG
701  CGCCTGCGAT TTTGGGCGTG agcgTGGCGC AAATTTCTTT
     GgttATCAAC
751  ACGATTTTCG CGTCTTATCT GCAATCGGGC AGCGTTTCAT
     GGATGTatta
801  cgCCGACCGC ATGATGGAGc tgcgccGGGG CGTGCTGGGG
     GCTGCACTCG
851  GTACAATTTT GCTGCCGACT TTGTCCAAAC ACTCGGCAAA
     CCAAGATACG
901  GAACAGTTTT CCGCCCTGCT CGACTGGGGT TTGCGCCTGT
     GCATGCTGCT
951  GACGCTGCCG GCGGCGGccg GACTGGCGGT ATTGTCGTTC
     CCGCTGGTGG
1001 CGACGCTGTT TATGTACCGA GAATTCACGC TGTTTGACGC
     ACAAATGACG
1051 CAACACGCGC TGATTGCCTA TTCTTTCGGT TTAATCGGTT
     TAATTATGAT
1101 TAAAGTGTTG GCATCCGGCT TTTATGCGCG GCAAAACATC
     AAAACGCCCG
1151 TCAAAATCGC CATCTTCACG CTCATCTGCA CGCAGTTGAT
     GAACCTCGCC
1201 TTTATCGGTC CGTTGAAACA CGCCGGGCTT TCGCTCGCCA
     TCGGCCTGGG
1251 CGCGTGCATC AACGCCGGAT TGTTGTTCTT CCTGTTGCGC
     AAACACGGTA
1301 TTTACCGGCC cggcaggggt tgggcggcgt TCTTGGCGAA
     AATGCTGCTC
1351 GCGCTCGCCG TGATGTGCGG CGGACTGTGG GCGGCGCAGG
     CTTGCCTGCC
1401 GTTCGAATGG GCGCACGCCG GCGGAATGCG GAAAGCGGGG
     CAGCTCTGCA
1451 TCCTGATTGC CGTCGGCGGC GGACTGTATT TCGCATCTCT
     GGCGGCTTTG
1501 GGCTTCCGTC CGCGCCATTT CAAACGCGTG GAAAGCTGA

This encodes the following amino acid sequence <SEQ ID 122; ORF20ng-1>:

1   MNMLGALAKV GSLTMVSRVL GFVRDTVIAR AFGAGMATDA
    FFVAFKLPNL
51  LRRVFAEGAF AQAFVPILAE YKETRSKEAT EAFIRHVAGM
    LSFVLIVVTA
101 LGILAAPWVI YVSAPGFTKD ADKFQLSISL LRITFPYILL
    ISLSSFVGSI
151 LNSYHKFGIP AFTPTFLNIS FIVFALFFVP YFDPPVTALA
    WAVFVGGILQ
201 LGFQLPWLAK LGFLKLPKLN FKDAAVNRVM KQMAPAILGV
    SVAQISLVIN
251 TIFASYLQSG SVSWMYYADR MMELRRGVLG AALGTILLPT
    LSKHSANQDT
301 EQFSALLDWG LRLCMLLTLP AAAGLAVLSF PLVATLFMYR
    EFTLFDAQMT
351 QHALIAYSFG LIGLIMIKVL ASGFYARQNI KTPVKIAIFT
    LICTQLMNLA
401 FIGPLKHAGL SLAIGLGACI NAGLLFFLLR KHGIYRPGRG
    WAAFLAKMLL
451 ALAVMCGGLW AAQACLPFEW AHAGGMRKAG QLCILIAVGG
    GLYFASLAAL
501 GFRPRHFKRV ES*

ORF20ng-1 and ORF20-1 show 95.7% identity in 512 aa overlap:

In addition, ORF20ng-1 shows significant homology with a virulence factor of S. typhimurium:

sp|P37169|MVIN_SALTY VIRULENCE FACTOR MVIN pir||S40271 mviN protein -
Salmonella typhimurium gi|438252 (Z26133) mviB gene product
[Salmonella typhimurium] gnl|PID|d1005521 (D25292) ORF2
[Salmonella typhimurium] Length = 524
Score = 1573 (750.1 bits), Expect = 1.1e−220, Sum P(2) = 1.1e−220
Identities = 309/467 (66%), Positives = 368/467 (78%)
Query:	1	MNMLGALAKVGSLTMVSRVLGFVRDTVIARAFGAGMATDAFFVAFKLPNLLRRVFAEGAF	60
		MN+L +LA V S+TM SRVLGF RD ++AR FGAGMATDAFFVAFKLPNLLRR+FAEGAF
Sbjct:	14	MNLLKSLAAVSSMTMFSRVLGFARDAIVARIFGAGMATDAFFVAFKLPNLLRRIFAEGAF	73
Query:	61	AQAFVPILAEYKETRSKEATEAFIRHVAGMLSFVLIVVTALGILAAPWVIYVSAPGFTKD	120
		+QAFVPILAEYK  + +EAT  F+ +V+G+L+  L VVT  G+LAAPWVI V+APGF
Sbjct:	74	SQAFVPILAEYKSKQGEEATRIFVAYVSGLLTLALAVVTVAGMLAAPWVIMVTAPGFADT	133
Query:	121	ADKFQLSISLLRITFPYILLISLSSFVGSILNSYHKFGIPAFTPTFLNISFIVFALFFVP	180
		ADKF L+  LLRITFPYILLISL+S VG+ILN++++F IPAF PTFLNIS I FALF  P
Sbjct:	134	ADKFALTTQLLRITFPYILLISLASLVGAILNTWNRFSIPAFAPTFLNISMIGFALFAAP	193
Query:	181	YFDPPVTALAWAVFVGGILQLGFQLPWLAKLGFLKLPKLNFKDAAVNRVMKQMAPAILGV	240
		YF+PPV ALAWAV VGG+LQL +QLP+L K+G L LP++NF+D    RV+KQM PAILGV
Sbjct:	194	YFNPPVLALAWAVTVGGVLQLVYQLPYLKKIGMLVLPRINFRDTGAMRVVKQMGPAILGV	253
Query:	241	SVAQISLVINTIFASYLQSGSVSWMYYADRMMELRRGVLGAALGTILLPTLSKHSANQDT	300
		SV+QISL+INTIFAS+L SGSVSWMYYADR+ME   GVLG ALGTILLP+LSK  A+ +
Sbjct:	254	SVSQISLIINTIFASFLASGSVSWMYYADRLMEFFSGVLGVALGTILLPSLSKSFASGNH	313
Query:	301	EQFSALLDWGLRLCMLLTLPAAAGLAVLSFPLVATLFMYREFTLFDAQMTQHALIAYSFG	360
		+++  L+DWGLRLC LL LP+A  L +L+ PL  +LF Y +FT FDA MTQ ALIAYS G
Sbjct:	314	DEYCRLMDWGLRLCFLLALPSAVALGILAKPLTVSLFQYGKFTAFDAAMTQRALIAYSVG	373
Query:	361	LIGLIMIKVLASGFYARQNIKTPVKIAIFTLICTQLMNLAFIGPLKHAGLSLAIGLGACI	420
		LIGLI++KVLA GFY+RQ+IKTPVKIAI TLI TQLMNLAFIGPLKHAGLSL+IGL AC+
Sbjct:	374	LIGLIVVKVLAPGFYSRQDIKTPVKIAIVTLIMTQLMNLAFIGPLKHAGLSLSIGLAACL	433
Query:	421	NAGLLFFLLRKHGIYRPGRGWXXXXXXXXXXXXVMCGGLWAAQACLP	467
		NA LL++ LRK  I+ P  GW            VM   L+     +P
Sbjct:	434	NASLLYWQLRKQNIFTPQPGWMWFLMRLIISVLVMAAVLFGVLHIMP	480
Score = 70 (33.4 bits), Expect = 1.1e−220, Sum P(2) = 1.1e−220
Identities = 14/41 (34%), Positives = 23/41 (56%)
Query:	469	EWAHAGGMRKAGQLCILIAVGGGLYFASLAALGFRPRHFKR	509
		EW+    + +  +L  ++  G   YFA+LA LGF+ + F R
Sbjct:	481	EWSQGSMLWRLLRLMAVVIAGIAAYFAALAVLGFKVKEFVR	521

Based on this analysis, including the homology with a virulence factor from S. typhimurium, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 15

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 123>:

1   atGATTAAAA TCAAAAAAGG TCTAAACCTG CCCATCGCGG
    GCAGACCGGA
51  GCAAGCCGTT tACGACGGCC CGGCCaTTAC CGAAGtCGCG
    TTGCTTGGCG
101 AAGAATATGC CGGTATGCGC CCCTCGATGA AAGTCAAGGA
    AGGCGATGCC
151 GTcAAAAAAG GCCAAGTGCT GTTTGAAGAC AAAAAGAATC
    CGGGCGTGGT
201 GTTTACTGCG CCGGCTTCAG GcAAAATCGC CGCGATTCAC
    CGTGGCGAAA
251 AGCGCGTACT TCAGTCAGTC GTGATTGCCG TTGAArGCAA
    CGACGAAATC
301 GAGTTTGAAC GCTACGCACC TGAAGCGCTG GCAAACTTAA
    GCGGCGAAGA
351 AGTGCGCCGC AACCTGATCC AATCCGGTTT GTGGACTGCG
    CTGCGCACCC
401 GTCCGTTCAG CAAAATTCCT GCCGTCGATG CCGAGCCGTT
    CGCCATCTTC
451 GTCAATGCGA tGGACACCAA TCCG..

This corresponds to the amino acid sequence <SEQ ID 124; ORF22>:

1   MIKIKKGLNL PIAGRPEQAV YDGPAITEVA LLGEEYAGMR
    PSMKVKEGDA
51  VKKGQVLFED KKNPGVVFTA PASGKIAAIH RGEKRVLQSV
    VIAVEXNDEI
101 EFERYAPEAL ANLSGEEVRR NLIQSGLWTA LRTRPFSKIP
    AVDAEPFAIF
151 VNAMDTNP..

Further work revealed the complete nucleotide sequence <SEQ ID 125>:

1    ATGATTAAAA TCAAAAAAGG TCTAAACCTG CCCATCGCGG
     GCAGACCGGA
51   GCAAGCCGTT TACGACGGCC CGGCCATTAC CGAAGTCGCG
     TTGCTTGGCG
101  AAGAATATGC CGGTATGCGC CCCTCGATGA AAGTCAAGGA
     AGGCGATGCC
151  GTCAAAAAAG GCCAAGTGCT GTTTGAAGAC AAAAAGAATC
     CGGGCGTGGT
201  GTTTACTGCG CCGGCTTCAG GCAAAATCGC CGCGATTCAC
     CGTGGCGAAA
251  AGCGCGTACT TCAGTCAGTC GTGATTGCCG TTGAAGGCAA
     CGACGAAATC
301  GAGTTTGAAC GCTACGCACC TGAAGCGCTG GCAAACTTAA
     GCGGCGAAGA
351  AGTGCGCCGC AACCTGATCC AATCCGGTTT GTGGACTGCG
     CTGCGCACCC
401  GTCCGTTCAG CAAAATTCCT GCCGTCGATG CCGAGCCGTT
     CGCCATCTTC
451  GTCAATGCGA TGGACACCAA TCCGCTGGCT GCCGACCCTA
     CGGTCATTAT
501  CAAAGAAGCC GCCGAGGATT TCAAACGCGG CCTGTTGGTA
     TTGAGCCGTT
551  TGACCGAACG CAAAATCCAT GTTTGTAAGG CAGCTGGCGC
     AGACGTGCCG
601  TCTGAAAATG CTGCCAACAT CGAAACACAT GAATTCGGCG
     GCCCGCATCC
651  TGCCGGTTTG AGTGGCACGC ACATTCATTT CATCGAGCCG
     GTCGGCGCGA
701  ATAAAACCGT GTGGACCATC AATTATCAAG ATGTAATTAC
     CATTGGCCGT
751  TTGTTTGCAA CAGGCCGTCT GAACACCGAG CGCGTGATTG
     CCCTAGGTGG
801  TTCTCAAGTC AACAAACCGC GCCTCTTGCG TACCGTTTTG
     GGTGCGAAAG
851  TATCGCAAAT TACTGCGGGC GAATTGGTTG ACACAGACAA
     CCGCGTGATT
901  TCCGGTTCGG TATTGAACGG CGCGATTACA CAAGGCGCGC
     ACGATTATTT
951  GGGACGCTAC CACAATCAGA TTTCCGTTAT CGAAGAAGGC
     CGCAGCAAAG
1001 AGCTGTTCGG CTGGGTTGCG CCGCAGCCGG ACAAATACTC
     CATCACGCGT
1051 ACAACCCTCG GCCATTTCCT GAAAAACAAA CTCTTCAAGT
     TCAACACAGC
1101 CGTCAACGGC GGCGACCGCG CCATGGTGCC GATTGGTACT
     TACGAGCGCG
1151 TGATGCCCTT GGATATCCTG CCCACCCTGC TTTTGCGCGA
     TTTAATCGTC
1201 GGCGATACCG ACAGCGCGCA GGCATTGGGT TGCTTGGAAT
     TGGACGAAGA
1251 AGACCTCGCT TTGTGCAGCT TCGTCTGCCC GGGCAAATAC
     GAATACGGCC
1301 CGCTGTTGCG CAAAGTGCTG GAAACCATTG AGAAGGAAGG
     CTGA

This corresponds to the amino acid sequence <SEQ ID 126; ORF22-1>:

1   MIKIKKGLNL PIAGRPEQAV YDGPAITEVA LLGEEYAGMR
    PSMKVKEGDA
51  VKKGQVLFED KKNPGVVFTA PASGKIAAIH RGEKRVLQSV
    VIAVEGNDEI
101 EFERYAPEAL ANLSGEEVRR NLIQSGLWTA LRTRPFSKIP
    AVDAEPFAIF
151 VNAMDTNPLA ADPTVIIKEA AEDFKRGLLV LSRLTERKIH
    VCKAAGADVP
201 SENAANIETH EFGGPHPAGL SGTHIHFIEP VGANKTVWTI
    NYQDVITIGR
251 LFATGRLNTE RVIALGGSQV NKPRLLRTVL GAKVSQITAG
    ELVDTDNRVI
301 SGSVLNGAIT QGAHDYLGRY HNQISVIEEG RSKELFGWVA
    PQPDKYSITR
351 TTLGHFLKNK LFKFNTAVNG GDRAMVPIGT YERVMPLDIL
    PTLLLRDLIV
401 GDTDSAQALG CLELDEEDLA LCSFVCPGKY EYGPLLRKVL
    ETIEKEG*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 127>:

1    ATGATTAAAA TCAAAAAAGG TCTAAACCTG CCCATCGCGG
     GCAGACCGGA
51   GCAAGTCATT TATGACGGGC CCGTCATTAC CGAAGTCGCG
     TTGCTTGGCG
101  AAGAATATGC CGGTATGCGC CCCTNGATGA AAGTCAAGGA
     AGGCGATGCC
151  GTCAAAAAAG GCCAAGTGCT GTTTGAAGAC AAAAAGNATC
     CGGGCGTGGT
201  GTTTACCGCG CCNGTTTCAG GCAAAATCGC CGCCATCCAT
     CGCGGCGAAA
251  AGCGCGTACT TCAGTCGGTC GTGATTGCCG TTGAAGGCAA
     CGACGAAATC
301  GAGTTCGAAC GCTACGCGCC CGAAGCGTTG GCAAACTTAA
     GCGGCGANGA
351  ANTNNGNNGC AATCTGATCC AATCCGGTTT GTGGACTGCG
     CTGCGTANCC
401  GTCCGTTCAG CAAAATCCCT GCCGTCGATG CCGAGCCGTT
     CGCCATCTTC
451  GTCAATGCGA TGGACACCAA TCCGCTNGCG GCAGACCCTG
     TGGTTGTGAT
501  CAAAGAAGCC GNCGANGATT TCAGACGANG TNTGCTGGTA
     TTGAGCCGTT
551  TGACCGAGCG TAAAATCCAT GTGTGTAAGG CAGCTGGCGC
     AGACGTGCCG
601  TCTGAAAATG CTGCCAACAT CGAAACACAT GAATTCGGCG
     GCCCGCATCC
651  GGCCGGTTTG AGTGGCACGC ACATTCATTT CATTGAGCCG
     GTCGGTGCAA
701  ACAAAACCGT TTGGACCATC AATTATCAAG ATGTAATTGC
     CATCGGACGT
751  TTGTTTGCAA CAGGCCGTCT GAACACCGAG CGCGTGATTG
     CTTTGGGTGG
801  TTCTCAAGTC AACAAACCAC GCCTCTTGCG TACCGTTTTG
     GGTGCGAAAG
851  TATCGCAAAT TACTGCGGGC GAATTGGTTG ACGCAGACAA
     CCGCGTGATT
901  TCCGGTTCGG TATTGAACGG CGCGATTACA CAAGGCGCGC
     ACGATTATTT
951  GGGACGCTAC CACAATCAGA TTTCCGTTAT CGAAGAAGGC
     CGCAGCAAAG
1001 AGCTGTTCGG CTGGGTTGCG CCGCAGCCGG ACAAATACTC
     CATCACGCGT
1051 ACGACCCTCG GCCATTTCCT GAAAAACAAA CTCTTCAAGT
     TCACGACAGC
1101 CGTCAACGGT GGCGACCGCG CCATGGTGCC GATTGGTACT
     TACGAGCGCG
1151 TAATGCCGCT AGACATCCTG CCTACCCTGC TTTTGCGCGA
     TTTAATCGTC
1201 GGCGATACCG ACAGCGCGCA AGCATTGGGT TGCTTGGAAT
     TGGACGAAGA
1251 AGACCTCGCT TTGTGCAGCT TCGTCTGCCC GGGCAAATAC
     GAATANGGCC
1301 CGCTGTTGCG TAAGGTGCTG GAAACCNTTG AGAAGGAAGG
     CTGA

This encodes a protein having amino acid sequence <SEQ ID 128; ORF22a>:

1   MIKIKKGLNL PIAGRPEQVI YDGPVITEVA LLGEEYAGMR
    PXMKVKEGDA
51  VKKGQVLFED KKXPGVVFTA PVSGKIAAIH RGEKRVLQSV
    VIAVEGNDEI
101 EFERYAPEAL ANLSGXEXXX NLIQSGLWTA LRXRPFSKIP
    AVDAEPFAIF
151 VNAMDTNPLA ADPVVVIKEA XXDFRRXXLV LSRLTERKIH
    VCKAAGADVP
201 SENAANIETH EFGGPHPAGL SGTHIHFIEP VGANKTVWTI
    NYQDVIAIGR
251 LFATGRLNTE RVIALGGSQV NKPRLLRTVL GAKVSQITAG
    ELVDADNRVI
301 SGSVLNGAIT QGAHDYLGRY HNQISVIEEG RSKELFGWVA
    PQPDKYSITR
351 TTLGHFLKNK LFKFTTAVNG GDRAMVPIGT YERVMPLDIL
    PTLLLRDLIV
401 GDTDSAQALG CLELDEEDLA LCSFVCPGKY EXGPLLRKVL
    ETXEKEG*

The originally-identified partial strain B sequence (ORF22) shows 94.2% identity over a 158aa overlap with ORF22a:

The complete strain B sequence (ORF22-1) and ORF22a show 94.9% identity in 447 aa overlap:

Further work identified a partial gene sequence <SEQ ID 129> from N. gonorrhoeae, which encodes the following amino acid sequence <SEQ ID 130; ORF22ng>:

1   MIKIKKGLNL PIAGRPEQVI YDGPAITEVA LLGEEYVGMR
    PSMKIKEGEA
51  VKKGQVLFED KKNPGVVFTA PASGKIAAIH RGEKRVLQSV
    VIAVEGNDEI
101 EFERYVPEAL AKLSSEKVRR NLIQSGLWTA LRTRPFSKIP
    AVDAEPFAIF
151 VNAMDTNPLA ADPTVIIKEA AEDFKRGLLV LSRLTERKIH
    VCKAAGADVP
201 SENAANIETH EFGGPHPAGL SGTHIHFIEP VGANKTVWTI
    NYQDVIAIGR
251 LFVTGRLNTE RVVALGGLQV NKPRLLRTVL GAKVSQLTAG
    ELVDADNRVI
301 SGSVLNGAIA QGAHDYLGRY HN*

Further work identified complete gonococcal gene <SEQ ID 131>:

1    ATGATTAAAA TCAAAAAAGG TCTAAATCTG CCCATCGCGG
     GCAGACCGGA
51   GCAAGTCATT TATGACGGCC CGGCCATTAC CGAAGTCGCG
     TTGCTTGGCG
101  AAGAATATGT CGGCATGCGC CCCTCGATGA AAATCAAGGA
     AGGTGAAGCC
151  GTCAAAAAAG GCCAAGTGCT GTTTGAAGAC AAAAAGAATC
     CGGGCGTAGT
201  ATTTACTGCG CCGGCTTCAG GCAAAATCGC CGCTATTCAC
     CGTGGCGAAA
251  AGCGCGTACT TCAGTCAGTC GTGATTGCCG TTGAAGGCAA
     CGACGAAATC
301  GAGTTCGAAC GCTACGTACC TGAAGCGCTG GCAAAATTGA
     GCAGCGAAAA
351  AGTGCGCCGC AACCTGATTC AATCAGGCTT ATGGACTGCG
     CTTCGCACCC
401  GTCCGTTCAG CAAAATCCCT GCCGTAGATG CCGAGCCGTT
     CGCCATCTTC
451  GTCAATGCGA TGGACACCAA TCCGCTGGCT GCCGACCCTA
     CGGTCATCAT
501  CAAAGAAGCC GCCGAAGACT TCAAACGCGG CCTGTTGGTA
     TTGAGCCGCC
551  TGACCGAACG TAAAATCCAT GTGTGTAAAG CAGCAGGCGC
     AGACGTGCCG
601  TCTGAAAATG CTGCCAATAT CGAAACACAT GAATTTGGCG
     GCCCGCATCC
651  TGCCGGCTTG AGTGGCACGC ACATTCATTT CATCGAGCCA
     GTCGGCGCGA
701  ATAAAACCGT GTGGACCATC AATTATCAAG ACGTGATTGC
     TATCGGACGT
751  TTGTTCGTAA CAGGCCGTCT GAATACCGAG CGCGTGGTTG
     CCTTGGGCGG
801  CCTGCAAGTC AACAAACCGC GCCTCTTGCG TACCGTTTTG
     GGTGCGAAGG
851  TGTCTCAACT TACCGCCGGC GAATTGGTTG ACGCGGACAA
     CCGCGTGATT
901  TCCGGTTCGG TATTGAACGG TGCGATTGCA CAAGGCGCGC
     ATGATTATTT
951  GGGACGCTAC CACAATCAGA TTTCCGTTAT CGAAGAAGGC
     CGCAGCAAAG
1001 AGCTGTTCGG CTGGGTTGCG CCGCAGCCGG ACAAATACTC
     CATCACGCGC
1051 ACCACTCTCG GCCATTTCCT AAAAAACAAA CTCTTCAAGT
     TCACGACAGC
1101 CGTCAACGGC GGCGACCGCG CCATGGTACC GATCGGCACT
     TATGAGCGCG
1151 TAATGCCGTT GGACATCCTG CCTACCTTGC TTTTGCGCGA
     TTTAATCGTC
1201 GGCGATACCG ACAGCGCGCA GGCTTTGGGT TGCTTGGAAT
     TGGACGAAGA
1251 AGACCTCGCT TTGTGCAGCT TCGTCTGCCC GGGCAAATAC
     GAATACGGCC
1301 CGCTGTTGCG CAAAGTGCTG GAAACCATTG AGAAGGAAGG
     CTGA

This encodes a protein having amino acid sequence <SEQ ID 132; ORF22ng-1>:

1   MIKIKKGLNL PIAGRPEQVI YDGPAITEVA LLGEEYVGMR
    PSMKIKEGEA
51  VKKGQVLFED KKNPGVVFTA PASGKIAAIH RGEKRVLQSV
    VIAVEGNDEI
101 EFERYVPEAL AKLSSEKVRR NLIQSGLWTA LRTRPFSKIP
    AVDAEPFAIF
151 VNAMDTNPLA ADPTVIIKEA AEDFKRGLLV LSRLTERKIH
    VCKAAGADVP
201 SENAANIETH EFGGPHPAGL SGTHIHFIEP VGANKTVWTI
    NYQDVIAIGR
251 LFVTGRLNTE RVVALGGLQV NKPRLLRTVL GAKVSQLTAG
    ELVDADNRVI
301 SGSVLNGAIA QGAHDYLGRY HNQISVIEEG RSKELFGWVA
    PQPDKYSITR
351 TTLGHFLKNK LFKFTTAVNG GDRAMVPIGT YERVMPLDIL
    PTLLLRDLIV
401 GDTDSAQALG CLELDEEDLA LCSFVCPGKY EYGPLLRKVL
    ETIEKEG*

The originally-identified partial strain B sequence (ORF22) shows 93.7% identity over a 158aa overlap with ORF22ng:

The complete sequences from strain B (ORF22-1) and gonococcus (ORF22ng) show 96.2% identity in 447 aa overlap:

Computer analysis of these sequences gave the following results:

Homology with 48 kDa Outer Membrane Protein of Actinobacillus pleuropneumoniae (Accession Number U24492).

ORF22 and this 48 kDa protein show 72% aa identity in 158aa overlap:

Orf22	1	MIKIKKGLNLPIAGRPEQAVYDGPAITEVALLGEEYAGMRPSMKVKEGDAVKKGQVLFED	60
		MI IKKGL+LPIAG P Q +++G  + EVA+LGEEY GMRPSMKV+EGD VKKGQVLFED
48kDa	1	MITIKKGLDLPIAGTPAQVIHNGNTVNEVAMLGEEYVGMRPSMKVREGDVVKKGQVLFED	60
orf22	61	KKNPGVVFTAPASGKIAAIHRGEKRVLQSVVIAVEXNDEIEFERYAPEALANLSGEEVRR	120
		KKNPGVVFTAPASG +  I+RGEKRVLQSVVI VE +++I F RY    LA+LS E+V++
48kDa	61	KKNPGVVFTAPASGTVVTINRGEKRVLQSVVIKVEGDEQITFTRYEAAQLASLSAEQVKQ	120
orf22	121	NLIQSGLWTALRTRPFSKIPAVDAEPFAIFVNAMDTNP	158
		NLI+SGLWTA RTRPFSK+PA+DA P +IFVNAMDTNP
48kDa	121	NLIESGLWTAFRTRPFSKVPALDAIPSSIFVNAMDTNP	158

ORF22a also shows homology to the 48 kDa Actinobacillus pleuropneumoniae protein:

gi|1185395 (U24492) 48 kDa outer membrane protein [Actinobacillus
pleuropneumoniae]
Length = 449
Score = 530 bits (1351), Expect = e−150
Identities = 274/450 (60%), Positives = 323/450 (70%), Gaps = 4/450 (0%)
Query:	1	MIKIKKGLNLPIAGRPEQVIYDGPVITEVALLGEEYAGMRPXMKVKEGDAVKKGQVLFED	60
		MI IKKGL+LPIAG P QVI++G  + EVA+LGEEY GMRP MKV+EGD VKKGQVLFED
Sbjct:	1	MITIKKGLDLPIAGTPAQVIHNGNTVNEVAMLGEEYVGMRPSMKVREGDVVKKGQVLFED	60
Query:	61	KKXPGVVFTAPVSGKIAAIHRGEKRVLQSVVIAVEGNDEIEFERYAPEALANLSGXEXXX	120
		KK PGVVFTAP SG +  I+RGEKRVLQSVVI VEG+++I F RY    LA+LS  +
Sbjct:	61	KKNPGVVFTAPASGTVVTINRGEKRVLQSVVIKVEGDEQITFTRYEAAQLASLSAEQVKQ	120
Query:	121	NLIQSGLWTALRXRPFSKIPAVDAEPFAIFVNAMDTNPLAADPVVVIKEAXXDFRRXXLV	180
		NLI+SGLWTA R RPFSK+PA+DA P +IFVNAMDTNPLAADP VV+KE   DF+    V
Sbjct:	121	NLIESGLWTAFRTRPFSKVPALDAIPSSIFVNAMDTNPLAADPEVVLKEYETDFKDGLTV	180
Query:	181	LSRL--TERKIHVCKAAGADVP-SENAANIETHEFGGPHPAGLSGTHIHFIEPVGANKTV	237
		L+RL   ++ +++CK A +++P S     I    F G HPAGL GTHIHF++PVGA K V
Sbjct:	181	LTRLFNGQKPVYLCKDADSNIPLSPAIEGITIKSFSGVHPAGLVGTHIHFVDPVGATKQV	240
Query:	238	WTINYQDVIAIGRLFATGRLNTERVIALGGSQVNKPRLLRTVLGAKVSQITAGELVDADN	297
		W +NYQDVIAIG+LF TG L T+R+I+L G QV  PRL+RT LGA +SQ+TA EL   +N
Sbjct:	241	WHLNYQDVIAIGKLFTTGELFTDRIISLAGPQVKNPRLVRTRLGANLSQLTANELNAGEN	300
Query:	298	RVISGSVLNGAITQGAHDYLGRYHNQISVIEEGRSKELFGWVAPQPDKYSITRTTLGHFL	357
		RVISGSVL+GA   G  DYLGRY  Q+SV+ EGR KELFGW+ P  DK+SITRT LGHF
Sbjct:	301	RVISGSVLSGATAAGPVDYLGRYALQVSVLAEGREKELFGWIMPGSDKFSITRTVLGHFG	360
Query:	358	KNKLFKFTTAVNGGDRAMVPIGTYERVMXXXXXXXXXXXXXXVGDTDSAQXXXXXXXXXX	417
		K KLF FTTAV+GG+RAMVPIG YERVM               GDTDSAQ
Sbjct:	361	K-KLFNFTTAVHGGERAMVPIGAYERVMPLDIIPTLLLRDLAAGDTDSAQNLGCLELDEE	419
Query:	418	XXXXXSFVCPGKYEXGPLLRKVLETXEKEG	447
		++VCPGK   GP+LR  LE  EKEG

ORF22ng-1 also shows homology with the OMP from A. pleuropneumoniae:

gi|1185395 (U24492) 48 kDa outer membrane protein [Actinobacillus
pleuropneumoniae] Length = 449
Score = 555 bits (1414), Expect = e−157
Identities = 284/450 (63%), Positives = 337/450 (74%), Gaps = 4/450 (0%)
Query:	27	MIKIKKGLNLPIAGRPEQVIYDGPAITEVALLGEEYVGMRPSMKIKEGEAVKKGQVLFED	86
		MI IKKGL+LPIAG P QVI++G  + EVA+LGEEYVGMRPSMK++EG+ VKKGQVLFED
Sbjct:	1	MITIKKGLDLPIAGTPAQVIHNGNTVNEVAMLGEEYVGMRPSMKVREGDVVKKGQVLFED	60
Query:	87	KKNPGVVFTAPASGKIAAIHRGEKRVLQSVVIAVEGNDEIEFERYVPEALAKLSSEKVRR	146
		KKNPGVVFTAPASG +  I+RGEKRVLQSVVI VEG+++I F RY    LA LS+E+V++
Sbjct:	61	KKNPGVVFTAPASGTVVTINRGEKRVLQSVVIKVEGDEQITFTRYEAAQLASLSAEQVKQ	120
Query:	147	NLIQSGLWTALRTRPFSKIPAVDAEPFAIFVNAMDTNPLAADPTVIIKEAAEDFKRGLLV	206
		NLI+SGLWTA RTRPFSK+PA+DA P +IFVNAMDTNPLAADP V++KE   DFK GL V
Sbjct:	121	NLIESGLWTAFRTRPFSKVPALDAIPSSIFVNAMDTNPLAADPEVVLKEYETDFKDGLTV	180
Query:	207	LSRL--TERKIHVCKAAGADVP-SENAANIETHEFGGPHPAGLSGTHIHFIEPVGANKTV	263
		L+RL   ++ +++CK A +++P S     I    F G HPAGL GTHIHF++PVGA K V
Sbjct:	181	LTRLFNGQKPVYLCKDADSNIPLSPAIEGITIKSFSGVHPAGLVGTHIHFVDPVGATKQV	240
Query:	264	WTINYQDVIAIGRLFVTGRLNTERVVALGGLQVNKPRLLRTVLGAKVSQLTAGELVDADN	323
		W +NYQDVIAIG+LF TG L T+R+++L G QV  PRL+RT LGA +SQLTA EL   +N
Sbjct:	241	WHLNYQDVIAIGKLFTTGELFTDRIISLAGPQVKNPRLVRTRLGANLSQLTANELNAGEN	300
Query:	324	RVISGSVLNGAIAQGAHDYLGRYHNQISVIEEGRSKELFGWVAPQPDKYSITRTTLGHFL	383
		RVISGSVL+GA A G  DYLGRY  Q+SV+ EGR KELFGW+ P  DK+SITRT LGHF
Sbjct:	301	RVISGSVLSGATAAGPVDYLGRYALQVSVLAEGREKELFGWIMPGSDKFSITRTVLGHFG	360
Query:	384	KNKLFKFTTAVNGGDRAMVPIGTYERVMXXXXXXXXXXXXXXVGDTDSAQXXXXXXXXXX	443
		K KLF FTTAV+GG+RAMVPIG YERVM               GDTDSAQ
Sbjct:	361	K-KLFNFTTAVHGGERAMVPIGAYERVMPLDIIPTLLLRDLAAGDTDSAQNLGCLELDEE	419
Query:	444	XXXXXSFVCPGKYEYGPLLRKVLETIEKEG	473
		++VCPGK  YGP+LR  LE IEKEG
Sbjct:	420	DLALCTYVCPGKNNYGPMLRAALEKIEKEG	449

Based on this analysis, including the homology with the outer membrane protein of Actinobacillus pleuropneumoniae, it was predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF22-1 (35.4 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 5A shows the results of affinity purification of the GST-fusion protein, and FIG. 5B shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for ELISA (positive result) and FACS analysis (FIG. 5C). These experiments confirm that ORF22-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 16

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 133>:

1   ..GCGnCGnAAA TCATCCATCC CC..nACGTC GTAGGCCCTG
    AAGCCAACTG
51  GTTTTTTATG GTAGCCAGTA CGTTTGTGAT TGCTTTGATT
    GGTTATTTTG
101 TTACTGAAAA AATCGTCGAA CCGCAATTGG GCCCTTATCA
    ATCAGATTTG
151 TCACAAGAAG AAAAAGACAT TCGGCATTCC AATGAAATCA
    CGCCTTTGGA
201 ATATAAAGGA TTAATTTGGG CTGGCGTGGT GTTTGTTGCC
    TTATCCGCCC
251 TATTGGCTTG GAGCATCGTC CCTGCCGACG GTATTTTGCG
    TCATCCTGAA
301 ACAGGATTGG TTTCCGGTTC GCCGTTTTTA AAATCGATTG
    TTGTTTTTAT
351 TTTCTTGTTG TTTGCACTGC CGGGCATTGT TTATGGCCGG
    GTAACCCGAA
401 GTTTGCGCGG CGAACAGGAA GTCGTTAATG CGmyGGCCGA
    ATCGATGAGT
451 ACTCTGGsGC TTTmTTTGsw CAkcATCTTT TTTGCCGCAC
    AGTTTGTCGC
501 ATTTTTTAAT TGGACGAATA TTGGGCAATA TATTGCCGTT
    AAAGGGGCGA
551 CGTTCTTAAA AGAAGTCGGC TTGGGCGGCA GCGTGTTGTT
    TATCGGTTTT
601 ATTTTAATTT GTGCTTTTAT CAATCTGATG ATAGGCTCCG
    CCTCCGCGCA
651 ATGGGCGGTA ACTGCGCCGA TTTTCGTCCC TATGCTGATG
    TTGGCCGGCT
701 ACGCGCCCGA AGTCATTCAA GCCGCTTACC GCATCGGTGA
    TTCCGTTACC
751 AATATTATTA CGCCGATGAT GAGTTATTTC GGGCTGATTA
    TGGCGACGGT
801 GrkCmmmTAC AAAAAAGATG CGGGCGTGGG TaCGcTGATT
    wCTATGATGT
851 TGCCGTATTC CGCTTTCTTC TTGATTGCgT GGATTGCCTT
    ATTCTGCATT
901 TGGGTATTTg TTTTGGGCCT GCCCGTCGGT CCCGGCGCGC
    CCACATTCTA
951 TCCCGCACCT TAA

This corresponds to the amino acid sequence <SEQ ID 134; ORF12>:

1   ..AXXIIHPXXV VGPEANWFFM VASTFVIALI GYFVTEKIVE
    PQLGPYQSDL
51  SQEEKDIRHS NEITPLEYKG LIWAGVVFVA LSALLAWSIV
    PADGILRHPE
101 TGLVSGSPFL KSIVVFIFLL FALPGIVYGR VTRSLRGEQE
    VVNAXAESMS
151 TLXLXLXXIF FAAQFVAFFN WTNIGQYIAV KGATFLKEVG
    LGGSVLFIGF
201 ILICAFINLM IGSASAQWAV TAPIFVPMLM LAGYAPEVIQ
    AAYRIGDSVT
251 NIITPMMSYF GLIMATVXXY KKDAGVGTLI XMMLPYSAFF
    LIAWIALFCI
301 WVFVLGLPVG PGAPTFYPAP *

Further sequence analysis revealed the complete DNA sequence <SEQ ID 135> to be:

1    ATGAGTCAAA CCGATACGCA ACGGGACGGA CGATTTTTAC
     GCACAGTCGA
51   ATGGCTGGGC AATATGTTGC CGCATCCGGT TACGCTTTTT
     ATTATTTTCA
101  TTGTGTTATT GCTGATTGCC TCTGCCGTCG GTGCGTATTT
     CGGACTATCC
151  GTCCCCGATC CGCGCCCTGT TGGTGCGAAA GGACGTGCCG
     ATGACGGTTT
201  GATTTACATT GTCAGCCTGC TCAATGCCGA CGGTTTTATC
     AAAATCCTGA
251  CGCATACCGT TAAAAATTTC ACCGGTTTCG CGCCGTTGGG
     AACGGTGTTG
301  GTTTCTTTAT TGGGCGTGGG GATTGCGGAA AAATCGGGCT
     TGATTTCCGC
351  ATTAATGCGC TTATTGCTCA CAAAATCGCC ACGCAAACTC
     ACTACTTTTA
401  TGGTTGTTTT TACAGGGATT TTATCTAATA CCGCTTCTGA
     ATTGGGCTAT
451  GTCGTCCTAA TCCCTTTGTC CGCCATCATC TTTCATTCCC
     TCGGCCGCCA
501  TCCGCTTGCC GGTCTGGCTG CGGCTTTCGC CGGCGTTTCG
     GGCGGTTATT
551  CGGCCAATCT GTTCTTAGGC ACAATCGATC CGCTCTTGGC
     AGGCATCACC
601  CAACAGGCGG CGCAAATCAT CCATCCCGAC TACGTCGTAG
     GCCCTGAAGC
651  CAACTGGTTT TTTATGGTAG CCAGTACGTT TGTGATTGCT
     TTGATTGGTT
701  ATTTTGTTAC TGAAAAAATC GTCGAACCGC AATTGGGCCC
     TTATCAATCA
751  GATTTGTCAC AAGAAGAAAA AGACATTCGG CATTCCAATG
     AAATCACGCC
801  TTTGGAATAT AAAGGATTAA TTTGGGCTGG CGTGGTGTTT
     GTTGCCTTAT
851  CCGCCCTATT GGCTTGGAGC ATCGTCCCTG CCGACGGTAT
     TTTGCGTCAT
901  CCTGAAACAG GATTGGTTTC CGGTTCGCCG TTTTTAAAAT
     CGATTGTTGT
951  TTTTATTTTC TTGTTGTTTG CACTGCCGGG CATTGTTTAT
     GGCCGGGTAA
1001 CCCGAAGTTT GCGCGGCGAA CAGGAAGTCG TTAATGCGAT
     GGCCGAATCG
1051 ATGAGTACTC TGGGGCTTTA TTTGGTCATC ATCTTTTTTG
     CCGCACAGTT
1101 TGTCGCATTT TTTAATTGGA CGAATATTGG GCAATATATT
     GCCGTTAAAG
1151 GGGCGACGTT CTTAAAAGAA GTCGGCTTGG GCGGCAGCGT
     GTTGTTTATC
1201 GGTTTTATTT TAATTTGTGC TTTTATCAAT CTGATGATAG
     GCTCCGCCTC
1251 CGCGCAATGG GCGGTAACTG CGCCGATTTT CGTCCCTATG
     CTGATGTTGG
1301 CCGGCTACGC GCCCGAAGTC ATTCAAGCCG CTTACCGCAT
     CGGTGATTCC
1351 GTTACCAATA TTATTACGCC GATGATGAGT TATTTCGGGC
     TGATTATGGC
1401 GACGGTGATC AAATACAAAA AAGATGCGGG CGTGGGTACG
     CTGATTTCTA
1451 TGATGTTGCC GTATTCCGCT TTCTTCTTGA TTGCGTGGAT
     TGCCTTATTC
1501 TGCATTTGGG TATTTGTTTT GGGCCTGCCC GTCGGTCCCG
     GCGCGCCCAC
1551 ATTCTATCCC GCACCTTAA

This corresponds to the amino acid sequence <SEQ ID 136; ORF12-1>:

1   MSQTDTQRDG RFLRTVEWLG NMLPHPVTLF IIFIVLLLIA
    SAVGAYFGLS
51  VPDPRPVGAK GRADDGLIYI VSLLNADGFI KILTHTVKNF
    TGFAPLGTVL
101 VSLLGVGIAE KSGLISALMR LLLTKSPRKL TTFMVVFTGI
    LSNTASELGY
151 VVLIPLSAII FHSLGRHPLA GLAAAFAGVS GGYSANLFLG
    TIDPLLAGIT
201 QQAAQIIHPD YVVGPEANWF FMVASTFVIA LIGYFVTEKI
    VEPQLGPYQS
251 DLSQEEKDIR HSNEITPLEY KGLIWAGVVF VALSALLAWS
    IVPADGILRH
301 PETGLVSGSP FLKSIVVFIF LLFALPGIVY GRVTRSLRGE
    QEVVNAMAES
351 MSTLGLYLVI IFFAAQFVAF FNWTNIGQYI AVKGATFLKE
    VGLGGSVLFI
401 GFILICAFIN LMIGSASAQW AVTAPIFVPM LMLAGYAPEV
    IQAAYRIGDS
451 VTNIITPMMS YFGLIMATVI KYKKDAGVGT LISMMLPYSA
    FFLIAWIALF
501 CIWVFVLGLP VGPGAPTFYP AP*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF12 shows 96.3% identity over a 320aa overlap with an ORF (ORF12a) from strain A of N. meningitidis:

The complete length ORF12a nucleotide sequence <SEQ ID 137> is:

1    ATGAGTCAAA CCGATACGCA ACGGGACGGA CGATTTTTAC
     GCACAGTCGA
51   ATGGCTGGGC AATATGTTGC CGCACCCGGT TACGCTTTTT
     ATTATTTTCA
101  TTGTGTTATT GCTGATTGCC TCTGCCGCCG GTGCGTATTT
     CGGACTATCC
151  GTCCCCGATC CGCGCCCTGT TGGTGCGAAA GGACGTGCCG
     ATGACGGTTT
201  GATTCACGTT GTCAGCCTGC TCGATGCTGA CGGTTTGATC
     AAAATCCTGA
251  CGCATACCGT TAAAAATTTC ACCGGTTTCG CGCCGTTGGG
     AACGGTGTTG
301  GTTTCTTTAT TGGGCGTGGG GATTGCGGAA AAATCGGGCT
     TGATTTCCGC
351  ATTAATGCGC TTATTGCTCA CAAAATCTCC ACGCAAACTC
     ACTACTTTTA
401  TGGTTGTTTT TACAGGGATT TTATCTAATA CCGCTTCTGA
     ATTGGGCTAT
451  GTCGTCCTAA TCCCTTTGTC CGCCATCATC TTTCATTCCC
     TCGGCCGCCA
501  TCCGCTTGCC GGTCTGGCTG CGGCTTTCGC CGGCGTTTCG
     GGCGGTTATT
551  CGGCCAATCT GTTCTTAGGC ACAATCGATC CGCTCTTGGC
     AGGCATCACC
601  CAACAGGCGG CGCAAATCAT CCATCCCGAC TACGTCGTAG
     GCCCTGAAGC
651  CAACTGGTTT TTTATGGTAG CCAGTACGTT TGTGATTGCT
     TTGATTGGTT
701  ATTTTGTTAC TGAAAAAATC GTCGAACCGC AATTGGGCCC
     TTATCAATCA
751  GATTTGTCAC AAGAAGAAAA AGACATTCGA CATTCCAATG
     AAATCACGCC
801  TTTGGAATAT AAAGGATTAA TTTGGGCTGG CGTGGTGTTT
     GTTGCCTTAT
851  CCGCCCTATT GGCTTGGAGC ATCGTCCCTG CCGACGGTAT
     TTTGCGTCAT
901  CCTGAAACAG GATTGGTTTC CGGTTCGCCG TTTTTAAAAT
     CAATTGTTGT
951  TTTTATTTTC TTGTTGTTTG CACTGCCGGG CATTGTTTAT
     GGCCGGGTAA
1001 CCCGAAGTTT GCGCGGCGAA CAGGAAGTCG TTAATGCGAT
     GGCCGAATCG
1051 ATGAGTACTC TGGGGCTTTA TTTGGTCATC ATCTTTTTTG
     CCGCACAGTT
1101 TGTCGCATTT TTTAATTGGA CGAATATTGG GCAATATATT
     GCCGTTAAAG
1151 GGGCGACGTT CTTAAAAGAA GTCGGCTTGG GCGGCAGCGT
     GTTGTTTATC
1201 GGTTTTATTT TAATTTGTGC TTTTATCAAT CTGATGATAG
     GCTCCGCCTC
1251 CGCGCAATGG GCGGTAACTG CGCCGATTTT CGTCCCTATG
     CTGATGTTGG
1301 CCGGCTACGC GCCCGAAGTC ATTCAAGCCG CTTACCGCAT
     CGGTGATTCC
1351 GTTACCAATA TTATTACGCC GATGATGAGT TATTTCGGGC
     TGATTATGGC
1401 GACGGTGATC AAATACAAAA AAGATGCGGG CGTGGGTACG
     CTGATTTCTA
1451 TGATGTTGCC GTATTCCGCT TTCTTCTTGA TTGCGTGGAT
     TGCCTTATTC
1501 TGCATTTGGG TATTTGTTTT GGGCCTGCCC GTCGGTCCCG
     GCGCGCCCAC
1551 ATTCTATCCC GCACCTTAA

This encodes a protein having amino acid sequence <SEQ ID 138>:

1   MSQTDTQRDG RFLRTVEWLG NMLPHPVTLF IIFIVLLLIA
    SAAGAYFGLS
51  VPDPRPVGAK GRADDGLIHV VSLLDADGLI KILTHTVKNF
    TGFAPLGTVL
101 VSLLGVGIAE KSGLISALMR LLLTKSPRKL TTFMVVFTGI
    LSNTASELGY
151 VVLIPLSAII FHSLGRHPLA GLAAAFAGVS GGYSANLFLG
    TIDPLLAGIT
201 QQAAQIIHPD YVVGPEANWF FMVASTFVIA LIGYFVTEKI
    VEPQLGPYQS
251 DLSQEEKDIR HSNEITPLEY KGLIWAGVVF VALSALLAWS
    IVPADGILRH
301 PETGLVSGSP FLKSIVVFIF LLFALPGIVY GRVTRSLRGE
    QEVVNAMAES
351 MSTLGLYLVI IFFAAQFVAF FNWTNIGQYI AVKGATFLKE
    VGLGGSVLFI
401 GFILICAFIN LMIGSASAQW AVTAPIFVPM LMLAGYAPEV
    IQAAYRIGDS
451 VTNIITPMMS YFGLIMATVI KYKKDAGVGT LISMMLPYSA
    FFLIAWIALF
501 CIWVFVLGLP VGPGAPTFYP AP*

ORF12a and ORF12-1 show 99.0% identity in 522 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF12 shows 92.5% identity over a 320aa overlap with a predicted ORF (ORF12.ng) from N. gonorrhoeae:

The complete length ORF12ng nucleotide sequence <SEQ ID 139> is:

1    ATGAGTCAAA CCGACGCGCG TCGTAGCGGA CGATTTTTAC
     GCACAGTCGA
51   ATGGCTGGGC AATATGTTGC CGCACCCGGT TACGCTTTTT
     ATTATTTTCA
101  TTGTGTTATT GCTGATTGcc tctgCCGTCG GTGCGTATTT
     CGGACTATCC
151  GTCCCCGATC CGCGTCCTGT TGGGGCGAAA GGACGTGCCG
     ATGACGGTTT
201  GATTCACGTT GTCAGCCTGC TCGATGCCGA CGGTTTGATC
     AAAATCCTGA
251  CGCATACCGT TAAAAATTTC ACCGGTTTCG CGCCGTTGGG
     AACGGTGTTG
301  GTTTCTTTAT TGGGCGTGGG GATTGCGGAA AAATCGGGCT
     TGATTTCCGC
351  ATTAATGCGC TTATTGCTCA CAAAATCCCC ACGCAAACTC
     ACTACTTTTA
401  TGGTTGTTTT TACAGGGATT TTATCCAATA CGGCTTCTGA
     ATTGGGCTAT
451  GTCGTCCTAA TCCCTTTGTC CGCCGTCATC TTTCATTCGC
     TCGGCCGCCA
501  TCCGCTTGCC GGTTTGGCTG CGGCTTTCGC CGGCGTTTCG
     GGCGGTTATT
551  CGGCCAATCT GTTCTTAGGC ACAATCGATC CGCTCTTGGC
     AGGCATCACC
601  CAACAGGCGG CGCAAATCAT CCATCCCGAC TACGTCGTAG
     GCCCTGAAGC
651  CAACTGGTTT TTTATGGCAG CCAGTACGTT TGTGATTGCT
     TTGATTGGTT
701  ATTTTGTTAC TGAAAAAATC GTCGAACCGC AATTGGGCCC
     TTATCAATCA
751  GATTTGTCAC AAGAAGAAAA AGACATTCGG CATTCCAATG
     AAATCACGCC
801  TTTGGAATAT AAAGGATTAA TTTGGGCAGG CGTGGTGTTT
     GTTGCCTTAT
851  CCGCCCTATT GGCTTGGAGC ATCGTCCCTG CCGACGGTAT
     TTTGCGTCAT
901  CCTGAAACAG GATTGGTTGC CGGTTCGCCG TTTTTAAAAT
     CGATTGTTGT
951  TTTTATTTTC TTGTTGTTTG CGCTGCCGGG CATTGTTTAT
     GGCCGGATAA
1001 CCCGAAGTTT GCGCGGCGAA CGGGAAGTCG TTAATGCGAT
     GGCCGAATCG
1051 ATGAGTACTT TGGGACTTTA TTTGGTCATC ATCTTTTTTG
     CCGCACAGTT
1101 TGTCGCATTT TTTAATTGGA CGAATATTGG GCAATATATT
     GCCGTTAAAG
1151 GGGCGGTGTT CTTAAAAGAA GTCGGCTTGG GCGGCAGTGT
     GTTGTTTATC
1201 GGTTTTATTT TAATTTGTGC TTTTATCAAT CTGATGATAG
     GCTCCGCCTC
1251 CGCGCAATGG GCGGTAACTG CGCCGATTTT CGTCCCTATG
     CTGATGTTGG
1301 CCGGCTACGC GCCCGAAGTC ATTCAAGCCG CTTACCGCAT
     CGGTGATTCC
1351 GTTACCAATA TTATTACGCC GATGATGAGT TATTTCGGGC
     TGATTATGGC
1401 GACGGTAATC AAATACAAAA AAGATGCGGG CGTAGGCACG
     CTGATTTCTA
1451 TGATGTTGCC GTATTCCGCT TTCTTCTTAA TTGCATGGAT
     CGCCTTATTC
1501 TGCATTTGGG TATTTGTTTT GGGTCTGCCC GTCGGTCCCG
     GCACACCCAC
1551 ATTCTATCCG GTGCCTTAA

This encodes a protein having amino acid sequence <SEQ ID 140>:

1   MSQTDARRSG RFLRTVEWLG NMLPHPVTLF IIFIVLLLIA
    SAVGAYFGLS
51  VPDPRPVGAK GRADDGLIHV VSLLDADGLI KILTHTVKNF
    TGFAPLGTVL
101 VSLLGVGIAE KSGLISALMR LLLTKSPRKL TTFMVVFTGI
    LSNTASELGY
151 VVLIPLSAVI FHSLGRHPLA GLAAAFAGVS GGYSANLFLG
    TIDPLLAGIT
201 QQAAQIIHPD YVVGPEANWF FMAASTFVIA LIGYFVTEKI
    VEPQLGPYQS
251 DLSQEEKDIR HSNEITPLEY KGLIWAGVVF VALSALLAWS
    IVPADGILRH
301 PETGLVAGSP FLKSIVVFIF LLFALPGIVY GRITRSLRGE
    REVVNAMAES
351 MSTLGLYLVI IFFAAQFVAF FNWTNIGQYI AVKGAVFLKK
    FRLGGSVLFI
401 GFILICAFIN LMIGSASAQW AVTAPIFVPM LMLAGNAPQV
    IQAAYRIGDS
451 VTNIITPMMS YFGLIMATVI KYKKDAGVGT LISMMLPYSA
    FFLIAWIALF
501 CIWVFVLGLP VGPGTPTFYP VP*

ORF12ng shows 97.1% identity in 522 aa overlap with ORF12-1:

In addition, ORF12ng shows significant homology with a hypothetical protein from E. coli:

sp|P46133|YDAH_ECOLI HYPOTHETICAL 55.1 KD PROTEIN IN OGT-DBPA
INTERGENIC REGION
>gi|1787597 (AE000231) hypothetical protein in ogt 5′region
[Escherichia coli]
Length = 510
Score = 329 bits (835), Expect = 2e−89
Identities = 178/507 (35%), Positives = 281/507 (55%), Gaps = 15/507 (2%)
Query:	8	RSGRFLRTVEWLGNMLPHPVTXXXXXXXXXXXASAVGAYFGLSVPDPRPVGAKGRADDGL	67
		+SG+    VE +GN +PHP              +A+ + FG+S  +P         D
Sbjct:	13	QSGKLYGWVERIGNKVPHPFLLFIYLIIVLMVTTAILSAFGVSAKNP--------TDGTP	64
Query:	68	IHVVSLLDADGLIKILTHTVKNFTGFAPXXXXXXXXXXXXIAEKSGLISALMRLLLTKSP	127
		+ V +LL  +GL   L + +KNF+GFAP            +AE+ GL+ ALM  + +
Sbjct:	65	VVVKNLLSVEGLHWFLPNVIKNFSGFAPLGAILALVLGAGLAERVGLLPALMVKMASHVN	124
Query:	128	RKLTTFMVVFTGILSNTASELGYVVLIPLSAVIFHSLGRHPLAGLAAAFAGVSGGYSANL	187
		+  ++MV+F    S+ +S+   V++ P+ A+IF ++GRHP+AGL AA AGV  G++ANL
Sbjct:	125	ARYASYMVLFIAFFSHISSDAALVIMPPMGALIFLAVGRHPVAGLLAAIAGVGCGFTANL	184
Query:	188	FLGTIDPLLAGITQQAAQIIHPDYVVGPEANWFFMAASTFVIALIGYFVTEKIVEPQLGP	247
		+ T D LL+GI+ +AA   +P   V    NW+FMA+S  V+ ++G  +T+KI+EP+LG
Sbjct:	185	LIVTTDVLLSGISTEAAAAFNPQMHVSVIDNWYFMASSVVVLTIVGGLITDKIIEPRLGQ	244
Query:	248	YQSDLSQEEKDIRHSNEITPLEYKGLIWAGVVFVALSALLAWSIVPADGILRHPETGLVA	307
		+Q +  ++ + +  S         GL  AGVV +   A +A  ++P +GILR P    V
Sbjct:	245	WQGNSDEKLQTLTESQRF------GLRIAGVVSLLFIAAIALMVIPQNGILRDPINHTVM	298
Query:	308	GSPFLKSIVVFIFLLFALPGIVYGRITRSLRGEREVVNAMAESMSTLGLYLXXXXXXXXX	367
		SPF+K IV  I L F +  + YG  TR++R + ++ + M E M  +  ++
Sbjct:	299	PSPFIKGIVPLIILFFFVVSLAYGIATRTIRRQADLPHLMIEPMKEMAGFIVMVFPLAQF	358
Query:	368	XXXXNWTNIGQYIAVKGAVFLKEVGLGGSVLFIGFILICAFINLMIGSASAQWAVTAPIF	427
		NW+N+G++IAV     L+  GL G   F+G  L+ +F+ +I S SA W++ APIF
Sbjct:	359	VAMFNWSNMGKFIAVGLTDILESSGLSGIPAFVGLALLSSFLCMFIASGSAIWSILAPIF	418
Query:	428	VPMLMLAGYAPEVIQAAYRIGDSVTNIITPMMSYFGLIMATVIKYKKDAGVGTLISMMLP	487
		VPM ML G+ P   Q  +RI DS    + P+  +  L +  + +YK DA +GT  S++LP
Sbjct:	419	VPMFMLLGFHPAFAQILFRIADSSVLPLAPVSPFVPLFLGFLQRYKPDAKLGTYYSLVLP	478
Query:	488	YSAFFLIAWIALFCIWVFVLGLPVGPG	514
		Y   FL+ W+ +   W +++GLP+GPG
Sbjct:	479	YPLIFLVVWLLMLLAW-YLVGLPIGPG	504

Based on this analysis, including the presence of several putative transmembrane domains and the predicted actinin-type actin-binding domain signature (shown in bold) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 17

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 141>:

1   ..ACAGCCGGCG CAGCAGGTTn CnCGGTCTTC GTTTTCGTAA
    CGGACAGTCA
51  GGTGGAGGTG TTCGGGAACA TCCAGACCGC AGTGGAAACA
    GGTTTTTTTC
101 ATGGCATTTC GGTTTCGTCT GTGTTTGGTG CGGCGGCACA
    AGACTCGGCA
151 ATgGCTTCGC GCAGTGCGTC TATACCGGTA TTTTCAGCAA
    CGGAAATGCG
201 GACGGcGgCA ATTTTTCCCG CAGCGTCGCG CCATATGCCC
    GTGTTTTgTT
251 CTTCAGACGG CAGCAGGTCG GTTTTGTTGT ACACCTTgAT
    GCACGGAaTA
301 TCGCCGGCAT GGATTTCTTG CAGTACGTTT TCCACGTCTT
    CAATCTGCTG
351 TCCGCTGTTC GGAGCGGCGG CATCGACGAC GTGCAGCAGC
    ACATCgGcTT
401 gCGCGGTTTC TTCCAGCGTG GCgGAAAAGG CGGAAATCAG
    TTTgTGCGGC
451 agATyGCTnA CGAATCCGAC GGTATCGGTC AGGATAATGC
    TGCATTCGGG
501 ACT..

This corresponds to the amino acid sequence <SEQ ID 142; ORF14>:

1   ..TAGAAGXXVF VFVTDSQVEV FGNIQTAVET GFFHGISVSS
    VFGAAAQDSA
51  MASRSASIPV FSATEMRTAA IFPAASRHMP VFCSSDGSRS
    VLLYTLMHGI
101 SPAWISCSTF STSSICCPLF GAAASTTCSS TSACAVSSSV
    AEKAEISLCG
151 RXLTNPTVSV RIMLHSG..

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF From N. meningitidis (Strain A)

ORF14 shows 94.0% identity over a 167aa overlap with an ORF (ORF14a) from strain A of N. meningitidis:

The complete length ORF14a nucleotide sequence <SEQ ID 143> is:

1    ATGGAGGATT TGCAGGAAAT CGGGTTCGAT GTCGCCGCCG
     TAAAGGTAGG
51   TCGGCAGCGC GAACATCATC GTCTGCATCA TCCCCAGCCC
     GGCAACGGCG
101  AGGCGGACGA TGTATTGTTT GCGTTCTTTT TGGTTGGCGG
     CTTCGATTTT
151  TTGCGCGTCA TAGGGTGCGG CGGTGTAGCC TATCTGCCTG
     ATTTTCAACA
201  GAATGTCGGA AAGGCGGATT TTGCCGTCGT CCCAGACGAC
     GCGGCAGCGG
251  TGCGTGCTGT AATTGAGGTC GATGCGGACG ATGCCGTCTG
     TACGCAAAAG
301  CTGCTGTTCG ATCAGCCAGA CGCAGGCGGC GCAGGTGATG
     CCGCCGAGCA
351  TTAAAACCGC CTCGCGCGTG CCGCCGTGGG TTTCCACAAA
     GTCGGACTGG
401  ACTTCGGGCA GGTCGTACAG GCGGATTTGG TCGAGGATTT
     CTTGGGGCGG
451  CAGCTCGGTT TTTTGCGCGT CGGCGGTGCG TTGTTTGTAA
     TAACTGCCCA
501  AGCCCGCGTC AATAATGCTT TGTGCGACTG CCTGACAACC
     GGCGCAGCAG
551  GTTTCGCGGT CTTCGTTTTC GTAACGGACG GTCAGATGCA
     GGTTTTCGGG
601  AACGTCCAGC CCGCAGTGGA AACAGGTTTT TTTCATGGCA
     TTTCGGTTTC
651  GTCTGTGTTT GGTGCGGCGG CACAATACTC GGCAATGGCT
     TCGCGCAGTG
701  CGTCTATACC GGTATTTTCA GCAACGGAAA TGCGGACGGC
     GGCAATTTTT
751  CCCGCAGCGT CGCGCCATAT GCCCGTGTTT TGTTCTTCAG
     ACGGCAGCAG
801  GTCGGTTTTG TTGTACACCT TGATGCACGG AATATCGCCG
     GCATGGATTT
851  CTTGCAGTAC GTTTTCCACG TCTTCAATCT GCTGTCCGCT
     GTTCGGAGCG
901  GCGGCATCGA CGACGTGCAG CAGCACATCG GCTTGCGCGG
     TTTCTTCCAG
951  CGTGGCGGAA AAGGCGGAAA TCAGTTTGTG CGGCAGATCG
     CTGACGAATC
1001 CGACGGTATC GGTCAGGATA ATGCTGCATT CGGGACTGAT
     GTACAGCCGC
1051 CGCGCCGTCG TGTCGAGTGT GGCGAAAAGC TGGTCTTTCG
     CATATATGCC
1101 CGACTTGGTC AGCCGGTTGA ACAGACTGGA TTTGCCGACA
     TTGGTATAG

This encodes a protein having amino acid sequence <SEQ ID 144>:

1   MEDLQEIGFD VAAVKVGRQR EHHRLHHPQP GNGEADDVLF
    AFFLVGGFDF
51  LRVIGCGGVA YLPDFQQNVG KADFAVVPDD AAAVRAVIEV
    DADDAVCTQK
101 LLFDQPDAGG AGDAAEH*NR LARAAVGFHK VGLDFGQVVQ
    ADLVEDFLGR
151 QLGFLRVGGA LFVITAQARV NNALCDCLTT GAAGFAVFVF
    VTDGQMQVFG
201 NVQPAVETGF FHGISVSSVF GAAAQYSAMA SRSASIPVFS
    ATEMRTAAIF
251 PAASRHMPVF CSSDGSRSVL LYTLMHGISP AWISCSTFST
    SSICCPLFGA
301 AASTTCSSTS ACAVSSSVAE KAEISLCGRS LTNPTVSVRI
    MLHSGLMYSR
351 RAVVSSVAKS WSFAYMPDLV SRLNRLDLPT LV*

It should be noted that this sequence includes a stop codon at position 118.

Homology with a Predicted ORF from N. gonorrhoeae

ORF14 shows 89.8% identity over a 167aa overlap with a predicted ORF (ORF14.ng) from N. gonorrhoeae:

The complete length ORF14ng nucleotide sequence <SEQ ID 145> is predicted to encode a protein having amino acid sequence <SEQ ID 146>:

1   MEDLQEIGFD VAAVKVGRQR EHHRLHHTQS GNGKADDVLF
    AFFLVGGFDF
51  LRVIGCGGVA CLPDFQQNVG EADFAVVPDD AAAVRAVIEV
    DADDAVCAQK
101 LLFDQPDAGG AGNAAEHQHC FVRAIMGFHK VGLDFGQVVQ
    ADLVEDFLGR
151 QFGFFRVGGA SFVITAQAGI DDALCDCLTA DAAGFAVFAF
    VADGQMQVFG
201 NVQPAVETGF FHGISVSSVF GAAAQYSAMA SRSASIPVFS
    ATEMRTAAIF
251 PAASRHMPVF CSSDGSRSVL LYTLMHGISW AWISCSTFST
    SSICCPLFRA
301 AASTTCSSTS ACTVSSKVAE KAEISLCGRS LTNPTVSVRI
    MLHAGLMYSR
351 RAVVSRVAKS WSFAYMPDLV SRLNRLDLPT LV*

Based on the putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 18

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 147>:

1   ..GGCCATTACT CCGACCGCAC TTGGAAGCCG CGTTTGGNCG
    GCCGCCGTCT
51  GCCGTATCTG CTTTATGGCA CGCTGATTGC GGTTATTGTG
    ATGATTTTGA
101 TGCCGAACTC GGGCAGCTTC GGTTTCGGCT ATGCGTCGCT
    GGCGGCTTTG
151 TCGTTCGGCG CGCTGATGAT TGCGCTGTTA GACGTGTCGT
    CAAATATGGC
201 GATGCAGCCG TTTAAGATGA TGGTCGGCGA CATGGTCAAC
    GAGGAGCAGA
251 AAA.NTACGC CTACGGGATT CAAAGTTTCT TAGCAAATAC
    GGGCGCGGTC
301 GTGGCGGCGA TTCTGCCGTT TGTGTTTGCG TATATCGGTT
    TGGCGAACAC
351 CGCCGANAAA GGCGTTGTGC CGCAGACCGT GGTCGTGGCG
    TTTTATGTGG
401 GTGCGGCGTT GCTGGTGATT ACCAGCGCGT TCACGATTTT
    CAAAGTGAAG
451 GAATACGANC CGGAAACCTA CGCCCGTTAC CACGGCATCG
    ATGTCGCCGC
501 GAATCAGGAA AAAGCCAACT GGATCGCACT CTTAAAA.CC
    GCGC..

This corresponds to the amino acid sequence <SEQ ID 148; ORF16>:

1   ..GHYSDRTWKP RLXGRRLPYL LYGTLIAVIV MILMPNSGSF
    GFGYASLAAL
51  SFGALMIALL DVSSNMAMQP FKMMVGDMVN EEQKXYAYGI
    QSFLANTGAV
101 VAAILPFVFA YIGLANTAXK GVVPQTVVVA FYVGAALLVI
    TSAFTIFKVK
151 EYXPETYARY HGIDVAANQE KANWIALLKX A..

Further work revealed the complete nucleotide sequence <SEQ ID 149>:

1    ATGTCGGAAT ATACGCCTCA AACAGCAAAA CAAGGTTTGC
     CCGCGCTGGC
51   AAAAAGCACG ATTTGGATGC TCAGTTTCGG CTTTCTCGGC
     GTTCAGACGG
101  CCTTTACCCT GCAAAGCTCG CAAATGAGCC GCATTTTTCA
     AACGCTAGGC
151  GCAGACCCGC ACAATTTGGG CTGGTTTTTC ATCCTGCCGC
     CGCTGGCGGG
201  GATGCTGGTG CAGCCGATTG TCGGCCATTA CTCCGACCGC
     ACTTGGAAGC
251  CGCGTTTGGG CGGCCGCCGT CTGCCGTATC TGCTTTATGG
     CACGCTGATT
301  GCGGTTATTG TGATGATTTT GATGCCGAAC TCGGGCAGCT
     TCGGTTTCGG
351  CTATGCGTCG CTGGCGGCTT TGTCGTTCGG CGCGCTGATG
     ATTGCGCTGT
401  TAGACGTGTC GTCAAATATG GCGATGCAGC CGTTTAAGAT
     GATGGTCGGC
451  GACATGGTCA ACGAGGAGCA GAAAGGCTAC GCCTACGGGA
     TTCAAAGTTT
501  CTTAGCAAAT ACGGGCGCGG TCGTGGCGGC GATTCTGCCG
     TTTGTGTTTG
551  CGTATATCGG TTTGGCGAAC ACCGCCGAGA AAGGCGTTGT
     GCCGCAGACC
601  GTGGTCGTGG CGTTTTATGT GGGTGCGGCG TTGCTGGTGA
     TTACCAGCGC
651  GTTCACGATT TTCAAAGTGA AGGAATACGA TCCGGAAACC
     TACGCCCGTT
701  ACCACGGCAT CGATGTCGCC GCGAATCAGG AAAAAGCCAA
     CTGGATCGAA
751  CTCTTGAAAA CCGCGCCTAA GGCGTTTTGG ACGGTTACTT
     TGGTGCAATT
801  CTTCTGCTGG TTCGCCTTCC AATATATGTG GACTTACTCG
     GCAGGCGCGA
851  TTGCGGAAAA CGTCTGGCAC ACCACCGATG CGTCTTCCGT
     AGGTTATCAG
901  GAGGCGGGTA ACTGGTACGG CGTTTTGGCG GCGGTGCAGT
     CGGTTGCGGC
951  GGTGATTTGT TCGTTTGTAT TGGCGAAAGT GCCGAATAAA
     TACCATAAGG
1001 CGGGTTATTT CGGCTGTTTG GCTTTGGGCG CGCTCGGCTT
     TTTCTCCGTT
1051 TTCTTCATCG GCAACCAATA CGCGCTGGTG TTGTCTTATA
     CCTTAATCGG
1101 CATCGCTTGG GCGGGCATTA TCACTTATCC GCTGACGATT
     GTGACCAACG
1151 CCTTGTCGGG CAAGCATATG GGCACTTACT TGGGCTTGTT
     TAACGGCTCT
1201 ATCTGTATGC CTCAAATCGT CGCTTCGCTG TTGAGTTTCG
     TGCTTTTCCC
1251 TATGCTGGGC GGCTTGCAGG CCACTATGTT CTTGGTAGGG
     GGCGTCGTCC
1301 TGCTGCTGGG CGCGTTTTCC GTGTTCCTGA TTAAAGAAAC
     ACACGGCGGG
1351 GTTTGA

This corresponds to the amino acid sequence <SEQ ID 150; ORF16-1>:

1   MSEYTPQTAK QGLPALAKST IWMLSFGFLG VQTAFTLQSS
    QMSRIFQTLG
51  ADPHNLGWFF ILPPLAGMLV QPIVGHYSDR TWKPRLGGRR
    LPYLLYGTLI
101 AVIVMILMPN SGSFGFGYAS LAALSFGALM IALLDVSSNM
    AMQPFKMMVG
151 DMVNEEQKGY AYGIQSFLAN TGAVVAAILP FVFAYIGLAN
    TAEKGVVPQT
201 VVVAFYVGAA LLVITSAFTI FKVKEYDPET YARYHGIDVA
    ANQEKANWIE
251 LLKTAPKAFW TVTLVQFFCW FAFQYMWTYS AGAIAENVWH
    TTDASSVGYQ
301 EAGNWYGVLA AVQSVAAVIC SFVLAKVPNK YHKAGYFGCL
    ALGALGFFSV
351 FFIGNQYALV LSYTLIGIAW AGIITYPLTI VTNALSGKHM
    GTYLGLFNGS
401 ICMPQIVASL LSFVLFPMLG GLQATMFLVG GVVLLLGAFS
    VFLIKETHGG
451 V*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. Meningitidis (Strain A)

ORF16 shows 96.7% identity over a 181 as overlap with an ORF (ORF16a) from strain A of N. meningitidis:

The complete length ORF16a nucleotide sequence <SEQ ID 151> is:

1    ATGTCGGAAT ATACGCCTCA AACAGCAAAA CAAGGTTTGC
     CCGCGCTGGC
51   AAAAAGCACG ATTTGGATGC TCAGTTTCGG CTTTCTCGGC
     GTTCAGACGG
101  CCTTTACCCT GCAAAGCTCG CAGATGAGCC GCATCTTCCA
     GACGCTCGGT
151  GCCGATCCGC ACAGCCTCGG CTGGTTCTTT ATCCTGCCGC
     CGCTGGCGGG
201  GATGCTGGTG CAGCCGATTG TCGGCCATTA CTCCGACCGC
     ACTTGGAAGC
251  CGCGTTTGGG CGGCCGCCGT CTGCCGTATC TGCTTTATGG
     CACGCTGATT
301  GCGGTTATTG TGATGATTTT GATGCCGAAC TCGGGCAGCT
     TCGGTTTCGG
351  CTATGCGTCG CTGGCGGCTT TGTCGTTCGG CGCGCTGATG
     ATTGCGCTGT
401  TAGACGTGTC GTCAAATATG GCGATGCAGC CGTTTAAGAT
     GATGGTCGGC
451  GACATGGTCA ACGAGGAGCA GAAAGGCTAC GCCTACGGGA
     TTCAAAGTTT
501  CTTAGCGAAT ACGGGCGCGG TCGTGGCGGC GATTCTGCCG
     TTTGTGTTTG
551  CGTATATCGG TTTGGCGAAC ACCGCCGAGA AAGGCGTTGT
     GCCGCAGACC
601  GTGGTCGTGG CGTTTTATGT GGGTGCGGCG TTGCTGGTGA
     TTACCAGCGC
651  GTTCACGATT TTCAAAGTGA AGGAATACAA TCCGGAAACC
     TACGCCCGTT
701  ACCACGGCAT CGATGTCGCC GCGAATCAGG AAAAAGCCAA
     CTGGATCGAA
751  CTCTTGAAAA CCGCGCCTAA GGCGTTTTGG ACGGTTACTT
     TGGTGCAATT
801  CTTCTGCTGG TTCGCCTTCC AATATATGTG GACTTACTCG
     GCAGGCGCGA
851  TTGCGGAAAA CGTCTGGCAC ACCACCGATG CGTCTTCCGT
     AGGTTATCAG
901  GAGGCGGGTA ACTGGTACGG CGTTTTGGCG GCGGTGCAGT
     CGGTTGCGGC
951  GGTGATTTGT TCGTTTGTAT TGGCGAAAGT GCCGAATAAA
     TACCATAAGG
1001 CGGGTTATTT CGGCTGTTTG GCTTTGGGCG CGCTCGGCTT
     TTTCTCCGTT
1051 TTCTTCATCG GCAACCAATA CGCGCTGGTG TTGTCTTATA
     CCTTAATCGG
1101 CATCGCTTGG GCGGGCATTA TCACTTATCC GCTGACGATT
     GTGACCAACG
1151 CCTTGTCGGG CAAGCATATG GGCACTTACT TGGGCCTGTT
     TAACGGCTCT
1201 ATCTGTATGC CGCAAATCGT CGCTTCGCTG TTGAGTTTCG
     TGCTTTTCCC
1251 TATGCTGGGC GGCTTGCAGG CCACTATGTT CTTGGTAGGG
     GGCGTCGTCC
1301 TGCTGCTGGG CGCGTTTTCC GTGTTCCTGA TTAAAGAAAC
     ACACGGCGGG
1351 GTTTGA

This encodes a protein having amino acid sequence <SEQ ID 152>:

1   MSEYTPQTAK QGLPALAKST IWMLSFGFLG VQTAFTLQSS
    QMSRIFQTLG
51  ADPHSLGWFF ILPPLAGMLV QPIVGHYSDR TWKPRLGGRR
    LPYLLYGTLI
101 AVIVMILMPN SGSFGFGYAS LAALSFGALM IALLDVSSNM
    AMQPFKMMVG
151 DMVNEEQKGY AYGIQSFLAN TGAVVAAILP FVFAYIGLAN
    TAEKGVVPQT
201 VVVAFYVGAA LLVITSAFTI FKVKEYNPET YARYHGIDVA
    ANQEKANWIE
251 LLKTAPKAFW TVTLVQFFCW FAFQYMWTYS AGAIAENVWH
    TTDASSVGYQ
301 EAGNWYGVLA AVQSVAAVIC SFVLAKVPNK YHKAGYFGCL
    ALGALGFFSV
351 FFIGNQYALV LSYTLIGIAW AGIITYPLTI VTNALSGKHM
    GTYLGLFNGS
401 ICMPQIVASL LSFVLFPMLG GLQATMFLVG GVVLLLGAFS
    VFLIKETHGG
451 V*

ORF16a and ORF16-1 show 99.6% identity in 451 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF16 shows 93.9% identity over a 181aa overlap with a predicted ORF (ORF16.ng) from N. gonorrhoeae:

The complete length ORF16ng nucleotide sequence <SEQ ID 153> is:

1    ATGATAGGGG ATCGCCGCGC CGGCAACCAT TTCGGATTTT
     CCAAAGCAAA
51   TACTTTTCAA ATCAAAAAAA AGGATTTACT TTATGTCGGA
     ATATACGCCT
101  CAAACAGCAA AACAAGGTTT GCCCGCGCCG GCAAAAAGCA
     CGATTTGGAT
151  GTTGAGCTTC GGCTATCTCG GCGTTCAGAC GGCCTTTACC
     CTGCAAAGCT
201  CGCAGATGAG CCGCATTTTT CAAACGCTAG GCGCAGACCC
     GCACAATTTG
251  GGCTGGTTTT TCATCCTGCC GCCGCTGGCG GGGATGCTGG
     TTCAGCCGAT
301  AGTGGCTACT ACTCAGACCG CACTTGGAAG CCGCGCTTGG
     GCGGCCGCCG
351  CCTGCCGTAT CTGCTTTACG GCACGCTGAT TGCGGTCATC
     GTGATGATTT
401  TGATGCCGAA CTCGGGCAGC TTCGGTTTCG GCTATGCGTC
     GCTGGCGGCC
451  TTGTCGTTCG GCGCGCTGAT GATTGCGCTG TTGGACGTGT
     CGTCGAATAT
501  GGCGATGCAG CCGTTTAAGA TGATGGTCGG CGATATGGTC
     AACGAGGAGC
551  AGAAAAGCTA CGCCTACGGG ATTCAAAGTT TCTTAGCGAA
     TACGGACGCG
601  GTTGTGGCAG CGATTCTGCC GTTTGTGTTC GCGTATATCG
     GTTTGGCGAA
651  CACTGCCGAG AAAGGCGTTG TGCCACAAAC CGTGGTCGTA
     GCATTCTATG
701  TGGGTGCGGC GTTACTGATT ATTACCAGTG CGTTCACAAT
     CTCCAAAGTC
751  AAAGAATACG ACCCGGAAAC CTACGCCCGT TACCACGGCA
     TCGATGTCGC
801  CGCGAATCAG GAAAAAGCCA ACTGGTTCGA ACTCTTAAAA
     ACCGCGCCTA
851  AAGTGTTTTG GACGGTTACT CCGGTACAGT TTTTCTGCTG
     GTTCGCCTTC
901  CGGTATATGT GGACTTACTC GGCAGGCGCG ATTGCAGAAA
     ACGTCTGGCA
951  CACTACCGAT GCGTCTTCCG TAGGCCATCA GGAGGCGGGC
     AACCGGTACG
1001 GCGTTTTGGC GGCGGTGTAG

This encodes a protein having amino acid sequence <SEQ ID 154>:

1   MIGDRRAGNH FGFSKANTFQ IKKKDLLYVG IYASNSKTRF
    ARAGKKHDLD
51  VELRLSRRSD GLYPAKLADE PHFSNARRRP AQFGLVFHPA
    AAGGDAGSAD
101 SGYYSDRTWK PRLGGRRLPY LLYGTLIAVI VMILMPNSGS
    FGFGYASLAA
151 LSFGALMIAL LDVSSNMAMQ PFKMMVGDMV NEEQKSYAYG
    IQSFLANTDA
201 VVAAILPFVF AYIGLANTAE KGVVPQTVVV AFYVGAALLI
    ITSAFTISKV
251 KEYDPETYAR YHGIDVAANQ EKANWFELLK TAPKVFWTVT
    PVQFFCWFAF
301 RYMWTYSAGA IAENVWHTTD ASSVGHQEAG NRYGVLAAV*

ORF16ng and ORF16-1 show 89.3% identity in 261 aa overlap:

Based on this analysis, including the presence of several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 19

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 155>:

1   ATGTTGTTCC GTAAAACGAC CGCCGCCGTT TTGGCGCATA
    CCTTGATGCT
51  GAACGGCTGT ACGTTGATGT TGTGGGGAAT GAACAACCCG
    GTCAGCGAAA
101 CAATCACCCG NAAACACGTT GNCAAAGACC AAATCCGNGN
    CTTCGGTGTG
151 GTTGCCGAAG ACAATGCCCA ATTGGAAAAG GGCAGCCTGG
    TGATGATGGG
201 CGGAAAATAC TGGTTCGTCG TCAATCCCGA AGATTCGGCG
    AA.NTGACGG
251 GNATTTTGAN GGCAGGGCTG GACAAACCCT TCCAAATAGT
    TNAGGATACC
301 CCGAGCTATG C.TGCCACCA AGCCCTGCCG GTCAAACTCG
    GATCGNCTGG
351 CAGCCAGAAT...

This corresponds to the amino acid sequence <SEQ ID 156; ORF28>:

1   MLFRKTTAAV LAHTLMLNGC TLMLWGMNNP VSETITRKHV
    XKDQIRXFGV
51  VAEDNAQLEK GSLVMMGGKY WFVVNPEDSA XXTGILXAGL
    DKPFQIVXDT
101 PSYXCHQALP VKLGSXGSQN...

Further work revealed the complete nucleotide sequence <SEQ ID 157>:

1   ATGTTGTTCC GTAAAACGAC CGCCGCCGTT TTGGCGGCAA
    CCTTGATGCT
51  GAACGGCTGT ACGTTGATGT TGTGGGGAAT GAACAACCCG
    GTCAGCGAAA
101 CAATCACCCG CAAACACGTT GACAAAGACC AAATCCGCGC
    CTTCGGTGTG
151 GTTGCCGAAG ACAATGCCCA ATTGGAAAAG GGCAGCCTGG
    TGATGATGGG
201 CGGAAAATAC TGGTTCGTCG TCAATCCCGA AGATTCGGCG
    AAGCTGACGG
251 GCATTTTGAA GGCAGGGCTG GACAAACCCT TCCAAATAGT
    TGAGGATACC
301 CCGAGCTATG CTCGCCACCA AGCCCTGCCG GTCAAACTCG
    AATCGCCTGG
351 CAGCCAGAAT TTCAGTACCG AAGGCCTTTG CCTGCGCTAC
    GATACCGACA
401 AGCCTGCCGA CATCGCCAAG CTGAAACAGC TCGGGTTTGA
    AGCGGTCAAA
451 CTCGACAATC GGACCATTTA CACGCGCTGC GTATCCGCCA
    AAGGCAAATA
501 CTACGCCACA CCGCAAAAAC TGAACGCCGA TTACCATTTT
    GAGCAAAGTG
551 TGCCTGCCGA TATTTATTAC ACGGTTACTG AAGAACATAC
    CGACAAATCC
601 AAGCTGTTTG CAAATATCTT ATATACGCCC CCCTTTTTGA
    TACTGGATGC
651 GGCGGGCGCG GTACTGGCCT TGCCTGCGGC GGCTCTGGGT
    GCGGTCGTGG
701 ATGCCGCCCG CAAATGA

This corresponds to the amino acid sequence <SEQ ID 158; ORF28-1>:

1   MLFRKTTAAV LAATLMLNGC TLMLWGMNNP VSETITRKHV
    DKDQIRAFGV
51  VAEDNAQLEK GSLVMMGGKY WFVVNPEDSA KLTGILKAGL
    DKPFQIVEDT
101 PSYARHQALP VKLESPGSQN FSTEGLCLRY DTDKPADIAK
    LKQLGFEAVK
151 LDNRTIYTRC VSAKGKYYAT PQKLNADYHF EQSVPADIYY
    TVTEEHTDKS
201 KLFANILYTP PFLILDAAGA VLALPAAALG AVVDAARK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF28 shows 79.2% identity over a 120aa overlap with an ORF (ORF28a) from strain A of N. meningitidis:

The complete length ORF28a nucleotide sequence <SEQ ID 159> is:

1   ATGTTGTTCC GTAAAACGAC CGCCGCCGTT TTGGCGGCAA
    CCTTGATGTT
51  GAACGGCTGT ACGGTAATGA TGTGGGGTAT GAACAGCCCG
    TTCAGCGAAA
101 CGACCGCCCG CAAACACGTT GACAAGGACC AAATCCGCGC
    CTTCGGTGTG
151 GTTGCCGAAG ACAATGCCCA ATTGGAAAAG GGCAGCCTGG
    TGATGATGGG
201 CGGGAAATAC TGGTTCGTCG TCAATCCTGA AGATTCGGCG
    AAGCTGACGG
251 GCATTTTGAA GGCCGGGTTG GACAAGCAGT TTCAAATGGT
    TGAGCCCAAC
301 CCGCGCTTTG CCTACCAAGC CCTGCCGGTC AAACTCGAAT
    CGCCCGCCAG
351 CCAGAATTTC AGTACCGAAG GCCTTTGCCT GCGCTACGAT
    ACCGACAGAC
401 CTGCCGACAT CGCCAAGCTG AAACAGCTTG AGTTTGAAGC
    GGTCGAACTC
451 GACAATCGGA CCATTTACAC GCGCTGCGTC TCCGCCAAAG
    GCAAATACTA
501 CGCCACACCG CAAAAACTGA ACGCCGATTA TCATTTTGAG
    CAAAGTGTGC
551 CTGCCGATAT TTATTACACG GTTACGAAAA AACATACCGA
    CAAATCCAAG
601 TTGTTTGAAA ATATTGCATA TACGCCCACC ACGTTGATAC
    TGGATGCGGT
651 GGGCGCGGTG CTGGCCTTGC CTGTCGCGGC GTTGATTGCA
    GCCACGAATT
701 CCTCAGACAA ATGA

This encodes a protein having amino acid sequence <SEQ ID 160>:

1   MLFRKTTAAV LAATLMLNGC TVMMWGMNSP FSETTARKHV
    DKDQIRAFGV
51  VAEDNAQLEK GSLVMMGGKY WFVVNPEDSA KLTGILKAGL
    DKQFQMVEPN
101 PRFAYQALPV KLESPASQNF STEGLCLRYD TDRPADIAKL
    KQLEFEAVEL
151 DNRTIYTRCV SAKGKYYATP QKLNADYHFE QSVPADIYYT
    VTKKHTDKSK
201 LFENIAYTPT TLILDAVGAV LALPVAALIA ATNSSDK*

ORF28a and ORF28-1 show 86.1% identity in 238 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF28 shows 84.2% identity over a 120aa overlap with a predicted ORF (ORF28.ng) from N. gonorrhoeae:

The complete length ORF28ng nucleotide sequence <SEQ ID 161> is

1   ATGTTGTTCC GTAAAACGAC CGCCGCCGTT TTGGCGGCAA
    CCTTGATACT
51  GAACGGCTGT ACGATGATGT TGCGGGGGAT GAACAACCCG
    GTCAGCCAAA
101 CAATCACCCG CAAACACGTT GACAAAGACC AAATCCGCGC
    CTTCGGTGTG
151 GTTGCCGAAG ACAATGCCCA ATTGGAAAAG GGCAGCCTGG
    TGATGATGGG
201 CGGGAAATAC TGGTTCGCCG TCAATCCCGA AGATTCGGCG
    AAGCTGACGG
251 GCCTTTTGAA GGCCGGGTTG GACAAGCCCT TCCAAATAGT
    TGAGGATACC
301 CCGAGCTATG CCCGCCACCA AGCCCTGCCG GTCAAATTCG
    AAGCGCCCGG
351 CAGCCAGAAT TTCAGTACCG GAGGTCTTTG CCTGCGCTAT
    GATACCGGCA
401 GACCTGACGA CATCGCCAAG CTGAAACAGC TTGAGTTTAA
    AGCGGTCAAA
451 CTCGACAATC GGACCATTTA CACGCGCTGC GTATCCGCCA
    AAGGCAAATA
501 CTACGCCACG CCGCAAAAAC TGAACGCCGA TTATCATTTT
    GAGCAAAGTG
551 TGCCCGCCGA TATTTATTAT ACGGTTACTG AAAAACATAC
    CGACAAATCC
601 AAGCTGTTTG GAAATATCTT ATATACGCCC CCCTTGTTGA
    TATTGGATGC
651 GGCGGCCGCG GTGCTGGTCT TGCCTATGGC TCTGATTGCA
    GCCGCGAATT
701 CCTCAGACAA ATGA

This encodes a protein having amino acid sequence <SEQ ID 162>:

1   MLFRKTTAAV LAATLILNGC TMMLRGMNNP VSQTITRKHV
    DKDQIRAFGV
51  VAEDNAQLEK GSLVMMGGKY WFAVNPEDSA KLTGLLKAGL
    DKPFQIVEDT
101 PSYARHQALP VKFEAPGSQN FSTGGLCLRY DTGRPDDIAK
    LKQLEFKAVK
151 LDNRTIYTRC VSAKGKYYAT PQKLNADYHF EQSVPADIYY
    TVTEKHTDKS
201 KLFGNILYTP PLLILDAAAA VLVLPMALIA AANSSDK*

ORF28ng and ORF28-1 share 90.0% identity in 231 aa overlap:

Based on this analysis, including the presence of a putative transmembrane domain in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF28-1 (24 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 6A shows the results of affinity purification of the GST-fusion protein, and FIG. 6B shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for ELISA, which gave a positive result. These experiments confirm that ORF28-1 is a surface-exposed protein, and that it may be a useful immunogen.

Example 20

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 163>:

1   ..GTCAGTCCTG TACTGCCTAT TACACACGAA CGGACAGGGT
    TTGAAGGTGT
51  TATCGGTTAT GAAACCCATT TTTCAGGGCA CGGACATGAA
    GTACACAGTC
101 CGTTCGATCA TCATGATTCA AAAAGCACTT CTGATTTCAG
    CGGCGGTGTA
151 GACGGCGGTT TTACTGTTTA CCAACTTCAT CGAACATGGT
    CGGAAATCCA
201 TCCGGAGGAT GAATATGACG GGCCGCAAGC AGCG.ATTAT
    CCGCCCCCCG
251 GAGGAGCAAG GGATATATAC AGCTATTATG TCAAAGGAAC
    TTCAACAAAA
301 ACAAAGACTA GTATTGTCCC TCAAGCCCCA TTTTCAGACC
    GTTGGCTAGA
351 AGAAAATGCC GGTGCCGCCT CTGGT..

This corresponds to the amino acid sequence <SEQ ID 164; ORF29>:

1   ..VSPVLPITHE RTGFEGVIGY ETHFSGHGHE VHSPFDHHDS
    KSTSDFSGGV
51  DGGFTVYQLH RTWSEIHPED EYDGPQAAXY PPPGGARDIY
    SYYVKGTSTK
101 TKTSIVPQAP FSDRWLEENA GAASG..

Further work revealed the complete nucleotide sequence <SEQ ID 165>:

1    ATGAATTTGC CTATTCAAAA ATTCATGATG CTGTTTGCAG
     CAGCAATATC
51   GTTGCTGCAA ATCCCCATTA GTCATGCGAA CGGTTTGGAT
     GCCCGTTTGC
101  GCGATGATAT GCAGGCAAAA CACTACGAAC CGGGTGGTAA
     ATACCATCTG
151  TTTGGTAATG CTCGCGGCAG TGTTAAAAAG CGGGTTTACG
     CCGTCCAGAC
201  ATTTGATGCA ACTGCGGTCA GTCCTGTACT GCCTATTACA
     CACGAACGGA
251  CAGGGTTTGA AGGTGTTATC GGTTATGAAA CCCATTTTTC
     AGGGCACGGA
301  CATGAAGTAC ACAGTCCGTT CGATCATCAT GATTCAAAAA
     GCACTTCTGA
351  TTTCAGCGGC GGTGTAGACG GCGGTTTTAC TGTTTACCAA
     CTTCATCGAA
401  CAGGGTCGGA AATCCATCCG GAGGATGGAT ATGACGGGCC
     GCAAGGCAGC
451  GATTATCCGC CCCCCGGAGG AGCAAGGGAT ATATACAGCT
     ATTATGTCAA
501  AGGAACTTCA ACAAAAACAA AGACTAATAT TGTCCCTCAA
     GCCCCATTTT
551  CAGACCGTTG GCTAAAAGAA AATGCCGGTG CCGCCTCTGG
     TTTTTTCAGC
601  CGTGCGGATG AAGCAGGAAA ACTGATATGG GAAAGCGACC
     CCAATAAAAA
651  TTGGTGGGCT AACCGTATGG ATGATGTTCG CGGCATCGTC
     CAAGGTGCGG
701  TTAATCCTTT TTTAATGGGT TTTCAAGGAG TAGGGATTGG
     GGCAATTACA
751  GACAGTGCAG TAAGCCCGGT CACAGATACA GCCGCGCAGC
     AGACTCTACA
801  AGGTATTAAT GATTTAGGAA AATTAAGTCC GGAAGCACAA
     CTTGCTGCCG
851  CGAGCCTATT ACAGGACAGT GCTTTTGCGG TAAAAGACGG
     TATCAACTCT
901  GCCAAACAAT GGGCTGATGC CCATCCAAAT ATAACAGCTA
     CTGCCCAAAC
951  TGCCCTTTCC GCAGCAGAGG CCGCAGGTAC GGTTTGGAGA
     GGTAAAAAAG
1001 TAGAACTTAA CCCGACTAAA TGGGATTGGG TTAAAAATAC
     CGGTTATAAA
1051 AAACCTGCTG CCCGCCATAT GCAGACTTTA GATGGGGAGA
     TGGCAGGTGG
1101 GAATAAACCT ATTAAATCTT TACCAAACAG TGCCGCTGAA
     AAAAGAAAAC
1151 AAAATTTTGA GAAGTTTAAT AGTAACTGGA GTTCAGCAAG
     TTTTGATTCA
1201 GTGCACAAAA CACTAACTCC CAATGCACCT GGTATTTTAA
     GTCCTGATAA
1251 AGTTAAAACT CGATACACTA GTTTAGATGG AAAAATTACA
     ATTATAAAAG
1301 ATAACGAAAA CAACTATTTT AGAATCCATG ATAATTCACG
     AAAACAGTAT
1351 CTTGATTCAA ATGGTAATGC TGTGAAAACC GGTAATTTAC
     AAGGTAAGCA
1401 AGCAAAAGAT TATTTACAAC AACAAACTCA TATCAGGAAC
     TTAGACAAAT
1451 GA

This corresponds to the amino acid sequence <SEQ ID 166; ORF29-1>:

1   MNLPIQKFMM LFAAAISLLQ IPISHANGLD ARLRDDMQAK
    HYEPGGKYHL
51  FGNARGSVKK RVYAVQTFDA TAVSPVLPIT HERTGFEGVI
    GYETHFSGHG
101 HEVHSPFDHH DSKSTSDFSG GVDGGFTVYQ LHRTGSEIHP
    EDGYDGPQGS
151 DYPPPGGARD IYSYYVKGTS TKTKTNIVPQ APFSDRWLKE
    NAGAASGFFS
201 RADEAGKLIW ESDPNKNWWA NRMDDVRGIV QGAVNPFLMG
    FQGVGIGAIT
251 DSAVSPVTDT AAQQTLQGIN DLGKLSPEAQ LAAASLLQDS
    AFAVKDGINS
301 AKQWADAHPN ITATAQTALS AAEAAGTVWR GKKVELNPTK
    WDWVKNTGYK
351 KPAARHMQTL DGEMAGGNKP IKSLPNSAAE KRKQNFEKFN
    SNWSSASFDS
401 VHKTLTPNAP GILSPDKVKT RYTSLDGKIT IIKDNENNYF
    RIHDNSRKQY
451 LDSNGNAVKT GNLQGKQAKD YLQQQTHIRN LDK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF29 shows 88.0% identity over a 125aa overlap with an ORF (ORF29a) from strain A of N. meningitidis:

The complete length ORF29a nucleotide sequence <SEQ ID 167> is:

1    ATGAATTNGC CTATTCAAAA ATTCATGATG CTGTTTGCAG
     CAGCAATATC
51   GTNGCTGCAA ATCCCNATTA GTCATGCGAA CGGTTTGGAT
     GCCCGTTTGC
101  GCGATGATAT GCAGGCAAAA CACTACGAAC CGGGTGGTAA
     ATACCATCTG
151  TTTGGTAATG CTCGCGGCAG TGTTAAAAAT CGGGTTTACG
     CCGTCCAAAC
201  ATTTGATGCA ACTGCGGTCG GCCCCATACT GCCTATTACA
     CACGAACGGA
251  CAGGATTTGA AGGCATTATC GGTTATGAAA CCCATTTTTC
     AGGACATGGA
301  CATGAAGTAC ACAGTCCGTT CGATAATCAT GATTCAAAAA
     GCACTTCTGA
351  TTTCAGCGGC GGCGTAGACG GTGGTTTTAC CGTTTACCAA
     CTTCATCGGA
401  CAGGGTCGGA AATCCATCCG GAGGATGGAT ATGACGGGCC
     GCAAGGCAGC
451  GATTATCCGC CCCCCGGAGG AGCAAGGGAT ATATACANNT
     ANTATGTCAA
501  AGGAACTTCA ACAAAAACAA AGAGTAATAT TGTTCCCCGA
     GCCCCATTTT
551  CAGACCGCTG GCTAAAAGAA AATGCCGGTG CCGCCTCTGG
     TTTTTTCAGC
601  CGTGCTGATG AAGCAGGAAA ACTGATATGG GAAAGCGACC
     CCAATAAAAA
651  TTGGTGGGCT AACCGTATGG ATGATATTCG CGGCATCGTC
     CAAGGTGCGG
701  TTAATCCTTT TTTAATGGGT TTTCAAGGAG TAGGGATTGG
     GGCAATTACA
751  GACAGTGCAG TAAGCCCGGT CACAGATACA GCCGCGCAGC
     AGACTCTACA
801  AGGTATNAAT CATTTAGGAA ANTTAAGTCC CGAAGCACAA
     CTTGCGGCTG
851  CAACCGCATT ACAAGACAGT GCTTTTGCGG TAAAAGACGG
     TATCAATTCC
901  GCCAGACAAT GGGCTGATGC CCATCCGAAT ATAACTGCAA
     CAGCCCAAAC
951  TGCCCTTGCC GTAGCAGANG CCGCAACTAC GGTTTGGGGC
     GGTAAAAAAG
1001 TAGAACTTAA CCCGACCAAA TGGGATTGGG TTAAAAATAC
     NGGCTATAAN
1051 ACACCTGCTG TTCGCACCAT GCATACTTTG GATGGGGAAA
     TGGCCGGTGG
1101 GAATAGACCG CCTAAATCTA TAACGTCCAA CAGCAAAGCA
     GATGCTTCCA
1151 CACAACCGTC TTTACAAGCG CAACTAATTG GAGAACAAAT
     TANNNNNGGG
1201 CATGCTTATA ACAAGCATGT CATAAGACAA CAAGAATTTA
     CGGATTTAAA
1251 TATCAATTCA CCAGCAGATT TTGCTCGGCA TATTGAAAAT
     ATTGTTAGCC
1301 ATCCANCAAA TATGAAAGAG TTACCTCGCG GTAGAACTGC
     GTATTGGGAT
1351 NATAAAACAG GGACNATAGT TATCCGAGAT AAAAATTCTG
     ACGATGGAGG
1401 TACAGCATTT AGACCAACAT CAGGTAAAAA ATATTATGAT
     GATTTATAG

This encodes a protein having amino acid sequence <SEQ ID 168>:

1   MNXPIQKFMM LFAAAISXLQ IPISHANGLD ARLRDDMQAK
    HYEPGGKYHL
51  FGNARGSVKN RVYAVQTFDA TAVGPILPIT HERTGFEGII
    GYETHFSGHG
101 HEVHSPFDNH DSKSTSDFSG GVDGGFTVYQ LHRTGSEIHP
    EDGYDGPQGS
151 DYPPPGGARD IYXXYVKGTS TKTKSNIVPR APFSDRWLKE
    NAGAASGFFS
201 RADEAGKLIW ESDPNKNWWA NRMDDIRGIV QGAVNPFLMG
    FQGVGIGAIT
251 DSAVSPVTDT AAQQTLQGXN HLGXLSPEAQ LAAATALQDS
    AFAVKDGINS
301 ARQWADAHPN ITATAQTALA VAXAATTVWG GKKVELNPTK
    WDWVKNTGYX
351 TPAVRTMHTL DGEMAGGNRP PKSITSNSKA DASTQPSLQA
    QLIGEQIXXG
401 HAYNKHVIRQ QEFTDLNINS PADFARHIEN IVSHPXNMKE
    LPRGRTAYWD
451 XKTGTIVIRD KNSDDGGTAF RPTSGKKYYD DL*

ORF29a and ORF29-1 show 90.1% identity in 385 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF29 shows 88.8% identity over a 125aa overlap with a predicted ORF (ORF29.ng) from N. gonorrhoeae:

The complete length ORF29ng nucleotide sequence <SEQ ID 169> is predicted to encode a protein having amino acid sequence <SEQ ID 170>:

1 MNLPIQKFMM LFAAAISLLQ IPISHANGLD ARLRDDMQAK HYEPGGKYHL
51 FGNARGSVKN RVCAVQTFDA TAVGPILPIT HERTGFEGVI GYETHFSGHG
101 HEVHSPFDNH DSKSTSDFSG GVDGGFTVYQ LHRTGSEIHP EDGYDGPQGG
151 GYPPPGGARD IYSYHIKGTS TKTKINTVPQ APFSDRWLKE NAGAASGFLS
201 RADEAGKLIW ENDPDKNWRA NRMDDIRGIV QGAVNPFLTG FQGLGVGAIT
251 DSAVSPVTYA AARKTLQGIH NLGNLSPEAQ LAAATALQDS AFAVKDSINS
301 ARQWADAHPN ITATAQTALA VTEAATTVWG GKKVELNPAK WDWVKNTGYK
351 KPAARHMQTV DGEMAGGNKP LESKNTVTTN NFFENTGYTE KVLRQASNGD
401 YHGFPQSVDA FSENGTVIQI VGGDNIVRHK LYIPGSYKGK DGNFEYIREA
451 DGKINHRLFV PNQQLPEK*

In a second experiment, the following DNA sequence <SEQ ID 171> was identified:

1 atgAATTTGC CTATTCAAAA ATTCATGATG ctgttggcAg cggcaatatc
51 gatgctGCat ATCCCCATTA GTCATGCGAA CGGTTTGGAT GCCCGTTTGC
101 GCGATGATAT GCAGGCAAAA CACTACGAAC CGGGTGGCAA ATACCATCTG
151 TTTGGTAATG CTCGCGGCAG TGTTAAAAAT CGGGTTTGCG CCGTCCAAAC
201 ATTTGATGCA ACTGCGGTCG GCCCCATACT GCCTATTACA CACGAACGGA
251 CAGGATTTGA AGGTGTTATC GGCTATGAAA CCCATTTTTC AGGACACGGA
301 CACGAAGTAC ACAGTCCGTT CGATAATCAT GATTCAAAAA GCACTTCTGA
351 TTTCAGCGGC GGCGTAGACG GCGGTTTTAC CGTTTACCAA CTTCATCGGA
401 CAGGGTCGGA AATACATCCC GCAGACGGAT ATGACGGGCC TCAAGGCGGC
451 GGTTATCCGG AACCACAAGG GGCAAGGGAT ATATACAGCT ACCATATCAA
501 AGGAACTTCA ACCAAAACAA AGATAAACAC TGTTCCGCAA GCCCCTTTTT
551 CAGACCGCTG GCTAAAAGAA AATGCCGGTG CCGCTTCCGG TTTTCTCAGC
601 CGTGCGGATG AAGCAGGAAA ACTGATATGG GAAAACGACC CCGATAAAAA
651 TTGGCGGGCT AACCGTATGG ATGATATTCG CGGCATCGTC CAAGGTGCGG
701 TTAATCCTTT TTTAACGGGT TTTCAAGGGG TAGGGATTGG GGCAATTACA
751 GACAGTGCGG TAAGCCCGGT CACAGATACA GCCGCTCAGC AGACTCTACA
801 AGGTATTAAT GATTTAGGAA ATTTAAGTCC GGAAGCACAA CTTGCCGCCG
851 CGAGCCTATT ACAGGACAGT GCCTTTGCGG TAAAAGACGG CATCAATTCC
901 GCCAGACAAT GGGCTGATGC CCATCCGAAT ATAACAGCAA CAGCCCAAAC
951 TGCCCTTGCC GTAGCAGAGG CCGCAGGTAC GGTTTGGCGC GGTAAAAAAG
1001 TAGAACTTAA CCCGACCAAA TGGGATTGGG TTAAAAATAC CGGCTATAAA
1051 AAACCTGCTG CCCGCCATAT GCAGACTGTA GATGGGGAGA TGGCAGGGGG
1101 GAATAGACCG CCTAAATCTA TAACGTCGGA AGGAAAAGCT AATGCTGCAA
1151 CCTATCCTAA GTTGGTTAAT CAGCTAAATG AGCAAAACTT AAATAACATT
1201 GCGGCTCAAG ATCCAAGATT GAGTCTAGCT ATTCATGAGG GTAAAAAAAA
1251 TTTTCCAATA GGAACTGCAA CTTATGAAGA GGCAGATAGA CTAGGTAAAA
1301 TTTGGGTTGG TGAGGGTGCA AGACAAACTA GTGGAGGCGG ATGGTTAAGT
1351 AGAGATGGCA CTCGACAATA TCGGCCACCA ACAGAAAAAA AATCACAATT
1401 TGCAACTACA GGTATTCAAG CAAATTTTGA AACTTATACT ATTGATTCAA
1451 ATGAAAAAAG AAATAAAATT AAAAATGGAC ATTTAAATAT TAGGTAA

This encodes a protein having amino acid sequence <SEQ ID 172; ORF29ng-1>:

1 MNLPIQKFMM LLAAAISMLH IPISHANGLD ARLRDDMQAK HYEPGGKYHL
51 FGNARGSVKN RVCAVQTFDA TAVGPILPIT HERTGFEGVI GYETHFSGHG
101 HEVHSPFDNH DSKSTSDFSG GVDGGFTVYQ LHRTGSEIHP ADGYDGPQGG
151 GYPEPQGARD IYSYHIKGTS TKTKINTVPQ APFSDRWLKE NAGAASGFLS
201 RADEAGKLIW ENDPDKNWRA NRMDDIRGIV QGAVNPFLTG FQGVGIGAIT
251 DSAVSPVTDT AAQQTLQGIN DLGNLSPEAQ LAAASLLQDS AFAVKDGINS
301 ARQWADAHPN ITATAQTALA VAEAAGTVWR GKKVELNPTK WDWVKNTGYK
351 KPAARHMQTV DGEMAGGNRP PKSITSEGKA NAATYPKLVN QLNEQNLNNI
401 AAQDPRLSLA IHEGKKNFPI GTATYEEADR LGKIWVGEGA RQTSGGGWLS
451 RDGTRQYRPP TEKKSQFATT GIQANFETYT IDSNEKRNKI KNGHLNIR*

ORF29ng-1 and ORF29-1 show 86.0% identity in 401 aa overlap:

Based on this analysis, including the presence of a putative leader sequence in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 21

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 173>:

1 ATGAAAAAAC AAATCACCGC AGCCGTAATG ATGCTGTCTA TGATTGCCCC
51 CGCAATGGCA AACGGCTTGG ACAATCAGGC ATTTGAAGAC CAAATGTTCC
101 ACACGCGGGC AGATGCACCG ATGCAG...

This corresponds to the amino acid sequence <SEQ ID 174; ORF30>:

1 MKKQITAAVM MLSMIAPAMA NGLDNQAFED QMFHTRADAP MQ..

Further work revealed the complete nucleotide sequence <SEQ ID 175>:

1 ATGAAAAAAC AAATCACCGC AGCCGTAATG ATGCTGTCTA TGATTGCCCC
51 CGCAATGGCA AACGGCTTGG ACAATCAGGC ATTTGAAGAC CAAGTGTTCC
101 ACACGCGGGC AGATGCACCG ATGCAGTTGG CGGAGCTTTC TCAAAAGGAG
151 ATGAAGGAGA CAGAGGGGGC GTTTCTTCCA TTGGCTATCT TGGGTGGTGC
201 TGCCATTGGT ATGTGGACAC AGCATGGTTT TAGTTATGCA ACGACAGGCA
251 GACCAGCTTC TGTTAGAGAT GTTGCTATTG CTGGCGGATT AGGCGCAATT
301 CCTGGTGGTG TAGGCGCCGC AGGAAAGGTT GTTTCCTTTG CTAAATATGG
351 ACGTGAGATT AAAATCGGCA ATAATATGCG GATAGCCCCT TTCGGTAATA
401 GAACAGGTCA TCCTATTGGA AAATTTCCCC ATTATCATCG TCGAGTTACG
451 GATAATACGG GCAAGACTTT GCCTGGACAG GGAATTGGTC GTCATCGCCC
501 TTGGGAATCA AAATCTACGG ACAGATCATG GAAAAACCGC TTCTAA

This corresponds to the amino acid sequence <SEQ ID 176; ORF30-1>:

1 MKKQITAAVM MLSMIAPAMA NGLDNQAFED QVFHTRADAP MQLAELSQKE
51 MKETEGAFLP LAILGGAAIG MWTQHGFSYA TTGRPASVRD VAIAGGLGAI
101 PGGVGAAGKV VSFAKYGREI KIGNNMRIAP FGNRTGHPIG KFPHYHRRVT
151 DNTGKTLPGQ GIGRHRPWES KSTDRSWKNR F*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF30 shows 97.6% identity over a 42aa overlap with an ORF (ORF30a) from strain A of N. meningitidis:

The complete length ORF30a nucleotide sequence <SEQ ID 177> is:

1 ATGAAAAAAC AAATCACCGC AGCCGTAATG ATGCTGTCTA TGATTGCCCC
51 CGCAATGGCA AACGGCTTGG ACAATCAGGC ATTTGAAGAC CAAGTGTTCC
101 ACACGCGGGC AGATGCACCG ATGCAGTTGG CGGAGCTTTC TCAAAAGGAG
151 ATGAAGGANA CAGNGGGGGC GTTTCTTCCA TTGGNTATCT TGGGTGGTGC
201 TGCCATTGGT ATGTGGACAC AGCATGGTTT TAGTTATGCA ACGACAGGCA
251 GACCAGCTTC TGTTAGAGAT GTTGCTATTG CTGGCGGATT AGGCGCAATT
301 CCTGGTGNTG TAGGCGCCGC AGGAAAGGTT GTTTCCTTTG CTAAATATGG
351 ACGTGAGATT AAAATCGGCA ATAATATGCG GATAGCCCCT TTCGGTAATA
401 GAACAGGTCA TCCTATTGGN AAATTTCCCC ATTATCATCG TCGAGTTACG
451 GATAATACGG GCAAGACTTT GCCTGGACAG GGAATTGGTC GTCATCGCCC
501 TTGGGAATCA AAATCTACGG ACAGATCATG GAAAAACCGC TTCTAA

This encodes a protein having amino acid sequence <SEQ ID 178>:

1 MKKQITAAVM MLSMIAPAMA NGLDNQAFED QVFHTRADAP MQLAELSQKE
51 MKXTXGAFLP LXILGGAAIG MWTQHGFSYA TTGRPASVRD VAIAGGLGAI
101 PGXVGAAGKV VSFAKYGREI KIGNNMRIAP FGNRTGHPIG KFPHYHRRVT
151 DNTGKTLPGQ GIGRHRPWES KSTDRSWKNR F*

ORF30a and ORF30-1 show 97.8% identity in 181 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF30 shows 97.6% identity over a 42aa overlap with a predicted ORF (ORF30.ng) from N. gonorrhoeae:

The complete length ORF30ng nucleotide sequence <SEQ ID 179> is

1 ATGAAAAAAC AAATCACCGC AGCCGTAATG ATGCTGTCTA TGATCGCCCC
51 CGCAATGGCA AACGGATTGG ACAATCAGGC ATTTGAAGAC CAAGTGTTCC
101 ACACGCGGGC AGATGCGCCG ATGCAGTTGG CGGAGCTTTC TCAGAAGGAG
151 ATGAAGGAGA CTGAAGGGGC TTTTCTTCCA TTGGCTATCT TGGGTGGTGC
201 TGCCATTGGT ATGTGGACAC AGCATGGTTT TAGTTATGCA ACGACAGGCA
251 GACCAGCTTC TGTTAGAGAT GTTGCTGGCG GATTAGGCGC AATTCCTGGT
301 GATGTAGGTG CTGCAGGAAA GGTTGTTTCC TTTGCTAAAT ATGGACGTGA
351 GATTAAAATC GGCAATAATA TGCGGATAGC CCCTTTCGGT AATAGAACAG
401 GTCATCCTAT TGGAAAATTT CCCCATTATC ATCGTCGAGT TACGGATAAT
451 ACGGGCAAGA CTTTGCCTGG ACAGGGAATT GGTCGTCATC GCCCTTGGGA
501 ATCAAAATCT ACGGACAGAT CATGGAAAAA CCGCTTCTAA

This encodes a protein having amino acid sequence <SEQ ID 180>:

1 MKKQITAAVM MLSMIAPAMA NGLDNQAFED QVFHTRADAP MQLAELSQKE
51 MKETEGAFLP LAILGGAAIG MWTQHGFSYA TTGRPASVRD VAGGLGAIPG
101 DVGAAGKVVS FAKYGREIKI GNNMRIAPFG NRTGHPIGKF PHYHRRVTDN
151 TGKTLPGQGI GRHRPWESKS TDRSWKNRF*

ORF30ng and ORF30-1 show 98.3% identity in 181 aa overlap:

Based on this analysis, including the presence of a putative leader sequence in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 22

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 181>:

1 ATGAATAAAA CTCTCTATCG TGTAATTTTC AACCGCAAAC GTGGGGCTGT
51 GrTAGCCGTT GCTGAAACTA CCAAGCGCGA AGGTAAAAGC TGTGCCGATA
101 GTGATTCAGG CAGCGCTCAT GTGAAATCTG TTCCTTTTGG TACTACTCAT
151 GCACCTGTTT GTg.CGTTaC AAATATCTTT TCTTTTTCTT TATTGGGCTT
201 TTCTTTATGT TTGGCTGTAG GtacGGyCAA TATTGCTTTT GCTGATGGCA
251 TT..

This corresponds to the amino acid sequence <SEQ ID 182; ORF31>:

1 MNKTLYRVIF NRKRGAVXAV AETTKREGKS CADSDSGSAH VKSVPFGTTH
51 APVCXVTNIF SFSLLGFSLC LAVGTXNIAF ADGI..

Further work revealed a further partial nucleotide sequence <SEQ ID 183>:

1 ATGAATAAAA CTCTCTATCG TGTAATTTTC AACCGCAAAC GTGGGGCTGT
51 GGTAGCCGTT GCTGAAACTA CCAAGCGCGA AGGTAAAAGC TGTGCCGATA
101 GTGATTCAGG CAGCGCTCAT GTGAAATCTG TTCCTTTTGG TACTACTCAT
151 GCACCTGTTT GTCGTTCAAA TATCTTTTCT TTTTCTTTAT TGGGCTTTTC
201 TTTATGTTTG GCTGTAGGTA CGGCCAATAT TGCTTTTGCT GATGGCATT..

This corresponds to the amino acid sequence <SEQ ID 184; ORF31-1>:

• • 1 MNKTLYRVIF NRKRGAVVAV AETTKREGKS CADSDSGSAH VKSVPFGTTH
  • 51 APVCRSNIFS FSLLGFSLCL AVGTANIAFA DGI . . . .

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. gonorrhoeae

ORF31 shows 76.2% identity over a 84aa overlap with a predicted ORF (ORF31.ng) from N. gonorrhoeae:

The complete length ORF31ng nucleotide sequence <SEQ ID 185> is:

1 ATGAACAAAA CCCTCTATCG TGTGATTTTC AACCGCAAAC GCGGTGCTGT
51 GGTAGCTGTT GCCGAAACCA CCAAGCGCGA AGGTAAAAGC TGTGCCGATA
101 GTGGTTCGGG CAGCGTTTAT GTGAAATCCG TTTCTTTCAT TCCTACTCAT
151 TCCAAAGCCT TTTGTTTTTC TGCATTAGGC TTTTCTTTAT GTTTGGCTTT
201 GGGTACGGTC AATATTGCTT TTGCTGACGG CATTATTACT GATAAAGCTG
251 CTCCTAAAAC CCAACAAGCC ACGATTCTGC AAACAGGTaa cGGCATACCG
301 CAAGTCAATA TTCAAACCCC TACTTCGGCA GGGGTTTCTG TTAATCAATA
351 TGCCCAGTTT GATGTGGGTA ATCGCGGGGC GATTTTAAAC AACAGTCGCA
401 GCAACACCCA AACACAGCTA GGCGGTTGGA TTCAAGGCAA TCCTTGGTTG
451 ACAAGGGGCG AAGCACGTGT GGTTGTAAAC CAAATCAACA GCAGCCATCC
501 TTCACAACTG AATGGCTATA TTGAAGTGGG TGGACGACGT GCAGAAGTCG
551 TTATTGCCAA TCCGGCAGGG ATTGCAGTCA ATGGTGGTGG TTTTATCAAT
601 GCTTCCCGTG CCACTTTGAC GACAGGCCAA CCGCAATATC AAGCAGGAGA
651 CTTTAGCGGC TTTAAGATAA GGCAAGGCAA TGCTGTAATC GCCGGACACG
701 GTTTGGATGC CCGTGATACC GATTTCACAC GTATTCTTGT ATGCCAACAA
751 AATCACCTTG ATCAGTACGG CCGAACAAGC AGGCATTCGT AA

This encodes a protein having amino acid sequence <SEQ ID 186>:

1   MNKTLYRVIF NRKRGAVVAV AETTKREGKS CADSGSGSVY
    VKSVSFIPTH
51  SKAFCFSALG FSLCLALGTV NIAFADGIIT DKAAPKTQQA
    TILQTGNGIP
101 QVNIQTPTSA GVSVNQYAQF DVGNRGAILN NSRSNTQTQL
    GGWIQGNPWL
151 TRGEARVVVN QINSSHPSQL NGYIEVGGRR AEVVIANPAG
    IAVNGGGFIN
201 ASRATLTTGQ PQYQAGDFSG FKIRQGNAVI AGHGLDARDT
    DFTRILVCQQ
251 NHLDQYGRTS RHS*

This gonococcal protein shares 50% identity over a 149aa overlap with the pore-forming hemolysins-like HecA protein from Erwinia chrysanthemi (accession number L39897):

orf31ng	96	GNGIPQVNIQTPTSAGVSVNQYAQFDVGNRGAILNNSRSN-TQTQLGGWIQGNPWLTRGE	154
		GNG+P VNI TP ++G+S N+Y  F+V NRG ILNN  +  T +QLGG IQ NP L
HecA	45	GNGVPVVNIATPDASGLSHNRYHDFNVDNRGLILNNGTARLTPSQLGGLIQNNPNLNGRA	104
Orf31ng	155	ARVVVNQINSSHPSQLNGYIEVGGRRAEVVIANPAGIAVNGGGFINASRATLTTGQPQYQ	214
		A  ++N++ S + S+L GY+EV G+ A VV+ANP GI  +G GF+N  R TLTTG PQ+
HecA	105	AAAILNEVVSPNRSRLAGYLEVAGQAANVVVANPYGITCSGCGFLNTPRLTLTTGTPQFD	164
Orf31ng	215	-AGDFSGFKIRQGNAVIAGHGLDARDTDF	242
		AG  SG  +R G+ +I G GLDA  +D+
HecA	165	AAGGLSGLDVRGGDILIDGAGLDASRSDY	193

Furthermore, ORF31ng and ORF31-1 show 79.5% identity in 83 aa overlap:

On this basis, including the homology with hemolysins, and also with adhesins, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 23

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 187>:

1   ATGAATACTC CTCCTTTTGT CTGTTGGATT TTTTGCAAGG
    TCATCGACAA
51  TTTCGGCGAC ATCGGCGTTT CGTGGCGGCT CGCCCGTGTT
    TTGCACCGCG
101 AACTCGGTTG GCAGGTGCAT TTGTGGACGG ACGATGTGTC
    CGCCTTGCGT
151 GCGCTTTGCC CTGATTTGCC CGATGTTCCC TGCGTTCATC
    AGGATATTCA
201 TGTCCGCACT TGGCATTCCG ATGCGGCAGA TATTGATACC
    GCG..

This corresponds to the amino acid sequence <SEQ ID 188; ORF32>:

1  MNTPPFVCWI FCKVIDNFGD IGVSWRLARV LHRELGWQVH
   LWTDDVSALR
51 ALCPDLPDVP CVHQDIHVRT WHSDAADIDT A..

Further work revealed the complete nucleotide sequence <SEQ ID 189>:

1    ATGAATACTC CTCCTTTTGT CTGTTGGATT TTTTGCAAGG
     TCATCGACAA
51   TTTCGGCGAC ATCGGCGTTT CGTGGCGGCT CGCCCGTGTT
     TTGCACCGCG
101  AACTCGGTTG GCAGGTGCAT TTGTGGACGG ACGATGTGTC
     CGCCTTGCGT
151  GCGCTTTGCC CTGATTTGCC CGATGTTCCC TGCGTTCATC
     AGGATATTCA
201  TGTCCGCACT TGGCATTCCG ATGCGGCAGA TATTGATACC
     GCGCCTGTTC
251  CCGATGTCGT CATCGAAACT TTTGCCTGCG ACCTGCCCGA
     AAATGTGCTG
301  CACATTATCC GCCGACACAA GCCGCTTTGG CTGAATTGGG
     AATATTTGAG
351  CGCGGAGGAA AGCAATGAAA GGCTGCATCT GATGCCTTCG
     CCGCAGGAGG
401  GTGTTCAAAA ATATTTTTGG TTTATGGGTT TCAGCGAAAA
     AAGCGGCGGG
451  TTGATACGCG AACGTGATTA CTGCGAAGCC GTCCGTTTCG
     ATACTGAAGC
501  CCTGCGAGAG CGGCTGATGC TGCCCGAAAA AAACGCCTCC
     GAATGGCTGC
551  TTTTCGGCTA TCGGAGCGAT GTTTGGGCAA AGTGGCTGGA
     AATGTGGCGA
601  CAGGCAGGCA GCCCGATGAC ACTGTTGCTG GCGGGGACGC
     AAATCATCGA
651  CAGCCTCAAA CAAAGCGGCG TTATTCCGCA AGATGCCCTG
     CAAAACGACG
701  GCGATGTTTT TCAGACGGCA TCCGTCCGCC TCGTCAAAAT
     CCCTTTCGTG
751  CCGCAACAGG ACTTCGACCA ACTGCTGCAC CTTGCCGACT
     GCGCCGTCAT
801  CCGCGGCGAA GACAGTTTCG TGCGCGCCCA GCTTGCGGGC
     AAACCCTTCT
851  TTTGGCACAT CTACCCGCAA GACGAGAATG TCCATCTCGA
     CAAACTCCAC
901  GCCTTTTGGG ATAAGGCACA CGGTTTCTAC ACGCCCGAAA
     CCGTGTCGGC
951  ACACCGCCGT CTTTCGGACG ACCTCAACGG CGGAGAGGCT
     TTATCCGCAA
1001 CACAACGCCT CGAATGTTGG CAAACCCTGC AACAACATCA
     AAACGGCTGG
1051 CGGCAAGGCG CGGAGGATTG GAGCCGTTAT CTTTTCGGGC
     AGCCGTCAGC
1101 TCCTGAAAAA CTCGCTGCCT TTGTTTCAAA GCATCAAAAA
     ATACGCTAG

This corresponds to the amino acid sequence <SEQ ID 190; ORF32-1>:

1   MNTPPFVCWI FCKVIDNFGD IGVSWRLARV LHRELGWQVH
    LWTDDVSALR
51  ALCPDLPDVP CVHQDIHVRT WHSDAADIDT APVPDVVIET
    FACDLPENVL
101 HIIRRHKPLW LNWEYLSAEE SNERLHLMPS PQEGVQKYFW
    FMGFSEKSGG
151 LIRERDYCEA VRFDTEALRE RLMLPEKNAS EWLLFGYRSD
    VWAKWLEMWR
201 QAGSPMTLLL AGTQIIDSLK QSGVIPQDAL QNDGDVFQTA
    SVRLVKIPFV
251 PQQDFDQLLH LADCAVIRGE DSFVRAQLAG KPFFWHIYPQ
    DENVHLDKLH
301 AFWDKAHGFY TPETVSAHRR LSDDLNGGEA LSATQRLECW
    QTLQQHQNGW
351 RQGAEDWSRY LFGQPSAPEK LAAFVSKHQK IR*w

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF32 shows 93.8% identity over a 81 as overlap with an ORF (ORF32a) from strain A of N. meningitidis:

The complete length ORF32a nucleotide sequence <SEQ ID 191> is:

1    ATGAATACTC CTCCTTTTTC TGCTGGANTT TTTTGCAAGG
     TCATCGACAA
51   TTTCGGCGAC ATCGGCGTTT CGTGGCGGCT TGCCCGTGTT
     TTGCACCGCG
101  AACTCGGTTG GCAGGTGCAT TTGTGGACGG ACGATGTGTC
     CGCCTTGCGT
151  GCGCTTTGCC CTGATTTGCC CGATGTTCNC TGCGTTCATC
     AGGATATTCA
201  TGTCCGCACT TGGCATTCCG ATGCGGCAGA TATTGATACC
     GCGCCTGTTC
251  NCGATGTCGT CATCGAAACT TTTGCCTGCG ACCTGCCCGA
     AAATGTGCTG
301  CACATCATCC GCCGACACAA GCCGCTTTGG CTGAANTGGG
     AATATTTGAG
351  CGCGGAGGAN AGCAATGAAA GGCTGCACNT GATGCCTTCG
     CCGCAGGAGA
401  GTGTTCNAAA ATANTTTTGG TTTATGGGTT TCAGCGAANN
     NAGCGGCGGA
451  CTGATACGCG AACGCGATTA CTGCGAAGCC GTCCGTTTCG
     ATAGCGGAGC
501  CTTGCGCAAG AGGCTGATGC TTCCCGAAAA AAACGNCCCC
     GAATGGCTGC
551  TTTTCGGCTA TCGGAGCGAT GTTTGGGCAA AGTGGCTGGA
     AATGTGGCGA
601  CAGGCAGGCA GTCCGTTGAC ACTTTTGCTG GCNGGGGCGC
     ANATTATCGA
651  CAGCCTCAAA CAAAACGGCG TTATTCCGCA AGATGCCCTG
     CAAAACGACG
701  GCGATGTTTT TCAGACGGCA TCCGTCCGCC TCGTCAAAAT
     CCCTTTCGTG
751  CCGCAACAGG ACTTCGACAA ACTGCTGCAC CTTGCCGACT
     GCGCCGTCAT
801  CCGCGGCGAA GACAGTTTCG TGCGCGCCCA GCTTGCGGGC
     AAACCCTTCT
851  TTTGGCACAT CTACCCGCAA GATGAGAATG TCCATCTCGA
     CAAACTCCAC
901  GCCTTTTGGG ATAAGGCACA CGGTTTCTAC ACGCCCGAAA
     CCGCATCGGC
951  ACACCGCCGC CTTTCAGACG ACCTCAACGG CGGAGAGGCT
     TTATCCGCAA
1001 CACAACGCCT CGAATGTTGG CAAATCCTGC AACAACATCA
     AAACGGCTGG
1051 CGGCAAGGCG CGGAGGATTG GAGCCGTTAT CTTTTTGGGC
     AGCCTTCCGC
1101 ATCCGAAAAA CTCGCCGCCT TTGTTTCAAA GCATCAAAAA
     ATACGCTAG

This encodes a protein having amino acid sequence <SEQ ID 192>:

1   MNTPPFSAGX FCKVIDNFGD IGVSWRLARV LHRELGWQVH
    LWTDDVSALR
51  ALCPDLPDVX CVHQDIHVRT WHSDAADIDT APVXDVVIET
    FACDLPENVL
101 HIIRRHKPLW LXWEYLSAEX SNERLHXMPS PQESVXKXFW
    FMGFSEXSGG
151 LIRERDYCEA VRFDSGALRK RLMLPEKNXP EWLLFGYRSD
    VWAKWLEMWR
201 QAGSPLTLLL AGAXIIDSLK QNGVIPQDAL QNDGDVFQTA
    SVRLVKIPFV
251 PQQDFDKLLH LADCAVIRGE DSFVRAQLAG KPFFWHIYPQ
    DENVHLDKLH
301 AFWDKAHGFY TPETASAHRR LSDDLNGGEA LSATQRLECW
    QILQQHQNGW
351 RQGAEDWSRY LFGQPSASEK LAAFVSKHQK IR*

ORF32a and ORF32-1 show 93.2% identity in 382 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF32 shows 95.1% identity over a 82aa overlap with a predicted ORF (ORF32.ng) from N. gonorrhoeae:

An ORF32ng nucleotide sequence <SEQ ID 193> was predicted to encode a protein having amino acid sequence <SEQ ID 194>:

1   MVMNTYAFPV CWIFCKVIDN FGDIGVSWRL ARVLHRELGW
    QVHLWTDDVS
51  ALRALCPDLP DVPFVHQDIH VRTWHSDAAD IDTAPVPDAV
    IETFACDLPE
101 NVLNIIRRHK PLWLNWEYLS AEESNERLHL MPSPQEGVQK
    YFWFMGFSEK
151 SGGLIRERDY REAVRFDTEA LRRRLVLPEK NAPEWLLFGY
    RGDVWAKWLD
201 MWQQAGSLMT LLLAGAQIID SLKQSGVIPQ NALQNEGGVF
    QTASVRLVKI
251 PFVPQQDFDK LLHLADCAVI RGEDSFVRTQ LAGKPFFWHI
    YPQDENVHLD
301 KLHAFWDKAY GFYTPETASV HRLLSDDLNG GEALSATQRL
    ECGVL*

Further sequencing revealed the following DNA sequence <SEQ ID 195>:

1    ATGAATACAT ACGCTTTTCC TGTCTGTTGG ATTTTTTGCA
     AGGTCATCGA
51   CAATTTCGGC GACATCGGCG TTTCGTGGCG GCTCGCCCGT
     GTTTTGCACC
101  GCGAACTCGG TTGGCAGGTG CATTTGTGGA CGGACGACGT
     GTCCGCCTTG
151  CGCGCGCTTT GTCCCGATTT GCCCGATGTT CCCTTCGTTC
     ATCAGGATAT
201  TCATGTCCGC ACTTGGCATT CCGATGCGGC AGACATTGAT
     ACCGCGCCCG
251  TTCCCGATGC CGTTATCGAA ACTTTTGCCT GCGACCTGCC
     CGAAAATGTG
301  CTGAACATCA TCCGCCGACA CAAACCGCTT TGGCTGAATT
     GGGAATATTT
351  GAGCGCGGAG GAAAGCAATG AAAGGCTGCA CCTGATGCCT
     TCGCCGCAGG
401  AGGGCGTTCA AAAATATTTT TGGTTTATGG GTTTCAGCGA
     AAAAAGCGGC
451  GGGTTGATAC GCGAACGCGA TTACCGCGAA GCCGTCCGTT
     TCGATACCGA
501  AGCCCTGCGC CGGCGGCTGG TGCTGCCCGA AAAAAACGCC
     CCCGAATGGC
551  TGCTTTTCGG CTATCGGGGC GATGTTTGGG CAAAGTGGCT
     GGACATGTGG
601  CAACAGGCAG GCAGCCTGAT GACCCTACTG CTGGCGGGGG
     CGCAAATTAT
651  CGACAGCCTC AAACAAAGCG GCGTTATTCC GCAAAACGCC
     CTGCAAAAtg
701  aaggcgGTGT CTTTCagacG gcatccgTcC gccttGTCAA
     AAtcCCGTTC
751  GTGCcGCAAC AGGAcTTCGA CAAATTGCTG CAcctcgcCG
     ACTGCGCCGT
801  GATACGCGGC GAAGACAGTT TCGTGCGTAC CCAGCTTGCC
     GGAAAACCCT
851  TTTTTTGGCA CATCTACCCG CAAGACGAGA ATGTCCATCT
     CGACAAACTC
901  CACGCCTTTT GGGATAAGGC ATACGGCTTC TACACGCCCG
     AAACCGCATC
951  GGTGCACCGC CTCCTTTCGG ACGACCTCAA CGGCGGAGAG
     GCTTTATCCG
1001 CAACACAACG CCTCGAATGT TGGCAAACCC TGCAACAACA
     TCAAAACGGC
1051 TGGCGGCAAG GCGCGGAGGA TTGGAGCCGT TATCTTTTCG
     GGCAGCCTTC
1101 CGCATCCGAA AAACTCGCCG CCTTTGTTTC AAAGCATCAA
     AAAATACGCT
1151 AG

This encodes a protein having amino acid sequence <SEQ ID 196; ORF32ng-1>:

1   MNTYAFPVCW IFCKVIDNFG DIGVSWRLAR VLHRELGWQV
    HLWTDDVSAL
51  RALCPDLPDV PFVHQDIHVR TWHSDAADID TAPVPDAVIE
    TFACDLPENV
101 LNIIRRHKPL WLNWEYLSAE ESNERLHLMP SPQEGVQKYF
    WFMGFSEKSG
151 GLIRERDYRE AVRFDTEALR RRLVLPEKNA PEWLLFGYRG
    DVWAKWLDMW
201 QQAGSLMTLL LAGAQIIDSL KQSGVIPQNA LQNEGGVFQT
    ASVRLVKIPF
251 VPQQDFDKLL HLADCAVIRG EDSFVRTQLA GKPFFWHIYP
    QDENVHLDKL
301 HAFWDKAYGF YTPETASVHR LLSDDLNGGE ALSATQRLEC
    WQTLQQHQNG
351 WRQGAEDWSR YLFGQPSASE KLAAFVSKHQ KIR*

ORF32ng-1 and ORF32-1 show 93.5% identity in 383 aa overlap:

On this basis, including the RGD sequence in the gonococcal protein, characteristic of adhesins, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF32-1 (42 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 7A shows the results of affinity purification of the His-fusion protein, and FIG. 7B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA, giving a positive result. These experiments confirm that ORF32-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 24

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 197>:

1   ..TTGTTCCTGC GTGTNAAAGT GGGGCGTTTT TTCAGCAGTC
    CGGCGACGTG
51  GTTTCGGGNC AAAGACCCTG TAAATCAGGC GGTGTTGCGG
    CTGTATNCGG
101 ACGAGTGGCG GCA.ACTTCG GTACGTTGGA AAATAGNCGC
    AACGTCGCAC
151 AGCCTGTGGC TCTGCACGCT GCTCGGAATG CTGGTGTCGG
    TATTGTTGCT
201 GCTTTTGGTG CGGCAATATA CGTTCAACTG GGAAAGCACG
    CTGTTGAGCA
251 ATGCCGCTTC GGTACGCGCG GTGGAAATGT TGGCATGGCT
    GCCGTCGAAA
301 CTCGGTTTCC CTGTCCCCGA TGCGCGGTCG GTCATCGAAG
    GCCGTCTGAA
351 CGGCAATATT GCCGATGCGC GGGCTTGGTC GGGGCTGCTG
    GTCGNCAGTA
401 TCGCCTGCTA NGGCATCCTG CCGCGCCTG..

This corresponds to the amino acid sequence <SEQ ID 198; ORF33>:

1   ..LFLRVKVGRF FSSPATWFRX KDPVNQAVLR LYXDEWRXTS
    VRWKIXATSH
51  SLWLCTLLGM LVSVLLLLLV RQYTFNWEST LLSNAASVRA
    VEMLAWLPSK
101 LGFPVPDARS VIEGRLNGNI ADARAWSGLL VXSIACXGIL
    PRL..

Further work revealed the complete nucleotide sequence <SEQ ID 199>:

1    ATGTTGAATC CATCCCGAAA ACTGGTTGAG CTGGTCCGTA
     TTTTGGACGA
51   AGGCGGTTTT ATTTTCAGCG GCGATCCCGT ACAGGCGACG
     GAGGCTTTGC
101  GCCGCGTGGA CGGCAGTACG GAGGAAAAAA TCATCCGTCG
     GGCGGAGATG
151  ATTGACAGGA ACCGTATGCT GCGGGAGACG TTGGAACGTG
     TGCGTGCGGG
201  GTCGTTCTGG TTGTGGGTGG TGGCGGCGAC GTTTGCATTT
     TTTACCGGTT
251  TTTCAGTCAC TTATCTTCTA ATGGACAATC AGGGTCTGAA
     TTTCTTTTTG
301  GTTTTGGCGG GCGTGTTGGG CATGAATACG CTGATGCTGG
     CAGTATGGTT
351  GGCAATGTTG TTCCTGCGTG TGAAAGTGGG GCGTTTTTTC
     AGCAGTCCGG
401  CGACGTGGTT TCGGGGCAAA GACCCTGTAA ATCAGGCGGT
     GTTGCGGCTG
451  TATGCGGACG AGTGGCGGCA ACCTTCGGTA CGTTGGAAAA
     TAGGCGCAAC
501  GTCGCACAGC CTGTGGCTCT GCACGCTGCT CGGAATGCTG
     GTGTCGGTAT
551  TGTTGCTGCT TTTGGTGCGG CAATATACGT TCAACTGGGA
     AAGCACGCTG
601  TTGAGCAATG CCGCTTCGGT ACGCGCGGTG GAAATGTTGG
     CATGGCTGCC
651  GTCGAAACTC GGTTTCCCTG TCCCCGATGC GCGGGCGGTC
     ATCGAAGGCC
701  GTCTGAACGG CAATATTGCC GATGCGCGGG CTTGGTCGGG
     GCTGCTGGTC
751  GGCAGTATCG CCTGCTACGG CATCCTGCCG CGCCTGCTGG
     CTTGGGTAGT
801  GTGTAAAATC CTTTTGAAAA CAAGCGAAAA CGGATTGGAT
     TTGGAAAAGC
851  CCTATTATCA GGCGGTCATC CGCCGCTGGC AGAACAAAAT
     CACCGATGCG
901  GATACGCGTC GGGAAACCGT GTCCGCCGTT TCACCGAAAA
     TCATCTTGAA
951  CGATGCGCCG AAATGGGCGG TCATGCTGGA GACCGAGTGG
     CAGGACGGCG
1001 AATGGTTCGA GGGCAGGCTG GCGCAGGAAT GGCTGGATAA
     GGGCGTTGCC
1051 ACCAATCGGG AACAGGTTGC CGCGCTGGAG ACAGAGCTGA
     AGCAGAAACC
1101 GGCGCAACTG CTTATCGGCG TGCGCGCCCA AACTGTGCCG
     GACCGCGGCG
1151 TGTTGCGGCA GATTGTCCGA CTCTCGGAAG CGGCGCAGGG
     CGGCGCGGTG
1201 GTGCAGCTTT TGGCGGAACA GGGGCTTTCA GACGACCTTT
     CGGAAAAGCT
1251 GGAACATTGG CGTAACGCGC TGGCCGAATG CGGCGCGGCG
     TGGCTTGAGC
1301 CTGACAGGGC GGCGCAGGAA GGGCGTTTGA AAGACCAATA
     A

This corresponds to the amino acid sequence <SEQ ID 200; ORF33-1>:

1   MLNPSRKLVE LVRILDEGGF IFSGDPVQAT EALRRVDGST
    EEKIIRRAEM
51  IDRNRMLRET LERVRAGSFW LWVVAATFAF FTGFSVTYLL
    MDNQGLNFFL
101 VLAGVLGMNT LMLAVWLAML FLRVKVGRFF SSPATWFRGK
    DPVNQAVLRL
151 YADEWRQPSV RWKIGATSHS LWLCTLLGML VSVLLLLLVR
    QYTFNWESTL
201 LSNAASVRAV EMLAWLPSKL GFPVPDARAV IEGRLNGNIA
    DARAWSGLLV
251 GSIACYGILP RLLAWVVCKI LLKTSENGLD LEKPYYQAVI
    RRWQNKITDA
301 DTRRETVSAV SPKIILNDAP KWAVMLETEW QDGEWFEGRL
    AQEWLDKGVA
351 TNREQVAALE TELKQKPAQL LIGVRAQTVP DRGVLRQIVR
    LSEAAQGGAV
401 VQLLAEQGLS DDLSEKLEHW RNALAECGAA WLEPDRAAQE
    GRLKDQ*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF33 shows 90.9% identity over a 143aa overlap with an ORF (ORF33a) from strain A of N. meningitidis:

The complete length ORF33a nucleotide sequence <SEQ ID 201> is:

1    ATGTTGAATC CATCCCGAAA ACTGGTTGAG CTGGTCCGTA
     TTTTGGAAGA
51   AGGCGGCTTT ATTTTCAGCG GCGATCCCGT GCAGGCGACG
     GAGGCTTTGC
101  GCCGCGTGGA CGGCAGTACG GAGGAAAAAA TCATCCGTCG
     GGCGAAGATG
151  ATCGACAGGA ACCGTATGCT GCGGGAGACG TTGGAACGTG
     TGCGTGCGGG
201  GTCGTTCTGG TTGTGGGTGG CGGCGGCGAC GTTTGCGTTT
     NTTACCGNTT
251  TTTCAGTTAC TTATCTTCTA ATGGACAATC AGGGTCTGAA
     TTTCTTTTTG
301  GTTTTGGCGG GCGTGNTGGG CATGAATACG CTGATGCTGG
     CAGTATGGTT
351  GGCAATGTTG TTCCTGCGCG TGAAAGTGGG GCGTTTTTTC
     AGCAGTCCGG
401  CGACGTGGTT TCGGGGCAAA GACCCTGTCA ATCAGGCGGT
     GTTGCGGCTG
451  TATGCGGACG AGTGGCGGCN ACCTTCGGTA CGTTGGAAAA
     TAGGCGCAAC
501  GTCGCACAGC CTGTGGCTCT GCACGCTGCT CGGAATGCTG
     GTGTCGGTAT
551  TGTTGCTGCT TTTGGTGCGG CAATATACGT TCAACTGGGA
     AAGCACGCTG
601  TTGGGCGATT CGTCTTCGGT ACGGCTGGTG GAAATGTTGG
     CATGGCTGCC
651  TGCGAAACTG GGTTTTCCCG TGCCTGATGC GCGGGCGGTC
     ATCGAAGGTC
701  GTCTGAACGG CAATATTGCC GATGCGCGGG CTTGGTCGGG
     GCTGCTGGTC
751  GGCAGTATCG CCTGCTACGG CATCCTGCCG CGCCTCTTGG
     CTTGGGCGGT
801  ATGCAAAATC CTTNTGNAAA CAAGCGAAAA CGGCTTGGAT
     TTGGAAAAGC
851  NCNNNNNTCN NNCGNTCATC CGCCGCTGGC AGAACAAAAT
     CACCGATGCG
901  GATACGCGTC GGGAAACCGT GTCCGCCGTT TCGCCGAAAA
     TCGTCTTGAA
951  CGATGCGCCG AAATGGGCGG TCATGCTGGA GACCGAATGG
     CAGGACGGCG
1001 AATGGTTCGA GGGCAGGCTG GCGCAGGAAT GGCTGGATAA
     GGGCGTTGCC
1051 GCCAATCGGG AACAGGTTGC CGCGCTGGAG ACAGAGCTGA
     AGCAGAAACC
1101 GGCGCAACTG CTTATCGGCG TGCGCGCCCA AACTGTGCCC
     GACCGCGGCG
1151 TGTTGCGGCA GATCGTCCGA CTTTCGGAAG CGGCGCAGGG
     CGGCGCGGTG
1201 GTGCANCTTT TGGCGGAACA GGGGCTTTCA GACGACCTTT
     CGGAAAAGCT
1251 GGAACATTGG CGTAACGCGC TGACCGAATG CGGCGCGGCG
     TGGCTGGAAC
1301 CCGACAGAGC GGCGCAGGAA GGCCGTCTGA AAACCAACGA
     CCGCACTTGA

This encodes a protein having amino acid sequence <SEQ ID 202>:

1   MLNPSRKLVE LVRILEEGGF IFSGDPVQAT EALRRVDGST
    EEKIIRRAKM
51  IDRNRMLRET LERVRAGSFW LWVAAATFAF XTXFSVTYLL
    MDNQGLNFFL
101 VLAGVXGMNT LMLAVWLAML FLRVKVGRFF SSPATWFRGK
    DPVNQAVLRL
151 YADEWRXPSV RWKIGATSHS LWLCTLLGML VSVLLLLLVR
    QYTFNWESTL
201 LGDSSSVRLV EMLAWLPAKL GFPVPDARAV IEGRLNGNIA
    DARAWSGLLV
251 GSIACYGILP RLLAWAVCKI LXXTSENGLD LEKXXXXXXI
    RRWQNKITDA
301 DTRRETVSAV SPKIVLNDAP KWAVMLETEW QDGEWFEGRL
    AQEWLDKGVA
351 ANREQVAALE TELKQKPAQL LIGVRAQTVP DRGVLRQIVR
    LSEAAQGGAV
401 VXLLAEQGLS DDLSEKLEHW RNALTECGAA WLEPDRAAQE
    GRLKTNDRT*

ORF33a and ORF33-1 show 94.1% identity in 444 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF33 shows 91.6% identity over a 143aa overlap with a predicted ORF (ORF33.ng) from N. gonorrhoeae:

An ORF33ng nucleotide sequence <SEQ ID 203> was predicted to encode a protein having amino acid sequence <SEQ ID 204>:

1   MIDRDRMLRD TLERVRAGSF WLWVVVASMM FTAGFSGTYL
    LMDNQGLNFF
51  LVLAGVLGMN TLMLAVWLAT LFLRVKVGRF FSSPATWFRG
    KGPVNQAVLR
101 LYADQWRQPS VRWKIGATAH SLWLCTLLGM LVSVLLLLLV
    RQYTFNWEST
151 LLSNAASVRA VEMLAWLPSK LGFPVPDARA VIEGRLNGNI
    ADARAWSGLL
201 VGSIVCYGIL PRLLAWVVCK ILLKTSENGL DLEKTYYQAV
    IRRWQNKITD
251 ADTRRETVSA VSPKIVLNDA PKWALMLETE WQDGQWFEGR
    LAQEWLDKGV
301 AANREQVAAL ETELKQKPAQ LLIGVRAQTV PDRGVLRQIV
    RLSEAAQGGA
351 VVQLLAEQGL SDDLSEKLEH WRNALTECGA AWLEPDRVAQ
    EGRLKDQ*

Further sequence analysis revealed the following DNA sequence <SEQ ID 205>:

1    ATGTTGaatC CATCCCgaAA ACTGgttgag ctGgTCCgtA
     Ttttgaataa
51   agggggtTTT attttcagcg gcgatcctgt gcaggcgacg
     gaggctttgc
101  gccgcgtgga cggcAGTACG GAggAaaaaa tcttccgtcg
     GGCGGAGAtg
151  atcgACAGGg accgtatgtt gcgggACaCg TtggaacGTG
     TGCGTGCggg
201  gtcgtTctgG TTATGGGTGG TggtggCAtC gATGATGTtt
     aCCGCCGGAT
251  TTTCAGgcac ttatCttCTG ATGGACaatC AGGGGCtGAA
     TtTCTTTTTA
301  GTTTTggcgG GAGTGTtggG CATGaatacG ctgATGCTGG
     CAGTATGGtt
351  gGCAACGTTG TTCCTGCGCG TGAAAGTGGG ACGGTTTTTC
     AGCAGTCCGG
401  CGACGTGGTT TCGGGGCAAA GGCCCTGTAA ATCAGGCGGT
     GTTGCGGCTG
451  TATGCGGACC AGTGGCGGCA ACCTTCGGTA CGATGGAAAA
     TAGGCGCAAC
501  GGCGCACAGC TTGTGGCTCT GCACGCTGCT CGGAATGCTG
     GTGTCGGTAT
551  TGCTGCTGCT TTTGGTGCGG CAATATACGT TCAACTGGGA
     AAGCACGCTG
601  TTGAGCAATG CCGCTTCGGT ACGCGCGGTG GAAATGTTGG
     CATGGCTGCC
651  GTCGAAACTC GGTTTCCCTG TCCCCGATGC GCGGGCGGTC
     ATCGAAGGTC
701  GTCTGAACGG CAATATTGCC GATGCGCGGG CTTGGTCGGG
     GCTGCTGGTC
751  GGCAGTATCG TCTGCTACGG CATCCTGCCG CGCCTCTTGG
     CTTGGGTAGT
801  GTGTAAAATC CTTTTGAAAA CAAGCGAAAA CGGattgGAT
     TTGGAAAAAA
851  CCTATTATCA GGCGGTCATC CGCCGCTGGC AGAACAAAAT
     CACCGATGCG
901  GATACGCGTC GGGAAACCGT GTCCGCCGTT TCGCcgaAAA
     TCGTCTTGAA
951  CGATGCGCCG AAATGGGCGC TCATGCTGGA GACCGAGTGG
     CAGGACGGCC
1001 AATGGTTCGA GGGCAGGCTG GCGCAGGAAT GGCTGGATAA
     GGGCGTTGCC
1051 GCCAATCGGG AACAGGTTGC CGCGCTGGAG ACAGAGCTGA
     AGCAGAAACC
1101 GGCGCAACTG CTTATCGGCG TACGCGCCCA AACTGTGCCG
     GACCGGGGCG
1151 TGCTGCGGCA GATTGTGCGG CTTTCGGAAG CGGCGCAGGG
     CGGCGCGGTG
1201 GTGCAGCTTT TGGCGGAACA GGGGCTTTCA GACGACCTTT
     CGGAAAAGCT
1251 GGAACATTGG CGTAACGCGC TGACCGAATG CGGCGCGGCG
     TGGCTTGAGC
1301 CTGACAGGGT GGCGCAGGAA GGCCGTTTGA AAGACCAATA
     A

This encodes a protein having amino acid sequence <SEQ ID 206; ORF33ng-1>:

1   MLNPSRKLVE LVRILNKGGF IFSGDPVQAT EALRRVDGST
    EEKIFRRAEM
51  IDRDRMLRDT LERVRAGSFW LWVVVASMMF TAGFSGTYLL
    MDNQGLNFFL
101 VLAGVLGMNT LMLAVWLATL FLRVKVGRFF SSPATWFRGK
    GPVNQAVLRL
151 YADQWRQPSV RWKIGATAHS LWLCTLLGML VSVLLLLLVR
    QYTFNWESTL
201 LSNAASVRAV EMLAWLPSKL GFPVPDARAV IEGRLNGNIA
    DARAWSGLLV
251 GSIVCYGILP RLLAWVVCKI LLKTSENGLD LEKTYYQAVI
    RRWQNKITDA
301 DTRRETVSAV SPKIVLNDAP KWALMLETEW QDGQWFEGRL
    AQEWLDKGVA
351 ANREQVAALE TELKQKPAQL LIGVRAQTVP DRGVLRQIVR
    LSEAAQGGAV
401 VQLLAEQGLS DDLSEKLEHW RNALTECGAA WLEPDRVAQE
    GRLKDQ*

ORF33ng-1 and ORF33-1 show 94.6% identity in 446 aa overlap:

Based on the presence of several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 25

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 207>:

1   ..CAGAAGAGTT TGTCGAGAAT TTCTTTATGG GGTTTGGGCG
    GCGTGTTTTT
51  CGGGGTGTCC GGTCTGGTAT GGTTTTCTTT GGGCGTTTCT
    TT.GAGTGCG
101 CCTGTTTTTC GGGTGTTTCT TTTCGGGGTT CGGGACGGGG
    GACGTTTGTG
151 GGCAGTACGG GGGTTTCTTT GAGTGTGTTT TCAGCTTGTG
    TTCC.GGCGT
201 CGTCCGGCTG CCTGTCGGTT TGAGCTGTGT CGGCAGGTTG
    CG..GTTTGA
251 CCCGGTTTTT CTTGGGTGCG GCAGGGGACG TCATTCTCCT
    GCCGCTTTCG
301 TCTGTGCCGT CCGGCTGTGC GGGTTCGGAT GAGGCGGCGT
    GGTGGTGTTC
351 GGGTTGGGCG GCATCTTGTT CCGACTACGC CGTTTGGCAG
    CCAGAATTCG
401 GTTTCGCGGG GGCTGTCGGT GTGTTGCGGT TCGGCTTGAA
    GGGTTTTGTC
451 GTCC..

This corresponds to the amino acid sequence <SEQ ID 208; ORF34>:

1   ..QKSLSRISLW GLGGVFFGVS GLVWFSLGVS XECACFSGVS
    FRGSGRGTFV
51  GSTGVSLSVF SACVXGVVRL PVGLSCVGRL XXLTRFFLGA
    AGDVILLPLS
101 SVPSGCAGSD EAAWWCSGWA ASCPTTPFGS QNSVSRGLSV
    CCGSA*RVLS
151 S..

Further work revealed the complete nucleotide sequence <SEQ ID 209>:

1    ATGATGATGC CGTTCATAAT GCTTCCTTGG ATTGCkGGTG
     TGCCTGCCGT
51   GCCGGGTCAG AATAGGTTGT CCAGAATTTC TTTATGGGGT
     TTGGGCGGCG
101  TGTTTTTCGG GGTGTCCGGT TTGGTATGGT TTTCTTTGGG
     CGTTTCTTTG
151  GGCTGCGCCT GTTTTTCGGG TGTTTCTTTT CGGGGTTCGG
     GACGGGGGAC
201  GTTTGTGGGC AGTACGGGGG TTTCTTTGAG TGTGTTTTCA
     GCTTGTGTTC
251  CGGCGTCGTC CGGCTGCCTG TCGGTTTGAG CTGTGTCGGC
     AGGTTGCGGT
301  TTGACCCGGT TTTTCTTGGG TGCGGCAGGG GACGGCAGTC
     CGCTGCCGCT
351  TTCGTCTGTG CCGTCCGGCT GTGCGGGTTC GGATGAGGCG
     GCGTGGTGGT
401  GTTCGGGTTG GGCGGCATCT TGTCCGACTA CGCCGTTTGG
     CAGCCAGAAT
451  TCGGTTTCGC GGGGGCTGTC GGTGTGTTGC GGTTCGGCTT
     GAAGGGTTTT
501  GTCGCCGTTC GGGTTGAATG TGCTGACGAT GCCTATTGCC
     AATGCGCCGA
551  TGGCGGCGAT ACAGATGAGC AATACGGCGC GTATCAGGAG
     TTTGGGGGTC
601  AGCCTGAAGG GTTTGTTCGG TTTTTTTGCC ATTTTGATTG
     TGCTTTTGGG
651  GTGTCGGGCA ATGCCGTCTG AAGGCGGTTC AGACGGCATT
     GCCGAGTCAG
701  CGTTGGACGT AGTTTTGGTA GAGGGTGATG ACTTTTTGTA
     CGCCGACGGT
751  GGTGCTGACT TTTTGGGTAA TCTGCGCCTG TTCTTCGGGG
     GTGAGGATGC
801  CCATAACGTA GGTTACGTTG CCGTAGGTAA CGATTTTGAC
     GCGCGCCTGT
851  GTGGCGGGGC TGATGCCCAA CAGCGTGGCG CGGACTTTGG
     ATGTGTTCCA
901  AGTGTCGCCG GCGATGTCGC CGGCAGTGCG CGGCAGGGAG
     GCGACGGTAA
951  TATAGTTGTA CACGCCTTCG GCGGCCTGTT CGGAACGTGC
     AATCTGACCG
1001 ACGAACTGTT TTTCGCCTTC GGTGGCGACT TGTCCGAGCA
     GCAGCAGGTG
1051 GCGGTTGTAG CCGACGACGG AGATTTGGGG CGTGTAGCCT
     TTGGTTTGGT
1101 TGTTTTGGCG CAGATAGGAA CGGGCGGTGG TTTCGATACG
     CAACGCCATA
1151 ACGTTGTCGT CGGTTTGCGC GCCGGTGGTT CGGCGGTCGA
     CGGCGGATTT
1201 CGCGCCGACG GCGGCGCTTC CGATTACTGC GCTGACGCAG
     CCGCTAAGGG
1251 CAAGGCTGAA AATGGCGGCA ATCAGGGTGC GGACGGTGTG
     CGGTTTGGGT
1301 TTCATCGGGT GCTTCCTTTC TTGGGCGTTT CAGACGGCAT
     TGCTTTGCGC
1351 CATGCCGTCT GA

This corresponds to the amino acid sequence <SEQ ID 210; ORF34-1>:

1   MMMPFIMLPW IAGVPAVPGQ NRLSRISLWG LGGVFFGVSG
    LVWFSLGVSL
51  GCACFSGVSF RGSGRGTFVG STGVSLSVFS ACVPASSGCL
    SV*AVSAGCG
101 LTRFFLGAAG DGSPLPLSSV PSGCAGSDEA AWWCSGWAAS
    CPTTPFGSQN
151 SVSRGLSVCC GSA*RVLSPF GLNVLTMPIA NAPMAAIQMS
    NTARIRSLGV
201 SLKGLFGFFA ILIVLLGCRA MPSEGGSDGI AESALDVVLV
    EGDDFLYADG
251 GADFLGNLRL FFGGEDAHNV GYVAVGNDFD ARLCGGADAQ
    QRGADFGCVP
301 SVAGDVAGSA RQGGDGNIVV HAFGGLFGTC NLTDELFFAF
    GGDLSEQQQV
351 AVVADDGDLG RVAFGLVVLA QIGTGGGFDT QRHNVVVGLR
    AGGSAVDGGF
401 RADGGASDYC ADAAAKGKAE NGGNQGADGV RFGFHRVLPF
    LGVSDGIALR
451 HAV*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF34 shows 73.3% identity over a 161aa overlap with an ORF (ORF34a) from strain A of N. meningitidis:

The complete length ORF34a nucleotide sequence <SEQ ID 211> is:

1    ATGATGATNC CGTTNATAAT GCTTCCTTGG ATTGCGGGTG
     TGCCTGCCGT
51   GCCGGGTCAG AAGAGGTTGT CGAGAANTTC TTTATGGGGT
     TTAGGCGGCN
101  TGTTTTTCGG GGTGTCCGGT TTGGTATGGT TTTCTTTGGG
     CGTTTCTNTT
151  TCTTTGGGTG TTTCTNTGGG CTGTGCCTGT TTTTCGGGTG
     TTTCTTTTCG
201  GGGTTCGGGA CGGGGGACGT TTGTGGGCAG TACNGGGGTT
     TCTTTGAGTG
251  TGTTTTCAGC TTGTGCTCCG GCGTCGTCCG GCTGCCTGTC
     GGTTTNAGCT
301  GTGTCGGCAG GTTGCGGTTT GACCCGGNTT TTCTTNGGTG
     CGGCAGGGGA
351  CGGCAGTCCG CTGCCGCTTT CGTCTGTGCC GTCCGGCTGT
     GCGGGTGCGG
401  ATGAGGAGGC GTNGTNGTGT TCGGGTTGGG CGGCATCTTG
     TCCGACTACG
451  CCGTTTGGCA GCCAGAATTC GGTTTCGCGG GGGCTGTCGG
     TGTGTTGCGG
501  TTCGGTNTGG AGGGTTTTGT CNCCGTTCGG GTNGAATGTG
     CTGACGATGC
551  CTATTGCCAA TGCGCCGATG GCGGTGATAC AGATGAGCAA
     TACGGCGCGT
601  ATCAGGAGTT TGGGGGTCAG CCTGAAGGGT TTGTTCNGTT
     TTTTTGCCAT
651  TTTGATTGTG CTTTTGGGGT GTCGGGCAAT GCCGTCTGAA
     GGCGGTTCAG
701  ACGGCATTGC CGAGTCAGCG TTGGACGTAG TTTNGGTAGA
     GGGTGATGAC
751  TTTTTGTACG CCGACGGTGG TGCTGACTTT TTGGGTAATC
     TGCGCCTGTT
801  CTTCGGGGGT GAGGATGCCC ATAACGTAGG TTACGTTGCC
     GTAGGTAACG
851  ATTTTGACGC GCGCCTGTGT GGCGGGGCTG ATGCCCAACA
     GCGTGGCGCG
901  GACTTTGGAT GTGTTCCAAG TGTCGCCGGC GATGTCGCCG
     GCAGTGCGCG
951  GCAGGGAGGC GACGGTAATG TANTTGTACA CGCCTTCGGC
     GGCCTGTTCG
1001 GAACGTGCAA TCTGACCGAC GAACTGTTTC TCGCCTTCGG
     TGGCGACTTG
1051 TCCGAGCAGC AGCAGGTGGC GGTTGTAGCC GACAACGGAG
     ATTTGGGGCG
1101 TGTANCCTTT GGTTTGGTTG TTTTGGCGCA GATAGGAGCG
     GGCGGTGGTT
1151 TCGATACGCA GCGCCATTAC GTTGTCGTCG GTTNGCGCGC
     CGGTGGTTCG
1201 GCGGTCGACG GCGGATTTCG CGCCGACCGC CGCGCCGCCG
     ACGACTGCGC
1251 TGACGCAGCC GCCGAGGGCA AGGCTGAGGA CGGCGGCAGT
     CAGGGTGCGG
1301 ACGGTGTGCG GTTTGGGTTT CATCGGGTGC TTCCTTTCTT
     GGGCGTTTCA
1351 GACGGCATTG CTTTGCGCCA TGCCGTCTGA

This encodes a protein having amino acid sequence <SEQ ID 212>:

1   MMXPXIMLPW IAGVPAVPGQ KRLSRXSLWG LGGXFFGVSG
    LVWFSLGVSX
51  SLGVSXGCAC FSGVSFRGSG RGTFVGSTGV SLSVFSACAP
    ASSGCLSVXA
101 VSAGCGLTRX FXGAAGDGSP LPLSSVPSGC AGADEEAXXC
    SGWAASCPTT
151 PFGSQNSVSR GLSVCCGSVW RVLSPFGXNV LTMPIANAPM
    AVIQMSNTAR
201 IRSLGVSLKG LFXFFAILIV LLGCRAMPSE GGSDGIAESA
    LDVVXVEGDD
251 FLYADGGADF LGNLRLFFGG EDAHNVGYVA VGNDFDARLC
    GGADAQQRGA
301 DFGCVPSVAG DVAGSARQGG DGNVXVHAFG GLFGTCNLTD
    ELFLAFGGDL
351 SEQQQVAVVA DNGDLGRVXF GLVVLAQIGA GGGFDTQRHY
    VVVGXRAGGS
401 AVDGGFRADR RAADDCADAA AEGKAEDGGS QGADGVRFGF
    HRVLPFLGVS
451 DGIALRHAV*

ORF34a and ORF34-1 show 91.3% identity in 459 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF34 shows 77.6% identity over a 161 as overlap with a predicted ORF (ORF34.ng) from N. gonorrhoeae:

The complete length ORF34ng nucleotide sequence <SEQ ID 213> is:

1    ATGATGATGC CGTTCATAAT GCTTCCTTGG ATTGCGGGTG
     TGCCTGCCGT
51   GCCGGGTCAA AAGAGGTTGT CGAGAATCTC TTTATGGGGT
     TTGGCCGGCG
101  TGTTTTTCGG GGTGTCCGGT TTGGTATGGT TTTCTTTGGG
     CGTTTCTTTT
151  TCTTTGGGTG TTTCTTTGGG CTGCGCCTGT TTTTCGGGTG
     TTTCTTTTCG
201  GGGTTCGGGA TGGGGGGCGT TTGTGGGCAG TACGGGGGTT
     TCTTTGAGTG
251  TGTTTTCAGC TTGTGTTCCG GTGCCGGTTA ACGAATCGGC
     TGCCCGGGCC
301  GCATCCGAAG GGCGCGGTTT gACCCGGTTT TTCTTGGGTG
     CGGCAGGGGA
351  CGGCAGTCCG CTGCCGCTTT CTTCTGTGCC GTCCGGCTGT
     GCGGGTTCGG
401  ATGAGGCGGC GTGGTGGTGT TCGGGTTGGG CGGCATCTTG
     TCCGACGGCG
451  CCGTTTGGCA GCCAGAATTC GGTTTCGCGG GGGCTGTCGG
     TGTGTTGCGG
501  TTCGGTTTGG AGGGTTTTGT CGCCGTTCGG GTTGAATGTG
     CTGACGATGC
551  CTACTGCCAA TGCGCCGATG GCGGTGATAC AGATGAGCAA
     TACGGCGCGT
601  ATCAGGAGTT TGGGGGTCAG CCTGAAGGGT TTGTTCGGTT
     TTTTTGCCAT
651  TTTGATTGTG CTTTTGGGGT GTCGGGCAAT GCCGTCTGAA
     GGCGGTTCAG
701  ACGGCATTGC CGAGTCAGCG TTGGACGTAG TTTTGGTAGA
     GGGTAATGAC
751  TTTTTGTACG CCGAcggTGG TGCTGACTTT TTGGGTAATC
     TGCGCCTGTT
801  CTTCGGGGGT GAGGATGCCC ATAACGTAGG TTACATTGCC
     GTAGGTAATG
851  ATTTTGACGC GCGCCTGTGT AGCGGGGCTG ATGCCCAGCA
     GcgtgGCGCG
901  GACTTTGGAC GTGTTCCAAG TGTCGCCGGC GATGTCGCCC
     GCAGTGCGCG
951  GCAGGGAGGC GACGGTAATG TAGTTGTATA CGCCTTCGGC
     GGCCTGTTCG
1001 GAACGTGCAA TCTGACCGAC GAACTGTTTT TCGCCTTCGG
     TGGCGACTTG
1051 TCCGAGCAGC AGCAGGTGGC GGTTGTAGCC GACGACGGAG
     ATTTGGGGCG
1101 TGTAGCCTTT GGTTTGGTTG TTTTGGCGCA GGTAGGAACG
     GGCGGTGGTT
1151 TCGATACGCA ACGCCATAAC GTtgtCATCG GTTtgcgcgc
     CGGTGGTTcg
1201 gCGGTCGATG ACGGATTTTG CGCCGACGGC GGCCCCGCCG
     ACGACTGCGC
1251 TGAAGCAGCC GCCGAGGGCA AGGCTGAGGA CGGCGGCAAT
     CAGGGTGCGG
1301 ACGGTGTGTG GTTTGGGTTT CATCGGGGAC TTCCTTTCTT
     GGGCGTTTCA
1351 GACGGCATTG CTTTGCGCCA TGCCGTCTGA

This encodes a protein having amino acid sequence <SEQ ID 214>:

1   MMMPFIMLPW IAGVPAVPGQ KRLSRISLWG LAGVFFGVSG
    LVWFSLGVSF
51  SLGVSLGCAC FSGVSFRGSG WGAFVGSTGV SLSVFSACVP
    VPVNESAARA
101 ASEGRGLTRF FLGAAGDGSP LPLSSVPSGC AGSDEAAWWC
    SGWAASCPTA
151 PFGSQNSVSR GLSVCCGSVW RVLSPFGLNV LTMPTANAPM
    AVIQMSNTAR
201 IRSLGVSLKG LFGFFAILIV LLGCRAMPSE GGSDGIAESA
    LDVVLVEGND
251 FLYADGGADF LGNLRLFFGG EDAHNVGYIA VGNDFDARLC
    SGADAQQRGA
301 DFGRVPSVAG DVARSARQGG DGNVVVYAFG GLFGTCNLTD
    ELFFAFGGDL
351 SEQQQVAVVA DDGDLGRVAF GLVVLAQVGT GGGFDTQRHN
    VVIGLRAGGS
401 AVDDGFCADG GPADDCAEAA AEGKAEDGGN QGADGVWFGF
    HRGLPFLGVS
451 DGIALRHAV*

ORF34ng and ORF34-1 show 90.0% identity in 459 aa overlap:

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 26

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 215>:

1   ATGAAAACCT TCTTCAAAAC CCTTTCCGCC GCCGCACTCG
    CGCTCATCCT
51  CGCCGCCTGC GGATT.CAAA AAGACAGCGC GCCCGCCGCA
    TCCGCTTCTG
101 CCGCCGCCGA CAACGGCGCG GCGTAAAAAA GAAATCGTCT
    TCGGCACGAC
151 CGTCGGCGAC TTCGGCGATA TGGTCAAAGA ACAAATCCAA
    GCCGAGCTGG
201 AGAAAAAAGG CTACACCGTC AAACTGGTCG AGTTTACCGA
    CTATGTACGC
251 CCGAATCTGG CATTGGCTGA GGGCGAGTTG

This corresponds to the amino acid sequence <SEQ ID 216; ORF4>:

1  MKTFFKTLSA AALALILAAC G.QKDSAPAA SASAAADNGA
   AKKEIVFGTT
51 VGDFGDMVKE QIQAELEKKG YTVKLVEFTD YVRPNLALAE
   GEL

Further sequence analysis revealed the complete nucleotide sequence <SEQ ID 217>:

1   ATGAAAACCT TCTTCAAAAC CCTTTCCGCC GCCGCACTCG
    CGCTCATCCT
51  CGCCGCCTGC GGCGGTCAAA AAGACAGCGC GCCCGCCGCA
    TCCGCTTCTG
101 CCGCCGCCGA CAACGGCGCG GCGAAAAAAG AAATCGTCTT
    CGGCACGACC
151 GTCGGCGACT TCGGCGATAT GGTCAAAGAA CAAATCCAAG
    CCGAGCTGGA
201 GAAAAAAGGC TACACCGTCA AACTGGTCGA GTTTACCGAC
    TATGTACGCC
251 CGAATCTGGC ATTGGCTGAG GGCGAGTTGG ACATCAACGT
    CTTCCAACAC
301 AAACCCTATC TTGACGACTT CAAAAAAGAA CACAATCTGG
    ACATCACCGA
351 AGTCTTCCAA GTGCCGACCG CGCCTTTGGG ACTGTACCCG
    GGCAAGCTGA
401 AATCGCTGGA AGAAGTCAAA GACGGCAGCA CCGTATCCGC
    GCCCAACGAC
451 CCGTCCAACT TCGCCCGCGT CTTGGTGATG CTCGACGAAC
    TGGGTTGGAT
501 CAAACTCAAA GACGGCATCA ATCCGTTGAC CGCATCCAAA
    GCGGACATCG
551 CCGAGAACCT GAAAAACATC AAAATCGTCG AGCTTGAAGC
    CGCGCAACTG
601 CCGCGTAGCC GCGCCGACGT GGATTTTGCC GTCGTCAACG
    GCAACTACGC
651 CATAAGCAGC GGCATGAAGC TGACCGAAGC CCTGTTCCAA
    GAACCGAGCT
701 TTGCCTATGT CAACTGGTCT GCCGTCAAAA CCGCCGACAA
    AGACAGCCAA
751 TGGCTTAAAG ACGTAACCGA GGCCTATAAC TCCGACGCGT
    TCAAAGCCTA
801 CGCGCACAAA CGCTTCGAGG GCTACAAATC CCCTGCCGCA
    TGGAATGAAG
851 GCGCAGCCAA ATAA

This corresponds to the amino acid sequence <SEQ ID 218; ORF4-1>:

1   MKTFFKTLSA AALALILAAC GGQKDSAPAA SASAAADNGA
    AKKEIVFGTT
51  VGDFGDMVKE QIQAELEKKG YTVKLVEFTD YVRPNLALAE
    GELDINVFQH
101 KPYLDDFKKE HNLDITEVFQ VPTAPLGLYP GKLKSLEEVK
    DGSTVSAPND
151 PSNFARVLVM LDELGWIKLK DGINPLTASK ADIAENLKNI
    KIVELEAAQL
201 PRSRADVDFA VVNGNYAISS GMKLTEALFQ EPSFAYVNWS
    AVKTADKDSQ
251 WLKDVTEAYN SDAFKAYAHK RFEGYKSPAA WNEGAAK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF4 shows 93.5% identity over a 93aa overlap with an ORF (ORF4a) from strain A of N. meningitidis:

The complete length ORF4a nucleotide sequence <SEQ ID 219> is:

1   ATGAAAACCT TCTTCAAAAC CCTTTCCGCC GCCGCACTCG
    CGCTCATCCT
51  CGCCGCCTGC GGCGGTCAAA AAGATAGCGC GCCCGCCGCA
    TCCGCTTCTG
101 CCGCCGCCGA CAACGGCGCG GCGAANAAAG AAATCGTCTT
    CGGCACGACC
151 GTCGGCGACT TCGGCGATAT GGTCAAAGAA CANATCCAAC
    CCGAGCTGGA
201 GAAAAAAGGC TACACCGTCA AACTGGTCGA GTNTACCGAC
    TATGTGCGCN
251 CGAATCTGGC ATTGGCTGAG GGCGAGTTGG ACATCAACGT
    CTTNCAACAC
301 ANACNCTATC TTGACGACTN CAAAAAANAA CACAATCTGG
    ACATCACCNN
351 AGTCTTNCAA GTGCCGACCG CGCCTTTGGG ACTGTACCCG
    GGCAAGCTGA
401 AATCGCTGGA NNAAGTCAAA GANGGCAGCA CCGTATCCGC
    GCCCAACGAC
451 CCGTNNNACT TCGNCCGCGT CTTGGTGATG CTCGACGAAC
    TGGGTTNGAT
501 CAAACTCAAA GACNGCATCA NNNNGNNGNN NNNANCNANA
    NNNGANANNN
551 NNNNANNNNT NNNNNNNNNN NNNNNCNNCG NNNNNNNANN
    NNNNNNNNNN
601 NCGNNTNNNN NNGCNNNNNT NNANNNTNNN NNCNNCNNNN
    NNNNNTNNNN
651 NANNANNAGC GGCATGAAGC TGACCGAAGC CCTGTTCCAA
    GAACCGAGCT
701 TTGCCTATGT CAACTGGTCT GCCGTCAAAA CCGCCGACAA
    AGACAGCCAA
751 TGGCTTAAAG ACGTAACCGA GGCCTATAAC TCCGACGCGT
    TCAAAGCCTA
801 CGCGCACAAA CGCTTCGAGG GCTACAAATC CCCTGCCGCA
    TGGAATGAAG
851 GCGCAGCCAA ATAA

This is predicted to encode a protein having amino acid sequence <SEQ ID 220>:

1   MKTFFKTLSA AALALILAAC GGQKDSAPAA SASAAADNGA
    AXKEIVFGTT
51  VGDFGDMVKE XIQPELEKKG YTVKLVEXTD YVRXNLALAE
    GELDINVXQH
101 XXYLDDXKKX HNLDITXVXQ VPTAPLGLYP GKLKSLXXVK
    XGSTVSAPND
151 PXXFXRVLVM LDELGXIKLK DXIXXXXXXX XXXXXXXXXX
    XXXXXXXXXX
201 XXXXAXXXXX XXXXXXXXXS GMKLTEALFQ EPSFAYVNWS
    AVKTADKDSQ
251 WLKDVTEAYN SDAFKAYAHK RFEGYKSPAA WNEGAAK*

A leader peptide is underlined.

Further analysis of these strain A sequences revealed the complete DNA sequence <SEQ ID 221>:

1   ATGAAAACCT TCTTCAAAAC CCTTTCCGCC GCCGCACTCG
    CGCTCATCCT
51  CGCCGCCTGC GGCGGTCAAA AAGATAGCGC GCCCGCCGCA
    TCCGCTTCTG
101 CCGCCGCCGA CAACGGCGCG GCGAAAAAAG AAATCGTCTT
    CGGCACGACC
151 GTCGGCGACT TCGGCGATAT GGTCAAAGAA CAAATCCAAC
    CCGAGCTGGA
201 GAAAAAAGGC TACACCGTCA AACTGGTCGA GTTTACCGAC
    TATGTGCGCC
251 CGAATCTGGC ATTGGCTGAG GGCGAGTTGG ACATCAACGT
    CTTCCAACAC
301 AAACCCTATC TTGACGACTT CAAAAAAGAA CACAATCTGG
    ACATCACCGA
351 AGTCTTCCAA GTGCCGACCG CGCCTTTGGG ACTGTACCCG
    GGCAAGCTGA
401 AATCGCTGGA AGAAGTCAAA GACGGCAGCA CCGTATCCGC
    GCCCAACGAC
451 CCGTCCAACT TCGCCCGCGT CTTGGTGATG CTCGACGAAC
    TGGGTTGGAT
501 CAAACTCAAA GACGGCATCA ATCCGCTGAC CGCATCCAAA
    GCGGACATTG
551 CCGAAAACCT GAAAAACATC AAAATCGTCG AGCTTGAAGC
    CGCGCAACTG
601 CCGCGTAGCC GCGCCGACGT GGATTTTGCC GTCGTCAACG
    GCAACTACGC
651 CATAAGCAGC GGCATGAAGC TGACCGAAGC CCTGTTCCAA
    GAACCGAGCT
701 TTGCCTATGT CAACTGGTCT GCCGTCAAAA CCGCCGACAA
    AGACAGCCAA
751 TGGCTTAAAG ACGTAACCGA GGCCTATAAC TCCGACGCGT
    TCAAAGCCTA
801 CGCGCACAAA CGCTTCGAGG GCTACAAATC CCCTGCCGCA
    TGGAATGAAG
851 GCGCAGCCAA ATAA

This encodes a protein having amino acid sequence <SEQ ID 222; ORF4a-1>:

1   MKTFFKTLSA AALALILAAC GGQKDSAPAA SASAAADNGA
    AKKEIVFGTT
51  VGDFGDMVKE QIQPELEKKG YTVKLVEFTD YVRPNLALAE
    GELDINVFQH
101 KPYLDDFKKE HNLDITEVFQ VPTAPLGLYP GKLKSLEEVK
    DGSTVSAPND
151 PSNFARVLVM LDELGWIKLK DGINPLTASK ADIAENLKNI
    KIVELEAAQL
201 PRSRADVDFA VVNGNYAISS GMKLTEALFQ EPSFAYVNWS
    AVKTADKDSQ
251 WLKDVTEAYN SDAFKAYAHK RFEGYKSPAA WNEGAAK*

ORF4a-1 and ORF4-1 show 99.7% identity in 287 aa overlap:

Homology with an Outer Membrane Protein of Pasteurella haemolitica (Accession q08869).

ORF4 and this outer membrane protein show 33% aa identity in 91aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF4 shows 93.6% identity over a 94aa overlap with a predicted ORF (ORF4.ng) from N. gonorrhoeae:

The complete length ORF4ng nucleotide sequence <SEQ ID 223> was predicted to encode a protein having amino acid sequence <SEQ ID 224>:

1   MKTFFKTLST ASLALILAAC GGQKDSAPAA SAAAPSADNG
    AAKKEIVFGT
51  TVGDFGDMVK EQIQAELEKK GYTVKLVEFT DYVRPNLALA
    EGELDINVFQ
101 HKPYLDDFKK EHNLDITEAF QVPTAPLGLY PGKLKSLEEV
    KDGSTVSAPN
151 DPSNFARALV MLNELGWIKL KDGINPLTAS KADIAENLKN
    IKIVELEAAQ
201 LPRSRADVDF AVVNGNYAIS SGMKLTEALF QEPSFAYVNW
    SAVKTADKDS
251 QWLKDVTEAY NSDAFKAYAH KRFEGYKYPA AWNEGAAK*

Further analysis revealed the complete length ORF4ng DNA sequence <SEQ ID 225> to be:

1   atgAAAACCT TCTTCAAAAC cctttccgcc gccgcaCTCG
    CGCTCATCCT
51  CGCAGCCTGc ggCggtcaAA AAGACAGCGC GCCCgcagcc
    tctgcCGCCG
101 CCCCTTCTGC CGATAACGgc gCgGCGAAAA AAGAAAtcgt
    ctTCGGCACG
151 Accgtgggcg acttcggcgA TAtggTCAAA GAACAAATCC
    AagcCGAgct
201 gGAGAAAAAA GgctACACcg tcAAattggt cgaatttacc
    gactatgtGC
251 gCCCGAATCT GGCATTGGCG GAGGGCGAGT TGGACATCAA
    CGTCTTCCAA
301 CACAAACCCT ATCTTGACGA TTTCAAAAAA GAACACAACC
    TGGACATCAC
351 CGAAGCCTTC CAAGTGCCGA CCGCGCCTTT GGGACTGTAT
    CCGGGCAAAC
401 TGAAATCGCT GGAAGAAGTC AAAGACGGCA GCACCGTATC
    CGCGCCCAac
451 gACccgTCCA ACTTCGCACG CGCCTTGGTG ATGCTGAACG
    AACTGGGTTG
501 GATCAAACTC AAAGACGGCA TCAATCCGCT GACCGCATCC
    AAAGCCGACA
551 TCGCGGAAAA CCTGAAAAAC ATCAAAATCG TCGAGCTTGA
    AGCCGCACAA
601 CTGCCGCGCA GCCGCGCCGA CGTGGATTTT GCCGTCGTCA
    ACGGCAACTA
651 CGCCATAAGC AGCGGCATGA AGCTGACCGA AGCCCTGTTC
    CAAGAGCCGA
701 GCTTTGCCTA TGTCAACTGG TCTGCCgtcA AAACCGCCGA
    CAAAGACAGC
751 CAATGGCTTA AAGACGTAAC CGAGGCCTAT AACTCCGACG
    CGTTCAAAGC
801 CTACGCGCAC AAACGCTTCG AGGGCTACAA ATACCCTGCC
    GCATGGAATG
851 AAGGCGCAGC CAAATAA

This encodes a protein having amino acid sequence <SEQ ID 226; ORF4ng-1>:

1   MKTFFKTLSA AALALILAAC GGQKDSAPAA SAAAPSADNG
    AAKKEIVFGT
51  TVGDFGDMVK EQIQAELEKK GYTVKLVEFT DYVRPNLALA
    EGELDINVFQ
101 HKPYLDDFKK EHNLDITEAF QVPTAPLGLY PGKLKSLEEV
    KDGSTVSAPN
151 DPSNFARALV MLNELGWIKL KDGINPLTAS KADIAENLKN
    IKIVELEAAQ
201 LPRSRADVDF AVVNGNYAIS SGMKLTEALF QEPSFAYVNW
    SAVKTADKDS
251 QWLKDVTEAY NSDAFKAYAH KRFEGYKYPA AWNEGAAK*

This shows 97.6% identity in 288 aa overlap with ORF4-1:

In addition, ORF4ng-1 shows significant homology with an outer membrane protein from the database:

Based on this analysis, including the homology with the outer membrane protein of Pasteurella haemolitica, and on the presence of a putative prokaryotic membrane lipoprotein lipid attachment site in the gonococcal protein, it was predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF4-1 (30 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIGS. 8A and 8B show, respectively, the results of affinity purification of the His-fusion and GST-fusion proteins. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA (positive result), Western blot (FIG. 8C), FACS analysis (FIG. 8D), and a bactericidal assay (FIG. 8E). These experiments confirm that ORF4-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 8F shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF4-1.

Example 27

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 227>:

1   CCTCGTCGTC CTCGGCATGC TCCAGTTTCA AGGGGCGATT
    TACTCCAAGG
51  CGGTGGAACG TATGCTCGGC ACGGTCATCG GGCTGGGCGC
    GGGTTTGGGC
101 GTTTTATGGC TGAACCAGCA TTATTTCCAC GGCAACCTCC
    TCTTCTACCT
151 CACCGTCGGC ACGGCAAGCG CACTGGCCGG CTGGGCGGCG
    GTCGGCAAAA
201 ACGGCTACGT CCCTmTGCTG GCAGGGCTGA CGATGTGTAT
    GCTCATCGGC
251 GACAACGGCA GCGAATGGCT CGACAGCGGA CTCATGCGCG
    CCATGAACGT
301 CCTCATCGGC GyGGCCATCG CCATCGCCGC CGCCAAACTG
    CTGCCGCTGA
351 AATCCACACT GATGTGGCGT TTCATGCTTG CCGACAACCT
    GGCCGACTGC
401 AGCAAAATGA TTGCCGAAAT CAGCAACGGC AGGCGCATGA
    CCCGCGAACG
451 CCTCGAGGAG AACATGGCGA AAATGCGCCA AATCAACGCA
    CGCATGGTCA
501 AAAGCCGCAG CCATCTCGCC GCCACATCGG GCGAAAGCTG
    CATCAGCCCC
551 GCCATGATGG AAGCCATGCA GCACGCCCAC CGTAAAATCG
    TCAACACCAC
601 CGAGCTGCTC CTGACCACCG CCGCCAAGCT GCAATCTCCC
    AAACTCAACG
651 GCAGCGAAAT CCGGCTGCTT GACCGCCACT TCACACTGCT
    CCAAAC....
701 ............................. GC AGACACGCCC
    GCCGCATCCG
751 CATCGACACC GCCATCAACC CCGAACTGGA AGCCCTCGCC
    GAACACCTCC
801 ACTACCAATG GCAGGGCTTC CTCTGGCTCA GCACCGATAT
    GCGTCAGGAA
851 ATTTCCGCCC TCGTCATCCT GCTGCAACGC ACCCGCCGCA
    AATGGCTGGA
901 TGCCCACGAA CGCCAACACC TGCGCCAAAG CCTGCTTGA

This corresponds to the amino acid sequence <SEQ ID 228; ORF8>:

1   ......PRRP RHAPVSRGDL LQGGGTYARH GHRAGRGFGR
    FMAEPALFPR
51  QPPLLPHRRH GKRTGRLGGG RQKRLRPXAG RADDVYAHRR
    QRQRMARQRT
101 HARHERPHRR GHRHRRRQTA AAEIHTDVAF HACRQPGRLQ
    QNDCRNQQRQ
151 AHDPRTPRGE HGENAPNQRT HGQKPQPSRR HIGRKLHQPR
    HDGSHAARPP
201 XNRQHHRAAP DHRRQAAISQ TQRQRNPAAX PPLHTAPN..
    .........Q
251 TRPPHPHRHR HQPRTGSPRR TPPLPMAGLP LAQHRYASGN
    FRPRHPAATH
301 PPQMAGCPRT PTPAPKPA*

Computer analysis of this amino acid sequence gave the following results:

Sequence Motifs

ORF8 is proline-rich and has a distribution of proline residues consistent with a surface localization. Furthermore the presence of an RGD motif may indicate a possible role in bacterial adhesion events.

Homology with a Predicted ORF from N. gonorrhoeae

ORF8 shows 86.5% identity over a 312aa overlap with a predicted ORF (ORF8.ng) from N. gonorrhoeae:

The complete length ORF8ng nucleotide sequence <SEQ ID 229> is predicted to encode a protein having amino acid sequence <SEQ ID 230>:

1   MDRDDRLRRP RHAPVPRRDL LQRGGTYARY GHRAGRGFGR
    FMAEPALFPR
51  QPPLLPDHRH GKRTGRLGGG RQKRLRPYVG GADDVHAHRR
    QRQRMARQRP
101 DARDERPHRR RHRHCRRQTA AAEIHTDVAF HACRQPGRLQ
    QNDCRNQQRQ
151 AYDARTFGAE YGQNAPNQRT HGQKPQPPRR HIGRKPHQPL
    HDGSHAARPP
201 QNRQHHRAAP DHRRQAAISQ TQRQRNPAAR PPLHTAPNRP
    ATNRRPHQRQ
251 TRPPHPHRHR HQPRTGSPRR TPPLPMAGFP LAQHQYASGN
    FRPRHPPATH
301 PPQMAGCPRT PTPAPKPA*

Based on the sequence motifs in these proteins, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 28

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 231>:

1   ..GAAATCAGCC TGCGGTCCGA CNACAGGCCG GTTTCCGTGN
    CGAAGCGGCG
51  GGATTCGGAA CGTTTTCTGC TGTTGGACGG CGGCAACAGC
    CGGCTCAAGT
101 GGGCGTGGGT GGAAAACGGC ACGTTCGCAA CCGTCGGTAG
    CGCGCCGTAC
151 CGCGATTTGT CGCCTTTGGG CGCGGAGTGG GCGGAAAAGG
    CGGATGGAAA
201 TGTCCGCATC GTCGGTTGCG CTGTGTGCGG AGAATTCAAA
    AAGGCACAAG
251 TGCAGGAACA GCTCGCCCGA AAAATCGAGT GGCTGCCGTC
    TTCCGCACAG
301 GCTTT.GGCA TACGCAACCA CTACCGCCAC CCCGAAGAAC
    ACGGTTCCGA
351 CCGCTGGTTC AACGCCTTGG GCAGCCGCCG CTTCAGCCGC
    AACGCCTGCG
401 TCGTCGTCAG TTGCGGCACG GCGGTAACGG TTGACGCGCT
    CACCGATGAC
451 GGACATTATC TCGGAGA.GG AACCATCATG CCCGGTTTCC
    ACCTGATGAA
501 AGAATCGCTC GCCGTCCGAA CCGCCAACCT CAACCGGCAC
    GCCGGTAAGC
551 GTTATCCTTT CCCGACCGG..

This corresponds to the amino acid sequence <SEQ ID 232; ORF61>:

1   ..EISLRSDXRP VSVXKRRDSE RFLLLDGGNS RLKWAWVENG
    TFATVGSAPY
51  RDLSPLGAEW AEKADGNVRI VGCAVCGEFK KAQVQEQLAR
    KIEWLPSSAQ
101 AXGIRNHYRH PEEHGSDRWF NALGSRRFSR NACVVVSCGT
    AVTVDALTDD
151 GHYLGXGTIM PGFHLMKESL AVRTANLNRH AGKRYPFPT..

Further work revealed the complete nucleotide sequence <SEQ ID 233>:

1    ATGACGGTTT TGAAGCTTTC GCACTGGCGG GTGTTGGCGG
     AGCTTGCCGA
51   CGGTTTGCCG CAACACGTCT CGCAACTGGC GCGTATGGCG
     GATATGAAGC
101  CGCAGCAGCT CAACGGTTTT TGGCAGCAGA TGCCGGCGCA
     CATACGCGGG
151  CTGTTGCGCC AACACGACGG CTATTGGCGG CTGGTGCGCC
     CATTGGCGGT
201  TTTCGATGCC GAAGGTTTGC GCGAGCTGGG GGAAAGGTCG
     GGTTTTCAGA
251  CGGCATTGAA GCACGAGTGC GCGTCCAGCA ACGACGAGAT
     ACTGGAATTG
301  GCGCGGATTG CGCCGGACAA GGCGCACAAA ACCATATGCG
     TGACCCACCT
351  GCAAAGTAAG GGCAGGGGGC GGCAGGGGCG GAAGTGGTCG
     CACCGTTTGG
401  GCGAGTGTCT GATGTTCAGT TTTGGCTGGG TGTTTGACCG
     GCCGCAGTAT
451  GAGTTGGGTT CGCTGTCGCC TGTTGCGGCA GTGGCGTGTC
     GGCGCGCCTT
501  GTCGCGTTTA GGTTTGGATG TGCAGATTAA GTGGCCCAAT
     GATTTGGTTG
551  TCGGACGCGA CAAATTGGGC GGCATTCTGA TTGAAACGGT
     CAGGACGGGC
601  GGCAAAACGG TTGCCGTGGT CGGTATCGGC ATCAATTTTG
     TCCTGCCCAA
651  GGAAGTAGAA AATGCCGCTT CCGTGCAATC GCTGTTTCAG
     ACGGCATCGC
701  GGCGGGGCAA TGCCGATGCC GCCGTGCTGC TGGAAACGCT
     GTTGGTGGAA
751  CTGGACGCGG TGTTGTTGCA ATATGCGCGG GACGGATTTG
     CGCCTTTTGT
801  GGCGGAATAT CAGGCTGCCA ACCGCGACCA CGGCAAGGCG
     GTATTGCTGT
851  TGCGCGACGG CGAAACCGTG TTCGAAGGCA CGGTTAAAGG
     CGTGGACGGA
901  CAAGGCGTTT TGCACTTGGA AACGGCAGAG GGCAAACAGA
     CGGTCGTCAG
951  CGGCGAAATC AGCCTGCGGT CCGACGACAG GCCGGTTTCC
     GTGCCGAAGC
1001 GGCGGGATTC GGAACGTTTT CTGCTGTTGG ACGGCGGCAA
     CAGCCGGCTC
1051 AAGTGGGCGT GGGTGGAAAA CGGCACGTTC GCAACCGTCG
     GTAGCGCGCC
1101 GTACCGCGAT TTGTCGCCTT TGGGCGCGGA GTGGGCGGAA
     AAGGCGGATG
1151 GAAATGTCCG CATCGTCGGT TGCGCTGTGT GCGGAGAATT
     CAAAAAGGCA
1201 CAAGTGCAGG AACAGCTCGC CCGAAAAATC GAGTGGCTGC
     CGTCTTCCGC
1251 ACAGGCTTTG GGCATACGCA ACCACTACCG CCACCCCGAA
     GAACACGGTT
1301 CCGACCGCTG GTTCAACGCC TTGGGCAGCC GCCGCTTCAG
     CCGCAACGCC
1351 TGCGTCGTCG TCAGTTGCGG CACGGCGGTA ACGGTTGACG
     CGCTCACCGA
1401 TGACGGACAT TATCTCGGGG GAACCATCAT GCCCGGTTTC
     CACCTGATGA
1451 AAGAATCGCT CGCCGTCCGA ACCGCCAACC TCAACCGGCA
     CGCCGGTAAG
1501 CGTTATCCTT TCCCGACCAC AACGGGCAAT GCCGTCGCCA
     GCGGCATGAT
1551 GGATGCGGTT TGCGGCTCGG TTATGATGAT GCACGGGCGT
     TTGAAAGAAA
1601 AAACCGGGGC GGGCAAGCCT GTCGATGTCA TCATTACCGG
     CGGCGGCGCG
1651 GCAAAAGTTG CCGAAGCCCT GCCGCCTGCA TTTTTGGCGG
     AAAATACCGT
1701 GCGCGTGGCG GACAACCTCG TCATTTACGG GTTGTTGAAC
     ATGATTGCCG
1751 CCGAAGGCAG GGAATATGAA CATATTTAA

This corresponds to the amino acid sequence <SEQ ID 234; ORF61-1>:

1   MTVLKLSHWR VLAELADGLP QHVSQLARMA DMKPQQLNGF
    WQQMPAHIRG
51  LLRQHDGYWR LVRPLAVFDA EGLRELGERS GFQTALKHEC
    ASSNDEILEL
101 ARIAPDKAHK TICVTHLQSK GRGRQGRKWS HRLGECLMFS
    FGWVFDRPQY
151 ELGSLSPVAA VACRRALSRL GLDVQIKWPN DLVVGRDKLG
    GILIETVRTG
201 GKTVAVVGIG INFVLPKEVE NAASVQSLFQ TASRRGNADA
    AVLLETLLVE
251 LDAVLLQYAR DGFAPFVAEY QAANRDHGKA VLLLRDGETV
    FEGTVKGVDG
301 QGVLHLETAE GKQTVVSGEI SLRSDDRPVS VPKRRDSERF
    LLLDGGNSRL
351 KWAWVENGTF ATVGSAPYRD LSPLGAEWAE KADGNVRIVG
    CAVCGEFKKA
401 QVQEQLARKI EWLPSSAQAL GIRNHYRHPE EHGSDRWFNA
    LGSRRFSRNA
451 CVVVSCGTAV TVDALTDDGH YLGGTIMPGF HLMKESLAVR
    TANLNRHAGK
501 RYPFPTTTGN AVASGMMDAV CGSVMMMHGR LKEKTGAGKP
    VDVIITGGGA
551 AKVAEALPPA FLAENTVRVA DNLVIYGLLN MIAAEGREYE
    HI*

FIG. 9 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF61-1. Further computer analysis of this amino acid sequence gave the following results:

Homology with the Baf Protein of B. pertussis (Accession Number U12020).

ORF61 and baf protein show 33% aa identity in 166aa overlap:

orf61  23 LLLDGGNSRLKWAWVE-NGTFATVGSAPYR----DLSPLGAEWAEKADGNVRIVGCAVCG  77
+L+D GNSRLK  W + +   A    AP      DL  LG   A      R +G V   G
baf     3 ILIDSGNSRLKVGWFDPDAPQAAREPAPVAFDNLDLDALGRWLATLPRRPQRALGVNVAG  62
orf61  78 EFKKAQVQEQLAR---KIEWLPSSAQAXGIRNHYRHPEEHGSDRW---FNALGSRRFSRN 131
+   +   L      I WL +   A G+RN YR+P++ G+DRW      L  +
baf    63 LARGEAIAATLRAGGCDIRWLRAQPLAMGLRNGYRNPDQLGADRWACMVGVLARQPSVHP 122
orf61 132 ACVVVSCGTAVTVDALTDDGHYLGXGTIMPGFHLMKESLAVRTANL               177
+V S GTA T+D + D   + G G I+PG  +M+ +LA  TA+L
baf   123 PLLVASFGTATTLDTIGPDNVFPG-GLILPGPAMMRGALAYGTAHL               167

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF61 shows 97.4% identity over a 189aa overlap with an ORF (ORF61a) from strain A of N. meningitidis:

The complete length ORF61a nucleotide sequence <SEQ ID 235> is:

1    ATGACGGTTT TGAAGCCTTC GCACTGGCGG GTGTTGGCGG
     AGCTTGCCGA
51   CGGTTTGCCG CAACACGTCT CGCAACTGGC GCGTATGGCG
     GATATGAAGC
101  CGCAGCAGCT CAACGGTTTT TGGCAGCAGA TGCCGGCGCA
     CATACGCGGG
151  CTGTTGCGCC AACACGACGG CTATTGGCGG CTGGTGCGCC
     CATTGGCGGT
201  TTTCGATGCC GAAGGTTTGC GCGAGCTGGG GGAAAGGTCG
     GGTTTTCAGA
251  CGGCATTGAA GCACGAGTGC GCGTCCAGCA ACGACGAGAT
     ACTGGAATTG
301  GCGCGGATTG CGCCGGACAA GGCGCACAAA ACCATATGTG
     TGACCCACCT
351  GCAAAGTAAG GGCAGGGGGC GGCAGGGGCG GAAGTGGTCG
     CACCGTTTGG
401  GCGAGTGTCT GATGTTCAGT TTTGGCTGGG TGTTTGACCG
     GCCGCAGTAT
451  GAGTTGGGTT CGCTGTCGCC TGTTGCGGCA GTGGCGTGCC
     GGCGCGCCTT
501  GTCGCGTTTG GGTTTGAAAA CGCAAATCAA GTGGCCAAAC
     GATTTGGTCG
551  TCGGACGCGA CAAATTGGGC GGCATTCTGA TTGAAACGGT
     CAGGACGGGC
601  GGCAAAACGG TTGCCGTGGT CGGTATCGGC ATCAATTTCG
     TGCTGCCCAA
651  GGAAGTGGAA AACGCCGCTT CCGTGCAATC GCTGTTTCAG
     ACGGCATCGC
701  GGCGGGGAAA TGCCGATGCC GCCGTGTTGC TGGAAACGCT
     GTTGGCGGAA
751  CTTGATGCGG TGTTGTTGCA ATATGCGCGG GACGGATTTG
     CGCCTTTTGT
801  GGCGGAATAT CAGGCTGCCA ACCGCGACCA CGGCAAGGCG
     GTATTGCTGT
851  TGCGCGACGG CGAAACCGTG TTCGAAGGCA CGGTTAAAGG
     CGTGGACGGA
901  CAAGGCGTTC TGCACTTGGA AACGGCAGAG GGCAAACAGA
     CGGTCGTCAG
951  CGGCGAAATC AGCCTGCGGT CCGACGACAG GCCGGTTTCC
     GTGCCGAAGC
1001 GGCGGGATTC GGAACGTTTT CTGCTGTTGG ACGGCGGCAA
     CAGCCGGCTC
1051 AAGTGGGCGT GGGTGGAAAA CGGCACGTTC GCAACCGTCG
     GTAGCGCGCC
1101 GTACCGCGAT TTGTCGCCTT TGGGCGCGGA GTGGGCGGAA
     AAGGTGGATG
1151 GAAATGTCCG CATCGTCGGT TGCGCCGTGT GCGGAGAATT
     CAAAAAGGCA
1201 CAAGTGCAGG AACAGCTCGC CCGAAAAATC GAGTGGCTGC
     CGTCTTCCGC
1251 ACAGGCTTTG GGCATACGCA ACCACTACCG CCACCCCGAA
     GAACACGGTT
1301 CCGACCGCTG GTTCAACGCC TTGGGCAGCC GCCGCTTCAG
     CCGCAACGCC
1351 TGCGTCGTCG TCAGTTGCGG CACGGCGGTA ACGGTTGACG
     CGCTCACCGA
1401 TGACGGACAT TATCTCGGGG GAACCATCAT GCCCGGTTTC
     CACCTGATGA
1451 AAGAATCGCT CGCCGTCCGA ACCGCCAACC TCAACCGGCA
     CGCCGGTAAG
1501 CGTTATCCTT TCCCGACCAC AACGGGCAAT GCCGTCGCCA
     GCGGCATGAT
1551 GGATGCGGTT TGCGGCTCGG TTATGATGAT GCACGGGCGT
     TTGAAAGAAA
1601 AAACCGGGGC GGGCAAGCCT GTCGATGTCA TCATTACCGG
     CGGCGGCGCG
1651 GCAAAAGTTG CCGAAGCCCT GCCGCCTGCA TTTTTGGCGG
     AAAATACCGT
1701 GCGCGTGGCG GACAACCTCG TCATTCACGG GCTGCTGAAC
     CTGATTGCCG
1751 CCGAAGGCGG GGAATCGGAA CATACTTAA

This encodes a protein having amino acid sequence <SEQ ID 236>:

1   MTVLKPSHWR VLAELADGLP QHVSQLARMA DMKPQQLNGF
    WQQMPAHIRG
51  LLRQHDGYWR LVRPLAVFDA EGLRELGERS GFQTALKHEC
    ASSNDEILEL
101 ARIAPDKAHK TICVTHLQSK GRGRQGRKWS HRLGECLMFS
    FGWVFDRPQY
151 ELGSLSPVAA VACRRALSRL GLKTQIKWPN DLVVGRDKLG
    GILIETVRTG
201 GKTVAVVGIG INFVLPKEVE NAASVQSLFQ TASRRGNADA
    AVLLETLLAE
251 LDAVLLQYAR DGFAPFVAEY QAANRDHGKA VLLLRDGETV
    FEGTVKGVDG
301 QGVLHLETAE GKQTVVSGEI SLRSDDRPVS VPKRRDSERF
    LLLDGGNSRL
351 KWAWVENGTF ATVGSAPYRD LSPLGAEWAE KVDGNVRIVG
    CAVCGEFKKA
401 QVQEQLARKI EWLPSSAQAL GIRNHYRHPE EHGSDRWFNA
    LGSRRFSRNA
451 CVVVSCGTAV TVDALTDDGH YLGGTIMPGF HLMKESLAVR
    TANLNRHAGK
501 RYPFPTTTGN AVASGMMDAV CGSVMMMHGR LKEKTGAGKP
    VDVIITGGGA
551 AKVAEALPPA FLAENTVRVA DNLVIHGLLN LIAAEGGESE
    HT*

ORF61a and ORF61-1 show 98.5% identity in 591 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF61 shows 94.2% identity over a 189aa overlap with a predicted ORF (ORF61.ng) from N. gonorrhoeae:

An ORF61ng nucleotide sequence <SEQ ID 237> was predicted to encode a protein having amino acid sequence <SEQ ID 238>:

1   MFSFGWAFDR PQYELGSLSP VAALACRRAL GCLGLETQIK
    WPNDLVVGRD
51  KLGGILIETV RAGGKTVAVV GIGINFVLPK EVENAASVQS
    LFQTASRRGN
101 ADAAVLLETL LAELGAVLEQ YAEEGFAPFL NEYETANRDH
    GKAVLLLRDG
151 ETVCEGTVKG VDGRGVLHLE TAEGEQTVVS GEISLRPDNR
    SVSVPKRPDS
201 ERFLLLEGGN SRLKWAWVEN GTFATVGSAP YRDLSPLGAE
    WAEKADGNVR
251 IVGCAVCGES KKAQVKEQLA RKIEWLPSSA QALGIRNHYR
    HPEEHGSDRW
301 FNALGSRRFS RNACVVVSCG TAVTVDALTD DGHYLGGTIM
    PGFHLMKESL
351 AVRTANLNRP AGKRYPFPTT TGNAVASGMM DAVCGSIMMM
    HGRLKEKNGA
401 GKPVDVIITG GGAAKVAEAL PPAFLAENTV RVADNLVIHG
    LLNLIAAEGG
451 ESEHA*

Further analysis revealed the complete gonococcal DNA sequence <SEQ ID 239> to be:

1    ATGACGGTTT TGAAGCCTTC GCATTGGCGG GTGTTGGCGG
     AGCTTGCCGA
51   CGGTTTGCCG CAACACGTAT CGCAATTGGC GCGTGAGGCG
     GACATGAAGC
101  CGCAGCAGCT CAACGGTTTT TGGCAGCAGA TGCCGGCGCA
     TATACGCGGG
151  CTGTTGCGCC AACACGACGG CTATTGGCGG CTGGTGCGCC
     CCTTGGCGGT
201  TTTCGATGCC GAAGGTTTGC GCGATCTGGG GGAAAGGTCG
     GGTTTTCAGA
251  CGGCATTGAA GCACGAGTGC GCGTCCAGCA ACGACGAGAT
     ACTGGAATTG
301  GCGCGGATTG CGCCGGACAA GGCGCACAAA ACCATATGCG
     TGACCCACCT
351  GCAAAGTAAG GGCAGGGGGC GGCAGGGGCG GAAGTGGTCG
     CACCGTTTGG
401  GCGAGTGCCT GATGTTCAGT TTCGGCTGGG CGTTTGACCG
     GCCGCAGTAT
451  GAGTTGGGTT CGCTGTCGCC TGTTGCGGCA CTTGCGTGCC
     GGCGCGCTTT
501  GGGGTGTTTG GGTTTGGAAA CGCAAATCAA GTGGCCAAAC
     GATTTGGTCG
551  TCGGACGCGA CAAATTGGGC GGCATTCTGA TTGAAACAGT
     CAGGGCGGGC
601  GGTAAAACGG TTGCCGTGGT CGGTATCGGC ATCAATTTCG
     TGCTGCCCAA
651  GGAAGTGGAA AACGCCGCTT CCGTGCAGTC GCTGTTTCAG
     ACGGCATCGC
701  GGCGGGGCAA TGCCGATGCC GCCGTATTGC TGGAAACATT
     GCTTGCGGAA
751  CTGGGCGCGG TGTTGGAACA ATATGCGGAA GAAGGGTTCG
     CGCCATTTTT
801  AAATGAGTAT GAAACGGCCA ACCGCGACCA CGGCAAGGCG
     GTATTGCTGT
851  TGCGCGACGG CGAAACCGTG TGCGAAGGCA CGGTTAAAGG
     CGTGGACGGA
901  CGAGGCGTTC TGCACTTGGA AACGGCAgaa ggcgaACAGa
     cggtcgtcag
951  cggcgaaaTC AGcctGCggc ccgacaacaG GTCGGtttcc
     gtgccgaagc
1001 ggccggatTC GgaacgtTTT tTGCtgttgg aaggcgggaa
     cagccgGCTC
1051 AAGTGGGCGT GggtggAAAa cggcacgttc gcaaccgtgg
     gcagcgcgCc
1101 gtaCCGCGAT TTGTCGCCTT TGGGCGCGGA GTGGGCGGAA
     AAGGCGGATG
1151 GAAATGTCCG CATCGTCGGT TGCGCCGTGT GCGGAGAATC
     CAAAAAGGCA
1201 CAAGTGAAGG AACAGCTCGC CCGAAAAATC GAGTGGCTGC
     CGTCTTCCGC
1251 ACAGGCTTTG GGCATACGCA ACCACTACCG CCACCCCGAA
     GAACACGGTT
1301 CCGACCGTTG GTTCAACGCC TTGGGCAGCC GCCGCTTCAG
     CCGCAACGCC
1351 TGCGTCGTCG TCAGTTGCGG CACGGCGGTA ACGGTTGACG
     CGCTCACCGA
1401 TGACGGACAT TATCTCGGCG GAACCATCAT GCCCGGCTTC
     CACCTGATGA
1451 AAGAATCGCT CGCCGTCCGA ACCGCCAACC TCAACCGCCC
     CGCCGGCAAA
1501 CGTTACCCTT TCCCGACCAC AACGGGCAAC GCCGTCGCAA
     GCGGCATGAT
1551 GGACGCGGTT TGCGGCTCGA TAATGATGAT GCACGGCCGT
     TTGAAAGAAA
1601 AAAACGGCGC GGGCAAGCCT GTCGATGTCA TCATTACCGG
     CGGCGGCGCG
1651 GCGAAAGTCG CCGAAGCCCT GCCGCCTGCA TTTTTGGCGG
     AAAATACCGT
1701 GCGCGTGGCG GACAACCTCG TCATCCACGG GCTGCTGAAC
     CTGATTGCCG
1751 CCGAAGGCGG GGAATCGGAA CACGCTTAA

This corresponds to the amino acid sequence <SEQ ID 240; ORF61ng-1>:

1   MTVLKPSHWR VLAELADGLP QHVSQLAREA DMKPQQLNGF
    WQQMPAHIRG
51  LLRQHDGYWR LVRPLAVFDA EGLRDLGERS GFQTALKHEC
    ASSNDEILEL
101 ARIAPDKAHK TICVTHLQSK GRGRQGRKWS HRLGECLMFS
    FGWAFDRPQY
151 ELGSLSPVAA LACRRALGCL GLETQIKWPN DLVVGRDKLG
    GILIETVRAG
201 GKTVAVVGIG INFVLPKEVE NAASVQSLFQ TASRRGNADA
    AVLLETLLAE
251 LGAVLEQYAE EGFAPFLNEY ETANRDHGKA VLLLRDGETV
    CEGTVKGVDG
301 RGVLHLETAE GEQTVVSGEI SLRPDNRSVS VPKRPDSERF
    LLLEGGNSRL
351 KWAWVENGTF ATVGSAPYRD LSPLGAEWAE KADGNVRIVG
    CAVCGESKKA
401 QVKEQLARKI EWLPSSAQAL GIRNHYRHPE EHGSDRWFNA
    LGSRRFSRNA
451 CVVVSCGTAV TVDALTDDGH YLGGTIMPGF HLMKESLAVR
    TANLNRPAGK
501 RYPFPTTTGN AVASGMMDAV CGSIMMMHGR LKEKNGAGKP
    VDVIITGGGA
551 AKVAEALPPA FLAENTVRVA DNLVIHGLLN LIAAEGGESE
    HA*

ORF61ng-1 and ORF61-1 show 93.9% identity in 591 aa overlap:

Based on this analysis, including the homology with the baf protein of B. pertussis and the presence of a putative prokaryotic membrane lipoprotein lipid attachment site, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 29

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 241>:

1   ATGTTTTACC AAATCCTTGC CCTGATTATC TGGAGCAGCT
    CGTTTATTGC
51  CGCCAAATAT GTCTATGGCG GCATCGATCC CGCATTGATG
    GTCGGCGTGC
101 GCCTGCTAAT TGCCGCGCTG CCTGCACTGC CCGCCTGCCG
    CCGTCATGTC
151 GGCAAGATTC CGCGTGAGGA ATGGAAGCCG TTGCTGATTG
    TGTCGTTCGT
201 CAACTATGTG CTGACCCTGC TGCTTCAGTT TGTCGGGTTG
    AAATACACTT
251 CCGCCGCCAG CGCATCGGTC ATTGTCGGAC TCGAGCCGCT
    GCTGATGGTG
301 TTTGTCGGAC ACTTTTTCTT CAACGACAAA GCGCGTGCCT
    ACCACTGGAT
351 ATGCGGCGCG GCGGCATTTG CCGGTGTCGC GCTGCTGATG
    GCGGGCGGTG
401 CGGaAGAGGG CGGCGaAGTC GGCTGGTTCG GCTGCCTGCT
    GGTGTTGTTG
451 GCGGGCGCGG GCTTTTGTGC CGCTATGCGT CCGACGCAAA
    GGCTGATTGC
501 ACGCATCGGC GCACCGGCAT TCACATCTGT TTCCATTGCC
    GCCGCATCGT
551 TGATGTGCCT GCCGTTTTCG CTTGCTTTGG CGCAAAGTTA
    TACCGTGGAC
601 TGGAGCGTCG GGATGGTATT GTCGCTGCTG TATTTGGGTT
    TGGGGTGC..

This corresponds to the amino acid sequence <SEQ ID 242; ORF62>:

1   MFYQILALII WSSSFIAAKY VYGGIDPALM VGVRLLIAAL
    PALPACRRHV
51  GKIPREEWKP LLIVSFVNYV LTLLLQFVGL KYTSAASASV
    IVGLEPLLMV
101 FVGHFFFNDK ARAYHWICGA AAFAGVALLM AGGAEEGGEV
    GWFGCLLVLL
151 AGAGFCAAMR PTQRLIARIG APAFTSVSIA AASLMCLPFS
    LALAQSYTVD
201 WSVGMVLSLL YLGLGC..

Further work revealed the complete nucleotide sequence <SEQ ID 243>:

1   ATGTTTTACC AAATCCTTGC CCTGATTATC TGGAGCAGCT
    CGTTTATTGC
51  CGCCAAATAT GTCTATGGCG GCATCGATCC CGCATTGATG
    GTCGGCGTGC
101 GCCTGCTAAT TGCCGCGCTG CCTGCACTGC CCGCCTGCCG
    CCGTCATGTC
151 GGCAAGATTC CGCGTGAGGA ATGGAAGCCG TTGCTGATTG
    TGTCGTTCGT
201 CAACTATGTG CTGACCCTGC TGCTTCAGTT TGTCGGGTTG
    AAATACACTT
251 CCGCCGCCAG CGCATCGGTC ATTGTCGGAC TCGAGCCGCT
    GCTGATGGTG
301 TTTGTCGGAC ACTTTTTCTT CAACGACAAA GCGCGTGCCT
    ACCACTGGAT
351 ATGCGGCGCG GCGGCATTTG CCGGTGTCGC GCTGCTGATG
    GCGGGCGGTG
401 CGGAAGAGGG CGGCGAAGTC GGCTGGTTCG GCTGCCTGCT
    GGTGTTGTTG
451 GCGGGCGCGG GCTTTTGTGC CGCTATGCGT CCGACGCAAA
    GGCTGATTGC
501 ACGCATCGGC GCACCGGCAT TCACATCTGT TTCCATTGCC
    GCCGCATCGT
551 TGATGTGCCT GCCGTTTTCG CTTGCTTTGG CGCAAAGTTA
    TACCGTGGAC
601 TGGAGCGTCG GGATGGTATT GTCGCTGCTG TATTTGGGTT
    TGGGGTGCGG
651 CTGGTACGCC TATTGGCTGT GGAACAAGGG GATGAGCCGT
    GTTCCTGCCA
701 ATGTTTCGGG ACTGTTGATT TCGCTCGAAC CCGTCGTCGG
    CGTGCTGCTG
751 GCGGTTTTGA TTTTGGGCGA ACACCTGTCG CCCGTGTCCG
    CCTTGGGCGT
801 GTTTGTCGTC ATCGCCGCCA CCTTGGTTGC CGGCCGGCTG
    TCGCATCAAA
851 AATAA

This corresponds to the amino acid sequence <SEQ ID 244; ORF62-1>:

1   MFYQILALII WSSSFIAAKY VYGGIDPALM VGVRLLIAAL
    PALPACRRHV
51  GKIPREEWKP LLIVSFVNYV LTLLLQFVGL KYTSAASASV
    IVGLEPLLMV
101 FVGHFFFNDK ARAYHWICGA AAFAGVALLM AGGAEEGGEV
    GWFGCLLVLL
151 AGAGFCAAMR PTQRLIARIG APAFTSVSIA AASLMCLPFS
    LALAQSYTVD
201 WSVGMVLSLL YLGLGCGWYA YWLWNKGMSR VPANVSGLLI
    SLEPVVGVLL
251 AVLILGEHLS PVSALGVFVV IAATLVAGRL SHQK*

Computer analysis of this amino acid sequence gave the following results:

Homology with Hypothetical Transmembrane Protein H10976 of H. influenzae (Accession Number Q57147)

ORF62 and HI0976 show 50% aa identity in 114aa overlap:

Orf62	1	MFYQILALIIWSSSFIAAKYVYGGIDPALMVGVRXXXXXXXXXXXCRRHVGKIPREEWKP	60
		M YQILAL+IWSSS I  K  Y  +DP L+V VR             R   KI +   K
HI0976	1	MLYQILALLIWSSSLIVGKLTYSMMDPVLVVQVRLIIAMIIVMPLFLRRWKKIDKPMRKQ	60
Orf62	61	LLIVSFVNYVLTLLLQFVGLKYTSAASASVIVGLEPLLMVFVGHFFFNDKARAY	114
		L  ++F NY    LLQF+GLKYTSA+SA  ++GLEPLL+VFVGHFFF  K   +
HI0976	61	LWWLAFFNYTAVFLLQFIGLKYTSASSAVTMIGLEPLLVVFVGHFFFKTKQNGF	114

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF62 shows 99.5% identity over a 216aa overlap with an ORF (ORF62a) from strain A of N. meningitidis:

The complete length ORF62a nucleotide sequence <SEQ ID 245> is:

1   ATGTTTTACC AAATCCTTGC CCTGATTATC TGGAGCAGCT
    CGTTTATTGC
51  CGCCAAATAT GTCTATGGCG GCATCGATCC CGCATTGATG
    GTCGGCGTGC
101 GCCTGCTGAT TGCTGCGCTG CCTGCACTGC CCGCCTGCCG
    CCGTCATGTC
151 GGCAAGATTC CGCGTGAGGA ATGGAAGCCG TTGCTGATTG
    TGTCGTTCGT
201 CAACTATGTG CTGACCCTGC TACTTCAGTT TGTCGGGTTG
    AAATACACTT
251 CCGCCGCCAG CGCATCGGTC ATTGTCGGAC TCGAGCCACT
    GCTGATGGTG
301 TTTGTCGGAC ACTTTTTCTT CAACGACAAA GCGCGTGCCT
    ACCACTGGAT
351 ATGCGGCGCG GCGGCATTTG CCGGTGTCGC GCTGCTGATG
    GCGGGCGGTG
401 CGGAAGAGGG CGGCGAAGTC GGCTGGTTCG GCTGCCTGCT
    GGTGTTGTTG
451 GCGGGCGCGG GCTTTTGTGC CGCTATGCGT CCGACGCAAA
    GGCTGATTGC
501 ACGCATCGGC GCACCGGCAT TCACATCTGT TTCCATTGCC
    GCCGCATCGT
551 TGATGTGCCT GCCGTTTTCG CTTGCTTTGG CGCAAAGTTA
    TACCGTGGAC
601 TGGAGCGTCG GAATGGTATT GTCGCTGCTG TATTTGGGCG
    TGGGGTGCAG
651 CTGGTACGCC TATTGGCTGT GGAACAAGGG GATGAGCCGT
    GTTCCTGCCA
701 ACGTTTCGGG ACTGTTGATT TCGCTCGAAC CCGTCGTCGG
    CGTGCTGCTG
751 GCGGTTTTGA TTTTGGGCGA ACACCTGTCG CCCGTGTCCG
    TCTTGGGCGT
801 GTTTGTCGTC ATCGCCGCCA CCTTGGTTGC CGGCCGGCTG
    TCGCATCAAA
851 AATAA

This encodes a protein having amino acid sequence <SEQ ID 246>:

1   MFYQILALII WSSSFIAAKY VYGGIDPALM VGVRLLIAAL
    PALPACRRHV
51  GKIPREEWKP LLIVSFVNYV LTLLLQFVGL KYTSAASASV
    IVGLEPLLMV
101 FVGHFFFNDK ARAYHWICGA AAFAGVALLM AGGAEEGGEV
    GWFGCLLVLL
151 AGAGFCAAMR PTQRLIARIG APAFTSVSIA AASLMCLPFS
    LALAQSYTVD
201 WSVGMVLSLL YLGVGCSWYA YWLWNKGMSR VPANVSGLLI
    SLEPVVGVLL
251 AVLILGEHLS PVSVLGVFVV IAATLVAGRL SHQK*

ORF62a and ORF62-1 show 98.9% identity in 284 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF62 shows 99.5% identity over a 216aa overlap with a predicted ORF (ORF62.ng) from N. gonorrhoeae:

The complete length ORF62ng nucleotide sequence <SEQ ID 247> is:

1   ATGTTTTACC AAATCCTTGC CCTGATTATC TGGGGCAGCT
    CGTTTATTGC
51  CGCCAAATAT GTCTATGGCG GCATCGATCC CGCATTGATG
    GTCGGCGTGC
101 GCCTGCTGAT TGCCGCGCTG CCTGCACTGC CCGCCTGCCG
    CCGTCATGTC
151 GGCAAGATTC CGCGTGAGGA ATGGAAGCCG TTGCTGATTG
    TGTCGTTCGT
201 CAACTATGTG CTGACCCTGC TGCTTCAGTT TGTCGGGTTG
    AAATACACTT
251 CCGCCGCCAG CGCATCGGTC ATTGTCGGAC TCGAGCCGCT
    GCTGATGGTG
301 TTTGTCGGAC ACTTTTTCTT CAACGACAAA GCGCGTGCCT
    ACCACTGGAT
351 ATGCGGCGCG GCGGCATTTG CCGGTGTCGC GCTGCTGATG
    GCGGGCGGTG
401 CGGAAGAGGG CGGCGAAGTC GGCTGGTTCG GCTGCCTGCT
    GGTGTTGTTG
451 GCGGGCGCGG GCTTTTGTGC CGCTATGCGT CCGACGCAAA
    GGCTGATTGC
501 CCGCATCGGC GCACCGGCAT TCACATCTGT TTCCATTGCC
    GCCGCATCGT
551 TGATGTGCCT GCCGTTTTCG CTTGCTTTGG CGCAAAGTTA
    TACCGTGGAC
601 TGGAGCGTCG GGATGGTATT GTCGCTGTTG TATTTGGGTT
    TGGGGTGCGG
651 CTGGTACGCC TATTGGCTGT GGAACAAGGG GATGAGCCGT
    GTTCCTGCCA
701 ACGCGTCGGG ACTGTTGATT TCGCTCGAAC CCGTCGTCGG
    CGTGCTGTTG
751 GCGGTTTTGA TTTTGGGCGA ACATTTATCG CCCGTGTCCG
    CCTTGGGCGT
801 GTTTGTCGTC ATCGCCGCCA CTTTCGCCGC CGGCCGGCTG
    TCGCGCAGGG
851 ACGCGCAAAA CGGCAATGCC GTCTGA

This encodes a protein having amino acid sequence <SEQ ID 248>:

1   MFYQILALII WGSSFIAAKY VYGGIDPALM VGVRLLIAAL
    PALPACRRHV
51  GKIPREEWKP LLIVSFVNYV LTLLLQFVGL KYTSAASASV
    IVGLEPLLMV
101 FVGHFFFNDK ARAYHWICGA AAFAGVALLM AGGAEEGGEV
    GWFGCLLVLL
151 AGAGFCAAMR PTQRLIARIG APAFTSVSIA AASLMCLPFS
    LALAQSYTVD
201 WSVGMVLSLL YLGLGCGWYA YWLWNKGMSR VPANASGLLI
    SLEPVVGVLL
251 AVLILGEHLS PVSALGVFVV IAATFAAGRL SRRDAQNGNA
    V*

ORF62ng and ORF62-1 show 97.9% identity in 283 aa overlap:

Furthermore, ORF62ng shows significant homology to a hypothetical H. influenzae protein:

sp|Q57147|Y976_HAEIN HYPOTHETICAL PROTEIN HI0976 >gi|1074589|pir||B64163
hypothetical protein HI0976 - Haemophilus influenzae (strain Rd KW20)
>gi|1574004 (U32778) hypothetical [Haemophilus influenzae] Length = 128
Score = 106 bits (262), Expect = 2e−22
Identities = 56/114 (49%), Positives = 68/114 (59%)
Query:	1	MFYQILALIIWGSSFIAAKYVYGGIDPALMVGVRXXXXXXXXXXXCRRHVGKIPREEWKP	60
		M YQILAL+IW SS I  K  Y  +DP L+V VR             R   KI +   K
Sbjct:	1	MLYQILALLIWSSSLIVGKLTYSMMDPVLVVQVRLIIAMIIVMPLFLRRWKKIDKPMRKQ	60
Query:	61	LLIVSFVNYVLTLLLQFVGLKYTSAASASVIVGLEPLLMVFVGHFFFNDKARAY	114
		L  ++F NY    LLQF+GLKYTSA+SA  ++GLEPLL+VFVGHFFF  K   +
Sbjct:	61	LWWLAFFNYTAVFLLQFIGLKYTSASSAVTMIGLEPLLVVFVGHFFFKTKQNGF	114

Based on this analysis, including the homology with the transmembrane protein of H. influenzae and the putative leader sequence and several transmembrane domains in the gonococcal protein, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 30

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 249>:

1    ATGCGCCGTT TTCTACCGAT CGCAGCCATA TGCGCmGwms
     TCCTGkkGTA
51   sGGACTGACG GCGGCAACCG GCAGCACCAG TTCGCTGGCG
     GATTATTTCT
101  GGTGGATTGT TGCGTTCAGC GCAATGCTGC TGCTGGTGTT
     GTCCGCCGTT
151  TTGGCACGTT ATGTCATATT GCTGTTGAAA GACAGGCGCG
     ACGGCGTATT
201  CGGTTCGCtA srTyGCCAAA gsGCCTgkks TGGG.ATGTT
     TACGCTGGTT
251  GCCGkACTGC CCGGCGTGTT TCTGTTCGGC TTTCCCGCAC
     AGTTCATCAA
301  CGGCACGATT AATTCGTGGT TCGGCAACGA TACCCACGAG
     GCGCTTGAAC
351  GCAGCCTCAA TTTGAGCAAG TCCGCATTGA ATTTGGCGGC
     AGACAACGCC
401  CTCGGCAACG CCGTCCCCGT GCAGATAGAC CTCATCGGCG
     CGGCTTCCCT
451  GCCCGGGGAT ATGGGCAGGG TGCTGGAACA TTACGCCGGC
     AGCGGTTTTG
501  CCCAGCTTGC CCTGTACAAy ksCGCAAGCG GCAAAATCGA
     AAAAAGCATC
551  AACCCGCACA AGCTCGATCA GCCGTTTCCA GGTAAGGCGC
     GTTGGGAaAa
601  AATCCaACGG GCGGGTTCGG TCAGGGATTT GGAAAGCATA
     GGCGGCGTAT
651  TGTaCGCGCA GGGCTGGCTG TCGGCGGGTA CGCACwACGG
     GCGCGATTAC
701  GCCTTGTTTT TCCGTCAGCC GGTTCCCAAA GGCGTGGCAG
     AGGATGCCGT
751  yTTAATCGAA AAGGCAAGGG CGAAATATGC TGAGTTGAGT
     TACAGCAAAA
801  AAGGTTTGCA GACCTTTTTC CTGGCAACCC TGCTGATTGC
     CTCGCTGCTG
851  TCGATTTTTC TTGCACTGGT CATGGCACTG TATTTCGCCC
     GCCGTTTCGT
901  CGAACCCGTC CTATCGCTTG CCGAGGGGGC GAAGGCGGTG
     GCGCAAGGCG
951  ATTTCAGCCA GACGCGCCCC GTGTTGCGCA ACGACGAGTT
     CGGACGCTTG
1001 ACCArGTTGT TCAACCACAT GACCGAGCAG CTTTCCATCG
     CCAAAGATGC
1051 AGACGAGCGC AACCGCCGGC GCGAGGAAGC CGCCAGGCAT
     TATCTTGAAT
1101 GCGTGTTGGA GGGGCTGACC ACGGGCGTGG TGGTGTTTGA
     CGAACAAGGC
1151 TGTCTGAAAA CCTTCAACAA AGCGGCGGGT ACC..

This corresponds to the amino acid sequence <SEQ ID 250; ORF64>:

1   MRRFLPIAAI CAXXLXXGLT AATGSTSSLA DYFWWIVAFS
    AMLLLVLSAV
51  LARYVILLLK DRRDGVFGSX XAKXPXXXMF TLVAXLPGVF
    LFGFPAQFIN
101 GTINSWFGND THEALERSLN LSKSALNLAA DNALGNAVPV
    QIDLIGAASL
151 PGDMGRVLEH YAGSGFAQLA LYNXASGKIE KSINPHKLDQ
    PFPGKARWEK
201 IQRAGSVRDL ESIGGVLYAQ GWLSAGTHXG RDYALFFRQP
    VPKGVAEDAV
251 LIEKARAKYA ELSYSKKGLQ TFFLATLLIA SLLSIFLALV
    MALYFARRFV
301 EPVLSLAEGA KAVAQGDFSQ TRPVLRNDEF GRLTXLFNHM
    TEQLSIAKDA
351 DERNRRREEA ARHYLECVLE GLTTGVVVFD EQGCLKTFNK
    AAGT..

Further work revealed the complete nucleotide sequence <SEQ ID 251>:

1    ATGCGCCGTT TTCTACCGAT CGCAGCCATA TGCGCCGTCG
     TCCTGTTGTA
51   CGGACTGACG GCGGCAACCG GCAGCACCAG TTCGCTGGCG
     GATTATTTCT
101  GGTGGATTGT TGCGTTCAGC GCAATGCTGC TGCTGGTGTT
     GTCCGCCGTT
151  TTGGCACGTT ATGTCATATT GCTGTTGAAA GACAGGCGCG
     ACGGCGTATT
201  CGGTTCGCAG ATTGCCAAAC GCCTTTCTGG GATGTTTACG
     CTGGTTGCCG
251  TACTGCCCGG CGTGTTTCTG TTCGGCGTTT CCGCACAGTT
     CATCAACGGC
301  ACGATTAATT CGTGGTTCGG CAACGATACC CACGAGGCGC
     TTGAACGCAG
351  CCTCAATTTG AGCAAGTCCG CATTGAATTT GGCGGCAGAC
     AACGCCCTCG
401  GCAACGCCGT CCCCGTGCAG ATAGACCTCA TCGGCGCGGC
     TTCCCTGCCC
451  GGGGATATGG GCAGGGTGCT GGAACATTAC GCCGGCAGCG
     GTTTTGCCCA
501  GCTTGCCCTG TACAATGCCG CAAGCGGCAA AATCGAAAAA
     AGCATCAACC
551  CGCACAAGCT CGATCAGCCG TTTCCAGGTA AGGCGCGTTG
     GGAAAAAATC
601  CAACGGGCGG GTTCGGTCAG GGATTTGGAA AGCATAGGCG
     GCGTATTGTA
651  CGCGCAGGGC TGGCTGTCGG CGGGTACGCA CAACGGGCGC
     GATTACGCCT
701  TGTTTTTCCG TCAGCCGGTT CCCAAAGGCG TGGCAGAGGA
     TGCCGTCTTA
751  ATCGAAAAGG CAAGGGCGAA ATATGCTGAG TTGAGTTACA
     GCAAAAAAGG
801  TTTGCAGACC TTTTTCCTGG CAACCCTGCT GATTGCCTCG
     CTGCTGTCGA
851  TTTTTCTTGC ACTGGTCATG GCACTGTATT TCGCCCGCCG
     TTTCGTCGAA
901  CCCGTCCTAT CGCTTGCCGA GGGGGCGAAG GCGGTGGCGC
     AAGGCGATTT
951  CAGCCAGACG CGCCCCGTGT TGCGCAACGA CGAGTTCGGA
     CGCTTGACCA
1001 AGTTGTTCAA CCACATGACC GAGCAGCTTT CCATCGCCAA
     AGAAGCAGAC
1051 GAGCGCAACC GCCGGCGCGA GGAAGCCGCC AGGCATTATC
     TTGAATGCGT
1101 GTTGGAGGGG CTGACCACGG GCGTGGTGGT GTTTGACGAA
     CAAGGCTGTC
1151 TGAAAACCTT CAACAAAGCG GCGGAACAGA TTTTGGGGAT
     GCCGCTTACC
1201 CCCCTGTGGG GCAGCAGCCG GCACGGTTGG CACGGCGTTT
     CGGCGCAGCA
1251 GTCCCTGCTT GCCGAAGTGT TTGCCGCCAT CGGCGCGGCG
     GCAGGTACGG
1301 ACAAACCGGT CCATGTGAAA TATGCCGCGC CGGACGATGC
     CAAAATCCTG
1351 CTGGGCAAGG CAACCGTCCT GCCCGAAGAC AACGGCAACG
     GCGTGGTAAT
1401 GGTGATTGAC GACATCACCG TTTTGATACA CGCGCAAAAA
     GAAGCCGCGT
1451 GGGGCGAAGT GGCGAAGCGG CTGGCACACG AAATCCGCAA
     TCCGCTCACG
1501 CCCATCCAGC TTTCCGCCGA ACGGCTGGCG TGGAAATTGG
     GCGGGAAGCT
1551 GGATGAGCAG GATGCGCAAA TCCTGACGCG TTCGACCGAC
     ACCATCGTCA
1601 AACAGGTGGC GGCATTGAAG GAAATGGTCG AAGCATTCCG
     CAATTATGCG
1651 CGTTCCCCTT CGCTCAAATT GGAAAATCAG GATTTGAACG
     CCTTAATCGG
1701 CGATGTGTTG GCATTGTATG AAGCCGGTCC GTGCCGGTTT
     GCGGCGGAGC
1751 TTGCCGGCGA ACCGCTGACG GTGGCGGCGG ATACGACCGC
     CATGCGGCAG
1801 GTGCTGCACA ATATTTTCAA AAATGCCGCC GAAGCGGCGG
     AAGAAGCCGA
1851 TGTGCCCGAA GTCAGGGTAA AATCGGAAAC AGGGCAGGAC
     GGTCGGATTG
1901 TCCTGACGGT TTGCGACAAC GGCAAAGGGT TCGGCAGGGA
     AATGCTGCAC
1951 AACGCCTTCG AGCCGTATGT AACGGACAAA CCGGCGGGAA
     CGGGATTGGG
2001 TCTGCCTGTG GTGAAAAAAA TCATTGAAGA ACACGGCGGC
     CGCATCAGCC
2051 TGAGCAATCA GGATGCGGGT GGCGCGTGTG TCAGAATCAT
     CTTGCCAAAA
2101 ACGGTAAAAA CTTATGCGTA G

This corresponds to the amino acid sequence <SEQ ID 252; ORF64-1>:

1   MRRFLPIAAI CAVVLLYGLT AATGSTSSLA DYFWWIVAFS
    AMLLLVLSAV
51  LARYVILLLK DRRDGVFGSQ IAKRLSGMFT LVAVLPGVFL
    FGVSAQFING
101 TINSWFGNDT HEALERSLNL SKSALNLAAD NALGNAVPVQ
    IDLIGAASLP
151 GDMGRVLEHY AGSGFAQLAL YNAASGKIEK SINPHKLDQP
    FPGKARWEKI
201 QRAGSVRDLE SIGGVLYAQG WLSAGTHNGR DYALFFRQPV
    PKGVAEDAVL
251 IEKARAKYAE LSYSKKGLQT FFLATLLIAS LLSIFLALVM
    ALYFARRFVE
301 PVLSLAEGAK AVAQGDFSQT RPVLRNDEFG RLTKLFNHMT
    EQLSIAKEAD
351 ERNRRREEAA RHYLECVLEG LTTGVVVFDE QGCLKTFNKA
    AEQILGMPLT
401 PLWGSSRHGW HGVSAQQSLL AEVFAAIGAA AGTDKPVHVK
    YAAPDDAKIL
451 LGKATVLPED NGNGVVMVID DITVLIHAQK EAAWGEVAKR
    LAHEIRNPLT
501 PIQLSAERLA WKLGGKLDEQ DAQILTRSTD TIVKQVAALK
    EMVEAFRNYA
551 RSPSLKLENQ DLNALIGDVL ALYEAGPCRF AAELAGEPLT
    VAADTTAMRQ
601 VLHNIFKNAA EAAEEADVPE VRVKSETGQD GRIVLTVCDN
    GKGFGREMLH
651 NAFEPYVTDK PAGTGLGLPV VKKIIEEHGG RISLSNQDAG
    GACVRIILPK
701 TVKTYA*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF64 shows 92.6% identity over a 392aa overlap with an ORF (ORF64a) from strain A of N. meningitidis:

The complete length ORF64a nucleotide sequence <SEQ ID 253> is:

1    ATGCGCCGTT TTCTACCGAT CGCAGCCATA TGCGCCGTCG
     TCCTGTTGTA
51   CGGACTGACG GCGGCAACCG GCAGCACCAG TTCGCTGGCG
     GATTATTTCT
101  GGTGGATTGT TGCGTTCAGC GCAATGCTGC TGCTGGTGTT
     GTCCGCCGTT
151  TTGGCACGTT ATGTCATATT GCTGTTGAAA GACAGGCGCG
     ACGGCGTATT
201  CGGTTCGCAG ATTGCCAAAC GCCTTTCCGG GATGTTTACG
     CTGGTTGCCG
251  TACTGCCCGG CGTGTTTCTG TTCGGCGTTT CCGCACAGTT
     TATCAACGGC
301  ACGATTAATT CGTGGTTCGG CAACGATACC CACGAGGCGC
     TTGAACGCAG
351  CCTCAATTTG AGCAAGTCCG CATTGAATCT GGCGGCAGAC
     AACGCCCTTG
401  GCAACGCCAT CCCCGTGCAG ATAGACNTCA TCGGCGCGGC
     TTCCCTGCCC
451  NGGGATATGG GCAGGGTGCT GGAACATTAC GCCGGCAGCG
     GTTTTGCCCA
501  GCTTGCCCTG TACAATGCCG CAAGCGGCAA AATCGAAAAA
     AGCATCAACC
551  CGCACAAGCT CGATCAGCCG TTTCCAGGTA AGGCGCGTTG
     GGAAAAAATC
601  CAACAGGCGG GTTCGGTCAG GGATNNGGAA AGCATAGGCG
     GCGTATTGTA
651  CGCGCANGGC TGGCTGTCGG CAGNNACGCA CAACGGGCGC
     GATTACGCCT
701  TGTTTTTCCG TCAGCCGGTT CCCAAAGGCG TGGCAGAGGA
     TGCCGTCTTA
751  ATCGAAAAGG CAAGGGCGNA ANANNNTNAG TTGAGTTACA
     GCAAAAAAGG
801  TTTGCAGACC TTTTTCCTNG CAACCCTGCT GATTGCCTCN
     CTGCTGTCGA
851  TTTTTCTTGC ACTGGTCATG GCACTGTATT TCGCCCGCCG
     TTTCGTCGAA
901  CCCGTCCTAT CGCTTGCCGA GGGGGCGAAG GCGGTGGCGC
     AAGGCGATTT
951  CAGCCAGACG CGCCCCGTGT TGCGCAACGA CGAGTTCGGA
     CGCTTGACCA
1001 AGTTGTTCAA CCACATGACC GAGCAGCTTT CCATCGCCAA
     AGAAGCAGAC
1051 GAGCGCAACC GCCGGCGCGA GGAAGCCGCC AGACATTATC
     TCGAATGCGT
1101 GTTGGAGGGG CTGACCACGG GCGTGGTGGT GTTTGACGAA
     CAAGGCTGTC
1151 TGAAAACCTT CAACAAAGCG GCGGAACAGA TTTTGGGGAT
     GCCGCTTACC
1201 CCCCTGTGGG GCAGCAGCCG GCACGGTTGG CACGGCGTTT
     CGGCGCAGCA
1251 GTCCCTGCTT GCCGAAGTGT TTGCCGCCAT CGGCGCGGCG
     GCAGGTACGG
1301 ACAAACCGGT CCATGTGAAA TATGCCGCGC CGGACGATGC
     CAAAATCCTG
1351 CTGGGCAAGG CAACCGTCCT GCCCGAAGAC AACNGCAACG
     GCGTGGTAAT
1401 GGTGATTGAC GACATCACCG TTTTGATACA CGCGCAAAAA
     GAAGCCGCGT
1451 GGGGCGAAGT GGCAAAACGG CTGGCACACG AAATCCGCAA
     TCCGCTCACG
1501 CCCATCCAGC TTTCTGCCGA ACGGCTGGCG TGGAAATTGG
     GCGGGAAGCT
1551 GGACGAGCAN GACGCGCAAA TCCTGACACG TTCGACCGAC
     ACCATCATCA
1601 AACAAGTGGC GGCATTAAAA GAAATGGTCG AGGCATTCCG
     CAATTACNCG
1651 CGTTCCCCTT CGNCTCAATT GGAAAATCAG GATTTGAACG
     CCTTAATCGG
1701 CGATGTGTTG GCATTGTACG AAGCTGGTCC GTGCCGGTTT
     GCGGCGGAAC
1751 TTGCCGGCGA ACCGCTGATG ATGGCGGCGG ATACGACCGC
     CATGCGGCAG
1801 GTGCTGCACA ATATTTTCAA AAATGCCGCC GAAGCGGCGG
     AAGAAGCCGA
1851 TGTGCCCGAA GTCAGGGTAA AATCGGAAGC GGGGCAGGAC
     GGACGGATTG
1901 TCCTGACAGT TTGCGACAAC GGCAAGGGGT TCGGCAGGGA
     AATGCTGCAC
1951 AATGCCTTCG AGCCGTATGT AACGGACAAA CCGGCTGGAA
     CGGGATTGNG
2001 ACTGCCCGTG GTGAAAAAAA TCATTGAAGA ACACGGCGGC
     CNCATCAGCC
2051 TGAGCAATCA GGATGCGGGC GGCGCGTNTG TCAGAATCAT
     CTTGCCAAAA
2101 ACGGTAGAAA CTTATGCGTA G

This encodes a protein having amino acid sequence <SEQ ID 254>:

1   MRRFLPIAAI CAVVLLYGLT AATGSTSSLA DYFWWIVAFS
    AMLLLVLSAV
51  LARYVILLLK DRRDGVFGSQ IAKRLSGMFT LVAVLPGVFL
    FGVSAQFING
101 TINSWFGNDT HEALERSLNL SKSALNLAAD NALGNAIPVQ
    IDXIGAASLP
151 XDMGRVLEHY AGSGFAQLAL YNAASGKIEK SINPHKLDQP
    FPGKARWEKI
201 QQAGSVRDXE SIGGVLYAXG WLSAXTHNGR DYALFFRQPV
    PKGVAEDAVL
251 IEKARAXXXX LSYSKKGLQT FFLATLLIAS LLSIFLALVM
    ALYFARRFVE
301 PVLSLAEGAK AVAQGDFSQT RPVLRNDEFG RLTKLFNHMT
    EQLSIAKEAD
351 ERNRRREEAA RHYLECVLEG LTTGVVVFDE QGCLKTFNKA
    AEQILGMPLT
401 PLWGSSRHGW HGVSAQQSLL AEVFAAIGAA AGTDKPVHVK
    YAAPDDAKIL
451 LGKATVLPED NXNGVVMVID DITVLIHAQK EAAWGEVAKR
    LAHEIRNPLT
501 PIQLSAERLA WKLGGKLDEX DAQILTRSTD TIIKQVAALK
    EMVEAFRNYX
551 RSPSXQLENQ DLNALIGDVL ALYEAGPCRF AAELAGEPLM
    MAADTTAMRQ
601 VLHNIFKNAA EAAEEADVPE VRVKSEAGQD GRIVLTVCDN
    GKGFGREMLH
651 NAFEPYVTDK PAGTGLXLPV VKKIIEEHGG XISLSNQDAG
    GAXVRIILPK
701 TVETYA*

ORF64a and ORF64-1 show 96.6% identity in 706 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF64 shows 86.6% identity over a 387aa overlap with a predicted ORF (ORF64.ng) from N. gonorrhoeae:

An ORF64ng nucleotide sequence <SEQ ID 255> was predicted to encode a protein having amino acid sequence <SEQ ID 256>:

1   MRRFLPIAAI CAVVLLYGLT AATGSTSSLA DYFWWIVSFS
    AMLLLVLSAV
51  LARYVILLLK DRRNGVFGSQ IAKRLSGMFT LVAVLPGLFL
    FGISAQFING
101 TINSWFGNDT HEALERSLNL SKSALDLAAD NAVSNAVPVQ
    IDLIGTASLS
151 GNMGSVLEHY AGSGFAQLAL YNAASGKIEK SINPHQFDQP
    LPDKEHWEQI
201 QQTGSVRSLE SIGGVLYAQG WLSAGTHNGR DYALFFRQPI
    PENVAQDAVL
251 IEKARAKYAE LSYSKKGLQT FFLVTLLIAS LLSIFLALVM
    ALYFARRFVE
301 PILSLAEGAK AVAQGDFSQT RPVLRNDEFG RLTKLFNHMT
    EQLSIAKEAD
351 ERNRRREEAA RHYLECVLDG LTTGVVVSYP LSCCRTAVFS
    TCHSSPLSYF*

Further work revealed the complete gonococcal DNA sequence <SEQ ID 257>:

1    ATGCGCCGCT TCCTACCGAT CGCAGCCATA TGCGCCGTCG
     TCCTGCTGTA
51   CGGATTGACG GCGGCGACCG GCAGCACCAG TTCGCTGGCG
     GATTATTTCT
101  GGTGGATAGT CTCGTTCAGC GCAATGCTGC TGCTGGTGTT
     GTCCGCCGTT
151  TTGGCACGTT ATGTCATATT GCTGTTGAAA GACAGGCGCA
     ACGGCGTGTT
201  CGGTTCGCAG ATTGCCAAAC GCCTTTCCGG GATGTTCACG
     CTGGTCGCCG
251  TACTGCCCGG CTTGTTCCTG TTCGGCATTT CCGCGCAGTT
     TATCAACGGC
301  ACGATTAATT CGTGGTTCGG CAACGACACC CACGAAGCCC
     TCGAACGCAG
351  CCTTAATTTG AGCAAGTCCG CACTGGATTT GGCGGCAGAC
     AATGCCGTCA
401  GCAACGCCGT TCCCGTACAG ATAGACCTCA TCGGCACCGC
     CTCCCTGTCG
451  GGCAATATGG GCAGTGTGCT GGAACACTAC GCCGGCAGCG
     GTTTTGCCCA
501  GCTTGCCCTG TACAATGCCG CAAGCGGGAA AATCGAAAAA
     AGCATCAATC
551  CGCACCAATT CGACCAGCCG CTTCCCGACA AAGAACATTG
     GGAACAGATT
601  CAGCAGACCG GTTCGGTTCG GAGTTTGGAA AGCATAGGCG
     GCGTATTGTA
651  CGCGCAGGGA TGGTTGTCGG CAGGTACGCA CAACGGGCGC
     GATTACGCGC
701  TGTTCTTCCG CCAGCCGATT CCCGAAAATG TGGCACAGGA
     TGCCGTTCTG
751  ATTGAAAAGG CGCGGGCGAA ATATGCCGAA TTGAGTTACA
     GCAAAAAAGG
801  TTTGCAGACC TTTTTTCTGG TAACCCTGCT GATTGCCTCG
     CTGCTGTCGA
851  TTTTTCTTGC GCTGGTAATG GCACTGTATT TTGCCCGCCG
     TTTCGTCGAA
901  CCCATTCTGT CGCTTGCCGA GGGCGCAAAG GCGGTGGCGC
     AGGGTGATTT
951  CAGCCAGACG CGCCCCGTAT TGCGCAACGA CGAGTTCGGA
     CGTTTGACCA
1001 AGCTGTTCAA CCATATGACC GAGCAGCTTT CCATCGCCAA
     AGAAGCAGAC
1051 GAACGCAACC GCCGGCGCGA GGAAGCCGCC CGTCACTACC
     TCGAGTGCGT
1101 GTTGGATGGG TTGACTACCG GTGTGGTGGT GTTTGACGAA
     AAAGGCCGTT
1151 TGAAAACCTT CAACAAGGCG GCGGAACAGA TTTTGGGGAT
     GCCGCTCGCC
1201 CCCCTGTGGG GCAGCAGCCG GCACGGTTGG CACGGCGTTT
     CGGCGCAGCA
1251 GTCCCTGCTT GCCGAAGTGT TtgccgccAT CGGTGCGGCG
     GCAGGTACGG
1301 ACAAACCGGT CCAGGTGGAA TATGCCGCGC CGGACGATGC
     CAAAATCCTG
1351 CTGGGCAAGG CGACGGTATT GCCCGAAGAC AACGGCAACG
     GCGTGGTGAT
1401 GGTGATTGAC GACATCACCG TGCTGATACG CGCGCAAAAA
     GAAGCCGCGT
1451 GGGGTGAAGT GGCGAAGCGG CTGGCACACG AAATCCGCAA
     TCCGCTCACG
1501 CCCATCCAGC TTTCCGCCGA ACGGCTGGCG TGGAAATTGG
     GCGGGAAGCT
1551 GGACGATCAG GACGCGCAAA TCCTGACGCG TtcgACCGAC
     ACCATCATCA
1601 AACAGgtggc gGCGTTAAAA GAAATGGTCG AGGCATTCCG
     CAATTACGCG
1651 CGCGCCCCTT CGCTCAAACT GGAAAATCAG GATTTGAACG
     CCTTAATCGG
1701 CGATGTTTTG GCCCTGTACG AAGCCGGCCC GTGCCGGTTT
     GAGGCGGAAC
1751 TTGCCGGCGA ACCGCTGATG ATGGCGGCGG ATACGACCGC
     CATGCGGCAG
1801 GTGCTGCACA ATATTTTCAA AAATGCCGCC GAAGCGGCGG
     AAGAAGCCGA
1851 TATGCCCGAA GTCAGGGTAA AATCGGAAAC GGGGCAGGAC
     GGACGGATTG
1901 TCCTGACGGT TTGCGACAAC GGCAAGGGAT TCGGCAAGGA
     AATGCTGCAC
1951 AATGCTTTCG AGCCGTATGT GACGGATAAG CCGGCGGGAA
     CGGGACTGGG
2001 TCTGCCTGTA GTGAAAAAAA TCATTGGAGA ACACGGCGGC
     CGCATCAGCC
2051 TGAGCAATCA GGATGCGGGT GGGGCGTGTG TCAGAATCAT
     CTTGCCAAAA
2101 ACGGTAGAAA CTTATGCGTA G

This corresponds to the amino acid sequence <SEQ ID 258; ORF64ng-1>:

1   MRRFLPIAAI CAVVLLYGLT AATGSTSSLA DYFWWIVSFS
    AMLLLVLSAV
51  LARYVILLLK DRRNGVFGSQ IAKRLSGMFT LVAVLPGLFL
    FGISAQFING
101 TINSWFGNDT HEALERSLNL SKSALDLAAD NAVSNAVPVQ
    IDLIGTASLS
151 GNMGSVLEHY AGSGFAQLAL YNAASGKIEK SINPHQFDQP
    LPDKEHWEQI
201 QQTGSVRSLE SIGGVLYAQG WLSAGTHNGR DYALFFRQPI
    PENVAQDAVL
251 IEKARAKYAE LSYSKKGLQT FFLVTLLIAS LLSIFLALVM
    ALYFARRFVE
301 PILSLAEGAK AVAQGDFSQT RPVLRNDEFG RLTKLFNHMT
    EQLSIAKEAD
351 ERNRRREEAA RHYLECVLDG LTTGVVVFDE KGRLKTFNKA
    AEQILGMPLA
401 PLWGSSRHGW HGVSAQQSLL AEVFAAIGAA AGTDKPVQVE
    YAAPDDAKIL
451 LGKATVLPED NGNGVVMVID DITVLIRAQK EAAWGEVAKR
    LAHEIRNPLT
501 PIQLSAERLA WKLGGKLDDQ DAQILTRSTD TIIKQVAALK
    EMVEAFRNYA
551 RAPSLKLENQ DLNALIGDVL ALYEAGPCRF EAELAGEPLM
    MAADTTAMRQ
601 VLHNIFKNAA EAAEEADMPE VRVKSETGQD GRIVLTVCDN
    GKGFGKEMLH
651 NAFEPYVTDK PAGTGLGLPV VKKIIGEHGG RISLSNQDAG
    GACVRIILPK
701 TVETYA*

ORF64ng-1 and ORF64-1 show 93.8% identity in 706 aa overlap:

Furthermore, ORF64ng-1 shows significant homology to a protein from A. caulinodans:

sp|Q04850|NTRY_AZOCA NITROGEN REGULATION PROTEIN NTRY
>gi|77479|pir||S18624 ntrY protein - Azorhizobium caulinodans >gi|38737
(X63841) NtrY gene product [Azorhizobium caulinodans] Length = 771
Score = 218 bits (550), Expect = 7e−56
Identities = 195/720 (27%), Positives = 320/720 (44%), Gaps = 58/720 (8%)
Query:	7	IAAICAVVLLYGLTAATGSTSSLADYFWWIXXXXXXXXXXXXXXXXRYVILLLKDRRNGV	66
		I+A+   ++L GLT    +   +      +                R +  + K R  G
Sbjct:	35	ISALATFLILMGLTPVVPTHQVVIS----VLLVNAAAVLILSAMVGREIWRIAKARARGR	90
Query:	67	FGSQIAKRLSGMFTLVAVLPGLFLFGISAQFINGTINSWFGNDTHEALERSLNLSKSALD	126
		+++  R+ G+F +V+V+P + +  +++  ++  ++ WF   T E +  S++++++ +
Sbjct:	91	AAARLHIRIVGLFAVVSVVPAILVAVVASLTLDRGLDRWFSMRTQEIVASSVSVAQTYVR	150
Query:	127	LAADNAVSNAVPVQIDLIGTASLSGNMGSVLEHYAG--SGFAQLALYNAASGKIEKSINP	184
		A N   + + +  DL    S+          Y G  S F Q+    AA   +  ++
Sbjct:	151	EHALNIRGDILAMSADLTRLKSV----------YEGDRSRFNQILTAQAALRNLPGAMLI	200
Query:	185	HQFDQPLPDKEHWEQIQQTGSVRSLESIGGVLYAQGWLSAGTHNGRDYA-----------	233
		+ D  + ++ +   I +   V +  +IG     Q  +     N  DY
Sbjct:	201	RR-DLSVVERAN-VNIGREFIVPANLAIGDATPDQPVIYLP--NDADYVAAVVPLKDYDD	256
Query:	234	--LFFRQPIPENVAQDAVLIEKARAKYAELSYSKKGLQTFFLVTXXXXXXXXXXXXXVMA	291
		L+  + I   V       ++  A Y  L   + G+Q  F +               +
Sbjct:	257	LYLYVARLIDPRVIGYLKTTQETLADYRSLEERRFGVQVAFALMYAVITLIVLLSAVWLG	316
Query:	292	LYFARRFVEPILSLAEGAKAVAQGDFSQTRPVLRND-EFGRLTKLFNHMTEQLSIXXXXX	350
		L F++  V PI  L   A  VA+G+     P+ R + +   L + FN MT +L
Sbjct:	317	LNFSKWLVAPIRRLMSAADHVAEGNLDVRVPIYRAEGDLASLAETFNKMTHELRSQREAI	376
Query:	351	XXXXXXXXXXXHYLECVLDGLTTGVVVFDEKGRLKTFNKAAEQILGMPLAPLWGSSRHGW	410
		+ E VL G+  GV+  D + R+   N++AE++LG  L+ +    RH
Sbjct:	377	LTARDQIDSRRRFTEAVLSGVGAGVIGLDSQERITILNRSAERLLG--LSEVEALHRHLA	434
Query:	411	HGVSAQQSLLAEVFXXXXXXXXTDKPVQVEYAAPDDAKILLGKATVLPEDNG---NGVVM	467
		V     LL E            + VQ       D +  +    V  E +    +G V+
Sbjct:	435	EVVPETAGLLEEA------EHARQRSVQGNITLTRDGRERVFAVRVTTEQSPEAEHGWVV	488
Query:	468	VIDDITVLIRAQKEAAWGEVAKRLAHEIRNPLTPIQLSAERLAWKLGGKLDDQDAQILTR	527
		+DDIT LI AQ+ +AW +VA+R+AHEI+NPLTPIQLSAERL  K G  +  QD +I  +
Sbjct:	489	TLDDITELISAQRTSAWADVARRIAHEIKNPLTPIQLSAERLKRKFGRHV-TQDREIFDQ	547
Query:	528	STDTIIKQVAALKEMVEAFRNYARAPSLKLENQDLNALIGDVLALYEAGPCRFEAELAGE	587
		TDTII+QV  +  MV+ F ++AR P   +++QD++ +I   + L   G      +
Sbjct:	548	CTDTIIRQVGDIGRMVDEFSSFARMPKPVVDSQDMSEIIRQTVFLMRVGHPEVVFDSEVP	607
Query:	588	PLMMAA-DTTAMRQVLHNIFKNXXXXXXXXDMPEVRVK-------SETGQDGRIVLTVCD	639
		P M A  D   + Q L NI KN          P+VR +       +  G+D  +V+ + D
Sbjct:	608	PAMPARFDRRLVSQALTNILKNAAEAIEAVP-PDVRGQGRIRVSANRVGED--LVIDIID	664
Query:	640	NGKGFGKEMLHNAFEPYVTDKPAGTGLGLPVVKKIIGEHGGRISLSNQDAG-GACVRIIL	698
		NG G  +E  +   EPYVT +  GTGLGL +V KI+ EHGG I L++   G GA +R+ L
Sbjct:	665	NGTGLPQESRNRLLEPYVTTREKGTGLGLAIVGKIMEEHGGGIELNDAPEGRGAWIRLTL	724

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 31

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 259>:

1   ATGTACGCAT TTACCGCCGC ACAGCAACAG AAGGCACTCT
    TCCGGCTGGT
51  GCTTTTTCAT ATCCTCATCA TCGCCGCCAG CAACTATCTG
    GTGCAGTTCC
101 CTTTCCAAAT TTTCGGCATC CACACCACTT GGGGCGCATT
    TTCCTTTCCC
151 TTCATCTTCC TTGCCACCGA CCTGACCGTC CGCATTTTCG
    GTTCTCACTT
201 GGCACGGCGG ATTATCTTTT GGGTGATGTT CCCCGCCCTT
    TTGCTTTCCT
251 ACGTCTTTTC CGTTTTGTTC CACAACGGCA GTTGGACAGG
    CTTGGGCGCG
301 CTGTCCGAAT TCAACACCTT TGTCGGACGC ATCGCCTTAG
    CCAGCTTTGC
351 CGCCTACGCG ATCGGACAAA TCCTTGATAT TTTTGTATTC
    AACAAATTAC
401 GCCGTCTGAA AGCGTGGTGG ATTGCACCGA ACGCATCAAC
    CGTCATCGGG
451 CACGCGTTGG ATACG...

This corresponds to the amino acid sequence <SEQ ID 260; ORF66>:

1   MYAFTAAQQQ KALFRLVLFH ILIIAASNYL VQFPFQIFGI
    HTTWGAFSFP
51  FIFLATDLTV RIFGSHLARR IIFWVMFPAL LLSYVFSVLF
    HNGSWTGLGA
101 LSEFNTFVGR IALASFAAYA IGQILDIFVF NKLRRLKAWW
    IAPNASTVIG
151 HALDT...

Further work revealed the complete nucleotide sequence <SEQ ID 261>:

1   ATGTACGCAT TTACCGCCGC ACAGCAACAG AAGGCACTCT
    TCCGGCTGGT
51  GCTTTTTCAT ATCCTCATCA TCGCCGCCAG CAACTATCTG
    GTGCAGTTCC
101 CTTTCCAAAT TTTCGGCATC CACACCACTT GGGGCGCATT
    TTCCTTTCCC
151 TTCATCTTCC TTGCCACCGA CCTGACCGTC CGCATTTTCG
    GTTCTCACTT
201 GGCACGGCGG ATTATCTTTT GGGTGATGTT CCCCGCCCTT
    TTGCTTTCCT
251 ACGTCTTTTC CGTTTTGTTC CACAACGGCA GTTGGACAGG
    CTTGGGCGCG
301 CTGTCCGAAT TCAACACCTT TGTCGGACGC ATCGCCTTAG
    CCAGCTTTGC
351 CGCCTACGCG ATCGGACAAA TCCTTGATAT TTTTGTATTC
    AACAAATTAC
401 GCCGTCTGAA AGCGTGGTGG ATTGCACCGA CCGCATCAAC
    CGTCATCGGC
451 AACGCCTTGG ATACGCTGGT ATTTTTCGCC GTTGCCTTCT
    ACGCAAGCAG
501 CGATGGATTT ATGGCGGCAA ACTGGCAGGG CATCGCTTTT
    GTCGATTACC
551 TGTTCAAACT TACCGTCTGC ACCCTCTTCT TCCTGCCCGC
    CTACGGCGTG
601 ATACTGAATC TGCTGACGAA AAAACTGACA ACCCTGCAAA
    CCAAACAGGC
651 GCAAGACCGC CCCGCGCCCT CGCTGCAAAA TCCGTAA

This corresponds to the amino acid sequence <SEQ ID 262; ORF66-1>:

1   MYAFTAAQQQ KALFRLVLFH ILIIAASNYL VQFPFQIFGI
    HTTWGAFSFP
51  FIFLATDLTV RIFGSHLARR IIFWVMFPAL LLSYVFSVLF
    HNGSWTGLGA
101 LSEFNTFVGR IALASFAAYA IGQILDIFVF NKLRRLKAWW
    IAPTASTVIG
151 NALDTLVFFA VAFYASSDGF MAANWQGIAF VDYLFKLTVC
    TLFFLPAYGV
201 ILNLLTKKLT TLQTKQAQDR PAPSLQNP*

Computer analysis of this amino acid sequence gave the following results:

Homology with the Hypothetical Protein o221 of E. coli (Accession Number P37619)

ORF66 and o221 protein show 67% aa identity in 155aa overlap:

orf66	1	MYAFTAAQQQKALFRLVLFHILIIAASNYLVQFPFQIFGIHTTWGAFSFPFIFLATDLTV	60
		M  F+  Q+ KALF L LFH+L+I +SNYLVQ P  I G HTTWGAFSFPFIFLATDLTV
o221	1	MNVFSQTQRYKALFWLSLFHLLVITSSNYLVQLPVSILGFHTTWGAFSFPFIFLATDLTV	60
orf66	61	RIFGSHLARRIIFWVMFPALLLSYVFSVLFHNGSWTGLGALSEFNTFVGRIALASFAAYA	120
		RIFG+ LARRIIF VM PALL+SYV S LF+ GSW G GAL+ FN FV RIA ASF AYA
o221	61	RIFGAPLARRIIFAVMIPALLISYVISSLFYMGSWQGFGALAHFNLFVARIATASFMAYA	120
orf66	121	IGQILDIFVFNKLRRLKAWWIAPNASTVIGHALDT	155
		+GQILD+ VFN+LR+ + WW+AP AST+ G+  DT
o221	121	LGQILDVHVFNRLRQSRRWWLAPTASTLFGNVSDT	155

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF66 shows 96.1% identity over a 155aa overlap with an ORF (ORF66a) from strain A of N. meningitidis:

The complete length ORF66a nucleotide sequence <SEQ ID 263> is:

1   ATGTACGCAT TTACCGCCGC ACAGCAACAG AAGGCACTCT
    TCTGGCTGGT
51  GCTTTTTCAT ATCCTCATCA TCGCCGCCAG CAACTATCTG
    GTGCAGTTCC
101 CCTTCCAAAT TTCCGGCATC CACACCACTT GGGGCGCGTT
    TTCCTTTCCC
151 TTCATCTTCC TCGCCACCGA CCTGACCGTC CGCATTTTCG
    GTTCGCACTT
201 GGCACGGCGG ATTATCTTTT GGGTCATGTT CCCCGCCCTT
    TTGCTTTCCT
251 ACGTCTTTTC CGTTTTGTTC CACAACGGCA GTTGGACGGG
    CTTGGGCGCG
301 CTGTCCGAAT TCAACACCTT TGTCGGACGC ATCGCGCTGG
    CAAGTTTTGC
351 CGCCTACGCG CTCGGACAAA TCCTTGATAT TTTTGTGTTC
    AACAAATTAC
401 GCCGTCTGAA AGCGTGGTGG GTTGCCCCGA CTGCATCAAC
    CGTCATCGGC
451 AACGCCTTAG ATACGTTGGT ATTTTTCGCC GTTGCCTTCT
    ACGCAAGCAG
501 CGATGGATTT ATGGCGGCAA ACTGGCAGGG CATCGCTTTT
    GTCGATTACC
551 TGTTCAAACT CACCGTCTGC GGTCTGTTTT TCCTGCCCGC
    CTACGGCGTG
601 ATTCTGAATC TGCTGACGAA AAAACTGACG ACCCTGCAAA
    CCAAACAGGC
651 GCAAGACCGC CCCGCGCCCT CGCTGCAAAA TCCGTAA

This encodes a protein having amino acid sequence <SEQ ID 264>:

1   MYAFTAAQQQ KALFWLVLFH ILIIAASNYL VQFPFQISGI
    HTTWGAFSFP
51  FIFLATDLTV RIFGSHLARR IIFWVMFPAL LLSYVFSVLF
    HNGSWTGLGA
101 LSEFNTFVGR IALASFAAYA LGQILDIFVF NKLRRLKAWW
    VAPTASTVIG
151 NALDTLVFFA VAFYASSDGF MAANWQGIAF VDYLFKLTVC
    GLFFLPAYGV
201 ILNLLTKKLT TLQTKQAQDR PAPSLQNP*

ORF66a and ORF66-1 show 97.8% identity in 228 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF66 shows 94.2% identity over a 155aa overlap with a predicted ORF (ORF66.ng) from N. gonorrhoeae:

The complete length ORF66ng nucleotide sequence <SEQ ID 265> is:

1   ATGTACGCAT TGACCGCCGC ACAGCAACAG AAGGCACTCT
    TCCGGCTGGT
51  GCTTTTCCAT ATCCTCATCA TCGCCGCCAG CAACTATCTG
    GTGCAGTTCC
101 CCTTCCGGAT TTTCGGCATC CACACCACTT GGGGCGCGTT
    TTCCTTTCCC
151 TTCATCTTCC TCGCCACCGA CCTGACCGTC CGCATTTTCG
    GTTCGCACTT
201 GGCGCGGCGG ATTATCTTTT GGGTGATGTT CCCCGCCCTT
    ttgCTTTcat
251 aCGTCTTTTC CGTTTTGTTC CACAACGGCA GTTGGACGGG
    CTTGGGCGCG
301 ctgTCCCAAT TCAACACCTT TGTCGGACGC ATCGCGCTGG
    CAAGTTTTGC
351 CGCCTACGCG CTCGGACAAA TCCTTGATAT TTTCGTATTC
    GACAAATTAC
401 GCCGTCTGAA AGCGTGGTGG ATTGCCCCGG CCGCATCAAC
    CGTCATCGGC
451 AATGCACTGG ACACGTTAGT ATTTTTTGCC GTTGCCTTTT
    ACGCAAGCAG
501 CGATGAATTT ATGGCGGCAA ACTGGCAGGG CATCGCTTTT
    GTCGATTACC
551 TGTTCAAACT TACCGTCTGC ACCCTCTTCT TCCTGCCCGC
    CTACGGCGTG
601 ATACTGAATC TGCTGACGAA AAAACTGACG GCCCTGCAAA
    CCAAACAGGC
651 GCAAGACCGC CCCGTGCCCT CGCTGCAAAA TCCGTAA

This encodes a protein having amino acid sequence <SEQ ID 266>:

1   MYALTAAQQQ KALFRLVLFH ILIIAASNYL VQFPFRIFGI
    HTTWGAFSFP
51  FIFLATDLTV RIFGSHLARR IIFWVMFPAL SLSYVFSVLF
    HNGSWTGLGA
101 PSQFNTFVGR IALASFAAYA LGQILDIFVF DKLRRLKAWW
    IAPAASTVIG
151 NALDTLVFFA VAFYASSDEF MAANWQGIAF VDYLFKLTVC
    TLFFLPAYGV
201 ILNLLTKKLT ALQTKQAQDR PVPSLQNP*

An alternative annotated sequence is:

1   MYALTAAQQQ KALFRLVLFH ILIIAASNYL VQFPFRIFGI
    HTTWGAFSFP
51  FIFLATDLTV RIFGSHLARR IIFWVMFPAL LLSYVFSVLF
    HNGSWTGLGA
101 LSQFNTFVGR IALASFAAYA LGQILDIFVF DKLRRLKAWW
    IAPAASTVIG
151 NALDTLVFFA VAFYASSDEF MAANWQGIAF VDYLFKLTVC
    TLFFLPAYGV
201 ILNLLTKKLT ALQTKQAQDR PVPSLQNP*

ORF66ng and ORF66-1 show 96.1% identity in 228 aa overlap:

Furthermore, ORF66ng shows significant homology with an E. coli ORF:

sp|P37619|YHHQ_ECOLI HYPOTHETICAL 25.3 KD PROTEIN IN FTSY-NIKA INTERGENIC
REGION (O221)
>gi|1073495|pir||S47690 hypothetical protein o221 - Escherichia coli
>gi|466607 (U00039) No definition line found [Escherichia coli]
>gi|1789882 (AE000423) hypothetical 25.3 kD protein in ftsY-nikA
intergenic region [Escherichia coli]
Length = 221
Score = 273 bits (692), Expect = 5e−73
Identities = 132/203 (65%), Positives = 155/203 (76%)
Query:	1	MYALTAAQQQKALFRLVLFHILIIAASNYLVQFPFRIFGIHTTWGAFSFPFIFLATDLTV	60
		M   +  Q+ KALF L LFH+L+I +SNYLVQ P  I G HTTWGAFSFPFIFLATDLTV
Sbjct:	1	MNVFSQTQRYKALFWLSLFHLLVITSSNYLVQLPVSILGFHTTWGAFSFPFIFLATDLTV	60
Query:	61	RIFGSHLARRIIFWVMFPALLLSYVFSVLFHNGSWTGLGALSQFNTFVGRIALASFAAYA	120
		RIFG+ LARRIIF VM PALL+SYV S LF+ GSW G GAL+ FN FV RIA ASF AYA
Sbjct:	61	RIFGAPLARRIIFAVMIPALLISYVISSLFYMGSWQGFGALAHFNLFVARIATASFMAYA	120
Query:	121	LGQILDIFVFDKLRRLKAWWIAPAASTVIGNALDTLVFFAVAFYASSDEFMAANWQGIAF	180
		LGQILD+ VF++LR+ + WW+AP AST+ GN  DTL FF +AF+ S D FMA +W  IA
Sbjct:	121	LGQILDVHVFNRLRQSRRWWLAPTASTLFGNVSDTLAFFFIAFWRSPDAFMAEHWMEIAL	180
Query:	181	VDYLFKLTVCTLFFLPAYGVILN	203
		VDY FK+ +  +FFLP YGV+LN
Sbjct:	181	VDYCFKVLISIVFFLPMYGVLLN	203

Based on this analysis, including the homology with the E. coli protein and the presence of several putative transmembrane domains in the gonococcal protein, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 32

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 267>:

1   ATGGTCATAA AATATACAAA TTTGAATTTT GCGAAATTGT
    CGATAATTGC
51  AATTTTGATG ATGTATTCGT TTGAAGCGAA TGCAAAyGCA
    GTmwrAATAT
101 CTGAAACTGT TTCAGTTGAT ACCGGACAAG GTGCGAAAAT
    TCATAAGTTT
151 GTACCTAAAA ATAGTAAAAC TTATTCATCT GATTTAATAA
    AAACGGTAGA
201 TTTAACACAC AyyCCTACGG GCGCAAAAGC CCGAATCAAC
    GCCAAAATAA
251 CCGCCAGCGT ATCCCGCGCC GGCGTATTGG CGGGGGTCGG
    CAAACTTGCC
301 CGCTTAGgCG CGAAATTCAG CACAAGGGCG GTtCCCTATG
    TCGGAACAGC
351 CcTTTTAGCC CACGACGTAT ACGAAAcTTT CAAAGAAGAC
    ATACAGGCAC
401 GAGGCTACCA ATACGACCCC GAAACCGACA AATTTGTAAA
    AGGCTACGAA
451 TATAGTAATT GCCTTTGGTA CGAAGACAAA AGACGTATTA
    ATAGAACCTA
501 TGGCTGCTAC GGCGTTGAT..

This corresponds to the amino acid sequence <SEQ ID 268; ORF72>:

1   MVIKYTNLNF AKLSIIAILM MYSFEANANA VXISETVSVD
    TGQGAKIHKF
51  VPKNSKTYSS DLIKTVDLTH XPTGAKARIN AKITASVSRA
    GVLAGVGKLA
101 RLGAKFSTRA VPYVGTALLA HDVYETFKED IQARGYQYDP
    ETDKFVKGYE
151 YSNCLWYEDK RRINRTYGCY GVD..

Further work revealed the complete nucleotide sequence <SEQ ID 269>:

1   ATGGTCATAA AATATACAAA TTTGAATTTT GCGAAATTGT
    CGATAATTGC
51  AATTTTGATG ATGTATTCGT TTGAAGCGAA TGCAAATGCA
    GTAAAAATAT
101 CTGAAACTGT TTCAGTTGAT ACCGGACAAG GTGCGAAAAT
    TCATAAGTTT
151 GTACCTAAAA ATAGTAAAAC TTATTCATCT GATTTAATAA
    AAACGGTAGA
201 TTTAACACAC ATCCCTACGG GCGCAAAAGC CCGAATCAAC
    GCCAAAATAA
251 CCGCCAGCGT ATCCCGCGCC GGCGTATTGG CGGGGGTCGG
    CAAACTTGCC
301 CGCTTAGGCG CGAAATTCAG CACAAGGGCG GTTCCCTATG
    TCGGAACAGC
351 CCTTTTAGCC CACGACGTAT ACGAAACTTT CAAAGAAGAC
    ATACAGGCAC
401 GAGGCTACCA ATACGACCCC GAAACCGACA AATTTGCAAA
    GGTCTCAGGC
451 TAA

This corresponds to the amino acid sequence <SEQ ID 270; ORF72-1>:

1   MVIKYTNLNF AKLSIIAILM MYSFEANANA VKISETVSVD
    TGQGAKIHKF
51  VPKNSKTYSS DLIKTVDLTH IPTGAKARIN AKITASVSRA
    GVLAGVGKLA
101 RLGAKFSTRA VPYVGTALLA HDVYETFKED IQARGYQYDP
    ETDKFAKVSG
151 *

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF72 shows 98.0% identity over a 147aa overlap with an ORF (ORF72a) from strain A of N. meningitidis:

The complete length ORF72a nucleotide sequence <SEQ ID 271> is:

1   ATGGTCATAA AATATACAAA TTTGAATTTT GCGAAATTGT
    CGATAATTGC
51  AATTTTGATG ATGTATTCGT TTGAAGCGAA TGCAAATGCA
    GTAAAAATAT
101 CTGAAACTGT TTCAGTTGAT ACCGGACAAG GTGCGAAAAT
    TCATAAGTTT
151 GTACCTAAAA ATAGTAAAAC TTATTCATCT GATTTAATAA
    AAACGGTAGA
201 TTTAACACAC ATCCCTACGG GCGCAAAAGC CCGAATCAAC
    GCCAAAATAA
251 CCGCCAGCGT ATCCCGCGCC GGCGTATTGG CGGGGGTCGG
    CAAACTTGCC
301 CGCTTAGGCG CGAAATTCAG CACAAGGGCG GTTCCCTATG
    TCGGAACAGC
351 CCTTTTAGCC CACGACGTAT ACGAAACTTT CAAAGAAGAC
    ATACAGGCAC
401 GAGGCTACCA ATACGACCCC GAAACCGACA AATTTGCAAA
    GGTCTCAGGC
451 TAA

This encodes a protein having amino acid sequence <SEQ ID 272>:

1   MVIKYTNLNF AKLSIIAILM MYSFEANANA VKISETVSVD
    TGQGAKIHKF
51  VPKNSKTYSS DLIKTVDLTH IPTGAKARIN AKITASVSRA
    GVLAGVGKLA
101 RLGAKFSTRA VPYVGTALLA HDVYETFKED IQARGYQYDP
    ETDKFAKVSG
151 *

ORF72a and ORF72-1 show 100.0% identity in 150 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF72 shows 89% identity over a 173aa overlap with a predicted ORF (ORF72.ng) from N. gonorrhoeae:

An ORF72ng nucleotide sequence <SEQ ID 273> was predicted to encode a protein having amino acid sequence <SEQ ID 274>:

1   MVTKHTNLNF AKLSIIAILM MYSFEANANA VKISETLSVD
    TGQGAKVHKF
51  VPKSSNIYSS DLTKAVDLTH IPTGAKARIN AKITASVSRA
    GVLSGVGKLV
101 RQGAKFGTRA VPYVGTALLA HDVYETFKED IQARGCRYDP
    ETDKFVKGYE
151 YANCLWYEDE RRINRTYGCY GVDSSIMRLM PDRSRFPEVK
    QLMESQMYRL
201 ARPFWNWRKE ELNKLSSLDW NNFVLNRCTF DWNGGGCAVN
    KGDDFRAGAS
251 FSLGRNPKYK EEMDAKKPEE ILSLKVDADP DKYIEATGYP
    GYSEKVEVAP
301 GTKVNMGPVT DRNGNPVQVA ATFGRDAQGN TTADVQVIPR
    PDLTPASAEA
351 PHAQPLPEVS PAENPANNPD PDENPGTRPN PEPDPDLNPD
    ANPDTDGQPG
401 TSPDSPAVPD RPNGRHRKER KEGEDGGLSC DYFPEILACQ
    EMGKPSDRMF
451 HDISIPQVTD DKTWSSHNFL PSNGVCPQPK TFHVFGRQYR
    ASYEPLCVFA
501 EKIRFAVLLA FIIMSAFVVF GSLGGE*

After further analysis, the following gonococcal DNA sequence <SEQ ID 275> was identified:

1   ATGGTCACAA AACATACAAA TTTGAATTTT GCGAAATTGT
    CGATAATTGC
51  AATTTTGATG ATGTATTCGT TTGAAGCGAA TGCAAATGCA
    GTAAAAATAT
101 CTGAAACTCT TTCGGTTGAT ACCGGACAAG GCGCGAAAGT
    TCATAAGTTC
151 GTTCCTAAAT CAAGTAATAT TTATTCATCT GATTTAACAA
    AAGCGGTAGA
201 TTTAACGCAT ATCCCCACGG GCGCAAAAGC CCGAATCAAC
    GCCAAAATAA
251 CCGCCAGCGT ATCCCGCGCC GGCGTATTGT CGGGGGTCGG
    CAAACTTGTC
301 CGCCAAGGCG CGAAATTCGG CACAAGGGCG GTTCCCTATG
    TCGGAACAGC
351 CCTTTTAGCC CACGACGTAT ACGAAACTTT CAAAGAAGAC
    ATACAGGCAC
401 GAGGCTGCCG ATACGATCCC GAAACCGACA AATTT

This corresponds to the amino acid sequence <SEQ ID 276; ORF72ng-1>:

1   MVTKHTNLNF AKLSIIAILM MYSFEANANA VKISETLSVD
    TGQGAKVHKF
51  VPKSSNIYSS DLTKAVDLTH IPTGAKARIN AKITASVSRA
    GVLSGVGKLV
101 RQGAKFGTRA VPYVGTALLA HDVYETFKED IQARGCRYDP
    ETDKF

ORF72ng-1 and ORF721-1 show 89.7% identity in 145 aa overlap:

Based on this analysis, including the presence of a putative leader sequence and transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 33

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 277>:

1   ATGAGATTTT TCGGTATCGG TTTTTTGGTG CTGCTGTTTT
    TGGAGATTAT
51  GTCGATTGTG TGGGTTGCCG ATTGGCTGGG CGGCGGCTGG
    ACGTTGTTTT
101 TGATGGCGGC AGGTTTTGCC GCCGGCGTGC TGATGCTCAG
    GCAAACCGGG
151 GCTGACCGGT CTTTTATTGG CGGGCGCGGC AATGAGAAGC
    GGCGGGAAGG
201 TATCCGTTTA TCAGATGTTG TGGCCTATC..

This corresponds to the amino acid sequence <SEQ ID 278; ORF73>:

1  MRFFGIGFLV LLFLEIMSIV WVADWLGGGW TLFLMAAGFA
   AGVLMLRQTG
51 LTGLLLAGAA MRSGGKVSVY QMLWPI..

Further work revealed the complete nucleotide sequence <SEQ ID 279>:

1   ATGAGATTTT TCGGTATCGG TTTTTTGGTG CTGCTGTTTT
    TGGAGATTAT
51  GTCGATTGTG TGGGTTGCCG ATTGGCTGGG CGGCGGCTGG
    ACGTTGTTTT
101 TGATGGCGGC AGGTTTTGCC GCCGGCGTGC TGATGCTCAG
    GCATACGGGG
151 CTGTCCGGTC TTTTATTGGC GGGCGCGGCA ATGAGAAGCG
    GCGGGAGGGT
201 ATCCGTTTAT CAGATGTTGT GGCCTATCCG TTATACGGTG
    GCGGCTGTGT
251 GTCTGATGAG TCCGGGATTC GTATCCTCGG TGTTGGCGGT
    ATTGCTGCTG
301 CTGCCGTTTA AGGGAGGGGC AGTGTTGCAG GCAGGAGGTG
    CGGAAAATTT
351 TTTCAACATG AACCAATCGG GCAGAAAAGA GGGCTTTTCC
    CGCGATGACG
401 ATATTATCGA GGGAGAATAT ACGGTTGAAG AGCCTTACGG
    CGGCAATCGT
451 TCCCGAAACG CCATCGAACA CAAAAAAGAC GAATAA

This corresponds to the amino acid sequence <SEQ ID 280; ORF73-1>:

1   MRFFGIGFLV LLFLEIMSIV WVADWLGGGW TLFLMAAGFA
    AGVLMLRHTG
51  LSGLLLAGAA MRSGGRVSVY QMLWPIRYTV AAVCLMSPGF
    VSSVLAVLLL
101 LPFKGGAVLQ AGGAENFFNM NQSGRKEGFS RDDDIIEGEY
    TVEEPYGGNR
151 SRNAIEHKKD E*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF73 shows 90.8% identity over a 76aa overlap with an ORF (ORF73a) from strain A of N. meningitidis:

The complete length ORF73a nucleotide sequence <SEQ ID 281> is:

1   ATGAGATTTT TCGGTATCGG TTTTTTGGTG CTGCTGTTTT
    TGGAGATTAT
51  GTCGATTGTG TGGGTTGCCG ATTGGTTGGG CGGCGGTTGG
    ACGCTGTTTC
101 TAATGGCGGC AACCTTTGCC GCCGGCGTGG TGATGCTCAG
    GCATACGGGG
151 CTGTCCGGTC TTTTATTGGC GGGCGCGGCA ATGAGAAGCG
    GCGGGAGGGT
201 ATCCGTTTAT CANATGTTGT GGCNTATCCG TTATACGGTG
    GCGGCGGTGT
251 GTCNGATGAG TCCGGGATTC GTATCCTCGG TGTNGGCGGT
    ATTGCTGNTG
301 CTNCCGTTTA AGGGAGGTGC AGTGTTGCAG GCAGGAGGTG
    CGGAAAATTT
351 TTTCAACATG AACCANTCGG GCAGAAAAGA NGGCNTTTCC
    CGCGATGACG
401 ATATTATCGA GGGGGAATAT ACGGTTGAAG ANCCTTACGG
    CGGCANTCGT
451 TTCCGAAACG CCNTNGAACA CAAAAAAGAC GAATAA

This encodes a protein having amino acid sequence <SEQ ID 282>:

1   MRFFGIGFLV LLFLEIMSIV WVADWLGGGW TLFLMAATFA
    AGVVMLRHTG
51  LSGLLLAGAA MRSGGRVSVY XMLWXIRYTV AAVCXMSPGF
    VSSVXAVLLX
101 LPFKGGAVLQ AGGAENFFNM NXSGRKXGXS RDDDIIEGEY
    TVEXPYGGXR
151 FRNAXEHKKD E*

ORF73a and ORF73-1 show 91.3% identity in 161 aa overlap

Homology with a Predicted ORF from N. gonorrhoeae

ORF73 shows 92.1% identity over a 76aa overlap with a predicted ORF (ORF73.ng) from N. gonorrhoeae:

The complete length ORF73ng nucleotide sequence <SEQ ID 283> is:

1   ATGAGATTTT TCGGTATCGG TTTTTTGGTG CTGCTGTTTT
    TGGAAATTAT
51  GTCGATTGTG TGGGTTGCCG ATTGGCTGGG CGGCGGTTGG
    AcgcTGTTTC
101 TAATGGCGGC AACCTTTGCC GCCGGTGTGC TGATGCTCAG
    GCATAcggGG
151 CTGTCCGGTC TTTTATTGGC TGGCGCGGCG GTAAAAagta
    gtgGGAAGGT
201 ATCTGTTTAT CagatgtTGT GGCCTATCCG TTATAcggtg
    gcggcggtgT
251 GTCTGatgag tCcggGATTC GTATCCTccg tgttggCGGT
    ATTGCTGCTG
301 CTGCcgttta aggGaggGgc agtgttgcag gcaggaggtg
    cggaaaATTT
351 TTTCAACATg aaCcaatcgg gcagaaAaga gggatttttc
    cacgatgacg
401 atattatcga gggagaatat acggttgaaa aacctgacgg
    cggcaatcgt
451 tcccgaAAcg ccatcgaaca cgaaaAagac gaataA

This encodes a protein having amino acid sequence <SEQ ID 284>:

1   MRFFGIGFLV LLFLEIMSIV WVADWLGGGW TLFLMAATFA
    AGVLMLRHTG
51  LSGLLLAGAA VKSSGKVSVY QMLWPIRYTV AAVCLMSPGF
    VSSVLAVLLL
101 LPFKGGAVLQ AGGAENFFNM NQSGRKEGFF HDDDIIEGEY
    TVEKPDGGNR
151 SRNAIEHEKD E*

ORF73ng and ORG73-1 show 93.8% identity in 161 aa overlap

Based on this analysis, including the presence of a putative leader sequence and putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 34

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 285>:

1   ATGTTTGTTT TTCAGACGGC ATTCTT.ATG TTTCAGAAAC
    ATTTGCAGAA
51  AGCCTCCGAC AGCGTCGTCG GAGGGACATT ATACGTGGTT
    GCCACGCCCA
101 TCGGCAATTT GGCGGACATT ACCCTGCGCG CTTTGGCGGT
    ATTGCAAAAG
151 GCG....... .....GCCGA AGACACGCGC GTTACCGCAC
    AGCTTTTGAG
201 CGCGTACGGC ATTCAGGGCA AACTCGTCAG TGTGCGCGAA
    CACAACGAAC
251 GGCAGATGGC GGACAAGATT GTCGGCTATC TTTCAGACGG
    CATGGTTGTG
301 GCACAGGTTT CCGATGCGGG TACGCCGGCC GTGTGCGACC
    CGGGCGCGAA
351 ACTCGCCCGC CGCGTGCGTG AGGCCGGGTT TAAAGTCGTT
    CCCGTCGTGG
401 GCGCAAC.GC GGTGATGGCG GCTTTGAGCG TGGCCGGTGT
    GGAAGGATCC
451 GATTTTTATT TCAACGGTTT TGTACCGCCG AAATCGGGAG
    AACGCAGGAA
501 ACTGTTTGCC AAATGGGTGC GGGCGGCGTT TCCTATCGTC
    ATGTTTGAAA
551 CGCCGCACCG CATCGGTGCA GCGCTTGCCG ATATGGCGGA
    ACTGTTCCCC
601 GAACGCCGAT TAATGCTGGC GCGCGAAATT ACGAAAACGT
    TTGAAACGTT
651 CTTAAGCGGC ACGGTTGGGG AAATTCAGAC GGCATTGTCT
    GCCGACGGCG
701 ACCAATCGCG CGGCGAGATG GTGTTGGTGC TTTATCCGGC
    GCAGGATGAA
751 AAACACGAAG GCTTGTCCGA GTCCGCGCAA AACATCATGA
    AAATCCTCAC
801 AGCCGAGCTG CCGACCAAAC AGGCGGCGGA GCTTGCTGCC
    AAAATCACGG
851 GCGAGGGAAA GAAAGCTTTG TACGAT..

This corresponds to the amino acid sequence <SEQ ID 286; ORF75>:

1   MFVFQTAFXM FQKHLQKASD SVVGGTLYVV ATPIGNLADI
    TLRALAVLQK
51  A....AEDTR VTAQLLSAYG IQGKLVSVRE HNERQMADKI
    VGYLSDGMVV
101 AQVSDAGTPA VCDPGAKLAR RVREAGFKVV PVVGAXAVMA
    ALSVAGVEGS
151 DFYFNGFVPP KSGERRKLFA KWVRAAFPIV MFETPHRIGA
    ALADMAELFP
201 ERRLMLAREI TKTFETFLSG TVGEIQTALS ADGDQSRGEM
    VLVLYPAQDE
251 KHEGLSESAQ NIMKILTAEL PTKQAAELAA KITGEGKKAL
    YD..

Further work revealed the complete nucleotide sequence <SEQ ID 287>:

1   ATGTTTCAGA AACATTTGCA GAAAGCCTCC GACAGCGTCG
    TCGGAGGGAC
51  ATTATACGTG GTTGCCACGC CCATCGGCAA TTTGGCGGAC
    ATTACCCTGC
101 GCGCTTTGGC GGTATTGCAA AAGGCGGACA TCATCTGTGC
    CGAAGACACG
151 CGCGTTACCG CACAGCTTTT GAGCGCGTAC GGCATTCAGG
    GCAAACTCGT
201 CAGTGTGCGC GAACACAACG AACGGCAGAT GGCGGACAAG
    ATTGTCGGCT
251 ATCTTTCAGA CGGCATGGTT GTGGCACAGG TTTCCGATGC
    GGGTACGCCG
301 GCCGTGTGCG ACCCGGGCGC GAAACTCGCC CGCCGCGTGC
    GTGAGGCCGG
351 GTTTAAAGTC GTTCCCGTCG TGGGCGCAAG CGCGGTGATG
    GCGGCTTTGA
401 GCGTGGCCGG TGTGGAAGGA TCCGATTTTT ATTTCAACGG
    TTTTGTACCG
451 CCGAAATCGG GAGAACGCAG GAAACTGTTT GCCAAATGGG
    TGCGGGCGGC
501 GTTTCCTATC GTCATGTTTG AAACGCCGCA CCGCATCGGT
    GCGACGCTTG
551 CCGATATGGC GGAACTGTTC CCCGAACGCC GATTAATGCT
    GGCGCGCGAA
601 ATTACGAAAA CGTTTGAAAC GTTCTTAAGC GGCACGGTTG
    GGGAAATTCA
651 GACGGCATTG TCTGCCGACG GCAACCAATC GCGCGGCGAG
    ATGGTGTTGG
701 TGCTTTATCC GGCGCAGGAT GAAAAACACG AAGGCTTGTC
    CGAGTCCGCG
751 CAAAACATCA TGAAAATCCT CACAGCCGAG CTGCCGACCA
    AACAGGCGGC
801 GGAGCTTGCT GCCAAAATCA CGGGCGAGGG AAAGAAAGCT
    TTGTACGATC
851 TGGCTCTGTC TTGGAAAAAC AAATAG

This corresponds to the amino acid sequence <SEQ ID 288; ORF75-1>:

1   MFQKHLQKAS DSVVGGTLYV VATPIGNLAD ITLRALAVLQ
    KADIICAEDT
51  RVTAQLLSAY GIQGKLVSVR EHNERQMADK IVGYLSDGMV
    VAQVSDAGTP
101 AVCDPGAKLA RRVREAGFKV VPVVGASAVM AALSVAGVEG
    SDFYFNGFVP
151 PKSGERRKLF AKWVRAAFPI VMFETPHRIG ATLADMAELF
    PERRLMLARE
201 ITKTFETFLS GTVGEIQTAL SADGNQSRGE MVLVLYPAQD
    EKHEGLSESA
251 QNIMKILTAE LPTKQAAELA AKITGEGKKA LYDLALSWKN
    K*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF75 shows 95.8% identity over a 283aa overlap with an ORF (ORF75a) from strain A of N. meningitidis:

The complete length ORF75a nucleotide sequence <SEQ ID 289> is:

1   ATGTTTCAGA AACATTTGCA GAAAGCCTCC GACAGCGTCG
    TCGGAGGGAC
51  ATTATACGTG GTTGCCACGC CCATCGGCAA TTTGGCGGAC
    ATTACCCTGC
101 GCGCTTTGGC GGTATTGCAA AAGGCGGACA TCATCTGTGC
    CGAAGACACG
151 CGCGTTACCG CGCAGCTTTT GAGCGCGTAC GGCATTCAGG
    GCAAACTCGT
201 CAGCGTGCGC GAACACAACG AACGGCAGAT GGCGGACAAG
    ATTGTCGGCT
251 ATCTTTCAGA CGGCATGGTT GTGGCACAGG TTTCCGATGC
    GGGTACGCCG
301 GCCGTGTGCG ACCCGGGCGC GAAACTCGCC CGCCGCGTGC
    GTGAGGTCGG
351 GTTTAAAGTT GTCCCTGTTG TCGGCGCAAG CGCGGTGATG
    GCGGCTTTGA
401 GTGTGGCTGG TGTGGCGGGA TCCGATTTTT ATTTCAACGG
    TTTTGTACCG
451 CCGAAATCGG GCGAACGTAG GAAATTGTTT GCCAAATGGG
    TGCGGGTGGC
501 GTTTCCCGTC GTGATGTTTG AAACGCCGCA CCGCATCGGG
    GCGACGCTTG
551 CCGATATGGC GGAACTGTTC CCCGAACGCC GATTAATGCT
    GGCGCGCGAA
601 ATCACGAAAA CGTTTGAAAC GTTCTTAAGC GGCACGGTTG
    GGGAAATTCA
651 GACGGCATTG GCGGCGGACG GCAACCAATC GCGCGGCGAG
    ATGGTGTTGG
701 TGCTTTATCC GGCGCAGGAT GAAAAACACG AAGGCTTGTC
    CGAGTCCGCG
751 CAAAACATCA TGAAAATCCT CACAGCCGAG CTGCCGACCA
    AACAGGCGGC
801 GGAGCTTGCC GCCAAAATCA CGGGCGAGGG AAAAAAAGCT
    TTGTACGATC
851 TGGCACTGTC TTGGAAAAAC AAATGA

This encodes a protein having amino acid sequence <SEQ ID 290>:

1   MFQKHLQKAS DSVVGGTLYV VATPIGNLAD ITLRALAVLQ
    KADIICAEDT
51  RVTAQLLSAY GIQGKLVSVR EHNERQMADK IVGYLSDGMV
    VAQVSDAGTP
101 AVCDPGAKLA RRVREVGFKV VPVVGASAVM AALSVAGVAG
    SDFYFNGFVP
151 PKSGERRKLF AKWVRVAFPV VMFETPHRIG ATLADMAELF
    PERRLMLARE
201 ITKTFETFLS GTVGEIQTAL AADGNQSRGE MVLVLYPAQD
    EKHEGLSESA
251 QNIMKILTAE LPTKQAAELA AKITGEGKKA LYDLALSWKN
    K*

ORF75a and ORF75-1 show 98.3% identity in 291 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF75 shows 93.2% identity over a 292aa overlap with a predicted ORF (ORF75.ng) from N. gonorrhoeae:

An ORF75ng nucleotide sequence <SEQ ID 291> was predicted to encode a protein having amino acid sequence <SEQ ID 292>:

1   MSVFQTAFFM FQKHLQKASD SVVGGTLYVV ATPIGNLADI
    TLRALAVLQK
51  ADIICAEDTR VTAQLLSAYG IQGRLVSVRE HNERQMADKV
    IGFLSDGLVV
101 AQVSDAGTPA VCDPGAKLAR RVREAGFKVV PVVGASAVMA
    ALSVAGVAES
151 DFYFNGFVPP KSGERRKLFA KWVRAAFPVV MFETPHRIGA
    TLADMAELFP
201 ERRLMLAREI TKTFETFLSG TVGEIQTALA ADGNQSRGEM
    VLVLYPAQDE
251 KHEGLSESAQ NAMKILAAEL PTKQAAELAA KITGEGKKAL
    YDLALSWKNK
301 *

After further analysis, the following gonococcal DNA sequence <SEQ ID 293> was identified:

1   ATGTTTCAGA AACACTTGCA GAAAGCCTCC GACAGCGTCG
    TCGGAGGGAC
51  ATTATACGTG GTTGCCACGC CCATCGGCAA TTTGGCAGAC
    ATTACCCTGC
101 GCGCTTTGGC GGTATTGCAA AAGGCGGACA TCATTTGTGC
    CGAAGACACG
151 CGCGTTACTG CGCAGCTTTT GAGCGCGTAC GGCATTCAGG
    GCAGGTTGGT
201 CAGTGTGCGC GAACACAACG AGCGGCAGAT GGCGGACAAG
    GTAATCGGTT
251 TCCTTTCAGA CGGCCTGGTT GTGGCGCAGG TTTCCGATGC
    GGGTACGCCG
301 GCCGTGTGCG ACCCGGGCGC GAAACTCGCC CGCCGCGTGC
    GCGAAGCAGG
351 GTTCAAAGTC GTTCCCGTCG TGGGCGCAAG CGCGGTAATG
    GCGGCGTTGA
401 GTGTGGCCGG TGTGGCGGAA TCCGATTTTT ATTTCAACGG
    TTTTGTACCG
451 CCGAAATCGG GCGAACGTAG GAAATTGTTT GCCAAATGGG
    TGCGGGCGGC
501 ATTTCCTGTC GTCATGTTTG AAACGCCGCA CCGAATCGGG
    GCAACGCTTG
551 CCGATATGGC GGAATTGTTC CCCGAACGCC GTCTGATGCT
    GGCGCGCGAA
601 ATCACGAAAA CGTTTGAAAC GTTCTTAAGC GGCACGGTTG
    GGGAAATTCA
651 GACGGCATTG GCGGCGGACG GCAACCAATC GCGCGGCGAG
    ATGGTGTTGG
701 TGCTTTATCC GGCGCAGGAT GAAAAACACG AAGGCTTGTC
    CGAGTCTGCG
751 CAAAATGCGA TGAAAATCCT TGCGGCCGAG CTGCCGACCA
    AGCAGGCGGC
801 GGAGCTTGCC GCCAAGATTA CAGGTGAGGG CAAAAAGGCT
    TTGTACGATT
851 TGGCACTGTC GTGGAAAAAC AAATGA

This corresponds to the amino acid sequence <SEQ ID 294; ORF75ng-1>:

1   MFQKHLQKAS DSVVGGTLYV VATPIGNLAD ITLRALAVLQ
    KADIICAEDT
51  RVTAQLLSAY GIQGRLVSVR EHNERQMADK VIGFLSDGLV
    VAQVSDAGTP
101 AVCDPGAKLA RRVREAGFKV VPVVGASAVM AALSVAGVAE
    SDFYFNGFVP
151 PKSGERRKLF AKWVRAAFPV VMFETPHRIG ATLADMAELF
    PERRLMLARE
201 ITKTFETFLS GTVGEIQTAL AADGNQSRGE MVLVLYPAQD
    EKHEGLSESA
251 QNAMKILAAE LPTKQAAELA AKITGEGKKA LYDLALSWKN
    K*

ORF75ng-1 and ORF75-1 show 96.2% identity in 291 aa overlap:

Furthermore, ORG75ng-1 shows significant homology to a hypothetical E. coli protein:

sp|P45528|YRAL_ECOLI HYPOTHETICAL 31.3 KD PROTEIN IN AGAI-MTR INTERGENIC
REGION (F286)
>gi|606086 (U18997) ORF_f286 [Escherichia coli]
>gi|1789535 (AE000395) hypothetical 31.3 kD protein in agai-mtr
intergenic region [Escherichia coli] Length = 286
Score = 218 bits (550), Expect = 3e−56
Identities = 128/284 (45%), Positives = 171/284 (60%), Gaps = 4/284 (1%)
Query:	4	KHLQKASDSVVGGTLYVVATPIGNLADITLRALAVLQKADIICAEDTRVTAQLLSAYGIQ	63
		K  Q A +S   G LY+V TPIGNLADIT RAL VLQ  D+I AEDTR T  LL  +GI
Sbjct:	2	KQHQSADNSQ--GQLYIVPTPIGNLADITQRALEVLQAVDLIAAEDTRHTGLLLQHFGIN	59
Query:	64	GRLVSVREHNERQMADKVIGFLSDGLVVAQVSDAGTPAVCDPGAKLARRVREAGFKVVPV	123
		RL ++ +HNE+Q A+ ++  L +G  +A VSDAGTP + DPG  L R  REAG +VVP+
Sbjct:	60	ARLFALHDHNEQQKAETLLAKLQEGQNIALVSDAGTPLINDPGYHLVRTCREAGIRVVPL	119
Query:	124	VGASAVMAALSVAGVAESDFYFNGFVPPKSGERRKLFAKWVRAAFPVVMFETPHRIGATL	183
		G  A + ALS AG+    F + GF+P KS  RR            ++ +E+ HR+  +L
Sbjct:	120	PGPCAAITALSAAGLPSDRFCYEGFLPAKSKGRRDALKAIEAEPRTLIFYESTHRLLDSL	179
Query:	184	ADMAELFPERR-LMLAREITKTFETFLSGTVGEIQTALAADGNQSRGEMVLVLYPAQDEK	242
		D+  +  E R ++LARE+TKT+ET     VGE+   +  D N+ +GEMVL++      +
Sbjct:	180	EDIVAVLGESRYVVLARELTKTWETIHGAPVGELLAWVKEDENRRKGEMVLIV-EGHKAQ	238
Query:	243	HEGLSESAQNAMKILAAELPTKQAAELAAKITGEGKKALYDLAL	286
		E L   A   + +L AELP K+AA LAA+I G  K ALY  AL
Sbjct:	239	EEDLPADALRTLALLQAELPLKKAAALAAEIHGVKKNALYKYAL	282

Based on this analysis, including the presence of a putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 35

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 295>:

This corresponds to the amino acid sequence <SEQ ID 296; ORF76>:

Further work revealed the complete nucleotide sequence <SEQ ID 297>:

1   ATGAAACAGA AAAAAACCGC TGCCGCAGTT ATTGCTGCAA
    TGTTGGCAGG
51  TTTTGCGGCA GCCAAAGCAC CCGAAATCGA CCCGGCTTTG
    GTGGATACGC
101 TGGTGGCGCA GATCATGCAG CAGGCAGACC GGCATGCGGA
    GCAGTCCCAA
151 AAACCGGACG GGCAGGCAAT CCGAAACGAT GCCGTCCGCC
    GGCTACAAAC
201 TTTGGAAGTT TTGAAAAACA GGGCATTGAA GGAAGGTTTG
    GATAAGGATA
251 AGGATGTCCA AAACCGCTTT AAAATCGCCG AAGCGTCTTT
    TTATGCCGAG
301 GAGTACGTCC GTTTTCTGGA ACGTTCGGAA ACGGTTTCCG
    AAGACGAGCT
351 GCACAAGTTT TACGAACAGC AAATCCGCAT GATCAAATTG
    CAGCAGGTCA
401 GCTTCGCAAC CGAAGAGGAG GCGCGTCAGG CGCAGCAGCT
    CCTGCTCAAA
451 GGGCTGTCTT TTGAAGGGCT GATGAAGCGT TATCCGAACG
    ACGAGCAGGC
501 TTTTGACGGT TTCATTATGG CGCAGCAGCT TCCCGAGCCG
    CTGGCTTCGC
551 AGTTTGCCGC GATGAATCGG GGCGACGTTA CCCGCGATCC
    GGTCAAATTG
601 GGCGAACGCT ATTATCTGTT CAAACTCAGC GAGGTCGGGA
    AAAACCCCGA
651 CGCGCAGCCT TTCGAGTTGG TCAGAAACCA GTTGGAGCAG
    GGTTTGAGAC
701 AGGAAAAAGC CCGCTTGAAA ATCGATGCCC TTTTGGAAGA
    AAACGGTGTC
751 AAACCGTAA

This corresponds to the amino acid sequence <SEQ ID 298; ORF76-1>:

1   MKQKKTAAAV IAAMLAGFAA AKAPEIDPAL VDTLVAQIMQ
    QADRHAEQSQ
51  KPDGQAIRND AVRRLQTLEV LKNRALKEGL DKDKDVQNRF
    KIAEASFYAE
101 EYVRFLERSE TVSEDELHKF YEQQIRMIKL QQVSFATEEE
    ARQAQQLLLK
151 GLSFEGLMKR YPNDEQAFDG FIMAQQLPEP LASQFAAMNR
    GDVTRDPVKL
201 GERYYLFKLS EVGKNPDAQP FELVRNQLEQ GLRQEKARLK
    IDALLEENGV
251 KP*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF76 shows 96.7% identity over a 30aa overlap and 96.8% identity over a 31aa overlap with an ORF (ORF76a) from strain A of N. meningitidis:

The complete length ORF76a nucleotide sequence <SEQ ID 299> is:

1   ATGAAACAGA AAAAAACCGC TGCCGCAGTT ATTGCTGCAA
    TGTTGGCAGG
51  TTTTGCGGCA GCCAAAGCAC CCGAAATCGA CCCGGCTTTG
    GTGGATACGC
101 TGGTGGCGCA GATCATGCAG CAGGCAGACC GGCATGCGGA
    GCAGTCCCAA
151 AAACCGGACG GGCAGGCAAT CCGAAACGAT GCCGTCCGTC
    GGCTGCAAAC
201 TTTGGAAGTT TTGAAAAACA GGGCATTGAA GGAAGGTTTG
    GATAAGGATA
251 AGGATGTCCA AAACCGCTTT AAAATCGCCG AAGCGTCTTT
    TTATGCCGAG
301 GAGTACGTCC GTTTTCTGGA ACGTTCGGAA ACGGTTTCCG
    AAAGCGCACT
351 GCGTCAGTTT TATGAGCGGC AAATCCGCAT GATCAAATTG
    CAGCAGGTCA
401 GCTTCGCAAC CGAAGAGGAG GCGCGTCAGG CGCAGCAGCT
    CCTGCTCAAA
451 GGGCTGTCTT TTGAAGGGCT GATGAAGCGT TATCCGAACG
    ACGAGCAGGC
501 TTTTGACGGT TTCATTATGG CGCAGCAGCT TCCCGAGCCG
    CTGGCTTCGC
551 AGTTTGCAGC GATGAATCGG GGCGACGTTA CCCGCGATCC
    GGTCAAATTG
601 GGCGAACGCT ATTATCTGTT CAAACTCAGC GAGGTCGGGA
    AAAACCCCGA
651 CGCGCAGCCT TTCGAGTTGG TCAGAAACCA GTTGGAACAA
    GGTTTGAGAC
701 AGGAAAAAGC CCGCTTGAAA ATCGATGCCA TTTTGGAAGA
    AAACGGTGTC
751 AAACCGTAA

This encodes a protein having amino acid sequence <SEQ ID 300>:

1   MKQKKTAAAV IAAMLAGFAA AKAPEIDPAL VDTLVAQIMQ
    QADRHAEQSQ
51  KPDGQAIRND AVRRLQTLEV LKNRALKEGL DKDKDVQNRF
    KIAEASFYAE
101 EYVRFLERSE TVSESALRQF YERQIRMIKL QQVSFATEEE
    ARQAQQLLLK
151 GLSFEGLMKR YPNDEQAFDG FIMAQQLPEP LASQFAAMNR
    GDVTRDPVKL
201 GERYYLFKLS EVGKNPDAQP FELVRNQLEQ GLRQEKARLK
    IDAILEENGV
251 KP*

ORF76a and ORF76-1 show 97.6% identity in 252 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

The aligned aa sequences of ORF76 and a predicted ORF (ORF76.ng) from N. gonorrhoeae of the N- and C-termini show 96.7% and 100% identity in 30 and 31 overlap, respectively:

The complete length ORF76ng nucleotide sequence <SEQ ID 301> is:

1   ATGAAACAGA AAAAGACCGC TGCCGCAGTT ATTGCTGCAA
    TGTTGGCAGG
51  TTTTGCGGCA GCCAAAGCAC CCGAAATCGA CCCGGCTTTG
    GTGGATACGC
101 TGGTGGCGCA GATCATGCAG CAGGCAGACC GGCATGCGGA
    GCAGTCCCAA
151 AGACCGGACG GGCAGGCAAT CCGAAACGAT GCCGTCCGCC
    GGCTGCAAAC
201 TTTGGAAGTT TTGAAAAACA GGGCATTGAA GGAAGGTTTG
    GATAAGGATA
251 AGGATGTCCA AAACCGCTTT AAAATCGCCG AAGCGTCTTT
    TTATGCCGAG
301 GAGTACGTCC GTTTTCTGGA ACGTTCGGAA ACGGTTTCCG
    AAAGCGCACT
351 GCGTCAGTTT TATGAGCGGC AAATCCGCAT GATCAAATTG
    CAGCAGGTCA
401 GCTTCGCAAC CGAAGAGGAG GCGCGTCAGG CGCAGCAGCT
    CCTGCTCAAA
451 GGGCTGTCTT TTGAAGGGCT GATGAAGCGT TATCCGAACG
    ACGAGCAGGC
501 GTTCGACGGT TTCATTATGG CGCAGCAGCT TCCCGAGCCG
    CTGGCTTcgc
551 agtttgCCGG TATGAACCGT GGCGACGTTA CCCGCAATCC
    GGTCAAATTG
601 GGCGAACGCT ATTACCTGTT CAAACTCGGC GCGGTCGGGA
    AAAACCCCGA
651 CGCGCAGCCT TTCGAGTTGG TCAGAAACCA GTTGGAACAA
    GGTTTGAGGC
701 AGGAAAAAGC CCGCTTGAAA ATCGATGCCC TTTTGGAaga
    Aaacggtgtc
751 AaacCGTAA

This encodes a protein having amino acid sequence <SEQ ID 302>:

1   MKQKKTAAAV IAAMLAGFAA AKAPEIDPAL VDTLVAQIMQ
    QADRHAEQSQ
51  RPDGQAIRND AVRRLQTLEV LKNRALKEGL DKDKDVQNRF
    KIAEASFYAE
101 EYVRFLERSE TVSESALRQF YERQIRMIKL QQVSFATEEE
    ARQAQQLLLK
151 GLSFEGLMKR YPNDEQAFDG FIMAQQLPEP LASQFAGMNR
    GDVTRNPVKL
201 GERYYLFKLG AVGKNPDAQP FELVRNQLEQ GLRQEKARLK
    IDALLEENGV
251 KP*

ORF76ng and ORF76-1 show 96.0% identity in 252 aa overlap

Furthermore, ORF76ng shows significant homology to a B. subtilis export protein precursor:

sp|P24327|PRSA_BACSU PROTEIN EXPORT PROTEIN PRSA
PRECURSOR >gi|98227|pir||S15269 33K lipoprotein - Bacillus subtilis
>gi|39782 (X57271) 33 kDa lipoprotein [Bacillus subtilis]
>gi|2226124|gnl|PID|e325181 (Y14077) 33 kDa lipoprotein
[Bacillus subtilis]
>gi|2633331|gnl|PID|e1182997 (Z99109) molecular chaperonin
[Bacillus subtilis]
Length = 292
Score = 50.4 bits (118), Expect = 1e−05
Identities = 48/199 (24%), Positives = 82/199 (41%), Gaps = 32/199 (16%)
Query:	70	VLKNRALKEGLDK-----DKDVQNRFKIAEASF----------YAEEYVRFLERSETVSE	114
		VL     ++ LDK     DK++ N+ K  +             Y ++Y++   + E +++
Sbjct:	53	VLTQLVQEKVLDKKYKVSDKEIDNKLKEYKTQLGDQYTALEKQYGKDYLKEQVKYELLTQ	112
Query:	115	SA-----------LRQFYERQIRMIKLQQVSFATEEEARQAQQLLLKGLSFEGLMKRYPN	163
		A           +++++E     I+   +  A ++ A + ++ L KG  FE L K Y
Sbjct:	113	KAAKDNIKVTDADIKEYWEGLKGKIRASHILVADKKTAEEVEKKLKKGEKFEDLAKEYST	172
Query:	164	DEQAFDG-----FIMAQQLPEPLASQFAAMNRGDVTRDPVKLGERYYLFKLSEVGKNPDA	218
		D  A  G     F    Q+ E  +     +  G+V+ DPVK    Y++ K +E     D
Sbjct:	173	DSSASKGGDLGWFAKEGQMDETFSKAAFKLKTGEVS-DPVKTQYGYHIIKKTEERGKYDD	231
Query:	219	QPFELVRNQLEQGLRQEKA	237
		EL    LEQ L    A
Sbjct:	232	MKKELKSEVLEQKLNDNAA	250

Based on this analysis, including the presence of a putative leader sequence and a RGD motif in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF76-1 (27.8 kDa) was cloned in the pET vector and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 10A shows the results of affinity purification of the His-fusion protein, Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot (FIG. 10B), ELISA (positive result), and FACS analysis (FIG. 10C). These experiments confirm that ORF76-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 36

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 303>:

This corresponds to the amino acid sequence <SEQ ID 304; ORF81>:

Further work revealed the complete nucleotide sequence <SEQ ID 305>:

1    ATGAAAAAAT CTTTCCTTAC GCTTGTTCTG TATTCGTCTT
     TACTTACCGC
51   CAGCGAAATT GCCTATCGCT TTGTATTTGG GATTGAAACC
     TTACCGGCGG
101  CAAAAATTGC GGAAACGTTT GCGCTGACAT TTGTGATTGC
     TGCGCTGTAT
151  CTGTTTGCGC GTTATAAGGT GACGCGTTTG TTGATTGCGG
     TGTTTTTTGC
201  GTTCAGCATT ATTGCCAACA ATGTGCATTA CGCGGTTTAT
     CAAAGCTGGA
251  TGACGGGCAT CAATTATTGG CTGATGCTGA AAGAGGTTAC
     CGAAGTCGGC
301  AGCGCGGGTG CGTCGATGTT GGATAAGTTG TGGCTGCCTG
     TGTTGTGGGG
351  CGTGTTGGAA GTCATGTTGT TTTGCAGCCT TGCCAAGTTC
     CGCCGTAAGA
401  CGCATTTTTC TGCCGATATA CTGTTTGCCT TCCTAATGCT
     GATGATTTTC
451  GTGCGTTCGT TCGACACGAA ACAAGAGCAC GGTATTTCGC
     CCAAACCGAC
501  ATACAGCCGC ATCAAAGCCA ATTATTTCAG CTTCGGTTAT
     TTTGTCGGAC
551  GCGTGTTGCC GTATCAGTTG TTTGATTTAA GCAGGATTCC
     CGCCTTTAAG
601  CAGCCTGCTC CAAGCAAAAT CGGGCAGGGC AGTGTTCAAA
     ATATCGTCCT
651  GATTATGGGC GAAAGCGAAA GCGCGGCGCA TTTGAAGCTG
     TTTGGCTACG
701  GACGCGAAAC TTCGCCGTTT TTAACCCGGC TGTCGCAAGC
     CGATTTTAAG
751  CCGATTGTGA AACAAAGTTA TTCCGCAGGC TTTATGACTG
     CAGTGTCCCT
801  GCCCAGTTTT TTCAATGCGA TACCGCACGC CAACGGCTTG
     GAACAAATCA
851  GCGGCGGCGA TACCAATATG TTCCGCCTCG CCAAAGAGCA
     GGGCTATGAA
901  ACGTATTTTT ACAGCGCGCA GGCGGAAAAC GAGATGGCGA
     TTTTGAACTT
951  AATCGGTAAG AAATGGATAG ACCATCTGAT TCAGCCGACG
     CAACTTGGCT
1001 ACGGCAACGG CGACAATATG CCCGATGAGA AGCTGCTGCC
     GTTGTTCGAC
1051 AAAATCAATT TGCAGCAGGG CAAGCATTTT ATCGTGTTGC
     ACCAACGCGG
1101 TTCGCACGCC CCATACGGCG CATTGTTGCA GCCTCAAGAT
     AAAGTATTCG
1151 GCGAAGCCGA TATTGTGGAT AAGTACGACA ACACCATCCA
     CAAAACCGAC
1201 CAAATGATTC AAACCGTATT CGAGCAGCTG CAAAAGCAGC
     CTGACGGCAA
1251 CTGGCTGTTT GCCTATACCT CCGATCATGG CCAGTATGTT
     CGCCAAGATA
1301 TCTACAATCA AGGCACGGTG CAGCCCGACA GCTATCTCGT
     GCCGCTAGTG
1351 TTGTACAGCC CGGATAAGGC CGTGCAACAG GCTGCCAACC
     AGGCTTTTGC
1401 GCCTTGCGAG ATTGCCTTCC ATCAGCAGCT TTCAACGTTC
     CTGATTCACA
1451 CGTTGGGCTA CGATATGCCG GTTTCAGGTT GTCGCGAAGG
     CTCGGTAACG
1501 GGCAACCTGA TTACGGGTGA TGCAGGCAGC TTGAACATTC
     GCGACGGCAA
1551 GGCGGAATAT GTTTATCCGC AATGA

This corresponds to the amino acid sequence <SEQ ID 306; ORF81-1>:

1   MKKSFLTLVL YSSLLTASEI AYRFVFGIET LPAAKIAETF
    ALTFVIAALY
51  LFARYKVTRL LIAVFFAFSI IANNVHYAVY QSWMTGINYW
    LMLKEVTEVG
101 SAGASMLDKL WLPVLWGVLE VMLFCSLAKF RRKTHFSADI
    LFAFLMLMIF
151 VRSFDTKQEH GISPKPTYSR IKANYFSFGY FVGRVLPYQL
    FDLSRIPAFK
201 QPAPSKIGQG SVQNIVLIMG ESESAAHLKL FGYGRETSPF
    LTRLSQADFK
251 PIVKQSYSAG FMTAVSLPSF FNAIPHANGL EQISGGDTNM
    FRLAKEQGYE
301 TYFYSAQAEN EMAILNLIGK KWIDHLIQPT QLGYGNGDNM
    PDEKLLPLFD
351 KINLQQGKHF IVLHQRGSHA PYGALLQPQD KVFGEADIVD
    KYDNTIHKTD
401 QMIQTVFEQL QKQPDGNWLF AYTSDHGQYV RQDIYNQGTV
    QPDSYLVPLV
451 LYSPDKAVQQ AANQAFAPCE IAFHQQLSTF LIHTLGYDMP
    VSGCREGSVT
501 GNLITGDAGS LNIRDGKAEY VYPQ*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF81 shows 84.7% identity over a 85aa overlap and 99.2% identity over a 121aa overlap with an ORF (ORF81a) from strain A of N. meningitidis:

The complete length ORF81a nucleotide sequence <SEQ ID 307> is:

1    ATGAAAAAAT CCCTTTTCGT TCTCTTTCTG TATTCGTCCC
     TACTTACTGC
51   CAGCGAAATT GCTTATCGCT TTGTATTCGG AATTGAAACC
     TTACCGGCTG
101  CAAAAATGGC AGAAACGTTT GCGCTGACAT TTGTGATTGC
     TGCGCTGTAT
151  CTGTTTGCGC GTTATAAGGC AACGCGTTTG TTGATTGCGG
     TGTTTTTCGC
201  GTTCAGCATT ATTGCCAACA ATGTGCATTA CGCGGTTTAT
     CAAAGCTGGA
251  TAACGGGCAT TAATTATTGG CTGATGCTGA AAGAGATTAC
     CGAAGTTGGC
301  GGCGCAGGGG CGTCGATGTT GGATAAGTTG TGGCTGCCTG
     CGTTGTGGGG
351  CGTGTTGGAA GTCATGTTGT TTTGCAGCCT TGCCAAGTTC
     CGCCGTAAGA
401  CGCATTTTTC TGCCGATATA CTGTTTGCCT TCCTAATGCT
     GATGATTTTC
451  GTGCGTTCGT TCGACACGAA ACAAGAACAC GGTATTTCGC
     CCAAACCGAC
501  ATACAGCCGC ATCAAAGCCA ATTATTTCAG CTTCGGTTAT
     TTTGTCGGAC
551  GCGTGTTGCC GTATCAGTTG TTTGATTTAA GCAAGATTCC
     TGTGTTCAAA
601  CAGCCTGCTC CAAGCAGAAT CGGGCAAGGC AGTATTCAAA
     ATATCGTCCT
651  GATTATGGGC GAAAGCGAAA GCGCGGCGCA TTTGAAATTG
     TTTGGCTACG
701  GGCGCGAAAC TTCGCCGTTT TTGACCCAGC TTTCGCAAGC
     CGATTTTAAG
751  CCGATTGTGA AACAAAGTTA TTCCGCAGGC TTTATGACGG
     CAGTATCCCT
801  GCCCAGTTTC TTTAACGTCA TACCGCATGC CAACGGCTTG
     GAACAAATCA
851  GCGGCGGCGA TATTGTGGAT AAGTACGACA ACACCATCCA
     CAAAACCGAC
901  CAAATGATTC AAACCGTATT CGAGCAGCTG CAAAAGCAGC
     CTGACGGCAA
951  CTGGCTGTTT GCCTATACCT CCGATCATGG CCAGTATGTT
     CGCCAAGATA
1001 TCTACAATCA AGGCACGGTG CAGCCCGACA GCTATCTCGT
     GCCGCTGGTG
1051 TTGTACAGCC CGGATAAGGC CGTGCAACAG GCTGCCAACC
     AGGCTTTTGC
1101 GCCTTGCGAG ATTGCCTTCC ATCAGCAGCT TTCAACGTTC
     CTGATTCACA
1151 CGTTGGGCTA CGATATGCCG GTTTCAGGTT GTCGCGAAGG
     CTCGGTAACG
1201 GGCAACCTGA TTACGGGTGA TGCAGGCAGC TTGAACATTC
     GCGACGGCAA
1251 GGCGGAATAT GTTTATCCGC AATGA

This encodes a protein having amino acid sequence <SEQ ID 308>:

1   MKKSLFVLFL YSSLLTASEI AYRFVFGIET LPAAKMAETF
    ALTFVIAALY
51  LFARYKATRL LIAVFFAFSI IANNVHYAVY QSWITGINYW
    LMLKEITEVG
101 GAGASMLDKL WLPALWGVLE VMLFCSLAKF RRKTHFSADI
    LFAFLMLMIF
151 VRSFDTKQEH GISPKPTYSR IKANYFSFGY FVGRVLPYQL
    FDLSKIPVFK
201 QPAPSRIGQG SIQNIVLIMG ESESAAHLKL FGYGRETSPF
    LTQLSQADFK
251 PIVKQSYSAG FMTAVSLPSF FNVIPHANGL EQISGGDIVD
    KYDNTIHKTD
301 QMIQTVFEQL QKQPDGNWLF AYTSDHGQYV RQDIYNQGTV
    QPDSYLVPLV
351 LYSPDKAVQQ AANQAFAPCE IAFHQQLSTF LIHTLGYDMP
    VSGCREGSVT
401 GNLITGDAGS LNIRDGKAEY VYPQ*

ORF81a and ORF81-1 show 77.9% identity in 524 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

The aligned aa sequences of ORF81 and a predicted ORF (ORF81.ng) from N. gonorrhoeae of the N- and C-termini show 82.4% and 97.5% identity in 85 and 121 overlap, respectively:

The complete length ORF81ng nucleotide sequence <SEQ ID 309> is:

1    ATGAAAAAAT CCCTTTTCGT TCTCTTTCTG TATTCATCCC
     TACTTACCGC
51   CAGCGAAATC GCCTATCGCT TTGTATTCGG AATTGAAACC
     TTACCGGCTG
101  CAAAAATGGC GGAAACGTTT GCGCTGACAT TTATGATTGC
     TGCGCTGTAT
151  CTGTTTGCGC GTTATAAGGC TTCGCGGCTG CTGATTGCGG
     TGTTTTTCGC
201  GTTCAGCATG ATTGCCAACA ATGTGCATTA CGCGGTTTAT
     CAAAGCTGGA
251  TGACGGGTAT TAACTATTGG CTGATGCTGA AAGAGGTTAC
     CGAAGTCGGC
301  AGCGCGGGCG CGTCGATGTT GGATAAGTTG TGGCTGCCTG
     CTTTGTGGGG
351  CGTGGCGGAA GTCATGTTGT TTTGCAGCCT TGCCAAGTTC
     CGCCGTAAGA
401  CGCATTTTTC TGCCGATATA CTGTTTGCCT TCCTAATGCT
     GATGATTTTC
451  GTGCGTTCGT TCGACACGAA ACAAGAGCAC GGTATTTCGC
     CCAAACCGAC
501  ATACAGCCGC ATCAAAGCCA ATTATTTCAG CTTCGGTTAT
     TTTGTCGGGC
551  GCGTGTTGCC GTATCAGTTG TTTGATTTAA GCAAGATCCC
     TGTGTTCAAA
601  CAGCCTGCTC CAAGCAAAAT CGGGCAAGGC AGTATTCAAA
     ATATCGTCCT
651  GATTATGGGC GAAAGCGAAA GCGCGGCGCA TTTGAAATTG
     TTTGGTTACG
701  GGCGCGAAAC TTCGCCGTTT TTAACCCGGC TGTCGCAAGC
     CGATTTTAAG
751  CCGATTGTGA AACAAAGTTA TTCCGCAGGC TTTATGACGG
     CAGTATCCCT
801  GCCCAGTTTC TTTAACGTCA TACCGCACGC CAACGGCTTG
     GAACAAATCA
851  GCGGCGGCGA TACCAATATG TTCCGCCTCG CCAAAGAGCA
     GGGCTATGAA
901  ACGTATTTTT ACAGTGCCCA GGCTGAAAAC CAAATGGCAA
     TTTTGAACTT
951  AATCGGTAAG AAATGGATAG ACCATCTGAT TCAGCCGACG
     CAACTTGGCT
1001 ACGGCAACGG CGACAATATG CCCGATGAGA AGCTGCTGCC
     GTTGTTCGAC
1051 AAAATCAATT TGCAGCAGGG CAGGCATTTT ATCGTGTTGC
     ACCAACGCGG
1101 TTCGCACGCC CCATACGGCG CATTGTTGCA GCCTCAAGAT
     AAAGTATTCG
1151 GCGAAGCCGA TATTGTGGAT AAGTACGACA ACACCATCCA
     CAAAACCGAC
1201 CAAATGATTC AAACCGTATT CGAGCAGCTG CAAAAGCAGC
     CTGACGGCAA
1251 CTGGCTGTTT GCCTATACCT CCGATCATGG CCAGTATGTG
     CGCCAAGATA
1301 TCTACAATCA AGGCACGGTG CAGCCCGACA GCTATATTGT
     GCCTCTGGTT
1351 TTGTACAGCC CGGATAAGGC CGTGCAACAG GCTGCCAACC
     AGGCTTTTGC
1401 GCCTTGCGAG ATTGCCTTCC ATCAGCAGCT TTCAACGTTC
     CTGATTCACA
1451 CGTTGGGCTA CGATATGCCG GTTTCAGGTT GTCGCGAAGG
     CTCGGTAACA
1501 GGCAACCTGA TTACGGGCGA TGCAGGCAGC TTGAACATTC
     GCAACGGCAA
1551 GGCGGAATAT GTTTATCCGC AATAA

This encodes a protein having amino acid sequence <SEQ ID 310>:

1   MKKSLFVLFL YSSLLTASEI AYRFVFGIET LPAAKMAETF
    ALTFMIAALY
51  LFARYKASRL LIAVFFAFSM IANNVHYAVY QSWMTGINYW
    LMLKEVTEVG
101 SAGASMLDKL WLPALWGVAE VMLFCSLAKF RRKTHFSADI
    LFAFLMLMIF
151 VRSFDTKQEH GISPKPTYSR IKANYFSFGY FVGRVLPYQL
    FDLSKIPVFK
201 QPAPSKIGQG SIQNIVLIMG ESESAAHLKL FGYGRETSPF
    LTRLSQADFK
251 PIVKQSYSAG FMTAVSLPSF FNVIPHANGL EQISGGDTNM
    FRLAKEQGYE
301 TYFYSAQAEN QMAILNLIGK KWIDHLIQPT QLGYGNGDNM
    PDEKLLPLFD
351 KINLQQGRHF IVLHQRGSHA PYGALLQPQD KVFGEADIVD
    KYDNTIHKTD
401 QMIQTVFEQL QKQPDGNWLF AYTSDHGQYV RQDIYNQGTV
    QPDSYIVPLV
451 LYSPDKAVQQ AANQAFAPCE IAFHQQLSTF LIHTLGYDMP
    VSGCREGSVT
501 GNLITGDAGS LNIRNGKAEY VYPQ*

ORF81ng and ORF81-1 show 96.4% identity in 524 aa overlap:

Furthermore, ORF81ng shows significant homology to an E. coli OMP:

gi|1256380 (U50906) outer membrane adherence protein-associated
protein [E. coli] Length = 547
Score = 87.4 bits (213), Expect = 2e−16
Identities = 122/468 (26%), Positives = 198/468 (42%),
Gaps = 70/468 (14%)
Query:	25	VFGIETLPAAKMAETFA-LTFMIAALYLFARYKAS--RLLIAVFFAFSMIANNVHYAVYQ	81
		VFGI  L A+  A     L F +  + +  R  +   RLL+A  F   + A ++  ++Y
Sbjct:	29	VFGITNLVASSGAHMVQRLLFFVLTILVVKRISSLPLRLLVAAPFVL-LTAADMSISLY-	86
Query:	82	SWMT-------GINYWLMLKEVTEVGSAGASMLDKLWLPALWGVAEVMLFCSLAKFRRKT	134
		SW T       G    ++  +  EV    A ML  ++ P L   A + L       +
Sbjct:	87	SWCTFGTTFNDGFAISVLQSDPDEV----AKMLG-MYSPYLCAFAFLSLLFLAVIIKYDV	141
Query:	135	HFSADILFAFLMLMIFVRSF---------DTKQEHGISPKPTYSRIKAN--YFSFGYFVG	183
		+   L+L++   S          D K ++  SP    SR      +F+  YF
Sbjct:	142	SLPTKKVTGILLLIVISGSLFSACQFAYKDAKNKNAFSPYILASRFATYTPFFNLNYFAL	201
Query:	184	RVLPYQ--LFDLSKIPVFKQPAPSKIGQGSIQNIVLIMGESESAAHLKLFGYGRETSPFL	241
		+Q  L   + +P F+      +    I   VLI+GES    ++ L+GY R T+P +
Sbjct:	202	AAKEHQRLLSIANTVPYFQL----SVRDTGIDTYVLIVGESVRVDNMSLYGYTRSTTPQV	257
Query:	242	TRLSQADFKPIVKQSYSAGFMTAVSLP---SFFNVIPHANGLEQISGGDTNMFRLAKEQG	298
		+Q     +  Q+ S    TA+S+P   +  +V+ H      I     N+  +A + G
Sbjct:	258	E--AQRKQIKLFNQAISGAPYTALSVPLSLTADSVLSH-----DIHNYPDNIINMANQAG	310
Query:	299	YETYFYSAQA---ENQMAILNLIGKKWIDHLIQPTQLGYGNGDNMPDEKLLPLFDKINLQ	355
		++T++ S+Q+   +N  A+ ++         ++  +  Y  G    DE LLP   +   Q
Sbjct:	311	FQTFWLSSQSAFRQNGTAVTSI--------AMRAMETVYVRGF---DELLLPHLSQALQQ	359
Query:	356	--QGRHFIVLHQRGSHAPYGALLQPQDKVFGEADIVDK-YDNTIHKTDQMIQTVFEQLQK	412
		Q +  IVLH  GSH P  +       VF   D  D  YDN+IH TD ++  VFE L+
Sbjct:	360	NTQQKKLIVLHLNGSHEPACSAYPQSSAVFQPQDDQDACYDNSIHYTDSLLGQVFELLK-	418
Query:	413	QPDGNWLFAYTSDHG---QYVRQDIYNQG--TVQPDSYIVPL-VLYSP	454
		D      Y +DHG      ++++Y  G       +Y VP+ + YSP
Sbjct:	419	--DRRASVMYFADHGLERDPTKKNVYFHGGREASQQAYHVPMFIWYSP	464

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 37

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 311>:

1   ...ACCCTGCTCC TCTTCATCCC CCTCGTCCTC ACAC.GTGCG
    GCACACTGAC
51  CGGCATACTC GCCCaCGGCG GCGGCAAACG CTTTGCCGTC
    GAACAAGAAC
101 TCGTCGCCGC ATCGTCCCGC GCCGCCGTCA AAGAAATGGA
    TTTGTCCGCC
151 yTAAAAGGAC GCAAAGCCGC CyTTTACGTC TCCGTTATGG
    GCGACCAAGG
201 TTCGGGCAAC ATAAGCGGCG GACGCTACTC TATCGACGCA
    CTGATACGCG
251 GCGGCTACCA CAACAACCCC GAAAGTGCCA CCCAATACAG
    CTACCCCGCC
301 TACGACACTA CCGCCACCAC CAAATCCGAC GCGCTCTCCA
    GCGTAACCAC
351 TTCCACATCG CTTTTGAACG CCCCCGCCGC CGyCyTGACG
    AAAAACAGCG
401 GACGCAAAGG CGAACGcTCC GCCGGACTGT CCGTCAACGG
    CACGGGCGAC
451 TACCGCAACG AAACCCTGCT CGCCAACCCC CGCGACGTTT
    CCTTCCTGAC
501 CAACCTCATC CAAACCGTCT TCTACCTGCG CGGCATCGAA
    GTCgTACCGC
551 CCGrATACGC CGACACCGAC GTATTCGTAA CCGTCGACGT
    A...

This corresponds to the amino acid sequence <SEQ ID 312; ORF83>:

1   ..TLLLFIPLVL TXCGTLTGIL AHGGGKRFAV EQELVAASSR
    AAVKEMDLSA
51  LKGRKAAXYV SVMGDQGSGN ISGGRYSIDA LIRGGYHNNP
    ESATQYSYPA
101 YDTTATTKSD ALSSVTTSTS LLNAPAAXLT KNSGRKGERS
    AGLSVNGTGD
151 YRNETLLANP RDVSFLTNLI QTVFYLRGIE VVPPXYADTD
    VFVTVDV..

Further work revealed the complete nucleotide sequence <SEQ ID 313>:

1   ATGAAAACCC TGCTCCTCCT CATCCCCCTC GTCCTCACAG
    CCTGCGGCAC
51  ACTGACCGGC ATACCCGCCC ACGGCGGCGG CAAACGCTTT
    GCCGTCGAAC
101 AAGAACTCGT CGCCGCATCG TCCCGCGCCG CCGTCAAAGA
    AATGGATTTG
151 TCCGCCCTAA AAGGACGCAA AGCCGCCCTT TACGTCTCCG
    TTATGGGCGA
201 CCAAGGTTCG GGCAACATAA GCGGCGGACG CTACTCTATC
    GACGCACTGA
251 TACGCGGCGG CTACCACAAC AACCCCGAAA GTGCCACCCA
    ATACAGCTAC
301 CCCGCCTACG ACACTACCGC CACCACCAAA TCCGACGCGC
    TCTCCAGCGT
351 AACCACTTCC ACATCGCTTT TGAACGCCCC CGCCGCCGCC
    CTGACGAAAA
401 ACAGCGGACG CAAAGGCGAA CGCTCCGCCG GACTGTCCGT
    CAACGGCACG
451 GGCGACTACC GCAACGAAAC CCTGCTCGCC AACCCCCGCG
    ACGTTTCCTT
501 CCTGACCAAC CTCATCCAAA CCGTCTTCTA CCTGCGCGGC
    ATCGAAGTCG
551 TACCGCCCGA ATACGCCGAC ACCGACGTAT TCGTAACCGT
    CGACGTATTC
601 GGCACCGTCC GCAGCCGTAC CGAACTGCAC CTCTACAACG
    CCGAAACCCT
651 TAAAGCCCAA ACCAAGCTCG AATATTTCGC CGTTGACCGC
    GACAGCCGGA
701 AACTGCTGAT TACCCCTAAA ACCGCCGCCT ACGAATCCCA
    ATACCAAGAA
751 CAATACGCCC TTTGGACCGG CCCTTACAAA GTCAGCAAAA
    CCGTCAAAGC
801 CTCAGACCGC CTGATGGTCG ATTTCTCCGA CATTACCCCC
    TACGGCGACA
851 CAACCGCCCA AAACCGTCCC GACTTCAAAC AAAACAACGG
    TAAAAAACCC
901 GATGTCGGCA ACGAAGTCAT CCGCCGCCGC AAAGGAGGAT
    AA

This corresponds to the amino acid sequence <SEQ ID 314; ORF83-1>:

1   MKTLLLLIPL VLTACGTLTG IPAHGGGKRF AVEQELVAAS
    SRAAVKEMDL
51  SALKGRKAAL YVSVMGDQGS GNISGGRYSI DALIRGGYHN
    NPESATQYSY
101 PAYDTTATTK SDALSSVTTS TSLLNAPAAA LTKNSGRKGE
    RSAGLSVNGT
151 GDYRNETLLA NPRDVSFLTN LIQTVFYLRG IEVVPPEYAD
    TDVFVTVDVF
201 GTVRSRTELH LYNAETLKAQ TKLEYFAVDR DSRKLLITPK
    TAAYESQYQE
251 QYALWTGPYK VSKTVKASDR LMVDFSDITP YGDTTAQNRP
    DFKQNNGKKP
301 DVGNEVIRRR KGG*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF83 shows 96.4% identity over a 197aa overlap with an ORF (ORF83a) from strain A of N. meningitidis:

The complete length ORF83a nucleotide sequence <SEQ ID 315> is:

1   ATGAAAACCC TGCTCNTCCT CATCCCCCTC GTCCTCACAG
    CCTGCGGCAC
51  ACTGACCGGC ATACCCGCCC ACGGCGGCGG CAAACGCTTT
    GCCGTCGAAC
101 AAGAACTCGT CGCCGCATCG TCCCGCGCCG CCGTCAAAGA
    AATGGACTTG
151 TCCGCCCTGA AAGGACGCAA AGCCGCCCTT TACGTCTCCG
    TTATGGGCGA
201 CCAAGGTTCG GGCAACATAA GCGGCGGACG CTACTCTATC
    GACGCACTGA
251 TACGCGGCGG CTACCACAAC AACCCCGAAA GTGCCACCCA
    ATACAGCTAC
301 CCCGCCTACG ACACTACCGC CACCACCAAA TCCGACGCGC
    TCTCCAGCGT
351 AACCACTTCC ACATCGCTTT TGAACGCCCC CGCCGCCGCC
    CTGACGAAAA
401 ACAGCGGACG CAAAGGCGAA CGCTCCGCCG GACTGTCCGT
    CAACGGCACG
451 GGCGACTACC GCAACGAAAC CCTGCTCGCC AACCCCCGCG
    ACGTTTCCTT
501 CCTGACCAAC CTCATCCAAA CCGTCTTCTA CCTGCGCGGC
    ATCGAAGTCG
551 TACCGCCCGA ATACGCCGAC ACCGACGTAT TCGTAACCGT
    CGACGTATTC
601 GGCACCGTCC GCAGCCGCAC CGAACTGCAC CTCTACAACG
    CCGAAACCCT
651 TAAAGCCCAA ACCAAGCTCG AATATTTCGC CGTTGACCGC
    GACAGCCGGA
701 AACTGCTGAT TGCCCCTAAA ACCGCCGCCT ACGAATCCCA
    ATACCAAGAA
751 CAATACGCCC TCTGGATGGG ACCTTACAGC GTCGGCAAAA
    CCGTCAAAGC
801 CTCAGACCGC CTGATGGTCG ATTTCTCCGA CATCACCCCC
    TACGGCGACA
851 CAACCGCCCA AAACCGTCCC GACTTCAAAC AAAACAACGG
    TAAAAAACCC
901 GATGTCGGCA ACGAAGTCAT CCGCCGCCGC AAAGGAGGAT
    AA

This encodes a protein having amino acid sequence <SEQ ID 316>:

1   MKTLLXLIPL VLTACGTLTG IPAHGGGKRF AVEQELVAAS
    SRAAVKEMDL
51  SALKGRKAAL YVSVMGDQGS GNISGGRYSI DALIRGGYHN
    NPESATQYSY
101 PAYDTTATTK SDALSSVTTS TSLLNAPAAA LTKNSGRKGE
    RSAGLSVNGT
151 GDYRNETLLA NPRDVSFLTN LIQTVFYLRG IEVVPPEYAD
    TDVFVTVDVF
201 GTVRSRTELH LYNAETLKAQ TKLEYFAVDR DSRKLLIAPK
    TAAYESQYQE
251 QYALWMGPYS VGKTVKASDR LMVDFSDITP YGDTTAQNRP
    DFKQNNGKKP
301 DVGNEVIRRR KGG*

ORF83a and ORF83-1 show 98.4% identity in 313 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF83 shows 94.9% identity over a 197aa overlap with a predicted ORF (ORF83.ng) from N. gonorrhoeae:

The complete length ORF83ng nucleotide sequence <SEQ ID 317> is:

1   ATGAAAACCC TGCTCCTCCT CATCCCCCTC GTACTCACCG
    CCTGCGGCAC
51  ACTGACCGGC ATACCCGCCC ACGGCGGCGG CAAACGCTTT
    GCCGTCGAAC
101 AGGAACTCGT CGCCGCATCG TCCCGCGCCG CCGTCAAAGA
    AATGGACTTG
151 TCCGCCCTGA AAGGACGCAA AGCCGCCCTT TACGTCTCCG
    TTATGGGCGA
201 CCAAGGTTCG GGCAACATAA GCGGCGGACG CTACTCCATC
    GACGCACTGA
251 TACGCGGCGG CTACCACAAC AACCCCGACA GCGCCACCCG
    ATACAGCTAC
301 CCCGCCTATG ACACTACCGC CACCACCAAA TCCGACGCGC
    TCTCCGGCGT
351 AACCACTTCC ACATCGCTTT TGAACGCCCC CGCCGCCGCC
    CTGACGAAAA
401 ACAACGGACG CAAAGGCGAA CGCTCCGCCG GACTGTCCGT
    CAACGGCACG
451 GGCGACTACC GCAACGAAAC CCTGCTCGCC AACCCCCGCG
    ACGTTTCCTT
501 CCTGACCAAC CTCATCCAAA CCGTCTTCTA CCTGCGCGGC
    ATCGAAGTCG
551 TACCGCCCGA ATACGCCGAC ACCGACGTAT TCGTAACCGT
    CGACGTATTC
601 GGCACCGTCC GCAGCCGTAC CGAACTGCAC CTCTACAACG
    CCGAAACCCT
651 TAAAGCCCAA ACCAAGCTCG AATATTTCGC CGTCGACCGC
    GACAGCCGGA
701 AACTGCTGAT TGCCCCTAAA ACCGCCGCCT ACGAATCCCA
    ATACCAAGAA
751 CAATACGCCC TCTGGATGGG ACCTTACAGC GTCGGCAAAA
    CCGTCAAAGC
801 CTCAGACCGC CTGATGGTCG ATTTCTCCGA CATCACCCCC
    TACGGCGACA
851 CAACCGCCCA AAACCGTCCC GACTTCAAAC AAAACAACGG
    TAAAAACCCC
901 GATGTCGGCA ACGAAGTCAT CCGCCGCCGC AAAGGAGGAT
    AA

This encodes a protein having amino acid sequence <SEQ ID 318>:

1   MKTLLLLIPL VLTACGTLTG IPAHGGGKRF AVEQELVAAS
    SRAAVKEMDL
51  SALKGRKAAL YVSVMGDQGS GNISGGRYSI DALIRGGYHN
    NPDSATRYSY
101 PAYDTTATTK SDALSGVTTS TSLLNAPAAA LTKNNGRKGE
    RSAGLSVNGT
151 GDYRNETLLA NPRDVSFLTN LIQTVFYLRG IEVVPPEYAD
    TDVFVTVDVF
201 GTVRSRTELH LYNAETLKAQ TKLEYFAVDR DSRKLLIAPK
    TAAYESQYQE
251 QYALWMGPYS VGKTVKASDR LMVDFSDITP YGDTTAQNRP
    DFKQNNGKNP
301 DVGNEVIRRR KGG*

ORF83ng and ORF83-1 show 97.1% identity in 313 aa overlap

Based on this analysis, including the presence of a putative ATP/GTP-binding site motif A (P-loop) in the gonococcal protein (double-underlined) and a putative prokaryotic membrane lipoprotein lipid attachment site (single-underlined), it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 38

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 319>:

1    ATGGCAGAGA TCTGTTTGAT AACCGGCACG CCCGGTTCAG
     GGAAAACATT
51   AAAAATGGTT TCCATGATGG CGAATGATGA AATGTTTAAG
     CCTGATGAAA
101  AAGCCATACG CCGTAAAGTA TTTACGAACA TAAAAGGCTT
     GAAAATACCG
151  CACACCTACA TAGAAACGGA CGCAAAAAAG CTGCCGAAAT
     CGACAGATGA
201  GCAGCTTTCG GCGCATGATA TGTACGAATG GATAAAGAAG
     CCCGAAAATA
251  TCGGGTCTAT TGTCATTGTA GATGAAGCTC AAGACGTATG
     GCCGGCACGC
301  TCGGCAGGTT CAAAAATCCC TGAAAATGTC CAATGGCTGA
     ATACGCACAG
351  ACATCAGGGC ATTGATATAT TTGTTTTGAC TCAAGGTCCT
     AAGCTTCTAG
401  ATCAAAATCT TAGAACGCTT GTACGGAAAC ATTACCACAT
     CGCTTCAAAC
451  AAGATGGGTA TGCGTACGCT TTTAGAATGG AAAATATGCG
     CGGACGATCC
501  CGTAAAAATG GCATCAAGCG CATTCTCCAG TATCTATACA
     CTGGATAAAA
551  AAGTTTATGA CTTGTAysrr TmmGCGGAAG TTCATACCGT
     AAATAAGGTC
601  AAGCGGTCAA AGTGGTTTTA CACTCTGCCa GTAATAGTAT
     TGCTGATTCC
651  CGTGTTTGTC GGCCTGTCCT ATAAAATGTT GagCaGTTAC
     GGAAAAAAAC
701  aGGAAGAACC CGCAGCACAA GAATCGGCGG CAACAGAACA
     GCAGGCAGTA
751  CTTCCGGATA AAACAGAAGG CGAGCCGGTA AATAACGGCA
     ACCTTACCGC
801  AGATATGTTT GTTCCGACAT TGTCCGAaAA ACCCGrAAGC
     AAGCcgaTTT
851  ATAACGGTGT AAGGCAGGTA AGAACCTTTG AATATATAGC
     AGGCTGTATA
901  GAAGGCGGAA GAACCGGATG CGCCTGCTAT TCGCaTCAAG
     GGACGGCATt
951  gaAAGAAGTG ACGGaGTTGA TGTGccaAgG aCTATGTaAA
     AAacGGCTTG
1001 CCGTTTAACC CaTACAAAGA AGAAAGCCAA GGGCAGGAAG
     TTCAGCAAAG
1051 CGCGCAgCAA CATTCGGACA GGGCGcCAAG TTGCCACATT
     GGGCGGAAAA
1101 CCGTAGCAGA ACCTAATGTA CGATAATTGG GAAGAACGCG
     GGAAACCGTT
1151 TGAAGGAATC GGaCGQGGGC GTGGTCGGAT CGGCAAACTG
     A

This corresponds to the amino acid sequence <SEQ ID 320; ORF84>:

1   MAEICLITGT PGSGKTLKMV SMMANDEMFK PDEKAIRRKV
    FTNIKGLKIP
51  HTYIETDAKK LPKSTDEQLS AHDMYEWIKK PENIGSIVIV
    DEAQDVWPAR
101 SAGSKIPENV QWLNTHRHQG IDIFVLTQGP KLLDQNLRTL
    VRKHYHIASN
151 KMGMRTLLEW KICADDPVKM ASSAFSSIYT LDKKVYDLYX
    XAEVHTVNKV
201 KRSKWFYTLP VIVLLIPVFV GLSYKMLSSY GKKQEEPAAQ
    ESAATEQQAV
251 LPDKTEGEPV NNGNLTADMF VPTLSEKPXS KPIYNGVRQV
    RTFEYIAGCI
301 EGGRTGCACY SHQGTALKEV TELMCKDYVK NGLPFNPYKE
    ESQGQEVQQS
351 AQQHSDRAQV ATLGGKPXQN LMYDNWEERG KPFEGIGGGV
    VGSAN*

Further work revealed the complete nucleotide sequence <SEQ ID 321>:

1    ATGGCAGAGA TCTGTTTGAT AACCGGCACG CCCGGTTCAG
     GGAAAACATT
51   AAAAATGGTT TCCATGATGG CGAATGATGA AATGTTTAAG
     CCTGATGAAA
101  ACGGCATACG CCGTAAAGTA TTTACGAACA TAAAAGGCTT
     GAAAATACCG
151  CACACCTACA TAGAAACGGA CGCAAAAAAG CTGCCGAAAT
     CGACAGATGA
201  GCAGCTTTCG GCGCATGATA TGTACGAATG GATAAAGAAG
     CCCGAAAATA
251  TCGGGTCTAT TGTCATTGTA GATGAAGCTC AAGACGTATG
     GCCGGCACGC
301  TCGGCAGGTT CAAAAATCCC TGAAAATGTC CAATGGCTGA
     ATACGCACAG
351  ACATCAGGGC ATTGATATAT TTGTTTTGAC TCAAGGTCCT
     AAGCTTCTAG
401  ATCAAAATCT TAGAACGCTT GTACGGAAAC ATTACCACAT
     CGCTTCAAAC
451  AAGATGGGTA TGCGTACGCT TTTAGAATGG AAAATATGCG
     CGGACGATCC
501  CGTAAAAATG GCATCAAGCG CATTCTCCAG TATCTATACA
     CTGGATAAAA
551  AAGTTTATGA CTTGTACGAA TCAGCGGAAG TTCATACCGT
     AAATAAGGTC
601  AAGCGGTCAA AGTGGTTTTA CACTCTGCCA GTAATAGTAT
     TGCTGATTCC
651  CGTGTTTGTC GGCCTGTCCT ATAAAATGTT GAGCAGTTAC
     GGAAAAAAAC
701  AGGAAGAACC CGCAGCACAA GAATCGGCGG CAACAGAACA
     GCAGGCAGTA
751  CTTCCGGATA AAACAGAAGG CGAGCCGGTA AATAACGGCA
     ACCTTACCGC
801  AGATATGTTT GTTCCGACAT TGTCCGAAAA ACCCGAAAGC
     AAGCCGATTT
851  ATAACGGTGT AAGGCAGGTA AGAACCTTTG AATATATAGC
     AGGCTGTATA
901  GAAGGCGGAA GAACCGGATG CGCCTGCTAT TCGCATCAAG
     GGACGGCATT
951  GAAAGAAGTG ACGGAGTTGA TGTGCAAGGA CTATGTAAAA
     AACGGCTTGC
1001 CGTTTAACCC ATACAAAGAA GAAAGCCAAG GGCAGGAAGT
     TCAGCAAAGC
1051 GCGCAGCAAC ATTCGGACAG GGCGCAAGTT GCCACATTGG
     GCGGAAAACC
1101 GTAGCAGAAC CTAATGTACG ATAATTGGGA AGAACGCGGG
     AAACCGTTTG
1151 AAGGAATCGG CGGGGGCGTG GTCGGATCGG CAAACTGA

This corresponds to the amino acid sequence <SEQ ID 322; ORF84-1>:

1   MAEICLITGT PGSGKTLKMV SMMANDEMFK PDENGIRRKV
    FTNIKGLKIP
51  HTYIETDAKK LPKSTDEQLS AHDMYEWIKK PENIGSIVIV
    DEAQDVWPAR
101 SAGSKIPENV QWLNTHRHQG IDIFVLTQGP KLLDQNLRTL
    VRKHYHIASN
151 KMGMRTLLEW KICADDPVKM ASSAFSSIYT LDKKVYDLYE
    SAEVHTVNKV
201 KRSKWFYTLP VIVLLIPVFV GLSYKMLSSY GKKQEEPAAQ
    ESAATEQQAV
251 LPDKTEGEPV NNGNLTADMF VPTLSEKPES KPIYNGVRQV
    RTFEYIAGCI
301 EGGRTGCACY SHQGTALKEV TELMCKDYVK NGLPFNPYKE
    ESQGQEVQQS
351 AQQHSDRAQV ATLGGKP*QN LMYDNWEERG KPFEGIGGGV
    VGSAN*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF84 shows 93.9% identity over a 395aa overlap with an ORF (ORF84a) from strain A of N. meningitidis:

The complete length ORF84a nucleotide sequence <SEQ ID 323> is:

1    ATGGCAGAGA TCTGTTTGAT AACCGGCACG CCCGGTTCAG
     GGAAAACATT
51   AAAAATGGTT TCCATGATGG CAAACGATGA AATGTTTAAG
     CCGGATGAAA
101  ACGGCATACG CCGTAAAGTA TTTACGAACA TCAAAGGCTT
     GAAGATACCG
151  CACACCTACA TAGAAACGGA CGCGAAAAAG CTGCCGAAAT
     CGACAGATGA
201  GCAGCTTTCG GCGCATGATA TGTACGAATG GATAAAGAAG
     CCCGAAAATA
251  TCGGGTCTAT TGTCATTGTA GATGAAGCTC AAGACGTATG
     GCCGGCACGC
301  TCGGCAGGTT CAAAAATCCC TGAAAATGTC CAATGGCTGA
     ATACGCACAG
351  ACATCAGGGC ATTGATATAT TTGTTTTGAC TCAAGGCTCT
     AAGCTTCTAG
401  ATCAAAATCT TAGAACGCTT GTACGGAAAC ATTACCACAT
     CGCTTCAAAC
451  AAGATGGGTA TGCGTACGCT TTTAGAATGG AAAATATGCG
     CGGACGATCC
501  CGTAAAAATG GCATCAAGCG CATTCTCCAG TATCTATACA
     CTGGATAAAA
551  AAGTTTATGA CTTGTACGAA TCAGCGGAAG TTCATACCGT
     AAATAAGGTC
601  AAGCGGTCAA AATGGTTTTA TACTCTGCCA GTAATAATAT
     TGCTGATTCC
651  CGTTTTTGTC GGCCTGTCCT ATAAAATGTT AAGTAGTTAT
     GGAAAAAAAC
701  AGGAAGAACC CGCAGCACAA GAATCGGCGG CAACAGAACA
     TCAGGCAGTA
751  TTTCAGGATA AAACAGAAGG CGAGCCGGTA AACAACGGTA
     ACCTTACCGC
801  AGATATGTTT GTTCCGACAT TGTCCGAAAA ACCCGAAAGC
     AAGCCGATTT
851  ATAACGGTGT AAGGCAGGTA AGAACCTTTG AATATATAGC
     AGGCTGTGTA
901  GAAGGCGGAA GAACCGGATG CACATGCTAT TCGCATCAAG
     GGACGGCATT
951  GAAAGAAATT ACAAAGGAAA TGTGCAAGGA TTACGCAAGA
     AACGGATTGC
1001 CGTTTAACCC ATATAAAGAA GAAAGCCAAG GGCGGGATGT
     CCAGCAAAGT
1051 GAGCAGCACC ATTCGGACAG ACCGCAAGTT GCCACGTTGG
     GCGGAAAGCC
1101 GTGGCAAAAT CTTATGTATG ATAATTGGCA GGAGCGCGGA
     AAACCGTTTG
1151 AAGGAATCGG CGGGGGCGTG GTCGGATCGG CAAACTGA

This encodes a protein having amino acid sequence <SEQ ID 324>:

1   MAEICLITGT PGSGKTLKMV SMMANDEMFK PDENGIRRKV
    FTNIKGLKIP
51  HTYIETDAKK LPKSTDEQLS AHDMYEWIKK PENIGSIVIV
    DEAQDVWPAR
101 SAGSKIPENV QWLNTHRHQG IDIFVLTQGS KLLDQNLRTL
    VRKHYHIASN
151 KMGMRTLLEW KICADDPVKM ASSAFSSIYT LDKKVYDLYE
    SAEVHTVNKV
201 KRSKWFYTLP VIILLIPVFV GLSYKMLSSY GKKQEEPAAQ
    ESAATEHQAV
251 FQDKTEGEPV NNGNLTADMF VPTLSEKPES KPIYNGVRQV
    RTFEYIAGCV
301 EGGRTGCTCY SHQGTALKEI TKEMCKDYAR NGLPFNPYKE
    ESQGRDVQQS
351 EQHHSDRPQV ATLGGKPWQN LMYDNWQERG KPFEGIGGGV
    VGSAN*

ORF84a and ORF84-1 show 95.2% identity in 395 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF84 shows 94.2% identity over a 395aa overlap with a predicted ORF (ORF84.ng) from N. gonorrhoeae:

The complete length ORF84ng nucleotide sequence <SEQ ID 325> is:

1    ATGGCAGAAA TCTGTTTGAT AACCGGCACG CCCGGTTCAG
     GGAAAACATT
51   AAAAATGGTT TCCATGATGG CAAACGATGA AATGTTTAAG
     CCAGATGAAA
101  ACGGCGTACG CCGTAAAGTA TTTACGAACA TCAAAGGTTT
     GAAGATACCG
151  CACACCCACA TAGAAACAGA CGCAAAGAAG CTGCCGAAAT
     CAACCGATGA
201  ACAGCTTTCG GCGCATGATA TGTATGAATG GATCAAGAAG
     CCTGAAAacg
251  tcggcgCAAT CGTTATTGTC GATGAGGCGC AAGACGTATG
     GCCCGCACGC
301  TccgCAGGTT CGAAAATCCC CGAAAACGTC CAATGGCTGA
     ACACACACAG
351  GCATCAGGGC ATAGATATAT TTGTATTGAC ACAAGGTCCT
     AAACTCTTAG
401  ATCAGAACTT GCGAACATTG GTTAAAAGAC ATTACCACAT
     TGCGGCCAAC
451  AAAATGGGTT TGCGTACCCT GCTTGAATGG AAAGTATGCG
     CGGATGACCC
501  GGTAAAAATG GCATCAAGTG CATTTTCCAG TATCTACACA
     CTGGATAAAA
551  AAGTTTATGA CTTGTACGAA TCCGCAGAAA TTCACACGGT
     AAACAAAGTC
601  AAGCGTTCAA AATGGTTTTA TGCATTGCCC GTCATCATAT
     TATTGATTCC
651  GCTATTTGTC GGTTTGTCTT ACAAAATGTT GGGCAGTTAC
     GGAAAAAAAC
701  AGGAAGAACC CGCAGCACAA GAATCGGCGG CAACAGAACA
     GCAGGCAGTA
751  CTTCCGGATA AAACAGAAGG AGAATCGGTG AATAACGGAA
     ACCTTACGGC
801  AGATATGTTT GTTCCGACAT TGCCCGAAAA ACCCGAAAGC
     AAGCCGATTT
851  ATAACGGTGT AAGGCAGGTA AGGACCTTTG AATATATAGC
     AGGCTGTATA
901  GAAGGCGGAA GAACCGGATG CACCTGCTAT TCGCATCAAG
     GGACGGCATT
951  GAAAGAAGTG ACGGAGTTGA TGTGCAAGGA CTATGTAAAA
     AACGGCTTGC
1001 CGTTTAACCC ATACAAAGAA GAAAGCCAAG GGCAGGAAGT
     TCAGCAAAGC
1051 GCGCAGCAAC ATTCGGACAG GGCGCAAGTT GCCACCTTGG
     GCGGAAAACC
1101 GCAGCAGAAC CTAATGTACG ACAATTGGGA AGAACGCGGG
     AAACCGTTTG
1151 AAGGAATCGG CGGGGGCGTG GTCGGATCGG CAAACTGA

This encodes a protein having amino acid sequence <SEQ ID 326>:

1   MAEICLITGT PGSGKTLKMV SMMANDEMFK PDENGVRRKV
    FTNIKGLKIP
51  HTHIETDAKK LPKSTDEQLS AHDMYEWIKK PENVGAIVIV
    DEAQDVWPAR
101 SAGSKIPENV QWLNTHRHQG IDIFVLTQGP KLLDQNLRTL
    VKRHYHIAAN
151 KMGLRTLLEW KVCADDPVKM ASSAFSSIYT LDKKVYDLYE
    SAEIHTVNKV
201 KRSKWFYALP VIILLIPLFV GLSYKMLGSY GKKQEEPAAQ
    ESAATEQQAV
251 LPDKTEGESV NNGNLTADMF VPTLPEKPES KPIYNGVRQV
    RTFEYIAGCI
301 EGGRTGCTCY SHQGTALKEV TELMCKDYVK NGLPFNPYKE
    ESQGQEVQQS
351 AQQHSDRAQV ATLGGKPQQN LMYDNWEERG KPFEGIGGGV
    VGSAN*

ORF84ng and ORF84-1 show 95.4% identity in 395 aa overlap:

Based on this analysis, includng the presence of a putative transmembrane domain (single-underlined) in the gonococcal protein, and a putative ATP/GTP-binding site motif A (P-loop, double-underlined), it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 39

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 327>:

1    GTGGTTTTCC TGAATGCCGA CAACGGGATA TTGGTTCAGG
     ACTTGCCTTT
51   TGAAGTCAAA CTGAAAAAAT TCCATATCGA TTTTTACAAT
     ACGGGTATGC
101  CGCGTGATTT CGCCAGCGAT ATTGAAGTGA CGGACAAGGC
     AACCGGTGAG
151  AAACTCGAGC GCACCATCCG CGTGAACCAT CCTTTGACCT
     TGCACGGCAT
201  CACGATTTAT CAGGCGAGTT TTGCCGACGG CGGTTCGGAT
     TTGACATTCA
251  AGGCGTGGAA TTTGGGTGAT GCTTCGCGCG AGCCTGTCGT
     GTTGAAGGCA
301  ACATCCATAC ACCAGTTTCC GTTGGAAATT GGCAAACACA
     AATATCGTCT
351  TGAGTTCGAT CAGTTCACTT CTATGAATGT GGAGGACATG
     AGCGAGGGCG
401  CGGAACGGGA AAAAAGCCTG AAATCCACGC TGCCCGATGT
     CCGCGCCGTT
451  ACTCAGGAAG GTCACAAATA CACCAAT... ..........
     .....TACCG
501  TATCCGTGAT GCGCCAGGCC AGGCGGTCGA ATATAAAAAC
     TATATGCTGC
551  CGGTTTTGCA GGAACAGGAT TATTTTTGGA TTACCGGCAC
     GCGCAGCGC.
601  TTGCAGCAGC AATACCGCTG GCTGCGTATC CCCTTGGACA
     AGCAGTTGAA
651  AGCGGACACC TTTATGGCAT TGCGTGAGTT TTTGAAAGAT
     GGGGAAGGGC
701  GCAAACGTCT .GTTGCCGAC GCAACCAAAG GCGCACCTGC
     CGAAATCCGC
751  GAACAATTCA TGCTGGCTGC GGAAAACACG CTGAACATCT
     TTGCACAAAA
801  AGGCTATTTG GGATTGGACG AATTTATTAC GTCCAATATC
     CCGAAAGAGC
851  AGCAGGATAA GATGCAGGGC TATTTCTACG AAATGCTTTA
     CGGCGTGATG
901  AACGCTGCTT TGGATGAAAC CAT.ACCCGG TACGGCTTGC
     CCGAATGGCA
951  GCAGGATGAA GCGCGGAATC GTTTCCTGCT GCACAGTATG
     GATGCGTACA
1001 CGGGTTTGAC CGAATATCCC GCGCCTATGC TGCTGCAACT
     TGATGGGTTT
1051 TCCGAGGTGC GTTCGTCGGG TTTGCAGATG ACCCGTTCCC
     C.GGTCCGCT
1101 TTTGGTCTAT CTC...

This corresponds to the amino acid sequence <SEQ ID 328; ORF88>:

1   MVFLNADNGI LVQDLPFEVK LKKFHIDFYN TGMPRDFASD
    IEVTDKATGE
51  KLERTIRVNH PLTLHGITIY QASFADGGSD LTFKAWNLGD
    ASREPVVLKA
101 TSIHQFPLEI GKHKYRLEFD QFTSMNVEDM SEGAEREKSL
    KSTLPDVRAV
151 TQEGHKYTNX XXXXXYRIRD APGQAVEYKN YMLPVLQEQD
    YFWITGTRSX
201 LQQQYRWLRI PLDKQLKADT FMALREFLKD GEGRKRXVAD
    ATKGAPAEIR
251 EQFMLAAENT LNIFAQKGYL GLDEFITSNI PKEQQDKMQG
    YFYEMLYGVM
301 NAALDETXTR YGLPEWQQDE ARNRFLLHSM DAYTGLTEYP
    APMLLQLDGF
351 SEVRSSGLQM TRSXGPLLVY L...

Further work revealed the complete nucleotide sequence <SEQ ID 329>:

1    ATGAGTAAAT CCCGTAGATC TCCCCCACTT CTTTCCCGTC
     CGTGGTTCGC
51   TTTTTTCAGC TCCATGCGCT TTGCAGTCGC TTTGCTCAGT
     CTGCTGGGTA
101  TTGCATCGGT TATCGGTACG GTGTTGCAGC AAAACCAGCC
     GCAGACGGAT
151  TATTTGGTCA AATTCGGATC GTTTTGGGCG CAGATTTTTG
     GTTTTCTGGG
201  ACTGTATGAC GTCTATGCTT CGGCATGGTT TGTCGTTATC
     ATGATGTTTT
251  TGGTGGTTTC TACCAGTTTG TGCCTGATTC GCAATGTGCC
     GCCGTTCTGG
301  CGCGAAATGA AGTCTTTTCG GGAAAAGGTT AAAGAAAAAT
     CTCTGGCGGC
351  GATGCGCCAT TCTTCGCTGT TGGATGTAAA AATTGCGCCC
     GAGGTTGCCA
401  AACGTTATCT GGAAGTACAA GGTTTTCAGG GAAAAACCAT
     TAACCGTGAA
451  GACGGGTCGG TTCTGATTGC CGCCAAAAAA GGCACAATGA
     ACAAATGGGG
501  CTATATCTTT GCCCATGTTG CTTTGATTGT CATTTGCCTG
     GGCGGGTTGA
551  TAGACAGTAA CCTGCTGTTG AAACTGGGTA TGCTGACCGG
     TCGGATTGTT
601  CCGGACAATC AGGCGGTTTA TGCCAAGGAT TTCAAGCCCG
     AAAGTATTTT
651  GGGTGCGTCC AATCTCTCAT TTAGGGGCAA CGTCAATATT
     TCCGAGGGGC
701  AGAGTGCGGA TGTGGTTTTC CTGAATGCCG ACAACGGGAT
     ATTGGTTCAG
751  GACTTGCCTT TTGAAGTCAA ACTGAAAAAA TTCCATATCG
     ATTTTTACAA
801  TACGGGTATG CCGCGTGATT TCGCCAGCGA TATTGAAGTG
     ACGGACAAGG
851  CAACCGGTGA GAAACTCGAG CGCACCATCC GCGTGAACCA
     TCCTTTGACC
901  TTGCACGGCA TCACGATTTA TCAGGCGAGT TTTGCCGACG
     GCGGTTCGGA
951  TTTGACATTC AAGGCGTGGA ATTTGGGTGA TGCTTCGCGC
     GAGCCTGTCG
1001 TGTTGAAGGC AACATCCATA CACCAGTTTC CGTTGGAAAT
     TGGCAAACAC
1051 AAATATCGTC TTGAGTTCGA TCAGTTCACT TCTATGAATG
     TGGAGGACAT
1101 GAGCGAGGGC GCGGAACGGG AAAAAAGCCT GAAATCCACG
     CTGAACGATG
1151 TCCGCGCCGT TACTCAGGAA GGTAAAAAAT ACACCAATAT
     CGGCCCTTCC
1201 ATTGTTTACC GTATCCGTGA TGCGGCAGGG CAGGCGGTCG
     AATATAAAAA
1251 CTATATGCTG CCGGTTTTGC AGGAACAGGA TTATTTTTGG
     ATTACCGGCA
1301 CGCGCAGCGG CTTGCAGCAG CAATACCGCT GGCTGCGTAT
     CCCCTTGGAC
1351 AAGCAGTTGA AAGCGGACAC CTTTATGGCA TTGCGTGAGT
     TTTTGAAAGA
1401 TGGGGAAGGG CGCAAACGTC TGGTTGCCGA CGCAACCAAA
     GGCGCACCTG
1451 CCGAAATCCG CGAACAATTC ATGCTGGCTG CGGAAAACAC
     GCTGAACATC
1501 TTTGCACAAA AAGGCTATTT GGGATTGGAC GAATTTATTA
     CGTCCAATAT
1551 CCCGAAAGAG CAGCAGGATA AGATGCAGGG CTATTTCTAC
     GAAATGCTTT
1601 ACGGCGTGAT GAACGCTGCT TTGGATGAAA CCATACGCCG
     GTACGGCTTG
1651 CCCGAATGGC AGCAGGATGA AGCGCGGAAT CGTTTCCTGC
     TGCACAGTAT
1701 GGATGCGTAC ACGGGTTTGA CCGAATATCC CGCGCCTATG
     CTGCTGCAAC
1751 TTGATGGGTT TTCCGAGGTG CGTTCGTCGG GTTTGCAGAT
     GACCCGTTCC
1801 CCGGGTGCGC TTTTGGTCTA TCTCGGCTCG GTGCTGTTGG
     TATTGGGTAC
1851 GGTATTGATG TTTTATGTGC GCGAAAAACG GGCGTGGGTA
     TTGTTTTCAG
1901 ACGGCAAAAT CCGTTTTGCC ATGTCTTCGG CCCGCAGCGA
     ACGGGATTTG
1951 CAGAAGGAAT TTCCAAAACA CGTCGAGAGT CTGCAACGGC
     TCGGCAAGGA
2001 CTTGAATCAT GACTGA

This corresponds to the amino acid sequence <SEQ ID 330; ORF88-1>:

1   MSKSRRSPPL LSRPWFAFFS SMRFAVALLS LLGIASVIGT
    VLQQNQPQTD
51  YLVKFGSFWA QIFGFLGLYD VYASAWFVVI MMFLVVSTSL
    CLIRNVPPFW
101 REMKSFREKV KEKSLAAMRH SSLLDVKIAP EVAKRYLEVQ
    GFQGKTINRE
151 DGSVLIAAKK GTMNKWGYIF AHVALIVICL GGLIDSNLLL
    KLGMLTGRIV
201 PDNQAVYAKD FKPESILGAS NLSFRGNVNI SEGQSADVVF
    LNADNGILVQ
251 DLPFEVKLKK FHIDFYNTGM PRDFASDIEV TDKATGEKLE
    RTIRVNHPLT
301 LHGITIYQAS FADGGSDLTF KAWNLGDASR EPVVLKATSI
    HQFPLEIGKH
351 KYRLEFDQFT SMNVEDMSEG AEREKSLKST LNDVRAVTQE
    GKKYTNIGPS
401 IVYRIRDAAG QAVEYKNYML PVLQEQDYFW ITGTRSGLQQ
    QYRWLRIPLD
451 KQLKADTFMA LREFLKDGEG RKRLVADATK GAPAEIREQF
    MLAAENTLNI
501 FAQKGYLGLD EFITSNIPKE QQDKMQGYFY EMLYGVMNAA
    LDETIRRYGL
551 PEWQQDEARN RFLLHSMDAY TGLTEYPAPM LLQLDGFSEV
    RSSGLQMTRS
601 PGALLVYLGS VLLVLGTVLM FYVREKRAWV LFSDGKIRFA
    MSSARSERDL
651 QKEFPKHVES LQRLGKDLNH D*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF88 shows 95.7% identity over a 371aa overlap with an ORF (ORF88a) from strain A of N. meningitidis:

The complete length ORF88a nucleotide sequence <SEQ ID 331> is:

1    ATGAGTAAAT CCCGTAGATC TCCCCCACTT CTTTCCCGTC
     CGTGGTTCGC
51   TTTTTTCAGC TCCATGCGCT TTGCGGTCGC TTTGCTCAGT
     CTGCTGGGTA
101  TTGCATCGGT TATCGGTACG GTGTTGCAGC AAAACCAGCC
     GCAGACGGAT
151  TATTTGGTCA AATTCGGATC GTTTTGGGCG CAGATTTTTG
     GTTTTCTGGG
201  ACTGTATGAC GTCTATGCTT CGGCATGGTT TGTCGTTATC
     ATGATGTTTT
251  TGGTGGTTTC TACCAGTTTG TGCCTGATTC GCAATGTGCC
     GCCGTTCTGG
301  CGCGAAATGA AGTCTTTTCG GGAAAAGGTT AAAGAAAAAT
     CTCTGGCGGC
351  GATGCGCCAT TCTTCGCTGT TGGATGTAAA AATTGCGCCC
     GAGGTTGCCA
401  AACGTTATCT GGAAGTACAA GGTTTTCAGG GAAAAACCAT
     TAACCGTGAA
451  GACGGGTCGG TTCTGATTGC CGCCAAAAAA GGCACAATGA
     ACAAATGGGG
501  CTATATCTTT GCCCATGTTG CTTTGATTGT CATTTGCCTG
     GGCGGGTTGA
551  TAGACAGTAA CCTGCTGTTG AAACTGGGTA TGCTGACCGG
     TCGGATTGTT
601  CCGGACAATC AGGCGGTTTA TGCCAAGGAT TTCAAGCCCG
     AAAGTATTTT
651  GGGTGCGTCC AATCTCTCAT TTAGGGGCAA CGTCAATATT
     TCCGAGGGGC
701  AGAGTGCGGA TGTGGTTTTC CTGAATGCCG ACAACGGGAT
     ATTGGTTCAG
751  GACTTGCCTT TTGAAGTCAA ACTGAAAAAA TTCCATATCG
     ATTTTTACAA
801  TACGGGTATG CCGCGCGATT TTGCCAGTGA TATTGAAGTA
     ACGGATAAGG
851  CAACCGGTGA GAAACTCGAG CGCACCATCC GCGTGAACCA
     TCCTTTGACC
901  TTGCACGGCA TCACGATTTA TCAGGCGAGT TTTGCCGACG
     GCGGTTCGGA
951  TTTGACATTC AAGGCGTGGA ATTTGGGTGA TGCTTCGCGC
     GAGCCTGTCG
1001 TGTTGAAGGC AACATCCATA CACCAGTTTC CGTTGGAAAT
     TGGCAAACAC
1051 AAATATCGTC TTGAGTTCGA TCAGTTTACT TCTATGAATG
     TGGAGGACAT
1101 GAGCGAGGGC GCGGAACGGG AAAAAAGCCT GAAATCCACG
     CTGAACGATG
1151 TCCGCGCCGT TACTCAGGAA GGTAAAAAAT ACACCAATAT
     CGGCCCTTCC
1201 ATTGTTTACC GTATCCGTGA TGCGGCAGGG CAGGCGGTCG
     AATATAAAAA
1251 CTATATGCTG CCGGTTTTGC AGGAACAGGA TTATTTTTGG
     ATTACCGGCA
1301 CGCGCAGCGG CTTGCAGCAG CAATACCGCT GGCTGCGTAT
     CCCCTTGGAC
1351 AAGCAGTTGA AAGCGGACAC CTTTATGGCA TTGCGTGAGT
     TTTTGAAAGA
1401 TGGGGAAGGG CGCAAACGTC TGGTTGCCGA CGCAACCAAA
     GGCGCACCTG
1451 CCGAAATCCG CGAACAATTC ATGCTGGCTG CGGAAAACAC
     GCTGAACATC
1501 TTTGCACAAA AAGGCTATTT GGGATTGGAC GAATTTATTA
     CGTCCAATAT
1551 CCCGAAAGAG CAGCAGGATA AGATGCAGGG CTATTTCTAC
     GAAATGCTTT
1601 ACGGCGTGAT GAACGCTGCT TTGGATGAAA CCATACGCCG
     GTACGGCTTG
1651 CCCGAATGGC AGCAGGATGA AGCGCGGAAT CGTTTCCTGC
     TGCACAGTAT
1701 GGATGCGTAC ACGGGTTTGA CCGAATATCC CGCGCCTATG
     CTGCTGCAAC
1751 TTGATGGGTT TTCCGAGGTG CGTTCGTCGG GTTTGCAGAT
     GACCCGTTCC
1801 CCGGGTGCGC TTTTGGTCTA TCTCGGCTCG GTGCTGTTGG
     TATTGGGTAC
1851 GGTATTGATG TTTTATGTGC GCGAAAAACG GGCGTGGGTA
     TTGTTTTCAG
1901 ACGGCAAAAT CCGTTTTGCC ATGTCTTCGG CCCGCAGCGA
     ACGGGATTTG
1951 CAGAAGGAAT TTCCAAAACA CGTCGAGAGT CTGCAACGGC
     TCGGCAAGGA
2001 CTTGAATCAT GACTGA

This encodes a protein having amino acid sequence <SEQ ID 332>:

1   MSKSRRSPPL LSRPWFAFFS SMRFAVALLS LLGIASVIGT
    VLQQNQPQTD
51  YLVKFGSFWA QIFGFLGLYD VYASAWFVVI MMFLVVSTSL
    CLIRNVPPFW
101 REMKSFREKV KEKSLAAMRH SSLLDVKIAP EVAKRYLEVQ
    GFQGKTINRE
151 DGSVLIAAKK GTMNKWGYIF AHVALIVICL GGLIDSNLLL
    KLGMLTGRIV
201 PDNQAVYAKD FKPESILGAS NLSFRGNVNI SEGQSADVVF
    LNADNGILVQ
251 DLPFEVKLKK FHIDFYNTGM PRDFASDIEV TDKATGEKLE
    RTIRVNHPLT
301 LHGITIYQAS FADGGSDLTF KAWNLGDASR EPVVLKATSI
    HQFPLEIGKH
351 KYRLEFDQFT SMNVEDMSEG AEREKSLKST LNDVRAVTQE
    GKKYTNIGPS
401 IVYRIRDAAG QAVEYKNYML PVLQEQDYFW ITGTRSGLQQ
    QYRWLRIPLD
451 KQLKADTFMA LREFLKDGEG RKRLVADATK GAPAEIREQF
    MLAAENTLNI
501 FAQKGYLGLD EFITSNIPKE QQDKMQGYFY EMLYGVMNAA
    LDETIRRYGL
551 PEWQQDEARN RFLLHSMDAY TGLTEYPAPM LLQLDGFSEV
    RSSGLQMTRS
601 PGALLVYLGS VLLVLGTVLM FYVREKRAWV LFSDGKIRFA
    MSSARSERDL
651 QKEFPKHVES LQRLGKDLNH D*

ORF88a and ORF88-1 100.0% identity in 671 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF88 shows 93.8% identity over a 371aa overlap with a predicted ORF (ORF88.ng) from N. gonorrhoeae:

An ORF88ng nucleotide sequence <SEQ ID 333> was predicted to encode a protein having amino acid sequence <SEQ ID 334>:

1   MVFLNADNGM LVQDLPFEVK LKKFHIDFYN TGMPRDFASD
    IEVTDKATGE
51  KLERTIRVNH PLTLHGITIY QASFADGGSD LTFKAWNLRD
    ASREPVVLKA
101 TSIHQFPLEI GKHKYRLEFD QFTSMNVEDM SEGAEREKSL
    KSTLNDVRAV
151 TQEGKKYTNI GPSIVYRIRD AAGQAVEYKN YMLPILQDKD
    YFWLTGTRSG
201 LQQQYRWLRI PLDKQLKADT FMALREFLKD GEGRKRLVAD
    ATKDAPAEIR
251 EQFMLAAENT LNIFAQKGYL GLDEFITSNI PKGQQDKMQG
    YFYEMLYGVM
301 NAALDETIRR YGLPEWQQDE ARNRFLLHSM DAYTGLTEYP
    APMLLQLDGF
351 SEVRSSGLQM TRSPGALLVY LGSVLLVLGT VFMFYVPKKR
    AWVLFSNXKI
401 RFAMSSARSE RDLQKEFPKH VESLQRLGKD LNHD*

Further work revealed the complete gonococcal DNA sequence <SEQ ID 335>:

1    ATGAGTAAAT CCCGTATATC TCCCACACTT CTTTCCCGTC
     CGTGGTTCGC
51   TTTTTTCAGC TCCATGCGCT TTGCGGTCGC TTTGCTCAGT
     CTGCTGGGTA
101  TTGCATCGGT TATCGGCACG GTGTTACAGC AAAACCAGCC
     GCAGACGGAT
151  TATTTGGTCA AATTCGGACC GTTTTGGACT CGGATTTTTG
     ATTTTTTGGG
201  TTTGTATGAT GTCTATGCTT CGGCATGGTT TGTCGTTATC
     ATGATGTTTC
251  TGGTGGTTTC TACCAGTTTG TGTTTAATCC GTAACGTTCC
     GCCGTTTTGG
301  CGCGAAATGA AGTCTTTCCG GGAAAAGGTT AAAGAAAAAT
     CTCTGGCGGC
351  GATGCGCCAT TCTTCGCTGT TGGATGTAAA AATTGCCCCC
     GAAGTTGCCA
401  AACGTTATCT GGAGGTGCGG GGTTTTCAGG GAAAAACCGT
     CAGCCGTGAG
451  GACGGGTCGG TTCTGATTGC CGCCAAAAAA GGCAcaatga
     acaaATGGGG
501  CTATATCTTT GCccaagtag ctTTGATTGT CATTTGCCTG
     GGCGGGTTGA
551  TAGACAGTAA CCTGCTGCTG AAGCTGGGTA TGCTGGCCGG
     TCGGATTGTT
601  CCGGACAATC AGGCGGTTTA TGCCAAGGAT TTCAAGCCCG
     AAAGTATTTT
651  GGGTGCGTCC AATCTCTCAT TTAGGGGCAA CGTCAATATT
     TCCGAGGGGC
701  AAAGTGCGGA TGTGGTTTTC CTGAATGCCG ACAACGGGAT
     GTTGGTTCAG
751  GACTTGCCTT TTGAAGTCAA ACTGAAAAAA TTCCATATCG
     ATTTTTACAA
801  TACGGGTATG CCGCGCGATT TTGCCAGCGA TATTGAAGTA
     ACGGACAAGG
851  CAACCGGTGA GAAACTCGAG CGCACCATCC GCGTGAACCA
     TCCTTTGACC
901  TTGCACGGCA TCACGATTTA TCAGGCGAGT TTTGCCGACG
     GCGGTTCGGA
951  TTTGACATTC AAGGCGTGGA ATTTGAGGGA TGCTTCGCGC
     GAACCTGTCG
1001 TGTTGAAGGC AACCTCCATA CACCAGTTTC CGTTGGAAAT
     CGGCAAACAC
1051 AAATATCGTC TTGAGTTCGA TCAGTTCACT TCTATGAATG
     TGGAGGACAT
1101 GAGCGAGGGT GCGGAACGGG AAAAAAGCCT GAAATCCACT
     CTGAACGATG
1151 TCCGCGCCGT TACTCAGGAA GGTAAAAAAT ACACCAATAT
     CGGCCCTTCC
1201 ATCGTGTACC GCATCCGTGA TGcggCAGGG CAGGCGGTCG
     AATATAAAAA
1251 CTATATGCTG CCGATTTTGC AGGACAAAGA TTATTTTTGG
     CTGACCGGCA
1301 CGCGCAGCGG CTTGCAGCAG CAATACCGCT GGCTGCGTAT
     CCCCTTGGAC
1351 AAGCAGTTGA AAGCGGACAC CTTTATGGCA TTGCGTGAGT
     TTTTGAAAGA
1401 TGGGGAAGGG CGCAAACGTC TGGTTGCCGA CGCAACCAAA
     GACGCACCTG
1451 CCGAAATCCG CGAACAATTC ATGCTGGCTG CGGAAAACAC
     GCTGAATATC
1501 TTTGCGCAAA AAGGCTATTT GGGATTGGAC GAATTTATTA
     CGTCCAATAT
1551 CCCGAAAGGG CAGCAGGATA AGATGCAGGG CTATTTCTAC
     GAAATGCTTT
1601 ACGGCGTGAT GAACGCTGCT TTGGATGAAA CCATACGCCG
     GTACGGCTTG
1651 CCCGAATGGC AGCAGGATGA AGCGCGGAAC CGTTTCCTGC
     TGCACAGTAT
1701 GGATGCCTAT ACGGGGCTGA CGGAATATCC CGCGCCTATG
     CTGCTCCAGC
1751 TTGACGGGTT TTCCGAGGTG CGTTCCTCAG GTTTGCAGAT
     GACCCGTTCG
1801 CCGGGTGCGC TTTTGGTCTA TCtcggctcg gtattgttgg
     TTTTGGgtac
1851 ggtaTttatg tTTTATGTGC GCGAAAAACG GGCGTGGgta
     tTGTTTTCag
1901 aCGGCAAAAT CCGTTTTGCT ATGtCTTcgg CCcgcagcga
     ACGGGATTTG
1951 cAGAaggaaT TTCCAAAACA CGtcgAGAGC CTGCAACggc
     tcggcaaggA
2001 CttgaaTCAT GACTga

This corresponds to the amino acid sequence <SEQ ID 336; ORF88ng-1>:

1   MSKSRISPTL LSRPWFAFFS SMRFAVALLS LLGIASVIGT
    VLQQNQPQTD
51  YLVKFGPFWT RIFDFLGLYD VYASAWFVVI MMFLVVSTSL
    CLIRNVPPFW
101 REMKSFREKV KEKSLAAMRH SSLLDVKIAP EVAKRYLEVR
    GFQGKTVSRE
151 DGSVLIAAKK GTMNKWGYIF AQVALIVICL GGLIDSNLLL
    KLGMLAGRIV
201 PDNQAVYAKD FKPESILGAS NLSFRGNVNI SEGQSADVVF
    LNADNGMLVQ
251 DLPFEVKLKK FHIDFYNTGM PRDFASDIEV TDKATGEKLE
    RTIRVNHPLT
301 LHGITIYQAS FADGGSDLTF KAWNLRDASR EPVVLKATSI
    HQFPLEIGKH
351 KYRLEFDQFT SMNVEDMSEG AEREKSLKST LNDVRAVTQE
    GKKYTNIGPS
401 IVYRIRDAAG QAVEYKNYML PILQDKDYFW LTGTRSGLQQ
    QYRWLRIPLD
451 KQLKADTFMA LREFLKDGEG RKRLVADATK DAPAEIREQF
    MLAAENTLNI
501 FAQKGYLGLD EFITSNIPKG QQDKMQGYFY EMLYGVMNAA
    LDETIRRYGL
551 PEWQQDEARN RFLLHSMDAY TGLTEYPAPM LLQLDGFSEV
    RSSGLQMTRS
601 PGALLVYLGS VLLVLGTVFM FYVREKRAWV LFSDGKIRFA
    MSSARSERDL
651 QKEFPKHVES LQRLGKDLNH D*

ORF88ng-1 and ORF88-1 show 97.0% identity in 671 aa overlap:

Furthermore, ORG88ng-1 shows homology with a hypothetical protein from Aquifex aeolicus:

gi|2984296 (AE000771) hypothetical protein [Aquifex aeolicus]
Length = 537 Score = 94.4 bits (231), Expect = 2e−18.
Identities = 91/334 (27%), Positives = 159/334 (47%), Gaps = 59/334 (17%)
Query:	16	FAFFSSMRFAVALLSLLGIASVIG-TVLQQNQPQTDYLVKFGPFWTRIFDFLGLYDVYAS	74
		+ F +S++ A+ ++ +LGI S++G T ++QNQ    YL +FG         L L DV+ S
Sbjct:	80	YDFLASLKLAIFIMLVLGILSMLGSTYIKQNQSFEWYLDQFGYDVGIWIWKLWLNDVFHS	139
Query:	75	AWFVVIMMFLVVSTSLCLIRNVPPFWREMKSFREKVKEKSLAAMRHSSLLDVKIAPEVAK	134
		++++ ++ L V+   C I+ +P  W++  S +E++ +    A +H   + VKI P+  K
Sbjct:	140	WYYILFIVLLAVNLIFCSIKRLPRVWKQAFS-KERILKLDEHAEKHLKPITVKI-PDKDK	197
Query:	135	--RYLEVRGFQGKTVSREDGSVLIAAKKGTMNKWGYIFAQVALIVICLGGLIDSNLLLKL	192
		++L  +GF+   V  E   + + A+KG  ++ G     +AL+VI  G LID
Sbjct:	198	VLKFLLKKGFK-VFVEEEGNKLYVFAEKGRFSRLGVYITHIALLVIMAGALID-------	249
Query:	193	GMLAGRIVPDNQAVYAKDFKPESILGASNLSFRGNVNISEGQSADVVFLNADNGMLVQDL	252
		+I+G      RG++ ++EG + DV+ + A+       L
Sbjct:	250	----------------------AIVGV-----RGSLIVAEGDTNDVMLVGAE--QKPYKL	280
Query:	253	PFEVKLKKFHIDFY---NTGMPRDFA-------SDIEVTDKATGEKLER--TIRVNHPLT	300
		PF V L  F I  Y   N  + + FA       SDIE+ +   G K+E   T++VN P
Sbjct:	281	PFAVHLIDFRIKTYAEENPNVDKRFAQAVSSYESDIEIIN---GGKVEAKGTVKVNEPFD	337
Query:	301	LHGITIYQASFA--DGGSDLTFKAWNLRDASREP	332
		++QA++   DG S +     + + A  +P
Sbjct:	338	FGRYRLFQATYGILDGTSGMGVIVVDRKKAHEDP	371

Based on this analysis, including the putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 40

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 337>:

1   ATGATGAGTA ATAmAATGGm ACAAAAAGGG TTTACATTGA
    TTGmGmTGAT
51  GATAGTCGTC GCGATACTCG GCATTATCAG CGTCATTGCC
    ATACCTTCTT
101 ATCmAAGTTA TATTGAAAAA GGCTATCAGT CCCAGCTTTA
    TACGGAGATG
151 GyCGGTATCA ACAATATTTC CAAACAGTTT ATTTTGAAAA
    ATCCCCTGGA
201 CGATAATCAG ACCATCGAGA ACAAACTGGA AATATTTGTC
    TCAGGCTATA
251 AGATGAATCC GAAAATTGCC AAAAAaTATA GTGTTTCGGT
    AAAGTTTGTC
301 GATAAGGAAA AATCAAGGGC ATACAGGTTG GTCGGCGTTC
    CGAAGGCGGG
351 GACGGGTTAT ACTTTGTCGG TATGGATGAA CAGCGTGGGC
    GACGGATACA
401 AATGCCGTGA TGCCGCTTCT GCCCAAGCCC ATTTGGAGAC
    CTTGTCCTCA
451 GATGTCGGCT GTGAAGCCTT CTCTAATCGT AAAAAATAA

This corresponds to the amino acid sequence <SEQ ID 338; ORF89>:

1   MMSNXMXQKG FTLIXXMIVV AILGIISVIA IPSYXSYIEK
    GYQSQLYTEM
51  XGINNISKQF ILKNPLDDNQ TIENKLEIFV SGYKMNPKIA
    KKYSVSVKFV
101 DKEKSRAYRL VGVPKAGTGY TLSVWMNSVG DGYKCRDAAS
    AQAHLETLSS
151 DVGCEAFSNR KK*

Further work revealed the complete nucleotide sequence <SEQ ID 339>:

1   ATGATGAGTA ATAAAATGGA ACAAAAAGGG TTTACATTGA
    TTGAGATGAT
51  GATAGTCGTC GCGATACTCG GCATTATCAG CGTCATTGCC
    ATACCTTCTT
101 ATCAAAGTTA TATTGAAAAA GGCTATCAGT CCCAGCTTTA
    TACGGAGATG
151 GTCGGTATCA ACAATATTTC CAAACAGTTT ATTTTGAAAA
    ATCCCCTGGA
201 CGATAATCAG ACCATCGAGA ACAAACTGGA AATATTTGTC
    TCAGGCTATA
251 AGATGAATCC GAAAATTGCC AAAAAATATA GTGTTTCGGT
    AAAGTTTGTC
301 GATAAGGAAA AATCAAGGGC ATACAGGTTG GTCGGCGTTC
    CGAAGGCGGG
351 GACGGGTTAT ACTTTGTCGG TATGGATGAA CAGCGTGGGC
    GACGGATACA
401 AATGCCGTGA TGCCGCTTCT GCCCAAGCCC ATTTGGAGAC
    CTTGTCCTCA
451 GATGTCGGCT GTGAAGCCTT CTCTAATCGT AAAAAATAA

This corresponds to the amino acid sequence <SEQ ID 340; ORF89-1>:

1   MMSNKMEQKG FTLIEMMIVV AILGIISVIA IPSYQSYIEK
    GYQSQLYTEM
51  VGINNISKQF ILKNPLDDNQ TIENKLEIFV SGYKMNPKIA
    KKYSVSVKFV
101 DKEKSRAYRL VGVPKAGTGY TLSVWMNSVG DGYKCRDAAS
    AQAHLETLSS
151 DVGCEAFSNR KK*

Computer analysis of this amino acid sequence gave the following results:

Homology with PilE of N. gonorrhoeae (Accession Number Z69260).

ORF89 and PilE protein show 30% aa identity in 120a overlap:

orf89	8	QKGFTLIXXMIVVAILGIISVIAIPSYXSYIEKGYQSQLYTEMXGINNISKQFILKNPL-	66
		QKGFTLI  MIV+AI+GI++ +A+P+Y  Y  +   S+      G  +   ++ L + +
PilE	5	QKGFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILLAEGQKSAVTEYYLNHGIW	64
orf89	67	-DDNQTIENKLEIFVSGYKMNPKIAKKYSVSVKFVDKEKSRAYRLVGVPKAGTGYTLSVW	125
		DN +         +G   + KI  KY  SV       +      GV K   G  LS+W
PilE	65	PKDNTS---------AGVASSDKIKGKYVQSVTVAKGVVTAEMASTGVNKEIQGKKLSLW	115

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF89 shows 83.3% identity over a 162aa overlap with an ORF (ORF89a) from strain A of N. meningitidis:

The complete length ORF89a nucleotide sequence <SEQ ID 341> is:

1   ATGATGAGTA ATAAAATGGA ACAAAAAGGG TTTACATTGA
    TTGNGANGNT
51  NATNGNCNTC GCGATACNCN GCNTTANCAG CGTCATTNCN
    ATNNNTNCNT
101 ATCNNAGTTA TATTGAAAAA GGCTATCAGT CCCAGCTTTA
    TACGGAGATG
151 GTCGGTATCA ACAATATTTC CAAACAGTNT ATTTTGAAAA
    ATCCCCTGGA
201 CGATAATCAG ACCATCAAGA GCAAACTGGA AATATTTGTC
    TCAGGCTATA
251 AGATGAATCC GAAAATTGCC GAAAAATATA ATGTTTCGGT
    GCATTTTGTC
301 AATGAGGAAA AACCNAGGGC ATACAGCTTG GTCGGCGTTC
    CAAAGACGGG
351 GACGGGTTAT ACTTTGTCGG TATGGATGAA CAGCGTGGGC
    GACGGATACA
401 AATGCCGTGA TGCCGCTTCT GCCCGAGCCC ATTTGGAGAC
    CTTGTCCTCA
451 GATGTCGGCT GTGAAGCCTT CTCTAATCGT AAAAAATAG

This encodes a protein having amino acid sequence <SEQ ID 342>:

1   MMSNKMEQKG FTLIXXXXXX AIXXXXSVIX XXXYXSYIEK
    GYQSQLYTEM
51  VGINNISKQX ILKNPLDDNQ TIKSKLEIFV SGYKMNPKIA
    EKYNVSVHFV
101 NEEKPRAYSL VGVPKTGTGY TLSVWMNSVG DGYKCRDAAS
    ARAHLETLSS
151 DVGCEAFSNR KK*

ORF89a and ORF89-1 show 83.3% identity in 162 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF89 shows 84.6% identity over a 162aa overlap with a predicted ORF (ORF89.ng) from N. gonorrhoeae:

The complete length ORF89ng nucleotide sequence <SEQ ID 343> is:

1   aTGATGAGCA ATAAAATGGA ACAAAAAGGG TTTACATTGA
    TTGAGATGAT
51  GATAGTTGTC ACGATACTCG GCATCATCAG CGTCATTGCC
    ATACCTTCTT
101 ATCAGAGTTA TATTGAAAAA GGCTATCAGT CCCAGCTTTA
    TACGGAGATG
151 GTCGGTATCA ACAATGTTCT CAAACAGTTT ATTTTGAAAA
    ATCCCCAGGA
201 CGATAATGAT ACCCTCAAGA GCAAACTGAA AATATTTGTC
    TCAGGCTATA
251 AGATGAATCC GAAAAttgCC AAAAAATATA GTGTTTCGGt
    aaggtttGTC
301 gatGCGGAAA AACCAAGGGC ATACAGGTTG GTCGGCGTTC
    CGAACGCGGG
351 GACGGGTTAT ACTTTGTCGG TATGGATGAA CAGCGTGGGC
    GACGGATACA
401 AATGCCGTGA TGCCACTTCT GCCCAGGCCT ATTCGGACAC
    CTTGTCCGCA
451 GATAGCGGCT GTGAAGCTTT CTCTAATCGT AAAAAATAG

This encodes a protein having amino acid sequence <SEQ ID 344>:

1   MMSNKMEQKG FTLIEMMIVV TILGIISVIA IPSYQSYIEK
    GYQSQLYTEM
51  VGINNVLKQF ILKNPQDDND TLKSKLKIFV SGYKMNPKIA
    KKYSVSVRFV
101 DAEKPRAYRL VGVPNAGTGY TLSVWMNSVG DGYKCRDATS
    AQAYSDTLSA
151 DSGCEAFSNR KK*

This gonococcal protein has a putative leader peptide (underlined) and N-terminal methylation site (NMePhe or type-4 pili, double-underlined). In addition, ORF89ng and ORF89-1 show 88.3% identity in 162 aa overlap:

Based on this analysis, including the gonococcal motifs and the homology with the known PilE protein, it was predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF89-1 (13.6 kDa) was cloned in the pGex vector and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 11A shows the results of affinity purification of the GST-fusion protein. Purified GST-fusion protein was used to immunise mice, whose sera gave a positive result in the ELISA test, confirming that ORF89-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 41

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 345>:

1   ATGAAAAAAT CCTCCCTCAT CAGCGCATTG GGCATCGGTA
    TTTTGAGCAT
51  CGGCATGGCA TTTGCCGCCC CTGCCGACGC GGTAAGCCAA
    ATCCGTCAAA
101 ACGCCACTCA AGTATTGAGC ATCTTAAAAA ACGGCGATGC
    CAACACCGCT
151 CGCCAAAAAG CCGAAGCCTA TGCGATTCCC TATTTCGATT
    TCCAACGTAT
201 GACCGCATTG GCGGTCGGCA ACCCTTGGsG CACCG.GTCC
    GACG.GCAAA
251 AACAAGCGTT GGCCn.AGAA TTTCAACCC...

This corresponds to the amino acid sequence <SEQ ID 346; ORF91>:

1  MKKSSLISAL GIGILSIGMA FAAPADAVSQ IRQNATQVLS
   ILKNGDANTA
51 RQKAEAYAIP YFDFQRMTAL AVGNPWXTXS DXQKQALAXE
   FQP...

Further work revealed the complete nucleotide sequence <SEQ ID 347>:

1   ATGAAAAAAT CCTCCCTCAT CAGCGCATTG GGCATCGGTA
    TTTTGAGCAT
51  CGGCATGGCA TTTGCCGCCC CTGCCGACGC GGTAAGCCAA
    ATCCGTCAAA
101 ACGCCACTCA AGTATTGAGC ATCTTAAAAA ACGGCGATGC
    CAACACCGCT
151 CGCCAAAAAG CCGAAGCCTA TGCGATTCCC TATTTCGATT
    TCCAACGTAT
201 GACCGCATTG GCGGTCGGCA ACCCTTGGCG CACCGCGTCC
    GACGCGCAAA
251 AACAAGCGTT GGCCAAAGAA TTTCAAACCC TGCTGATCCG
    CACCTATTCC
301 GGCACGATGC TGAAATTAAA AAACGCCAAC GTCAACGTCA
    AAGACAATCC
351 CATCGTCAAT AAAGGCGGCA AAGAAATCAT CGTCCGCGCC
    GAAGTCGGCG
401 TACCCGGGCA AAAACCCGTC AACATGGACT TCACCACCTA
    CCAAAGCGGC
451 GGTAAATACC GTACCTACAA CGTCGCCATC GAAGGCGCGA
    GCCTGGTTAC
501 CGTGTACCGC AACCAATTCG GCGAAATTAT CAAAGCGAAA
    GGCGTGGACG
551 GACTGATTGC CGAGTTGAAA GCCAAAAACG GCGGCAAATA
    A

This corresponds to the amino acid sequence <SEQ ID 348; ORF91-1>:

1   MKKSSLISAL GIGILSIGMA FAAPADAVSQ IRQNATQVLS
    ILKNGDANTA
51  RQKAEAYAIP YFDFQRMTAL AVGNPWRTAS DAQKQALAKE
    FQTLLIRTYS
101 GTMLKLKNAN VNVKDNPIVN KGGKEIIVRA EVGVPGQKPV
    NMDFTTYQSG
151 GKYRTYNVAI EGASLVTVYR NQFGEIIKAK GVDGLIAELK
    AKNGGK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF91 shows 92.4% identity over a 92aa overlap with an ORF (ORF91a) from strain A of N. meningitidis:

The complete length ORF91a nucleotide sequence <SEQ ID 349> is:

1   ATGAAAAAAT CCTCCTTCAT CAGCGCATTG GGCATCGGTA
    TTTTGAGCAT
51  CGGCATGGCA TTTGCCGCCC CTGCCGACGC GGTAAACCAA
    ATCCGTCAAA
101 ACGCCACTCA AGTATTGAGC ATCTTAAAAA GCGGTGATGC
    CAACACCGCC
151 CGCCAAAAAG CCGAAGCCTA TGCGATTCCC TATTTCGATT
    TCCAACGTAT
201 GACCGCATTG GCGGTCGGCA ACCCTTGGCG CACCGCGTCC
    GACGCGCAAA
251 AACAAGCGTT GGCCAAAGAA TTTCAAACCC TGCTGATCCG
    CACCTATTCC
301 GGCACGATGC TGAAATTAAA AAACGCCAAC GTCAACGTCA
    AAGACAATCC
351 CATCGTCAAT AAAGGCGGCA AAGAAATCAT CGTCCGCGCC
    GAAGTCGGCG
401 TACCCGGGCA AAAACCCGTC AACATGGACT TCACCACCTA
    CCAAAGCGGC
451 GGTAAATACC GTACCTACAA CGTCGCCATC GAAGGCGCGA
    GCCTGGTTAC
501 CGTGTACCGC AACCAATTCG GCGAAATTAT CAAAGCGAAA
    GGCGTGGACG
551 GACTGATTGC CGAGTTGAAG GCTAAAAACG GCAGCAAGTA
    A

This encodes a protein having amino acid sequence <SEQ ID 350>:

1   MKKSSFISAL GIGILSIGMA FAAPADAVNQ IRQNATQVLS
    ILKSGDANTA
51  RQKAEAYAIP YFDFQRMTAL AVGNPWRTAS DAQKQALAKE
    FQTLLIRTYS
101 GTMLKLKNAN VNVKDNPIVN KGGKEIIVRA EVGVPGQKPV
    NMDFTTYQSG
151 GKYRTYNVAI EGASLVTVYR NQFGEIIKAK GVDGLIAELK
    AKNGSK*

ORF91a and ORF91-1 show 98.0% identity in 196 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF91 shows 84.8% identity over a 92aa overlap with a predicted ORF (ORF91.ng) from N. gonorrhoeae:

The complete length ORF91ng nucleotide sequence <SEQ ID 351> is predicted to encode a protein having amino acid sequence <SEQ ID 352>:

1   VKKSSFISAL GIGILSIGMA FASPADAVGQ IRQNATQVLT
    ILKSGDAASA
51  RPKAEAYAVP YFDFQRMTAL AVGNPWRTAS DAQKQALAKE
    FQTLLIRTYS
101 GTMLKFKNAT VNVKDNPIVN KGGKEIVVRA EVGIPGQKPV
    NMDFTTYQSG
151 GKYRTYNVAI EGTSLVTVYR NQFGEIIKAK GIDGLIAELK
    AKNGGK*

Further work revealed the complete nucleotide sequence <SEQ ID 353>:

1   ATGAAAAAAT CCTCCTTCAT CAGCGCATTG GGCATCGGTA
    TTTTGAGCAT
51  CGGCATGGCA TTTGCCTCCC CGGCCGACGC AGTGGGACAA
    ATCCGCCAAA
101 ACGCCACACA GGTTTTGACC ATCCTCAAAA GCGGCGACGC
    GGCTTCTGCA
151 CGCCCAAAAG CCGAAGCCTA TGCGGTTCCC TATTTCGATT
    TCCAACGTAT
201 GACCGCATTG GCGGTCGGCA ACCCTTGGCG TACCGCGTCC
    GACGCGCAAA
251 AACAAGCGTT GGCCAAAGAA TTTCAAACCC TGCTGATCCG
    CACCTATTCC
301 GGCACGATGC TGAAATTCAA AAACGCGACC GTCAACGTCA
    AAGACAATCC
351 CATCGTCAAT AAGGGCGGCA AGGAAATCGT CGTCCGTGCC
    GAAGTCGGCA
401 TCCCCGGTCA GAAGCCCGTC AATATGGACT TTACCACCTA
    CCAAAGCGGC
451 GGCAAATACC GTACCTACAA CGTCGCCATC GAAGGCACGA
    GCCTGGTTAC
501 CGTGTACCGC AACCAATTCG GCGAAATCAT CAAAGCCAAA
    GGCATCGACG
551 GGCTGATTGC CGAGTTGAAA GCCAAAAACG GCGGCAAATA
    A

This corresponds to the amino acid sequence <SEQ ID 354; ORF91ng-1>:

1   MKKSSFISAL GIGILSIGMA FASPADAVGQ IRQNATQVLT
    ILKSGDAASA
51  RPKAEAYAVP YFDFQRMTAL AVGNPWRTAS DAQKQALAKE
    FQTLLIRTYS
101 GTMLKFKNAT VNVKDNPIVN KGGKEIVVRA EVGIPGQKPV
    NMDFTTYQSG
151 GKYRTYNVAI EGTSLVTVYR NQFGEIIKAK GIDGLIAELK
    AKNGGK*

ORF91ng-1 and ORF91-1 show 92.3% identity in 196 aa overlap:

In addition, ORF91ng-1 shows homology to a hypothetical E. coli protein:

sp|P45390|YRBC_ECOLI HYPOTHETICAL 24.0 KD PROTEIN IN MURA-RPON
INTERGENIC
REGION PRECURSOR (F211) >gi|606130 (U18997) ORF_f211 [Escherichia coli]
>gi|1789583 (AE000399) hypothetical 24.0 kD protein in murZ-rpoN
intergenic region [Escherichia coli]Length = 211
Score = 70.6 bits (170), Expect = 6e−12
Identities = 42/137 (30%), Positives = 76/137 (54%), Gaps = 6/137 (4%)
Query:	59	VPYFDFQRMTALAVGNPWRTASDAQKQALAKEFQTLLIRTYSGTMLKFKNATVNVKDNPI	118
		+PY   +   AL +G  +++A+ AQ++A    F+  L + Y   +  +   T  +   P
Sbjct:	65	LPYVQVKYAGALVLGQYYKSATPAQREAYFAAFREYLKQAYGQALAMYHGQTYQIA--PE	122
Query:	119	VNKGGKEIV-VRAEVGIP-GQKPVNMDFTTYQSG--GKYRTYNVAIEGTSLVTVYRNQFG	174
		G K IV +R  +  P G+ PV +DF   ++   G ++ Y++  EG S++T  +N++G
Sbjct:	123	QPLGDKTIVPIRVTIIDPNGRPPVRLDFQWRKNSQTGNWQAYDMIAEGVSMITTKQNEWG	182
Query:	175	EIIKAKGIDGLIAELKA	191
		+++ KGIDGL A+LK+
Sbjct:	183	TLLRTKGIDGLTAQLKS	199

Based on this analysis, including the presence of a putative leader sequence in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 42

The following DNA sequence was identified in N. meningitidis <SEQ ID 355>:

1   ATGAAACACA TACTCCCCCT GATTGCCGCA TCCGCACTCT
    GCATTTCAAC
51  CGCTTCGGCA CATCCTGCCA GCGAACCGTC CACTCAAAAC
    GAAACCGCTA
101 TGATCACGCA TACCCTCATC TCAAAATACA GTTTTGGnnn
    nnnnnnnnnn
151 nnnnnnnnnn nnGCCATAAA AAGCAAAGGG ATGGACATTT
    TTGCCGTCAT
201 CGACCATCAG GAAGCCGCAC GCCGAAACGG CTTAACGATG
    CAGCCGGCAA
251 AAGTCATCGT CTTCGGCACG CCCAAAGCCG GCACGCCGCT
    GATGGTCAAA
301 GACCCCGCCT TCGCCCTGCA ACTGCCCCTA CGCGTCCTCG
    TTACCGAAAC
351 GGACGGCAAA GTACGCGCCG CCTATACCGA TACGCGCGCC
    CTCATCGCCG
401 GCAGCCGCAT CGGTTTCGAC GAAGTGGCAA ACACTTTGGC
    AAACGCCGAA
451 AAACTGATAC AAAAAACCGT AGGCGAATAA

This corresponds to the amino acid sequence <SEQ ID 356; ORF97>:

1   MKHILPLIAA SALCISTASA HPASEPSTQN ETAMITHTLI
    SKYSFGXXXX
51  XXXXAIKSKG MDIFAVIDHQ EAARRNGLTM QPAKVIVFGT
    PKAGTPLMVK
101 DPAFALQLPL RVLVTETDGK VRAAYTDTRA LIAGSRIGFD
    EVANTLANAE
151 KLIQKTVGE*

Further work revealed the complete nucleotide sequence <SEQ ID 357>:

1   ATGAAACACA TACTCCCCCT GATTGCCGCA TCCGCACTCT
    GCATTTCAAC
51  CGCTTCGGCA CATCCTGCCA GCGAACCGTC CACCCAAAAC
    GAAACCGCTA
101 TGACCACGCA TACCCTCACC TCAAAATACA GTTTTGACGA
    AACCGTCAGC
151 CGCCTTGAAA CCGCCATAAA AAGCAAAGGG ATGGACATTT
    TTGCCGTCAT
201 CGACCATCAG GAAGCCGCCC GCCGAAACGG CTTAACGATG
    CAGCCGGCAA
251 AAGTCATCGT CTTCGGCACG CCCAAAGCCG GCACGCCGCT
    GATGGTCAAA
301 GACCCCGCCT TCGCCCTGCA ACTGCCCCTA CGCGTCCTCG
    TTACCGAAAC
351 GGACGGCAAA GTACGCGCCG CCTATACCGA TACGCGCGCC
    CTCATCGCCG
401 GCAGCCGCAT CGGTTTCGAC GAAGTGGCAA ACACTTTGGC
    AAACGCCGAA
451 AAACTGATAC AAAAAACCGT AGGCGAATAA

This corresponds to the amino acid sequence <SEQ ID 358; ORF97-1>:

1   MKHILPLIAA SALCISTASA HPASEPSTQN ETAMTTHTLT
    SKYSFDETVS
51  RLETAIKSKG MDIFAVIDHQ EAARRNGLTM QPAKVIVFGT
    PKAGTPLMVK
101 DPAFALQLPL RVLVTETDGK VRAAYTDTRA LIAGSRIGFD
    EVANTLANAE
151 KLIQKTVGE*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF97 shows 88.7% identity over a 159aa overlap with an ORF (ORF97a) from strain A of N. meningitidis:

The complete length ORF97a nucleotide sequence <SEQ ID 359> is:

1   ATGANACACA TACTCCCCCT GANTGNCGCA TCCGCACTCT
    GCATTTCAAC
51  CGCTTCGGNN CATCCTGCCA GCGAACCGCA AACCCAAAAC
    GAAACCGCTA
101 TGACCACGCA TACCCTCACC TCAAAATACA GTTTTGACGA
    AACCGTCAGC
151 CGCCTTGAAA CCGCCATAAA AAGCAAAGGG ATGGACATTT
    TTGCCGTCAT
201 CGACCATCAG GAAGCCGCCC GCCGAAACGG CTTAACGATG
    CAGCCGGCAA
251 AAGTCATCGT CTTCGGCACG CCCAAAGCCG GTACGCCGCT
    GATGGTCAAA
301 GACCCCGCCT TCGCCCTGCA ACTGCCCCTG CGCGTCNTCG
    TTACCGAAAC
351 GGACGGCAAA GTACGCGCCG CCTATACCGA TACGCGCGCC
    CTCATCGCCG
401 GCAGCCGCAT CGGTTTCGAC GAAGTGGCAA ACACTTTGGC
    AAACGCCGAA
451 AAACTGATAC AAAAAACCAT AGGCGAATAA

This encodes a protein having amino acid sequence <SEQ ID 360>:

1   MXHILPLXXA SALCISTASX HPASEPQTQN ETAMTTHTLT
    SKYSFDETVS
51  RLETAIKSKG MDIFAVIDHQ EAARRNGLTM QPAKVIVFGT
    PKAGTPLMVK
101 DPAFALQLPL RVXVTETDGK VRAAYTDTRA LIAGSRIGFD
    EVANTLANAE
151 KLIQKTIGE*

ORF97a and ORF97-1 show 95.6% identity in 159 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF97 shows 88.1% identity over a 159aa overlap with a predicted ORF (ORF97.ng) from N. gonorrhoeae:

The complete length ORF97ng nucleotide sequence <SEQ ID 361> is predicted to encode a protein having amino acid sequence <SEQ ID 362>:

1   MKHILPPIAA SAFCISTASA HPAGKPPTQN ETAMTTHTLT
    SKYSFDETVS
51  RLETAIKSKG MDIFAVIDHQ EAARRNGLTM QPAKVIVFGT
    PKAGTPLMVK
101 DPAFALQLPL RVLVTETDGK VRTAYTDTRA LIVGSRISFD
    EVANTLANAE
151 KLIQKTVGE*

Further work revealed the complete nucleotide sequence <SEQ ID 363>:

1   ATGAAACACA TACTCCCcct gatcgccgca TccgcactCT
    GCATTTCAAC
51  CGCTTCGGCA CACCCTGCCG GCAAACCGCC CACCCAAAAC
    GAAACCGCTA
101 TGACCACGCA CACCCTCACC TCGAAATACA GTTTTGACGA
    AACCGTCAGC
151 CGCCTTGAAA CCGCCATAAA AAGCAAAGGG ATGGACATTT
    TTGCCGTCAT
201 CGACCATCAG GAAGCGGCAC GCCGAAACGG CCTGACCATG
    CAGCCGGCAA
251 AAGTCATCGT CTTCGGCACG CCCAAGGCCG GTACGCCgct
    GATGGTCAAA
301 GACCCCGCCT TCGCCCTGCA ACTGCCCCTG CGCGTCCTCG
    TTACCGAAAC
351 GGACGGCAAA GTACGCACCG CCTATACCGA TACGCGCGCC
    CTCATCGTCG
401 GCAGCCGCAT CAGTTTCGAC GAAGTGGCAA ACACTTTGGC
    AAACGCCGAA
451 AAACTGATAC AAAAAACCGT AGGCGAATAA

This corresponds to the amino acid sequence <SEQ ID 364; ORF97ng-1>:

1   MKHILPLIAA SALCISTASA HPAGKPPTQN ETAMTTHTLT
    SKYSFDETVS
51  RLETAIKSKG MDIFAVIDHQ EAARRNGLTM QPAKVIVFGT
    PKAGTPLMVK
101 DPAFALQLPL RVLVTETDGK VRTAYTDTRA LIVGSRISFD
    EVANTLANAE
151 KLIQKTVGE*

ORF97ng-1 and ORF97-1 show 96.2% identity in 159 aa overlap:

Based on this analysis, including the presence of a putative leader sequence in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF97-1 (15.3 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIGS. 12A & 12B show, respectively, the results of affinity purification of the GST-fusion and His-fusion proteins. Purified GST-fusion protein was used to immunise mice, whose sera were used for Western Blot (FIG. 12C), ELISA (positive result), and FACS analysis (FIG. 12D). These experiments confirm that ORF97-1 is a surface-exposed protein, and that it is a useful immunogen. FIG. 12E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF97-1.

Example 43

The following DNA, believed to be complete, sequence was identified in N. meningitidis <SEQ ID 365>:

1   ATGGCTTTTA TTACGCGCTT ATTCAAAAGC AGTAAATGGC
    TGATTGTGCC
51  GCTGATGCTC CCCGCCTTTC AGAATGTGGC GGCGGAGGGG
    ATAGATGTGA
101 GCCGTGCCGA AGCGAGGATA ACCGACGGCG GGCAGCTTTC
    CATCAGCAGC
151 CGCTTCCAAA CCGAGCTGCC CGACCAGCTC CAACAGGCGT
    TGCGCCGGGg
201 CGTGCCGCTC AACTTTACCT TAAGCTGGCA GCTTTCCGCC
    CCGATAATCG
251 CTTCTTATCG GTTTAAATTG GGGCAACTGA TTGGCGATGA
    CGACaATATT
301 GACTACAAAC TGAGTTTCCA TCCGCTGACc AaACGCTACC
    GCGTTACCgT
351 CGgCGCGTTT TCGACAGACT ACGACACCTT GGATGCGGCA
    TTGCGCGCGA
401 CCGGCGCGGT TGCCAACTGG AAAGTCCTGA ACAAAGGCGC
    GCTGTCCGGT
451 GCGGAAGCAG GGGAAACCAA GGCGGAAATC CGCCTGACGC
    TGTCCACTTC
501 AAAACTGCCC AAGCCTTTTC AAATCAATGC ATTGACTTCT
    CAAAACTGGC
551 ATTTGGATTC GGGTTGGAAA CCTCTAAACA TCATCGGGAA
    CAAATAA

This corresponds to the amino acid sequence <SEQ ID 366; ORF106>:

1   MAFITRLFKS SKWLIVPLML PAFQNVAAEG IDVSRAEARI
    TDGGQLSISS
51  RFQTELPDQL QQALRRGVPL NFTLSWQLSA PIIASYRFKL
    GQLIGDDDNI
101 DYKLSFHPLT KRYRVTVGAF STDYDTLDAA LRATGAVANW
    KVLNKGALSG
151 AEAGETKAEI RLTLSTSKLP KPFQINALTS QNWHLDSGWK
    PLNIIGNK*

Further work revealed the following DNA sequence <SEQ ID 367>:

1   ATGGCTTTTA TTACGCGCTT ATTCAAAAGC AGTAAATGGC
    TGATTGTGCC
51  GCTGATGCTC CCCGCCTTTC AGAATGTGGC GGCGGAGGGG
    ATAGATGTGA
101 GCCGTGCCGA AGCGAGGATA ACCGACGGCG GGCAGCTTTC
    CATCAGCAGC
151 CGCTTCCAAA CCGAGCTGCC CGACCAGCTC CAACAGGCGT
    TGCGCCGGGG
201 CGTGCCGCTC AACTTTACCT TAAGCTGGCA GCTTTCCGCC
    CCGATAATCG
251 CTTCTTATCG GTTTAAATTG GGGCAACTGA TTGGCGATGA
    CGACAATATT
301 GACTACAAAC TGAGTTTCCA TCCGCTGACC AACCGCTACC
    GCGTTACCGT
351 CGGCGCGTTT TCGACAGACT ACGACACCTT GGATGCGGCA
    TTGCGCGCGA
401 CCGGCGCGGT TGCCAACTGG AAAGTCCTGA ACAAAGGCGC
    GCTGTCCGGT
451 GCGGAAGCAG GGGAAACCAA GGCGGAAATC CGCCTGACGC
    TGTCCACTTC
501 AAAACTGCCC AAGCCTTTTC AAATCAATGC ATTGACTTCT
    CAAAACTGGC
551 ATTTGGATTC GGGTTGGAAA CCTCTAAACA TCATCGGGAA
    CAAATAA

This corresponds to the amino acid sequence <SEQ ID 368; ORF106-1>:

1   MAFITRLFKS SKWLIVPLML PAFQNVAAEG IDVSRAEARI
    TDGGQLSISS
51  RFQTELPDQL QQALRRGVPL NFTLSWQLSA PIIASYRFKL
    GQLIGDDDNI
101 DYKLSFHPLT NRYRVTVGAF STDYDTLDAA LRATGAVANW
    KVLNKGALSG
151 AEAGETKAEI RLTLSTSKLP KPFQINALTS QNWHLDSGWK
    PLNIIGNK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF106 shows 87.4% identity over a 199aa overlap with an ORF (ORF106a) from strain A of N. meningitidis:

Due to the K→N substitution at residue 111, the homology between ORF106a and ORF106-1 is 87.9% over the same 199 aa overlap.

The complete length ORF106a nucleotide sequence <SEQ ID 369> is:

1   ATGGCTTTTA TTACGCGCTT ATTCAAAAGC ATTAAACAAT
    GGCTTGTGCT
51  GCTGCCGATG CTTTCCGTTT TGCCGGACGC GGCGGCGGAG
    GGGATAGATG
101 TGAGCCGCGC CGAAGCGAGG ATAANCGACG GCGGGCAGCT
    TTCCATNAGN
151 AGCCGCTTCC AAACCGAGCT GCCCGACCAG CTCCAANNNG
    CGNNGNGCCG
201 GGGCGTGNCG CTCAACTNTA CCTTAAGNTG GCAGCTTTCC
    GCCCCGATAA
251 TCGCTTCTTA TCGGTTTNAA TTGGGGCAAC TGATTGGCGA
    TGACGACNAT
301 ATTGACTACA AACTGAGTTT CCATCCGCTG ACCAACCGCT
    ACCGCGTTAC
351 CGTCGGCGCG TTTTCGACAG ANTACGACAC CTTGGATGCG
    GCATTGCGCG
401 CGACCGGCGC GGTTGCCAAC TGGAAAGTCC TGAACAAAGG
    CGCGCTGTCC
451 GGTGCGGAAG CAGGGGAAAC CAAGGCGGAA ATCCGCCTGA
    CGCTGTCCAC
501 TTCAAAACTG CCCAAGCCTT TTCAAATCAA TGCATTGACT
    TCTCAAAACT
551 GGCATTTGGA TTCGGGTTGG AAACCTCTAA ACATCATCGG
    GAACAAATAA

This encodes a protein having amino acid sequence <SEQ ID 370>:

1   MAFITRLFKS IKQWLVLLPM LSVLPDAAAE GIDVSRAEAR
    IXDGGQLSXX
51  SRFQTELPDQ LQXAXXRGVX LNXTLXWQLS APIIASYRFX
    LGQLIGDDDX
101 IDYKLSFHPL TNRYRVTVGA FSTXYDTLDA ALRATGAVAN
    WKVLNKGALS
151 GAEAGETKAE IRLTLSTSKL PKPFQINALT SQNWHLDSGW
    KPLNIIGNK*

Homology with a Predicted ORF from N. gonorrhoeae

ORF106 shows 90.5% identity over a 199aa overlap with a predicted ORF (ORF106.ng) from N. gonorrhoeae:

Due to the K→N substitution at residue 111, the homology between ORF106ng and ORF106-1 is 91.0% over the same 199 aa overlap.

The complete length ORF106ng nucleotide sequence <SEQ ID 371> is:

1   ATGGCTTTTA TTACGCGCTT ATTCAAAAGC ATTAAACAAT
    GGCTTGTGCT
51  GTTGCCGATA CTCTCCGTTT TGCCGGACGC GGCGGCGGAG
    GGCATTGCCG
101 CGACCCGCGC CGAAGCGAGG ATAACCGACG GCGGGCGGCT
    TTCCATCAGC
151 AGCCGCTTCC AAACCGAGCT GCCCGACCAG CTCCAACAGG
    CGTTGCGCCG
201 GGGCGTACCG CTCAACTTTA CCTTAAGCTG GCAGCTTTCC
    GCCCCGACAA
251 TCGCTTCTTA TCGGTTTAAA TTGGGGCAAC TGATTGGCGA
    TGACGACAAT
301 ATTGACTACA AACTAAGTTT CCATCCGCTG ACCAACCGCT
    ACCGCGTTAC
351 CGTCGGCGCA TTTTCCACCG ATTACGACAC TTTGGATGCG
    GCATTGCGCG
401 CGACCGGCGC GGTTGCCAAC TGGAAAGTCC TGAACAAAGG
    CGCGTTGTCC
451 GGTGCGGAAG CAGGGGAAAC CAAGGCGGAA ATCCGCCTGA
    CGCTGTCCAC
501 TTCAAAACTG CCCAAGCCTT TCCAAATCAA CGCATTGACT
    TCTCAAAACT
551 GGCATTTGGA TTCGGGTTGG AAACCTCTAA ACATCATCGG
    GAACAAATAA

This encodes a protein having amino acid sequence <SEQ ID 372>:

1   MAFITRLFKS IKQWLVLLPI LSVLPDAAAE GIAATRAEAR
    ITDGGRLSIS
51  SRFQTELPDQ LQQALRRGVP LNFTLSWQLS APTIASYRFK
    LGQLIGDDDN
101 IDYKLSFHPL TNRYRVTVGA FSTDYDTLDA ALRATGAVAN
    WKVLNKGALS
151 GAEAGETKAE IRLTLSTSKL PKPFQINALT SQNWHLDSGW
    KPLNIIGNK*

Based on this analysis, including the presence of a putative leader sequence in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF106-1 (18 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 13A shows the results of affinity purification of the His-fusion protein, and FIG. 13B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for FACS analysis (FIG. 13C) These experiments confirm that ORF106-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 44

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 373>:

1    ATGGACACAA AAGAAATCCT CGG.TACGCG GcAGGcTCGA
     TCGGCAGCGC
51   GGTTTTAGCC GTCATCATCc TGCCGCTGCT GTCGTGGTAT
     TTCCCCGCCG
101  ACGACATCGG GCGCATCGTG CTGATGCAGA CGGCGGCGGG
     GCTgACGGTG
151  TCGGTGTTGT GCCTCGGGCT GGATCAGGCA TACGTCCGCG
     AATACTATGC
201  CACCGCCGAC AAAGACAcCT TGTTCAAAAC CCTGTTCCTG
     CCGCCGCTGC
251  TGTCTGCCGC CGCGATAGCC GCCCTGCTGC TTTCCCGCCC
     GTCCCTGCCG
301  TCTGAAATCC TGTTTTCACT CGACGATGCC gCCGCCGGCa
     TCGGGCTGGT
351  GCTGTTTGAA CtGAGCTTCC TGCCCATCCG cTTTCTCTTA
     CTGGTTTTGC
401  GTATGGAAGG ACGCGCCcTT GCCTTTTCGT CCGCGCAACT
     CGTGCcCAAG
451  CTCGCCATCC TGCTGCTG.T GCCGCTGACG GTCGGGCTGC
     TGCACTTTCC
501  AGCGAACACC GCCGTCCTGA CCGCCGTTTA CGCGCTGGCA
     AACCTTGCCG
551  CCGCCGCCTT TTTGCTGTTT CAAAACCGAT GCCGTCTGAA
     GGCCGTCCGG
601  CACGCACCGT TTTCGCCCGC CGTCCTGCAC CGGGGG.TGC
     GCTACGGCAT
651  ACCGATCGCA CTGAGCAGCA TCGCCTATTG GGGGCTGGCA
     TCCGCCGACC
701  GTTTGTTCCT GAAAAAATAT GCCGGCCTGG AACAGCTCGG
     CGTTTATTCG
751  ATGGGTATTT CGTTCGGCGG GGCGGCATTA TTGTTCCAAA
     GCATCTTTTC
801  AACGGTCTGG ACACCGTATA TTTTCCGCGC AATCGAAGAA
     AACGCCCCGC
851  CCGCTCGCCT CTCGGCAACG GCAGAATCCG CCGCCGCCCT
     GCTTGCCTCC
901  GCCCTCTGC. TGACCGGCAT TTTCTCGCCC CTTGCCTCCC
     TCCTGCTGCC
951  GGAAAACTAC GCCGCCGTCC GGTTTATCGT CGTATCGTGT
     ATG.TGCCGC
1001 CGCTGTTTTG CACGCTGGCG GAAATCAGCG GCATCGGTTT
     GAACGTCGTT
1051 CGCAAAACGC GCCCGATCGC GCTCGCCACC TTGGGCGCGC
     TGGCGGCAAA
1101 CCTGCTGCTG CTGGGGCTTG ACCGTGCCGT ACCGGCGAGG
     CCGCC.GGCG
1151 CGGCGGTTGC CTGTGCCGCC TCATTCTGGC TGTTTTTTGC
     CTTCAAGACC
1201 GAAAGCTCyT GCCGCCTGTG GCAGCCGCTC AAACGCCTGC
     CGCTTTATCT
1251 GCACACATTG TTCTGCCTGA CCTCCTCGGC GGCCTACACC
     TGCTTCGGCA
1301 CGCCGGCAAA CTATCCCCTG TTTGCCGGCG TATGGGCGGC
     ATATCTGGCA
1351 GGCTGCATCC TGCGCCACCG GAAAGATTTG CACAAACTGT
     TTCATTATTT
1401 GAAAAAACAA GGTTTCCCAT TATGA

This corresponds to the amino acid sequence <SEQ ID 374; ORF10>:

1   MDTKEILXYA AGSIGSAVLA VIILPLLSWY FPADDIGRIV
    LMQTAAGLTV
51  SVLCLGLDQA YVREYYATAD KDTLFKTLFL PPLLSAAAIA
    ALLLSRPSLP
101 SEILFSLDDA AAGIGLVLFE LSFLPIRFLL LVLRMEGRAL
    AFSSAQLVPK
151 LAILLLXPLT VGLLHFPANT AVLTAVYALA NLAAAAFLLF
    QNRCRLKAVR
201 HAPFSPAVLH RGXRYGIPIA LSSIAYWGLA SADRLFLKKY
    AGLEQLGVYS
251 MGISFGGAAL LFQSIFSTVW TPYIFRAIEE NAPPARLSAT
    AESAAALLAS
301 ALCXTGIFSP LASLLLPENY AAVRFIVVSC MXPPLFCTLA
    EISGIGLNVV
351 RKTRPIALAT LGALAANLLL LGLDRAVPAR PXGAAVACAA
    SFWLFFAFKT
401 ESSCRLWQPL KRLPLYLHTL FCLTSSAAYT CFGTPANYPL
    FAGVWAAYLA
451 GCILRHRKDL HKLFHYLKKQ GFPL*

Further sequence analysis revealed the complete DNA sequence <SEQ ID 375> to be:

1    ATGGACACAA AAGAAATCCT CGGCTACGCG GCAGGCTCGA
     TCGGCAGCGC
51   GGTTTTAGCC GTCATCATCC TGCCGCTGCT GTCGTGGTAT
     TTCCCCGCCG
101  ACGACATCGG GCGCATCGTG CTGATGCAGA CGGCGGCGGG
     GCTGACGGTG
151  TCGGTGTTGT GCCTCGGGCT GGATCAGGCA TACGTCCGCG
     AATACTATGC
201  CACCGCCGAC AAAGACACCT TGTTCAAAAC CCTGTTCCTG
     CCGCCGCTGC
251  TGTCTGCCGC CGCGATAGCC GCCCTGCTGC TTTCCCGCCC
     GTCCCTGCCG
301  TCTGAAATCC TGTTTTCACT CGACGATGCC GCCGCCGGCA
     TCGGGCTGGT
351  GCTGTTTGAA CTGAGCTTCC TGCCCATCCG CTTTCTCTTA
     CTGGTTTTGC
401  GTATGGAAGG ACGCGCCCTT GCCTTTTCGT CCGCGCAACT
     CGTGCCCAAG
451  CTCGCCATCC TGCTGCTGCT GCCGCTGACG GTCGGGCTGC
     TGCACTTTCC
501  AGCGAACACC GCCGTCCTGA CCGCCGTTTA CGCGCTGGCA
     AACCTTGCCG
551  CCGCCGCCTT TTTGCTGTTT CAAAACCGAT GCCGTCTGAA
     GGCCGTCCGG
601  CACGCACCGT TTTCGCCCGC CGTCCTGCAC CGGGGGCTGC
     GCTACGGCAT
651  ACCGATCGCA CTGAGCAGCA TCGCCTATTG GGGGCTGGCA
     TCCGCCGACC
701  GTTTGTTCCT GAAAAAATAT GCCGGCCTGG AACAGCTCGG
     CGTTTATTCG
751  ATGGGTATTT CGTTCGGCGG GGCGGCATTA TTGTTCCAAA
     GCATCTTTTC
801  AACGGTCTGG ACACCGTATA TTTTCCGCGC AATCGAAGAA
     AACGCCCCGC
851  CCGCCCGCCT CTCGGCAACG GCAGAATCCG CCGCCGCCCT
     GCTTGCCTCC
901  GCCCTCTGCC TGACCGGCAT TTTCTCGCCC CTTGCCTCCC
     TCCTGCTGCC
951  GGAAAACTAC GCCGCCGTCC GGTTTATCGT CGTATCGTGT
     ATGCTGCCGC
1001 CGCTGTTTTG CACGCTGGCG GAAATCAGCG GCATCGGTTT
     GAACGTCGTC
1051 CGCAAAACGC GCCCGATCGC GCTCGCCACC TTGGGCGCGC
     TGGCGGCAAA
1101 CCTGCTGCTG CTGGGGCTTG CCGTGCCGTC CGGCGGCGCG
     CGCGGCGCGG
1151 CGGTTGCCTG TGCCGCCTCA TTCTGGCTGT TTTTTGCCTT
     CAAGACCGAA
1201 AGCTCCTGCC GCCTGTGGCA GCCGCTCAAA CGCCTGCCGC
     TTTATCTGCA
1251 CACATTGTTC TGCCTGACCT CCTCGGCGGC CTACACCTGC
     TTCGGCACGC
1301 CGGCAAACTA TCCCCTGTTT GCCGGCGTAT GGGCGGCATA
     TCTGGCAGGC
1351 TGCATCCTGC GCCACCGGAA AGATTTGCAC AAACTGTTTC
     ATTATTTGAA
1401 AAAACAAGGT TTCCCATTAT GA

This corresponds to the amino acid sequence <SEQ ID 376; ORF10-1>:

1   MDTKEILGYA AGSIGSAVLA VIILPLLSWY FPADDIGRIV
    LMQTAAGLTV
51  SVLCLGLDQA YVREYYATAD KDTLFKTLFL PPLLSAAAIA
    ALLLSRPSLP
101 SEILFSLDDA AAGIGLVLFE LSFLPIRFLL LVLRMEGRAL
    AFSSAQLVPK
151 LAILLLLPLT VGLLHFPANT AVLTAVYALA NLAAAAFLLF
    QNRCRLKAVR
201 HAPFSPAVLH RGLRYGIPIA LSSIAYWGLA SADRLFLKKY
    AGLEQLGVYS
251 MGISFGGAAL LFQSIFSTVW TPYIFRAIEE NAPPARLSAT
    AESAAALLAS
301 ALCLTGIFSP LASLLLPENY AAVRFIVVSC MLPPLFCTLA
    EISGIGLNVV
351 RKTRPIALAT LGALAANLLL LGLAVPSGGA RGAAVACAAS
    FWLFFAFKTE
401 SSCRLWQPLK RLPLYLHTLF CLTSSAAYTC FGTPANYPLF
    AGVWAAYLAG
451 CILRHRKDLH KLFHYLKKQG FPL*

Computer analysis of this amino acid sequence gave the following results:

Prediction

ORF10-1 is predicted to be the precursor of an integral membrane protein, since it comprises several (12-13) potential transmembrane segments, and a probable cleavable signal peptide

Homology with EpsM from Streptococcus thermophilus (Accession Number U40830).

ORF10 shows homology with the epsM gene of S. thermophilus, which encodes a protein of a size similar to ORF10 and is involved in expolysaccharide synthesis. Other homologies are with prokaryotic membrane proteins:

Identities = (25%)
Query:	213	LRYGIPLALSSLAYWGLASADRLFLKKYAGLEQLGVYSMGISFGGAALLLQSIFSTVW	270
		L Y +PL  SS+ +W L ++ R F+  + G    G+ ++         +  +IF+  W
Sbjct:	210	LYYALPLIPSSILWWLLNASSRYFVLFFLGAGANGLLAVATKIPSIISIFNTIFTQAW	267
Identities = 15/57 (26%), Positives = 31/57 (54%)
Query:	7	LGYAAGSIGSAVLAVIILPLLSWYFPADDIGRIVLMQTAAGLTVSVLCLGLDQAYVR	63
		L +  G++GS +L  +++PL ++     + G   L QT A L + ++ + +  A +R
Sbjct:	12	LVFTIGNLGSKLLVFLLVPLYTYAMTPQEYGMADLYQTTANLLLPLITMNVFDATLR	68
Identities = 16/96 (16%), Positives = 36/96 (37%)
Query:	307	IFSPLASLLLPENYAAVRFTVVSCMLPPLFYTLTEISGIGLNVVRKTRPIXXXXXXXXXX	366
		+  P+   ++  +YA+    V   ML  LF + ++  G      ++T+ +
Sbjct:	305	VLKPIVEKVVSSDYASSWQYVPFFMLSMLFSSFSDFFGTNYIAAKQTKGVFMTSIYGTIV	364

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF10 shows 95.4% identity over a 475aa overlap with an ORF (ORF10a) from strain A of N. meningitidis:

The complete length ORF10a nucleotide sequence <SEQ ID 377> is:

1    ATGGACACAA AAGAAATCCT CGGCTACGCG GCAGGCTCGA
     TCGGCAGCGC
51   GGTTTTAGCC GTCATCATCC TGCCGCTGCT GTCGTGGTAT
     TTCCCTGCCG
101  ACGACATCGG ACGCATCGTG CTGATGCAGA CGGCGGCGGG
     GCTGACGGTG
151  TCGGTGTTGT GCCTCGGGCT GGATCAGGCA TACGTCCGCG
     AATACTATGC
201  CGCCGCCGAC AAAGACACTT TGTTCAAAAC CCTGTTCCTG
     CCGCCGCTGC
251  TGTCTGCCGC CGCGATAGCC GCCCTGCTGC TTTCCCGCCC
     ATCCCTGCCG
301  TCTGAAATCC TGTTTTCGCT CGACGATGCC GCCGCCGGCA
     TCGGGCTGGT
351  GCTGTTTGAA CTGAGCTTCC TGCCCATCCG CTTTCTCTTA
     CTGGTTTTGC
401  GTATGGAAGG ACGCGCCCTT GCCTTTTCGT CCGCGCAACT
     CGTGTCCAAG
451  CTCGCCATCC TGCTGCTGCT GCCGCTGACG GTCGGGCTGC
     TGCACTTTCC
501  GGCGAACACC GCCGTCCTGA CCGCCGTTTA CGCGCTGGCA
     AACCTTGCCG
551  CCGCCGCCTT TTTGCTGTTT CAAAACCGAT GCCGTCTGAA
     GGCCGTCCGG
601  CGCGCACCGT TTTCATCCGC CGTCCTGCAT CGCGGCCTGC
     GCTACGGCAT
651  ACCGATCGCA CTAAGCAGCA TCGCCTATTG GGGGCTGGCA
     TCCGCCGACC
701  GTTTGTTCCT GAAAAAATAT GCCGGCCTAG AACAGCTCGG
     CGTTTATTCG
751  ATGGGTATTT CGTTCGGCGG AGCGGCATTA TTGTTCCAAA
     GCATCTTTTC
801  AACGGTCTGG ACACCGTATA TTTTCCGCGC AATCGAAGCA
     AACGCCCCGC
851  CCGCCCGCCT CTCGGCAACG GCAGAATCCG CCGCCGCCCT
     GCTTGCCTCC
901  GCCCTCTGCC TGACCGGCAT TTTCTCGCCC CTCGCCTCCC
     TCCTGCTGCC
951  GGAAAACTAC GCCGCCGTCC GGTTTATCGT CGTATCGTGT
     ATGCTGCCTC
1001 CGCTGTTTTG CACGCTGGTA GAAATCAGCG GCATCGGTTT
     GAACGTCGTC
1051 CGAAAAACAC GCCCGATCGC GCTCGCCACC TTGGGCGCGC
     TGGCGGCAAA
1101 CCTGCTGCTG CTGGGGCTTG CCGTACCGTC CGGCGGCGCG
     CGCGGCGCGG
1151 CGGTTGCCTG TGCCGCCTCA TTTTGGCTGT TTTTTGTTTT
     CAAGACCGAA
1201 AGCTCCTGCC GCCTGTGGCA GCCGCTCAAA CGCCTGCCGC
     TTTATATGCA
1251 CACATTGTTC TGCCTGGCCT CCTCGGCGGC CTACACCTGC
     TTCGGCACTC
1301 CGGCAAACTA CCCCCTGTTT GCCGGCGTAT GGGCGGTATA
     TCTGGCAGGC
1351 TGCATCCTGC GCCACCGGAA AGATTTGCAC AAACTGTTTC
     ATTATTTGAA
1401 AAAACAAGGT TTCCCATTAT GA

This encodes a protein having amino acid sequence <SEQ ID 378>:

1   MDTKEILGYA AGSIGSAVLA VIILPLLSWY FPADDIGRIV
    LMQTAAGLTV
51  SVLCLGLDQA YVREYYAAAD KDTLFKTLFL PPLLSAAAIA
    ALLLSRPSLP
101 SEILFSLDDA AAGIGLVLFE LSFLPIRFLL LVLRMEGRAL
    AFSSAQLVSK
151 LAILLLLPLT VGLLHFPANT AVLTAVYALA NLAAAAFLLF
    QNRCRLKAVR
201 RAPFSSAVLH RGLRYGIPIA LSSIAYWGLA SADRLFLKKY
    AGLEQLGVYS
251 MGISFGGAAL LFQSIFSTVW TPYIFRAIEA NAPPARLSAT
    AESAAALLAS
301 ALCLTGIFSP LASLLLPENY AAVRFIVVSC MLPPLFCTLV
    EISGIGLNVV
351 RKTRPIALAT LGALAANLLL LGLAVPSGGA RGAAVACAAS
    FWLFFVFKTE
401 SSCRLWQPLK RLPLYMHTLF CLASSAAYTC FGTPANYPLF
    AGVWAVYLAG
451 CILRHRKDLH KLFHYLKKQG FPL*

ORF10a and ORF10-1 show 95.4% identity in 475 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF10 shows 94.1% identity over a 475aa overlap with a predicted ORF (ORF10.ng) from N. gonorrhoeae:

The complete length ORF10ng nucleotide sequence <SEQ ID 379> is:

1    ATGGACACAA AAGAAATCCT CGGCTACGCG GCAGGCTCGA
     TCGGCAGCGC
51   GGTTTTAGCC GTCATCATCC TGCCGCTGCT GTCGTGGTAT
     TTCcccgCCG
101  ACGACATCGG GCGCATCGTG CTGATGCAGA CGGCGGCGGG
     ACTGACGGTG
151  TCGGTATTGT GCCTCGGGCT GGATCAGGCA TACGTCCGCG
     AATACTATGC
201  CGCCGCCGAC AAAGACACTT TGTTCAAAAC CCTGTTCCTG
     CCGCCGCTGC
251  TGTTTTCCGC CGCGATAGCC GCCCTGCTGC TTTCCCGCCC
     GTCCCTGCCG
301  TCTGAAATCC TGTTTTCGCT CGACGATGCC GCCGCCGGCA
     TCGGGCTGGT
351  GCTGTTTGAA CTGAGCTTCC TGCCCATCCG CTTTCTCTTA
     CTGGTTTTGC
401  GTATGGAAGG GCGCGCCCTT GCCTTTTCGT CCGCGCAACT
     CGTGCCCAAA
451  CTCGCCATTC TGCTGCTGTT GCCGCTGACG GTCGGGCTGC
     TGCACTTTCC
501  GGCGAACACC TCCGTCCTGA CCGCCGTTTA CGCGCTGGCA
     AACCTTGCCG
551  CCGCCGCCTT TTTGCTGTTT CAAAACCGAT GCCGTCTGAA
     GGCCGTCCGG
601  CGCGCGCCGT TTTCGCCCGC CGTCCTGCAC CGGGGGCTGC
     GCTACGGCAT
651  ACCGCTCGCA CTGAGCAGCC TTGCCTATTG GGGGCTGGCA
     TCCGCCGACC
701  GTTTGTTCCT GAAAAAATAT GCGGGCCTGG AACAGCTCGG
     CGTTTATTCG
751  ATGGGTATTT CGTTCGGCGG GGCGGCATTA TTGCTCCAAA
     GCATCTTTTC
801  AACGGTCTGG ACACCGTATA TTTTCCGTGC AATCGAAGAA
     AACGCCACGC
851  CCGCCCGCCT CTCGGCAACG GCAGAATCCG CCGCCGCCCT
     GCTTGCCTCC
901  GCCCTCTGCC TGACCGGAAT TTTCTCGCCC CTCGCCTCCC
     TCCTGCTGCC
951  GGAAAACTAC GCCGCCGTCC GGTTTACCGT CGTATCGTGT
     ATGCTGccgc
1001 cgctGTTTTA CACGCTGACC GAAATCAGCG GCATCGGTTT
     GAACGTCGTC
1051 CGCAAAACGC GTCCGATCGC GCTTGCCACC TTGGGCGCGC
     TGGCGGCAAA
1101 CCTGCTGCTG CTGGGGCTTG CCGTACCGTC CGGCGGCACG
     CGCGGCGCGG
1151 CGGTTGCCTG TGCCGCCTCA TTCTGGTTGT TTTTTGTTTT
     CAAGACAGAA
1201 AGCTCCTGCC GCCTGTGGCA GCCGCTCAAA CGCCTGCCGC
     TTTATATGCA
1251 CACATTGTTC TGCCTgGCCT CCTCGGCGGC CTACACCTGC
     TTCGGCACAC
1301 CGGCAAACTA CCCcctgttt gccggcgtAT GGGCGGCATA
     TCTGGCAGGC
1351 TGCATCCTGC GCCACCGGAA AAATTTGCAC AAACTGTTTC
     ATTATTTGAA
1401 AAAACAAGGT TTCCCATTAT GA

This encodes a protein having amino acid sequence <SEQ ID 380>:

1   MDTKEILGYA AGSIGSAVLA VIILPLLSWY FPADDIGRIV
    LMQTAAGLTV
51  SVLCLGLDQA YVREYYAAAD KDTLFKTLFL PPLLFSAAIA
    ALLLSRPSLP
101 SEILFSLDDA AAGIGLVLFE LSFLPIRFLL LVLRMEGRAL
    AFSSAQLVPK
151 LAILLLLPLT VGLLHFPANT SVLTAVYALA NLAAAAFLLF
    QNRCRLKAVR
201 RAPFSPAVLH RGLRYGIPLA LSSLAYWGLA SADRLFLKKY
    AGLEQLGVYS
251 MGISFGGAAL LLQSIFSTVW TPYIFRAIEE NATPARLSAT
    AESAAALLAS
301 ALCLTGIFSP LASLLLPENY AAVRFTVVSC MLPPLFYTLT
    EISGIGLNVV
351 RKTRPIALAT LGALAANLLL LGLAVPSGGT RGAAVACAAS
    FWLFFVFKTE
401 SSCRLWQPLK RLPLYMHTLF CLASSAAYTC FGTPANYPLF
    AGVWAAYLAG
451 CILRHRKNLH KLFHYLKKQG FPL*

ORF10ng and ORF10-1 show 96.4% identity in 473 aa overlap:

Based on this analysis, including the presence of a putative leader peptide and several transmembrane segments and the presence of a leucine-zipper motif(4 Leu residues spaced by 6 aa, shown in bold), it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 45

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 381>:

1   ..ATCCTGAAAC CGCATAACCA GCTTAAGGAA GACATCCAAC
    CTGATCCGGC
51  CGATCAAAAC GCCTTGTCCG AACCGGATGC TGCGACAGAG
    GCAGAGCAGT
101 CGGATGCGGA AAATGCTGCC GACAAGCAGC CCGTTGCCGA
    TAAAGCCGAC
151 GAGGTTGAAG AAAAGGCGGG CGAGCCGGAA CGGGAAGAGC
    CGGACGGACA
201 GGCAGTGCGT AAGAAAGCGC TGACGGAAGA GCGTGAACAA
    ACCGTCAGGG
251 AAAAAGCGCA GAAGAAAGAT GCCGAAACGG TTAAAATACA
    AGCGGTAAAA
301 CCGTCTAAAG AAACAGAGAA AAAAGCTTCA AAAGAAGAGA
    AAAAGGCGGC
351 GAAGGAAAAA GTTGCACCCA AACCAACCCC GGAACAAATC
    CTCAACAGCG
401 GCAgCATCGA AAAmGCGCGC AgTGCCGCCG CCAAAGAAGT
    GCAGAAAATG
451 AA.AACGTCC GACAAGGCGG AAGC.AACGC ATTATCTGCA
    AATGGGCGCG
501 TATGCCGACC GTCAGAGCGC GGAAGGGCAG CGTGCCAAAC
    TGGCAATCTT
551 GGGCATATCT TCCAAGGTGG TCGGTTATCA GGCGGGACAT
    AAAACGCTTT
601 ACCGGGTGCA AAGCGGCAAT ATGTCTGCCG ATGCGGTGA

This corresponds to the amino acid sequence <SEQ ID 382; ORF65>:

1   ..ILKPHNQLKE DIQPDPADQN ALSEPDAATE AEQSDAENAA
    DKQPVADKAD
51  EVEEKAGEPE REEPDGQAVR KKALTEEREQ TVREKAQKKD
    AETVKIQAVK
101 PSKETEKKAS KEEKKAAKEK VAPKPTPEQI LNSGSIEXAR
    SAAAKEVQKM
151 XNVRQGGSXR IICKWARMPT VRARKGSVPN WQSWAYLPRW
    SVIRRDIKRF
201 TGCKAAICLP MR*

Further work revealed the complete nucleotide sequence <SEQ ID 383>:

1   ATGTTTATGA ACAAATTTTC CCAATCCGGA AAAGGTCTGT
    CCGGTTTTTT
51  CTTCGGTTTG ATACTGGCGA CGGTCATTAT TGCCGGTATT
    TTGTTTTATC
101 TGAACCAGAG CGGTCAAAAT GCGTTCAAAA TCCCGGCTTC
    GTCGAAGCAG
151 CCTGCAGAAA CGGAAATCCT GAAACCGAAA AACCAGCCTA
    AGGAAGACAT
201 CCAACCTGAA CCGGCCGATC AAAACGCCTT GTCCGAACCG
    GATGCTGCGA
251 CAGAGGCAGA GCAGTCGGAT GCGGAAAAAG CTGCCGACAA
    GCAGCCCGTT
301 GCCGATAAAG CCGACGAGGT TGAAGAAAAG GCGGGCGAGC
    CGGAACGGGA
351 AGAGCCGGAC GGACAGGCAG TGCGTAAGAA AGCGCTGACG
    GAAGAGCGTG
401 AACAAACCGT CAGGGAAAAA GCGCAGAAGA AAGATGCCGA
    AACGGTTAAA
451 AAACAAGCGG TAAAACCGTC TAAAGAAACA GAGAAAAAAG
    CTTCAAAAGA
501 AGAGAAAAAG GCGGCGAAGG AAAAAGTTGC ACCCAAACCA
    ACCCCGGAAC
551 AAATCCTCAA CAGCGGCAGC ATCGAAAAAG CGCGCAGTGC
    CGCCGCCAAA
601 GAAGTGCAGA AAATGAAAAC GTCCGACAAG GCGGAAGCAA
    CGCATTATCT
651 GCAAATGGGC GCGTATGCCG ACCGTCAGAG CGCGGAAGGG
    CAGCGTGCCA
701 AACTGGCAAT CTTGGGCATA TCTTCCAAGG TGGTCGGTTA
    TCAGGCGGGA
751 CATAAAACGC TTTACCGGGT GCAAAGCGGC AATATGTCTG
    CCGATGCGGT
801 GAAAAAAATG CAGGACGAGT TGAAAAAACA TGAAGTCGCC
    AGCCTGATCC
851 GTTCTATCGA AAGCAAATAA

This corresponds to the amino acid sequence <SEQ ID 384; ORF65-1>:

1   MFMNKFSQSG KGLSGFFFGL ILATVIIAGI LFYLNQSGQN
    AFKIPASSKQ
51  PAETEILKPK NQPKEDIQPE PADQNALSEP DAATEAEQSD
    AEKAADKQPV
101 ADKADEVEEK AGEPEREEPD GQAVRKKALT EEREQTVREK
    AQKKDAETVK
151 KQAVKPSKET EKKASKEEKK AAKEKVAPKP TPEQILNSGS
    IEKARSAAAK
201 EVQKMKTSDK AEATHYLQMG AYADRQSAEG QRAKLAILGI
    SSKVVGYQAG
251 HKTLYRVQSG NMSADAVKKM QDELKKHEVA SLIRSIESK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF65 shows 92.0% identity over a 150aa overlap with an ORF (ORF65a) from strain A of N. meningitidis:

The complete length ORF65a nucleotide sequence <SEQ ID 385> is:

1   ATGTTTATGA ACAAATTTTC CCAATCCGGA AAAGGTCTGT
    CCGGTTTTTT
51  CTTCGGTTTG ATACTGGCGA CGGTCATTAT TGCCGGTATT
    TTGTTTTATC
101 TGAACCAGAG CGGTCAAAAT GCGTTCAAAA TCCCGGTTCC
    GTCGAAGCAG
151 CCTGCAGAAA CGGAAATCCT GAAACCGAAA AACCAGCCTA
    AGGAAGACAT
201 CCAACCTGAA CCGGCCGATC AAAACGCCTT GTCCGAACCG
    GATGCTGCGA
251 AAGAGGCAGA GCAGTCGGAT GCGGAAAAAG CTGCCGACAA
    GCAGCCCGTT
301 GCCGACAAAG CCGACGAGGT TGAGGAAAAG GCGGACGAGC
    CGGAGCGGGA
351 AAAGTCGGAC GGACAGGCAG TGCGCAAGAA AGCACTGACG
    GAAGAGCGTG
401 AACAAACCGT CGGGGAAAAA GCGCAGAAGA AAGATGCCGA
    AACGGTTAAA
451 AAACAAGCGG TAAAACCATC TAAAGAAACA GAGAAAAAAG
    CTTCAAAAGA
501 AGAGAAAAAG GCGGAGAAGG AAAAAGTTGC ACCCAAACCG
    ACCCCGGAAC
551 AAATCCTCAA CAGCGGCAGC ATCGAAAAAG CGCGCAGTGC
    CGCTGCCAAA
601 GAAGTGCAGA AAATGAAAAC GCCCGACAAG GCGGAAGCAA
    CGCATTATCT
651 GCAAATGGGC GCGTATGCCG ACCGCCGGAG CGCGGAAGGG
    CAGCGTGCCA
701 AACTGGCAAT CTTGGGCATA TCTTCCAAGG TGGTCGGTTA
    TCAGGCGGGA
751 CATAAAACGC TTTACCGGGT GCAAAGCGGC AATATGTCTG
    CCGATGCGGT
801 GAAAAAAATG CAGGACGAGT TGAAAAAACA TGAAGTCGCC
    AGCCTGATCC
851 GTTCTATCGA AAGCAAATAA

This encodes a protein having amino acid sequence <SEQ ID 386>:

1   MFMNKFSQSG KGLSGFFFGL ILATVIIAGI LFYLNQSGQN
    AFKIPVPSKQ
51  PAETEILKPK NQPKEDIQPE PADQNALSEP DAAKEAEQSD
    AEKAADKQPV
101 ADKADEVEEK ADEPEREKSD GQAVRKKALT EEREQTVGEK
    AQKKDAETVK
151 KQAVKPSKET EKKASKEEKK AEKEKVAPKP TPEQILNSGS
    IEKARSAAAK
201 EVQKMKTPDK AEATHYLQMG AYADRRSAEG QRAKLAILGI
    SSKVVGYQAG
251 HKTLYRVQSG NMSADAVKKM QDELKKHEVA SLIRSIESK*

ORF65a and ORF65-1 show 96.5% identity in 289 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF65 shows 89.6% identity over a 212aa overlap with a predicted ORF (ORF65.ng) from N. gonorrhoeae:

An ORF65ng nucleotide sequence <SEQ ID 387> was predicted to encode a protein having amino acid sequence <SEQ ID 388>:

1   MFMNKFSQSG KGLSGFFFGL ILATVIIAGI LLYLNQGGQN
    AFKIPAPSKQ
51  PAETEILKLK NQPKEDIQPE PADQNALSEP DVAKEAEQSD
    AEKAADKQPV
101 ADKADEVEEK AGEPEREEPD GQAVRKKALT EEREQTVREK
    AQKKDAETVK
151 KKAVKPSKET EKKASKEEKK AAKEKVAPKP TPEQILNSRS
    IEKARSAAAK
201 EVQKMKNFGQ GGSQRIICKW ARMPNPGARK GSVPNWQSWA
    YLPKWSAIRR
251 DIKRFTACKA AICPPMR*

After further analysis, the complete gonococcal DNA sequence <SEQ ID 389> was found to be:

1   ATGTTTATGA ACAAATTTTC CCAATCCGGA AAAGGTCTGT
    CCGGTTTCTT
51  CTTCGGTTTG ATACTGGCAA CGGTCATTAT TGCCGGTATT
    TTGCTTTATC
101 TGAACCAGGG CGGTCAAAAT GCGTTCAAAA TCCCGGCTCC
    GTCGAAGCAG
151 CCTGCAGAAA CGGAAATCCT GAAACTGAAA AACCAGCCTA
    AGGAAGACAT
201 CCAACCTGAA CCGGCCGATC AAAACGCCTT GTCCGAACCG
    GATGTTGCGA
251 AAGAGGCAGA GCAGTCGGAT GCGGAAAAAG CTGCCGACAA
    GCAGCCCGTT
301 GCCGACAAag ccgacgAGGT TGAAGAAAag GcGGgcgAgc
    cggaACGGga
351 aGAGCCGGAC ggACAGGCAG TGCGCAAGAA AGCACTGAcg
    gAAGAgcGTG
401 AACAAACcgt cagggAAAAA GCGCagaaga AAGATGCCGA
    AACGgTTAAA
451 AAacaaGCgg tAaaaccgtc tAAAGAAACa gagaaaaaag
    cTtcaaaaga
501 agagaaaaag gcggcgaaag aaaAAGttgc acccaaaccg
    accccggaaC
551 aaatcctcaa cagccgCagc atcgaaaaag cgcgtagtgc
    cgctgccaaa
601 gaAgtgcaGA AAatgaaaaa ctTtgggcaa ggcgGaagcc
    aacgcattaT
651 CTGcaaatgg gcgcgtatgc cgaccgtccg gagcgcggaA
    gggcagcgtg
701 ccaaACtggc aAtcttgGgc atatctTccg aagtggtcgG
    CTATCAGGCG
751 GGACATAAAA CGCTTTACCG CGTGCAAagc GGCAatatgt
    ccgccgatgc
801 gGTGAAAAAA ATGCAGGACG AGTTGAAAAA GCATGGGGtt
    gcCAGCCTGA
851 TCCGTGcgAT TGAAGGCAAA TAA

This encodes the following amino acid sequence <SEQ ID 390>:

1   MFMNKFSQSG KGLSGFFFGL ILATVIIAGI LLYLNQGGQN
    AFKIPAPSKQ
51  PAETEILKLK NQPKEDIQPE PADQNALSEP DVAKEAEQSD
    AEKAADKQPV
101 ADKADEVEEK AGEPEREEPD GQAVRKKALT EEREQTVREK
    AQKKDAETVK
151 KQAVKPSKET EKKASKEEKK AAKEKVAPKP TPEQILNSRS
    IEKARSAAAK
201 EVQKMKNFGQ GGSQRIICKW ARMPTVRSAE GQRAKLAILG
    ISSEVVGYQA
251 GHKTLYRVQS GNMSADAVKK MQDELKKHGV ASLIRAIEGK
    *

ORF65ng-1 and ORF65-1 show 89.0% identity in 290 aa overlap:

On this basis, including the presence of a putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 46

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 391>:

1   ATGAACCACG ACATCACTTT CCTCACCCTG TTCCTACTCG
    GTkTCTTCGG
51  CGGAAcGCAC TGCATCGGTA TGTGCGGCGG ATTAAGCAGC
    GcGTTTGs.s
101 TCCAACTCCC CCCGCATATC AACCGCTTTT GGCTGATCCT
    GCTGCTTAAC
151 ACAGGACGGG TAAGCAGCTA TACGGCAAtC GGCCTGATAC
    TCGGATTAAT
201 CGGACAGGTC GGCGTTTCAC TCGAcCAaAC CCGCGTCCTG
    CAGAATATTT
251 TATACACGGC CGCCAACCTC CTGCTGCTCT TTTTAGGCTT
    ATACTTGAGC
301 GGTATTTCTT CCTTGGCGGC AAAAATCGAG AAaATCGGCA
    AACCGATATG
351 GCGGAACCTG AACCCGATAC TCAACCGGCT GTTACCCATA
    AAATCCATAC
401 CCGCCTGCCT tGCGgTCGGA ATATTATGGG GCTGGCTGCC
    GTGCGGACTG
451 GTTTACAGCG CGTCGCTTTA CGCGCTGGGA AgCGGTAGTG
    CGGCAACGGG
501 CGGGTTATAT ATGCTTGCCT TTGCACTGGG TACGCTGCCC
    AATCTTtTAG
551 CAATCGGCAT TTTtTCCCTG CAACTGAAwA AAATCATGCA
    AAACCGATAT
601 ATCCGCCTGT GTACGGGATT ATCCGTATCA TTATGGGCAT
    TATGGAAACT
651 TGCCGTCCTG TGGCTGTAA

This corresponds to the amino acid sequence <SEQ ID 392; ORF103>:

1   MNHDITFLTL FLLGXFGGTH CIGMCGGLSS AFXXQLPPHI
    NRFWLILLLN
51  TGRVSSYTAI GLILGLIGQV GVSLDQTRVL QNILYTAANL
    LLLFLGLYLS
101 GISSLAAKIE KIGKPIWRNL NPILNRLLPI KSIPACLAVG
    ILWGWLPCGL
151 VYSASLYALG SGSAATGGLY MLAFALGTLP NLLAIGIFSL
    QLXKIMQNRY
201 IRLCTGLSVS LWALWKLAVL WL*

Further work elaborated the DNA sequence <SEQ ID 393> as:

1   ATGAACCACG ACATCACTTT CCTCACCCTG TTCCTACTCG
    GTTTCTTCGG
51  CGGAACGCAC TGCATCGGTA TGTGCGGCGG ATTAAGCAGC
    GCGTTTGCGC
101 TCCAACTCCC CCCGCATATC AACCGCTTTT GGCTGATCCT
    GCTGCTTAAC
151 ACAGGACGGG TAAGCAGCTA TACGGCAATC GGCCTGATAC
    TCGGATTAAT
201 CGGACAGGTC GGCGTTTCAC TCGACCAAAC CCGCGTCCTG
    CAGAATATTT
251 TATACACGGC CGCCAACCTC CTGCTGCTCT TTTTAGGCTT
    ATACTTGAGC
301 GGTATTTCTT CCTTGGCGGC AAAAATCGAG AAAATCGGCA
    AACCGATATG
351 GCGGAACCTG AACCCGATAC TCAACCGGCT GTTACCCATA
    AAATCCATAC
401 CCGCCTGCCT TGCGGTCGGA ATATTATGGG GCTGGCTGCC
    GTGCGGACTG
451 GTTTACAGCG CGTCGCTTTA CGCGCTGGGA AGCGGTAGTG
    CGGCAACGGG
501 CGGGTTATAT ATGCTTGCCT TTGCACTGGG TACGCTGCCC
    AATCTTTTAG
551 CAATCGGCAT TTTTTCCCTG CAACTGAAAA AAATCATGCA
    AAACCGATAT
601 ATCCGCCTGT GTACGGGATT ATCCGTATCA TTATGGGCAT
    TATGGAAACT
651 TGCCGTCCTG TGGCTGTAA

This corresponds to the amino acid sequence <SEQ ID 394; ORF103-1>:

1   MNHDITFLTL FLLGFFGGTH CIGMCGGLSS AFALQLPPHI
    NRFWLILLLN
51  TGRVSSYTAI GLILGLIGQV GVSLDQTRVL QNILYTAANL
    LLLFLGLYLS
101 GISSLAAKIE KIGKPIWRNL NPILNRLLPI KSIPACLAVG
    ILWGWLPCGL
151 VYSASLYALG SGSAATGGLY MLAFALGTLP NLLAIGIFSL
    QLKKIMQNRY
201 IRLCTGLSVS LWALWKLAVL WL*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF103 shows 93.8% identity over. a 222aa overlap with an ORF (ORF103a) from strain A of N. meningitidis:

The complete length ORF103a nucleotide sequence <SEQ ID 395> is:

1   ATGAACCANG ACATCACTTT CCTCACCCTG TTCCTACTCG
    GTTTCTTCGG
51  CGGAACGCAC TGCATCGGTA TGTGCGGCGG ATTAAGCAGC
    GCGTTTGCGC
101 TCCAACTCCC CCCGCATATC AACCGCTTNT GGCTGATCCT
    GCTGCTTAAC
151 ACAGGACGGG TAAGCAGCTA TACGGCAATC GGCCTGATAC
    TCGGATTAAT
201 CGGACAGGTC GGCGTTTCAC TCGACCAAAC CCGCGTCNTG
    CAGAATATTT
251 TATACACGGC CGCCAACCTC CTGCTGCTCT TTTTAGGCTT
    ATACTTGAGC
301 GGTATTTCTT CCTTGGCGGC AAAAATCGAG AAAATCGGCA
    AACCGATATG
351 GCGGAACCTG AACCCGATAC TCAACCGGCT GTTACCCATA
    AAATCCATAC
401 CCGCCTGCCT TGCGGTCGGA ATATTATGGG GCTGGCTGCC
    GTGCGGACTA
451 GTTTACAGCG CGTCGCTTTA CGCGCTGGGA AGCGGTAGTG
    CGGCAACGGG
501 CGGGTTATAT ATGCTTGCCT TTGCACTGGG TACGCTGCCC
    AATCTTTNGG
551 CAATCGGCAT TTTTTCCCTG CAACTGNAAA AAATCATGCA
    AAACCGATAT
601 ATCCGCCTGT GTACGGGATT ATCCGTATCA TTATGGGCAT
    TATGGAAACT
651 TGCCGTCCTG TGGCTGTAA

This encodes a protein having amino acid sequence <SEQ ID 396>:

1   MNXDITFLTL FLLGFFGGTH CIGMCGGLSS AFALQLPPHI
    NRXWLILLLN
51  TGRVSSYTAI GLILGLIGQV GVSLDQTRVX QNILYTAANL
    LLLFLGLYLS
101 GISSLAAKIE KIGKPIWRNL NPILNRLLPI KSIPACLAVG
    ILWGWLPCGL
151 VYSASLYALG SGSAATGGLY MLAFALGTLP NLXAIGIFSL
    QLXKIMQNRY
201 IRLCTGLSVS LWALWKLAVL WL*

ORF103a and ORF103-1 show 97.7% identity in 222 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF103 shows 95.5% identity over a 222aa overlap with a predicted ORF (ORF103.ng) from N. gonorrhoeae:

The complete length ORF103ng nucleotide sequence <SEQ ID 397> is:

1   ATGAACCACG ACATCACTTT CCTCACCCTG TTCCTGCTCG
    GTTTCTTCGG
51  CGGAACTCAC TGCATCGGTA TGTGCGGCGG ATTAAGCAGC
    GCGTTTGCGC
101 TCCAACTCCC CCCGCATATC AACCGCTTTT GGCTGATTCT
    GCTGCTTAAC
151 ACAGGACGGA TAAGCAGCTA TACGGCAATC GGCCTGATGC
    TCGGATTAAT
201 CGGACAACTC GGCATTTCAC TCGACCAAAc ccgcgTCCTG
    CAAAATATTT
251 tatacacagc ctccaaCCTC CTGCTGCTCT TTTTAGGCTT
    ATACTTGAGC
301 GGTATTTCTT CCTTGGCGGC AAAAATCGAG AAAATCGGCA
    AACCGATATG
351 GCGCAACCTG AACCCGATAC TCAACCGGCT GCTGCCCATA
    AAATCCATAC
401 CCGCCTGCCT TGCTGTCGGA ATATTATGGG GCTGGCTGCC
    GTGCGGACTG
451 GTTTACAGCG CATCACTTTA CGCGCTGGGA AGCGGTAGTG
    CGACAACCGG
501 CGGACTGTAT ATGCTTGCCT TTGCACTGGG TACGCTGCCC
    AATCTTTTGG
551 CAATCGGCAT TTTTTCCCTG CAACTGAAAA AAATCATGCA
    AAACCGATAT
601 ATCCGCCTGT GTACAGGATT ATCCGTATCA TTATGGGCAT
    TATGGAAGCT
651 TGCCGTCCTG TGGCTGTAA

This encodes a protein having amino acid sequence <SEQ ID 398>:

1   MNHDITFLTL FLLGFFGGTH CIGMCGGLSS AFALQLPPHI
    NRFWLILLLN
51  TGRISSYTAI GLMLGLIGQL GISLDQTRVL QNILYTASNL
    LLLFLGLYLS
101 GISSLAAKIE KIGKPIWRNL NPILNRLLPI KSIPACLAVG
    ILWGWLPCGL
151 VYSASLYALG SGSATTGGLY MLAFALGTLP NLLAIGIFSL
    QLKKIMQNRY
201 IRLCTGLSVS LWALWKLAVL WL*

In addition, ORF103ng and ORF103-1 show 97.3% identity in 222 aa overlap:

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 47

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 399>:

1   ATGGAAAACC AAAGGCCGCT CCTAGGCTTT CGCTTGGCAC
    TTTTGGCGGC
51  GATGACGTGG GGAACGCTGC CGAT.TCCGT GCGGCAGGTA
    TTGAAGTTTG
101 TCGATGCGCC GACGCTGGTG TGGGTGCGTT TTACCGTGGC
    GGCGGCGGTA
151 TTGTTTGTTT TGCTGGCACT GGGCGGGCGG CTGCcGAAGC
    GGCGaGGATT
201 TTTCTTGGTG CTCATTCAGG CTGCTGCTGC TCGGCGTGGC
    GGGCATTTCG
251 GCAAACTTTG TGCTGATTGC CCAAGGGCTG CATTATATTT
    CGCCGACCAC
301 GACGCAGGTT TTGTGGCAGA TTTCGCCGTT TACGATGATT
    GTwGTCGGTG
351 TGTTGGTGTT TAAAGACCGG ATGACTGCCG CTCAGAAAAT
    CGGCTTGGTT
401 TTGCTGCTTG CCGGTTTGCT TATGTATTTT AACGATAAAT
    TCGGCGAGTT
451 GTCGGGTTTG GGCGCGTATG C.AAGGGCGT GTTGCTGTGT
    GCGGCAGGCA
501 GTATGGCATG GGTGTGTAAT GCCGTGGCGC AAAAGCTGCT
    GTCGGCGCAA
551 TTCGGGCCGC AACAGATTCT GCTGTTGATT TATGCGGCAA
    GTGCCGCCGT
601 GTTCCTGCCG TTTGCCGAAC CGGCACACAT CGGAAGTATG
    GACGGTACGT
651 TGGCGTGGGT ATGTATTGCG TATTGCTGCT TGAATACGTT
    AATCGGTTAC
701 GGCTCGTTCG GCGAGGCGTT GAAACATTGG GAGGCTTCCA
    AAGTCAGCGC
751 GGTAACAACC TTGCTCCCCG TGTTTACCGT AATAAATACT
    TTGCTCGGGC
801 ATTATGTGAT GCCTGAAACT TTTGCCGCGC CGGA..

This corresponds to the amino acid sequence <SEQ ID 400; ORF104>:

1   MENQRPLLGF RLALLAAMTW GTLPXSVRQV LKFVDAPTLV
    WVRFTVAAAV
51  LFVLLALGGR LPKRRDFSWC SFRLLLLGVA GISANFVLIA
    QGLHYISPTT
101 TQVLWQISPF TMIVVGVLVF KDRMTAAQKI GLVLLLAGLL
    MYFNDKFGEL
151 SGLGAYXKGV LLCAAGSMAW VCNAVAQKLL SAQFGPQQIL
    LLIYAASAAV
201 FLPFAEPAHI GSMDGTLAWV CIAYCCLNTL IGYGSFGEAL
    KHWEASKVSA
251 VTTLLPVFTV INTLLGHYVM PETFAAP...

Further work revealed further, partial DNA sequence <SEQ ID 401>:

1   ATGGAAAACC AAAGGCCGCT CCTAGGCTTC GCGTTGGCAC
    TTTTGGCGGC
51  GATGACGTGG GGAACGCTGC CGATTGCCGT GCGGCAGGTA
    TTGAAGTTTG
101 TCGATGCGCC GACGCTGGTG TGGGTGCGTT TTACCGTGGC
    GGCGGCGGTA
151 TTGTTTGTTT TGCTGGCACT GGGCGGGCGG CTGCCGAAGC
    GGCGGGATTT
201 TTCTTGGTGC TCATTCAGGC TGCTGCTGCT CGGCGTGGCG
    GGCATTTCGG
251 CAAACTTTGT GCTGATTGCC CAAGGGCTGC ATTATATTTC
    GCCGACCACG
301 ACGCAGGTTT TGTGGCAGAT TTCGCCGTTT ACGATGATTG
    TTGTCGGTGT
351 GTTGGTGTTT AAAGACCGGA TGACTGCCGC TCAGAAAATC
    GGCTTGGTTT
401 TGCTGCTTGC CGGTTTGCTT ATGTTTTTTA ACGATAAATT
    CGGCGAGTTG
451 TCGGGTTTGG GCGCGTATGC GAAGGGCGTG TTGCTGTGTG
    CGGCAGGCAG
501 TATGGCATGG GTGTGTTATG CCGTGGCGCA AAAGCTGCTG
    TCGGCGCAAT
551 TCGGGCCGCA ACAGATTCTG CTGTTGATTT ATGCGGCAAG
    TGCCGCCGTG
601 TTCCTGCCGT TTGCCGAACC GGCACACATC GGAAGTTTGG
    ACGGTACGTT
651 GGCGTGGGTT TGTTTTGCGT ATTGCTGCTT GAATACGTTA
    ATCGGTTACG
701 GCTCGTTCGG CGAGGCGTTG AAACATTGGG AGGCTTCCAA
    AGTCAGCGCG
751 GTAACAACCT TGCTCCCCGT GTTTACCGTA ATAwTwwCTT
    TGCTCGGGCA
801 TTATGTGATG CCTGAAACTT TTGCCGCGCC GGA...

This corresponds to the amino acid sequence <SEQ ID 402; ORF104-1>:

1   MENQRPLLGF ALALLAAMTW GTLPIAVRQV LKFVDAPTLV
    WVRFTVAAAV
51  LFVLLALGGR LPKRRDFSWC SFRLLLLGVA GISANFVLIA
    QGLHYISPTT
101 TQVLWQISPF TMIVVGVLVF KDRMTAAQKI GLVLLLAGLL
    MFFNDKFGEL
151 SGLGAYAKGV LLCAAGSMAW VCYAVAQKLL SAQFGPQQIL
    LLIYAASAAV
201 FLPFAEPAHI GSLDGTLAWV CFAYCCLNTL IGYGSFGEAL
    KHWEASKVSA
251 VTTLLPVFTV IXXLLGHYVM PETFAAP...

Computer analysis of this amino acid sequence gave the following results:

Homology with Hypothetical HI0878 Protein of H. influenzae (Accession Number U32769)

ORF104 and HI0878 show 40% aa identity in 277aa overlap:

orf104	4	QRPLLGFRLALLAAMTWGTLPXSVRQVLKFVDAPTLVWXXXXXXXXXXXXXXXXXXXXP-	62
		Q+PLLGF  AL+ AM WG+LP +++QVL  ++A T+VW                    P
HI0878	3	QQPLLGFTFALITAMAWGSLPIALKQVLSVMNAQTIVWYRFIIAAVSLLALLAYKKQLPE	62
orf104	63	--KRRDFSWCSFRLLLLGVAGISANFVLIAQGLHYISPTTTQVLWQISPFTMIVVGVLVF	120
		K R ++W    ++L+GV G+++NF+L +  L+YI P+  Q+   +S F M++ GVL+F
HI0878	63	LMKVRQYAW----IMLIGVIGLTSNFLLFSSSLNYIEPSVAQIFIHLSSFGMLICGVLIF	118
orf104	121	KDRMTAAQKIXXXXXXXXXXMYFNDKFGELSGLGAYXKGVLLCAAGSMAWVCNAVAQKLL	180
		K+++   QKI          ++FND+F   +GL  Y  GV+L   G++ WV   +AQKL+
HI0878	119	KEKLGLHQKIGLFLLLIGLGLFFNDRFDAFAGLNQYSTGVILGVGGALIWVAYGMAQKLM	178
orf104	181	SAQFGPQQILLLIYAASAAVFLPFAEPAHIGSMDGTLAWVCIAYCCLNTLIGYGSFGEAL	240
		+F  QQILL++Y   A  F+P A+ + +  +   LA +C  YCCLNTLIGYGS+ EAL
HI0878	179	LRKFNSQQILLMMYLGCAIAFMPMADFSQVQELT-PLALICFIYCCLNTLIGYGSYAEAL	237
orf104	241	KHWEASKVSAVTTLLPVFTVINTLLGHYVMPETFAAP	277
		W+ SKVS V TL+P+FT++ + + HY  P  FAAP
HI0878	238	NRWDVSKVSVVITLVPLFTILFSHIAHYFSPADFAAP	274

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF104 shows 95.3% identity over a 277aa overlap with an ORF (ORF104a) from strain A of N. meningitidis:

The complete length ORF104a nucleotide sequence <SEQ ID 403> is:

1   ATGGAAAACC AAAGGCCGCT CCTAGGCTTC GCGTTGGCAC
    TTTTGGCGGC
51  GATGACGTGG GGAACGCTGC CGATTGCCGT GCGGCAGGTA
    TTGAAGTTTG
101 TCGATGCGCC GACGCTGGTG TGGGTGCGTT TTACCGTGGC
    GGCGGCGGTA
151 TTGTTTGTTT TGCTGGCATT GGGCGGGCGG CTGCCGAAGT
    GGCGGGATTT
201 TTCTTGGTGC TCATTCAGGC TGCTGCTGCT CGGCGTGGCG
    GGCATTTCGG
251 CAAACTTTGT GCTGATTGCC CAAGGGCTGC ATTATATTTC
    GCCGACCACG
301 ACGCAGGTTT TGTGGCAGAT TTCGCCGTTT ACGATGATTG
    TTGTCGGTGT
351 GTTGGTGTTT AAAGACCGGA TGACTGCCGC TCAGAAAATC
    GGCTTGGTTT
401 TGCTGCTTGC CGGTTTGCTT ATGTTTTTTA ACGATAAATT
    CGGCGAGTTG
451 TCGGGTTTGG GCGCGTATGC GAAGGGCGTG TTGCTGTGTG
    CGGCAGGCAG
501 TATGGCATGG GTGTGTTATG CCGTGGCGCA AAAGCTGCTG
    TCGGCGCAAT
551 TCGGGCCGCA ACAGATTCTG CTGTTGATTT ATGCGGCAAG
    TGCCGCCGTG
601 TTCCTGCCGT TTGCCGAACT GGCACACATC GGAAGTTTGG
    ACGGTACGTT
651 GGCGTGGGTT TGTTTTGCGT ATTGCTGCTT GAATACGTTA
    ATCGGTTACG
701 GCTCGTTCGG CGAGGCGTTG AAACATTGGG AGGCTTCCAA
    AGTCAGCGCG
751 GTAACAACCT TGCTCCCCGT GTTTACCGTA ATATTTTCTT
    TGCTCGGGCA
801 TTATGTGATG CCTGATACTT TTGCCGCGCC GGATATGAAC
    GGTTTGGGTT
851 ATGCCGGCGC ACTGGTCGTG GTCGGGGGTG CGGTTACGGC
    GGCGGTGGGG
901 GACAGGCTGT TCAAACGCCG CTAG

This encodes a protein having amino acid sequence <SEQ ID 404>:

1   MENQRPLLGF ALALLAAMTW GTLPIAVRQV LKFVDAPTLV
    WVRFTVAAAV
51  LFVLLALGGR LPKWRDFSWC SFRLLLLGVA GISANFVLIA
    QGLHYISPTT
101 TQVLWQISPF TMIVVGVLVF KDRMTAAQKI GLVLLLAGLL
    MFFNDKFGEL
151 SGLGAYAKGV LLCAAGSMAW VCYAVAQKLL SAQFGPQQIL
    LLIYAASAAV
201 FLPFAELAHI GSLDGTLAWV CFAYCCLNTL IGYGSFGEAL
    KHWEASKVSA
251 VTTLLPVFTV IFSLLGHYVM PDTFAAPDMN GLGYAGALVV
    VGGAVTAAVG
301 DRLFKRR*

ORF104a and ORF104-1 show 98.2% identity in 277 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF104 shows 93.9% identity over a 277aa overlap with a predicted ORF (ORF104.ng) from N. gonorrhoeae:

The complete length ORF104ng nucleotide sequence <SEQ ID 405> is predicted to encode a protein having amino acid sequence <SEQ ID 406>:

1   MENQRPLLGF ALALLAAMTW GTLPIAVRQV LKFVDAPTLV
    WVRFTVAAAV
51  LFVLLALGGR LPKRRDFSWH SFRLLLLGVT GISANFVLIA
    QGLHYISPTT
101 TQVLWQISPF TMIVVGVLVF KDRMTAAQKI GLVLLLVGLL
    MFFNDKFGEL
151 SGLGAYAKGV LLCAAGSMAW VCYAVAQKLL SAQFGPQQIL
    LLIYAASAAV
201 FLLXAEPAHI GSLDGTLAWV CFVYCCLNTL IGYGSFGEAL
    KHWEASKVSA
251 VTTLLPVFTV IFSLLGHYVM PDTFAAPDMN GLGYVGALVV
    VGGAVTAAVG
301 DRPFKRR*

Further work revealed the complete gonococcal nucleotide sequence <SEQ ID 407>:

1   ATGGAAAACC AAAGGCCGCT CCTAGGCTTC GCGTTGGCAC
    TTTTGGCGGC
51  GATGACGTGG GGGACGCTGC CGATTGCCGT GCGGCAGGTA
    TTGAAGTTTG
101 TCGATGCGCC GACGCTGGTG TGGGTGCGTT TTACCGTGGC
    GGCGGCGGTA
151 TTGTTTGTTT TGCTGGCATT GGGCGGGCGG CTGCCGAAGC
    GGCGGGATTT
201 TTCTTGGCAT TCATTCAGGC TGCTGCTGCT CGGCGTGACG
    GGCATTTCGG
251 CAAACTTTGT GCTGATTGCC CAAGGGCTGC ATTATATTTC
    GCCGACCACG
301 ACGCAGGTTT TGTGGCAGAT TTCGCCGTTT ACGATGATTG
    TTGTCGGCGT
351 GTTGGTGTTT AAAGACCGGA tgaCTGCCGC GCAGAAAATC
    GGTTTGGTTT
401 TGCTGCttgT CGGTttgCTT ATGTTTTtta ACGACAAATT
    CGGCGAGTTG
451 TCGGGTTTGG GCGCGTATGC GAAGGGCGTG TTGCTGTGTG
    CGGCAGGCAG
501 TATGGCCTGG GTGTGTTATG CCGTGGCGCA AAAGCTGCTG
    TCGGCGCAAT
551 TCGGGCCGCA ACAGATTCTG CTGTTGATTT ATGCGGcaag
    tgccgccGTG
601 TTCCtgccgT TTGccgaaCC GGCACACATC GGAAGTTTgg
    aCGGTACGtt
651 GGCGTGGGTT TGTTTTGTGT ATTGCTGCTT GAATACGTTA
    ATCGGTTACG
701 GCTCGTTCGG CGAGGCGTTG AAACATTGGG AGGCTTCCAA
    AGTCAGCGCG
751 GTAACAACCT TGCTCCCCGT GTTTACCGTA ATATTTTCTT
    TGCTCGGGCA
801 TTATGTGATG CCTGATACTT TTGCCGCGCC GGATATGAAC
    GGTTTGGGTT
851 ATGTCGGCGC ACTGGTCGTG GTCGGGGGTG CGGTTACGGC
    GGCGGTGGGG
901 GACAGGCCGT TCAAACGCCG CTAG

This corresponds to the amino acid sequence <SEQ ID 408; ORF104ng-1>:

1   MENQRPLLGF ALALLAAMTW GTLPIAVRQV LKFVDAPTLV
    WVRFTVAAAV
51  LFVLLALGGR LPKRRDFSWH SFRLLLLGVT GISANFVLIA
    QGLHYISPTT
101 TQVLWQISPF TMIVVGVLVF KDRMTAAQKI GLVLLLVGLL
    MFFNDKFGEL
151 SGLGAYAKGV LLCAAGSMAW VCYAVAQKLL SAQFGPQQIL
    LLIYAASAAV
201 FLPFAEPAHI GSLDGTLAWV CFVYCCLNTL IGYGSFGEAL
    KHWEASKVSA
251 VTTLLPVFTV IFSLLGHYVM PDTFAAPDMN GLGYVGALVV
    VGGAVTAAVG
301 DRPFKRR*

ORF104ng-1 and ORF104-1 show 97.5% identity in 277 aa overlap:

In addition, ORF104ng-1 shows significant homology with a hypothetical H. influenzae protein:

gi|1573895 (U32769) hypothetical [Haemophilus influenzae] Length = 306
Score = 237 bits (598), Expect = 8e−62
Identities = 114/280 (40%), Positives = 168/280 (59%), Gaps = 8/280 (2%)
Query:	30	QRPXXXXXXXXXXXMTWGTLPIAVRQVLKFVDAPTLVWXXXXXXXXXXXXXXXXXXXXP-	88
		Q+P           M WG+LPIA++QVL  ++A T+VW                    P
Sbjct:	3	QQPLLGFTFALITAMAWGSLPIALKQVLSVMNAQTIVWYRFIIAAVSLLALLAYKKQLPE	62
Query:	89	--KRRDFSWHSFRLLLLGVTGISANFVLIAQGLHYISPTTTQVLWQISPFTMIVVGVLVF	146
		K R ++W    ++L+GV G+++NF+L +  L+YI P+  Q+   +S F M++ GVL+F
Sbjct:	63	LMKVRQYAW----IMLIGVIGLTSNFLLFSSSLNYIEPSVAQIFIHLSSFGMLICGVLIF	118
Query:	147	KDRMTAAQKIXXXXXXXXXXMFFNDKFGELSGLGAYAKGVLLCAAGSMAWVCYAVAQKLL	206
		K+++   QKI          +FFND+F   +GL  Y+ GV+L   G++ WV Y +AQKL+
Sbjct:	119	KEKLGLHQKIGLFLLLIGLGLFFNDRFDAFAGLNQYSTGVILGVGGALIWVAYGMAQKLM	178
Query:	207	SAQFGPQQILLLIYAASAAVFLPFAEPAHIGSLDGTLAWVCFVYCCLNTLIGYGSFGEAL	266
		+F  QQILL++Y   A  F+P A+ + +  L   LA +CF+YCCLNTLIGYGS+ EAL
Sbjct:	179	LRKFNSQQILLMMYLGCAIAFMPMADFSQVQELT-PLALICFIYCCLNTLIGYGSYAEAL	237
Query:	267	KHWEASKVSAVTTLLPVFTVIFSLLGHYVMPDTFAAPDMN	306
		W+ SKVS V TL+P+FT++FS + HY  P  FAAP++N
Sbjct:	238	NRWDVSKVSVVITLVPLFTILFSHIAHYFSPADFAAPELN	277

Based on this analysis, including the presence of a putative leader sequence and several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 48

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 409>:

1   ATGGTAGCTC GTCGGGCTCA TAACCCGAAG GTCGTAGGTT
    CGAATCCTGT
51  .CCCGCAACC TAATTTCAAA CCCCTCGGTT CAATGCCGAG
    GG.GTTTTGT
101 T.TTGCCTGT TTCCTGTTTC CTGTTTCCTG CCGCCTCCGT
    TTTTTGCCGG
151 ATTTTCCTTC CGGCCGCAAT ATCGGAACGG CAGACCGCCG
    TCTGTTTGCG
201 GTTGCAAATT CAGGCAGTTT GGCTACAATC TTCCGCATTG
    TCTTCAAGAA
251 AGCCAACCAT GCCGACCGTC CGTTTTACCG AATCCGTCAG
    CAAACAAGAC
301 CTTGATGCTC TGTTCGAGTG GGCAAAAGCA AGTTACGGTG
    CAGAAAGTTG
351 CTGGAAAACG CTGTATCTGA ACGGTCysCC TTTGGGCAAC
    CTGTCGCCGG
401 AATGGGTGGA ACGCGTsmmA AAAGACTGGG AGGCAGGCTG
    CyCGGAGTCT
451 TCAGACGGCA TTTTTCTGAA TgCGGACGGc TGgCctGATA
    TGGgCGGAcg
501 cTTACAGCAC CTCGCCCTCG GTTGGCACTG TGCGGGGCTG
    TTGGACGgsT
551 GGCGCAACGA GTGTTTCGAC CTGACCGACG GCGGCGGCAA
    CCCCTTGTTC
601 ACGCTCGaAc GCGCCGyTTT mCGTCCTkTC GGACTGCTCA
    GCCGCGCCGT
651 CCATCTCAAC GGTCTGACCG AATCGGACGG CCGATGGCAT
    TTCTGGATAG
701 GCAGGCGCAG TCCGCACAAA GCAGTCGATC CCAACAAACT
    CGACAATACT
751 rCCGCCGGCG GTGTTTCCGG CGGCGAAATG CCGTCTGAAG
    CCGTGTGTCG
801 CGAAAGCAGC GAAGAAGCCG GTTTGGATAA AACGCTGcTT
    CCGCTCATCC
851 GCCCGGTATC GCAGCTGCAC AGCCTGCGCT CCGTCAGCCG
    GGGTGTACAC
901 AATGAAATCC TGTATGTATT CGATGCCGTC CTGCCG...

This corresponds to the amino acid sequence <SEQ ID 410; ORF105>:

1   MVARRAHNPK VVGSNPXPAT XFQTPRFNAE XVLXLPVSCF
    LFPAASVFCR
51  IFLPAAISER QTAVCLRLQI QAVWLQSSAL SSRKPTMPTV
    RFTESVSKQD
101 LDALFEWAKA SYGAESCWKT LYLNGXPLGN LSPEWVERVX
    KDWEAGCXES
151 SDGIFLNADG WPDMGGRLQH LALGWHCAGL LDGWRNECFD
    LTDGGGNPLF
201 TLERAXXRPX GLLSRAVHLN GLTESDGRWH FWIGRRSPHK
    AVDPNKLDNT
251 XAGGVSGGEM PSEAVCRESS EEAGLDKTLL PLIRPVSQLH
    SLRSVSRGVH
301 NEILYVFDAV LP...

Further work revealed the complete nucleotide sequence <SEQ ID 411>:

1   ATGCCGACCG TCCGTTTTAC CGAATCCGTC AGCAAACAAG
    ACCTTGATGC
51  TCTGTTCGAG TGGGCAAAAG CAAGTTACGG TGCAGAAAGT
    TGCTGGAAAA
101 CGCTGTATCT GAACGGTCTG CCTTTGGGCA ACCTGTCGCC
    GGAATGGGTG
151 GAACGCGTCA AAAAAGACTG GGAGGCAGGC TGCTCGGAGT
    CTTCAGACGG
201 CATTTTTCTG AATGCGGACG GCTGGCCTGA TATGGGCGGA
    CGCTTACAGC
251 ACCTCGCCCT CGGTTGGCAC TGTGCGGGGC TGTTGGACGG
    CTGGCGCAAC
301 GAGTGTTTCG ACCTGACCGA CGGCGGCGGC AACCCCTTGT
    TCACGCTCGA
351 ACGCGCCGCT TTCCGTCCTT TCGGACTGCT CAGCCGCGCC
    GTCCATCTCA
401 ACGGTCTGAC CGAATCGGAC GGCCGATGGC ATTTCTGGAT
    AGGCAGGCGC
451 AGTCCGCACA AAGCAGTCGA TCCCAACAAA CTCGACAATA
    CTGCCGCCGG
501 CGGTGTTTCC GGCGGCGAAA TGCCGTCTGA AGCCGTGTGT
    CGCGAAAGCA
551 GCGAAGAAGC CGGTTTGGAT AAAACGCTGC TTCCGCTCAT
    CCGCCCGGTA
601 TCGCAGCTGC ACAGCCTGCG CTCCGTCAGC CGGGGTGTAC
    ACAATGAAAT
651 CCTGTATGTA TTCGATGCCG TCCTGCCCGA AACCTTCCTG
    CCTGAAAATC
701 AGGATGGCGA AGTGGCGGGT TTTGAGAAAA TGGACATCGG
    CGGTCTGTTG
751 GATGCCATGT TGTCGGGAAA CATGATGCAC GACGCGCAAC
    TGGTTACGCT
801 GGACGCGTTT TGCCGTTACG GTCTGATTGA TGCCGCCCAT
    CCGCTGTCCG
851 AGTGGCTGGA CGGCATACGT TTATAG

This corresponds to the amino acid sequence <SEQ ID 412; ORF105-1>:

1   MPTVRFTESV SKQDLDALFE WAKASYGAES CWKTLYLNGL
    PLGNLSPEWV
51  ERVKKDWEAG CSESSDGIFL NADGWPDMGG RLQHLALGWH
    CAGLLDGWRN
101 ECFDLTDGGG NPLFTLERAA FRPFGLLSRA VHLNGLTESD
    GRWHFWIGRR
151 SPHKAVDPNK LDNTAAGGVS GGEMPSEAVC RESSEEAGLD
    KTLLPLIRPV
201 SQLHSLRSVS RGVHNEILYV FDAVLPETFL PENQDGEVAG
    FEKMDIGGLL
251 DAMLSGNMMH DAQLVTLDAF CRYGLIDAAH PLSEWLDGIR
    L*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF105 shows 89.4% identity over a 226aa overlap with an ORF (ORF105a) from strain A of N. meningitidis:

The complete length ORF105a nucleotide sequence <SEQ ID 413> is:

1   ATGCCGACCG TCCGTTTTAC CGAATCCGTC AGCAAACACG
    ACCTTGATGC
51  CCTATTCGAG TGGGCAAAGG CAAGTTACGG TGCGGAAAGT
    TGCTGGAAAA
101 CGCTGTATCT GAACGGTCTG CCTTTGGGCA ATCTGTCGCC
    GGAATGGGCG
151 GAGCGCGTCA AAAAAGACTG GGAGGCAGGC TGCTCGGAGT
    CTTCAGACGG
201 CATTTTCCTG AATGCGGACG GCTGGCCAGA TATGGGCAGA
    CGCTTGCAGC
251 ACCTCGCCCG AATATGGAAA GAAGCGGGAC TGCTTCACGG
    CTGGCGCGAC
301 GAGTGTTTCG ACCTGACCGA CGGCGGCAGC AATCCCTTGT
    TCGCGCTCGA
351 ACGCGCCGCT TTCCGTCCGT TCGGACTGCT CAGCCGCGCC
    GTCCATCTCA
401 ACGGTTTGGT CGAATCGGAC GGCCGATGGC ATTTCTGGAT
    AGGCAGGCGC
451 AGTCCGCACA AAGCAGTCGA TCCCGACAAA CTCGACAATA
    CTGCCGCCGG
501 CGGTGTTTCC AGCGGTGAAT TGCCGTCTGA AACCGTGTGT
    CGCGAAAGCA
551 GCGAAGAAGC CGGTTTGGAT AAAACGCTGC TTCCGCTCAT
    CCGCCCGGTA
601 TCGCAGCTGC ACAGCCTGCG CCCCGTCAGC CGGGGTGTGC
    ACAATGAAAT
651 CCTGTATGTA TTCGATGCCG TCCTGCCCGA AACCTTCCTG
    CCTGAAAATC
701 AGGATGGCGA AGTGGCGGGT TTTGAGAAAA TGGACATCGG
    CGGTCTGTTG
751 GCTGCCATGT TGTCGGGAAA CATGATGCAC GACGCGCAAC
    TGGTTACGCT
801 GGACGCGTTT TGCCGTTACG GTCTGATTGA TGCCGCCCAT
    CCGCTGTCCG
851 AGTGGCTGGA CGGCATACGT TTATAG

This encodes a protein having amino acid sequence <SEQ ID 414>:

1   MPTVRFTESV SKHDLDALFE WAKASYGAES CWKTLYLNGL
    PLGNLSPEWA
51  ERVKKDWEAG CSESSDGIFL NADGWPDMGR RLQHLARIWK
    EAGLLHGWRD
101 ECFDLTDGGS NPLFALERAA FRPFGLLSRA VHLNGLVESD
    GRWHFWIGRR
151 SPHKAVDPDK LDNTAAGGVS SGELPSETVC RESSEEAGLD
    KTLLPLIRPV
201 SQLHSLRPVS RGVHNEILYV FDAVLPETFL PENQDGEVAG
    FEKMDIGGLL
251 AAMLSGNMMH DAQLVTLDAF CRYGLIDAAH PLSEWLDGIR
    L*

ORF105a and ORF105-1 show 93.8% identity in 291 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF105 shows 87.5% identity over a 312aa overlap with a predicted ORF (ORF105.ng) from N. gonorrhoeae:

A complete length ORF105ng nucleotide sequence <SEQ ID 415> was predicted to encode a protein having amino acid sequence <SEQ ID 416>:

1   MVARRAHNPK VVGSNPAPAT KYQTPRFNAE GVLFFLFPAA
    SVFCRIFLPA
51  AISERQAAVC LRLQIQAVWL QSSALCSRKP AMPTVRFTES
    VSKQDLDALF
101 ERAKASYGAE SCWKTLYLNR LPLGNLSPEW AERIKKDWEA
    GCSESSNGIF
151 LNADGWPDMG GRLQHLARTW NKAGLLHGWR NECFDLTDGG
    GNPLFTLERA
201 AFRPFGLLIR AVHLNGLVES NGRWHFWIGR RSPHKAVDPG
    KLDNIAGGGV
251 SGGEMPSEAV CRESSEEAGL DKTLFPLIRP VSRLHSLRPV
    SRGVHNEILY
301 VFDAVLPETF LPENQDGEVA GFEKMDIGGL LDAMLSKNMM
    HDAQLVTLDA
351 FYRYGLIDAA HPLSEWLDGI RL*

Further work revealed the complete nucleotide sequence <SEQ ID 417>:

1   ATGCCGACCG TCCGTTTTAC CGAATCCGTC AGCAAACAAG
    ACCTTGATGC
51  CCTGTTCGAG CGGGCAAAAG CAAGTTACGG TGCCGAAAGT
    TGCTGGAAAA
101 CGCTGTATCT GAACCGTCTT CCTTTGGGCA ATCTGTCGCC
    GGAATGGGCT
151 GAGCGCATCA AAAAAGACTG GGAGGCAGGC TGCTCCGAGT
    CTTCAGACGG
201 CATTTTTCTG AATGCGGACG GCTGGCCGGA TATGGGCGGA
    CGCTTGCAGC
251 ACCTCGCCCG CACATGGAAC AAGGCGGGGC TGCTTCACGG
    ATGGCGCAAC
301 GAGTGTTTCG ACCTGACCGA CGGCGGCGGC AACCCCTTGT
    TCACGCTCGA
351 ACGCGCCGCT TTCCGTCCGT TCGGACTACT CAGCCGCGCC
    GTCCATCTCA
401 ACGGTTTGGT CGAATCGAAC GGCAGATGGC ATTTTTGGAT
    AGGCAGGCGC
451 AGTCCGCACA AAGCAGTCGa tcCCGGCAAG CTCGACAATA
    TTGCCGGCGG
501 CGGTGTTTCC GGCGGCGAAA TGCCGTCTGA AGCCGTGTGC
    CGCGAAAGCA
551 GCGAAGAAGC CGGTTTGGAT AAAACGCTGT TTCCGCTCAT
    CCGCCCAGTA
601 TCGCGGCTGC ACAGCCTTCG CCCCGTCAGC CGAGGTGTGC
    ACAATGAAAT
651 CCTGTATGTG TTCGATGCCG TCCTGCCCGA AACCTTCCTG
    CCTGAAAATC
701 AGGATGGCGA GGTAGCGGGT TTTGAAAAGA TGGACATTGG
    CGGCCTATTG
751 GATGCCATGT TGTCGAAAAA CATGATGCAC GACGCGCAAC
    TGGTTACGCT
801 GGACGCGTTT TACCGTTACG GTCTGATTGA TGCCGCCCAT
    CCGCTGTCCG
851 AGTGGCTGGA CGGCATACGT TTATAG

This corresponds to the amino acid sequence <SEQ ID 418; ORF105ng-1>:

1   MPTVRFTESV SKQDLDALFE RAKASYGAES CWKTLYLNRL
    PLGNLSPEWA
51  ERIKKDWEAG CSESSDGIFL NADGWPDMGG RLQHLARTWN
    KAGLLHGWRN
101 ECFDLTDGGG NPLFTLERAA FRPFGLLSRA VHLNGLVESN
    GRWHFWIGRR
151 SPHKAVDPGK LDNIAGGGVS GGEMPSEAVC RESSEEAGLD
    KTLFPLIRPV
201 SRLHSLRPVS RGVHNEILYV FDAVLPETFL PENQDGEVAG
    FEKMDIGGLL
251 DAMLSKNMMH DAQLVTLDAF YRYGLIDAAH PLSEWLDGIR
    L*

ORG105ng-1 and ORF105-1 show 93.5% identity in 291 aa overlap:

Furthermore, ORF105ng-1 shows homology with a yeast enzyme:

sp|P41888|TNR3_SCHPO THIAMIN PYROPHOSPHOKINASE (TPK) (THIAMIN KINASE)
>gi|1076928|pir||S52350 thiamin pyrophosphokinase (EC 2.7.6.2) - fission
yeast (Schizosaccharomyces pombe) >gi|666111 (X84417) thiamin
pyrophosphokinase [Schizosaccharomyces pombe]
>gi|2330852|gnl|PID|e334056 (Z98533) thiamin
pyrophosphokinase [Schizosaccharomyces pombe] Length = 569
Score = 105 bits (259), Expect = 4e−22
Identities = 64/192 (33%), Positives = 94/192 (48%), Gaps = 3/192 (1%)
Query:	268	NKAGLLHGWRNECFDLTDGGGNPLFTLERAAFRPFGLLSRAVHLNGLVESNGRW--HFWI	441
		N  G+   WRNE + +      P+  +ER  F  FG LS  VH    + +        W+
Sbjct:	96	NTFGIADQWRNELYTVYGKSKKPVLAVERGGFWLFGFLSTGVHCTMYIPATKEHPLRIWV	155
Query:	442	GRRSPHKAVDPGKLDNIAGGGVSGGEMPSEAVCRESSEEAGLDKTLFPLIRPVSRLHSLR	621
		RRSP K   P  LDN   GG++ G+     + +E SEEA LD +   LI P   +  ++
Sbjct:	156	PRRSPTKQTWPNYLDNSVAGGIAHGDSVIGTMIKEFSEEANLDVSSMNLI-PCGTVSYIK	214
Query:	622	PVSRG-VHNEILYVFDAVLPETFLPENQDGEVAGFEKMDIGGLLDAMLSKNMMHDAQLVT	798
		R  +  E+ YVFD  + +  +P   DGEVAGF  + +  +L  +  K+   +  LV
Sbjct:	215	MEKRHWIQPELQYVFDLPVDDLVIPRINDGEVAGFSLLPLNQVLHELELKSFKPNCALVL	274
Query:	799	LDAFYRYGLIDAAHP	843
		LD   R+G+I   HP
Sbjct:	275	LDFLIRHGIITPQHP	289

Based on this analysis, including the presence of a putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 49

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 419>:

1   ATGAATAGAC CCAAGCAACC CTTCTTCCGT CCCGAAGTCG
    CCGTTGCCCG
51  CCAAACCAGC CTGACGGGTA AAGTGATTCT GACACGACCG
    TTGTCATTTT
101 CCCTATGGAC GACATTTGCA TCGATATCTG CGTTATTGAT
    TATCCTGTTT
151 TTGATATTTG GTAACTATAC GCGAAAGACA ACAGTGGAGG
    GACAAATTTT
201 ACCTGCATCG GGCGTAATCA GGGTGTATGC ACCGgATACG
    rGkACAATTA
251 CAGCGAAATT CGTGGAAGAT GGmsAAAAGG TTAAGGCTGG
    CGACAAGCTA
301 TTTGCGCTTT CGACCTCACG TTTCGGCGCA GGAGGTAGCG
    TGCAGCAGCA
351 GTTGAAAACG GAGGCAGTTT TGAAGAAAAC GTTGGCAGAA
    CAGGAACTGG
401 GTCGTCTGAA GCTGATACAC GGGAATGAAA CGCGCAgCcT
    TAAAGCAACT
451 GTCGAACGTT TGGAAAACCA GGAACTCCAT ATTTCGCAAC
    AGATAGACGG
501 TCAGAAAAGG CGCATTAGAC TTGCGGAAGA AATGTTGCAG
    AAATATCGTT
551 TCCTATCCGC .CAATGA

This corresponds to the amino acid sequence <SEQ ID 420; ORF107>:

1   MNRPKQPFFR PEVAVARQTS LTGKVILTRP LSFSLWTTFA
    SISALLIILF
51  LIFGNYTRKT TVEGQILPAS GVIRVYAPDT XTITAKFVED
    GXKVKAGDKL
101 FALSTSRFGA GGSVQQQLKT EAVLKKTLAE QELGRLKLIH
    GNETRSLKAT
151 VERLENQELH ISQQIDGQKR RIRLAEEMLQ KYRFLSXQ*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF107 shows 97.8% identity over a 186aa overlap with an ORF (ORF107a) from strain A of N. meningitidis:

The complete length ORF107a nucleotide sequence <SEQ ID 421> is:

1   ATGAATAGAC CCAAGCAACC NTTCTTCCGT CCCGAAGTCG
    CCGTTGCCCG
51  CCAAACCAGC CTGACGGGTA AAGTGATTCT GACACGACCG
    TTGTCATTTT
101 CCCTATGGAC GACATTTGCA TCGATATCTG CGTTATTGAT
    TATCCTGTTT
151 TTGATATTTG GTAACTATAC GCGAAAGACA ACAGTGGAGG
    GACAAATTTT
201 ACCTGCATCG GGCGTAATCA GGGTGTATGC ACCGGATACG
    GGGACAATTA
251 CNGCGAAATT CNTGGAAGAT GGAGAAAAGG TTAAGGCTGG
    CGACAAGCTA
301 TTTGCGCTTT CGACCTCACG TTTCGGCGCA GGAGATAGCG
    TGCAGCAGCA
351 GTTGAAAACG GAGGCAGTTT TGAAGAAAAC GTTGGCAGAA
    CAGGAACTGG
401 GTCGTCTGAA GCTGATACAC GGGAATGAAA CGCGCAGCCT
    TAAAGCAACT
451 GTCGAACGTT TGGAAAACCA GGAACTCCAT ATTTCGCAAC
    AGATAGACGG
501 TCAGAAAAGG CGCATTAGAC TTGCGGAAGA AATGTTGCAG
    AAATATCGTT
551 TCCTATCCGC CAATGATGCA GTGCCAAAAC AAGAAATGAT
    GAATGTCAAG
601 GCAGAGCTTT TAGAGCAGAA AGCCAAACTT GATGCCTACC
    GCCGAGAAGA
651 AGTCGGGCTG CTTCAGGAAA TCCGCACGCA GAATCTGACA
    TTGGNNAGCC
701 TCCCCCAAGC GGCATGA

This encodes a protein having amino acid sequence <SEQ ID 422>:

1   MNRPKQPFFR PEVAVARQTS LTGKVILTRP LSFSLWTTFA
    SISALLIILF
51  LIFGNYTRKT TVEGQILPAS GVIRVYAPDT GTITAKFXED
    GEKVKAGDKL
101 FALSTSRFGA GDSVQQQLKT EAVLKKTLAE QELGRLKLIH
    GNETRSLKAT
151 VERLENQELH ISQQIDGQKR RIRLAEEMLQ KYRFLSANDA
    VPKQEMMNVK
201 AELLEQKAKL DAYRREEVGL LQEIRTQNLT LXSLPQAA*

Homology with a Predicted ORF from N. gonorrhoeae

ORF107 shows 95.7% identity over a 188aa overlap with a predicted ORF (ORF107.ng) from N. gonorrhoeae:

The complete length ORF107ng nucleotide sequence <SEQ ID 423> is predicted to encode a protein having amino acid sequence <SEQ ID 424>:

1   MNRPKQPFFR PEVAIARQTS LTGKVILTRP LSFSLWTTFA
    SISALLIILF
51  LIFGNYTRKT TMEGQILPAS GVIRVYAPDT GTITAKFVED
    GEKVKAGDKL
101 FALSTSRFGA GGSVQQQLKT EAVLKKTLAE QELGRLKLIH
    ENETRSLKAT
151 VERLENQKLH ISQQIDGQKR RIRLAEEMLR KYRFLSAQ*

Based on the presence of a putative transmembrane domain in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 50

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 425>:

1   ATGCTGAATA CTTTTTTTGC CGTATTGGGC GGCTGCCTGC
    TGCT.TTGCC
51  GTGCGGCAAA TCCGTAAATA CGGCGGTACA GCCGCAAAAC
    GCGGTACAAA
101 GCGCGCCGAA ACCGGTTTTC AAAGTCATAT ATATCGACAA
    TACGGCGATT
151 GCCGGTTTGG ATTTGGGACA AAGCAGCGAA GGCAAAACCA
    ACGACGGCAA
201 AAAACAAATC AGTTATCCGA TTAAAGGCTT GCCGGAACAA
    AATGTTATCC
251 GACTGATCGG CAAGCATCCC GGCGACTTGG AAGCCGTCAG
    CGGCAAATGT
301 ATGGAAACCG ATGATAAGGA CAGTCCGGCA GGTTGGGCAG
    AAAACGGCGT
351 GTGCCATACC TTGTTTGCCA AACTGGTGGG CAATATCGCC
    GAAGACGGCG
401 GCAAACTGAC GGATTACCTA GTTTCGCATG CCGCCCTGCA
    ACCCTATCAG
451 GCAGGCAAAA GCGGCTATGC CGCCGTGCAG AACGGACGCT
    ATGTGCTGGA
501 AATCGACAGC GAAGGGGCGT TTTATTTCCG CCGCCGCCAT
    TATTGA

This corresponds to the amino acid sequence <SEQ ID 426; ORF108>:

1   MLNTFFAVLG GCLLXLPCGK SVNTAVQPQN AVQSAPKPVF
    KVIYIDNTAI
51  AGLDLGQSSE GKTNDGKKQI SYPIKGLPEQ NVIRLIGKHP
    GDLEAVSGKC
101 METDDKDSPA GWAENGVCHT LFAKLVGNIA EDGGKLTDYL
    VSHAALQPYQ
151 AGKSGYAAVQ NGRYVLEIDS EGAFYFRRRH Y*

Further work revealed the following DNA sequence <SEQ ID 427>:

1   ATGCTGAAAA CATCTTTTGC CGTATTGGGC GGCTGCCTGC
    TGCTTGCCGC
51  CTGCGGCAAA TCCGAAAATA CGGCGGAACA GCCGCAAAAC
    GCGGTACAAA
101 GCGCGCCGAA ACCGGTTTTC AAAGTCAAAT ATATCGACAA
    TACGGCGATT
151 GCCGGTTTGG ATTTGGGACA AAGCAGCGAA GGCAAAACCA
    ACGACGGCAA
201 AAAACAAATC AGTTATCCGA TTAAAGGCTT GCCGGAACAA
    AATGTTATCC
251 GACTGATCGG CAAGCATCCC GGCGACTTGG AAGCCGTCAG
    CGGCAAATGT
301 ATGGAAACCG ATGATAAGGA CAGTCCGGCA GGTTGGGCAG
    AAAACGGCGT
351 GTGCCATACC TTGTTTGCCA AACTGGTGGG CAATATCGCC
    GAAGACGGCG
401 GCAAACTGAC GGATTACCTA GTTTCGCATG CCGCCCTGCA
    ACCCTATCAG
451 GCAGGCAAAA GCGGCTATGC CGCCGTGCAG AACGGACGCT
    ATGTGCTGGA
501 AATCGACAGC GAAGGGGCGT TTTATTTCCG CCGCCGCCAT
    TATTGA

This corresponds to the amino acid sequence <SEQ ID 428; ORF108-1>:

1   MLKTSFAVLG GCLLLAACGK SENTAEQPQN AVQSAPKPVF
    KVKYIDNTAI
51  AGLDLGQSSE GKTNDGKKQI SYPIKGLPEQ NVIRLIGKHP
    GDLEAVSGKC
101 METDDKDSPA GWAENGVCHT LFAKLVGNIA EDGGKLTDYL
    VSHAALQPYQ
151 AGKSGYAAVQ NGRYVLEIDS EGAFYFRRRH Y*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. gonorrhoeae

ORF108 shows 88.4% identity over a 181 aa overlap with a predicted ORF (ORF 108.ng) from N. gonorrhoeae:

ORF108-1 shows 92.3% identity with ORF108ng over the same 181 aa overlap:

The complete length ORF108ng nucleotide sequence <SEQ ID 429> is:

1   ATGCTGAAAa tacctTTTGC CGTGTtgggc ggCtgcctGC
    TGCTTGCCGC
51  CTGCGGCAAA TCCGAAAATa cggcggaACA GCCGCAAAAT
    gcggCACAAA
101 GCGCGCCGAA ACCGGTTTTC AAAGTCAAAT ACATCGACAA
    TACGGCGATT
151 GCCGGTTTGG CTTTGGGACA AAGTAGCGAA GGCAAAACCA
    acgacgGCAA
201 AAAACAAATC AGTTATccgA TTAAAGGCTT GCCGGAACAA
    Aacgccgtcc
251 gGCTGACCGG AAAGCATCCC AACGACTTGG AagccgtcgT
    CGGCAAATGT
301 ATGGAAACCG ACGGAAAGGA CGCGCCTTCG GGCTGGGCGG
    AAAACGGCGT
351 GTGCCATACC TTGTTTGCCA AACTGGTGGG CAATATCGCC
    GAAGACGGCG
401 GCAAACTGAC TGATTACCTG ATTTCGCATT CCGCCCTGCA
    ACCCTATCAG
451 GCAGGCAAAA GCGGCTATGC CGCCGTGCAG AACGGACGCT
    ATGTGCTGGA
501 AATCGACAGC GagggGGCGT TTTATttccg ccgccgccat
    tattgA

This encodes a protein having amino acid sequence <SEQ ID 430>:

1   MLKIPFAVLG GCLLLAACGK SENTAEQPQN AAQSAPKPVF
    KVKYIDNTAI
51  AGLALGQSSE GKTNDGKKQI SYPIKGLPEQ NAVRLTGKHP
    NDLEAVVGKC
101 METDGKDAPS GWAENGVCHT LFAKLVGNIA EDGGKLTDYL
    ISHSALQPYQ
151 AGKSGYAAVQ NGRYVLEIDS EGAFYFRRRH Y*

Based on this analysis, including the presence of a predicted prokaryotic membrane lipoprotein lipid attachment site (underlined) and a putative ATP/GTP-binding site motif A (P-loop, double-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 51

The following DNA sequence was identified in N. meningitidis <SEQ ID 431>:

1   ATGGAAGATT TATATATAAT ACTCGCTTTG GGTTTGGTTG
    CGATGATTGC
51  CGgATTTATC GATgcgatTg cGggCGGGGG TGGTTTGATT
    ACGCTGCCCG
101 CACTCTTGTT GGCAGGTATT CCTCCCGTGT CGGCAATTGC
    CACCAACAAG
151 CTGCAAgCAG CCGCTGCTAC GTTTTCAGCT ACGGTTTCTT
    TTGCACGCAA
201 AGGTTTGATT GATTGGAAGA AAGGTCTCCC GATTGCCGCA
    GCATCGTTTG
251 TAGGCGGCGT GGcCGGTGCA TTATCGGTCA GCTTGGTTTC
    CAAAGATATT
301 CTgCTgGCGG TCGTGCCGGT TTTGTTGATA TTTGTCGCAC
    TGTATTTTGT
351 GTTTTCGCCC AAGCTCGACG GCAGTAAGGA AGGCAAAGCC
    AGAATGTCTT
401 TTTTTCTGTT cGGGCTGACG GTCGC.ACCG CTTTTGGGTT
    TTTACGACGG
451 TGTGTTCGGA CCGGGTGTCG GCTCGTTTTT TCTGATTGCC
    TTTATTGTTT
501 TGCTCGGCTG CAAgCTGTTG AACGCGATGT CTTACACCAA
    ATTGGCGAAC
551 GTTGCCTGCA ATCTTGGTTC GCTATCGGTA TTCCTGCTGC
    ACGGTTCGAT
601 TATTTTCCCG ATTGCGGCAA CGaTGGCGGT CGGTGCGTTT
    GTCGGtGCGA
651 ATTTAgGTGC GAGATTTGCC GTaCgctTCG GTTCGAAGCT
    GATTAA

This corresponds to the amino acid sequence <SEQ ID 432; ORF109>:

1   MEDLYIILAL GLVAMIAGFI DAIAGGGGLI TLPALLLAGI
    PPVSAIATNK
51  LQAAAATFSA TVSFARKGLI DWKKGLPIAA ASFVGGVAGA
    LSVSLVSKDI
101 LLAVVPVLLI FVALYFVFSP KLDGSKEGKA RMSFFLFGLT
    VXTAFGFLRR
151 CVRTGCRLVF SDCLYCFARL QAVERDVLHQ IGERCLQSWF
    AIGIPAARFD
201 YFPDCGNDGG RCVCRCEFRC EICRTLRFEA D*

Further work revealed the following DNA sequence <SEQ ID 433>:

1   ATGGAAGATT TATATATAAT ACTCGCTTTG GGTTTGGTTG
    CGATGATTGC
51  CGGATTTATC GATGCGATTG CGGGCGGGGG TGGTTTGATT
    ACGCTGCCCG
101 CACTCTTGTT GGCAGGTATT CCTCCCGTGT CGGCAATTGC
    CACCAACAAG
151 CTGCAAGCAG CCGCTGCTAC GTTTTCAGCT ACGGTTTCTT
    TTGCACGCAA
201 AGGTTTGATT GATTGGAAGA AAGGTCTCCC GATTGCCGCA
    GCATCGTTTG
251 TAGGCGGCGT GGCCGGTGCA TTATCGGTCA GCTTGGTTTC
    CAAAGATATT
301 CTGCTGGCGG TCGTGCCGGT TTTGTTGATA TTTGTCGCAC
    TGTATTTTGT
351 GTTTTCGCCC AAGCTCGACG GCAGTAAGGA AGGCAAAGCC
    AGAATGTCTT
401 TTTTTCTGTT CGGGCTGACG GTCGCACCGC TTTTGGGTTT
    TTACGACGGT
451 GTGTTCGGAC CGGGTGTCGG CTCGTTTTTT CTGATTGCCT
    TTATTGTTTT
501 GCTCGGCTGC AAGCTGTTGA ACGCGATGTC TTACACCAAA
    TTGGCGAACG
551 TTGCCTGCAA TCTTGGTTCG CTATCGGTAT TCCTGCTGCA
    CGGTTCGATT
601 ATTTTCCCGA TTGCGGCAAC GATGGCGGTC GGTGCGTTTG
    TCGGTGCGAA
651 TTTAGGTGCG AGATTTGCCG TCCGCTTCGG TTCGAAGCTG
    ATTAAGCCGC
701 TGCTGATTGT CATCAGCATT TCGATGGCTG TGAAATTGTT
    GATAGACGAG
751 AGAAATCCGC TGTATCAGAT GATTGTTTCG ATGTTTTAA

This corresponds to the amino acid sequence <SEQ ID 434; ORF109-1>:

1   MEDLYIILAL GLVAMIAGFI DAIAGGGGLI TLPALLLAGI
    PPVSAIATNK
51  LQAAAATFSA TVSFARKGLI DWKKGLPIAA ASFVGGVAGA
    LSVSLVSKDI
101 LLAVVPVLLI FVALYFVFSP KLDGSKEGKA RMSFFLFGLT
    VAPLLGFYDG
151 VFGPGVGSFF LIAFIVLLGC KLLNAMSYTK LANVACNLGS
    LSVFLLHGSI
201 IFPIAATMAV GAFVGANLGA RFAVRFGSKL IKPLLIVISI
    SMAVKLLIDE
251 RNPLYQMIVS MF*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF109 shows 95.9% identity over a 147aa overlap with an ORF (ORF109a) from strain A of N. meningitidis:

The complete length ORF109a nucleotide sequence <SEQ ID 435> is:

1   ATGGAAGATT TATACATAAT ACTCGCTTTG GGTTTGGTTG
    CGATGATTGC
51  CGGATTTATC GATGCGATTG CGGGTGGGGG TGGTTTGATT
    ACGCTGCCTG
101 CACTCTTGTT GGCAGGTATT CCTCCCGTGT CGGCAATTGC
    CACCAACAAG
151 CTGCAAGCAG CCGCTGCTAC GTTTTCGGCT ACGGTTTCTT
    TTGCACGCAA
201 AGGTTTGATT GATTGGAAGA AAGGTCTCCC GATTGCGGCA
    GCATCGTTTG
251 CAGGCGGCGT GGTCGGTGCA TTATCGGTCA GCTTGGTTTC
    CAAAGATATT
301 CTGCTGGCGG TCGTGCCGGT TTTGTTGATA TTTGTCGCGC
    TGTATTTTGT
351 GTTTTCGCCC AAGCTCGACG GCAGTAAGGA AGGCAAAGCC
    AGAATGTCTT
401 TTTTTCTGTT CGGTCTGACG GTTGCACCAC TTTTGGGTTT
    TTACGACGGT
451 GTGTTCGGAC CGGGTGTCGG CTCGTTTTTT CTGATTGCCT
    TTATTGTTTT
501 GCTCGGCTGC AAGCTGTTGA ACGCGATGTC TTACACCAAA
    TTGGCGAACG
551 TTGCCTGCAA TCTTGGTTCG CTATCGGTAT TCCTGCTGCA
    CGGTTCGATT
601 ATTTTCCCGA TTGCGGCAAC GATGGCGGTC GGTGCGTTTG
    TCGGTGCGAA
651 TTTAGGTGCG AGATTTGCCG TCCGCTTCGG TTCGAAGCTG
    ATTAAGCCGC
701 TGCTGATTGT CATCAGCATT TCGATGGCTG TGAAATTGTT
    GATAGACGAG
751 AGAAATCCGC TGTATCAGAT GATTGTTTCG ATGTTTTAA

This encodes a protein having amino acid sequence <SEQ ID 436>:

1   MEDLYIILAL GLVAMIAGFI DAIAGGGGLI TLPALLLAGI
    PPVSAIATNK
51  LQAAAATFSA TVSFARKGLI DWKKGLPIAA ASFAGGVVGA
    LSVSLVSKDI
101 LLAVVPVLLI FVALYFVFSP KLDGSKEGKA RMSFFLFGLT
    VAPLLGFYDG
151 VFGPGVGSFF LIAFIVLLGC KLNAMSYTK LANVACNLGS
    LSVFLLHGSI
201 IFPIAATMAV GAFVGANLGA RFAVRFGSKL IKPLLIVISI
    SMAVKLLIDE
251 RNPLYQMIVS MF*

ORF109a and ORF109-1 show 99.2% identity in 262 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF109 shows 98.3% identity over a 231aa overlap with a predicted ORF (ORF109.ng) from N. gonorrhoeae:

An ORF109ng nucleotide sequence <SEQ ID 437> was predicted to encode a protein having amino acid sequence <SEQ ID 438>:

1   MEDLYIILAL GLVAMIAGFI DAIAGGGGLI TLPALLLAGI
    PPVSAIATNK
51  LQAAAATFSA TVSFARKGLI DWKKGLPIAA ASFAGGVVGA
    LSVSLVSKDI
101 LLAVVPVLLI FVALYFVFSP KLDGSKEGKA RMSFFLFGLT
    VATAFGFLRR
151 CVRTGCRLVF SDCLYCFARL QAVERDVLHQ IGERCLQSWF
    AIGIPAARFD
201 YFPDCGNDGG RCVCRCEFRC EICRPLRFEA D*

Further work revealed the following gonococcal DNA sequence <SEQ ID 439>:

1   ATGGAAGATT TATACATAAT ACTCGCTTTG GGTTTGGTTG
    CGATGATCGC
51  CGGATTTATC GATGCGATTG CGGGCGGGGG TGGTTTGATT
    ACGCTGCCTG
101 CACTCTTGTT GGCAGGTATT CCTCCCGTGT CGGCAATTGC
    CACCAACAAG
151 CTGCAAGCAG CCGCTGCTAC GTTTTCGGCT ACGGTTTCTT
    TTGCACGCAA
201 AGGTTTGATT GATTGGAAGA AAGGTCTCCC GATTGCCGCA
    GCATCGTTTG
251 CAGGCGGCGT GGTCGGTGCA TTATCGGTCA GCTTGGTTTC
    CAAAGATATT
301 TTGCTGGCGG TCGTGCCGGT TTTGTTGATA TTTGTCGCGC
    TGTATTTTGT
351 GTTTTCGCCC AAGCTCGACG GCAGTAAGGA AGGCAAAGCC
    AGAATGTCTT
401 TTTTTCTATT CGGGCTGACG GTTGCACCGC TTTTGGGTTT
    TTACGACGGT
451 GTGTTCGGAC CGGGTGTCGG CTCGTTTTTT CTGATTGCCT
    TTATTGTTTT
501 GCTCGGCTGC AAGCTGTTGA ACGCGATGTC TTACACCAAA
    TTGGCGAACG
551 TTGCTTGCAA TCTTGGTTCG CTATCGGTAT TCCTGCTGCA
    CGGTTCGATT
601 ATTTTCCCGA TTGTGGCAAC GATGGCGGTC GGTGCGTTTG
    TCGGTGCGAA
651 TTTAGGTGCG AGATTTGCCG TCCGCTTCGG TTCGAAGCTG
    ATTAAGCCGC
701 TGCTGATTGT CATCAGCATT TCGATGGCTG TGAAATTGTT
    GATAGACGAG
751 AGAAATCCGC TGTATCAGAT GATTGTTTCG ATGTTTTAA

This corresponds to the amino acid sequence <SEQ ID 440; ORF109ng-1>:

1   MEDLYIILAL GLVAMIAGFI DAIAGGGGLI TLPALLLAGI
    PPVSAIATNK
51  LQAAAATFSA TVSFARKGLI DWKKGLPIAA ASFAGGVVGA
    LSVSLVSKDI
101 LLAVVPVLLI FVALYFVFSP KLDGSKEGKA RMSFFLFGLT
    VAPLLGFYDG
151 VFGPGVGSFF LIAFIVLLGC KLLNAMSYTK LANVACNLGS
    LSVFLLHGSI
201 IFPIVATMAV GAFVGANLGA RFAVRFGSKL IKPLLIVISI
    SMAVKLLIDE
251 RNPLYQMIVS MF*

ORF109ng-1 and ORF109-1 show 98.9% identity in 262 aa overlap:

In addition, ORF109ng-1 shows homology to a hypothetical Pseudomonas protein:

sp|P29942|YCB9_PSEDE HYPOTHETICAL 27.4 KD PROTEIN IN COBO 3′REGION (ORF9)
>gi|94984|pir||I38164 hypothetical protein 9 - Pseudomonas sp >gi|551929
(M62866) ORF9 [Pseudomonas denitrificans] Length = 261
Score = 175 bits (439), Expect = 3e−43
Identities = 83/214 (38%), Positives = 131/214 (60%), Gaps = 1/214 (0%)
Query:	41	PPVSAIATNKLQXXXXXXXXXXXXXRKGLIDWKKGLPIXXXXXXXXXXXXXXXXXXXKDI	100
		PP+  + TNKLQ             R+G ++ K+ LP+                    D+
Sbjct:	43	PPLQTLGTNKLQGLFGSGSATLSYARRGHVNLKEQLPMALMSAAGAVLGALLATIVPGDV	102
Query:	101	LLAVVPVLLIFVALYFVFSPKLDGSKEGKARMSFFLFGLTVAPLLGFYDGVFGPGVGSFF	160
		L A++P LLI +ALYF   P + G  +  +R++ F+F LT+ PL+GFYDGVFGPG GSFF
Sbjct:	103	LKAILPFLLIAIALYFGLKPNM-GDVDQHSRVTPFVFTLTLVPLIGFYDGVFGPGTGSFF	161
Query:	161	LIAFIVLLGCKLLNAMSYTKLANVACNLGSLSVFLLHGSIIFPIVATMAVGAFVGANLGA	220
		++ F+ L G  +L A ++TK  N   N+G+  VFL  G++++ +   M +G F+GA +G+
Sbjct:	162	MLGFVTLAGFGVLKATAHTKFLNFGSNVGAFGVFLFFGAVLWKVGLLMGLGQFLGAQVGS	221
Query:	221	RFAVRFGSKLIKPLLIVISISMAVKLLIDERNPL	254
		R+A+  G+K+IKPLL+++SI++A++LL D  +PL
Sbjct:	222	RYAMAKGAKIIKPLLVIVSIALAIRLLADPTHPL	255

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 52

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 441>:

1   ..CTGCTAGGGT ATTGCATCGG TTATCGGTAC GGCTGTTGCA
    GCAAAACCAG
51  CCGCAGACGG ATTATTTGGT CAAATTCGGA TCGTTTTGGG
    CGAG.ATTTT
101 TGGTTTTCTG GGACTGTATG ACGTCTATGC TTCGGCATGG
    TTTGTCGTTA
151 TCATGATGTT TTTGGTGGTT TCTACCAGTT TGTGCCTGAT
    TCGCAATGTG
201 CCGCCGTTCT GGCGCGAAAT GAAGTCTTTT CGGGAAAAGG
    TTAAAGAAAA
251 ATCTCTGGCG GCGATGCGCC ATTCTTCGCT GTTGGATGTA
    AAAATTGCGC
301 CCGAGGTTGC CAAACGTTAT CTGGAAGTAC AAGGTTTTCA
    GGGGAAAACC
351 ATTAACCGTG AAGACGGGTC GGTTCTGATT GCCGCCAAAA
    AAGGCACAAT
401 GAACAAATGG GGCTATATCT TTGCCCATGT TGCTTTGATT
    GTCATTTGCC
451 TGGGCGGGTT GATAGACAGT AACCTGCTGT TGAAACTGGG
    TATGCTGACC
501 GGTCGGATTG TTCCGGACAA TCAGGCGGTT TATGCCAAGG
    ATTTC.AAGC
551 CCGAAAGTAT .TTTGGGTGC gTCCAATCTC TCATTTAGGG
    GCAACGTCAA
601 TATTTCCG.A GGGGCAGAgT GCGGATGTGG TTTTCCTGA

This corresponds to the amino acid sequence <SEQ ID 442; ORF110>:

1   ..LLGIASVIGT LLQQNQPQTD YLVKFGSFWA XIFGFLGLYD
    VYASAWFVVI
51  MMFLVVSTSL CLIRNVPPFW REMKSFREKV KEKSLAAMRH
    SSLLDVKIAP
101 EVAKRYLEVQ GFQGKTINRE DGSVLIAAKK GTMNKWGYIF
    AHVALIVICL
151 GGLIDSNLLL KLGMLTGRIF RTIRRFMPRI XKPESXFGCV
    QSLI*GQRQY
201 FXRGRVRMWF S*

Computer analysis of this amino acid sequence gave the following results:

Homology with ORF88a from N. meningitidis (Strain A)

ORF110 shows 91.5% identity over a 188aa overlap with ORF88a from strain A of N. meningitidis:

However, ORF88 and ORF110 do not align, because they represent two different fragments of the same protein.

Homology with a Predicted ORF from N. gonorrhoeae

ORF110 shows 88.6% identity over a 211 as overlap with a predicted ORF (ORF110.ng) from N. gonorrhoeae:

The complete length ORF110ng nucleotide sequence <SEQ ID 443> is predicted to encode a protein having amino acid sequence <SEQ ID 444>:

1   MSKSRISPTL LSRPWFAFFS SMRFAVALLS LLGIASVIGT
    VLQQNQPQTD
51  YLVKFGPFWT RIFDFLGLYD VYASAWFVVI MMFLVVSTSL
    CLIRNVPPFW
101 REMKSFREKV KEKSLAAMRH SSLLDVKIAP EVAKRYLEVR
    GFQGKTVSRE
151 DGSVLIAAKK GTMNKWGYIX AHVALIVICL GRLINXNLLL
    KLGMLAGSIF
201 RNNRRVMPRI SKPESIWGGV QSLIKGQRQY FQRGKVRMWF
    S*

Based on the putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 53

The following DNA sequence was identified in N. meningitidis <SEQ ID 445>:

1    ATGCCGTCTG AAACACGCCT GCCGAACTTT ATCCGCGTCT
     TGATATTTGC
51   CCTGGGTTTC ATCTTCCTGA ACGCCTGTTC GGAACAAACC
     GCGCAAACCG
101  TTACCCTGCA AGGCGAAACG ATGGGCACGA CCTATACCGT
     CAAATACCTT
151  TCAAATAATC GGGACAAACT CCCCTCACCT GCCGAAATAC
     AAAAACGCAT
201  CGATGACGCG CTTAAAGAAG TCAACCGGCA GATGTCCACC
     TATCAGCCCG
251  ACTCCGAAAT CAGCCGGTTC AACCAACACA CAGCCGGCAA
     GCCCCTCCGC
301  ATTTCAAGCG ACTTCGCACA CGTTACTGCC GAAGCCGTCC
     GCCTGAACCG
351  CCTGACACAC GGCGCGCTGG ACGTAACCGT CGGCCCCTTG
     GTCAACCTTT
401  GGGGATTCGG CCCCGACAAA TCCGTTACCC GTGAACCGTC
     GCCGGAACAA
451  ATCAAACAGG CGGCATCTTA TACGGGCATA GACAAAATCA
     TTTTGAAACA
501  AGGCAAAGAT TACGCTTCCT TGAGCAAAAC CCACCCCAAG
     GCCTATTTGG
551  ATTTATCTTC GATTGCCAAA GGCTTCGGCG TTGATAAAGT
     TGCGGGCGAA
601  CTGGAAAAAT ACGGCATTCA AAATTATCTG GTCGAAATCG
     GCGGCGAGTT
651  GCACGGCAAA GGCAAAAACG CGCGCGGCGA ACCGTGGCGC
     ATCGGTATCG
701  AGCAGCCCAA TATCGTCCAA GGCGGCAATA CGCAGATTAT
     CGTCCCGCTG
751  AACAACCGTT CGCTTGCCAC TTCCGGCGAT TACCGTATTT
     TCCACGTCGA
801  TAAAAACGGC AAACGCCTCT CCCATATCAT CAACCCGAAC
     AACAAACGAC
851  CCATCAGCCA CAACCTCGCC TCCATCAGCG TGGTCGCAGA
     CAGTGCGATG
901  ACGGCGGACG GCTTGTCCAC AGGATTATTC GTATTGGGCG
     AAACCGAAGC
951  CTTAAAGCTG GCAGAGCGCG AAAAACTCGC TGTTTTCCTG
     ATTGTCAGGG
1001 ATAAAGGCGG CTACCGCACC GCCATGTCTT CCGAATTTGA
     AAAACTGCTC
1051 CGCTAA

This corresponds to the amino acid sequence <SEQ ID 446; ORF111>:

1   MPSETRLPNF IRVLIFALGF IFLNACSEQT AQTVTLQGET
    MGTTYTVKYL
51  SNNRDKLPSP AEIQKRIDDA LKEVNRQMST YQPDSEISRF
    NQHTAGKPLR
101 ISSDFAHVTA EAVRLNRLTH GALDVTVGPL VNLWGFGPDK
    SVTREPSPEQ
151 IKQAASYTGI DKIILKQGKD YASLSKTHPK AYLDLSSIAK
    GFGVDKVAGE
201 LEKYGIQNYL VEIGGELHGK GKNARGEPWR IGIEQPNIVQ
    GGNTQIIVPL
251 NNRSLATSGD YRIFHVDKNG KRLSHIINPN NKRPISHNLA
    SISVVADSAM
301 TADGLSTGLF VLGETEALKL AEREKLAVFL IVRDKGGYRT
    AMSSEFEKLL
351 R*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF111 shows 96.9% identity over a 351aa overlap with an ORF (ORF111a) from strain A of N. meningitidis:

The complete length ORF111a nucleotide sequence <SEQ ID 447> is:

1    ATGCCGTCTG AAACACGCCT GCCGAACTTT ATCCGCACCT
     TGATATTTGC
51   CCTGAGTTTT ATCTTCCTGA ACGCCTGTTC GGAACAAACC
     GCGCAAACCG
101  TTACCCTGCA AGGTGAAACG ATGGGCACGA CCTATACCGT
     CAAATACCTT
151  TCAAATAATC GGGACNAACT CCCNTCACCT GCCGAAATAC
     AAAANCGCAT
201  CGATGACGCG CTTAAAGAAG TCAACCGGCA GATGTCCACC
     TATCAGCCCG
251  ACTCCGAAAT CAGCCGGTTC AACCAACACA CAGCCGGCAA
     GCCCCTCCGC
301  ATTTCAAGCG ACTTCGCACA CGTTACTGCC GAAGCCGTCC
     ACCTGAACCG
351  CCTGACACAC GGCGCGCTGG ACGTAACCGT CGGCCCCTTG
     GTCAACCTTT
401  GGGGATTCGG CCCCGACAAA TCCGTTACCC GTGAACCGTC
     GCCGGAACAA
451  ATCAAACAAG CAGCATCTTA TACGGGCATA GACAAAATCA
     TTTTGAAACA
501  AGGCAAAGAT TACGCTTCCT TGAGCAAAAC CCACCCCAAG
     GCCTATTTGG
551  ATTTATCTTC GATTGCCAAA GGCTTCGGCG TTGATNANGT
     TGCGGGCGAA
601  CTGGAAAAAT ACGGCATTCA AAATTATCTG GTCGAAATCG
     GCGGNGAGTT
651  GCACGGCAAA GNCAAAAACG CGCGCGGCGA ACCTTGGCGC
     ATCGGCATCG
701  AACAGCCCAA CATCGTCCAA GGCGGCAATA CGCAGATTAT
     CGTCCCGCTG
751  AACAACCGTT CGNTTGCCAC TTCCGGCGAT TACCGTATTT
     TCCACGTCGA
801  TAAAAGCGGC AAACGCCTCT CCCATATCAT TAATCCGAAC
     AACAAACGAC
851  CCATCAGCCA CAACCTCGCC TCCATCAGCG TGNTCGCAGA
     CAGTGCGATG
901  ACGGCGGACG GCTTNTCCAC AGGATTATTC GTATTGGGCG
     AAACCGAAGC
951  CTTAAAGCTG GCAGAGCGCG AAAAACTCGC TGTTTTCCTG
     ATTGTCAGGG
1001 ATAAAGGCGG CTACCGCACC GCCATGTCTT CCGAATTTGA
     AAAACTGCTC
1051 CGCTAA

This encodes a protein having amino acid sequence <SEQ ID 448>:

1   MPSETRLPNF IRTLIFALSF IFLNACSEQT AQTVTLQGET
    MGTTYTVKYL
51  SNNRDXLPSP AEIQXRIDDA LKEVNRQMST YQPDSEISRF
    NQHTAGKPLR
101 ISSDFAHVTA EAVHLNRLTH GALDVTVGPL VNLWGFGPDK
    SVTREPSPEQ
151 IKQAASYTGI DKIILKQGKD YASLSKTHPK AYLDLSSIAK
    GFGVDXVAGE
201 LEKYGIQNYL VEIGGELHGK XKNARGEPWR IGIEQPNIVQ
    GGNTQIIVPL
251 NNRSXATSGD YRIFHVDKSG KRLSHIINPN NKRPISHNLA
    SISVXADSAM
301 TADGXSTGLF VLGETEALKL AEREKLAVFL IVRDKGGYRT
    AMSSEFEKLL
351 R*

Homology with a Predicted ORF from N. gonorrhoeae

ORF111 shows 96.6% identity over a 351aa overlap with a predicted ORF (ORF111.ng) from N. gonorrhoeae:

The complete length ORF111ng nucleotide sequence <SEQ ID 449> is:

1    ATGCCGTCTG AAACACGCCT GCCGAACCTT ATCCGCGCCT
     TGATATTTGC
51   CCTGGGTTTC ATCTTCCTGA ACGCCTGTTC GGaacaaacC
     GCGCAaaccg
101  TTACCCTGCA AGGCGAAAcg aTGGGTACGA CCTATACCGT
     CAAATACCTT
151  TCAAATAATC GGGACAAACT CCCCTCCCCT GCCAAAATAC
     AAAAGCGCAT
201  TGATGATGCG CTTAAAGAAG TCAACCGGCA GATGTCCACC
     TACCAGACCG
251  ATTCCGAAAT CAGCCGGTTC AACCAACACA CAGCCGGCAA
     GCCCCTCCGC
301  ATTTCAAGCG ATTTCGCACA CGTTACCGCC GAAGCCGTCC
     GCCTGAACCG
351  CCTGACTCAC GGCGCACTGG ACGTAACCGT CGGCCCTTTG
     GTCAACCTTT
401  GGGGGTTCGG CCCCGACAAA TCCGTTACCC GTGAACCGTC
     GCCGGAACAA
451  ATCAAACAGG CGGCATCTTA TACGGGCATA GACAAAATCA
     TTTTGCAACA
501  AGGCAAAGAT TACGCTTCCT TGAGCAAAAC CCACCCCAAA
     GCCTATTTGG
551  ATTTATCTTC GATTGCCAAA GGCTTCGGCG TTGATAAAGT
     TGCGGGCGAA
601  CTGGAAAAAT ACGGCATTCA AAATTATCTG GTCGAAAtcg
     gcggcGAGTT
651  GCACGGCAAA GGCAAAAATG CGCACGGCGA ACCGTGGCGC
     ATCGGTATAG
701  AGCAACCCAA TATCATCCAA GgcgGCAata CGCAGATTAt
     cgtcccgctg
751  aaCaaccgtt cgctTGCCAC TTCCGGCGAT TAccgtaTTT
     tccacgtcgA
801  TAAAAAcggc aaacgccttt cccacaTCAT CAATCCCaAC
     aacAAACgac
851  ccATCAGcca caacctcgcc tccatcagcg tggtctcAGA
     CAGTGCAATG
901  ACGGCGGACG GTTtatCCAC AGGATTATTT GTTTTAGGCG
     AAACCGAAGC
951  CTTAAGGCTG GCAGAACAAG AAAAACTCGC TGTTTTCCTA
     ATTGTCCGGG
1001 ATAAGGACGG CTACCGCACC GCCATGTCTT CCGAATTTGC
     CAAGCTGCTC
1051 CGCTAA

This encodes a protein having amino acid sequence <SEQ ID 450>:

1   MPSETRLPNL IRALIFALGF IFLNACSEQT AQTVTLQGET
    MGTTYTVKYL
51  SNNRDKLPSP AKIQKRIDDA LKEVNRQMST YQTDSEISRF
    NQHTAGKPLR
101 ISSDFAHVTA EAVRLNRLTH GALDVTVGPL VNLWGFGPDK
    SVTREPSPEQ
151 IKQAASYTGI DKIILQQGKD YASLSKTHPK AYLDLSSIAK
    GFGVDKVAGE
201 LEKYGIQNYL VEIGGELHGK GKNAHGEPWR IGIEQPNIIQ
    GGNTQIIVPL
251 NNRSLATSGD YRIFHVDKNG KRLSHIINPN NKRPISHNLA
    SISVVSDSAM
301 TADGLSTGLF VLGETEALRL AEQEKLAVFL IVRDKDGYRT
    AMSSEFAKLL
351 R*

This protein shows homology with a hypothetical lipoprotein precursor from H. influenzae:

sp|P44550|YOJL_HAEIN HYPOTHETICAL LIPOPROTEIN HI0172 PRECURSOR >gi|1074292|pir|4
hypothetical protein HI0172 - Haemophilus influenzae (strain Rd KW20)
>gi|1573128 (U32702) hypothetical [Haemophilus influenzae] Length = 346
Score = 353 bits (896), Expect = 9e−97
Identities = 181/344 (52%), Positives = 247/344 (71%), Gaps = 4/344 (1%)
Query:	7	LPNLIRALIFALGFIFLNACSEQTAQTVTLQGETMGTTYTVKYLSNNRDKLPSPAKIQKR	66
		+  LI  +I     + L AC ++T + ++L G+TMGTTY VKYL +      S  K  +
Sbjct:	1	MKKLISGIIAVAMALSLAACQKET-KVISLSGKTMGTTYHVKYLDDGSITATSE-KTHEE	58
Query:	67	IDDALKEVNRQMSTYQTDSEISRFNQHT-AGKPLRISSDFAHVTAEAVRLNRLTHGALDV	125
		I+  LK+VN +MSTY+ DSE+SRFNQ+T    P+ IS+DFA V AEA+RLN++T GALDV
Sbjct:	59	IEAILKDVNAKMSTYKKDSELSRFNQNTQVNTPIEISADFAKVLAEAIRLNKVTEGALDV	118
Query:	126	TVGPLVNLWGFGPDKSVTREPSPEQIKQAASYTGIDKIILQQGKDYASLSKTHPKAYLDL	185
		TVGP+VNLWGFGP+K   ++P+PEQ+ +  ++ GIDKI L   K+ A+LSK  P+ Y+DL
Sbjct:	119	TVGPVVNLWGFGPEKRPEKQPTPEQLAERQAWVGIDKITLDTNKEKATLSKALPQVYVDL	178
Query:	186	SSIAKGFGVDKVAGELEKYGIQNYLVEIGGELHGKGKNAHGEPWRIGIEQPNIIQGGNTQ	245
		SSIAKGFGVD+VA +LE+   QNY+VEIGGE+  KGKN  G+PW+I IE+P        +
Sbjct:	179	SSIAKGFGVDQVAEKLEQLNAQNYMVEIGGEIRAKGKNIEGKPWQIAIEKPTTTGERAVE	238
Query:	246	IIVPLNNRSLATSGDYRIFHVDKNGKRLSHIINPNNKRPISHNLASISVVSDSAMTADGL	305
		++ LNN  +A+SGDYRI+  ++NGKR +H I+P    PI H+LASI+V++ ++MTADGL
Sbjct:	239	AVIGLNNMGMASSGDYRIY-FEENGKRFAHEIDPKTGYPIQHHLASITVLAPTSMTADGL	297
Query:	306	STGLFVLGETEALRLAEQEKLAVFLIVRDKDGYRTAMSSEFAKL	349
		STGLFVLGE +AL +AE+  LAV+LI+R  +G+ T  SS F KL
Sbjct:	298	STGLFVLGEDKALEVAEKNNLAVYLIIRTDNGFVTKSSSAFKKL	341

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 54

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 451>:

1   ..CCGTGCCGCC GACAGGGCGA CGACGTGTAT GCGGCGCACG
    CGTCCCGTCA
51  AAAATTGTGG CTGCGCTTCA TCGGCGGCCG GTCGCATCAA
    AATATACGGG
101 GCGGCGCGGC TGCGGACGGG TGGCGCAAAG GCGTGCAAAT
    CGGCGGCGAG
151 GTGTTTGTAC GGCAAAATGA AGGCAGCCkA yTGGCAATCG
    GCGTGATGGG
201 CGGCAGGGCC GGCCAGCACG CwTCAGTCAA CGGCAAAGGC
    GGTGCGGCAG
251 gCAGTGATTT GTATGGTTAT GgCGGGGgTG TTTATGCTgC
    GTGGCATCAG
301 TTGCGCGATA AACAAACGGG TgCGTATTTG GACGGCTGGT
    TGCAATACCA
351 ACGTTTCAAA CACCGCATCA ATGATGAAAA CCGTGCGGAA
    CgCTACAAAA
401 CCAAAGGTTG GACGGCTTCT GTCGAAGGCG GCTACAACGC
    GCTTGTGGCG
451 GAAGGCATTG TCGGAAAAGG CAATAATGTG CGGTTTTACC
    TACAACCGCA
501 GgCGCAGTTT ACCTACTTGG GCGTAAACGG CGGCTTTACC
    GACAGCGAGG
551 GGACGGCGGT CGGACTGCTC GGCAGCGGTC AGTGGCAAAG
    CCGCGCCGGC
601 AtTCGGGCAA AAACCCGTTT TGCTTTGCGT AACGGTGTCA
    ATCTTCAGCC
651 TTTTGCCGCT TTTAATGTtt TGCACAGGTC AAAATCTTTC
    GGCGTGGAAA
701 TGGACGGCGA AAAACAGACG CTGGCAGGCA GGACGGCACT
    CGAAGGGCGG
751 TTCGGTATTG AAGCCGGTTG GAAAGGCCAT ATGTCCGCA..

This corresponds to the amino acid sequence <SEQ ID 452; ORF35>:

1   ..PCRRQGDDVY AAHASRQKLW LRFIGGRSHQ NIRGGAAADG
    WRKGVQIGGE
51  VFVRQNEGSX LAIGVMGGRA GQHASVNGKG GAAGSDLYGY
    GGGVYAAWHQ
101 LRDKQTGAYL DGWLQYQRFK HRINDENRAE RYKTKGWTAS
    VEGGYNALVA
151 EGIVGKGNNV RFYLQPQAQF TYLGVNGGFT DSEGTAVGLL
    GSGQWQSRAG
201 IRAKTRFALR NGVNLQPFAA FNVLHRSKSF GVEMDGEKQT
    LAGRTALEGR
251 FGIEAGWKGH MSA..

Computer analysis of this amino acid sequence gave the following results:

Homology with Putative Secreted VirG-Homolgue of N. meningitidis (Accession Number A32247)

ORF and virg-h protein show 51% aa identity in 261aa overlap:

Orf35	5	QGDDVYAAHASRQKLWLRFIGGRSHQNIRGGAA-ADGWRKGVQIGGEVFVRQNEGSXLAI	63
		+  D++     R+ LWLR I G S+Q ++G  A  +G+RKGVQ+GGEVF  QNE + L+I
virg-h	396	KNSDIFDRTLPRKGLWLRVIDGHSNQWVQGKTAPVEGYRKGVQLGGEVFTWQNESNQLSI	455
Orf35	64	GVMGGRAGQHASVNGKG--GAAGSDLYGYGGGVYAAWHQLRDKQTGAYLDGWLQYQRFKH	121
		G+MGG+A Q ++ +          ++ G+G GVYA WHQL+DKQTGAY D W+QYQRF+H
virg-h	456	GLMGGQAEQRSTFHNPDTDNLTTGNVKGFGAGVYATWHQLQDKQTGAYADSWMQYQRFRH	515
Orf35	122	RINDENRAERYKTKGWTASVEGGYNALVAEGIVGKGNNVRFYLQPQAQFTYLGVNGGFTD	181
		RIN E+  ER+ +KG TAS+E GYNAL+AE    KGN++R YLQPQAQ TYLGVNG F+D
virg-h	516	RINTEDGTERFTSKGITASIEAGYNALLAEHFTKKGNSLRVYLQPQAQLTYLGVNGKFSD	575
Orf35	182	SEGTAVGLLGSGQWQSRAGIRAKTRFALRNGVNLQPFAAFNVLHRSKSFGVEMDGEKQTL	241
		SE   V LLGS Q Q+R G++AK +F+L   + ++PFAA N L+ +K FGVEMDGE++ +
virg-h	576	SENAHVNLLGSRQLQTRVGVQAKAQFSLYKNIAIEPFAAVNALYHNKPFGVEMDGERRVI	635
Orf35	242	AGRTALEGRFGIEAGWKGHMS	262
		+TA+E + G+    K H++
virg-h	636	NNKTAIESQLGVAVKIKSHLT	656

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF35 shows 96.9% identity over a 259aa overlap with an ORF (ORF35a) from strain A of N. meningitidis:

The complete length ORF35a nucleotide sequence <SEQ ID 453> is:

1    ATGTTCAGAG CTCAGCTTGG TTCAAATACT CGTTCTACCA
     AAATCGGCGA
51   CGATGCCGAT TTTTCATTTT CAGACAAGCC GAAACCCGGC
     ACTTCCCATT
101  ATTTTTCCAG CGGTAAAACC GATCAAAATT CATCCGAATA
     TGGGTATGAC
151  GAAATCAATA TCCAAGGTAA AAACTACAAT AGCGGCATAC
     TCGCCGTCGA
201  TAATATGCCC GTTGTTAAGA AATATATTAC AGATACTTAC
     GGGGATAATT
251  TAAAGGATGC GGTTAAGAAG CAATTACAGG ATTTATACAA
     AACAAGACCC
301  GAAGCTTGGG AAGAAAATAA AAAACGGACT GAGGAGGCGT
     ATATAGAACA
351  GCTTGGACCA AAATTTAGTA TACTCAAACA GAAAAACCCC
     GATTTAATTA
401  ATAAATTGGT AGAAGATTCC GTACTCACTC CTCATAGTAA
     TACATCACAG
451  ACTAGTCTCA ACAACATCTT CAATAAAAAA TTACACGTCA
     AAATCGAAAA
501  CAAATCCCAC GTCGCCGGAC AGGTGTTGGA ACTGACCAAG
     ATGACGCTGA
551  AAGATTCCCT TTGGGAACCG CGCCGCCATT CCGACATCCA
     TATGCTGGAA
601  ACTTCCGATA ATGCCCGCAT CCGCCTGAAC ACGAAAGATG
     AAAAACTGAC
651  CGTCCATAAA GCGTATCAGG GCGGTGCGGA TTTCCTGTTC
     GGCTACGACG
701  TGCGGGAGTC GGACAAACCC GCCCTGACCT TTGAAGAAAA
     AGTCAGCGGA
751  CAATCCGGCG TGGTTTTGGA ACGCCGGCCG GAAAATCTGA
     AAACGCTCGA
801  CGGGCGCAAA CTGATTGCGG CGGAAAAGGC AGACTCTAAT
     TCGTTTGCGT
851  TTAAACAAAA TTACCGGCAG GGACTGTACG AATTATTGCT
     CAAGCAATGC
901  GAAGGCGGAT TTTGCTTGGG CGTGCAGCGT TTGGCTATCC
     CCGAGGCGGA
951  AGCGGTTTTA TATGCCCAAC AGGCTTATGC GGCAAATACT
     TTGTTCGGGC
1001 TGCGTGCCGC CGACAGGGGC GACGACGTGT ATGCCGCCGA
     TCCGTCCCGT
1051 CAAAAATTGT GGCTGCGCTT CATCGGCGGC CGGTCGCATC
     AAAATATACG
1101 GGGCGGCGCG GCTGCGGACG GGCGGCGCAA AGGCGTGCAA
     ATCGGCGGCG
1151 AGGTGTTTGT ACGGCAAAAT GAAGGCAGCC GGCTGGCAAT
     CGGCGTGATG
1201 GGCGGCAGGG CTGGCCAGCA CGCATCAGTC AACGGCAAAG
     GCGGTGCGGC
1251 AGGCAGTTAT TTGCATGGTT ATGGCGGGGG TGTTTATGCT
     GCGTGGCATC
1301 AGTTGCGCGA TAAACAAACG GGTGCGTATT TGGACGGCTG
     GTTGCAATAC
1351 CAACGTTTCA AACACCGCAT CAATGATGAA AACCGTGCGG
     AACGCTACAA
1401 AACCAAAGGT TGGACGGCTT CTGTCGAAGG CGGCTACAAC
     GCGCTTGTGG
1451 CGGAAGGCGT TGTCGGAAAA GGCAATAATG TGCGGTTTTA
     CCTGCAACCG
1501 CAGGCGCAGT TTACCTACTT GGGCGTAAAC GGCGGCTTTA
     CCGACAGCGA
1551 GGGGACGGCG GTCGGACTGC TCGGCAGCGG TCAGTGGCAA
     AGCCGCGCCG
1601 GCATTCGGGC AAAAACCCGT TTTGCTTTGC GTAACGGTGT
     CAATCTTCAG
1651 CCTTTTGCCG CTTTTAATGT TTTGCACAGG TCAAAATCTT
     TCGGCGTGGA
1701 AATGGACGGC GAAAAACAGA CGCTGGCAGG CAGGACGGCG
     CTCGAAGGGC
1751 GGTTCGGCAT TGAAGCCGGT TGGAAAGGCC ATATGTCCGC
     ACGCATCGGA
1801 TACGGCAAAA GGACGGACGG CGACAAAGAA GCCGCATTGT
     CGCTCAAATG
1851 GCTGTTTTGA

This encodes a protein having amino acid sequence <SEQ ID 454>:

1   MFRAQLGSNT RSTKIGDDAD FSFSDKPKPG TSHYFSSGKT
    DQNSSEYGYD
51  EINIQGKNYN SGILAVDNMP VVKKYITDTY GDNLKDAVKK
    QLQDLYKTRP
101 EAWEENKKRT EEAYIEQLGP KFSILKQKNP DLINKLVEDS
    VLTPHSNTSQ
151 TSLNNIFNKK LHVKIENKSH VAGQVLELTK MTLKDSLWEP
    RRHSDIHMLE
201 TSDNARIRLN TKDEKLTVHK AYQGGADFLF GYDVRESDKP
    ALTFEEKVSG
251 QSGVVLERRP ENLKTLDGRK LIAAEKADSN SFAFKQNYRQ
    GLYELLLKQC
301 EGGFCLGVQR LAIPEAEAVL YAQQAYAANT LFGLRAADRG
    DDVYAADPSR
351 QKLWLRFIGG RSHQNIRGGA AADGRRKGVQ IGGEVFVRQN
    EGSRLAIGVM
401 GGRAGQHASV NGKGGAAGSY LHGYGGGVYA AWHQLRDKQT
    GAYLDGWLQY
451 QRFKHRINDE NRAERYKTKG WTASVEGGYN ALVAEGVVGK
    GNNVRFYLQP
501 QAQFTYLGVN GGFTDSEGTA VGLLGSGQWQ SRAGIRAKTR
    FALRNGVNLQ
551 PFAAFNVLHR SKSFGVEMDG EKQTLAGRTA LEGRFGIEAG
    WKGHMSARIG
601 YGKRTDGDKE AALSLKWLF*

Homology with a Predicted ORF from N. gonorrhoeae

ORF35 shows 51.7% identity over a 261aa overlap with a predicted ORF (ORF35ngh) from N. gonorrhoeae:

A partial ORF35ngh nucleotide sequence <SEQ ID 455> is predicted to encode a protein having partial amino acid sequence <SEQ ID 456>:

1   ..KKLRDRNSEY WKEETYHIKS NGRTYPNIPA LFPKHPFDPF
    ENINNSKKIS
51  FYDKEYTEDY LVGFARGFGV EKRNGEEEKP LRQYFKDCVN
    TENSNNDNCK
101 ISSFGNYGPI LIKSDIFALA SQIKNSHINS EILSVGNYIE
    WLRPTLNKLT
151 GWQEHLYAGL DPFHYIEVTD NSHVIGQTID LGALELTNSL
    WKPRWNSNID
201 YLITKNAEIR FNTKNESLLV KEDYAGGARF RFAYDLKDKV
    PEIPVLTFEK
251 NITGTSDIIF EGKALDNLKH LDGHQIVKVN DTADKDAFRL
    SSKYRKGIYT
301 LSLQQRPEGF FTKVQERDDI AIYAQQAQAA NTLFALRLND
    KNSDIFDRTL
351 PRKGLWLRVI DGHSNQWVQG KTAPVEGYRK GVQLGGEVFT
    WQNESNQLSI
401 GLMGGQAEQR STFRNPDTDN LTTGNVKGFG AGVYATWHQL
    QDKQTGAYVD
451 SWMQYQRFRH RINTEYATER FTSKGITASI EAGYNALLAE
    HFTKKGNSLR
501 VYLQPQAQLT YLGVNGKFSD SENAQVNLLG SRQLQSRVGV
    QAKAQFAFTN
551 GVTFQPFVAV NSIYQQKPFG VEIDGDRRVI NNKTVIETQL
    GVAAKIKSHL
601 TLQASFNRQT SKHHHAKQGA LNLQWTF*

Based on this prediction, these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 55

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 457>:

1   ..GCGGAATATG TTCAGTTCTC TATAGATTTG TTCAGTGTGG
    GTAAATCGGG
51  GGGCGGTATA CCTAAGGCTA AGCCTGTGTT TGATGCGAAA
    CCGAGATGGG
101 AGGTTGATAG GAAGCTTAAT AAATTGACAA CTCGTGAGCA
    GGTGGAGAAA
151 AATGTTCAGG AAACGAGAAG AAGGAGTCAG AGTAGTCAGT
    TTAAAGCCCA
201 TGCGCAACGA GAATGGGAAA ATAAAACAGG GTTAGATTTT
    AATCATTTTA
251 TAGGTGGTGA TATCAATAAA AAAGGCACAG TAACAGGAGG
    GCATAGTCTA
301 ACCCGTGGTG ATGTACGGGT GATACAACAA ACCTCGGCAC
    CTGATAAACA
351 TGGGGT.TTA TCAAGCGACA GTGGAAATTN A

This corresponds to the amino acid sequence <SEQ ID 458; ORF46>:

1   ..AEYVQFSIDL FSVGKSGGGI PKAKPVFDAK PRWEVDRKLN
    KLTTREQVEK
51  NVQETRRRSQ SSQFKAHAQR EWENKTGLDF NHFIGGDINK
    KGTVTGGHSL
101 TRGDVRVIQQ TSAPDKHGXL SSDSGNX

Further work revealed further partial nucleotide sequence <SEQ ID 459>:

1   ..GCAGTGTGCC TnCCGATGCA TGCACACGCC TCAnATTTGG
    CAAACGATTC
51  TTTTATCCGG CAGGTTCTCG ACCGTCAGCA TTTCGAACCC
    GACGGGAAAT
101 ACCACCTATT CGGCAGCAGG GGGGAACTTG CCGAGCGCCA
    GTCTCATATC
151 GGATTGGGAA AAATACAAAG CCATCAGTTG GGCAACCTGA
    TGATTCAACA
201 GGCGGCCATT AAAGGAAATA TCGGCTACAT TGTCCGCTTT
    TCCGATCACG
251 GGCACGAAGT CCATTCCCCs TTCGACAACC ATGCCTCACA
    TTCCGATTCT
301 GATGAAGCCG GTAGTCCCGT TGACGGATTT AGCCTTTACC
    GCATCCATTG
351 GGACGGATAC GAACACCATC CCGCCGACGG CTATGACGGG
    CCACAGGGCG
401 GCGGCTATCC CGCTCCCAAA GGCGCGAGGG ATATATACAG
    TTACGACATA
451 AAAGGCGTTG CCCAAAATAT CCGCCTCAAC CTGACCGACA
    ACCGCAGCAC
501 CGGACAACGG CTTGCCGACC GTTTCCACAA TGCCGGTAGT
    ATGCTGACGC
551 AAGGAGTAGG CGACGGATTC AAACGCGCCA CCCGATACAG
    CCCCGAGCTG
601 GACAGATCGG GCAATGCCGC CGAAGCCTTC AACGGCACTG
    CAGATATCGT
651 TAAAAACATC ATCGGCGCTG CAGGAGAAAT TGT

This corresponds to the amino acid sequence <SEQ ID 460; ORF46-1>:

1   ..AVCLPMHAHA SXLANDSFIR QVLDRQHFEP DGKYHLFGSR
    GELAERQSHI
51  GLGKIQSHQL GNLMIQQAAI KGNIGYIVRF SDHGHEVHSP
    FDNHASHSDS
101 DEAGSPVDGF SLYRIHWDGY EHHPADGYDG PQGGGYPAPK
    GARDIYSYDI
151 KGVAQNIRLN LTDNRSTGQR LADRFHNAGS MLTQGVGDGF
    KRATRYSPEL
201 DRSGNAAEAF NGTADIVKNI IGAAGEI

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. gonorrhoeae

ORF46 shows 98.2% identity over, a 111 aa overlap with a predicted ORF (ORF46ng) from N. gonorrhoeae:

A partial ORF46ng nucleotide sequence <SEQ ID 461> is predicted to encode a protein having partial amino acid sequence <SEQ ID 462>:

1   ..RRLKHCCHAR LGSAFHRKQD GAHQRFGRYG ATQRLCRSSH
    PRLGSPKPQC
51  RTRHRSRQQY LYGSHPHQRD WSCPGKIQLG RHHGTSCRAV
    ADXRDRICER
101 EIRRQRQXCR CRLGKIPSLS IPKYPLKLEQ RYGKENITSS
    TVPPSNGKNV
151 KLADQRHPKT GVPFDGKGFP NFEKHVKYDT KLDIQELSGG
    GIPKAKPVFD
201 AKPRWEVDRK LNKLTTREQV EKNVQETRRR SQSSQFKAHA
    QREWENKTGL
251 DFNHFIGGDI NKKGAVTGGH SLTRGDVRVI QQTSAPDKHG
    VLSSDSGN*

Further work revealed the complete gonococcal DNA sequence <SEQ ID 463>:

1    TTGGGCATTT CCCGCAAAAT ATCCCTTATT CTGTCCATAC
     TGGCAGTGTG
51   CCTGCCGATG CATGCACACG CCTCAGATTT GGcaAACGAT
     CCCTTTATCC
101  GgCaggttcT CGaccGTCAG CATTTCGaac ccgacggGAa
     ATACCaCCTA
151  TTcggCaGCA GGGGGGAGCT TgccnagcGC aacggccATa
     tcggattggG
201  aaacaTAcaa Agccatcagt tGggccacct gatgattcaa
     caggcggccg
251  ttgaaggaaA TAtcgGctac attgtccgct tttccgatca
     cgggcacaaa
301  ttccattcgc ccttcGAcaa ccaTGCCTCA CATTCCGATT
     CTGACGAAGC
351  CGGTAGTCCC GTTGACGGAT TCAGCCTTTA CCGCATCCAT
     TGGGACGGAT
401  ACGAACACCA TCCCGCCGAC GGCTATGACG GGCCACAGGG
     CGGCGGCTAT
451  CCCGCTCCCA AAGGCGCGAG GGATATATAC AGCTACGACA
     TAAAAGGCGT
501  TGCCCAAAAT ATCCGCCTCA ACCTGACCGA CAACCGCAGC
     ACCGGACAAC
551  GGCTTGCCGA CCGTTTCCAC AATGCCGGCG CTATGCTGAC
     GCAAGGAGTA
601  GGCGACGGAT TCAAACGCGC CACCCGATAC AGCCCCGAGC
     TGGACAGATC
651  GGGCAATGCc gccGAAGCCT TCAACGGCAC TGCAGATATC
     GTCAAAAACA
701  TCATCGGCGC GGCAGGAGAA ATTGTCGGCG CAGGCGATGC
     CGTGCagGGT
751  ATAAGCGAAG GCTCAAACAT TGCTGTCATG CACGGCTTGG
     GTCTGCTTTC
801  CACCGAAAAC AAGATGGCGC GCATCAACGA TTTGGCAGAT
     ATGGCGCAAC
851  TCAAAGACTA TGCCGCAGCA GCCATCCGCG ATTGGGCAGT
     CCAAAACCCC
901  AATGCCGCAC AAGGCATAGA AGCCGTCAGC AATATCTTTA
     TGGCAGCCAT
951  CCCCATCAAA GGGATTGGAG CTGTCCGGGG AAAATACGGC
     TTGGGCGGCA
1001 TCACGGCACA TCCTGTCAAG CGGTCGCAGA TGGGCGCGAT
     CGCATTGCCG
1051 AAAGGGAAAT CCGCCGTCAG CGACAATTTT GCCGATGCGG
     CATACGCCAA
1101 ATACCCGTCC CCTTACCATT CCCGAAATAT CCGTTCAAAC
     TTGGAGCAGC
1151 GTTACGGCAA AGAAAACATC ACCTCCTCAA CCGTGCCGCC
     GTCAAACGGC
1201 AAAAATGTCA AACTGGCAGA CCAACGCCAC CCGAAGACAG
     GCGTACCGTT
1251 TGACGGTAAA GGGTTTCCGA ATTTTGAGAA GCACGTGAAA
     TATGATACGA
1301 AGCTCGATAT TCAAGAATTA TCGGGGGGCG GTATACCTAA
     GGCTAAGCCT
1351 GTGTTTGATG CGAAACCGAG ATGGGAGGTT GATAGGAAGC
     TTAATAAATT
1401 GACAACTCGT GAGCAGGTGG AGAAAAATGT TCAGGAAACG
     AGAAGAAGGA
1451 GTCAGAGTAG TCAGTTTAAA GCCCATGCGC AACGAGAATG
     GGAAAATAAA
1501 ACAGGGTTAG ATTTTAATCA TTTTATAGGT GGTGATATCA
     ATAAGAAAGG
1551 CACAGTAACA GGAGGGCATA GTCTAACCCG TGGTGATGTA
     CGGGTGATAC
1601 AACAAACCTC GGCACCTGAT AAACATGGGG TTTATCAAGC
     GACAGTGGAA
1651 ATTAAAAAGC CTGATGGAAG TTGGGAGGTG AAAACGAAAA
     AAGGTGGGAA
1701 AGTGATGACC AAGCACACCA TGTTCCCAAA AGATTGGGAT
     GAGGCTAGAA
1751 TTAGGGCTGA AGTTACTTCG GCTTGGGAAA GTAGAATAAT
     GCTTAAGGAT
1801 AATAAATGGC AGGGTACAAG TAAATCGGGT ATTAAAATAG
     AAGGATTTAC
1851 CGAACCTAAT AGAACAGCAT ATCCCATTTA TGAATAG

This corresponds to the amino acid sequence <SEQ ID 464; ORF46ng-1>:

1   LGISRKISLI LSILAVCLPM HAHASDLAND PFIRQVLDRQ
    HFEPDGKYHL
51  FGSRGELAXR NGHIGLGNIQ SHQLGHLMIQ QAAVEGNIGY
    IVRFSDHGHK
101 FHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD
    GYDGPQGGGY
151 PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLADRFH
    NAGAMLTQGV
201 GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE
    IVGAGDAVQG
251 ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA
    AIRDWAVQNP
301 NAAQGIEAVS NIFMAAIPIK GIGAVRGKYG LGGITAHPVK
    RSQMGAIALP
351 KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI
    TSSTVPPSNG
401 KNVKLADQRH PKTGVPFDGK GFPNFEKHVK YDTKLDIQEL
    SGGGIPKAKP
451 VFDAKPRWEV DRKLNKLTTR EQVEKNVQET RRRSQSSQFK
    AHAQREWENK
501 TGLDFNHFIG GDINKKGTVT GGHSLTRGDV RVIQQTSAPD
    KHGVYQATVE
551 IKKPDGSWEV KTKKGGKVMT KHTMFPKDWD EARIRAEVTS
    AWESRIMLKD
601 NKWQGTSKSG IKIEGFTEPN RTAYPIYE*

ORF46ng-1 and ORF46-1 show 94.7% identity in 227 aa overlap:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF46ng-1 shows 87.4% identity over a 486aa overlap with an ORF (ORF46a) from strain A of N. meningitidis:

The complete length ORF46a DNA sequence <SEQ ID 465> is:

1    TTGGGCATTT CCCGCAAAAT ATCCCTTATT CTGTCCATAC
     TGGCAGTGTG
51   CCTGCCGATG CATGCACACG CCTCAGATTT GGCAAACGAT
     TCTTTTATCC
101  GGCAGGTTCT CGACCGTCAG CATTTCGAAC CCGACGGGAA
     ATACCACCTA
151  TTCGGCAGCA GGGGGGAACT TGCCGAGCGC AGCGGTCATA
     TCGGATTGGG
201  AAACATACAA AGCCATCAGT TGGGCAACCT GTTCATCCAG
     CAGGCGGCCA
251  TTAAAGGAAA TATCGGCTAC ATTGTCCGCT TTTCCGATCA
     CGGGCACGAA
301  GTCCATTCCC CCTTCGACAA CCATGCCTCA CATTCCGATT
     CTGATGAAGC
351  CGGTAGTCCC GTTGACGGAT TCAGCCTTTA CCGCATCCAT
     TGGGACGGAT
401  ACGAACACCA TCCCGCCGAC GGCTATGACG GGCCACAGGG
     CGGCGGCTAT
451  CCCGCTCCCA AAGGCGCGAG GGATATATAC AGCTACGACA
     TAAAAGGCGT
501  TGCCCAAAAT ATCCGCCTCA ACCTGACCGA CAACCGCAGC
     ACCGGACAAC
551  GGCTTGTCGA CCGTTTCCAC AATACCGGTA GTATGCTGAC
     GCAAGGAGTA
601  GGCGACGGAT TCAAACGCGC CACCCGATAC AGCCCCGAGC
     TGGACAGATC
651  GGGCAATGCC GCCGAAGCTT TCAACGGCAC TGCAGATATC
     GTCAAAAACA
701  TCATCGGCGC GGCAGGAGAA ATTGTCGGCG CAGGCGATGC
     CGTGCAGGGT
751  ATAAGCGAAG GCTCAAACAT TGCTGTTATG CACGGCTTGG
     GTCTGCTTTC
801  CACCGAAAAC AAGATGGCGC GCATCAACGA TTTGGCAGAT
     ATGGCGCAAC
851  TCAAAGACTA TGCCGCAGCA GCCATCCGCG ATTGGGCAGT
     CCAAAACCCC
901  AATGCCGCAC AAGGCATAGA AGCCGTCAGC AATATCTTTA
     CGGCAGTCAT
951  CCCCGTCAAA GGGATTGGAG CTGTTCGGGG AAAATACGGC
     TTGGGCGGCA
1001 TCACGGCACA TCCTGTCAAG CGGTCGCAGA TGGGCGAGAT
     CGCATTGCCG
1051 AAAGGGAAAT CCGCCGTCAG CGACAATTTT GCCGATGCGG
     CATACGCCAA
1101 ATACCCGTCC CCTTACCATT CCCGAAATAT CCGTTCAAAC
     TTGGAGCAGC
1151 GTTACGGCAA AGAAAACATC ACCTCCTCAA CCGTGCCGCC
     GTCAAACGGA
1201 AAGAATGTGA AACTGGCAAA CAAACGCCAC CCGAAGACCA
     AAGTGCCGTT
1251 TGACGGTAAA GGGTTTCCGA ATTTTGAAAA AGACGTAAAA
     TACGATACGA
1301 GAATTAATAC CGCTGTACCA CAAGTGAATC CTATAGATGA
     ACCCGTCTTT
1351 AATCCTAAAG GTTCTGTCGG ATCGGCTCAT TCTTGGTCTA
     TAACTGCCAG
1401 AATTCAATAC GCAAAATTAC CAAGGCAAGG TAGAATCAGA
     TATATCCCAC
1451 CTAAAAATTA CTCTCCTTCA GCACCGCTAC CAAAAGGACC
     TAATAATGGA
1501 TATTTGGATA AATTTGGTAA TGAATGGACT AAAGGTCCAT
     CAAGAACTAA
1551 AGGTCAAGAA TTTGAATGGG ATGTTCAATT GTCTAAAACA
     GGAAGAGAGC
1601 AACTTGGATG GGCTAGTAGG GATGGTAAGC ATTTAAATAT
     ATCAATTGAT
1651 GGAAAGATTA CACACAAATG A

This corresponds to the amino acid sequence <SEQ ID 466>:

1   LGISRKISLI LSILAVCLPM HAHASDLAND SFIRQVLDRQ
    HFEPDGKYHL
51  FGSRGELAER SGHIGLGNIQ SHQLGNLFIQ QAAIKGNIGY
    IVRFSDHGHE
101 VHSPFDNHAS HSDSDEAGSP VDGFSLYRIH WDGYEHHPAD
    GYDGPQGGGY
151 PAPKGARDIY SYDIKGVAQN IRLNLTDNRS TGQRLVDRFH
    NTGSMLTQGV
201 GDGFKRATRY SPELDRSGNA AEAFNGTADI VKNIIGAAGE
    IVGAGDAVQG
251 ISEGSNIAVM HGLGLLSTEN KMARINDLAD MAQLKDYAAA
    AIRDWAVQNP
301 NAAQGIEAVS NIFTAVIPVK GIGAVRGKYG LGGITAHPVK
    RSQMGEIALP
351 KGKSAVSDNF ADAAYAKYPS PYHSRNIRSN LEQRYGKENI
    TSSTVPPSNG
401 KNVKLANKRH PKTKVPFDGK GFPNFEKDVK YDTRINTAVP
    QVNPIDEPVF
451 NPKGSVGSAH SWSITARIQY AKLPRQGRIR YIPPKNYSPS
    APLPKGPNNG
501 YLDKFGNEWT KGPSRTKGQE FEWDVQLSKT GREQLGWASR
    DGKHLNISID
551 GKITHK*

Based on this analysis, including the presence of a RGD sequence in the gonococcal protein, typical of adhesins, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 56

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 467>:

1   ATGAATATTC ACACCCTGCT CTCCAAACAA TGGACGCTGC
    CGCCATTCCT
51  GCCGAAACGG CTGCTGCTGT CCCTGCTGAT ACTGCTTGCC
    CCCAATGCGG
101 TGTTTTGGGT TTTGGCACTG CTGACCGCCA CCGCCCGCCC
    GATTGTCAAT
151 TTGGACTATC TTCCCGCCGC GCTGCTGATC GCCCTGCCTT
    GGCGTTTCGT
201 CAAAATTGCC GGCGTATTGG CGTTTTGGCT GGCGGTTTTG
    TTTGACGGGC
251 TGATGATGGT GATCCAACTC TTCCCTTTTA TGGATCTCAT
    CGGCGCCATC
301 AACCTCGTCC CCTTCATCCT GACCGCCCCC GCCCCTTATC
    AGATAATGAC
351 CGGGCTG...

This corresponds to the amino acid sequence <SEQ ID 468; ORF48>:

1   MNIHTLLSKQ WTLPPFLPKR LLLSLLILLA PNAVFWVLAL
    LTATARPIVN
51  LDYLPAALLI ALPWRFVKIA GVLAFWLAVL FDGLMMVIQL
    FPFMDLIGAI
101 NLVPFILTAP APYQIMTGL...

Further work revealed the complete nucleotide sequence <SEQ ID 469>:

1    ATGAATATTC ACACCCTGCT CTCCAAACAA TGGACGCTGC
     CGCCATTCCT
51   GCCGAAACGG CTGCTGCTGT CCCTGCTGAT ACTGCTTGCC
     CCCAATGCGG
101  TGTTTTGGGT TTTGGCACTG CTGACCGCCA CCGCCCGCCC
     GATTGTCAAT
151  TTGGACTATC TTCCCGCCGC GCTGCTGATC GCCCTGCCTT
     GGCGTTTCGT
201  CAAAATTGCC GGCGTATTGG CGTTTTGGCT GGCGGTTTTG
     TTTGACGGGC
251  TGATGATGGT GATCCAACTC TTCCCTTTTA TGGATCTCAT
     CGGCGCCATC
301  AACCTCGTCC CCTTCATCCT GACCGCCCCC GCCCCTTATC
     AGATAATGAC
351  CGGGCTGTTG CTGCTGTATA TGCTGGCGAT GCCGTTTGTG
     TTGCAGAAAG
401  CCGCCGCCAA AACCGACTTC CGGCACATTG CCGTCTGCGC
     CGCCGTTGTG
451  GCGGCAGCCG GCTATTTCAC CGGCCATTTG AGTTACTACG
     ACCGGGGTCG
501  GATGGCCAAT ATCTTCGGCG CAAACAACTT CTACTACGCC
     AAAAGTCAGG
551  CGATGCTCTA CACCGTCAGC CAGAATGCCG ACTTTATTAC
     CGCCGGCCTG
601  GTCGATCCCG TCTTCCTCCC CTTGGGCAAT CAACAGCGTG
     CCGCCACGCA
651  TCTGAACGAG CCGAAATCTC AAAAAATCCT CTTTATCGTC
     GCCGAATCTT
701  GGGGGCTGCC GGCCAATCCC GAACTTCAAA ACGCCACTTT
     TGCCAAACTG
751  CTGGCGCAAA AAGACCGTTT TTCGGTTTGG GAAAGCGGCA
     GTTTTCCCTT
801  CATCGGCGCG ACGGTCGAAG GCGAAATGCG CGAACTGTGT
     GCCTACGGCG
851  GTTTGCGCGG GTTCGCACTG CGCCGCGCGC CCGACGAAAA
     ATTTGCCCGC
901  TGCCTCCCCA ACCGTTTGAA ACAAGAAGGT TACGCCACCT
     TTGCGATGCA
951  CGGCGCGGGC AGTTCGCTTT ACGACCGCTT CAGCTGGTAT
     CCGAGGGCGG
1001 GCTTTCAAGA AATCAAAACC GCCGAAAACC TGATCGGTAA
     AAAAACCTGC
1051 GCCATTTTCG GCGGCGTGTG CGACAGCGAG CTGTTCGGCG
     AAGTGTCGGC
1101 ATTTTTCAAA AAACACGACA AGGGACTGTT TTACTGGATG
     ACGCTGACCA
1151 GCCACGCCGA CTATCCCGAA TCCGACATTT TCAACCACAG
     GCTCAAATGC
1201 ACCGAATATG GCCTGCCCGC CGAAACCGAC CTCTGCCGCA
     ATTTCAGCCT
1251 GCACACCCAA TTCTTCGACC AACTGGCGGA TTTGATCCAA
     CGCCCCGAAA
1301 TGAAAGGCAC GGAAGTCATC ATCGTCGGCG ACCATCCGCC
     GCCCGTCGGC
1351 AACCTCAATG AAACCTTCCG CTACCTCAAA CAGGGGCACG
     TCGCCTGGCT
1401 GAACTTCAAA ATCAAATAA

This corresponds to the amino acid sequence <SEQ ID 470; ORF48-1>:

1   MNIHTLLSKQ WTLPPFLPKR LLLSLLILLA PNAVFWVLAL
    LTATARPIVN
51  LDYLPAALLI ALPWRFVKIA GVLAFWLAVL FDGLMMVIQL
    FPFMDLIGAI
101 NLVPFILTAP APYQIMTGLL LLYMLAMPFV LQKAAAKTDF
    RHIAVCAAVV
151 AAAGYFTGHL SYYDRGRMAN IFGANNFYYA KSQAMLYTVS
    QNADFITAGL
201 VDPVFLPLGN QQRAATHLNE PKSQKILFIV AESWGLPANP
    ELQNATFAKL
251 LAQKDRFSVW ESGSFPFIGA TVEGEMRELC AYGGLRGFAL
    RRAPDEKFAR
301 CLPNRLKQEG YATFAMHGAG SSLYDRFSWY PRAGFQEIKT
    AENLIGKKTC
351 AIFGGVCDSE LFGEVSAFFK KHDKGLFYWM TLTSHADYPE
    SDIFNHRLKC
401 TEYGLPAETD LCRNFSLHTQ FFDQLADLIQ RPEMKGTEVI
    IVGDHPPPVG
451 NLNETFRYLK QGHVAWLNFK IK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF48 shows 94.1% identity over a 119aa overlap with an ORF (ORF48a) from strain A of N. meningitidis:

The complete length ORF48a nucleotide sequence <SEQ ID 471> is:

1    ATGAATATTC ACACCCTGCT CTCCAAACAA TGGACGCTGC
     CGCCATTCCT
51   GCCGAAACGG CTGCTGCTGT CCCTGCTGAT ACTGCTNNCC
     CCCAATGCGG
101  TGTTTTGGGT TTTGGCACTG CTGACCGCCA CCGCCCGCCC
     GATTGTCAAT
151  TTGGANTACC TTCCCGCCGC GCTGCTGATC GCCCTGCCTT
     GGCGTNTCGT
201  CAAAATTGNC GGCGTATTGG CGTNTTGGCT GGCGGTTTTG
     TTTGACGGGC
251  TGATGATGGT GATCCAACTC TTCCCTTTTA TGGATCTCAT
     CGGCGCCATC
301  AACCTCGTCC CCTTCATCNT GACCGCCCCC GCCCTTTATC
     AGATAATGAC
351  CGGGCTGTTA CTGCTGTATA TGCTGGCGAT GCCGTTTGTG
     TTGCAGAAAG
401  CCGCCGCCAA AACCGACTTC CGACACATTG CCGCCTGTGC
     CGCCGTTGTG
451  GTGGCAGCCG GCTATTTTAC CGGCCATTTG AGTTANTACG
     ACCGGGGGCG
501  GATGGCCAAT ATCTTCGGCG CAAACAACTT CTATTACGCC
     AAAAGTCAGG
551  CGATGCTCTA CACCGTCAGC CAGAATGCCG ACTTTATTAC
     CGCCGGCCTG
601  GTCGATCCCG TCTTCCTCCC CTTGGGCAAT CAACAGCGTG
     CCGCCACGCA
651  TCTGAACGAG CCGAAATCTC AAAAAATCCT CTTTATCGTC
     GCCGAATCTT
701  GGGGGCTGCC GGCCAATCCC GAACTTCAAA ACGCCACTTT
     TGCCAAACTG
751  CTGGCGCAAA AAGANCGTTT TTCGGTTTGG GAAAGCGGCA
     GTTTTCCCTT
801  CATCGGCGCG ACGATCGAAG GCGAAATGCG CGAACTGTGT
     GCCTACGGCG
851  GTTTGCGCGG GTTCGCACTG CGCCGCGCGC CCGACGAAAA
     ATTTGCCCGC
901  TGCCTCCCCA ACCGTTTGAA ACAAGAAGGT TACGCCACCT
     TTGCGATGCA
951  CGGCGCGGGC AGTTCGCTTT ACGACCGCTT CAGCTGGTAT
     CCGAGGGCGG
1001 GCTTTCAAGA AATCAAAACC GCCGAAAACC TGATCGGTAA
     AAAAACCTGC
1051 GCCATTTTCG GCGGCGTGTG CGACAGCGAG CTGTTCGGCG
     AAGTGTCGGC
1101 ANTTTTCAAA AAACACGACA AGGGACTGTT TTACTGGATG
     ACGCTGACCA
1151 GCCACGCCGA CTATCCCGAA TCNGACATTT TCAACCACAG
     GCTCAAATGC
1201 ACCGAATATG GCCTGCCCGC CGAAACCGAC NTCTGCCGCA
     ATTTCAGCCT
1251 GCACACCCAA TTCTTCGACC AACTGGCGGA TTTGATCCAA
     CGCCCCGAAA
1301 TGAAAGGCAC GGAAGTCATC ATCGTCGGCG ACCATCCGCC
     GCCCGTCGGC
1351 AACCTCAATG AAACCTTCCG CTACCTCAAA CAGGGGCACG
     TCGNCTGGCT
1401 GAACTTCAAA ATCAAATAA

This encodes a protein having amino acid sequence <SEQ ID 472>:

1   MNIHTLLSKQ WTLPPFLPKR LLLSLLILLX PNAVFWVLAL
    LTATARPIVN
51  LXYLPAALLI ALPWRXVKIX GVLAXWLAVL FDGLMMVIQL
    FPFMDLIGAI
101 NLVPFIXTAP ALYQIMTGLL LLYMLAMPFV LQKAAAKTDF
    RHIAACAAVV
151 VAAGYFTGHL SXYDRGRMAN IFGANNFYYA KSQAMLYTVS
    QNADFITAGL
201 VDPVFLPLGN QQRAATHLNE PKSQKILFIV AESWGLPANP
    ELQNATFAKL
251 LAQKXRFSVW ESGSFPFIGA TIEGEMRELC AYGGLRGFAL
    RRAPDEKFAR
301 CLPNRLKQEG YATFAMHGAG SSLYDRFSWY PRAGFQEIKT
    AENLIGKKTC
351 AIFGGVCDSE LFGEVSAXFK KHDKGLFYWM TLTSHADYPE
    SDIFNHRLKC
401 TEYGLPAETD XCRNFSLHTQ FFDQLADLIQ RPEMKGTEVI
    IVGDHPPPVG
451 NLNETFRYLK QGHVXWLNFK IK*

ORF48a and ORF48-1 show 96.8% identity in 472 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF48 shows 97.5% identity over a 119aa overlap with a predicted ORF (ORF48ng) from N. gonorrhoeae:

The ORF48ng nucleotide sequence <SEQ ID 473> was predicted to encode a protein having amino acid sequence <SEQ ID 474>:

1   MNIHALLSEQ WTLPPFLPKR LLLSLLILLA PNAVFWVLAL
    LTATARPIVN
51  LDYLPAALLI ALPWRFVKIA GVLAFWPAVL FDGLMMVIQL
    FPFMDLIGAI
101 NLVPFILTAP APYQIMTGLL LLYMLAMPFV LQKAAVKTDF
    RHIAVCAAVV
151 AAARYFTGPF ELLRTGGRWQ YVQHRRLLLS GSRASFRRRQ
    KADVLRRLGN
201 PYASMGNGG..

Further work identified the complete gonococcal DNA sequence <SEQ ID 475>:

1    ATGAATATTC ACGCCCTGCT CTCCGAACAA TGGACGCTGC
     CGCCATTCCT
51   GCCGAAACGG CTGCTGCTGT CCCTGCTGAT ACTGCTGGCC
     CCCAATGCGG
101  TGTTTTGGGT TTTGGCACTG CTGACCGCCA CCGCCCGCCC
     GATTGTCAAT
151  TTGGACTACC TTCCCGCCGC GCTGCTGATC GCCCTGCCTT
     GGCGTTTCGT
201  CAAAATTGCC GGCGTATTGG CGTTTTGGCC GGCGGTTTTG
     TTTGACGGGC
251  TGATGATGGT GATCCAACTC TTCCCTTTTA TGGACCTCAT
     CGGCGCCATC
301  AACCTCGTCC CCTTCATCCT GACCGCCCCC GCCCCTTATC
     AGATAATGAC
351  CGGGCTGTTG CTGCTGTATA TGCTGGCGAT GCCGTTTGTG
     TTGCAAAAAG
401  CCGCCGTCAA AACCGACTTC CGACACATTG CCGTCTGTGC
     CGCCGTTGTG
451  GCGGCAGCCG GCTATTTCAC CGGCCATTTG AGTTACTACG
     ACCGGGGGCG
501  GATGGCCAAT ATCTTCGGCG CAAACAACTT CTATTACGCc
     aAAAGTCAGG
551  CGATGCTCTA CACCGTCAGC CAGAATGCCG ACTTTATTAC
     CGCCGgcctG
601  GTCGACCCCG TCTTCCTCCC CTTGGGCAAT CAGCAGCGTG
     CCGCCACGCG
651  GCTGAGTGAG CCGAAATCTC AAAAAATCCT CTTTATCGTC
     GCCGAATCTT
701  GGGGGCTGCC GGGCAATCCC GAGCTTCAAA ACGCCACTTT
     TGCCAAACTG
751  CTGGCGCAAA AAGACCGTTT TTCGGTTTGG GAAAGCGGCA
     GTTTTCCCTT
801  CATCGGCGCG ACGGTCGAAG GCGAAATGCG CGAATTGTGC
     GCCTACGGCG
851  GTTTGCGCGG GTTCGCACTG CGCCGCGCGC CCGACGAAAA
     ATTTGCCCGC
901  TGCCTCCCCA ACCGTTTGAA ACAAGAAGGT TACGCCACCT
     TTGCGATGCA
951  CGGCGCGGGT AGTTCGCTTT ACGACCGCTT CAGCTGGTAT
     CCGAGGGCGG
1001 GCTTTCAAAA AATCAAAACC GCCGAAAACC TGATCGGTAA
     AAAAACCTGC
1051 GCCATTTTCG GCGGCGTGTG CGACAGCGAG CTGTTCGGCG
     AAGTGTCGGC
1101 ATTTTTCAAA AAACACGACA AGGGACTGTT TTACTGGATG
     ACGCTGACCA
1151 GCCACGCCGA CTATCCCGAA TCCGACATTT TCAACCACAG
     GCTCAAATGC
1201 ACCGAATACG GCCTGCCCGC CGAAACCGAC CTCTGCCGCA
     ATTTCAGCCT
1251 GCACACCCAA TtcttcgACC AACTGGCGGA TTTGATCCGA
     CGCCCCGAAA
1301 TGAAAGGCAC GGAAGTCATC ATCGTCGGCG ACCATCCGCC
     GCCCGTCGGC
1351 AACCTCAATG AAACCTTCCG CTACCTCAAA CAGGGACACG
     TCGCCTGGCT
1401 GCACTTCAAA ATCAAATAA

This encodes a protein having amino acid sequence <SEQ ID 476; ORF48ng-1>:

1   MNIHALLSEQ WTLPPFLPKR LLLSLLILLA PNAVFWVLAL
    LTATARPIVN
51  LDYLPAALLI ALPWRFVKIA GVLAFWPAVL FDGLMMVIQL
    FPFMDLIGAI
101 NLVPFILTAP APYQIMTGLL LLYMLAMPFV LQKAAVKTDF
    RHIAVCAAVV
151 AAAGYFTGHL SYYDRGRMAN IFGANNFYYA KSQAMLYTVS
    QNADFITAGL
201 VDPVFLPLGN QQRAATRLSE PKSQKILFIV AESWGLPGNP
    ELQNATFAKL
251 LAQKDRFSVW ESGSFPFIGA TVEGEMRELC AYGGLRGFAL
    RRAPDEKFAR
301 CLPNRLKQEG YATFAMHGAG SSLYDRFSWY PRAGFQKIKT
    AENLIGKKTC
351 AIFGGVCDSE LFGEVSAFFK KHDKGLFYWM TLTSHADYPE
    SDIFNHRLKC
401 TEYGLPAETD LCRNFSLHTQ FFDQLADLIR RPEMKGTEVI
    IVGDHPPPVG
451 NLNETFRYLK QGHVAWLHFK IK*

ORG48ng-1 and ORF48-1 show 97.9% identity in 472 aa overlap:

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and two putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 57

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 477>:

1   ..GTGAGCGGAC GTTACCGCGC TTTGGATCGC GTTTCCAAAA
    TCATCATCGT
51  TACTTTGAGT ATCGCCACGC TTGCCGCCGC CGGCATCGCT
    ATGTCGCGCG
101 GTATGCAGAT GCAGTCCGAT TTTATCGAGC CGACACCGTG
    GACGCTTGCC
151 GGTTTGGGCT TCCTGATCGC GCTGATGGGC TGGATGCCCG
    CGCCGATTGA
201 AATTTCCGCC ATCAATTCTT TGTGGGTAAC CGAAAAACAA
    CGCATCAATC
251 CTTCCGAATA CCGCGACGGG ATTTTTGAAT TCAACGTCGG
    TTATATCGCC
301 AGTGCGGTTT TGGCTTTGGT TTTCCTTGCA CTGGGCGC.G
    TAGCGCCGAA
351 CGGCAACGGC GA.ACAGTGC AGATGGCGGG CGGCAAATAT
    AACGGGCAAT
401 TGATCAATAT GTACGCC..

This corresponds to the amino acid sequence <SEQ ID 478; ORF53>:

1   ..VSGRYRALDR VSKIIIVTLS IATLAAAGIA MSRGMQMQSD
    FIEPTPWTLA
51  GLGFLIALMG WMPAPIEISA INSLWVTEKQ RINPSEYRDG
    IFEFNVGYIA
101 SAVLALVFLA LGXVAPNGNG XTVQMAGGKY NGQLINMYA..

Further work revealed the complete nucleotide sequence <SEQ ID 479>:

1    ATGTCCGAAC AACATATTTC GACTTGGAAA AGTAAAATCA
     ACGCATTGGG
51   TCCGGGGATC ATGATGGCTT CGGCGGCGGT CGGCGGTTCG
     CACCTGATTG
101  CCTCGACGCA GGCGGGCGCG CTTTACGGCT GGCAGATCGC
     GCTCATCATC
151  ATCCTGACCA ACCTCTTCAA ATACCCGTTT TTCCGCTTCA
     GCGCGCATTA
201  CACGCTGGAC ACGGGCAAGA GCCTGATTGA AGGTTATGCC
     GAGAAAAGCC
251  GCGTTTATTT GTGGGTATTC CTGATTTTGT GCATCCTCTC
     CGCCACGATT
301  AACGCGGGCG CGGTCGCCAT TGTAACCGCC GCCATCGTCA
     AAATGGCGAT
351  TCCCTCGCTG ATGTTTGATG CCGGCACGGT TGCCGCCTTG
     ATTATGGCAT
401  CCTGCCTGAT TATTTTGGTG AGCGGACGTT ACCGCGCTTT
     GGATCGCGTT
451  TCCAAAATCA TCATCGTTAC TTTGAGTATC GCCACGCTTG
     CCGCCGCCGG
501  CATCGCTATG TCGCGCGGTA TGCAGATGCA GTCCGATTTT
     ATCGAGCCGA
551  CACCGTGGAC GCTTGCCGGT TTGGGCTTCC TGATCGCGCT
     GATGGGCTGG
601  ATGCCCGCGC CGATTGAAAT TTCCGCCATC AATTCTTTGT
     GGGTAACCGA
651  AAAACAACGC ATCAATCCTT CCGAATACCG CGACGGGATT
     TTTGATTTCA
701  ACGTCGGTTA TATCGCCAGT GCGGTTTTGG CTTTGGTTTT
     CCTTGCACTG
751  GGCGCGTTTG TGCAATACGG CAACGGCGAA GCAGTGCAGA
     TGGCGGGCGG
801  CAAATATATC GGGCAATTGA TCAATATGTA CGCCGTTACC
     ATCGGCGGCT
851  GGTCGCGCCC GCTGGTGGCG TTTATCGCGT TTGCCTGTAT
     GTACGGCACG
901  ACGATTACCG TCGTGGACGG CTATGCCCGT GCCATTGCCG
     AACCCGTGCG
951  CCTGCTGCGC GGAAAAGACA AAACGGGCAA CGCCGAATTC
     TTTGCCTGGA
1001 ATATTTGGGT GGCGGGCAGC GGTTTGGCGG TGATTTTCTG
     GTTTGACGGC
1051 GTAATGGCGA ATCTGCTCAA ATTTGCGATG ATTGCCGCTT
     TTGTGTCCGC
1101 CCCTGTGTTT GCCTGGCTGA ATTACCGTTT GGTTAAAGGT
     GATGAAAAAC
1151 ACAAACTCAC ATCAGGTATG AATGCCCTTG CATTGGCAGG
     CTTGATTTAT
1201 CTGACCGGTT TTACCGTTTT GTTCTTATTG AATTTGGCGG
     GAATGTTCAA
1251 ATGA

This corresponds to the amino acid sequence <SEQ ID 480; ORF53-1>:

1   MSEQHISTWK SKINALGPGI MMASAAVGGS HLIASTQAGA
    LYGWQIALII
51  ILTNLFKYPF FRFSAHYTLD TGKSLIEGYA EKSRVYLWVF
    LILCILSATI
101 NAGAVAIVTA AIVKMAIPSL MFDAGTVAAL IMASCLIILV
    SGRYRALDRV
151 SKIIIVTLSI ATLAAAGIAM SRGMQMQSDF IEPTPWTLAG
    LGFLIALMGW
201 MPAPIEISAI NSLWVTEKQR INPSEYRDGI FDFNVGYIAS
    AVLALVFLAL
251 GAFVQYGNGE AVQMAGGKYI GQLINMYAVT IGGWSRPLVA
    FIAFACMYGT
301 TITVVDGYAR AIAEPVRLLR GKDKTGNAEF FAWNIWVAGS
    GLAVIFWFDG
351 VMANLLKFAM IAAFVSAPVF AWLNYRLVKG DEKHKLTSGM
    NALALAGLIY
401 LTGFTVLFLL NLAGMFK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF53 shows 93.5% identity over a 139aa overlap with an ORF (ORF53a) from strain A of N. meningitidis:

The complete length ORF53a nucleotide sequence <SEQ ID 481> is:

1    ATGTCCGAAC AACATATTTC GACTTGGAAA AGTAAAATCA
     ACGCATTGGG
51   ACCGGGGATT ATGATGGCTT CGGCGGCGGT CGGCGGTTCG
     CACCTGATTG
101  CCTCGACGCA GGCGGGCGCG CTTTACGGCT GGCAGATCGC
     GCTCATCATC
151  ATCCTGACCA ACCTCTTCAA ATACCCGTTT TTCCGCTTCA
     GCGCGCATTA
201  CACGCTGGAC ACGGGCAAGA GCCTGATTGA AGGTTATGCC
     GAGAAAAGCC
251  GCGTTTATTT GTGGGTATTC CTGATTTTGT GCATCCTCTC
     CGCCACGATT
301  AACGCGGGCG CGGTCGCCAT TGTAACCGCC GCCATCGTCA
     AAATGGCGAT
351  TCCCTCGCTG ATGTTTGATG CCGGCACGGT TGCCGCCTTG
     ATTATGGCAT
401  CCTGCCTGAT TATTTTGGTG AGCGGACGTT ACCGCGCTTT
     GGATCGCGTT
451  TCCAAAATCA TCATCGTTAC TTTGAGTATC GCCACGCTTG
     CCGCCGCCGG
501  CATCGCTATG TCGCGCGGTA TGCAGATGCA GTCCGATTTT
     ATCGAGCCGA
551  CACCGTGGAC GCTTGCCGGT TTGGGCTTCC TGATCGCGCT
     GATGGGCTGG
601  ATGCCCGCGC CGATTGAAAT TTCCGCCATC AATTCTTTGT
     GGGTAACCGA
651  AAAACAACGC ATCAATCCTT CCGAATACCG CGACGGGATT
     TTTGATTTCA
701  ACGTCGGTTA TATCGCCAGT GCGGTTTTGG CTTTGGTTTT
     CCTTGCACTG
751  GGCGCGTTTG TGCAATACGG CAACGGCGAA GCAGTGCAGA
     TGGCGGGCGG
801  CAAATATATC GGGCAATTGA TCAATATGTA CGCCGTTACC
     ATCGGCGGCT
851  GGTCGCGCCC GCTGGTGGCG TTTATCGCGT TTGCCTGTAT
     GTACGGCACG
901  ACGATTACCG TTGTGGACGG CTATGCCCGT GCCATTGCCG
     AACCCGTGCG
951  CCTGCTGCGC GGAAAAGACA AAACGGGCAA CGCCGAATTC
     TTTGCCTGGA
1001 ATATTTGGGT GGCGGGCAGC GGTTTGGCGG TGATTTTCTG
     GTTTGACGGC
1051 GTAATGGCGA ATCTGCTCAA ATTTGCGATG ATTGCCGCTT
     TTGTGTCCGC
1101 CCCTGTGTTT GCCTGGCTGA ATTACCGTTT GGTCAAAGGT
     GATGAAAAAC
1151 ACAAACTCAC ATCAGGTATG AATGCCCTTG CATTGGCAGG
     CTTGATTTAT
1201 CTGACCGGTT TTACCGTTTT GTTCTTATTG AATTTGGCGG
     GAATGTTCAA
1251 ATGA

This encodes a protein having amino acid sequence <SEQ ID 482>:

1   MSEQHISTWK SKINALGPGI MMASAAVGGS HLIASTQAGA
    LYGWQIALII
51  ILTNLFKYPF FRFSAHYTLD TGKSLIEGYA EKSRVYLWVF
    LILCILSATI
101 NAGAVAIVTA AIVKMAIPSL MFDAGTVAAL IMASCLIILV
    SGRYRALDRV
151 SKIIIVTLSI ATLAAAGIAM SRGMQMQSDF IEPTPWTLAG
    LGFLIALMGW
201 MPAPIEISAI NSLWVTEKQR INPSEYRDGI FDFNVGYIAS
    AVLALVFLAL
251 GAFVQYGNGE AVQMAGGKYI GQLINMYAVT IGGWSRPLVA
    FIAFACMYGT
301 TITVVDGYAR AIAEPVRLLR GKDKTGNAEF FAWNIWVAGS
    GLAVIFWFDG
351 VMANLLKFAM IAAFVSAPVF AWLNYRLVKG DEKHKLTSGM
    NALALAGLIY
401 LTGFTVLFLL NLAGMFK*

ORF 53a shows 100.0% identity in 417 aa overlap with ORF53-1:

Homology with a Predicted ORF from N. gonorrhoeae

ORF53 shows 92.1% identity over a 139aa overlap with a predicted ORF (ORF53ng) from N. gonorrhoeae:

An ORF53ng nucleotide sequence <SEQ ID 483> was predicted to encode a protein having amino acid sequence <SEQ ID 484>:

1   MPKKSCVYLW VFLILCIASA TINAGAVAIV TAAIVKMAIP
    SLMFDAGTVA
51  ALIMASCLII LVSGRYRALD RVSKIIIVTL SIATLAAAGI
    AMSRGMQMQP
101 DFIEPTPWTL AGLGFLIALM GWMPAPIEIS AINSLWVTEK
    QRINPSEYRD
151 GIFDFNVGYI ASAVLALVFL ALGAFVQYGN GEAVQMGGGK
    YIGQLINMYA
201 VTIGGGSRPL VAFIAFACMY GAASTVVDGY ARAIAEPVRL
    LRGKDKTARP
251 IVLLEKLGGR HRFGRDFLV*

Further analysis revealed further partial DNA gonococcal sequence <SEQ ID 485>:

1    ..aagaAAAGCT GCGTTTATTT GTGGGTTTTT TTGATTTTGT
     GTATCGCCTC
51   CGCCACGATT AACGCGGGCG CGGTCGCCAT TGTAACCGCC
     GCCATCGTCA
101  AAATGGCGAT TCCCTCGCTG ATGTTTGATG CCGGCACGGT
     TGCCGCCTTG
151  ATTATGGCAT CCTGCCTGAT TATTTTGGTG AGCGGACGTT
     ACCGCGCTTT
201  GGATCGTGTT TCCAAAATCA TCATTGTTAC TTTGAGCATC
     GCCACGCTTG
251  CCGCCGCCGG CATCGCTATG TCGCGCGGTA TGCAGATGCA
     GCCCGATTTT
301  ATCGAGCCGA CACCGTGGAC GCTTGCCGGT TTGGGCTTCC
     TGATCGCGCT
351  GATGGGCTGG ATGCCCGCGC CGATCGAAAT TTCCGCCATC
     AATTCTTTGT
401  GGGTAACCGA AAAACAACGC ATCAATCCTT CTGAATACCG
     CGACGGGATT
451  TTCGATTTCA ACGTCGGTTA TATCGCcagT GCGGTTTTGG
     CTTTGGTTTT
501  CCTTGCACTG GGCGCGTTTG TGCAATACGG CAACGGCGAA
     GCAGTGCAGA
551  TGGCGGGCGG CAAATATATC GGGCAATTGA TTAATATGTA
     TGCCGTAACC
601  ATCGGCGGCT GGTCTCGTCC GCTGGTGGCG TTTATCGCGT
     TTGCCTGTAT
651  GTACGGCACG ACGATTACCG TTGTGGACGG TTATGCGCGT
     GCCATTGCCG
701  AACCCGTGCG CCTGCTGCGC GGCAGGGATA AAACCGGCAA
     CGCCGAGTTG
751  TTtgccTGGA ATATTTGGGT GGCGGGCAGC GGTTTGGCGG
     TGATTTTCTG
801  GTTTGACggc gcaaTGGCgG AACtgcTCAA ATTTGCGATG
     ATtgccgcCT
851  TTGTGTCCGC CCCTGTGTTC GCCTGGCTCA ACTACCGCCT
     CGTCAAAGGG
901  GACAAACGCC ACAGGCTTAC CGCCGGTATG AACGCCCTTG
     CCATTGTCGG
951  CCTGCTCTAC CTGGCCGGGT TTGCCGTTTT GTTCCTGTTG
     AACCTTACCG
1001 GACTTTTGGC ATAG

This corresponds to the amino acid sequence <SEQ ID 486; ORF53ng-1>:

1   ..KKSCVYLWVF LILCIASATI NAGAVAIVTA AIVKMAIPSL
    MFDAGTVAAL
51  IMASCLIILV SGRYRALDRV SKIIIVTLSI ATLAAAGIAM
    SRGMQMQPDF
101 IEPTPWTLAG LGFLIALMGW MPAPIEISAI NSLWVTEKQR
    INPSEYRDGI
151 FDFNVGYIAS AVLALVFLAL GAFVQYGNGE AVQMAGGKYI
    GQLINMYAVT
201 IGGWSRPLVA FIAFACMYGT TITVVDGYAR AIAEPVRLLR
    GRDKTGNAEL
251 FAWNIWVAGS GLAVIFWFDG AMAELLKFAM IAAFVSAPVF
    AWLNYRLVKG
301 DKRHRLTAGM NALAIVGLLY LAGFAVLFLL NLTGLLA*

ORF53ng-1 and ORF53-1 show 94.0% identity in 336 aa overlap:

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 58

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 487>:

1   ..TTGCGGGAAA CGGCATATGT TTTGGATAGT TTTGATCGTT
    ATTTTGTTGT
51  TGCGCTTGCC GGCTTGTTTT TTGTCCGCGC ACAATCCGAA
    CGCGAGTGGA
101 TGCGCGAGGT TTCTGCGTGG CAGGAAAAGA AAGGGGAAAA
    ACAGGCGGAG
151 CTGCCTGAAA TCAAAGACGG TATGCCCGAT TTTCCCGAAC
    TTGCCCTGAT
201 GCTTTTCCAC GCCGTCAAAA CGGCAGTGTA TTGGCTGTTT
    GTCGGTGTCG
251 TCCGTTTCTG CCGAAACTAT CTGGCGCACG AATCCGAACC
    GGACAGGCCC
301 GTTCCGCCT..

This corresponds to the amino acid sequence <SEQ ID 488; ORF58>:

1   ..LRETAYVLDS FDRYFVVALA GLFFVRAQSE REWMREVSAW
    QEKKGEKQAE
51  LPEIKDGMPD FPELALMLFH AVKTAVYWLF VGVVRFCRNY
    LAHESEPDRP
101 VPP..

Further work revealed the complete nucleotide sequence <SEQ ID 489>:

1    ATGTTTTGGA TAGTTTTGAT CGTTATTTTG TTGCTTGCGC
     TTGCCGGCTT
51   GTTTTTTGTC CGCGCACAAT CCGAACGCGA GTGGATGCGC
     GAGGTTTCTG
101  CGTGGCAGGA AAAGAAAGGG GAAAAACAGG CGGAGCTGCC
     TGAAATCAAA
151  GACGGTATGC CCGATTTTCC CGAACTTGCC CTGATGCTTT
     TCCATGCCGT
201  CAAAACGGCA GTGTATTGGC TGTTTGTCGG TGTCGTCCGT
     TTCTGCCGAA
251  ACTATCTGGC GCACGAATCC GAACCGGACA GGCCCGTTCC
     GCCTGCTTCT
301  GCAAACCGTG CGGATGTTCC GACCGCATCC GACGGATATT
     CAGACAGTGG
351  AAACGGGACG GAAGAAGCGG AAACGGAAGA AGCAGAAGCT
     GCGGAGGAAG
401  AGGCTGCCGA TACGGAAGAC ATTGCAACTG CCGTAATCGA
     CAACCGCCGC
451  ATCCCATTCG ACCGGAGTAT TGCTGAAGGG TTGATGCCGT
     CTGAAAGCGA
501  AATTTCGCCC GTCCGTCCGG TTTTTAAAGA AATCACTTTG
     GAAGAAGCAA
551  CGCGTGCTTT AAACAGCGCG GCTTTAAGGG AAACGAAAAA
     ACGCTATATC
601  GATGCATTTG AGAAAAACGA AACAGCGGTC CCCAAAGTCC
     GCGTGTCCGA
651  TACCCCGATG GAAGGGCTGC AGATTATCGG TTTGGACGAC
     CCTGTGCTTC
701  AACGCACGTA TTCCCATATG TTCGATGCGG ACAAAGAAGC
     GTTTTCCGAG
751  TCTGCGGATT ACGGATTTGA GCCGTATTTT GAGAAGCAGC
     ATCCGTCTGC
801  CTTTTCTGCA GTCAAAGCCG AAAATGCACG GAATGCGCCG
     TTCCACCGTC
851  ATGCAGGGCA GGGGAAAGGG CAGGCGGAGG CAAAATCCCC
     GGATGTTTCC
901  CAAGGGCAGT CCGTTTCAGA CGGCACGGCC GTCCGCGATG
     CCCGCCGCCG
951  CGTTTCCGTC AATTTGAAAG AACCGAACAA GGCAACGGTT
     TCTGCGGAGG
1001 CGCGAATTTC TCGCCTGATT CCGGAAAGTC AGACGGTTGT
     CGGGAAACGG
1051 GATGTCGAAA TGCCGTCTGA AACCGAAAAT GTTTTCACGG
     AAACCGTTTC
1101 GTCTGTGGGA TACGGCGGTC CGGTTTATGA TGAAACTGCC
     GATATCCATA
1151 TTGAAGAACC TGCCGCGCCC GATGCTTGGG TGGTCGAACC
     ACCCGAAGTG
1201 CCGAAAGTTC CCATGACCGC AATCGATATT CAGCCGCCGC
     CTCCCGTATC
1251 GGAAATCTAC AACCGTACCT ATGAACCGCC GTCAGGATTC
     GAGCAGGTGC
1301 AACGCAGCCG CATTGCCGAG ACCGACCATC TTGCCGATGA
     TGTTTTGAAT
1351 GGAGGTTGGC AGGAGGAAAC CGCCGCTATT GCGGATGACG
     GCAGTGAAGG
1401 TGCGGCAGAG CGGTCAAGCG GGCAATATCT GTCGGAAACC
     GAAGCGTTCG
1451 GGCATGACAG TCAGGCGGTT TGTCCGTTTG AAAATGTGCC
     GTCTGAACGC
1501 CCGTCCTGCC GGGTATCGGA TACGGAAGCG GATGAAGGGG
     CGTTCCCATC
1551 TGAAGAAACC GGTGCGGTAT CCGAACACCT GCCGACAACC
     GACCTGCTTC
1601 TGCCTCCGCT GTTCAATCCC GAGGCGACGC AAACCGAAGA
     AGAACTGTTG
1651 GAAAACAGCA TCACCATCGA AGAAAAATTG GCGGAGTTCA
     AAGTCAAGGT
1701 CAAGGTTGTC GATTCTTATT CCGGCCCCGT AATTACGCGT
     TATGAAATCG
1751 AACCCGATGT CGGCGTGCGC GGCAATTCCG TTCTGAATCT
     GGAAAAAGAT
1801 TTGGCGCGTT CGCTCGGCGT GGCTTCCATC CGCGTTGTCG
     AAACCATCCC
1851 CGGCAAAACC TGCATGGGTT TGGAACTTCC GAACCCGAAA
     CGCCAAATGA
1901 TACGCCTGAG CGAAATCTTC AATTCGCCCG AGTTTGCCGA
     ATCCAAATCC
1951 AAGCTGACGC TCGCGCTCGG TCAGGACATC ACCGGACAGC
     CCGTCGTAAC
2001 CGACTTGGGA AAAGCACCGC ATTTGTTGGT TGCCGGCACG
     ACCGGTTCGG
2051 GCAAATCGGT GGGTGTCAAC GCGATGATTC TGTCTATGCT
     TTTCAAAGCC
2101 GCGCCGGAAG ACGTGCGTAT GATTATGATC GATCCGAAAA
     TGCTGGAATT
2151 GAGCATTTAC GAAGGCATCC CGCACCTGCT CGCCCCTGTC
     GTTACCGATA
2201 TGAAGCTGGC GGCAAACGCG CTGAACTGGT GTGTTAACGA
     AATGGAAAAA
2251 CGCTACCGCC TGATGAGCTT TATGGGCGTG CGTAATCTTG
     CGGGCTTCAA
2301 TCAAAAAATC GCCGAAGCCG CAGCAAGGGG AGAAAAAATC
     GGCAATCCGT
2351 TCAGCCTCAC GCCCGACGAT CCCGAACCTT TGGAAAAACT
     GCCGTTTATC
2401 GTGGTCGTGG TCGATGAGTT TGCCGACCTG ATGATGACGG
     CAGGCAAGAA
2451 AATCGAAGAA CTGATTGCCC GCCTCGCCCA AAAAGCCCGC
     GCGGCAGGCA
2501 TCCATTTGAT TCTTGCCACA CAACGCCCCA GCGTCGATGT
     CATCACGGGT
2551 CTGATTAAGG CGAACATCCC GACGCGTATC GCGTTCCAAG
     TGTCCAGCAA
2601 AATCGACAGC CGCACGATTC TCGACCAAAT GGGCGCGGAA
     AACCTGCTCG
2651 GTCAGGGCGA TATGCTGTTC CTGCTGCCGG GTACTGCCTA
     TCCGCAGCGC
2701 GTTCACGGCG CGTTTGCCTC GGATGAAGAG GTGCACCGCG
     TGGTCGAATA
2751 TTTGAAACAG TTTGGCGAAC CGGACTATGT TGACGATATT
     TTGAGCGGCG
2801 GCGGCAGCGA AGAGCTGCCC GGCATCGGGC GCAGCGGCGA
     CGACGAAACC
2851 GATCCGATGT ACGACGAGGC CGTATCCGTT GTCCTGAAAA
     CGCGCAAAGC
2901 CAGCATTTCG GGCGTACAGC GCGCCTTGCG TATCGGCTAC
     AACCGCGCCG
2951 CGCGTCTGAT TGACCAGATG GAGGCGGAAG GCATTGTGTC
     CGCACCGGAA
3001 CACAACGGCA ACCGTACGAT TCTCGTCCCC TTGGACAATG
     CTTGA

This corresponds to the amino acid sequence <SEQ ID 490; ORF58-1>:

1    MFWIVLIVIL LLALAGLFFV RAQSEREWMR EVSAWQEKKG
     EKQAELPEIK
51   DGMPDFPELA LMLFHAVKTA VYWLFVGVVR FCRNYLAHES
     EPDRPVPPAS
101  ANRADVPTAS DGYSDSGNGT EEAETEEAEA AEEEAADTED
     IATAVIDNRR
151  IPFDRSIAEG LMPSESEISP VRPVFKEITL EEATRALNSA
     ALRETKKRYI
201  DAFEKNETAV PKVRVSDTPM EGLQIIGLDD PVLQRTYSHM
     FDADKEAFSE
251  SADYGFEPYF EKQHPSAFSA VKAENARNAP FHRHAGQGKG
     QAEAKSPDVS
301  QGQSVSDGTA VRDARRRVSV NLKEPNKATV SAEARISRLI
     PESQTVVGKR
351  DVEMPSETEN VFTETVSSVG YGGPVYDETA DIHIEEPAAP
     DAWVVEPPEV
401  PKVPMTAIDI QPPPPVSEIY NRTYEPPSGF EQVQRSRIAE
     TDHLADDVLN
451  GGWQEETAAI ADDGSEGAAE RSSGQYLSET EAFGHDSQAV
     CPFENVPSER
501  PSCRVSDTEA DEGAFPSEET GAVSEHLPTT DLLLPPLFNP
     EATQTEEELL
551  ENSITIEEKL AEFKVKVKVV DSYSGPVITR YEIEPDVGVR
     GNSVLNLEKD
601  LARSLGVASI RVVETIPGKT CMGLELPNPK RQMIRLSEIF
     NSPEFAESKS
651  KLTLALGQDI TGQPVVTDLG KAPHLLVAGT TGSGKSVGVN
     AMILSMLFKA
701  APEDVRMIMI DPKMLELSIY EGIPHLLAPV VTDMKLAANA
     LNWCVNEMEK
751  RYRLMSFMGV RNLAGFNQKI AEAAARGEKI GNPFSLTPDD
     PEPLEKLPFI
801  VVVVDEFADL MMTAGKKIEE LIARLAQKAR AAGIHLILAT
     QRPSVDVITG
851  LIKANIPTRI AFQVSSKIDS RTILDQMGAE NLLGQGDMLF
     LLPGTAYPQR
901  VHGAFASDEE VHRVVEYLKQ FGEPDYVDDI LSGGGSEELP
     GIGRSGDDET
951  DPMYDEAVSV VLKTRKASIS GVQRALRIGY NRAARLIDQM
     EAEGIVSAPE
1001 HNGNRTILVP LDNA*

Computer analysis of this amino acid sequence predicts the indicated transmembrane region, and also gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF58 shows 96.6% identity over a 89aa overlap with an ORF (ORF58a) from strain A of N. meningitidis:

The complete length ORF58a nucleotide sequence <SEQ ID 491> is:

1    ATGTTTTGGA TAGTTTTGAT CGTTATTTTG TTGCTTGCGC
     TTGCCGGCTT
51   GTTTTTTGTC CGCGCACAAT CCGAACGCGA GTGGATGCGC
     GAGGTTTCTG
101  CGTGGCAGGA AAAGAAAGGG GAAAAACAGG CGGAGCTGCC
     TGAAATCAAA
151  GACGGTATGC CCGATTTTCC CGAACTTGCC CTGATGCTTT
     TCCATGCCGT
201  CAAAACGGCA GTGTATTGGC TGTTTGTCGG TGTCGTCCGT
     TTCTGCCGAA
251  ACTATCTGGC GCACGAATCC GAACCGGACA GGCCCGTTCC
     GCCTGCTTCT
301  GCAAATCGTG CGGATGTTCC GACCGCATCC GACGGATATT
     CAGACAGTGG
351  AAACGGGACG GAAGAAGCGG AAACGGAAGA AGCAGAAGCT
     GCGGAGGAAG
401  AGGCTGCCGA TACGGAAGAC ATTGCAACTG CCGTAATCGA
     CAACCGCCGC
451  ATCCCATTCG ACCGGAGTAT TGCTGAAGGG TTGATGCCGT
     CTGAAAGCGA
501  AATTTCGCCC GTCCGTCCGG TTTTTAAGGA AATCACTTTG
     GAAGAAGCAA
551  CGCGTGCTTT AAACAGCGCG GCTTTAAGGG AAACGAAAAA
     ACGCTATATC
601  GATGCATTTG AGAAAAACGA AACAGCGGTC CCCAAAGTCC
     GCGTGTCCGA
651  TACCCCGATG GAAGGGCTGC AGATTATCGG TTTGGACGAC
     CCTGTGCTTC
701  AACGCACGTA TTCCCGTATG TTCGATGCGG ACAAAGAAGC
     GTTTTCCGAG
751  TCTGCGGATT ACGGATTTGA GCCGTATTTT GAGAAGCAGC
     ATCCGTCTGC
801  CTTTTCTGCA GTCAAAGCCG AAAATGCACG GAATGCGCCG
     TTCCGCCGTC
851  ATGCAGGGCA GGGNAAAGGG CAGGCGGAGG CNAAATCCCC
     GGATGTTTCC
901  CAAGGGCAGT CCGTTTCAGA CGGCACAGCC GTCCGCGATG
     CCNGCCGCCG
951  CGTTTCCGTC AATTTGAAAG AACCGAACAA GGCAACGGTT
     TCTGCGGAGG
1001 CGCGGATTTC GCGCCTGATT CCGGAAAGTC GGACGGTTGT
     CGGGAAACGG
1051 GATGTCGAAA TGCCGTCTGA AACCGAAAAT GTTTTCACGG
     AAANTGTTTC
1101 GTCTGTGGGA TACGGCGNTC CGGTTTATGA TGAAACTGCC
     GATATCCATA
1151 TTGAAGAACC TGCCGCGCCC GATGCTTGGG TGGTCGAACC
     ACCCGAAGTG
1201 CCGAAAGTTC CCATGCCCGC AATNGATATT CCGCCGCCGC
     CTCCCGTATC
1251 GGAAATCTAC AACCGTACCT ATGAACCGCC GGCAGGATTC
     GAGCAGGTGC
1301 AACGCAGCCG CATTGCCGAA ACCGATCATC TTGCCGATGA
     TGTTTTGAAT
1351 GGAGGTTGGC AGGAGGAAAC CGCCGCTATT GCGAATGACG
     GCAGTGAGGG
1401 TGTGGCAGAG CGGTCAAGCG GGCAATATTT GTCGGAAACC
     GAAGCGTTCG
1451 GGCATGACAG TCAGGCGGTT TGTCCGTTTG AAAATGTGCC
     GTCTGAACGC
1501 CCGTCCCGCC GGGCATNGGA TACGGAAGCG GATGAAGGGG
     CGTTCCAATC
1551 TGAAGAAACC GGTGCGGTAT CCGAACACCT GCCGACAACC
     GACCTGCTTC
1601 TGCCGCCGCT GTTCAATCCC GGGGCGACGC AAACCGAAGA
     AGANCTGTTG
1651 GANAACAGCA TCACCATCGA AGAAAAATNG GCGGAGTTCA
     AAGTCAAGGT
1701 CAAGGTTGTC GATTCTTATT CCGGCCCCGT GATTACGCGT
     TATGAAATCG
1751 AACCCGATGT CGGCGTGCGC GGCAATTCCG TTCTAAATCT
     GGAAAAAGAN
1801 TTGGCGCGTT CGCTCGGCGT GGCTTCCATC CGCGTTGTCG
     AAACCATCCT
1851 CGGCAAAACC TGTATGGGTT TGGAACTTCC GAACCCGAAA
     CGCCAAATGA
1901 TACGCCTGAG CGAAATCTTC AATTCGCCCG AGTTTGCCGA
     ATCCAAATCC
1951 AAGCTGACGC TCGCGCTCGG TCAGGACATC ACCGGACAGC
     CCGTCGTAAC
2001 CGACTTGGGC AAAGCACCGC ATTTGTTGGT TGCCGGCACG
     ACCGGTTCGG
2051 GCAAATCGGT GGGTGTCAAC GCGATGATTC TGTCTATGCT
     TTTCAAAGCC
2101 GCGCCGGAAG ACGTGCGTAT GATTATGATC GATCCGAAAA
     TGCTGGAATT
2151 GAGCATTTAC GAAGGCATCC CGCACCTGCT CGCCCCTGTC
     GTTACCGATA
2201 TGAAGCTGGC GGCAAACGCG CTGAACTGGT GTGTTAACGA
     AATGGAAAAA
2251 CGCTACCGCC TGATGAGCTT TATGGGCGTG CGCAATCTTG
     CGGGTNTCAA
2301 TCAAAAAATC GCCGAAGCCG CAGCAAGGGG GGAGAAAATC
     GGCAACCCGT
2351 TCAGCCTCAC GCCCGACAAT CCCGAACCTT TGGANAAATT
     GCCGTTTATC
2401 GTGGTCGTGG TTGATGAGTT TGCCGACCTG ATGATGACGG
     CAGGCAAGAA
2451 AATCGAAGAA CTGATTGCCC GCCTCGCCCA AAAAGCCCGC
     GCGGCAGGCA
2501 TCCATCTTAT CCTTGCCACA CAACGCCCCA GTGTCGATGT
     CATCACGGGT
2551 CTGATTAAGG CGAACATCCC GACGCGTATC GCGTTCCAAG
     TGTCCAGCAA
2601 AATCGACAGC CGCACGATTC TTGACCAAAT GGGTGCGGAA
     AACCTGCTCG
2651 GGCAGGGCGA TATGCTGTTC CTGCCGCCGG GTACGGCCTA
     TCCGCAGCGC
2701 GTTCACGGCG CGTTTGCCTC GGATGAAGAG GTGCACCGCG
     TGGTCGAATA
2751 TCTGAAACAG TTTGGCGAAC CGGACTATGT TGACGATATN
     TTGAGCGGCG
2801 GTATGTCCGA CGATTTGCTG GGAATCAGCC GGAGCGGCGA
     CGGCGAAACC
2851 GATCCGATGT ACGACGAGGC CGTGTCNGTT GTTTTGAAAA
     CGCGCAAAGC
2901 CAGCATTTCT GGCGTGCAGC GCGCATTGCG TATCGGCTAT
     AATCGCGCCG
2951 CGCGTCTGAT TGACCAGATG GAGGCGGAAG GCATTGTGTC
     CGCACCGGAA
3001 CACAACGGCA ACCGTACGAT TCTCGTCCCC TTNGACAATG
     CTTGA

This encodes a protein having amino acid sequence <SEQ ID 492>:

1    MFWIVLIVIL LLALAGLFFV RAQSEREWMR EVSAWQEKKG
     EKQAELPEIK
51   DGMPDFPELA LMLFHAVKTA VYWLFVGVVR FCRNYLAHES
     EPDRPVPPAS
101  ANRADVPTAS DGYSDSGNGT EEAETEEAEA AEEEAADTED
     IATAVIDNRR
151  IPFDRSIAEG LMPSESEISP VRPVFKEITL EEATRALNSA
     ALRETKKRYI
201  DAFEKNETAV PKVRVSDTPM EGLQIIGLDD PVLQRTYSRM
     FDADKEAFSE
251  SADYGFEPYF EKQHPSAFSA VKAENARNAP FRRHAGQGKG
     QAEAKSPDVS
301  QGQSVSDGTA VRDAXRRVSV NLKEPNKATV SAEARISRLI
     PESRTVVGKR
351  DVEMPSETEN VFTEXVSSVG YGXPVYDETA DIHIEEPAAP
     wDAWVVEPPEV
401  PKVPMPAXDI PPPPPVSEIY NRTYEPPAGF EQVQRSRIAE
     TDHLADDVLN
451  GGWQEETAAI ANDGSEGVAE RSSGQYLSET EAFGHDSQAV
     CPFENVPSER
501  PSRRAXDTEA DEGAFQSEET GAVSEHLPTT DLLLPPLFNP
     GATQTEEXLL
551  XNSITIEEKX AEFKVKVKVV DSYSGPVITR YEIEPDVGVR
     GNSVLNLEKX
601  LARSLGVASI RVVETILGKT CMGLELPNPK RQMIRLSEIF
     NSPEFAESKS
651  KLTLALGQDI TGQPVVTDLG KAPHLLVAGT TGSGKSVGVN
     AMILSMLFKA
701  APEDVRMIMI DPKMLELSIY EGIPHLLAPV VTDMKLAANA
     LNWCVNEMEK
751  RYRLMSFMGV RNLAGXNQKI AEAAARGEKI GNPFSLTPDN
     PEPLXKLPFI
801  VVVVDEFADL MMTAGKKIEE LIARLAQKAR AAGIHLILAT
     QRPSVDVITG
851  LIKANIPTRI AFQVSSKIDS RTILDQMGAE NLLGQGDMLF
     LPPGTAYPQR
901  VHGAFASDEE VHRVVEYLKQ FGEPDYVDDX LSGGMSDDLL
     GISRSGDGET
951  DPMYDEAVSV VLKTRKASIS GVQRALRIGY NRAARLIDQM
     EAEGIVSAPE
1001 HNGNRTILVP XDNA*

ORF58a and ORF58-1 show 96.6% identity in 1014 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF58 shows complete identity over a 9aa overlap with a predicted ORF (ORF58ng) from N. gonorrhoeae:

The ORF58ng nucleotide sequence <SEQ ID 493> is predicted to encode a protein having partial amino acid sequence <SEQ ID 494>:

1   ..SEPDRPVPPA SANRADVPTA SDGYSDSGNG TEEAETEAAE
    AAEEEAADTE
51  DIATAVIDNR RIPFDRSIAE GLMQSESKTS PVRPVFKEIT
    LEEATRALSS
101 AALRETKKRY IDAFEKNGTA VPKVRVSDTP MEGLQIIGLD
    DPVLQRTYSR
151 MFDADKEAFS ESADYGFEPY FEKQHPSAFS AVKAENARNA
    PFRRHAGQEK
201 GQAEAKSPDV SQGQSVSDGT AVRDARRRVS VNLKEPNKAT
    VSAEARISRL
251 IPESRTVVGK RDVEMPSETE NVFTETVSSV GYGGPVYDEA
    ADIHIEEPAA
301 PDAWVVEPPE VPEVAVPEID ILPPPPVSEI YNRTYEPPAG
    FEQAQRSRIA
351 ETDHLAADVL NGGWQEETAA IADDGSEGAA ERSSGQYLSE
    TEAFGHDSQA
401 VCPFEDVPSE RPSCRVSDTE ADEGAFQSEE TGAVSEHLPT
    TDLLLPPLFN
451 PEATQTEEEL LENSITIEEK LAEFKVKVKV VDSYSGPVIT
    RYEIEPDVGV
501 RGNSVLNLEK DLARSLGVAS IRVVETIPGK TCMGLELPNP
    KRQMIRLSEI
551 FNSPEFAESK SKLTLALGQD ITGQPVVTDL GKAPHLLVAG
    TTGSGKSVGV
601 NAMILSMLFK AAPEDVRMIM IDPKMLELSI YEGITHLLAP
    VVTDMKLAAN
651 ALNWCVNEME KRYRLMSFMG VRNLAGFNQK IAEAAARGEK
    IGNPFSLTPD
701 DPEPLEKLPF IVVVVDEFAD LMMTAGKKIE ELIARLAQKA
    RAAGIHLILA
751 TQRPSVDVIT GLIKANIPTR IAFQVSSKID SRTILDQMGA
    ENLLGQGDML
801 FLPPGTAYPQ RVHGAFASDE EVHRVVEYLK QFGEPDYVDD
    ILSGGGSEEL
851 PGIGRSGDGE TDPMYDEAVS VVLKTRKASI SGVQRALRIG
    YNRAARLIDQ
901 MEAEGIVSAP EHNGNRTILV PLDNA*

This partial gonococcal sequence contains a predicted transmembrane region and a predicted ATP/GTP-binding site motif A (P-loop; double underlined). Furthermore, it has a domain homologous to the FTSK cell division protein of E. coli. Alignment of ORF58ng and FtsK (accession number p46889) show a 65% amino acid identity in 459 overlap:

ORF58ng:  467 IEEKLAEFKVKVKVVDSYSGPVITRYEIEPDVGVRGNSVLNLEKDLARSLGVASIRVVET  526
+E +LA+F++K  VV+   GPVITR+E+    GV+   + NL +DLARSL    ++RVVE
FtsK:     868 VEARLADFRIKADVVNYSPGPVITRFELNLAPGVKAARISNLSRDLARSLSTVAVRVVEV  927
ORF58ng:  527 IPGKTCMGLELPNPKRQMIRLSEIFNSPEFAESKSKLTLALGQDITGQPVVTDLGKAPHL  586
IPGK  +GLELPN KRQ + L E+ ++ +F ++ S LT+ LG+DI G+PVV DL K  PHL
FtSK:     928 IPGKPYVGLELPNKKRQTVYLREVLDNAKFRDNPSPLTVVLGKDIAGEPVVADLAKMPHL  987
ORF58ng:  587 LVAGTTGSGKSVGVNAMILSMLFKAAPEDVRMIMIDPKMLELSIYEGITHLLAPVVTDMK  646
LVAGTTGSGKSVGVNAMILSML+KA PEDVR IMIDPKMLELS+YEGI HLL  VVTDMK
FtsK:     988 LVAGTTGSGKSVGVNAMILSMLYKAQPEDVRFIMIDPKMLELSVYEGIPHLLTEVVTDMK 1047
ORF58ng:  647 LAANALNWCVNEMEKRYRLMSFMGVANLAGFNQKIAEAAARGEKIGNPFSLTPDDPEP--  704
AANAL WCVNEME+RY+LMS +GVRNLAG+N+KIAEA      I +P+     D  +
FtsK:    1048 DAANALRWCVNEMERRYKLMSALGVRNLAGYNEKIAEADRMMRPIPDPYWKPGDSMDAQH 1107
ORF58ng:  705 --LEKLPFIVVVVDEFADLMMTAGKKIEELIARLAQKARAAGIHLILATQRPSVDVITGL  762
L+K P+IVV+VDEFADLMMT GKK+EELIARLAQKARAAGIHL+LATQRPSVDVITGL
FtsK:    1108 PVLKKEPYIVVLVDEFADLMMTVGKKVEELIARLAQKARAAGIHLVLATQRPSVDVITGL 1167
ORF58ng:  763 IKANIPTRIAFQVSSKIDSRTILDQMGAENLLGQGDMLFLPPGTAYPQRVHGAFASDEEV  822
IKANIPTRIAF VSSKIDSRTILDQ GAE+LLG GDML+  P +  P RVHGAF  D+EV
FtsK:    1168 IKANIPTRIAFTVSSKIDSRTILDQAGAESLLGMGDMLYSGPNSTLPVRVHGAFVRDQEV 1227
ORF58ng:  823 HRVVEYLKQFGEPDYVDDILSGGGSEELPGIGRSGDGETDPMYDEAVSVVLKTRKASISG   882
H VV+  K  G P YVD I S   SE   G G  G  E DP++D+AV  V + RKASISG
FtsK:    1228 HAVVQDWKARGRPQYVDGITSDSESEGGAG-GFDGAEELDPLFDQAVQFVTEKRKASISG  1286
ORF58ng:  883 VQRALRIGYNRAARLIDQMEAEGIVSAPEHNGNRTILVP                       921
VQR  RIGYNRAAR+I+QMEA+GIVS   HNGNR +L P
FtsK:    1287 VQRQFRIGYNRAARIIEQMEAQGIVSEQGHNGNREVLAP                      1325

Further work on ORF58ng revealed the complete gonococcal DNA sequence to be <SEQ ID 495>:

1    ATGTTTTGGA TAGTTTTGAT CGTTATtgtg TTGCTTGCGC
     TTGCCGGCCT
51   GTTTTTTGTC CGCGCACAAT CCGAACGCGA GTGGATGCGC
     GAGGTTTCTG
101  CGTGGCAGGA AAAGAAAGGG GAAAAACAGG CGGAGCTGCC
     TGAAATCAAA
151  GACGGTATGC CCGATTTTCC CGAGTTTTCC CTGATGCTTT
     TCCATGCCGT
201  CAAAACGGCA GTGTATTGGC TGTTTGTCGG TGTCGTCCGT
     TTCTGCCGAA
251  ACTATCTGGC GCACGAATCC GAACCGGACA GGCCCGTTCC
     GCCTGCTTCT
301  GCAAACCGTG CGGATGTTCC GACCGCATCC GACGGGTATT
     CAGACAGTGG
351  AAACGGGACG GAAGAAGCGG AAACGGAAGC AGCAGAAGCT
     GCGGAGGAAG
401  AGGCTGCCgA TACgGAAGAC ATTGCAACTG CCGTAATCGA
     CAACCGCCGC
451  ATCCcatTCG ACCGGAGTAT TGCTGAAGGG TTGATGCAGT
     CTGAAAGCAA
501  AACTTCGCCC GTCCGTCCGG TTTTTAAGGA AATCACTTTG
     GAAGAAGCAA
551  CGCGTGCTTT AAGCAGCGCG GCTTTAAGGG AAACGAAAAA
     ACGCTATATC
601  GATGCATTTG AGAAAAACGG AACAGCCGTC CCCAAAGTAC
     GCGTGTCCGA
651  TACCCCGATG GAAGGGCTGC AGATTATCGG TTTGGACGAC
     CCTGTGCTTC
701  AACGCACGTA TTCCCGTATG TTTGATGCGG ACAAAGAAGC
     GTTTTCCGAG
751  TCTGCGGATT ACGGATTTGA GCCGTATTTT GAGAAGCAGC
     ATCCGTCTGC
801  CTTTTCTGCA GTCAAAGCCG AAAATGCACG GAATGCGCCG
     TTCCGCCGTC
851  ATGCAGGGCA GGAGAAAGGG CAGGCGGAGG CAAAATCCCC
     GGATGTTTCC
901  CAAGGGCAGT CCGTTTCAGA CGGCACAGCC GTCCGCGATG
     CCCGCCGCCG
951  CGTTTCCGTC AATTTGAAAG AACCGAACAA GGCAACGGTT
     TCTGCGGAGG
1001 CGCGGATTTC GCGCCTGATT CCGGAAAGTC GGACGGTTGT
     CGGGAAACGG
1051 GATGTCGAAA TGCCGTCTGA AACCGAAAAT GTTTTCACGG
     AAACCGTTTC
1101 GTCTGTGGGA TACGGCGGTC CGGTTTATGA TGAAGCTGCC
     GATATCCATA
1151 TTGAAGAGCC TGCCGCGCCC GATGCTTGGG TGGTCGAACC
     ACCCGAAGTG
1201 CCGGAGGTAG CCGTACCCGA AATCGATATT CTGCCGCCGC
     CTCCCGTATC
1251 GGAAATCTAC AACCGTACCT ATGAGCCGCC GGCAGGATTC
     GAGCAGGCGC
1301 AACGCAGCCG CATTGCCGAA ACCGACCATC TTGCCGCTGA
     TGTTTTGAAT
1351 GGAGGTTGGC AGGAGGAAAC CGCCGCTATT GCAGATGACG
     GCAGTGAGGG
1401 TGCGGCAGAG CGGTCAAGCG GGCAATATCT GTCGGAAACC
     GAAGCGTTCG
1451 GGCATGACAG TCAGGCGGTT TGTCCGTTTG AAGATGTGCC
     GTCTGAACGC
1501 CCGTCCTGCC GGGTATCGGA TACGGAAGCG GATGAAGGGG
     CGTTCCAATC
1551 GGAAGAGACC GGTGCGGTAT CCGAACACCT GCCGACAACC
     GACCTGCTTC
1601 TGCCTCCGCT GTTCAATCCC GAGGCGACGC AAACCGAAGA
     AGAACTGTTG
1651 GAAAACAGCA TCACCATCGA AGAAAAATTG GCGGAGTTCA
     AAGTCAAGGT
1701 CAAGGTTGTC GATTCTTATT CCGGCCCCGT GATTACGCGT
     TATGAAATCG
1751 AACCCGATGT CGGCGTGCGC GGCAATTCCG TTCTGAATTT
     GGAAAAAGAC
1801 TTGGCGCGTT CGCTCGGCGT GGCTTCCATC CGCGTTGTCG
     AAACCATCCC
1851 CGGCAAAACC TGCATGGGTT TGGAACTTCC GAACCCGAAA
     CGCCAAATGA
1901 TACGCCTGAG CGAAATTTTC AATTCGCCCG AGTTTGCCGA
     ATCCAAATCC
1951 AAGCTGACGC TCGCGCTCGG TCAGGACATT ACCGGACAGC
     CCGTCGTAAC
2001 CGACTTGGGC AAAGCACCGC ATTTGCTGGT TGCCGGCACG
     ACCGGTTCGG
2051 GCAAATCGGT GGGTGTCAAC GCGATGATTC TGTCTATGCT
     TTTCAAAGCC
2101 GCGCCGGAAG ACGTGCGTAT GATTATGATC GATCCGAAAA
     TGCTGGAATT
2151 GAGCATTTAC GAAGGCATCA CGCACCTGCT CGCCCCTGTC
     GTTACCGATA
2201 TGAAGCTGGC GGCAAACGCG CTGAACTGGT GTGTTAACGA
     AATGGAAAAA
2251 CGCTACCGCC TGATGAGCTT TATGGGCGTG CGCAATCTTG
     CGGGCTTCAA
2301 CCAAAAAATC GCCGAAGCCG CAGCAAGGGG AGAAAAAATC
     GGCAATCCGT
2351 TCAGCCTCAC GCCCGACGAT CCCGAACCTT TGGAAAAACT
     GCCGTTTATC
2401 GTGGTCGTGG TCGATGAGTT TGCCGATTTG ATGATGACGG
     CAGGCAAGAA
2451 AATCGAAGAA CTGATTGCGC GCCTCGCCCA AAAAGCCCGC
     GCGGCAGGCA
2501 TCCACCTTAT CCTTGCCACA CAACGCCCCA GCGTCGATGT
     CATCACGGGT
2551 CTGATTAAGG CGAACATCCC GACGCGTATC GCGTTCCAAG
     TGTCCAGCAA
2601 AATCGACAGC CGCACGATTC TCGACCAAAT GGGCGCGGAA
     AACCTGCTCG
2651 GTCAGGGCGA TATGCTGTTC CTGCCGCCGG GTACTGCCTA
     TCCGCAGCGC
2701 GTTCACGGCG CGTTTGCCTC GGATGAAGAG GTGCACCGCG
     TGGTCGAATA
2751 TCTGAAGCAG TTTGGCGAGC CGGACTATGT TGACGATATT
     TTGAGCGGCG
2801 GCGGCAGCGA AGAGCTGCCC GGCATCGGGC GCAGCGGCGA
     CGGCGAAACC
2851 GATCCGATGT ACGACGAGGC CGTATCCGTT GTCCTGAAAA
     CGCGCAAAGC
2901 CAGCATTTCG GGCGTACAGC GCGCCTTGCG CATCGGCTAC
     AACCGCGCCG
2951 CGCGTCTGAT TGACCAAATG GAAGCGGAAG GCATTGTGTC
     CGCACCGGAA
3001 CACAACGGCA ACCGTACGAT TCTCGTCCCC TTGGACAATG
     CTTGA

This corresponds to the amino acid sequence <SEQ ID 496; ORF58ng-1>:

1    MFWIVLIVIV LLALAGLFFV RAQSEREWMR EVSAWQEKKG
     EKQAELPEIK
51   DGMPDFPEFS LMLFHAVKTA VYWLFVGVVR FCRNYLAHES
     EPDRPVPPAS
101  ANRADVPTAS DGYSDSGNGT EEAETEAAEA AEEEAADTED
     IATAVIDNRR
151  IPFDRSIAEG LMQSESKTSP VRPVFKEITL EEATRALSSA
     ALRETKKRYI
201  DAFEKNGTAV PKVRVSDTPM EGLQIIGLDD PVLQRTYSRM
     FDADKEAFSE
251  SADYGFEPYF EKQHPSAFSA VKAENARNAP FRRHAGQEKG
     QAEAKSPDVS
301  QGQSVSDGTA VRDARRRVSV NLKEPNKATV SAEARISRLI
     PESRTVVGKR
351  DVEMPSETEN VFTETVSSVG YGGPVYDEAA DIHIEEPAAP
     DAWVVEPPEV
401  PEVAVPEIDI LPPPPVSEIY NRTYEPPAGF EQAQRSRIAE
     TDHLAADVLN
451  GGWQEETAAI ADDGSEGAAE RSSGQYLSET EAFGHDSQAV
     CPFEDVPSER
501  PSCRVSDTEA DEGAFQSEET GAVSEHLPTT DLLLPPLFNP
     EATQTEEELL
551  ENSITIEEKL AEFKVKVKVV DSYSGPVITR YEIEPDVGVR
     GNSVLNLEKD
601  LARSLGVASI RVVETIPGKT CMGLELPNPK RQMIRLSEIF
     NSPEFAESKS
651  KLTLALGQDI TGQPVVTDLG KAPHLLVAGT TGSGKSVGVN
     AMILSMLFKA
701  APEDVRMIMI DPKMLELSIY EGITHLLAPV VTDMKLAANA
     LNWCVNEMEK
751  RYRLMSFMGV RNLAGFNQKI AEAAARGEKI GNPFSLTPDD
     PEPLEKLPFI
801  VVVVDEFADL MMTAGKKIEE LIARLAQKAR AAGIHLILAT
     QRPSVDVITG
851  LIKANIPTRI AFQVSSKIDS RTILDQMGAE NLLGQGDMLF
     LPPGTAYPQR
901  VHGAFASDEE VHRVVEYLKQ FGEPDYVDDI LSGGGSEELP
     GIGRSGDGET
951  DPMYDEAVSV VLKTRKASIS GVQRALRIGY NRAARLIDQM
     EAEGIVSAPE
1001 HNGNRTILVP LDNA*

ORF58ng-1 and ORF58-1 show 97.2% identity in 1014 aa overlap:

Furthermore, ORF58ng-1 shows significant homology to the E. coli protein FtsK:

sp|P46889|FTSK_ECOLI CELL DIVISION PROTEIN FTSK >gi|1651412|gnl|PID|d1015290
(Dl division protein FtsK [Escherichia coli] >gi|1651418|gnl|PID|d1015296
(D90727) Cell division protein FtsK [Escherichia coli] >gi|1787117 (AE000191)
cell division protein FtsK [Escherichia coli] Length = 1329
Score = 576 bits (1469), Expect = e−163
Identities = 301/459 (65%), Positives = 353/459 (76%), Gaps = 5/459 (1%)
Query:	556	IEEKLAEFKVKVKVVDSYSGPVITRYEIEPDVGVRGNSVLNLEKDLARSLGVASIRVVET	615
		+E +LA+F++K  VV+   GPVITR+E+    GV+   + NL +DLARSL   ++RVVE
Sbjct:	868	VEARLADFRIKADVVNYSPGPVITRFELNLAPGVKAARISNLSRDLARSLSTVAVRVVEV	927
Query:	616	IPGKTCMGLELPNPKRQMIRLSEIFNSPEFAESKSKLTLALGQDITGQPVVTDLGKAPHL	675
		IPGK  +GLELPN KRQ + L E+ ++ +F ++ S LT+ LG+DI G+PVV DL K PHL
Sbjct:	928	IPGKPYVGLELPNKKRQTVYLREVLDNAKFRDNPSPLTVVLGKDIAGEPVVADLAKMPHL	987
Query:	676	LVAGTTGSGKSVGVNAMILSMLFKAAPEDVRMIMIDPKMLELSIYEGITHLLAPVVTDMK	735
		LVAGTTGSGKSVGVNAMILSML+KA  PEDVR IMIDPKMLELS+YEGI HLL  VVTDMK
Sbjct:	988	LVAGTTGSGKSVGVNAMILSMLYKAQPEDVRFIMIDPKMLELSVYEGIPHLLTEVVTDMK	1047
Query:	736	LAANALNWCVNEMEKRYRLMSFMGVRNLAGFNQKIAEAAARGEKIGNPFSLTPDDPEP--	793
		AANAL WCVNEME+RY+LMS +GVRNLAG+N+KIAEA      I +P+    D  +
Sbjct:	1048	DAANALRWCVNEMERRYKLMSALGVRNLAGYNEKIAEADRMMRPIPDPYWKPGDSMDAQH	1107
Query:	794	--LEKLPFIVVVVDEFADLMMTAGKKIEELIARLAQKARAAGIHLILATQRPSVDVITGL	851
		L+K P+IVV+VDEFADLMMT GKK+EELIARLAQKARAAGIHL+LATQRPSVDVITGL
Sbjct:	1108	PVLKKEPYIVVLVDEFADLMMTVGKKVEELIARLAQKARAAGIHLVLATQRPSVDVITGL	1167
Query:	852	IKANIPTRIAFQVSSKIDSRTILDQMGAENLLGQGDMLFLPPGTAYPQRVHGAFASDEEV	911
		IKANIPTRIAF VSSKIDSRTILDQ GAE+LLG GDML+  P +  P RVHGAF  D+EV
Sbjct:	1168	IKANIPTRIAFTVSSKIDSRTILDQAGAESLLGMGDMLYSGPNSTLPVRVHGAFVRDQEV	1227
Query:	912	HRVVEYLKQFGEPDYVDDILSGGGSEELPGIGRSGDGETDPMYDEAVSVVLKTRKASISG	971
		H VV+  K  G P YVD I S   SE   G G  G  E DP++D+AV  V + RKASISG
Sbjct:	1228	HAVVQDWKARGRPQYVDGITSDSESEGGAG-GFDGAEELDPLFDQAVQFVTEKRKASISG	1286
Query:	972	VQRALRIGYNRAARLIDQMEAEGIVSAPEHNGNRTILVP	1010
		VQR  RIGYNRAAR+I+QMEA+GIVS   HNGNR +L P
Sbjct:	1287	VQRQFRIGYNRAARIIEQMEAQGIVSEQGHNGNREVLAP	1325

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 59

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 497>:

This corresponds to the amino acid sequence <SEQ ID 498; ORF101>:

Further work revealed the complete nucleotide sequence <SEQ ID 499>:

1    ATGATTTATC AAAGAAACCT CATCAAAGAA CTCTCTTTTA
     CCGCCGTCGG
51   CATTTTCGTC GTCCTCTTGG CGGTATTGGT CTCCACGCAG
     GCAATCAACC
101  TGCTCGGCCG TGCCGCCGAC GGGCGTGTCG CCATCGATGC
     CGTGTTGGCA
151  TTGGTCGGCT TCTGGGTCAT CGGTATGACG CCGCTTTTGC
     TGGTGTTGAC
201  CGCATTTATC AGTACGTTGA CCGTGTTGAC CCGCTACTGG
     CGCGACAGCG
251  AAATGTCGGT CTGGCTATCC TGCGGATTGG CATTGAAACA
     ATGGATACGC
301  CCGGTGATGC AGTTTGCCGT GCCGTTTGCC GTTTTGGTTG
     CCGTCATGCA
351  GCTTTGGGTG ATACCGTGGG CAGAGCTACG CAGCCGCGAA
     TACGCTGAAA
401  TCCTGAAGCA GAAGCAGGAA TTGTCTTTGG TGGAGGCAGG
     CGAGTTCAAC
451  AGTTTGGGCA AGCGCAACGG CAGGGTTTAT TTTGTCGAAA
     CCTTCGATAC
501  CGAATCCGGC ATCATGAAAA ACCTGTTCCT GCGCGAACAG
     GACAAAAACG
551  GCGGCGACAA CATCATCTTC GCCAAAGAAG GTAACTTCTC
     GCTGAACGAC
601  AACAAACGCA CGCTCGAATT GCGCCACGGC TACCGTTACA
     GCGGCACGCC
651  CGGACGCGCC GACTACAATC AGGTTTCCTT CCAAAAACTC
     AACCTGATTA
701  TCAGCACCAC GCCCAAACTC ATCGACCCCG TTTCCCACCG
     CCGTACCATT
751  CCGACCGCCC AACTGATTGG CAGCAGCAAC CCGCAACATC
     AGGCGGAATT
801  GATGTGGCGC ATCTCGCTGA CCGTCAGCGT CCTCCTACTC
     TGCCTGCTTG
851  CCGTGCCGCT TTCCTATTTC AACCCGCGCA GCGGACATAC
     CTACAATATC
901  TTGATTGCCA TCGGTTTGTT TTTAATTTAC CAAAACGGGC
     TGACCCTGCT
951  TTTTGAAGCC GTGGAAGACG GCAAAATCCA TTTTTGGCTC
     GGACTGCTGC
1001 CTATGCACAT TATCATGTTT GCCGTTGCAC TCATCCTGTT
     GCGCGTCCGC
1051 AGTATGCCCA GCCAGCCCTT CTGGCAGGCG GTTGGCAAAA
     GTCTGACATT
1101 GAAAGGCGGA AAATGA

This corresponds to the amino acid sequence <SEQ ID 500; ORF101-1>:

1   MIYQRNLIKE LSFTAVGIFV VLLAVLVSTQ AINLLGRAAD
    GRVAIDAVLA
51  LVGFWVIGMT PLLLVLTAFI STLTVLTRYW RDSEMSVWLS
    CGLALKQWIR
101 PVMQFAVPFA VLVAVMQLWV IPWAELRSRE YAEILKQKQE
    LSLVEAGEFN
151 SLGKRNGRVY FVETFDTESG IMKNLFLREQ DKNGGDNIIF
    AKEGNFSLND
201 NKRTLELRHG YRYSGTPGRA DYNQVSFQKL NLIISTTPKL
    IDPVSHRRTI
251 PTAQLIGSSN PQHQAELMWR ISLTVSVLLL CLLAVPLSYF
    NPRSGHTYNI
301 LIAIGLFLIY QNGLTLLFEA VEDGKIHFWL GLLPMHIIMF
    AVALILLRVR
351 SMPSQPFWQA VGKSLTLKGG K*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF101 shows 91.2% identity over a 57aa overlap and 95.7% identity over a 69aa overlap with an ORF (ORF101a) from strain A of N. meningitidis:

The complete length ORF101a nucleotide sequence <SEQ ID 501> is:

1    ATGATTTATC AAAGAAACCT CATCAAAGAA CTCTCTTTTA
     CCGCCGTCGG
51   CATTTTCGTC GTCCTCTTGG CGGTATTGGT CTCCACGCAG
     GCAATCAACC
101  TGCTCGGCCN TGCCGCCGAC NGGCGTNTCG CCATCGATGC
     CGTGTTGGCA
151  TTGGTCGGCT TCTGGGTCNN NNGNATGACG CCGCTTTTGC
     TNGTGTTGAC
201  CGCATTTATC AGTACGTTGA CCGTGTTGAC CCGCTACTGG
     CGNGACAGCG
251  AAATGTCGGT CTGGNTATCC TGCGGATTGG CATTGAAACA
     ATGGATACGC
301  CCGGTGATGC AGTTTGCCGT GCCGTTTGCC GTTTTGGTTG
     CCGTCATGCA
351  GCTTTGGGTG ATACCGTGGG CAGAGCTACG CAGCCGCGAA
     TACGCTGAAA
401  TCCTGAAGCA GAAGCAGGAA TTGTCTTTGG TGGAGGCAGG
     CGGGTTCAAC
451  AGTTTGGGCA AGCGCAACGG CAGGGTTTAT TTTGTCGAAA
     CCTTCGATAC
501  CGAATCCGGC ATCATGAAAA ACCTGTTCCT GCGCGAACAG
     GACAAAAACG
551  GCGGCGACAA CATCATCTTC NCCAAAGAAA GTAACTTCTC
     GCTGAACGAC
601  AACAAACGCA CGCTCGAATT GCGCCACGGC TACCGTTACA
     GCGGCACGCC
651  CGGACGCGCC GACTACAATC AGGTTTCCTT CCNAAAACTC
     AACCTGATTA
701  TCAGCACCAC GCCCAAACTC ATCGACCCCG TTTCCCACCG
     CCGTACNATN
751  CCNACNGCCC AACTGATTGG CAGCAGCAAC CCGCAACATC
     ANGCGGAATT
801  GATGTGGCGC ATCTCGCTGA CCGTCAGCGT CCTCCTACTC
     TGCCTGCTTG
851  CCGTGCCGCT TTCCTATTTC AACCCGCGCA GCGGACATAC
     CTACAATATC
901  TTGANTGCCA TCGGTTTGTT TTTAATTTAC CAAAACGGGC
     TGACCCTGCT
951  TTTTGAAGCC GTGGAAGACG GCAAAATCCA TTTTTGGCTC
     GGACTGCTGC
1001 CTATGCACAT CATCATGTTC GTCATCGCAA TCGTACTTCT
     GCGCGTCCGC
1051 AGCATGCCCA GCCAGCCCTT CTGGCAGGCG GTTGGCAAAA
     GTCTGACATT
1101 GAAAGGCGGA AAATGA

This encodes a protein having amino acid sequence <SEQ ID 502>:

1   MIYQRNLIKE LSFTAVGIFV VLLAVLVSTQ AINLLGXAAD
    XRXAIDAVLA
51  LVGFWVXXMT PLLLVLTAFI STLTVLTRYW RDSEMSVWXS
    CGLALKQWIR
101 PVMQFAVPFA VLVAVMQLWV IPWAELRSRE YAEILKQKQE
    LSLVEAGGFN
151 SLGKRNGRVY FVETFDTESG IMKNLFLREQ DKNGGDNIIF
    XKESNFSLND
201 NKRTLELRHG YRYSGTPGRA DYNQVSFXKL NLIISTTPKL
    IDPVSHRRTX
251 PTAQLIGSSN PQHXAELMWR ISLTVSVLLL CLLAVPLSYF
    NPRSGHTYNI
301 LXAIGLFLIY QNGLTLLFEA VEDGKIHFWL GLLPMHIIMF
    VIAIVLLRVR
351 SMPSQPFWQA VGKSLTLKGG K*

ORF101a and ORF101-1 show 95.4% identity in 371 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF101 shows 96.5% identity in 57aa overlap at the N-terminal domain and 95.1% identity in 61 as overlap at the C-terminal domain, respectively, with a predicted ORF (ORF101ng) from N. gonorrhoeae:

The ORF101ng nucleotide sequence <SEQ ID 503> is predicted to encode a protein having partial amino acid sequence <SEQ ID 504>:

1   MIYQRNLIKE LSFTAVGIFV VLLAVLVSTQ AINLLGRAAD
    GRVAIDAVLA
51  LVGFWVIGMT PLLLVLTAFI STLTVLTRYW RDSEMSVWLS
    CGLALKQWIR
101 PVMQFAVPFA ILIAVMQLWV IPWAELRSRE YAEILKQKQE
    LSLVEAGEFN
151 NLGKRNGRVY FVETFDTESG IMKNLFLREQ DKNGGDNIIF
    AKEGNFSLKD
201 NKRTLELRHG YRYSGTPGRA DYNQVSFQKL NLIISTTPKL
    IDPVSHRRTI
251 STAQLIGSSN PQHQAELMWR ISLTVSVLLL CLLAVPLSYF
    NPRSGHTYNI
301 LIAIGLFLIY QNGLTLLFEA VEDGKIHFWL GLLPMHIIMF
    VIAIVLLRVR
351 SMPSQPFWQA VG...

Further work revealed the complete nucleotide sequence <SEQ ID 505>:

1    ATGATTTATC AAAGAAACCT CATCAAAGAA CTCTCTTTTA
     CCGCCGTCGG
51   CATTTTCGTC GTCCTCTTGG CGGTGTTGGT GTCCACGCAG
     GCGATCAACC
101  TGCTTGGCCG CGCAGCTGAC GGGCGTGTCG CCATCGATGC
     CGTGTTGGCC
151  TTAGTCGGCT TCTGGGTCAT CGGTATGACC CCGCTTTTGC
     TGGTGTTGAC
201  CGCATTCATC AGCACGCTGA CCGTATTGAC CCGCTACTGG
     CGCGACAGCG
251  AAATGTCGGT CTGGCTATCC TGCGGATTGG CGTTGAAACA
     GTGGATACGC
301  CCCGTCATGC AGTTTGCCGT GCCGTTTGCC ATCCTGATTG
     CCGTCATGCA
351  GCTTTGGGTG ATACCGTGGG CAGAGCTGCG CAGCCGCGAA
     TATGCCGAAA
401  TTTTGAAGCA GAAGCAGGAA TTGTCTTTGG TGGAAGCCGG
     CGAGTTCAAT
451  AACTTGGGCA AGCGCAACGG CAgggtttaT TtcgtcgaaA
     CCTTTGACAC
501  CGaatccgGC ATCATGAAAA ACCTGTtcct GcGCGAACAG
     GACAAAAACG
551  gcggcgacaA CATCATCTTC GCcaaaGAag gtaactTctc
     gctgaaggaC
601  AACAAAcgca cgctcgaATT GCGCCACGGC TACCGTTACA
     GCGGcacgcC
651  CGGacGCGCc gactaCAATC AGGTTtcctt cCAAAAacTc
     aacctgATta
701  TCAGCACCAC GCCCAAacTT ATCGaccCCG TTTCCCACCG
     CCGCACCATT
751  tcgacCGCCC AAcTGATTGG CAGCAGCAAT CCGCAACATC
     AGGCAGAATT
801  GATGTGGCGC ATCTCGCTGA CCGTCAGCGT CCTCCTGCTC
     TGCCTACTCG
851  CCGTGCCGCT TTCCTATTTC AACCCGCGCA GCGGACATAC
     CTACAATATC
901  TTGATTGCCA TCGGTTTGTT TTTAATTTAC CAAAACGGGC
     TGACCCTGCT
951  TTTTGAAGCC GTGGAAGACG GCAAAATCCA TTTTTGGCTC
     GGACTGCTGC
1001 CTATGCACAT CATCATGTTC GTCATCGCAA TCGTACTTCT
     GCGCGTCCGC
1051 AGTATGCCCA GCCAGCCCTT CTGGCAGGCG GTTGGCAAAA
     GTCTGACATT
1101 GAAAGgcgGA AAATGA

This corresponds to the amino acid sequence <SEQ ID 506; ORF101ng-1>:

1   MIYQRNLIKE LSFTAVGIFV VLLAVLVSTQ AINLLGRAAD
    GRVAIDAVLA
51  LVGFWVIGMT PLLLVLTAFI STLTVLTRYW RDSEMSVWLS
    CGLALKQWIR
101 PVMQFAVPFA ILIAVMQLWV IPWAELRSRE YAEILKQKQE
    LSLVEAGEFN
151 NLGKRNGRVY FVETFDTESG IMKNLFLREQ DKNGGDNIIF
    AKEGNFSLKD
201 NKRTLELRHG YRYSGTPGRA DYNQVSFQKL NLIISTTPKL
    IDPVSHRRTI
251 STAQLIGSSN PQHQAELMWR ISLTVSVLLL CLLAVPLSYF
    NPRSGHTYNI
301 LIAIGLFLIY QNGLTLLFEA VEDGKIHFWL GLLPMHIIMF
    VIAIVLLRVR
351 SMPSQPFWQA VGKSLTLKGG K*

ORF101ng-1 and ORF101-1 show 97.6% identity in 371 aa overlap:

Based on this analysis, including the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 60

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 507>:

1   ..GGTGGTGGTT TTATCAATGC TTCCTGTGCC ACTTTGACGA
    CAGCCAAACC
51  GCAATATCAA GCAGGAGACC TTAGCGCTTT TAAGATAAGG
    CAAGGCAATG
101 TTGTAATCGC CGGACACGGT TTGGATGCAC GTGATACCGA
    TTACACACGT
151 ATTCTCAGTT ATCATTCCAA AATCGATGCA CCCGTATGGG
    GACAAGATGT
201 TCGTGTCGTC GCGGGACAAA ACGATGTGGC CGCAACAGGT
    GATGCACATT
251 CGCCTATTCT CAATAATGCT GCTGCCAATA CGTCAAACAA
    TACAGCCAAC
301 AACGGCACAC ATATCCCTTT ATTTGCGATT GATACAGGCA
    AATTAGGAGG
351 TAT.GTATGC CAACAAAATC ACCTTGATCA GTACGGTCGA
    GCAAGCAGGC
401 ATTCGTAA

This corresponds to the amino acid sequence <SEQ ID 508; ORF113>:

1   ..GGGFINASCA TLTTAKPQYQ AGDLSAFKIR QGNVVIAGHG
    LDARDTDYTR
51  ILSYHSKIDA PVWGQDVRVV AGQNDVAATG DAHSPILNNA
    AANTSNNTAN
101 NGTHIPLFAI DTGKLGGXVC QQNHLDQYGR ASRHS*

Computer analysis of this amino acid sequence gave the following results:

Homology with pspA Putative Secreted Protein of N. meningitidis (Accession AF030941)

ORF and pspA show 44% aa identity in 179aa overlap:

orf113	GGGFINASCATLTTAKPQYQAGDLSAFKIRQGNVVIAGHGLDARDTDYTRILSYHSKIDA	60
	GGG INA+  TLT+  P    G+L+ F +  G VVI G GLD  D DYTRILS  ++I+A
pspa	GGGLINAASVTLTSGVPVLNNGNLTGFDVSSGKVVIGGKGLDTSDADYTRILSRAAEINA	256
orf113	PVWGQDVRVVAGQNDVAATGDAHSPILXXXXXXXXXXXXXXGTHIPLFAIDTGKLGGMYA	120
	VWG+DV+VV+G+N +   G                      +  P  AIDT  LGGMYA
pspa	GVWGKDVKVVSGKNKLDFDG---------SLAKTASAPSSSDSVTPTVAIDTATLGGMYA	307
orf113	NKITLISTVEQAGIRNQGQWFASAGNVAVNAEGKLVNTGMIAATGENHAVSLHARNVHN	179
	+KITLIST   A IRN+G+ FA+ G V ++A+GKL N+G I A      +++ A+ V N
pspa	DKITLISTDNGAVIRNKGRIFAATGGVTLSADGKLSNSGSIDAA----EITISAQTVDN	362

Homology with a Predicted ORF from N. gonorrhoeae

ORF113 shows 86.5% identity in 52aa overlap at the N-terminal part and 94.1% identity in 17aa overlap at the C-terminal part with a predicted ORF (ORF113ng) from N. gonorrhoeae:

The complete length ORF113ng nucleotide sequence <SEQ ID 509> is predicted to encode a protein having amino acid sequence <SEQ ID 510>:

1   MNKTLYRVIF NRKRGAVVAV AETTKREGKS CADSGSGSVY
    VKSVSFIPTH
51  SKAFCFSALG FSLCLALGTV NIAFADGIIT DKAAPKTQQA
    TILQTGNGIP
101 QVNIQTPTSA GVSVNQYAQF DVGNRGAILN NSRSNTQTQL
    GGWIQGNPWL
151 TRGEARVVVN QINSSHPSQL NGYIEVGGRR AEVVIANPAG
    IAVNGGGFIN
201 ASRATLTTGQ PQYQAGDFSG FKIRQGNAVI AGHGLDARDT
    DFTRILVCQQ
251 NHLDQYGRTS RHS*

Based on this analysis, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 61

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 511>:

1   ..TCAACGGGAC ATAGCGAACA AAATTACACT TTGCCGCGAG
    AAATCACACG
51  CAACATTTCA CTGGGTTCAT TTGCCTATGA ATCGCATCGC
    AAAGCATTAA
101 GCCATCATGC GCCCAGCCAA GGCACTGAGT TGCCGCAAAG
    CAACGGTATT
151 TCGCTACCCT ATACGTCCAA TTCTTTTACC CCATTACCCA
    GCAGCAGCTT
201 ATACATTATC AATCCTGTCA ATAAAGGCTA TCTTGTTGAA
    ACCGATCCAC
251 GCTTTGCCAA CTACCGTCAA TGGTTGGGTA GTGACTATAT
    GCtGGACAGC
301 CTCAAACTAG ACCCAAACAA TTTACATAAA CGTTTGGGTG
    ATGGTTATTA
351 CGAGCAACGT TTAATCAATG AACAAATCGC AGAGCTGACA
    GGGCATCGTC
401 GTTTAGAcGG TTATCAAAAC GACGAAGAAC AATTTAAAGC
    CTTAATGGAT
451 AATGGCGCGA CTGCGGCACG TTcGATGAAT CTCAGCGTTG
    GCATTGCATT
501 AAGTGCCGAG CAAGTAGCGC AACTGACCAG CGATATTGTT
    TGGTTGGTAC
551 AAAAAGAAGT TAAGCTTCCT GATGGCGGCA CACAAACCGT
    ATTGGTGCCA
601 CAGGTTTATG TACGCGTTAA AAATGGCGAC ATAGACGGTA
    AAGGTGCATT
651 GTTGTCAGGC AGCAATACAC AAATCAATGT TTCAGGCAGC
    CTGAAAAACT
701 CAGGCACGAT TGCAGGgCGC AATGCGCTTA TTATCAATAC
    CGATACGCTA
751 GACAATATCG GTGGGCGTAT TCATGCGCAA AAATCAGCGG
    TTACGGCCAC
801 ACAAGACATC AATAATATTG GCGGCATGCT TTCTGCCGAA
    CAGACATTAT
851 TGCTCAACGC AGGCAACAAC ATCAACAGCC AAAGCACCAC
    CGCCAGCAGT
901 CAAAATACAC AAGGCAGCAG CACCTACCTA GACCGAATGG
    CAGGTATTTA
951 TATCACAGGC AAAGAAAAAG GTGTTT..

This corresponds to the amino acid sequence <SEQ ID 512; ORF115>:

1   ..STGHSEQNYT LPREITRNIS LGSFAYESHR KALSHHAPSQ
    GTELPQSNGI
51  SLPYTSNSFT PLPSSSLYII NPVNKGYLVE TDPRFANYRQ
    WLGSDYMLDS
101 LKLDPNNLHK RLGDGYYEQR LINEQIAELT GHRRLDGYQN
    DEEQFKALMD
151 NGATAARSMN LSVGIALSAE QVAQLTSDIV WLVQKEVKLP
    DGGTQTVLVP
201 QVYVRVKNGD IDGKGALLSG SNTQINVSGS LKNSGTIAGR
    NALIINTDTL
251 DNIGGRIHAQ KSAVTATQDI NNIGGMLSAE QTLLLNAGNN
    INSQSTTASS
301 QNTQGSSTYL DRMAGIYITG KEKGV..

Computer analysis of this amino acid sequence gave the following results:

Homology with the pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF115 and pspA protein show 50% aa identity in 325aa overlap:

Orf115:	1	STGHSEQNYTLPREITRNISLGSFAYESHRKALSHHAPSQGTELPQSNGISLPYTSNSFT	60
		STG+S   Y    E++ +I +G  AY+ +    +   P     +   NGI   +T
pspA:	778	STGYSRSPYEPAPEVS-SIRMGISAYKGYAPQQASDIPGTVVPVVAENGIHPTFT-----	831
Orf115:	61	PLPSSSLYIINPVNKGYLVETDPRFANYRQWLGSDYMLDSLKLDPNNLHKRLGDGYYEQR	120
		LP+SSL+ I P NKGYL+ETDP F +YR+WLGS YML +L+ DPN++HKRLGDGYYEQ+
pspA:	832	-LPNSSLFAIAPNNKGYLIETDPAFTDYRKWLGSGYMLAALQQDPNHIHKRLGDGYYEQK	890
Orf115:	121	LINEQIAELTGHRRLDGYQNDEEQFKALMDNGATAARSMNLSVGIALSAEQVAQLTSDIV	180
		L+NEQIA+LTG+RRLDGY NDEEQFKALMDNG T A+ + L+ GIALSAEQVA+LTSDIV
pspA:	891	LVNEQIAKLTGYRRLDGYTNDEEQFKALMDNGITIAKELQLTPGIALSAEQVARLTSDIV	950
Orf115:	181	WLVQKEVKLPDGGTQTVLVPQVYVRVKNGDIDGKGALLSGSNTQINVSGSLKN-SGTIAG	239
		WL  + V LPDG TQTVL P+VYVR +  D++G+GALLSGS   I  SG+++N  G IAG
pspA:	951	WLENETVTLPDGTTQTVLKPKVYVRARPKDMNGQGALLSGSVVDIG-SGAIENRGGLIAG	1009
Orf115:	240	RNALIINTDTLDNIGGRIHAQKSAVTATQDINNIGGMLSAEQTLLLNAGXXXXXXXXXXX	299
		R ALI+N   + N+ G +  +     A  DI N G  + AE  LLL A
pspA:	1010	REALILNAQNIKNLQGDLQGKNIFAAAGSDITNTGS-IGAENALLLKASNNIESRSETRS	1068
Orf115:	300	XXXXXXXXXYLDRMAGIYITGKEKG	324
		+ R+AGIY+TG++ G
pspA:	1069	NQNEQGSVRNIGRVAGIYLTGRQNG	1093

Homology with a Predicted ORF from N. gonorrhoeae

ORF115 shows 91.9% identity over a 334aa overlap with a predicted ORF (ORF115ng) from N. gonorrhoeae:

An ORF115ng nucleotide sequence <SEQ ID 513> was predicted to encode a protein having amino acid sequence <SEQ ID 514>:

1   MLVQTEKDGL HNEQTFGEKK VFSENGKLHN YWRARRKGHD
    ETGHREQNYT
51  LPEEITRDIS LGSFAYESHS KALSRHAPSQ GTELPQSNRD
    NIRTAKSNGI
101 SLPYTPNSFT PLPGSSLYII NPANKGYLVE TDPRFANYRQ
    WLGSDYMLGS
151 LKLDPNNLHK RLGDGYYEQR LINEQIAELT GHRRLDGYQN
    DEEQFKALMD
201 NGATAARSMN LSVGIALSAE QAAQLTSDIV WLVQKEVKLP
    DGGTQTVLMP
251 QVYVRVKNGG IDGKGALLSG SNTQINVSGS LKNSGTIAGR
    NALIINTDTL
301 DNIGGRIHAQ KSAVTATQDI NNIGGILSAE QTLLLNAGNN
    INNQSTAKSS
351 QNAQGSSTYL DRMAGIYITG KEKGVLAAQA GKDINIIAGQ
    ISNQSDQGQT
401 RLQAGRDINL DTVQTGKYQE IHFDADNHTI RGSTNEVGSS
    IQTKGDVTLL
451 SGNNLNAKAA EVGSAKGTLA VYAKNDITIS SGIHAGQVDD
    ASKHTGRSGG
501 GNKLVITDKA QSHHETAQSS TFEGKQVVLQ AGNDANILGS
    NVISDNGTRI
551 QAGNHVRIGT TQTQSQSETY HQTQKSGLMS AGIGFTIGSK
    TNTQENQSQS
601 NEHTGSTVGS LKGDTTIVAS KHYEQTGSNV SSPEGNNLIS
    TQSMDIGAAQ
651 NQLNSKTTQT YEQKGLTVAF SSPVTDLAQQ AIAVAHKAAK
    QFDKAKTTAL
701 MPWRLPMQVG RLFKQAKAPK K*

Further work revealed the following partial gonococcal DNA sequence <SEQ ID 515>:

1    TTGCTTGTGC AAACAGAAAA AGACGGTTTG CATAACGAGC
     AAACCTTTGG
51   CGAGAAGAAA GTCTTCAGCG AAAATGGTAA GTTGCACAAC
     TACTGGCGTG
101  CGCGTCGTAA AGGACATGAT GAAACAGGGC ATCGTGAACA
     AAATTATACT
151  TTGCCGGAGG AAATCACACG CGACATTTCA CTGGGTTCAT
     TTGCCTATGA
201  ATCGCATAGC AAAGCATTAA GCCGTCATGC GCCCAGCCAA
     GGCACTGAGT
251  TGCCACAAAG TAACCGGGAT AATATCCGTA CTGCGAAAAG
     CAACGGTATT
301  TCGCTACCCT ATACGCCCAA TTCTTTTACC CCATTACCCG
     GCAGCAGCTT
351  ATACATTATC AATCCTGCCA ATAAAGGCTA TCTTGTTGAA
     ACCGATCCAC
401  GCTTTGCCAA CTACCGTCAA TGGTTGGGTA GTGACTATAT
     GCTGGGCAGC
451  CTCAAACTAG ACCCAAACAA TTTACATAAA CGTTTGGGTG
     ATGGTTATTA
501  CGAGCAACGT TTAATCAATG AACAAATCGC AGAGCTGACA
     GGGCATCGTC
551  GTTTAGACGG TTATCAAAAC GACGAAGAAC AATTTAAAGC
     CTTAATGGAT
601  AATGGCGCGA CTGCGGCACG TTCGATGAAT CTCAGCGTTG
     GCATTGCATT
651  AAGTGCCGAG CAAGCAGCGC AACTGACCAG CGATATTGTT
     TGGTTGGTAC
701  AAAAAGAAGT TAAACTTCCT GATGGCGGCA CACAAACCGT
     ATTGATGCCA
751  CAGGTTTATG TACGCGTTAA AAATGGCGGC ATAGACGGTA
     AAGGTGCATT
801  GTTGTCAGGC AGCAATACAC AAATCAATGT TTCAGGCAGC
     CTGAAAAACT
851  CAGGCACGAT TGCAGGGCGC AATGCGCTTA TTATCAATAC
     CGATACGCTA
901  GACAATATCG GTGGGCGTAT TCATGCGCAA AAATCAGCGG
     TTACGGCCAC
951  ACAAGACATC AATAATATTG GCGGCATTCT TTCTGCCGAA
     CAGACATTAT
1001 TGCTCAATGC GGGTAACAAC ATCAACAACC AAAGCACGGC
     CAAGAGCAGT
1051 CAAAATGCAC AAGGTAGCAG CACCTACCTA GACCGAATGG
     CAGGTATTTA
1101 TATCACAGGC AAAGAAAAAG GTGTTTTAGC AGCGCAGGCA
     GGCAAAGACA
1151 TCAACATCAT TGCCGGTCAA ATCAGCAATC AATCAGATCA
     AGGGCAAACC
1201 CGGCTGCAGG CAGGACGCGA CATTAACCTG GATACGGTAC
     AAACCGGCAA
1251 ATATCAAGAA ATCCATTTTG ATGCCGATAA CCATACCATC
     CGAGGTTCAA
1301 CGAACGAAGT CGGCAGCAGC ATTCAAACAA AAGGCGATGT
     TACCCtatTG
1351 TCAGGGAATA ATCTCAATGC CAAAGCTGCC GAAGTCGGCA
     GCGCAAAAGG
1401 CACACTTGCC GTGTATGCTA AAAATGACAT TACTATCAGC
     TCAGGCATCC
1451 ATGCCGGCCA AGTTGATGAT GCGTCCAAAC ATACAGGCAG
     AAGCGGCGGC
1501 GGTAATAAAT TAGTCATTAC CGATAAAGCC CAAAGTCATC
     ACGAAACTGC
1551 TCAAAGCAGC ACCTTTGAAG GCAAGCAAGT TGTATTGCAG
     GCAGGAAACG
1601 ATGCCAACAT CCTTGGCAGT AATGTTATTT CCGATAATGG
     CACCCGGATT
1651 CAAGCAGGCA ATCATGTTCG CATTGGTACA ACCCAAACTC
     AAAGCCAAAG
1701 CGAAACCTAT CATCAAACCC AAAAATCAGG ATTGATGAGT
     GCAGGTATCG
1751 GCTTCACTAT TGGCAGCAAG ACAAACACAC AAGAAAACCA
     ATCCCAAAGC
1801 AACGAACATA CAGGCAGTAC CGTAGGCAGC CTGAAAGGCG
     ATACCACCAT
1851 TGTTGCAAGC AAACACTACG AACAAACCGG CAGCAACGTT
     TCCAGCCCTG
1901 AGGGCAACAA CCTTATCAGC ACGCAAAGTA TGGATATTGG
     CGCAGCACAA
1951 AACCAATTAA ACAGCAAAAC CACCCAAACC TACGAACAAA
     AAGGCTTAAC
2001 GGTGGCATTC AGTTCGCCCG TTACCGATTT GGCACAACAA
     GCGATTGCCG
2051 TAGCACACAA AGCAGCAAAC AAGTCGGACA AAGCAAAAAC
     GACCGCGTTA
2101 ATGCCATGGC GGCTGCCAAT GCAGGTTGGC AGGCCTATCA
     AACAGGCAAA
2151 GGCGCACAAA ACTTAG

This corresponds to the amino acid sequence <SEQ ID 516; ORF115ng-1>:

1   LLVQTEKDGL HNEQTFGEKK VFSENGKLHN YWRARRKGHD
    ETGHREQNYT
51  LPEEITRDIS LGSFAYESHS KALSRHAPSQ GTELPQSNRD
    NIRTAKSNGI
101 SLPYTPNSFT PLPGSSLYII NPANKGYLVE TDPRFANYRQ
    WLGSDYMLGS
151 LKLDPNNLHK RLGDGYYEQR LINEQIAELT GHRRLDGYQN
    DEEQFKALMD
201 NGATAARSMN LSVGIALSAE QAAQLTSDIV WLVQKEVKLP
    DGGTQTVLMP
251 QVYVRVKNGG IDGKGALLSG SNTQINVSGS LKNSGTIAGR
    NALIINTDTL
301 DNIGGRIHAQ KSAVTATQDI NNIGGILSAE QTLLLNAGNN
    INNQSTAKSS
351 QNAQGSSTYL DRMAGIYITG KEKGVLAAQA GKDINIIAGQ
    ISNQSDQGQT
401 RLQAGRDINL DTVQTGKYQE IHFDADNHTI RGSTNEVGSS
    IQTKGDVTLL
451 SGNNLNAKAA EVGSAKGTLA VYAKNDITIS SGIHAGQVDD
    ASKHTGRSGG
501 GNKLVITDKA QSHHETAQSS TFEGKQVVLQ AGNDANILGS
    NVISDNGTRI
551 QAGNHVRIGT TQTQSQSETY HQTQKSGLMS AGIGFTIGSK
    TNTQENQSQS
601 NEHTGSTVGS LKGDTTIVAS KHYEQTGSNV SSPEGNNLIS
    TQSMDIGAAQ
651 NQLNSKTTQT YEQKGLTVAF SSPVTDLAQQ AIAVAHKAAN
    KSDKAKTTAL
701 MPWRLPMQVG RPIKQAKAHK T*

This gonococcal protein (ORF115ng-1) shows 91.9% identity with ORF115 over 334aa:

In addition, it shows homology with a secreted N. meningitidis protein in the database:

gi|2623258 (AF030941) putative secreted protein [Neisseria meningitidis]
Length = 2273
Score = 604 bits (1541), Expect = e−172
Identities = 325/678 (47%), Positives = 449/678 (65%), Gaps = 22/678 (3%)
Query:	1	LLVQTEKDGLHNEQTFGEKKVFSENGKLHNYWRARRKGHDETGHREQNYTLPEEITRDIS	60
		L+V T +  L N++T G K + ++ G LH Y R  +KG D TG+    Y    E++  I
Sbjct:	739	LIVGTPESALDNDETLGTKTI-TDKGDLHRYHRHHKKGRDSTGYSRSPYEPAPEVS-SIR	796
Query:	61	LGSFAYESHSKALSRHAPSQGTELPQSNRDNIRTAKSNGISLPYTPNSFTPLPGSSLYII	120
		+G  AY+ +       AP Q +++P +    +     NGI   +T      LP SSL+ I
Sbjct:	797	MGISAYKGY-------APQQASDIPGTV---VPVVAENGIHPTFT------LPNSSLFAI	840
Query:	121	NPANKGYLVETDPRFANYRQWLGSDYMLGSLKLDPNNLHKRLGDGYYEQRLINEQIAELT	180
		P NKGYL+ETDP F +YR+WLGS YML +L+ DPN++HKRLGDGYYEQ+L+NEQIA+LT
Sbjct:	841	APNNKGYLIETDPAFTDYRKWLGSGYMLAALQQDPNHIHKRLGDGYYEQKLVNEQIAKLT	900
Query:	181	GHRRLDGYQNDEEQFKALMDNGATAARSMNLSVGIALSAEQAAQLTSDIVWLVQKEVKLP	240
		G+RRLDGY NDEEQFKALMDNG T A+ + L+ GIALSAEQ A+LTSDIVWL  + V LP
Sbjct:	901	GYRRLDGYTNDEEQFKALMDNGITIAKELQLTPGIALSAEQVARLTSDIVWLENETVTLP	960
Query:	241	DGGTQTVLMPQVYVRVKNGGIDGKGALLSGSNTQINVSGSLKN-SGTIAGRNALIINTDT	299
		DG TQTVL P+VYVR +   ++G+GALLSGS   I  SG+++N  G IAGR ALI+N
Sbjct:	961	DGTTQTVLKPKVYVRARPKDMNGQGALLSGSVVDIG-SGAIENRGGLIAGREALILNAQN	1019
Query:	300	LDNIGGRIHAQKSAVTATQDINNIGGILSAEQTLLLNAGNNINNQSTAKSSQNAQGSSTY	359
		+ N+ G +  +     A  DI N G I  AE  LLL A NNI ++S  +S+QN QGS
Sbjct:	1020	IKNLQGDLQGKNIFAAAGSDITNTGSI-GAENALLLKASNNIESRSETRSNQNEQGSVRN	1078
Query:	360	LDRMAGIYITGKEKGVLAAQAGKDINIIAGQISNQSDQGQTRLQAGRDINLDTVQTGKYQ	419
		+ R+AGIY+TG++ G +   AG +I + A +++NQS+ GQT L AG DI  DT    + Q
Sbjct:	1079	IGRVAGIYLTGRQNGSVLLDAGNNIVLTASELTNQSEDGQTVLNAGGDIRSDTTGISRNQ	1138
Query:	420	EIHFDADNHTIRGSTNEVGSSIQTKGDVTLLSGNNLNAKAAEVGSAKGTLAVYAKNDITI	479
		FD+DN+ IR   NEVGS+I+T+G+++L +  ++  +AAEVGS + G L + A  DI +
Sbjct:	1139	NTIFDSDNYVIRKEQNEVGSTIRTRGNLSLNAKGDIRIRAAEVGSEQGRLKLAAGRDIKV	1198
Query:	480	SSGIHAGQVDDASKHTGRSGGGNKLVITDKAQSHHETAQSSTFEGKQVVLQAGNDANILG	539
		+G    + +DA K+TGRSGGG K  +T   ++ +  A S T +GK+++L +G D  + G
Sbjct:	1199	EAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQNGQAVSGTLDGKEIILVSGRDITVTG	1258
Query:	540	SNVISDNGTRIQAGNHVRIGTTQTQSQSETYHQTQKSGLM-SAGIGFTIGSKTNTQENQS	598
		SN+I+DN T + A N++ +   +T+S+S   ++ +KSGLM S GIGFT GSK +TQ N+S
Sbjct:	1259	SNIIADNHTILSAKNNIVLKAAETRSRSAEMNKKEKSGLMGSGGIGFTAGSKKDTQTNRS	1318
Query:	599	QSNEHTGSTVGSLKGDTTIVASKHYEQTGSNVSSPEGNNLISTQSMDIGAAQNQLNSKTT	658
		++  HT S VGSL G+T I A KHY QTGS +SSP+G+  IS+  + I AAQN+ + ++
Sbjct:	1319	ETVSHTESVVGSLNGNTLISAGKHYTQTGSTISSPQGDVGISSGKISIDAAQNRYSQESK	1378
Query:	659	QTYEQKGLTVAFSSPVTD	676
		Q YEQKG+TVA S PV +
Sbjct:	1379	QVYEQKGVTVAISVPVVN	1396

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 62

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 517>:

1   ..TCAGGGAATA ACCTCAATGC CAAAGCTGCC GAAGTCAGCA
    GCGCAAACGG
51  TACACTCGCT GTGTCTGCCA ATAATGACAT CAACATCAGC
    GCAGGCATCA
101 ACACGACCCA TGTTGATGAT GCGTCCAAAC ACACAGGCAG
    AAGCGGTGGT
151 GGCAATAAAT TAGTCATTAC CGATAAAGCC CAAAGTCATC
    ACGAAACCGC
201 CCAAAGCAGC ACCTTTGAAG GCAAGCAAGT TGTATTGCAG
    GCAGGAAACG
251 ATGCCAACAT CCTTGGCAGC AATGTTATTT CCGATAATGG
    CACCCAGATT
301 CAAGCAGGCA ATCATGTTCG CATTGGTACA ACCCAAACTC
    AAAGCCAAAG
351 CGAAACCTAT CATCAAACCC AGAAATCAGG ATTGATGAGT
    GCAGGTATCG
401 GCTTCACTAT TGGCAGCAAG ACAAACACAC AAGAAAACCA
    ATCCCAAAGC
451 AACGAACATA CAGGCAGTAC CGTAGGCAGC TTGAAAGGCG
    ATACCACCAT
501 TGTTGCAGGC AAACACTACG AACAAATCGG CAGTACCGTT
    TCCAGCCCGG
551 AAGGCAACAA TACCATCTAT GCCCAAAGCA TAGACATTCA
    AGCGGCACAC
601 AACAAATTAA ACAGTAATAC CACCCAAACC TATGAACAAA
    AAGG.CTAAC
651 GGTGGCATTC AGTTCGCCCG TTACCGATTT GGCACAACAA
    ...

This corresponds to the amino acid sequence <SEQ ID 518; ORF117>:

1   ..SGNNLNAKAA EVSSANGTLA VSANNDINIS AGINTTHVDD
    ASKHTGRSGG
51  GNKLVITDKA QSHHETAQSS TFEGKQVVLQ AGNDANILGS
    NVISDNGTQI
101 QAGNHVRIGT TQTQSQSETY HQTQKSGLMS AGIGFTIGSK
    TNTQENQSQS
151 NEHTGSTVGS LKGDTTIVAG KHYEQIGSTV SSPEGNNTIY
    AQSIDIQAAH
201 NKLNSNTTQT YEQKXLTVAF SSPVTDLAQQ ...

Computer analysis of this amino acid sequence gave the following results:

Homology with the pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF117 and pspA protein show 45% aa identity in 224aa overlap:

Orf117:	4	NLNAKAAEVSSANGTLAVSANNDINISAGINTTHVDDASKHTGRSGGGNKLVITDKAQSH	63
		++  +AAEV S  G L ++A  DI + AG   T  +DA K+TGRSGGG K  +T   ++
pspA:	1173	DIRIRAAEVGSEQGRLKLAAGRDIKVEAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQ	1232
Orf117:	64	HETAQSSTFEGKQVVLQAGNDANILGSNVISDNGTQIQAGNHVRIGTTQTQSQSETYHQT	123
		+  A S T +GK+++L +G D  + GSN+I+DN T + A N++ +   +T+S+S   ++
pspA:	1233	NGQAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILSAKNNIVLKAAETRSRSAEMNKK	1292
Orf117:	124	QKSGLM-SAGIGFTIGSKTNTQENQSQSNEHTGSTVGSLKGDTTIVAGKHYEQIGSTVSS	182
		+KSGLM S GIGFT GSK +TQ N+S++  HT S VGSL G+T I AGKHY Q GST+SS
pspA:	1293	EKSGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISS	1352
Orf117:	183	PEGNNTIYAQSIDIQAAHNKLNSNTTQTYEQKXLTVAFSSPVTD	226
		P+G+  I +  I I AA N+ +  + Q YEQK +TVA S PV +
pspA:	1353	PQGDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVN	1396

Homology with a Predicted ORF from N. gonorrhoeae

ORF117 shows 90% identity over a 230aa overlap with a predicted ORF (ORF117ng) from N. gonorrhoeae:

An ORF117ng nucleotide sequence <SEQ ID 519> was predicted to encode a protein having amino acid sequence <SEQ ID 520>:

1   ..LLVQTEKDGL HNEQTFGEKK VFSENGKLHN YWRARRKGHD
    ETGHREQNYT
51  LPEEITRDIS LGSFAYESHS KALSRHAPSQ GTELPQSNRD
    NIRTAKSNGI
101 SLPYTPNSFT PLPGSSLYII NPANKGYLVE TDPRFANYRQ
    WLGSDYMLGS
151 LKLDPNNLHK RLGDGYYEQR LINEQIAELT GHRRLDGYQN
    DEEQFKALMD
201 NGATAARSMN LSVGIALSAE QAAQLTSDIV WLVQKEVKLP
    DGGTQTVLMP
251 QVYVRVKNGG IDGKGALLSG SNTQINVSGS LKNSGTIAGR
    NALIINTDTL
301 DNIGGRIHAQ KSAVTATQDI NNIGGILSAE QTLLLNAGNN
    INNQSTAKSS
351 QNAQGSSTYL DRMAGIYITG KEKGVLAAQA GKDINIIAGQ
    ISNQSDQGQT
401 RLQAGRDINL DTVQTGKYQE IHFDADNHTI RGSTNEVGSS
    IQTKGDVTLL
451 SGNNLNAKAA EVGSAKGTLA VYAKNDITIS SGIHAGQVDD
    ASKHTGRSGG
501 GNKLVITDKA QSHHETAQSS TFEGKQVVLQ AGNDANILGS
    NVISDNGTRI
551 QAGNHVRIGT TQTQSQSETY HQTQKSGLMS AGIGFTIGSK
    TNTQENQSQS
601 NEHTGSTVGS LKGDTTIVAS KHYEQTGSNV SSPEGNNLIS
    TQSMDIGAAQ
651 NQLNSKTTQT YEQKGLTVAF SSPVTDLAQQ AIAVAHKAAK
    QFDKAKTTAL
701 MPWRLPMQVG RLFKQAKAPK K*

Further work revealed the following gonococcal partial DNA sequence <SEQ ID 521>:

1    TTGCTTGTGC AAACAGAAAA AGACGGTTTG CATAACGAGC
     AAACCTTTGG
51   CGAGAAGAAA GTCTTCAGCG AAAATGGTAA GTTGCACAAC
     TACTGGCGTG
101  CGCGTCGTAA AGGACATGAT GAAACAGGGC ATCGTGAACA
     AAATTATACT
151  TTGCCGGAGG AAATCACACG CGACATTTCA CTGGGTTCAT
     TTGCCTATGA
201  ATCGCATAGC AAAGCATTAA GCCGTCATGC GCCCAGCCAA
     GGCACTGAGT
251  TGCCACAAAG TAACCGGGAT AATATCCGTA CTGCGAAAAG
     CAACGGTATT
301  TCGCTACCCT ATACGCCCAA TTCTTTTACC CCATTACCCG
     GCAGCAGCTT
351  ATACATTATC AATCCTGCCA ATAAAGGCTA TCTTGTTGAA
     ACCGATCCAC
401  GCTTTGCCAA CTACCGTCAA TGGTTGGGTA GTGACTATAT
     GCTGGGCAGC
451  CTCAAACTAG ACCCAAACAA TTTACATAAA CGTTTGGGTG
     ATGGTTATTA
501  CGAGCAACGT TTAATCAATG AACAAATCGC AGAGCTGACA
     GGGCATCGTC
551  GTTTAGACGG TTATCAAAAC GACGAAGAAC AATTTAAAGC
     CTTAATGGAT
601  AATGGCGCGA CTGCGGCACG TTCGATGAAT CTCAGCGTTG
     GCATTGCATT
651  AAGTGCCGAG CAAGCAGCGC AACTGACCAG CGATATTGTT
     TGGTTGGTAC
701  AAAAAGAAGT TAAACTTCCT GATGGCGGCA CACAAACCGT
     ATTGATGCCA
751  CAGGTTTATG TACGCGTTAA AAATGGCGGC ATAGACGGTA
     AAGGTGCATT
801  GTTGTCAGGC AGCAATACAC AAATCAATGT TTCAGGCAGC
     CTGAAAAACT
851  CAGGCACGAT TGCAGGGCGC AATGCGCTTA TTATCAATAC
     CGATACGCTA
901  GACAATATCG GTGGGCGTAT TCATGCGCAA AAATCAGCGG
     TTACGGCCAC
951  ACAAGACATC AATAATATTG GCGGCATTCT TTCTGCCGAA
     CAGACATTAT
1001 TGCTCAATGC GGGTAACAAC ATCAACAACC AAAGCACGGC
     CAAGAGCAGT
1051 CAAAATGCAC AAGGTAGCAG CACCTACCTA GACCGAATGG
     CAGGTATTTA
1101 TATCACAGGC AAAGAAAAAG GTGTTTTAGC AGCGCAGGCA
     GGCAAAGACA
1151 TCAACATCAT TGCCGGTCAA ATCAGCAATC AATCAGATCA
     AGGGCAAACC
1201 CGGCTGCAGG CAGGACGCGA CATTAACCTG GATACGGTAC
     AAACCGGCAA
1251 ATATCAAGAA ATCCATTTTG ATGCCGATAA CCATACCATC
     CGAGGTTCAA
1301 CGAACGAAGT CGGCAGCAGC ATTCAAACAA AAGGCGATGT
     TACCCtatTG
1351 TCAGGGAATA ATCTCAATGC CAAAGCTGCC GAAGTCGGCA
     GCGCAAAAGG
1401 CACACTTGCC GTGTATGCTA AAAATGACAT TACTATCAGC
     TCAGGCATCC
1451 ATGCCGGCCA AGTTGATGAT GCGTCCAAAC ATACAGGCAG
     AAGCGGCGGC
1501 GGTAATAAAT TAGTCATTAC CGATAAAGCC CAAAGTCATC
     ACGAAACTGC
1551 TCAAAGCAGC ACCTTTGAAG GCAAGCAAGT TGTATTGCAG
     GCAGGAAACG
1601 ATGCCAACAT CCTTGGCAGT AATGTTATTT CCGATAATGG
     CACCCGGATT
1651 CAAGCAGGCA ATCATGTTCG CATTGGTACA ACCCAAACTC
     AAAGCCAAAG
1701 CGAAACCTAT CATCAAACCC AAAAATCAGG ATTGATGAGT
     GCAGGTATCG
1751 GCTTCACTAT TGGCAGCAAG ACAAACACAC AAGAAAACCA
     ATCCCAAAGC
1801 AACGAACATA CAGGCAGTAC CGTAGGCAGC CTGAAAGGCG
     ATACCACCAT
1851 TGTTGCAAGC AAACACTACG AACAAACCGG CAGCAACGTT
     TCCAGCCCTG
1901 AGGGCAACAA CCTTATCAGC ACGCAAAGTA TGGATATTGG
     CGCAGCACAA
1951 AACCAATTAA ACAGCAAAAC CACCCAAACC TACGAACAAA
     AAGGCTTAAC
2001 GGTGGCATTC AGTTCGCCCG TTACCGATTT GGCACAACAA
     GCGATTGCCG
2051 TAGCACACAA AGCAGCAAAC AAGTCGGACA AAGCAAAAAC
     GACCGCGTTA
2101 ATGCCATGGC GGCTGCCAAT GCAGGTTGGC AGGCCTATCA
     AACAGGCAAA
2151 GGCGCACAAA ACTTAG

This corresponds to the amino acid sequence <SEQ ID 522; ORF117ng-1>:

1   LLVQTEKDGL HNEQTFGEKK VFSENGKLHN YWRARRKGHD
    ETGHREQNYT
51  LPEEITRDIS LGSFAYESHS KALSRHAPSQ GTELPQSNRD
    NIRTAKSNGI
101 SLPYTPNSFT PLPGSSLYII NPANKGYLVE TDPRFANYRQ
    WLGSDYMLGS
151 LKLDPNNLHK RLGDGYYEQR LINEQIAELT GHRRLDGYQN
    DEEQFKALMD
201 NGATAARSMN LSVGIALSAE QAAQLTSDIV WLVQKEVKLP
    DGGTQTVLMP
251 QVYVRVKNGG IDGKGALLSG SNTQINVSGS LKNSGTIAGR
    NALIINTDTL
301 DNIGGRIHAQ KSAVTATQDI NNIGGILSAE QTLLLNAGNN
    INNQSTAKSS
351 QNAQGSSTYL DRMAGIYITG KEKGVLAAQA GKDINIIAGQ
    ISNQSDQGQT
401 RLQAGRDINL DTVQTGKYQE IHFDADNHTI RGSTNEVGSS
    IQTKGDVTLL
451 SGNNLNAKAA EVGSAKGTLA VYAKNDITIS SGIHAGQVDD
    ASKHTGRSGG
501 GNKLVITDKA QSHHETAQSS TFEGKQVVLQ AGNDANILGS
    NVISDNGTRI
551 QAGNHVRIGT TQTQSQSETY HQTQKSGLMS AGIGFTIGSK
    TNTQENQSQS
601 NEHTGSTVGS LKGDTTIVAS KHYEQTGSNV SSPEGNNLIS
    TQSMDIGAAQ
651 NQLNSKTTQT YEQKGLTVAF SSPVTDLAQQ AIAVAHKAAN
    KSDKAKTTAL
701 MPWRLPMQVG RPIKQAKAHK T*

ORF117ng-1 shows the same 90% identity over a 230aa overlap with ORF117. In addition, it shows homology with a secreted N. meningitidis protein in the database:

gi|2623258 (AF030941) putative secreted protein [Neisseria meningitidis]
Length = 2273
Score = 604 bits (1541), Expect = e−172
Identities = 325/678 (47%), Positives = 449/678 (65%), Gaps = 22/678 (3%)
Query:	1	LLVQTEKDGLHNEQTFGEKKVFSENGKLHNYWRARRKGHDETGHREQNYTLPEEITRDIS	60
		L+V T +  L N++T G K + ++ G LH Y R  +KG D TG+    Y    E++  I
Sbjct:	739	LIVGTPESALDNDETLGTKTI-TDKGDLHRYHRHHKKGRDSTGYSRSPYEPAPEVS-SIR	796
Query:	61	LGSFAYESHSKALSRHAPSQGTELPQSNRDNIRTAKSNGISLPYTPNSFTPLPGSSLYII	120
		+G  AY+ +       AP Q +++P +    +     NGI   +T      LP SSL+ I
Sbjct:	797	MGISAYKGY-------APQQASDIPGTV---VPVVAENGIHPTFT------LPNSSLFAI	840
Query:	121	NPANKGYLVETDPRFANYRQWLGSDYMLGSLKLDPNNLHKRLGDGYYEQRLINEQIAELT	180
		P NKGYL+ETDP F +YR+WLGS YML +L+ DPN++HKRLGDGYYEQ+L+NEQIA+LT
Sbjct:	841	APNNKGYLIETDPAFTDYRKWLGSGYMLAALQQDPNHIHKRLGDGYYEQKLVNEQIAKLT	900
Query:	181	GHRRLDGYQNDEEQFKALMDNGATAARSMNLSVGIALSAEQAAQLTSDIVWLVQKEVKLP	240
		G+RRLDGY NDEEQFKALMDNG T A+ + L+ GIALSAEQ A+LTSDIVWL  + V LP
Sbjct:	901	GYRRLDGYTNDEEQFKALMDNGITIAKELQLTPGIALSAEQVARLTSDIVWLENETVTLP	960
Query:	241	DGGTQTVLMPQVYVRVKNGGIDGKGALLSGSNTQINVSGSLKN-SGTIAGRNALIINTDT	299
		DG TQTVL P+VYVR +   ++G+GALLSGS   I  SG+++N  G IAGR ALI+N
Sbjct:	961	DGTTQTVLKPKVYVRARPKDMNGQGALLSGSVVDIG-SGAIENRGGLIAGREALILNAQN	1019
Query:	300	LDNIGGRIHAQKSAVTATQDINNIGGILSAEQTLLLNAGNNINNQSTAKSSQNAQGSSTY	359
		+ N+ G +  +     A  DI N G I  AE  LLL A NNI ++S  +S+QN QGS
Sbjct:	1020	IKNLQGDLQGKNIFAAAGSDITNTGSI-GAENALLLKASNNIESRSETRSNQNEQGSVRN	1078
Query:	360	LDRMAGIYITGKEKGVLAAQAGKDINIIAGQISNQSDQGQTRLQAGRDINLDTVQTGKYQ	419
		+ R+AGIY+TG++ G +   AG +I + A +++NQS+ GQT L AG DI  DT    + Q
Sbjct:	1079	IGRVAGIYLTGRQNGSVLLDAGNNIVLTASELTNQSEDGQTVLNAGGDIRSDTTGISRNQ	1138
Query:	420	EIHFDADNHTIRGSTNEVGSSIQTKGDVTLLSGNNLNAKAAEVGSAKGTLAVYAKNDITI	479
		FD+DN+ IR   NEVGS+I+T+G+++L +  ++  +AAEVGS +G L + A  DI +
Sbjct:	1139	NTIFDSDNYVIRKEQNEVGSTIRTRGNLSLNAKGDIRIRAAEVGSEQGRLKLAAGRDIKV	1198
Query:	480	SSGIHAGQVDDASKHTGRSGGGNKLVITDKAQSHHETAQSSTFEGKQVVLQAGNDANILG	539
		+G    + +DA K+TGRSGGG K  +T   ++ +  A S T +GK+++L +G D  + G
Sbjct:	1199	EAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQNGQAVSGTLDGKEIILVSGRDITVTG	1258
Query:	540	SNVISDNGTRIQAGNHVRIGTTQTQSQSETYHQTQKSGLM-SAGIGFTIGSKTNTQENQS	598
		SN+I+DN T + A N++ +   +T+S+S   ++ +KSGLM S GIGFT GSK +TQ N+S
Sbjct:	1259	SNIIADNHTILSAKNNIVLKAAETRSRSAEMNKKEKSGLMGSGGIGFTAGSKKDTQTNRS	1318
Query:	599	QSNEHTGSTVGSLKGDTTIVASKHYEQTGSNVSSPEGNNLISTQSMDIGAAQNQLNSKTT	658
		++  HT S VGSL G+T I A KHY QTGS +SSP+G+  IS+  + I AAQN+ + ++
Sbjct:	1319	ETVSHTESVVGSLNGNTLISAGKHYTQTGSTISSPQGDVGISSGKISIDAAQNRYSQESK	1378
Query:	659	QTYEQKGLTVAFSSPVTD	676
		Q YEQKG+TVA S PV +
Sbjct:	1379	QVYEQKGVTVAISVPVVN	1396

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 63

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 523>:

1   ATGATTTACA TCGTACTGTT TCTAGCTGTC GTCCTCGCCG
    TTGTCGCCTA
51  CAACATGTAT CAGGAAAACC AATACCGCAA AAAAGTGCGC
    GACCAGTTCG
101 GACACTCCGA CAAAGATGCC CTGCTCAACA GCAwAACCAG
    CCATGTCCGC
151 GACGGCAAAC CGTCCGGCGG GTCAGTCATG ATGCCGAAAC
    CCCAACCGGC
201 GGTCAAAAAA ACGGCAAAAC CCCAAGACCC CGyCATGCGC
    AACCTGCAAG
251 AACAGGATGC CGTCTACATC GCCAAGCAGA AACAGGCAAA
    AGCCTCCCCG
301 TTCAAAACCG AAATCGAAAC CGCCTTGGAA GAAAGCGGCA
    TTATCGGCAA
351 CTCCGCCCAC ACCGTTTCCG AACCCCAAAC CGGACATTCC
    GCAACGAAAC
401 CTGCCGACGC GTCGGCAAAA CCTGCACCCG TTCCGCAAAC
    ACCTGCAAAA
451 CCGCTGATTA CGCTCAAAGA ACTGTCAAAA GTCGAATTAT
    CCTGGTTTGA
501 CGTGCGCATC GACTTCATCT CCTAT...

This corresponds to the amino acid sequence <SEQ ID 524; ORF119>:

1   MIYIVLFLAV VLAVVAYNMY QENQYRKKVR DQFGHSDKDA
    LLNSXTSHVR
51  DGKPSGGSVM MPKPQPAVKK TAKPQDPXMR NLQEQDAVYI
    AKQKQAKASP
101 FKTEIETALE ESGIIGNSAH TVSEPQTGHS ATKPADASAK
    PAPVPQTPAK
151 PLITLKELSK VELSWFDVRI DFISY...

Further work revealed the complete nucleotide sequence <SEQ ID 525>:

1    ATGATTTACA TCGTACTGTT TCTAGCTGTC GTCCTCGCCG
     TTGTCGCCTA
51   CAACATGTAT CAGGAAAACC AATACCGCAA AAAAGTGCGC
     GACCAGTTCG
101  GACACTCCGA CAAAGATGCC CTGCTCAACA GCAAAACCAG
     CCATGTCCGC
151  GACGGCAAAC CGTCCGGCGG GTCAGTCATG ATGCCGAAAC
     CCCAACCGGC
201  GGTCAAAAAA ACGGCAAAAC CCCAAGACCC CGCCATGCGC
     AACCTGCAAG
251  AACAGGATGC CGTCTACATC GCCAAGCAGA AACAGGCAAA
     AGCCTCCCCG
301  TTCAAAACCG AAATCGAAAC CGCCTTGGAA GAAAGCGGCA
     TTATCGGCAA
351  CTCCGCCCAC ACCGTTTCCG AACCCCAAAC CGGACATTCC
     GCACCGAAAC
401  CTGCCGACGC GCCGGCAAAA CCTGCACCCG TTCCGCAAAC
     ACCTGCAAAA
451  CCGCTGATTA CGCTCAAAGA ACTGTCAAAA GTCGAATTAC
     CCTGGTTTGA
501  CGTGCGCTTC GACTTCATCT CCTATATCGC GCTGACCGAA
     GCCAAAGAAC
551  TGCACGCACT GCCGCGCCTT TCCAACCGCT GCCGCTACCA
     GATTGTCGGC
601  TGCACCATGG ACGACCATTT CCAGATTGCC GAACCCATCC
     CGGGCATCCG
651  CTATCAGGCA TTTATCGTGG GTATTCAGGC AGTCAGCCGC
     AACGGACTTG
701  CCTCGCAGGA AGAACTCTCC GCATTCAACC GCCAGGTGGA
     CGCATTCGCA
751  CAAAGCATGG GCGGTCAGAC GCTGCACACC GACCTTGCCG
     CCTTTATCGA
801  AGTGGCTTCC GCACTGGACG CATTCTGCGC GCGCGTCGAC
     CAGACCATCG
851  CCATCCATTT GGTTTCCCCG ACCAGCATCA GCGGCGTAGA
     ACTGCGTTCC
901  GCCGTAACGG GCGTGGGTTT CGTTTTGGAA GACGACGGCG
     CGTTCCACTA
951  TACCGACACG TCGGGCTCGA CCATGTTCTC CATCTGCTCG
     CTCAACAACG
1001 AGCCGTTTAC CAACGCCCTT TTGGACAACC AGTCCTACAA
     AGGCTTCAGT
1051 ATGCTGCTCG ACATCCCGCA CTCTCCGGCA GGCGAAAAAA
     CCTTCGACGA
1101 TTTGTTTATG GATTTGGCGG TACGCCTGTC CGGCCAGTTG
     AACCTGAATC
1151 TGGTCAACGA CAAAATGGAA GAAGTTTCGA CCCAATGGCT
     CAAAGACGTG
1201 CGCACTTATG TATTGGCGCG TCAGTCCGAG ATGCTCAAAG
     TCGGTATCGA
1251 ACCGGGCGGC AAAACCGCAT TGCGCCTGTT CTCCTAA

This corresponds to the amino acid sequence <SEQ ID 526; ORF119-1>:

1   MIYIVLFLAV VLAVVAYNMY QENQYRKKVR DQFGHSDKDA
    LLNSKTSHVR
51  DGKPSGGSVM MPKPQPAVKK TAKPQDPAMR NLQEQDAVYI
    AKQKQAKASP
101 FKTEIETALE ESGIIGNSAH TVSEPQTGHS APKPADAPAK
    PAPVPQTPAK
151 PLITLKELSK VELPWFDVRF DFISYIALTE AKELHALPRL
    SNRCRYQIVG
201 CTMDDHFQIA EPIPGIRYQA FIVGIQAVSR NGLASQEELS
    AFNRQVDAFA
251 QSMGGQTLHT DLAAFIEVAS ALDAFCARVD QTIAIHLVSP
    TSISGVELRS
301 AVTGVGFVLE DDGAFHYTDT SGSTMFSICS LNNEPFTNAL
    LDNQSYKGFS
351 MLLDIPHSPA GEKTFDDLFM DLAVRLSGQL NLNLVNDKME
    EVSTQWLKDV
401 RTYVLARQSE MLKVGIEPGG KTALRLFS*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF119 shows 93.7% identity over. a 175aa overlap with an ORF (ORF119a) from strain A of N. meningitidis:

The complete length ORF119a nucleotide sequence <SEQ ID 527> is:

1    ATGATTTACA TCGTACTGTT CCTCGCCGCC GTCCTCGCCG
     TTGTCGCCTA
51   CAATATGTAT CAGGAAAACC AATACCGCAA AAAAGTGCGC
     GACCAGTTCG
101  GGCACTCCGA CAAAGATGCC CTGCTCAACA GCAAAACCAG
     CCATGTCCGC
151  GACGGCAAAC CGTCCGGCGG GCCAGTCATG ATGCCGAAAC
     CCCAACCGGC
201  GGTCAAAAAA ACGGCAAAAT CCCAAGACCC CGCCATGCGC
     AACCTGCAAG
251  AGCAGGATGC CGTCTACATC GCCAAGCAGA AACAGGCAAA
     AGCCTCCCCG
301  TTCAAAACCG AAATCGAAAC CGCCTTGGAA GAAAGCGGCA
     TTATCGGCAA
351  CTCCGCCCAC ACCGTTCCCG AACCCCAAAC CGGACATTCC
     GCACCAAAAC
401  CTGCCGACGC GCCGGCAAAA CCTGTTCCCG TTCCGCAAAC
     GCCGGCAAAA
451  CCGCTGATTA CGCTCAAAGA GCTGTCGAAG GTCGAGCTGC
     CCTGGTTTGA
501  CGTGCGCTTC GACTTCATCT CTTATATCGC GCTGACCGAA
     GCCAAAGAAC
551  TGCACGCACT GCCGCGCCTT TCCAACCGCT GCCGCTACCA
     GATTGTCGGC
601  TGCACCATGG ACGACCATTT CCAGATTGCC GAACCCATCC
     CGGGCATCCG
651  CTATCAGGCA TTTATCGTGG GTATTCAGGC AGTCAGCCGC
     AACGGACTTG
701  CCTCGCAGGA AGAACTCTCC GCATTCAACC GCCAGGTGGA
     TGCATTCGCA
751  CACAGCATGG GCGGTCAGAC GCTGCACACC GACCTTGCCG
     CCTTTATCGA
801  AGTGGCTTCC GCACTGGACG CATTCTGCGC GCGCGTCGAC
     CAGACTATCG
851  CCATCCATTT GGTTTCCCCG ACCAGCATCA GCGGCGTAGA
     ACTGCGTTCC
901  GCCGTAACGG GCGTGGGTTT CGTTTTGGAA GACGACGGCG
     CGTTCCACTA
951  TACCGACACG TCGGGCTCGA CCATGTTCTC CATCTGCTCG
     CTCAACAACG
1001 AGCCGTTTAC CAATGCCCTT TTGGACAACC AGTCCTATAA
     AGGCTTCAGT
1051 ATGCTGCTCG ACATCCCGCA CTCTCCGGCA GGCGAAAAAA
     CCTTCGACGA
1101 TTTGTTTATG GATTTGGCGG TACGCCTGTC CGGCCAGTTG
     AACCTGAATC
1151 TGGTCAACGA CAAAATGGAA GAAGTTTCGA CCCAATGGCT
     CAAAGACGTG
1201 CGCACTTATG TATTGGCTCG TCAGTCCGAG ATGCTCAAAG
     TCGGTATCGA
1251 ACCGGGCGGC AAAACCGCAT TGCGCCTGTT CTCCTAA

This encodes a protein having amino acid sequence <SEQ ID 528>:

1   MIYIVLFLAA VLAVVAYNMY QENQYRKKVR DQFGHSDKDA
    LLNSKTSHVR
51  DGKPSGGPVM MPKPQPAVKK TAKSQDPAMR NLQEQDAVYI
    AKQKQAKASP
101 FKTEIETALE ESGIIGNSAH TVPEPQTGHS APKPADAPAK
    PVPVPQTPAK
151 PLITLKELSK VELPWFDVRF DFISYIALTE AKELHALPRL
    SNRCRYQIVG
201 CTMDDHFQIA EPIPGIRYQA FIVGIQAVSR NGLASQEELS
    AFNRQVDAFA
251 HSMGGQTLHT DLAAFIEVAS ALDAFCARVD QTIAIHLVSP
    TSISGVELRS
301 AVTGVGFVLE DDGAFHYTDT SGSTMFSICS LNNEPFTNAL
    LDNQSYKGFS
351 MLLDIPHSPA GEKTFDDLFM DLAVRLSGQL NLNLVNDKME
    EVSTQWLKDV
401 RTYVLARQSE MLKVGIEPGG KTALRLFS*

ORF119a and ORF119-1 show 98.6% identity in 428 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF119 shows 93.1% identity over a 175aa overlap with a predicted ORF (ORF119ng) from N. gonorrhoeae:

The complete length ORF119ng nucleotide sequence <SEQ ID 529> is:

1    ATGATTTACA TCGTACTGTT CCTCGCCGCC GTCCTCGCCG
     TTGTCGCCTA
51   CAATATGTAT CAGGAAAACC AATACCGCAA AAAAGTGCGC
     GACCAGTTCG
101  GACACTCCGA CAAAGATGCC CTGCTCAACA GCAAAACCAG
     CCATGTCCGC
151  GACGGCAAAC CGTCCGGCGG GCCAGTCATG ATGCCGAAAC
     CCCAACCGGC
201  GGTCAAAAAA CCGGCCAAAC CCCAAGACTC CGCCATGCGC
     AACCTGCAAG
251  AACAGGATGC CGTCTACATC GCCAAGCAGA AACAGGCAAA
     AGCCTCCCCG
301  TTCAAAACCG AAATCGAAAC CGCCTTGGAA GAAATCGGCA
     TTATCGGCAA
351  CTCCGCCCAC ACCGTTTCCG AACCCCAAAC CGGACATTCC
     GCACCGAAAC
401  CTGCCGACGC GCCGGCAAAA CCCGTTCCCG TTCCGCAAAC
     GCCGGCAAAA
451  CCGCTGATTA CGCTCAAAGA GCTGTCGAAG GTCGAGCTGC
     CCTGGTTTGA
501  CGTGCGCTtc gACTTCATCT CCTATATCGC GCTGACCGAA
     GCCAAAGAAC
551  TGCACGCACT GCCGCGCCTT tccAACCGCT GCCGCTACCA
     GATTGTCGGC
601  TGCACCATGG ACGACCATTT CCAGATTGCC GAACCCATCC
     CGGGCATCCG
651  CTATCAGGCA TTTATCGTGG GTATCCAGGC AGTCAGCCGC
     AACGGACTTG
701  CCTCGCAGGA AGAACTCTCC GCATTCAACC GCCAGGCGGA
     CGCATTCGCA
751  CAAAGCATGG GCGGTCAGAC GCTGCACACC GACCTTGCCG
     CCTTTATCGA
801  AGTGGCTTCC GCACTGGACG CATTCTGCGC GCGCGTCGAC
     CAGACCATCG
851  CCATCCATTT GGTTTCGCCG ACCAGCATCA GCGGCGTAGA
     ACTGCGTTCC
901  GCCGTAACGG GCGTGGGTTT CGTTTTGGAA GACGACGGCG
     CGTTCCACTA
951  TACCGACACG TCGGGCTCGA CCATGTTCTC CATCTGCTCG
     CTCAACAACG
1001 AGCCGTTTAC CAATGCCCTT TTGGACAACC AGTCCTACAA
     AGGCTTCAGT
1051 ATGCTGCTCG ACATCCCGCA CTCTCCGGCA GGCGAAAAAA
     CCTTCGACGA
1101 TTTGTTTATG GATTTGGCGG TACGCCTGTC CGGTCAGTTG
     AACCTGAATC
1151 TGGTCAACGA CAAAATGGAA GAAGTTTCGA CCCAATGGCT
     CAAAGACGTA
1201 CGCACTTATG TATTGGCGCG TCAGTCCGAG ATGCTCAAAG
     TCGGTATCGA
1251 ACCGGGCGGC AAAACCGCCC TGCGCCTGTT TTCATAA

This encodes a protein having amino acid sequence <SEQ ID 530>:

1   MIYIVLFLAA VLAVVAYNMY QENQYRKKVR DQFGHSDKDA
    LLNSKTSHVR
51  DGKPSGGPVM MPKPQPAVKK PAKPQDSAMR NLQEQDAVYI
    AKQKQAKASP
101 FKTEIETALE EIGIIGNSAH TVSEPQTGHS APKPADAPAK
    PVPVPQTPAK
151 PLITLKELSK VELPWFDVRF DFISYIALTE AKELHALPRL
    SNRCRYQIVG
201 CTMDDHFQIA EPIPGIRYQA FIVGIQAVSR NGLASQEELS
    AFNRQADAFA
251 QSMGGQTLHT DLAAFIEVAS ALDAFCARVD QTIAIHLVSP
    TSISGVELRS
301 AVTGVGFVLE DDGAFHYTDT SGSTMFSICS LNNEPFTNAL
    LDNQSYKGFS
351 MLLDIPHSPA GEKTFDDLFM DLAVRLSGQL NLNLVNDKME
    EVSTQWLKDV
401 RTYVLARQSE MLKVGIEPGG KTALRLFS*

ORF119ng and ORF119-1 show 98.4% identity over 428 aa overlap:

Based on this analysis, including the presence of a putative leader sequence in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 64

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 531>

1   ..GCGCGGCACG GCACGGAAGA TTTCTTCATG AACAACAGCG
    ACAC.ATCAG
51  GCAGATAGTC GAAAGCACCA CCGGTACGAT GAAGCTGCTG
    ATTTCCTCCA
101 TCGCCCTGAT TTCATTGGTA GTCGGCGGCA TCGGCGTGAT
    GAACATCATG
151 CTGGTGTCCG TTACCGAGCG CACCAAAGAA ATCGGCATAC
    GGATGGCAAT
201 CGGCGCGCGG CGCGGCAATA TTTyGCAGCA GTTTTTGATT
    GAGGCGGTGT
251 TAATCTGCGT CATCGGCGGT TTGGTCGGCG TGGGTTTGTC
    CGCCGCCGTC
301 AGCCTCGTGT TCAATCATTT TGTAACCGAC TTCCCGATGG
    ACATTTCCGC
351 CATGTCCGTC ATCGGCGCGG TCGCCTGTTC GACCGGAATC
    GGCATCGCGT
401 TCGGCTTTAT GCCTGCCAAT AAAGCAGCCA AACTCAATCC
    GATAGACGCA
451 TTGGCACAGG ATTGA

This corresponds to the amino acid sequence <SEQ ID 532; ORF134>:

1   ..ARHGTEDFFM NNSDXIRQIV ESTTGTMKLL ISSIALISLV
    VGGIGVMNIM
51  LVSVTERTKE IGIRMAIGAR RGNIXQQFLI EAVLICVIGG
    LVGVGLSAAV
101 SLVFNHFVTD FPMDISAMSV IGAVACSTGI GIAFGFMPAN
    KAAKLNPIDA
151 LAQD*

Further work revealed the complete nucleotide sequence <SEQ ID 533>:

1    ATGTCGGTGC AAGCAGTATT GGCGCACAAA ATGCGTTCGC
     TTCTGACGAT
51   GCTCGGCATC ATCATCGGTA TCGCGTCGGT GGTTTCCGTC
     GTCGCATTGG
101  GCAATGGTTC GCAGAAAAAA ATCCTTGAAG ACATCAGTTC
     GATAGGGACG
151  AACACCATCA GCATCTTCCC GGGGCGCGGC TTCGGCGACA
     GGCGCAGCGG
201  CAGGATTAAA ACCCTGACCA TAGACGACGC AAAAATCATC
     GCCAAACAAA
251  GCTACGTTGC TTCCGCCACG CCCATGACTT CGAGCGGCGG
     CACGCTGACT
301  TACCGCAACA CCGACCTGAC CGCCTCGCTT TACGGCGTGG
     GCGAACAATA
351  TTTCGACGTG CGCGGACTGA AGCTGGAAAC GGGGCGGCTG
     TTTGACGAAA
401  ACGATGTGAA AGAAGACGCG CAGGTCGTCG TCATCGACCA
     AAATGTCAAA
451  GACAAACTCT TTGCGGACTC GGATCCGTTG GGTAAAACCA
     TTTTGTTCAG
501  GAAACGCCCC TTGACCGTCA TCGGCGTGAT GAAAAAAGAC
     GAAAACGCTT
551  TCGGCAATTC CGACGTGCTG ATGCTTTGGT CGCCCTATAC
     GACGGTGATG
601  CACCAAATCA CAGGCGAGAG CCACACCAAC TCCATCACCG
     TCAAAATCAA
651  AGACAATGCC AATACCCAGG TTGCCGAAAA AGGGCTGACC
     GATCTGCTCA
701  AAGCGCGGCA CGGCACGGAA GATTTCTTCA TGAACAACAG
     CGACAGCATC
751  AGGCAGATAG TCGAAAGCAC CACCGGTACG ATGAAGCTGC
     TGATTTCCTC
801  CATCGCCCTG ATTTCATTGG TAGTCGGCGG CATCGGCGTG
     ATGAACATCA
851  TGCTGGTGTC CGTTACCGAG CGCACCAAAG AAATCGGCAT
     ACGGATGGCA
901  ATCGGCGCGC GGCGCGGCAA TATTTTGCAG CAGTTTTTGA
     TTGAGGCGGT
951  GTTAATCTGC GTCATCGGCG GTTTGGTCGG CGTGGGTTTG
     TCCGCCGCCG
1001 TCAGCCTCGT GTTCAATCAT TTTGTAACCG ACTTCCCGAT
     GGACATTTCC
1051 GCCATGTCCG TCATCGGCGC GGTCGCCTGT TCGACCGGAA
     TCGGCATCGC
1101 GTTCGGCTTT ATGCCTGCCA ATAAAGCAGC CAAACTCAAT
     CCGATAGACG
1151 CATTGGCACA GGATTGA

This corresponds to the amino acid sequence <SEQ ID 534; ORF134-1>:

1   MSVQAVLAHK MRSLLTMLGI IIGIASVVSV VALGNGSQKK
    ILEDISSIGT
51  NTISIFPGRG FGDRRSGRIK TLTIDDAKII AKQSYVASAT
    PMTSSGGTLT
101 YRNTDLTASL YGVGEQYFDV RGLKLETGRL FDENDVKEDA
    QVVVIDQNVK
151 DKLFADSDPL GKTILFRKRP LTVIGVMKKD ENAFGNSDVL
    MLWSPYTTVM
201 HQITGESHTN SITVKIKDNA NTQVAEKGLT DLLKARHGTE
    DFFMNNSDSI
251 RQIVESTTGT MKLLISSIAL ISLVVGGIGV MNIMLVSVTE
    RTKEIGIRMA
301 IGARRGNILQ QFLIEAVLIC VIGGLVGVGL SAAVSLVFNH
    FVTDFPMDIS
351 AMSVIGAVAC STGIGIAFGF MPANKAAKLN PIDALAQD*

Computer analysis of this amino acid sequence gave the following results:

Homology with the Hypothetical Protein o648 of E. coli (Accession Number AE000189)

ORF134 and o648 protein show 45% aa identity in 153aa overlap:

Orf134:   2 RHGTEDFFMNNSDXIRQIVESTTGTMKXXXXXXXXXXXVVGGIGVMNIMLVSVTERTKEI  61
RHG +DFFN   D + + VE TT T++            VVGGIGVMNIMLVSVTERT+EI
o648:   496 RHGKKDFFTWNMDGVLKTVEKTTRTLQLFLTLVAVISLVVGGIGVMNIMLVSVTERTREI 555
Orf134:  62 GIRMAIGARRGNIXQQFLIEAXXXXXXXXXXXXXXXXXXXXXFNHFVTDFPMDISAMSVI 121
GIRMA+GAR  ++ QQFLIEA                        F+ + +   S ++++
o648:   556 GIRMAVGARASDVLQQFLIEAVLVCLVGGALGITLSLLIAFTLQLFLPGWEIGFSPLALL 615
Orf134: 122 GAVACSTGIGIAFGFMPANKAAKLNPIDALAQD                            154
A  CST  GI FG++PA  AA+L+P+DALA++
o648:   616 LAFLCSTVTGILFGWLPARNAARLDPVDALARE                            648

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF134 shows 98.7% identity over a 154aa overlap with an ORF (ORF134a) from strain A of N. meningitidis:

The complete length ORF134a nucleotide sequence <SEQ ID 535> is:

1    ATGTCGGTGC AAGCAGTATT GGCGCACAAA ATGCGTTCGC
     TTCTGACGAT
51   GCTCGGCATC ATCATCGGTA TCGCTTCGGT TGTCTCCGTC
     GTCGCATTGG
101  GCAACGGTTC GCAGAAAAAA ATCCTTGAAG ACATCAGTTC
     GATAGGGACG
151  AACACCATCA GCATCTTCCC AGGGCGCGGC TTCGGCGACA
     GGCGCAGCGG
201  CAGGATTAAA ACCCTGACCA TAGACGACGC AAAAATCATC
     GCCAAACAAA
251  GCTACGTTGC TTCCGCCACG CCCATGACTT CGAGCGGCGG
     CACGCTGACT
301  TACCGCAATA CCGACCTGAC CGCTTCTTTG TACGGTGTGG
     GCGAACAATA
351  TTTCGACGTG CGCGGGCTGA AGCTGGAAAC GGGGCGGCTG
     TTTGACGAAA
401  ACGATGTGAA AGAAGACGCG CAGGTCGTCG TCATCGACCA
     AAATGTCAAA
451  GACAAACTCT TTGCGGACTC GGATCCGTTG GGTAAAACCA
     TTTTGTTCAG
501  GAAACGCCCC TTGACCGTCA TCGGCGTGAT GAAAAAAGAC
     GAAAACGCTT
551  TCGGCAATTC CGACGTGCTG ATGCTTTGGT CGCCCTATAC
     GACGGTGATG
601  CACCAAATCA CAGGCGAGAG CCACACCAAC TCCATCACCG
     TCAAAATCAA
651  AGACAATGCC AATACCCAGG TTGCCGAAAA AGGGCTGACC
     GATCTGCTCA
701  AAGCGCGGCA CGGCACGGAA GATTTCTTCA TGAACAACAG
     CGACAGCATC
751  AGGCAGATAG TCGAAAGCAC CACCGGTACG ATGAAGCTGC
     TGATTTCCTC
801  CATCGCCCTG ATTTCATTGG TAGTCGGCGG CATCGGCGTG
     ATGAACATCA
851  TGCTGGTGTC CGTTACCGAG CGCACCAAAG AAATCGGCAT
     ACGGATGGCA
901  ATCGGCGCGC GGCGCGGCAA TATTTTGCAG CAGTTTTTGA
     TTGAGGCGGT
951  GTTAATCTGC GTCATCGGCG GTTTGGTCGG CGTGGGTTTG
     TCCGCCGCCG
1001 TCAGCCTCGT GTTCAATCAT TTTGTAACCG ACTTCCCGAT
     GGACATTTCC
1051 GCCATGTCCG TCATCGGCGC GGTCGCCTGT TCGACCGGAA
     TCGGCATCGC
1101 GTTCGGCTTT ATGCCTGCCA ATAAAGCAGC CAAACTCAAT
     CCGATAGATG
1151 CATTGGCGCA GGATTGA

This encodes a protein having amino acid sequence <SEQ ID 536>:

1   MSVQAVLAHK MRSLLTMLGI IIGIASVVSV VALGNGSQKK
    ILEDISSIGT
51  NTISIFPGRG FGDRRSGRIK TLTIDDAKII AKQSYVASAT
    PMTSSGGTLT
101 YRNTDLTASL YGVGEQYFDV RGLKLETGRL FDENDVKEDA
    QVVVIDQNVK
151 DKLFADSDPL GKTILFRKRP LTVIGVMKKD ENAFGNSDVL
    MLWSPYTTVM
201 HQITGESHTN SITVKIKDNA NTQVAEKGLT DLLKARHGTE
    DFFMNNSDSI
251 RQIVESTTGT MKLLISSIAL ISLVVGGIGV MNIMLVSVTE
    RTKEIGIRMA
301 IGARRGNILQ QFLIEAVLIC VIGGLVGVGL SAAVSLVFNH
    FVTDFPMDIS
351 AMSVIGAVAC STGIGIAFGF MPANKAAKLN PIDALAQD*

ORF134a and ORF134-1 show 100.0% identity in 388 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF134 shows 96.8% identity over a 154aa overlap with a predicted ORF (ORF 134.ng) from N. gonorrhoeae:

The complete length ORF134ng nucleotide sequence <SEQ ID 537> is:

1    ATGTCGGTGC AAGCAGTATT GGCGCACAAA ATGCGTTCGC
     TTCTGACCAT
51   GCTCGGCATC ATCATCGGTA TCGCTTCGGT TGTCTCCGTC
     GTCGCGCTGG
101  GCAACGGTTC GCAGAAAAAA ATCCTCGAAG ACATCAGTTC
     GATGGGGACG
151  AACACCATCA GCATCTTCCC CGGGCGCGGC TTCGGCGACA
     GGCGCAGCGG
201  CAAAATCAAA ACCCTGACCA TAGACGACGC AAAAATCATC
     GCCAAACAAA
251  GCTACGTTGC CTCCGCCACG CCCATGACTT CGAGCGGCGG
     CACGCTGACC
301  TACCGCAATA CCGACCTGAC CGCTTCTTTG TACGGTGTGG
     GCGAACAATA
351  TTTCGACGTG CGCGGGCTGA AGCTGGAAAC GGGGCGGCTG
     TTTGATGAGA
401  ACGATGTGAA AGAAGACGCG CAAGTCGTCG TCATCGACCA
     AAATGTCAAA
451  GACAAACTCT TTGCGGACTC GGATCCGTTG GGTAAAACCA
     TTTTGTTCAG
501  GAAACGCCCC TTGACCGTCA TCGGCGTGAT GAAAAAAGAC
     GAAAACGCTT
551  TCGGCAATTC CGACGTGCTG ATGCTTTGGT CGCCCTATAC
     GACGGTGATG
601  CACCAAATCA CAGGCGAGAG CCACACCAAC TCCATCACCG
     TCAAAATCAA
651  AGACAATGCC AATACCCGGG TTGCCGAAAA AGGGCTGGCC
     GAGCTGCTCA
701  AAGCACGGCA CGGCACGGAA GACTTCTTTA TGAACAACAG
     CGACAGCATC
751  AGGCAGATGG TCGAAAGCAC CACCGGTACG ATGAAGCTGC
     TGATTTCCTC
801  CATCGCCCTG ATTTCATTGG TAGTCGGCGG CATCGGTGTG
     ATGAACATTA
851  TGCTGGTGTC CGTTACCGAG CGCACCAAAG AAATCGGCAT
     ACGGATGGCA
901  ATCGGCGCGC GGCGCGGCAA TATTTTGCAG CAGTTTTTGA
     TTGAGGCGGT
951  GTTAATCTGC ATCATCGGAG GCTTGGTCGG CGTAGGTTTG
     TCCGCCGCCG
1001 TCAGCCTCGT GTTCAATCAT TTTGTAACCG ATTTCCCGAT
     GGACATTTCG
1051 GCGGCATCCG TTATCGGGGC GGTCGCCTGT TCGACCGGAA
     TCGGCATCGC
1101 GTTCGGCTTT ATGCCTGCCA ATAAGGCAGC CAAACTCAAT
     CCGATAGATG
1151 CATTGGCGCA GGATTGA

This encodes a protein having amino acid sequence <SEQ ID 538>:

1   MSVQAVLAHK MRSLLTMLGI IIGIASVVSV VALGNGSQKK
    ILEDISSMGT
51  NTISIFPGRG FGDRRSGKIK TLTIDDAKII AKQSYVASAT
    PMTSSGGTLT
101 YRNTDLTASL YGVGEQYFDV RGLKLETGRL FDENDVKEDA
    QVVVIDQNVK
151 DKLFADSDPL GKTILFRKRP LTVIGVMKKD ENAFGNSDVL
    MLWSPYTTVM
201 HQITGESHTN SITVKIKDNA NTRVAEKGLA ELLKARHGTE
    DFFMNNSDSI
251 RQMVESTTGT MKLLISSIAL ISLVVGGIGV MNIMLVSVTE
    RTKEIGIRMA
301 IGARRGNILQ QFLIEAVLIC IIGGLVGVGL SAAVSLVFNH
    FVTDFPMDIS
351 AASVIGAVAC STGIGIAFGF MPANKAAKLN PIDALAQD*

ORF134ng and ORF134-1 show 97.9% identity in 388 aa overlap:

ORF134ng also shows homology to an E. coli ABC transporter:

sp|P75831|YBJZ_ECOLI HYPOTHETICAL ABC TRANSPORTER ATP-BINDING
PROTEIN YBJZ >gi5 (AE000189) o648; similar to YBBA_HAEIN SW: P45247
[Escherichia coli] Length = 648
Score = 297 bits (753), Expect = 6e−80
Identities = 162/389 (41%), Positives = 230/389 (58%), Gaps = 1/389 (0%)
Query:	1	MSVQAVLAHKMRSLLTMLXXXXXXXXXXXXXXLGNGSQKKILEDISSMGTNTISIFPGRG	60
		M+ +A+ A+KMR+LLTML              +G+ +++ +L DI S+GTNTI ++PG+
Sbjct:	260	MAWRALAANKMRTLLTMLGIIIGIASVVSIVVVGDAAKQMVLADIRSIGTNTIDVYPGKD	319
Query:	61	FGDRRSGKIKTLTIDDAKIIAKQSYVASATPMTSSGGTLTYRNTDLTASLYGVGEQYFDV	120
		FGD      + L  DD   I KQ +VASATP  S    L Y N D+ AS  GV   YF+V
Sbjct:	320	FGDDDPQYQQALKYDDLIAIQKQPWVASATPAVSQNLRLRYNNVDVAASANGVSGDYFNV	379
Query:	121	RGLKLETGRLFDENDVKEDAQVVVIDQNVKDKLFAD-SDPLGKTILFRKRPLTVIGVMKK	179
		G+    G  F++  +   AQVVV+D N + +LF   +D +G+ IL    P  VIGV ++
Sbjct:	380	YGMTFSEGNTFNQEQLNGRAQVVVLDSNTRRQLFPHKADVVGEVILVGNMPARVIGVAEE	439
Query:	180	DENAFGNSDVLMLWSPYTTVMHQITGESHTNSITVKIKDNANTRVAEKGLAELLKARHGT	239
		++ FG+S VL +W PY+T+  ++ G+S  NSITV++K+  ++  AE+ L  LL  REG
Sbjct:	440	KQSMFGSSKVLRVWLPYSTMSGRVMGQSWLNSITVRVKEGFDSAEAEQQLTRLLSLRHGK	499
Query:	240	EDFFMNNSDSIRQMVESTTGTMKXXXXXXXXXXXVVGGIGVMNIMLVSVTERTKEIGIRM	299
		+DFF  N D + + VE TT T++           VVGGIGVMNIMLVSVTERT+EIGIRM
Sbjct:	500	KDFFTWNMDGVLKTVEKTTRTLQLFLTLVAVISLVVGGIGVMNIMLVSVTERTREIGIRM	559
Query:	300	AIGARRGNILQQFLIEXXXXXXXXXXXXXXXXXXXXXXFNHFVTDFPMDISAASVIGAVA	359
		A+GAR  ++LQQFLIE                         F+  + +  S  +++ A
Sbjct:	560	AVGARASDVLQQFLIEAVLVCLVGGALGITLSLLIAFTLQLFLPGWEIGFSPLALLLAFL	619
Query:	360	CSTGIGIAFGFMPANKAAKLNPIDALAQD	388
		CST  GI FG++PA  AA+L+P+DALA++
Sbjct:	620	CSTVTGILFGWLPARNAARLDPVDALARE	648

Based on this analysis, including the presence of the leader peptide and transmembrane regions in the gonococcal protein, it is prediceted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 65

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 539>:

1   ..GGGACGGGAG CGATGCTGCT GCTGTTTTAC GCGGTAACGA
    T.CTGCCTTT
51  GGCCACTGGC GTTACCCTGA GTTACACCTC GTCGATTTTT
    TTGGCGGTAT
101 TTTCCTTCCT GATTTTGAAA GAACGGATTT CCGTTTACAC
    GCAGGCGGTG
151 CTGCTCCTTG GTTTTGCCGG CGTGGTATTG CTGCTTAATC
    CCTCGTTCCG
201 CAGCGGTCAG GAAACGGCGG CACTCGCCGG GCTGGCGGGC
    GGCGCGATGT
251 CCGGCTGGGC GTATTTGAAA GTGCGCGAAC TGTCTTTGGC
    GGGCGAACCC
301 GGCTGGCGCG TCGTGTTTTA CCTTTCCGTG ACAGGTGTGG
    CGATGTCGTC
351 GGTTTGGGCG ACGCTGACCG GCTGGCACAC CCTGTCCTTT
    CCATCGGCAG
401 TTTATCTGTC GTGCATCGGC GTGTCCGCGC TGATTGCCCA
    ACTGTCGATG
451 ACGCGCGCCT ACAAAGTCGG CGACAAATTC ACGGTTGCCT
    CGCTTTCCTA
501 TATGACCGTC GTTTTTTCCG CTCTGTCTGC CGCATTTTTT
    CTGGGCGAAG
551 AGCTTTTCTG GCAGGAAATA CTCGGTATGT GCATCATCAT
    CCTCAGCGGT
601 ATTTTGA

This corresponds to the amino acid sequence <SEQ ID 540; ORF135>:

1   ..GTGAMLLLFY AVTILPLATG VTLSYTSSIF LAVFSFLILK
    ERISVYTQAV
51  LLLGFAGVVL LLNPSFRSGQ ETAALAGLAG GAMSGWAYLK
    VRELSLAGEP
101 GWRVVFYLSV TGVAMSSVWA TLTGWHTLSF PSAVYLSCIG
    VSALIAQLSM
151 TRAYKVGDKF TVASLSYMTV VFSALSAAFF LGEELFWQEI
    LGMCIIISAV
201 F*

Further work revealed the complete nucleotide sequence <SEQ ID 541>:

1   ATGGATACCG CAAAAAAAGA CATTTTAGGA TCGGGCTGGA
    TGCTGGTGGC
51  GGCGGCCTGC TTTACCATTA TGAACGTATT GATTAAAGAG
    GCATCGGCAA
101 AATTTGCCCT CGGCAGCGGC GAATTGGTCT TTTGGCGCAT
    GCTGTTTTCA
151 ACCGTTGCGC TCGGGGCTGC CGCCGTATTG CGTCGGGACA
    mCTTCCGCAC
201 GCCCCATTGG AAAAACCACT TAAACCGCAG TATGGTCGGG
    ACGGGGGCGA
251 TGCTGCTGCT GTTTTACGCG GTAACGCATC TGCCTTTGGC
    CACTGGCGTT
301 ACCCTGAGTT ACACCTCGTC GATTTTTTTG GCGGTATTTT
    CCTTCCTGAT
351 TTTGAAAGAA CGGATTTCCG TTTACACGCA GGCGGTGCTG
    CTCCTTGGTT
401 TTGCCGGCGT GGTATTGCTG CTTAATCCCT CGTTCCGCAG
    CGGTCAGGAA
451 ACGGCGGCAC TCGCCGGGCT GGCGGGCGGC GCGATGTCCG
    GCTGGGCGTA
501 TTTGAAAGTG CGCGAACTGT CTTTGGCGGG CGAACCCGGC
    TGGCGCGTCG
551 TGTTTTACCT TTCCGTGACA GGTGTGGCGA TGTCGTCGGT
    TTGGGCGACG
601 CTGACCGGCT GGCACACCCT GTCCTTTCCA TCGGCAGTTT
    ATCTGTCGTG
651 CATCGGCGTG TCCGCGCTGA TTGCCCAACT GTCGATGACG
    CGCGCCTACA
701 AAGTCGGCGA CAAATTCACG GTTGCCTCGC TTTCCTATAT
    GACCGTCGTT
751 TTTTCCGCTC TGTCTGCCGC ATTTTTTCTG GGCGAAGAGC
    TTTTCTGGCA
801 GGAAATACTC GGTATGTGCA TCATCATCCT CAGCGGTATT
    TTGAGCAGCA
851 TCCGCCCCAC TGCCTTCAAA CAGCGGCTGC AATCCCTGTT
    CCGCCAAAGA
901 TAA

This corresponds to the amino acid sequence <SEQ ID 542; ORF135-1>:

1   MDTAKKDILG SGWMLVAAAC FTIMNVLIKE ASAKFALGSG
    ELVFWRMLFS
51  TVALGAAAVL RRDXFRTPHW KNHLNRSMVG TGAMLLLFYA
    VTHLPLATGV
101 TLSYTSSIFL AVFSFLILKE RISVYTQAVL LLGFAGVVLL
    LNPSFRSGQE
151 TAALAGLAGG AMSGWAYLKV RELSLAGEPG WRVVFYLSVT
    GVAMSSVWAT
201 LTGWHTLSFP SAVYLSCIGV SALIAQLSMT RAYKVGDKFT
    VASLSYMTVV
251 FSALSAAFFL GEELFWQEIL GMCIIILSGI LSSIRPTAFK
    QRLQSLFRQR
301 *

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF135 shows 99.0% identity over a 197aa overlap with an ORF (ORF135a) from strain A of N. meningitidis:

The complete length ORF135a nucleotide sequence <SEQ ID 543> is:

1   ATGGATACCG CAAAAAAAGA CATTTTAGGA TCGGGCTGGA
    TGCTGGTGGC
51  GGCGGCCTGC TTTACCATTA TGAACGTATT GATTAAAGAG
    GCATCGGCAA
101 AATTTGCCCT CGGCAGCGGC GAATTGGTCT TTTGGCGCAT
    GCTGTTTTCA
151 ACCGTTGCGC TCGGGGCTGC CGCCGTATTG CGTCGGGACA
    CCTTCCGCAC
201 GCCCCATTGG AAAAACCACT TAAACCGCAG TATGGTCGGG
    ACGGGGGCGA
251 TGCTGCTGCT GTTTTACGCG GTAACGCATC TGCCTTTGGC
    CACCGGCGTT
301 ACCCTGAGTT ACACCTCGTC GATTTTTTTG GCGGTATTTT
    CCTTCCTGAT
351 TTTGAAAGAA CGGATTTCCG TTTACACGCA GGCGGTGCTG
    CTCCTTGGTT
401 TTGCCGGCGT GGTATTGCTG CTTAATCCCT CGTTCCGCAG
    CGGTCAGGAA
451 ACGGCGGCAC TCGCCGGGCT GGCGGGCGGC GCGATGTCCG
    GCTGGGCGTA
501 TTTGAAAGTG CGCGAACTGT CTTTGGCGGG CGAACCCGGC
    TGGCGCGTCG
551 TGTTTTACCT TTCCGTGACA GGTGTGGCGA TGTCATCGGT
    TTGGGCGACG
601 CTGACCGGCT GGCACACCCT GTCCTTTCCA TCGGCAGTTT
    ATCTGTCGTG
651 CATCGGCGTG TCCGCGCTGA TTGCCCAACT GTCGATGACG
    CGCGCCTACA
701 AAGTCGGCGA CAAATTCACG GTTGCCTCGC TTTCCTATAT
    GACCGTCGTT
751 TTTTCCGCTC TGTCTGCCGC ATTTTTTCTG GCCGAAGAGC
    TTTTCTGGCA
801 GGAAATACTC GGTATGTGCA TCATCATCCT CAGCGGTATT
    TTGAGCAGCA
851 TCCGCCCCAC TGCCTTCAAA CAGCGGCTGC AATCCCTGTT
    CCGCCAAAGA
901 TAA

This encodes a protein having amino acid sequence <SEQ ID 544>:

1   MDTAKKDILG SGWMLVAAAC FTIMNVLIKE ASAKFALGSG
    ELVFWRMLFS
51  TVALGAAAVL RRDTFRTPHW KNHLNRSMVG TGAMLLLFYA
    VTHLPLATGV
101 TLSYTSSIFL AVFSFLILKE RISVYTQAVL LLGFAGVVLL
    LNPSFRSGQE
151 TAALAGLAGG AMSGWAYLKV RELSLAGEPG WRVVFYLSVT
    GVAMSSVWAT
201 LTGWHTLSFP SAVYLSCIGV SALIAQLSMT RAYKVGDKFT
    VASLSYMTVV
251 FSALSAAFFL AEELFWQEIL GMCIIILSGI LSSIRPTAFK
    QRLQSLFRQR
301 *

ORF135a and ORF135-1 show 99.3% identity in 300 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF135 shows 97% identity over a 201aa overlap with a predicted ORF (ORF135ng) from N. gonorrhoeae:

An ORF135ng nucleotide sequence <SEQ ID 545> was predicted to encode a protein having amino acid sequence <SEQ ID 546>:

1   MPSEKAFRRH LRTASFQGLH LHHFHQKVGK CGIIGFGIHI
    FPTLLPAAQG
51  ILDIQLGLFR IDFAALAVYR RTQVDFIHTV IDGIASDQAF
    SEVVQILRRL
101 NLGHFTDTHL IAQARRFIAD FGNIRPMRRG EAKTFCRCFR
    FDGIDGIHGD
151 FRQCGHINRL APGKDCRNGK RDKVFFHTRH YNQVCLEKTN
    CSARKIKFRH
201 QKQAKTHSTS LAARFTIRPS LSQRPFMDTA KKDILGSGWM
    LVAAACFTVM
251 NVLIKEASAK FALGSGELVF WRMLFSTVTL GAAAVLRRDT
    FRTPHWKNHL
301 NRSMVGTGAM LLLFYAVTHL PLTTGVTLSY TSSIFLAVFS
    FLILKERISV
351 YTQAVLLLGF AGVVLLLNPS FRSGQEPAAL AGLAGGAMSG
    WAYLKVRELS
401 LAGEPGWRVV FYLSATGVAM SSVWATLTGW HTLSFPSAVY
    LSGIGVSALI
451 AQLSMTRAYK VGDKFTVASL SYMTVVFSAL SAAFFLGEEL
    FWQEILGMCI
501 IISAAF*

Further work revealed the following gonococcal sequence <SEQ ID 547>:

1   ATGGATACCG CAAAAAAAGA CATTTTAGGA TCGGGCTGGA
    TGCTGGTGGC
51  GGCGGCCTGC TTCACCGTTA TGAACGTATT GATTAAAGAG
    GCATCGGCAA
101 AATTTGCCCT CGGCAGCGGC GAATTGGTCT TTTGGCGCAT
    GCTGTTTTCA
151 ACCGTTACGC TCGGTGCTGC CGCCGTATTG CGGCGCGACA
    CCTTCCGCAC
201 GCCCCATTGG AAAAACCACT TAAACCGCAG TATGGTCGGG
    ACGGGGGCGA
251 TGCTGCTGCT GTTTTACGCG GTAACGCATC TGCCTTTGAC
    AACCGGCGTT
301 ACCCTGAGTT ACACCTCGTC GATTTTTttg GCGGTATTTT
    CCTTCCTCAT
351 TTTGAAAGAA CGGATTTCCG TTTACACGCA GGCGGTGCTG
    CTCCTTGGTT
401 TTGCCGGCGT GGTATTGCTG CTTAATCCCT CGTTCCGCAG
    CGGTCAGGAA
451 CCGGCGGCAC TCGCCGGGCT GGCGGGCGGC GCGATGTCCG
    GCTGGGCGTA
501 TTTGAAAGTG CGCGAACTGT CTTTGGCGGG CGAACCCGGC
    TGGCGCGTCG
551 TGTTTTACCT TTCCGCAACC GGCGTGGCGA TGTCGTCggt
    ttgggcgacg
601 Ctgaccggct ggCACAcccT GTCCTTTcca tcggcagttt
    ATCtgtCGGG
651 CATCGGCGTG tccgcgCtgA TTGCCCAaCT GtcgatgAcg
    cGCGcctaca
701 aaGTCGGCGA CAAATTCACG GTTGCCTCGC tttcctaTAt
    gaccgtcGTC
751 TTTTCCGCCC TGTCTGCCGC ATTTTTTCTg ggcgaagagc
    tttTCtggCA
801 GGAAATACTC GGTATGTGCA TCATTAtccT CAGCGGCATT
    TTGAGCAGCA
851 TCCGCCCCAT TGCCTTCAAA CAGCGGCTGC AAGCCCTCTT
    CCGCCAAAGA
901 TAA

This corresponds to the amino acid sequence <SEQ ID 548; ORF135ng-1>:

1   MDTAKKDILG SGWMLVAAAC FTVMNVLIKE ASAKFALGSG
    ELVFWRMLFS
51  TVTLGAAAVL RRDTFRTPHW KNHLNRSMVG TGAMLLLFYA
    VTHLPLTTGV
101 TLSYTSSIFL AVFSFLILKE RISVYTQAVL LLGFAGVVLL
    LNPSFRSGQE
151 PAALAGLAGG AMSGWAYLKV RELSLAGEPG WRVVFYLSAT
    GVAMSSVWAT
201 LTGWHTLSFP SAVYLSGIGV SALIAQLSMT RAYKVGDKFT
    VASLSYMTVV
251 FSALSAAFFL GEELFWQEIL GMCIIILSGI LSSIRPIAFK
    QRLQALFRQR
301 *

ORF135ng-1 and ORF135-1 show 97.0% identity in 300 aa overlap:

Based on this analysis, including the presence of several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 66

The following DNA sequence was identified in N. meningitidis <SEQ ID 549>:

1   ATGAAGCGGC GTATAGCCGT CTTCGTCCTG TTCCCGCAGA
    TAATCCGAGT
51  TTTGGGACAA CTGTTGCCGA AAATCGTCAA TACAGTTCCG
    GCACATCGGA
101 TGCTCTTCCA GATTTTCGGG ATGTTCTTTT TCTTCATACA
    CCAGCAATAT
151 CTGCCCGGGA TCGCCGAAAT CGATTCCCCA TGCGGCATCG
    TGTTCGGTGC
201 GCTCCTCTTC CGTCATCTGC CCGCGCATTG CCTGTATGGT
    AAAGCCGCCG
251 TAGGGGATGC CgTTGCACAC GAACATCCAG TCGCTGATGT
    CGTCAACCGG
301 AACGCAAACG cTTTCGCCTT GTTCGACATT GGTCAGTTCG
    CCsGGTTCAT
351 TGTTCAGCAC ACCGTAAATA TAAAGACCGT CAAAATAAAT
    ATCGTCGATC
401 CACATATGTT CGCAAATTTC GCCGTCTTCG CCGTCTTGGA
    AAAAAGGGAC
451 TTTGACCATG GCAAAATCCA AGGCGGAAAT AATGCGGCGG
    CGTTCCCAAA
501 AAAGcTCGCG CCAAAAATAT TTGAATGTTT TACGGGCGCG
    TTCGTCGGCA
551 CGGTTTACCG GTTCGTCTGC CTGTTCTACA TAATAAATGA
    CGGAATCGCC
601 CATCATATCT GCTCCTCAAC GTGTACGGTA TCTGTTTGCA
    CCTTACTGCG
651 GCTTTCTgcC kTCGGCATCC GATTCGGATT TGAAAAGTTC
    mmrwyATTCG
701 GAATAG

This corresponds to the amino acid sequence <SEQ ID 550; ORF136>:

1   MKRRIAVFVL FPQIIRVLGQ LLPKIVNTVP AHRMLFQIFG
    MFFFFIHQQY
51  LPGIAEIDSP CGIVFGALLF RHLPAHCLYG KAAVGDAVAH
    EHPVADVVNR
101 NANAFALFDI GQFAXFIVQH TVNIKTVKIN IVDPHMFANF
    AVFAVLEKRD
151 FDHGKIQGGN NAAAFPKKLA PKIFECFTGA FVGTVYRFVC
    LFYIINDGIA
201 HHSAPQRVRY LFAPYCGFLP SASDSDLKSS XXSE*

Further work revealed the complete nucleotide sequence <SEQ ID 551>:

1   ATGATGAAGC GGCGTATAGC CGTCTTCGTC CTGTTCCCGC
    AGATAATCCG
51  AGTTTTGGGA CAACTGTTGC CGAAAATCGT CAATACAGTT
    CCGGCACATC
101 GGATGCTCTT CCAGATTTTC GGGATGTTCT TTTTCTTCAT
    ACACCAGCAA
151 TATCTGCCCG GGATCGCCGA AATCGATTCC CCATGCGGCA
    TCGTGTTCGG
201 TGCGCTCCTC TTCCGTCATC TGCCCGCGCA TTGCCTGTAT
    GGTAAAGCCG
251 CCGTAGGGGA TGCCGTTGCA CACGAACATC CAGTCGCTGA
    TGTCGTCAAC
301 CGGAACGCAA ACGCTTTCGC CTTGTTCGAC ATTGGTCAGT
    TCGCCGGGTT
351 CATTGTTCAG CACACCGTAA ATATAAAGAC CGTCAAAATA
    AATATCGTCG
401 ATCCACATAT GTTCGCAAAT TTCGCCGTCT TCGCCGTCTT
    GGAAAAAAGG
451 GACTTTGACC ATGGCAAAAT CCAAGGCGGA AATAATGCGG
    CGGCGTTCCC
501 AAAAAAGCTC GCGCCAAAAA TATTTGAATG TTTTACGGGC
    GCGTTCGTCG
551 GCACGGTTTA CCGGTTCGTC TGCCTGTTCT ACATAATAAA
    TGACGGAATC
601 GCCCATCATT CTGCTCCTCA ACGTGTACGG TATCTGTTTG
    CACCTTACTG
651 CGGCTTTCTG CCTTCGGCAT CCGATTCGGA TTTGAAAAGT
    TCCAAATATT
701 CGGAATAG

This corresponds to the amino acid sequence <SEQ ID 552; ORF136-1>:

1   MMKRRIAVFV LFPQIIRVLG QLLPKIVNTV PAHRMLFQIF
    GMFFFFIHQQ
51  YLPGIAEIDS PCGIVFGALL FRHLPAHCLY GKAAVGDAVA
    HEHPVADVVN
101 RNANAFALFD IGQFAGFIVQ HTVNIKTVKI NIVDPHMFAN
    FAVFAVLEKR
151 DFDHGKIQGG NNAAAFPKKL APKIFECFTG AFVGTVYRFV
    CLFYIINDGI
201 AHHSAPQRVR YLFAPYCGFL PSASDSDLKS SKYSE*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF136 shows 71.7% identity over. a 237aa overlap with an ORF (ORF136a) from strain A of N. meningitidis:

The complete length ORF136a nucleotide sequence <SEQ ID 553> is:

1   ATGATGAAGC GGCGTATAGC CGTCTTCGTC CTGCTCATGC
    AGAAAATCCG
51  GATTTTGGGA CAACTGTTGC CGAAAATCGT CAATACAGTT
    CCGGCACATC
101 GGATGCTCTT CCAGATNTTC GGGATGTTCT TTTTCTTCAT
    ACACCAGCAA
151 TACCTGCCCG GGATCGCCGA AATCGATTCC CCATGCGGCA
    TCGTGTTCGG
201 TACGCTCCTC TTCCGTCATC NGTCCACGCA TTGCCTGTAT
    GGTAAAGCCG
251 CCGTAGGGAA TGCCGTTGCA CACGAACATC CAGTCGCTGA
    TGTCGTCAAC
301 CGGAACGCAA ACGCTTTCGC CTTGTTCGAC ATTGGTCAGT
    TCGCCGGGTT
351 CATTGTTCAG CACGCCATAA ATGTAAAGAC CGTCAAAATA
    AATATCGTCG
401 ATCCACATAT GTTCGCAAAT TTCGCCNTCT TCGCCGTCTT
    GGAAAAAAGG
451 GCTTTGACCA TGGCAAAATC TAAGGNGNNA NNGATGCGGC
    GGCGTTCCCA
501 AAAAAGCTCG CGCCAAAAAT ATTTGAATGT TTTGCGGGCG
    CGTTCGCCGG
551 CACGGTTTAC CGGTTTGTCT GCCTGTTCTA CATAATAAAT
    GACGGAATCG
601 CCCATCATAT CTGCTCCTCA ACGTGTACGG TATCTGTTTG
    CACCTTACTG
651 CGGCTTTCTG CCTTCGGCAT CCGATTCGGA TTTGAAAAGT
    TCCAAATATT
701 CGGAATAG

This encodes a protein having amino acid sequence <SEQ ID 554>:

1   MMKRRIAVFV LLMQKIRILG QLLPKIVNTV PAHRMLFQXF
    GMFFFFIHQQ
51  YLPGIAEIDS PCGIVFGTLL FRHXSTHCLY GKAAVGNAVA
    HEHPVADVVN
101 RNANAFALFD IGQFAGFIVQ HAINVKTVKI NIVDPHMFAN
    FAXFAVLEKR
151 ALTMAKSKXX XMRRRSQKSS RQKYLNVLRA RSPARFTGLS
    ACST**MTES
201 PIISAPQRVR YLFAPYCGFL PSASDSDLKS SKYSE*

ORF136a and ORF136-1 show 73.1% identity in 238 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF136 shows 92.3% identity over a 234aa overlap with a predicted ORF (ORF136ng) from N. gonorrhoeae:

The complete length ORF136ng nucleotide sequence <SEQ ID 555> is:

1   ATGATGAAGC GGCGTATAGC CGTCTTCGTC CTGCTCATGC
    AGAAAATCCG
51  GATTTTGGGA CAACTGTTGC CGAAAATCGT CAATACAGTT
    CCGGCACATC
101 GGATGCTCTT CCAAATTTTC GGGATGTTCT TTTTCTTCAT
    ACACCGGCAA
151 TACCTGCCCG GGATCGCCGA AATCGATTCC CCAGGCGGTA
    TCGTGTTCGG
201 TACGCTCCTC TTCCGTCATC TGTCCGCGCA TTGCCTGTAC
    GGTAAAGCCG
251 CCGTAGGGGA TGCCGTTGCA CACGAACATC CAGTCGCTGA
    TGTCGCCAAC
301 CGGAACGCAA ACGCTTTCGC CTTGTTCGAC ATTGGTCAGT
    CCGCCGGGTT
351 CATTGTTCAG CACACCGTAA ATATAAAGAC CGTCAAAATA
    AATATCGTCG
401 ATCCACATAT GTTCGCAAAT TTCGCCGTCT TCGCCGTCTT
    GGAAAAAAGG
451 GACTTTGACC ATGGCAAAAT CCAAGGCGGA AATAATGCGG
    CGGCGTTCCC
501 AAAAAAGCTC GCGCCAAAAG TATTTGAATG TTTTACGGGC
    GCGTTCGCCG
551 GCACGGTTTA CCGGTTCGTC TGCCTGTTCT ACATAATAAA
    TGACGGAATC
601 GCCCATCATA CTGCTCCTCA ACGTGTACGG TATCTGTTTG
    CACCTTACCG
651 CGGTTTTCTA CCTCCGGCAT CCGATTCGGA TTTGAAAAGT
    TCCAAATATT
701 CGGAATAG

This encodes a protein having amino acid sequence <SEQ ID 556>:

1   MMKRRIAVFV LLMQKIRILG QLLPKIVNTV PAHRMLFQIF
    GMFFFFIHRQ
51  YLPGIAEIDS PGGIVFGTLL FRHLSAHCLY GKAAVGDAVA
    HEHPVADVAN
101 RNANAFALFD IGQSAGFIVQ HTVNIKTVKI NIVDPHMFAN
    FAVFAVLEKR
151 DFDHGKIQGG NNAAAFPKKL APKVFECFTG AFAGTVYRFV
    CLFYIINDGI
201 AHHTAPQRVR YLFAPYRGFL PPASDSDLKS SKYSE*

ORF136ng and ORF136-1 show 93.6% identity in 235 aa overlap:

Based on the presence of the putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 67

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 557>:

1   ATGGAAAATA TGGTAACGTT TTCAAAAATC AGACCGCTTT
    TGGCAATCGC
51  CGCCGCCGCG TTGCTTGCCG CC.TGCGGAC GGCGGGAAAT
    AATGCTGTCC
101 GCAAGCCGGT GCAAACCGCC AAACCCGCCG CAGTGGTCGG
    TTTGGCACTC
151 GGTGGCGGCG CATCTAAAGG ATTTGCCCAT GTAGGTATTA
    TTAAGGTTTT
201 GAAAGAAAAC GGTATTCCTG TGAAGGTGGT TACCGGCACC
    TCCGCAGGTT
251 CGATTGTCGG CAACCTTTTT GCATCGGGTA TGTCGCCCGA
    CCGCCTCGAA
301 TTGGAAGCCG AAATTTTAGG CAAAACCGAT TTGGTCGATT
    TAACCTTGTC
351 CACCAATGGG TTTATCAAAG GCGCAAAGCT GCAAAATTAC
    ATCAACCGAA
401 AACTCCGCGG CATGCAGATT CAGCAGTTTC CCATCAAATT
    TGCCGCC..

This corresponds to the amino acid sequence <SEQ ID 558; ORF137>:

1   MENMVTFSKI RPLLAIAAAA LLAAXRTAGN NAVRKPVQTA
    KPAAVVGLAL
51  GGGASKGFAH VGIIKVLKEN GIPVKVVTGT SAGSIVGNLF
    ASGMSPDRLE
101 LEAEILGKTD LVDLTLSTNG FIKGAKLQNY INRKLRGMQI
    QQFPIKFAA..

Further work revealed the complete nucleotide sequence <SEQ ID 559>:

1   ATGGAAAATA TGGTAACGTT TTCAAAAATC AGACCGCTTT
    TGGCAATCGC
51  CGCCGCCGCG TTGCTTGCCG CCTGCGGCAC GGCGGGAAAT
    AATGCTGTCC
101 GCAAGCCGGT GCAAACCGCC AAACCCGCCG CAGTGGTCGG
    TTTGGCACTC
151 GGTGGCGGCG CATCTAAAGG ATTTGCCCAT GTAGGTATTA
    TTAAGGTTTT
201 GAAAGAAAAC GGTATTCCTG TGAAGGTGGT TACCGGCACA
    TCGGCAGGTT
251 CGATTGTCGG CAGCCTTTTT GCATCGGGTA TGTCGCCCGA
    CCGCCTCGAA
301 TTGGAAGCCG AAATTTTAGG CAAAACCGAT TTGGTCGATT
    TAACCTTGTC
351 CACCAGTGGT TTTATCAAAG GCGAAAAGCT GCAAAATTAC
    ATCAACCGAA
401 AAGTCGGCGG CAGGCAGATT CAGCAGTTTC CCATCAAATT
    TGCCGCCGTT
451 GCTACTGATT TTGAAACCGG CAAGGCCGTC GCTTTCAATC
    AGGGGAATGC
501 CGGGCAGGCT GTGCGCGCTT CCGCCGCCAT TCCCAATGTG
    TTCCAACCCG
551 TTATCATCGG CAGGCATACA TATGTTGACG GCGGTCTGTC
    GCAGCCCGTG
601 CCCGTCAGTG CCGCCCGGCG GCAGGGGGCG AATTTCGTGA
    TTGCCGTCGA
651 TATTTCCGCC CGTCCGGGCA AAAACATCAG CCAAGGTTTC
    TTCTCTTATC
701 TCGATCAGAC GCTGAACGTA ATGAGCGTTT CTGCGTTGCA
    AAATGAGTTG
751 GGGCAGGCGG ATGTGGTTAT CAAACCGCAG GTTTTGGATT
    TGGGTGCAGT
801 CGGCGGATTC GATCAGAAAA AACGCGCCAT CCGGTTGGGT
    GAGGAGGCAG
851 CACGTGCCGC ATTGCCTGAA ATCAAACGCA AACTGGCGGC
    ATACCGTTAT
901 TGA

This corresponds to the amino acid sequence <SEQ ID 560; ORF137-1>:

1   MENMVTFSKI RPLLAIAAAA LLAACGTAGN NAVRKPVQTA
    KPAAVVGLAL
51  GGGASKGFAH VGIIKVLKEN GIPVKVVTGT SAGSIVGSLF
    ASGMSPDRLE
101 LEAEILGKTD LVDLTLSTSG FIKGEKLQNY INRKVGGRQI
    QQFPIKFAAV
151 ATDFETGKAV AFNQGNAGQA VRASAAIPNV FQPVIIGRHT
    YVDGGLSQPV
201 PVSAARRQGA NFVIAVDISA RPGKNISQGF FSYLDQTLNV
    MSVSALQNEL
251 GQADVVIKPQ VLDLGAVGGF DQKKRAIRLG EEAARAALPE
    IKRKLAAYRY
301 *

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF137 shows 93.3% identity over a 149aa overlap with an ORF (ORF137a) from strain A of N. meningitidis:

The complete length ORF137a nucleotide sequence <SEQ ID 561> is:

1   ATGGAAAATA TGGTAACGTT TTCAAAAATC AGACCGCTTT
    TGGCAATCGC
51  CGCCGCCGCG TTGCTTGCCG CCTGCGGCAC GGCGGGAAAT
    AATGCTGCCC
101 GCAAGCCGGT GCAAACCGCC AAACCCGCCG CAGTGGTCGG
    TTTGGCACTC
151 GGTGGCGGCG CATCTAAAGG ATTTGCCCAT GTAGGTATTA
    TTAAGGTTTT
201 GAAAGAAAAC GGTATTCCTG TGAAGGTGGT TACCGGCACA
    TCGGCAGGTT
251 CGATAGTCGG CAGCCTTTTT GCATCGGGTA TGTCGCCCGA
    CCGCCTCGAA
301 TTGGAAGCCG AAATTTTAGG TAAAACCGAT TTGGTCGATT
    TAACCTTGTC
351 CACCAGTGGT TTTATCAAAG GCGAAAAGCT GCAAAATTAC
    ATCAACCGAA
401 AAGTCGGCGG CAGGCGGATT CAGCAGTTTC CCATCAAATT
    TGCCGCCGTT
451 GCTACTGATT TTGAAACCGG CAAGGCCGTC GCTTTCAATC
    AAGGGAATGC
501 CGGGCAGGCT GTGCGCGCTT CCGCCGCCAT TCCCAATGTG
    TTCCAACCCG
551 TTATCATCGG CAGGCATACA TATGTTGACG GCGGTCTGTC
    GCAGCCCGTG
601 CCCGTCAGTG CCGCCCGGCG GCANGNNNNG NATNTCGTGA
    TTGCCGTCGA
651 TATTTCCGCC CGTCCGAGCA AAAACATCAG CCAAGGCTTC
    TTCTCTTATC
701 TCGATCAGAC GCTGAACGTA ATGAGCGTTT CCGCGTTGCA
    AAATGAGTTG
751 GGGCAGGCGG ATGTGGTTAT CAAACCGCAG GTTTTGGATT
    TGGGTGCAGT
801 CGGCGGATTC GATCAGAAAA AACGCGCCAT CCGGTTGGGT
    GAGGAGGCAG
851 CACGTGCCGC ATTGCCTGAA ATCAAACGCA AACTGGCGGC
    ATACCGTTAT
901 TGA

This encodes a protein having amino acid sequence <SEQ ID 562>:

1   MENMVTFSKI RPLLAIAAAA LLAACGTAGN NAARKPVQTA
    KPAAVVGLAL
51  GGGASKGFAH VGIIKVLKEN GIPVKVVTGT SAGSIVGSLF
    ASGMSPDRLE
101 LEAEILGKTD LVDLTLSTSG FIKGEKLQNY INRKVGGRRI
    QQFPIKFAAV
151 ATDFETGKAV AFNQGNAGQA VRASAAIPNV FQPVIIGRHT
    YVDGGLSQPV
201 PVSAARRXXX XXVIAVDISA RPSKNISQGF FSYLDQTLNV
    MSVSALQNEL
251 GQADVVIKPQ VLDLGAVGGF DQKKRAIRLG EEAARAALPE
    IKRKLAAYRY
301 *

ORF137a and ORF137-1 show 97.3% identity in 300 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF137 shows 89.9% identity over a 149aa overlap with a predicted ORF (ORF137ng) from N. gonorrhoeae:

The complete length ORF137ng nucleotide sequence <SEQ ID 563> is:

1   ATGGAAAATA TGGTAACGTT TTCAAAAATC AGATCATTTT
    TGGCAATCGC
51  CGCCGCCGCG TTGCTTGCCG CCTGCGGTAC GGCGGGAAAC
    AATGCCGCCC
101 GCAAGCCGGT GCAAACCGCC AAACCCGCCG CAGTGGTCGC
    TTTGGCACTC
151 GGTGGCGGCG CATCTAAAGG ATTTGCCCAT ATAGGAATTG
    TTAAGGTTTT
201 GAAAGAAAAC GGTATTCCTG TGAAGGTGGT TACCGGCACA
    TCGGCAGGTT
251 CGATAGTCGG CAGCCTTTTG GCATCGGGTA TGTCGCCCGA
    CCGCCTCGAA
301 TTGGAAGCCG AGATTTTAGG TAAAACCGAT TTAGTCGATT
    TAACCTTGTC
351 CACCAGTGGT TTTATCAAAG GCGAAAAGCT GCAAAATTAC
    ATCAACCGAA
401 AAGTCGGCGG CAGGCAGATT CAGCAGTTTC CCATCAAATT
    TGCCGCCGTT
451 GCCACTGATT TTGAAACCGG CAAGGCCGTC GCTTTCAATC
    AAGGGAATGC
501 CGGGCAGGCG GTTCGTGCTT CCGCCGCCAT TCCCAATGTG
    TTCCAGCCAG
551 TCATCATCGG CAGGCACAAA TATGTTGACG GCGGTCTGTC
    GCAGCCCGTG
601 CCCGTCAGTG CCGCTCGGCG GCAGGGGGCG AATTTCGTGA
    TTGCCGTCGA
651 TATTTCCGCA CGTCCGAGCA AAAATGTCGG TCAAGGTTTC
    TTCTCTTATC
701 TCGATCAGAC GCTGAACGTG ATGAGCGTTT CCGTGTTGCA
    AAACGAGTTG
751 gggcAGGCGG ATGTGGTTAT CAAACCGCag gtTTTGGATT
    TGGGTGCAGT
801 CGGCGGATTC GATCAGAAAA AGCGCGCCAT CCGGTTGGGC
    GAGGAGGCAG
851 CACGTGCCGC ATTGCCTGAA ATCAAACGCA AACTGGCGGC
    ATACCGTTAT
901 TGA

This encodes a protein having amino acid sequence <SEQ ID 564>:

1   MENMVTFSKI RSFLAIAAAA LLAACGTAGN NAARKPVQTA
    KPAAVVALAL
51  GGGASKGFAH IGIVKVLKEN GIPVKVVTGT SAGSIVGSLL
    ASGMSPDRLE
101 LEAEILGKTD LVDLTLSTSG FIKGEKLQNY INRKVGGRQI
    QQFPIKFAAV
151 ATDFETGKAV AFNQGNAGQA VRASAAIPNV FQPVIIGRHK
    YVDGGLSQPV
201 PVSAARRQGA NFVIAVDISA RPSKNVGQGF FSYLDQTLNV
    MSVSVLQNEL
251 GQADVVIKPQ VLDLGAVGGF DQKKRAIRLG EEAARAALPE
    IKRKLAAYRY
301 *

ORF137ng and ORF137-1 show 96.0% identity in 300 aa overlap:

Based on the presence of a predicted prokaryotic membrane lipoprotein lipid attachment site (underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 68

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 565>:

1   ATGTTTCGTT TACAATTCAG GCTGTTTCCC CCTTTGCGAA
    CCGCCATGCA
51  CATCCTGTTG ACCGCCCTGC TCAAATGCCT CTCCCTGcTG
    CCGCTTTCCT
101 GTCTGCACAC GCTGGGAAAC CGGCTCGGAC ATCTGGCGTT
    TTACCTTTTA
151 AAGGAAGACC GCGCGCGCAT CGTCGCCmAT ATGCGGCAGG
    CGGGTTTGAA
201 CCCCGACCCC AAAACGGTCA AAGCCGTTTT TGCGGAAACG
    GCAAAAGGCG
251 GTTTGGAACT TGCCCCCGCG TTTTTCAGAA AACCGGAAGA
    CATAGAAACA
301 ATGTTCAAAG CGGTACACGG CTGGGAACAT GTGCAGCAGG
    CTTTGGACAA
351 ACACGAAGGG CTGCTATTC..

This corresponds to the amino acid sequence <SEQ ID 566; ORF138>:

1   MFRLQFRLFP PLRTAMHILL TALLKCLSLL PLSCLHTLGN
    RLGHLAFYLL
51  KEDRARIVAX MRQAGLNPDP KTVKAVFAET AKGGLELAPA
    FFRKPEDIET
101 MFKAVHGWEH VQQALDKHEG LLF

Further work revealed the complete nucleotide sequence <SEQ ID 567>:

1   ATGTTTCGTT TACAATTCAG GCTGTTTCCC CCTTTGCGAA
    CCGCCATGCA
51  CATCCTGTTG ACCGCCCTGC TCAAATGCCT CTCCCTGCTG
    CCGCTTTCCT
101 GTCTGCACAC GCTGGGAAAC CGGCTCGGAC ATCTGGCGTT
    TTACCTTTTA
151 AAGGAAGACC GCGCGCGCAT CGTCGCCAAT ATGCGGCAGG
    CGGGTTTGAA
201 CCCCGACCCC AAAACGGTCA AAGCCGTTTT TGCGGAAACG
    GCAAAAGGCG
251 GTTTGGAACT TGCCCCCGCG TTTTTCAGAA AACCGGAAGA
    CATAGAAACA
301 ATGTTCAAAG CGGTACACGG CTGGGAACAT GTGCAGCAGG
    CTTTGGACAA
351 ACACGAAGGG CTGCTATTCA TCACGCCGCA CATCGGCAGC
    TACGATTTGG
401 GCGGACGCTA CATCAGCCAG CAGCTTCCGT TCCCGCTGAC
    CGCCATGTAC
451 AAACCGCCGA AAATCAAAGC GATAGACAAA ATCATGCAGG
    CGGGCAGGGT
501 TCGCGGCAAA GGAAAAACCG CGCCTACCAG CATACAAGGG
    GTCAAACAAA
551 TCATCAAAGC CCTGCGTTCG GGCGAAGCAA CCATCGTCCT
    GCCCGACCAC
601 GTCCCCTCCC CTCAAGAAGG CGGGGAAGGC GTATGGGTGG
    ATTTCTTCGG
651 CAAACCTGCC TATACCATGA CGCTGGCGGC AAAATTGGCA
    CACGTCAAAG
701 GCGTGAAAAC CCTGTTTTTC TGCTGCGAAC GCCTGCCTGG
    CGGACAAGGT
751 TTCGATTTGC ACATCCGCCC CGTCCAAGGG GAATTGAACG
    GCGACAAAGC
801 CCATGATGCC GCCGTGTTCA ACCGCAATGC CGAATATTGG
    ATACGCCGTT
851 TTCCGACGCA GTATCTGTTT ATGTACAACC GCTACAAAAT
    GCCGTAA

This corresponds to the amino acid sequence <SEQ ID 568; ORF138-1>:

1   MFRLQFRLFP PLRTAMHILL TALLKCLSLL PLSCLHTLGN
    RLGHLAFYLL
51  KEDRARIVAN MRQAGLNPDP KTVKAVFAET AKGGLELAPA
    FFRKPEDIET
101 MFKAVHGWEH VQQALDKHEG LLFITPHIGS YDLGGRYISQ
    QLPFPLTAMY
151 KPPKIKAIDK IMQAGRVRGK GKTAPTSIQG VKQIIKALRS
    GEATIVLPDH
201 VPSPQEGGEG VWVDFFGKPA YTMTLAAKLA HVKGVKTLFF
    CCERLPGGQG
251 FDLHIRPVQG ELNGDKAHDA AVFNRNAEYW IRRFPTQYLF
    MYNRYKMP*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF138 shows 99.2% identity over a 123aa overlap with an ORF (ORF138a) from strain A of N. meningitidis:

The complete length ORF138a nucleotide sequence <SEQ ID 569> is:

1   ATGTTTCGTT TACAATTCAG GCTGTTTCCC CCTTTGCGAA
    CCGCCATGCA
51  CATCCTGTTG ACCGCCCTGC TCAAATGCCT CTCCCTGCTG
    CCGCTTTCCT
101 GTCTGCACAC GCTGGGAAAC CGGCTCGGAC ATCTGGCGTT
    TTACCTTTTA
151 AAGGAAGACC GCGCGCGCAT CGTCGCCAAT ATGCGTCAGG
    CAGGCATGAA
201 TCCCGACCCC AAAACGGTCA AAGCCGTTTT TGCGGAAACG
    GCAAAAGGCG
251 GTTTGGAACT TGCCCCCGCG TTTTTCAGAA AACCGGAAGA
    CATAGAAACA
301 ATGTTCAAAG CGGTACACGG CTGGGAACAT GTGCAGCAGG
    CTTTGGACAA
351 ACACGAAGGG CTGCTATTCA TCACGCCGCA CATCGGCAGC
    TACGATTTGG
401 GCGGACGCTA CATCAGCCAG CAGCTTCCGT TCCCGCTGAC
    CGCCATGTAC
451 AAACCGCCGA AAATCAAAGC GATAGACAAA ATCATGCAGG
    CGGGCAGGGT
501 TCGCGGCAAA GGAAAAACCG CGCCTACCAG CATACAAGGG
    GTCAAACAAA
551 TCATCAAAGC CCTGCGTTCG GGCGAAGCAA CCATCGTCCT
    GCCCGACCAC
601 GTCCCCTCCC CTCAAGAAGG CGGGGAAGGC GTATGGGTGG
    ATTTCTTCGG
651 CAAACCTGCC TATACCATGA CGCTGGCGGC AAAATTGGCA
    CACGTCAAAG
701 GCGTGAAAAC CCTGTTTTTC TGCTGCGAAC GCCTGCCTGG
    CGGACAAGGT
751 TTCGATTTGC ACATCCGCCC CGTCCAAGGG GAATTGAACG
    GCGACAAAGC
801 CCATGATGCC GCCGTGTTCA ACCGCAATGC CGAATATTGG
    ATACGCCGTT
851 TTCCGACGCA GTATCTGTTT ATGTACAACC GCTACAAAAT
    GCCGTAA

This encodes a protein having amino acid sequence <SEQ ID 570>:

1 MFRLQFRLFP PLRTAMHILL TALLKCLSLL PLSCLHTLGN RLGHLAFYLL
51 KEDRARIVAN MRQAGLNPDP KTVKAVFAET AKGGLELAPA FFRKPEDIET
101 MFKAVHGWEH VQQALDKHEG LLFITPHIGS YDLGGRYISQ QLPFPLTAMY
151 KPPKIKAIDK IMQAGRVRGK GKTAPTSIQG VKQIIKALRS GEATIVLPDH
201 VPSPQEGGEG VWVDFFGKPA YTMTLAAKLA HVKGVKTLFF CCERLPGGQG
251 FDLHIRPVQG ELNGDKAHDA AVFNRNAEYW IRRFPTQYLF MYNRYKMP*

ORF138a and ORF138-1 show 99.7% identity over a 298aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF138 shows 94.3% identity over a 123aa overlap with a predicted ORF (ORF138ng) from N. gonorrhoeae:

The complete length ORF138ng nucleotide sequence <SEQ ID 571> is:

1   ATGTTTCGTT TACAATTCAG GCTGTTTCCC CCTTTGCGAA
    CCGCCATGCA
51  CATCCTGTTG ACCGCCCTGC TCAAATGCCT CTCCCTGCTG
    TCGCTTTCCT
101 GTCTGCACAC GCTGGGAAAC CGGCTCGGAC ATCTGGCGTT
    TTACCTTTTA
151 AAGGAAGACC GCGCGCGCAT CGTCGCCAAT ATGCGGCAGG
    CGGGTTTGAA
201 CCCCGACACG CAGACGGTCA AAGCCGTTTT TGCGGAAACG
    GCAAAATGCG
251 GTTTGGAACT TGCCCCCGCG TTTTTCAAAA AACCGGAAGA
    CATCGAAACA
301 ATGTTCAAAG CGGTACACGG CTGGGAACAC GTGCAGCAGG
    CTTTGGACAA
351 GGGCGAAGGG CTGCTGTTCA TCACGCCGCA CATCGGCAGC
    TACGATTTGG
401 GCGGACGCTA CATCAGCCAG CAGCTTCCGT TCCACCTGAC
    CGCCATGTAC
451 AAGCCGCCGA AAATCAAAGC GATAGACAAA ATCATGCAGG
    CGGGCAGGGT
501 GCGCGGCAAA GGCAAAACcg cgcccaccgg catACAAGGG
    GTCAAACAAA
551 tcatcaAGGC CCTGCGCGCG GGCGAGGCAA CCAtcATCCT
    GCCCGACCAC
601 GTCCCTTCTC CGCAGGAagg cggCGGCGTG TGGGCGGATT
    TTTTCGGCAA
651 ACCTGCATAC acCATGACAC TGGCGGCAAA ATTGGCACAC
    GTCAAAGGCG
701 TGAAAACCCT GTTTTTCTGC TGCGAACGCC TGCCCGACGG
    ACAAGGCTTC
751 GTGTTGCACA TCCGCCCCGT CCAAGGGGAA TTGAACGGCA
    ACAAAGCCCA
801 CGATGCCGCC GTGTTCAACC GCAATACCGA ATATTGGATA
    CGCCGTTTTC
851 CGACGCAGTA TCTGTTTATG TACAACCGCT ATAAAACGCC
    GTAA

This encodes a protein having amino acid sequence <SEQ ID 572>:

1   MFRLQFRLFP PLRTAMHILL TALLKCLSLL SLSCLHTLGN
    RLGHLAFYLL
51  KEDRARIVAN MRQAGLNPDT QTVKAVFAET AKCGLELAPA
    FFKKPEDIET
101 MFKAVHGWEH VQQALDKGEG LLFITPHIGS YDLGGRYISQ
    QLPFHLTAMY
151 KPPKIKAIDK IMQAGRVRGK GKTAPTGIQG VKQIIKALRA
    GEATIILPDH
201 VPSPQEGGGV WADFFGKPAY TMTLAAKLAH VKGVKTLFFC
    CERLPDGQGF
251 VLHIRPVQGE LNGNKAHDAA VFNRNTEYWI RRFPTQYLFM
    YNRYKTP*

ORF138ng and ORF138-1 show 94.3% identity over 299aa overlap:

In addition, ORF138ng is homologous to htrB protein from Pseudomonas fluorescens:

gnl|PID|e334283 (Y14568) htrB [Pseudomonas fluorescens] Length = 253
Score = 80.8 bits (196), Expect = 9e−15
Identities = 49/151 (32%), Positives = 79/151 (51%), Gaps = 6/151 (3%)
Query:	101	MFKAVHGWEHVQQALDKGEGLLFITPHIGSYD-LGGRYISQQLPFHLTAMYKPPKIKAID	159
		+ + V G E +++AL  G+G++ IT H+G+++ L   Y SQ  P      Y+PPK+KA+D
Sbjct:	94	LVREVEGLEVLKEALASGKGVVGITSHLGNWEVLNHFYCSQCKPI---IFYRPPKLKAVD	150
Query:	160	KIMQAGRVRGKGKTAPTGIQGVKQIIKALRAGEATIILPDHVPSPQEGGGVWADFFGKPA	219
		++++  RV+   K A +  +G+  +IK +R G    I  D  P P E  G++  FF   A
Sbjct:	151	ELLRKQRVQLGNKVAASTKEGILSVIKEVRKGGQVGIPAD--PEPAESAGIFVPFFATQA	208
Query:	220	YTMTLAAKLAHVKGVKTLFFCCERLPDGQGF	250
		T      +        +F    RLPDG G+
Sbjct:	209	LTSKFVPNMLAGGKAVGVFLHALRLPDGSGY	239

Based on this analysis, including the presence of a putative transmembrane domain in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF138-1 (57 kDa) was cloned in the pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 14A shows the results of affinity purification of the GST-fusion protein. Purified GST-fusion protein was used to immunise mice, whose sera were used for ELISA (positive result) and FACS analysis (FIG. 14B). These experiments confirm that ORF138-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 69

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 573>:

1   ..GCGTGGTCGG CCGGCGAATC GTGGCGTGTG TTAATGGAAA
    GTGAAACGTG
51  GCATGCGGTG TGGAATACTT TGCGCTTCTC GGCGGCGGCG
    GTGTATGCGG
101 CAGCGGTTTT GGGTGTGGTG TATGCGGCGC CGGCGCGGCG
    GTCGGCGTGG
151 ATGCGCGGGC TGATGTTTTA GCCGTTTATG GTGTCGCCGG
    TTTGTGTTTC
201 GGCGGGCGTG CTGCTGCTTT ATCCGCAGTG GACGGCTTCG
    TTGCCGTTGC
251 TGCTGGCGAT GTATGCGCTG CTGGCGTATC CGTTTGTGGC
    AAAAGATGTT
301 TTATCAGCCT GGGATGCACT GCCGCCGGAT TACGGCAGGG
    CGGCGGCGGG
351 TTTGGGTGCA AACGGCTTTC AGACGGCATG CCGCATCACG
    TTCCCCCTCT
401 TGAAACCGGC GTTGCGGCGC GGTCTGACTT TGGCGGCGGC
    AACCTGCGTG
451 GGCGAATTTG CGGCGACATT GTTTCTGTCG CGTCCGGAAT
    GGCAGACGCT
501 GACGACTTTG ATTTATGCCT ATTTGGGACG CGCGGGTGAG
    GATAATTACG
551 CGCGGGCGAT GGTGCTG..

This corresponds to the amino acid sequence <SEQ ID 574; ORF139>:

1   ..AWSAGESWRV LMESETWHAV WNTLRFSAAA VYAAAVLGVV
    YAAPARRSAW
51  MRGLMFXPFM VSPVCVSAGV LLLYPQWTAS LPLLLAMYAL
    LAYPFVAKDV
101 LSAWDALPPD YGRAAAGLGA NGFQTACRIT FPLLKPALRR
    GLTLAAATCV
151 GEFAATLFLS RPEWQTLTTL IYAYLGRAGE DNYARAMVL..

Further work revealed the complete nucleotide sequence <SEQ ID 575>:

1    ATGGATGGAC GGCGTTGGGT GGTATGGGGT GCTTTTGCCC
     TGCTGCCTTC
51   GGCTTTTTTG GCGGTAATGG TCGTTGCGCC TTTGTGGGCG
     GTGGCGGCGT
101  ATGACGGTTT GGCGTGGCGC GCGGTGCTGT CGGATGCCTA
     TATGCTCAAA
151  CGTTTGGCGT GGACGGTATT TCAGGCAGCG GCAACCTGTG
     TGCTGGTGCT
201  GCCTTTGGGC GTGCCTGTCG CGTGGGTGCT GGCGCGGCTG
     GCGTTTCCGG
251  GGCGGGCTTT GGTGCTGCGC CTGCTGATGC TGCCTTTTGT
     GATGCCCACG
301  TTGGTGGCGG GCGTGGGCGT GCTGGCCCTG TTCGGGGCGG
     ACGGGCTGTT
351  GTGGCGCGGC AGGCAGGATA CGCCGTATCT GTTGTTGTAC
     GGCAATGTGT
401  TTTTCAACCT TCCTGTGTTG GTCAGGGCGG CGTATCAGGG
     GTTTGTGCAA
451  GTGCCTGCGG CACGGCTTCA GACGGCACGG ACGTTGGGCG
     CGGGGGCGTG
501  GCGGCGGTTT TGGGACATTG AAATGCCCGT TTTGCGCCCG
     TGGCTTGCCG
551  GCGGCGTGTG CCTTGTCTTT CTGTATTGTT TTTCCGGGTT
     CGGGCTGGCG
601  CTGCTGCTGG GCGGCAGCCG TTATGCCACG GTCGAAGTGG
     AAATTTACCA
651  GTTGGTCATG TTCGAACTCG ATATGGCGGT TGCTTCGGTG
     CTGGTGTGGC
701  TGGTGTTGGG GGTAACGGCG GCGGCAGGGT TGCTGTATGC
     GTGGTTCGGC
751  AGGCGCGCGG TTTCGGATAA GGCGGTTTCC CCTGTGATGC
     CGTCGCCGCC
801  GCAGTCGGTC GGGGAATATG TGCTGCTGGC GTTTGCGGCG
     GCGGTGTTGT
851  CTGTGTGCTG CCTGTTTCCT TTGTTGGCAA TTGTTGTGAA
     AGCGTGGTCG
901  GCCGGCGAAT CGTGGCGTGT GTTAATGGAA AGTGAAACGT
     GGCAGGCGGT
951  GTGGAATACT TTGCGCTTCT CGGCGGCGGC GGTGTATGCG
     GCGGCGGTTT
1001 TGGGTGTGGT GTATGCGGCG GCGGCGCGGC GGTCGGCGTG
     GATGCGCGGG
1051 CTGATGTTTT TGCCGTTTAT GGTGTCGCCG GTTTGTGTTT
     CGGCGGGCGT
1101 GCTGCTGCTT TATCCGCAGT GGACGGCTTC GTTGCCGTTG
     CTGCTGGCGA
1151 TGTATGCGCT GCTGGCGTAT CCGTTTGTGG CAAAAGATGT
     TTTATCAGCC
1201 TGGGATGCAC TGCCGCCGGA TTACGGCAGG GCGGCGGCGG
     GTTTGGGTGC
1251 AAACGGCTTT CAGACGGCAT GCCGCATCAC GTTCCCCCTC
     TTGAAACCGG
1301 CGTTGCGGCG CGGTCTGACT TTGGCGGCGG CAACCTGCGT
     GGGCGAATTT
1351 GCGGCGACAT TGTTTCTGTC GCGTCCGGAA TGGCAGACGC
     TGACGACTTT
1401 GATTTATGCC TATTTGGGAC GCGCGGGTGA GGATAATTAC
     GCGCGGGCGA
1451 TGGTGCTGAC ATTGCTGTTG GCGGCGTTCG CGCTGGGTAT
     TTTCCTGCTG
1501 TTGGACGGCG GCGAAGGCGG AAAACAGACG GAAACGTTAT
     AA

This corresponds to the amino acid sequence <SEQ ID 576; ORF139-1>:

1   MDGRRWVVWG AFALLPSAFL AVMVVAPLWA VAAYDGLAWR
    AVLSDAYMLK
51  RLAWTVFQAA ATCVLVLPLG VPVAWVLARL AFPGRALVLR
    LLMLPFVMPT
101 LVAGVGVLAL FGADGLLWRG RQDTPYLLLY GNVFFNLPVL
    VRAAYQGFVQ
151 VPAARLQTAR TLGAGAWRRF WDIEMPVLRP WLAGGVCLVF
    LYCFSGFGLA
201 LLLGGSRYAT VEVEIYQLVM FELDMAVASV LVWLVLGVTA
    AAGLLYAWFG
251 RRAVSDKAVS PVMPSPPQSV GEYVLLAFAA AVLSVCCLFP
    LLAIVVKAWS
301 AGESWRVLME SETWQAVWNT LRFSAAAVYA AAVLGVVYAA
    AARRSAWMRG
351 LMFLPFMVSP VCVSAGVLLL YPQWTASLPL LLAMYALLAY
    PFVAKDVLSA
401 WDALPPDYGR AAAGLGANGF QTACRITFPL LKPALRRGLT
    LAAATCVGEF
451 AATLFLSRPE WQTLTTLIYA YLGRAGEDNY ARAMVLTLLL
    AAFALGIFLL
501 LDGGEGGKQT ETL*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF139 shows 94.7% identity over a 189aa overlap with an ORF (ORF139a) from strain A of N. meningitidis:

The complete length ORF139a nucleotide sequence <SEQ ID 577> is:

1    ATGGATGGAC GGCGTTGGGC GGTATGGGGT GCTTTTGCCC
     TGCTGCCTTC
51   GGCTTTTTTG GCGGCAATGG TCGTTGCGCC TTTGTGGGCG
     GTGGCGGCGT
101  ATGACGGTTT GGCGTGGCGC GCGGTGCTGT CGGATGCCTA
     TATGCTCAAA
151  CGTTTGGCGT GGACGGTATT TCAGGCAGCG GCAACCTGTG
     TGCTGGTGCT
201  GCCTTTGGGC GTGCCTGTCG CGTGGGTGCT GGCGCGGCTG
     GCGTTTCCGG
251  GGCGGGCTTT GGTGCTGCGC CTGCTGATGC TGCCTTTTGT
     GATGCCCACG
301  TTGGTGGCGG GCGTGGGCGT GCTGGCTCTG TTCGGGGCGG
     ACGGCCTGTN
351  GTGGCGCGGC TGGCAGGATA CGCCGTATCT GTTGTTGTAC
     GGCAATGTGT
401  TTTTTNACCT TCCTGTGTTG GTCAGGGCGG CATATCAGGG
     GTTTGTGCAA
451  GTGCCTGCGG CACGGCTTCA GACGGCACNG ACATTGGGCG
     CGGGGGCGTG
501  GCGGCGGTTT TGGGACATTG AAATGCCCGT TTTGCGCCCG
     TGGCTTGCCG
551  GCGGCGTGTG CCTTGTCTTC CTGTATTGTT TTTCGGGGTT
     CGGGCTGGCA
601  TTGCTGCTGG GCGGCAGCCG TTATGCCACG GTCGAAGTGG
     AAATTTACCA
651  GTTGGTCATG TTCGAACTCG ATATGGCGGT TGCTTCGGTG
     CTNGTGTGGC
701  TGGTGTNGGG GGTAACNGCG GCGGCAGGGT TGCTGTATGC
     GTGGTTCGGC
751  AGGCGCGCGG TTTCGGATAA GGCNGTTTCC CCTGTGATGC
     CGTCGCCGCC
801  GCAGTCGGTC GGGGAATATG TGCTNCTGGC GTTTGCGGCG
     GCGGTGTNGT
851  CTGTGTGCTG CCTGTTTCNT TTGTTGGCAA TTGTTGTGAA
     AGCGTGGTCG
901  GCCGGCGAAT CGTGGCGTGT GTTAATGGAA AGTGAAACGT
     GGCAGGCGGT
951  GTGGAATACT NTGCGCTTCT CGGCGGCGGC GGTGTATGCG
     GCGGCGGTTT
1001 TGGGTGTGGT GTATGCGGCG GCGGCGCGGC GGTCGGCGTG
     GATGCGCGGG
1051 CTGATGTTTT TGCCGTTTAT GGTGTCGCCG GTTTGTGTTT
     CGGCGGGCGT
1101 GCTGCTGCTT NATCCGCAGT GGACGGCTTC GTTGCCGCTG
     CTGCTGGCGA
1151 TGTATGCGCT GCTGGCGTAT CCGTTTGTGG CAAAAGATGT
     TTTATCAGCC
1201 TGNGATGCAC TGCCGCCGGA TTACGGCAGG GCGGCGGCGG
     GTTTGGGTGC
1251 AAACGGCTTT CAGACGGCAT GCCGCATCAC GTTCCCCCTC
     TTGAAACCGG
1301 CGTTGCGGCG CGGTCTGACT TTGGCGGCGG CAACCTGCGT
     GGGCGAATTT
1351 GCGGCAACCT TGTTCNTGTC GCGTCNCGAG TGGCAGACGC
     TGACGACTTT
1401 GATTTATGCC TATNTGGGAC GCGCGGGTGA NGATAATTAC
     GCGCGGGCGA
1451 TGGTGCTGAC ATTGCTGTTG GCGGCGTTCG CGCTGGGTAT
     NTTCCTGCTG
1501 TTGGACGGCG GCGAAGGCGG AAAACGGACG GAAACGTTAT
     AA

This encodes a protein having amino acid sequence <SEQ ID 578>:

1   MDGRRWAVWG AFALLPSAFL AAMVVAPLWA VAAYDGLAWR
    AVLSDAYMLK
51  RLAWTVFQAA ATCVLVLPLG VPVAWVLARL AFPGRALVLR
    LLMLPFVMPT
101 LVAGVGVLAL FGADGLXWRG WQDTPYLLLY GNVFFXLPVL
    VRAAYQGFVQ
151 VPAARLQTAX TLGAGAWRRF WDIEMPVLRP WLAGGVCLVF
    LYCFSGFGLA
201 LLLGGSRYAT VEVEIYQLVM FELDMAVASV LVWLVXGVTA
    AAGLLYAWFG
251 RRAVSDKAVS PVMPSPPQSV GEYVLLAFAA AVXSVCCLFX
    LLAIVVKAWS
301 AGESWRVLME SETWQAVWNT XRFSAAAVYA AAVLGVVYAA
    AARRSAWMRG
351 LMFLPFMVSP VCVSAGVLLL XPQWTASLPL LLAMYALLAY
    PFVAKDVLSA
401 XDALPPDYGR AAAGLGANGF QTACRITFPL LKPALRRGLT
    LAAATCVGEF
451 AATLFXSRXE WQTLTTLIYA YXGRAGXDNY ARAMVLTLLL
    AAFALGXFLL
501 LDGGEGGKRT ETL*

ORF139a and ORF139-1 show 96.5% homology over a 514aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF139 shows 95.2% identity over a 189aa overlap with a predicted ORF (ORF139ng) from N. gonorrhoeae:

The complete length ORF139ng nucleotide sequence <SEQ ID 579> is predicted to encode a protein having amino acid sequence <SEQ ID 580>:

1   MDGRCWAVRG AFSLLPSAFL AVMVVAPLWA VAAYDGLAWR
    AVLSDAYMLK
51  RLAWTVFQAA ATCVLVLPLG VPVAWVLARL AFPGRALVLR
    LLMLPFVMPT
101 LVAGVGVLAL FGADGLLWRG RQDTPYLLLY GNVFFNLPVL
    VRAAYQGFAQ
151 VPAARLQTAR TLGAGAWRPF WDIEMPVLRP WLAGGVCLVF
    LYCFSGFGLA
201 LLLGGSRYAT VEVEIYQLVM FELDMAGASA LVWLVLGVTA
    AAGLLYAWFG
251 RRAVSDKAVS PVMPSPPQSV GEYVLLAFSV AVLSVCCLFP
    LSAIVVKAWS
301 AGESRRVLME SETWQAVWNT LRFSAAAVFA AAVLGVVYAA
    AARRLVWMRG
351 LVFLPFMVSP VCVSAGVLLL YPGWTASLPL LLAMYALLAY
    PFVAKDVLSA
401 WDALPPDYGR AAAGLGANGF QTACRITFPL LKPALRRGLT
    LAAATCVGEF
451 AATLFLSRPE WQTLTTLIYA YLGRAGEDNY ARAMVLTLLL
    SAFAVCIFLL
501 LDNGEGGKRT ETL*

Further work revealed a variant gonococcal DNA sequence <SEQ ID 581>:

1    ATGGATGGAC GGTGTTGGGC GGTACGGGGT GCTTTTTCCC
     TGCTGCCTTC
51   GGCTTTTTTG GCGGTAATGG TCGTTGCGCC TTTGTGGGCG
     GTGGCGGCGT
101  ATGACGGTTT GGCGTGGCGC GCGGTGCTGT CGGATGCCTA
     TATGCTCAAA
151  CGTTTGGCGT GGACGGTGTT TCAGGCGGCG GCAACCTGTG
     TGCTGGTGCT
201  GCCTTTGGGC GTGCCTGTCG CGTGGGTGCT GGCGCGGCTG
     GCGTTCCCGG
251  GGCGGGCTTT GGTGCTGCGC CTGCTGATGC TGCCGTTTGT
     GATGCCCACG
301  CTGGTGGCGG GCGTGGGCGT GCTGGCTCTG TTCGGGGCGG
     ACGGGCTGTT
351  GTGGCGCGGC CGGCAGGATA CGCCGTATCT GTTGTTGTAC
     GGCAATGTGT
401  TTTTCAACCT GCCCGTGTTG GTCAGGGCGG CGTATCAGGG
     GTTTGCTCAA
451  GTGCCTGCGG CACGGCTTCA GACGGCACGG ACGTTGGGCG
     CGGGGGCGTG
501  GCGGCGGTTT TGGGACATTG AAATGCCCGT TTTGCGCCCG
     TGGCTTGCCG
551  GCGGCGTGTG CCTTGTCTTC CTGTATTGTT TTTCGGGGTT
     CGGGCTGGCA
601  TTGCTGTTGG GCGGCAGCCG TTATGCCACG GTCGAAGTGG
     AAATTTACCA
651  GTTGGTTATG TTCGAACTCG ATATGGCGGG GGCTTCGGCG
     CTGGTGTGGC
701  TGGTGTTGGG GGTAACGGCG GCGGCAGGGT TGCTGTATGC
     GTGGTTCGGC
751  AGGCGCGCGG TTTCGGATAA GGCGGTTTCC CCCGTGATGC
     CGTCGCCGCC
801  GCAATCGGTG GGGGAATATG TATTGCTGGC ATTTTCGGTG
     GCGGTGTTGT
851  CCGTGTGCTG CCTGTTTCCT TTGTCGGCAA TTGTTGTGAA
     AGCGTGGTCG
901  GCCGGCGAAT CGCGGCGTGT GTTAATGGAA AGTGAAACGT
     GGCAGGCAGT
951  GTGGAATACt ttGCGCTTTT CGGCGGCGGC GGTGTTTGCG
     GCGGCGGTTT
1001 TGGGTGTGGT GTATGCGGCG GCGGCGCGGC GGCTGGTGTG
     GATGCGCGGA
1051 CTGGTGTTTT TACCGTTTAT GGTGTCGCCG GTTTGTGTTT
     CGGCGGGCGT
1101 GCTGCTGCTT TATCCGGGGT GGACGGCTTC GTTACCGCTG
     CTGCTGGCGA
1151 TGTATGCGCT GCTGGCGTAT CCGTTTGTGG CAAAAGATGT
     TTTATCGGCC
1201 TGGGATGCAC TGCCGCCGGA TTACGGCAGG GCGGCGGCAG
     GTTTGGGCGC
1251 AAACGGCTTT CAGACGGCAT GCCGTATCAC GTTCCCCCTC
     TTGAAACCGG
1301 CGTTGCGGCG CGGTCTGACT TTGGCGGCGG CGACGTGTGT
     GGGCGAATTT
1351 GCGGCAACCT TGTTCCTGTC GCGTCCGGAA TGGCAGACGT
     TGACGACTTT
1401 GATTTATGCC TATTTGGGGC GTGCGGGTGA GGACAATTAT
     GCGCGGGCAA
1451 TGGTGTTGAC ATTGCTGTTG TCGGCATTTG CGGTGTGCAT
     TTTCCTGCTG
1501 TTGGACAACG GCGAAGGCGg aaaACGGACG GAAACGTTAT
     AA

This corresponds to the amino acid sequence <SEQ ID 582; ORF139ng-1>:

1   MDGRCWAVRG AFSLLPSAFL AVMVVAPLWA VAAYDGLAWR
    AVLSDAYMLK
51  RLAWTVFQAA ATCVLVLPLG VPVAWVLARL AFPGRALVLR
    LLMLPFVMPT
101 LVAGVGVLAL FGADGLLWRG RQDTPYLLLY GNVFFNLPVL
    VRAAYQGFAQ
151 VPAARLQTAR TLGAGAWRRF WDIEMPVLRP WLAGGVCLVF
    LYCFSGFGLA
201 LLLGGSRYAT VEVEIYQLVM FELDMAGASA LVWLVLGVTA
    AAGLLYAWFG
251 RRAVSDKAVS PVMPSPPQSV GEYVLLAFSV AVLSVCCLFP
    LSAIVVKAWS
301 AGESRRVLME SETWQAVWNT LRFSAAAVFA AAVLGVVYAA
    AARRLVWMRG
351 LVFLPFMVSP VCVSAGVLLL YPGWTASLPL LLAMYALLAY
    PFVAKDVLSA
401 WDALPPDYGR AAAGLGANGF QTACRITFPL LKPALRRGLT
    LAAATCVGEF
451 AATLFLSRPE WQTLTTLIYA YLGRAGEDNY ARAMVLTLLL
    SAFAVCIFLL
501 LDNGEGGKRT ETL*

ORF139ng-1 and ORF139-1 show 95.9% identity over 513aa overlap:

Based on the presence of a predicted binding-protein-dependent transport systems inner membrane component signature (underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 70

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 583>:

1   ATGGACGGCT GGACACAGAC GCTGTCCGCG CAAACCCTGT
    TGGGCATTTC
51  GGCGGCGGCA ATCATCCTCA TTCTGATTTT AATCGTCAGA
    TTCCGCATCC
101 ACGCGCTGCT GACACTGGTC ATCGTCAGCC TGCTGACGGC
    TTTGGCAACC
151 GGTTTGCCCA CAGGCAGCAT TGTCAAAGAC ATACTGGTCA
    AAAACTTCGG
201 CGGCACGCTC GGCGGCGTGG CGCTTCTGGT CGGCCTGGGC
    GCGATGCTCG
251 AACGTTTGGT C...

This corresponds to the amino acid sequence <SEQ ID 584; ORF140>:

1  MDGWTQTLSA QTLLGISAAA IILILILIVR FRIHALLTLV
   IVSLLTALAT
51 GLPTGSIVKD ILVKNFGGTL GGVALLVGLG AMLERLV..

Further work revealed the complete nucleotide sequence <SEQ ID 585>:

1    ATGGACGGCT GGACACAGAC GCTGTCCGCG CAAACCCTGT
     TGGGCATTTC
51   GGCGGCGGCA ATCATCCTCA TTCTGATTTT AATCGTCAAA
     TTCCGCATCC
101  ACGCGCTGCT GACACTGGTC ATCGTCAGCC TGCTGACGGC
     TTTGGCAACC
151  GGTTTGCCCA CAGGCAGCAT TGTCAACGAC ATACTGGTCA
     AAAACTTCGG
201  CGGCACGCTC GGCGGCGTGG CGCTTCTGGT CGGCCTGGGC
     GCGATGCTCG
251  GACGTTTGGT CGAAACATCC GGCGGCGCAC AGTCGCTGGC
     GGACGCGCTG
301  ATCCGGATGT TCGGCGAAAA ACGCGCACCG TTCGCGCTGG
     GCGTTGCCTC
351  GCTGATTTTC GGCTTCCCGA TTTTCTTCGA TGCCGGACTA
     ATCGTCATGC
401  TGCCCATCGT GTTCGCCACC GCACGGCGCA TGAAACAGGA
     CGTACTGCCC
451  TTCGCGCTTG CCTCCATCGG CGCATTTTCC GTCATGCACG
     TCTTCCTGCC
501  GCCCCATCCG GGCCCGATTG CCGCTTCCGA ATTTTACGGC
     GCGAACATCG
551  GCCAAGTTTT GATTTTGGGT CTGCCGACCG CCTTCATCAC
     ATGGTATTTC
601  AGCGGCTATA TGCTCGGCAA AGTGTTGGGG CGCACCATCC
     ATGTTCCCGT
651  TCCCGAACTG CTCAGCGGCG GCACGCAAGA CAACGACCTG
     CCGAAAGAAC
701  CTGCCAAAGC AGGAACGGTC GTCGCCATCA TGCTGATTCC
     CATGCTGCTG
751  ATTTTCCTGA ATACCGGCGT ATCGGCCCTC ATCAGCGAAA
     AACTCGTAAG
801  TGCGGACGAA ACCTGGGTTC AGACGGCAAA AATAATCGGT
     TCGACACCGA
851  TCGCCCTTCT GATTTCCGTA TTGGTCGCAC TGTTTGTCTT
     GGGACGCAAA
901  CGCGGCGAAA GCGGCAGCGC GTTGGAAAAA ACCGTGGACG
     GCGCACTCGC
951  CCCCGTCTGT TCCGTGATTC TGATTACCGG CGCGGGCGGT
     ATGTTCGGCG
1001 GCGTTTTGCG CGCTTCCGGC ATCGGCAAGG CACTCGCCGA
     CAGCATGGCG
1051 GATTTGGGCA TTCCCGTCCT TTTGGGCTGT TTCCTTGTCG
     CCTTGGCACT
1101 GCGTATCGCG CAAGGTTCGG CAACCGTCGC CCTGACCACC
     GCCGCCGCGC
1151 TGATGGCTCC TGCCGTTGCC GCCGCCGGCT TTACCGACTG
     GCAGCTCGCC
1201 TGTATCGTAT TGGCAACGGC GGCAGGTTCG GTCGGTTGCA
     GCCACTTCAA
1251 CGACTCCGGC TTCTGGCTGG TCGGCCGTCT CTTGGACATG
     GACGTACCGA
1301 CCACGCTGAA AACCTGGACG GTCAACCAAA CCCTCATCGC
     ACTCATCGGC
1351 TTTGCCTTGT CCGCACTGCT GTTCGCCATC GTCTGA

This corresponds to the amino acid sequence <SEQ ID 586; ORF140-1>:

1   MDGWTQTLSA QTLLGISAAA IILILILIVK FRIHALLTLV
    IVSLLTALAT
51  GLPTGSIVND ILVKNFGGTL GGVALLVGLG AMLGRLVETS
    GGAQSLADAL
101 IRMFGEKRAP FALGVASLIF GFPIFFDAGL IVMLPIVFAT
    ARRMKQDVLP
151 FALASIGAFS VMHVFLPPHP GPIAASEFYG ANIGQVLILG
    LPTAFITWYF
201 SGYMLGKVLG RTIHVPVPEL LSGGTQDNDL PKEPAKAGTV
    VAIMLIPMLL
251 IFLNTGVSAL ISEKLVSADE TWVQTAKIIG STPIALLISV
    LVALFVLGRK
301 RGESGSALEK TVDGALAPVC SVILITGAGG MFGGVLRASG
    IGKALADSMA
351 DLGIPVLLGC FLVALALRIA QGSATVALTT AAALMAPAVA
    AAGFTDWQLA
401 CIVLATAAGS VGCSHFNDSG FWLVGRLLDM DVPTTLKTWT
    VNQTLIALIG
451 FALSALLFAI V*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF140 shows 95.4% identity over a 87aa overlap with an ORF (ORF140a) from strain A of N. meningitidis:

The complete length ORF140a nucleotide sequence <SEQ ID 587> is:

1    ATGGACGGCT GGACACAGAC GCTGTCCGCG CAAACCCTGT
     TGGGCATTTC
51   GGCGGCGGCA ATCATCCTCA TTCTGATTTT AATCGTCAAA
     TTCCGCATCC
101  ACGCGCTGCT GACACTGGTC ATCGTCAGCC TGCTGACGGC
     TTTGGCAACC
151  GGTTTGCCCA CAGGCAGCAT TGTCAACGAC GTACTGGTCA
     AAAACTTCGG
201  CGGCACGCTC GGCGGCGTGG CGCTTCTGGT CGGCCTGGGC
     GCGATGCTCG
251  GACGTTTGGT CGAAACATCC GGCGGCGCAC AGTCGCTGGC
     GGACGCGCTG
301  ATCCGGATGT TCGGCGAAAA ACGCGCACCG TTCGCGCTGG
     GCGTTGCCTC
351  GCTGATTTTC GGCTTCCCGA TTTTCTTCGA TGCCGGACTA
     ATCGTCATGC
401  TGCCCATCGT GTTCGCCACC GCACGGCGCA TGAAACAGGA
     CGTACTGCCC
451  TTCGCGCTTG CCTCCATCGG CGCATTTTCC GTCATGCACG
     TCTTCCTGCC
501  GCCCCATCCG GGCCCGATTG CCGCTTCCGA ATTTTACGGC
     GCGAACATCG
551  GCCAAGTTTT GATTTTGGGT CTGCCGACCG CCTTCATCAC
     ATGGTATTTC
601  AGCGGCTATA TGCTCGGCAA AGTGTTGGGG CGCACCATCC
     ATGTTCCCGT
651  TCCCGAACTG CTCAGCGGCG GCACGCAAGA CAACGACCTG
     CCGAAAGAAC
701  CTGCCAAAGC AGGAACGGTC GTCGCCATCA TGCTGATTCC
     CATGCTGCTG
751  ATTTTCCTGA ATACCGGCGT ATCGGCCCTC ATCAGCGAAA
     AACTCGTAAG
801  TGCGGACGAA ACCTGGGTTC AGACGGCAAA AATAATCGGT
     TCGACACCGA
851  TCGCCCTTCT GATTTCCGTA TTGGTCGCAC TGTTTGTCTT
     GGGACGCAAA
901  CGCGGCGAAA GCGGCAGCGC GTTGGAAAAA ACCGTGGACG
     GCGCACTCGC
951  CCCCGTCTGT TCCGTGATTC TGATTACCGG CGCGGGCGGT
     ATGTTCGGCG
1001 GCGTTTTGCG CGCTTCCGGC ATCGGCAAGG CACTCGCCGA
     CAGCATGGCG
1051 GATTTGGGCA TTCCCGTCCT TTTGGGCTGT TTCCTTGTCG
     CCTTGGCACT
1101 GCGTATCGCG CAAGGTTCGG CAACCGTCGC CCTGACCACC
     GCCGCCGCGC
1151 TGATGGCTCC TGCCGTTGCC GCCGCCGGCT TTACCGACTG
     GCAGCTCGCC
1201 TGTATCGTAT TGGCAACGGC GGCAGGTTCG GTCGGTTGCA
     GCCACTTCAA
1251 CGACTCCGGC TTCTGGCTGG TCGGCCGCCT CTTGGACATG
     GACGTACCGA
1301 CCACGCTGAA AACCTGGACG GTCAACCAAA CCCTCATCGC
     ACTCATCGGC
1351 TTTGCCTTGT CCGCACTGCT GTTCGCCATC GTCTGA

This encodes a protein having amino acid sequence <SEQ ID 588>:

1   MDGWTQTLSA QTLLGISAAA IILILILIVK FRIHALLTLV
    IVSLLTALAT
51  GLPTGSIVND VLVKNFGGTL GGVALLVGLG AMLGRLVETS
    GGAQSLADAL
101 IRMFGEKRAP FALGVASLIF GFPIFFDAGL IVMLPIVFAT
    ARRMKQDVLP
151 FALASIGAFS VMHVFLPPHP GPIAASEFYG ANIGQVLILG
    LPTAFITWYF
201 SGYMLGKVLG RTIHVPVPEL LSGGTQDNDL PKEPAKAGTV
    VAIMLIPMLL
251 IFLPNTGVSAL ISEKLVSADE TWVQTAKIIG STPIALLISV
    LVALFVLGRK
301 RGESGSALEK TVDGALAPVC SVILITGAGG MFGGVLRASG
    IGKALADSMA
351 DLGIPVLLGC FLVALALRIA QGSATVALTT AAALMAPAVA
    AAGFTDWQLA
401 CIVLATAAGS VGCSHFNDSG FWLVGRLLDM DVPTTLKTWT
    VNQTLIALIG
451 FALSALLFAI V*

ORF140a and ORF140-1 show 99.8% identity over a 461aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF140 shows 92% identity over a 87aa overlap with a predicted ORF (ORF140ng) from N. gonorrhoeae:

The complete length ORF140ng nucleotide sequence <SEQ ID 589> was predicted to encode a protein having amino acid sequence <SEQ ID 590>:

1   MDGRTQTLSA QTLLGISAAA IILILILIVK FRIRALLTLV
    IASLLTALAT
51  GLPTGSIVND VLVKNFGGTL GGVALLVGLG AMLGRLVETS
    GGAQSLADAL
101 IRMFGEKRAP FAPGVASLIF GFPIFFDAGL IVMLPIVFAT
    ARRMKQDVLP
151 FALASVGAFS VMHVFLPPHP GPIAASEFYG ANIGQVLILG
    LPTAFITWYF
201 SGYMLGKVLG RAIHVPVPEL LSGGTQDSDP PKEPAKAGTV
    VAVMLIPMLL
251 IFLNTGVSAL ISEKLVSADE TWVQTAKMIG STPVALLISV
    LAALLVLGRK
301 RGESGSTLEK TVDGALAPAC SVILITGAGG MFGGVLRASG
    IGKALADSMA
351 DLGIPVLLGC FLVALALRIA QGSATVALTT AAALMAPAVA
    AAGFTDWQLA
401 CIVLATAAGS VGCSHFNDSG FWLVGRLSDM DVPTTLKTWT
    VNQTLIAFIG
451 FALSALLFAI V*

Further work revealed a variant gonococcal DNA sequence <SEQ ID 591>:

1    ATGGACGGCC GGACACAGAC GCTGTCCGCG CAAACCTTGT
     TGGGCATTTC
51   GGCGGCGGCA ATCATCCTCA TTCTGATTTT AATCGTCAAA
     TTCCGCATCC
101  GCGCGCTGCT GACACTGGTC ATCGCCAGCC TGCTGACGGC
     TTTGGCAACC
151  GGTTTGCCCA CAGGCAGCAT CGTCAACGAC GTACTGGTCA
     AAAACTTCGG
201  CGGCACGCTC GGCGGCGTGG CGCTTCTGGT CGGTCTGGGC
     GCAATGCTCG
251  GACGTTTGGT AGAAACATCC GGCGGCGCAC AGTCGCTGGC
     GGACGCGCTG
301  ATCCGGATGT TCGGCGAAAA ACGCGCACCG TTCGCTCCGG
     GCGTTGCCTC
351  GCTGATTTTC GGCTTCCCGA TTTTCTTCGA TGCCGGACTA
     ATCGTCATGC
401  TGCCCATCGT ATTCGCCACC GCACGGCGCA TGAAACAGGA
     CGTACTGCCC
451  TTCGCGCTTG CCTCCGTCGG CGCATTTTCC GTCATGCACG
     TCTTCCTGCC
501  GCCCCATCCG GGCCCGATTG CCGCTTCCGA ATTTTACGGC
     GCGAACATCG
551  GCCAGGTTTT GATTTTGGGT CTGCCGACCG CCTTCATCAC
     ATGGTATTTC
601  AGCGGCTATA TGCTCGGCAA AGTGTTGGGG CGCGCCATCC
     ATGTTCCCGT
651  TCCCGAACTG CTCAGCGGCG GCACGCAAGA CAGCGACCCG
     CCGAAAGAAC
701  CTGCCAAAGC AGGAACGGTC GTCGCCGTCA TGCTGATTCC
     CATGCTGCTG
751  ATTTTCCTGA ATACCGGCGT ATCAGCCCTC ATCAGCGAAA
     AACTCGTAAG
801  TGCGGACGAA ACTTGGGTTC AGACGGCAAA AATGATCGGT
     TCGACACCTG
851  TCGCCCTTCT GATTTCCGTA TTGGCCGCAC TGTTGGTCTT
     GGGACGCAAA
901  CGCGGCGAAA GCGGCAGCAC GTTGGAAAAA ACCGTGGACG
     GCGCACTCGC
951  CCCCGCCTGT TCCGTGATTC TGATTACCGG CGCGGGCGGT
     ATGTTCGGCG
1001 GCGTTTTGCG CGCTTCCGGC ATCGGCAAGG CACTCGCCGA
     CAGCATGGCG
1051 GATTTGGGCA TTCCCGTCCT TTTGGGCTGC TTCCTTGTCG
     CCTTGGCACT
1101 GCGTATCGCG CAAGGTTCGG CAACCGTCGC CCTGACCACA
     GCCGCCGCGC
1151 TGATGGCTCC TGCCGTTGCC GCCGCCGGCT TTACCGACTG
     GCAGCTCGCC
1201 TGTATCGTAT TGGCAACGGC GGCAGGTTCG GTCGGTTGCA
     GCCACTTCAA
1251 CGACTCCGGC TTCTGGCTGG TCGGCCGCCT CTTGGATATG
     GACGTACCGA
1301 CCACGCTGAA AACCTGGACG GTCAACCAAA CCCTCATCGC
     ATTCATCGGC
1351 TTTGCCTTGT CCGCACTGCT GTTTGCCATC GTCTGA

This corresponds to the amino acid sequence <SEQ ID 592; ORF140ng-1>:

1   MDGRTQTLSA QTLLGISAAA IILILILIVK FRIRALLTLV
    IASLLTALAT
51  GLPTGSIVND VLVKNFGGTL GGVALLVGLG AMLGRLVETS
    GGAQSLADAL
101 IRMFGEKRAP FAPGVASLIF GFPIFFDAGL IVMLPIVFAT
    ARRMKQDVLP
151 FALASVGAFS VMHVFLPPHP GPIAASEFYG ANIGQVLILG
    LPTAFITWYF
201 SGYMLGKVLG RAIHVPVPEL LSGGTQDSDP PKEPAKAGTV
    VAVMLIPMLL
251 IFLTGVSAL ISEKLVSADE TWVQTAKMIG STPVALLISV
    LAALLVLGRK
301 RGESGSTLEK TVDGALAPAC SVILITGAGG MFGGVLRASG
    IGKALADSMA
351 DLGIPVLLGC FLVALALRIA QGSATVALTT AAALMAPAVA
    AAGFTDWQLA
401 CIVLATAAGS VGCSHFNDSG FWLVGRLLDM DVPTTLKTWT
    VNQTLIAFIG
451 FALSALLFAI V*

ORF140ng-1 and ORF140-1 show 96.3% identity over 461aa overlap:

Furthermore, ORF140ng-1 is homologous to an E. coli protein:

gi|882633 (U29579) ORF_o454 [Escherichia coli] >gi|1789097 (AE000358) o454;
This 454 aa ORF is 34% identical (9 gaps) to 444 residues of an approx.
456 aa protein GNTP_BACLI SW: P46832 [Escherichia coli] Length = 454
Score = 210 bits (529), Expect = 1e−53
Identities = 130/384 (33%), Positives = 194/384 (49%), Gaps = 19/384 (4%)
Query:	88	ETSGGAQSLADALIRMFGEKRAPFAPGVASLIFGFPIFFDAGLIVMLPIVFATARRMKQD	147
		E SGGA+SLA+   R  G+KR   A  +A+   G P+FFD G I++ PI++  A+  K
Sbjct:	80	EHSGGAESLANYFSRKLGDKRTIAALTLAAFFLGIPVFFDVGFIILAPIIYGFAKVAKIS	139
Query:	148	VLPFALASVGAFSVMHVFLPPHPGPIAASEFYGANIGQVLILGLPTAFITWYFSGYMLGK	207
		L F L   G    +HV +PPHPGP+AA+    A+IG + I+G+  + I    GY   K
Sbjct:	140	PLKFGLPVAGIMLTVHVAVPPHPGPVAAAGLLHADIGWLTIIGIAIS-IPVGVVGYFAAK	198
Query:	208	VLGRAIHVPVPELL----------SGGTQDSDPPKEPAKAGTVVAVMLIPMLLIFLNTGV	257
		++ +  +    E+L           G T+ SD    P  A  V ++++IP+ +I   T
Sbjct:	199	IINKRQYAMSVEVLEQMQLAPASEEGATKLSDKINPPGVA-LVTSLIVIPIAIIMAGT--	255
Query:	258	SALISEKLVSADETWVQTAKMIGSTPXXXXXXXXXXXXXXGRKRGESGSTLEKTVDGALA	317
		+S  L+      + T ++IGS                  +RG S       +  AL
Sbjct:	256	---VSATLMPPSHPLLGTLQLIGSPMVALMIALVLAFWLLALRRGWSLQHTSDIMGSALP	312
Query:	318	PACSVILITGAGGMFGGVLRASGIGKALADSMADLGIPVLLGCFLVALALRIAQGSXXXX	377
		A  VIL+TGAGG+FG VL  SG+GKALA+ +  + +P+L   F+++LALR +QGS
Sbjct:	313	TAAVVILVTGAGGVFGKVLVESGVGKALANMLQMIDLPLLPAAFIISLALRASQGS--AT	370
Query:	378	XXXXXXXXXXXXXXXGFTDWQLACIVLATAAGSVGCSHFNDSGFWLVGRLLDMDVPTTLK	437
		G   Q   + LA   G +G SH NDSGFW+V + L + V   LK
Sbjct:	371	VAILTTGGLLSEAVMGLNPIQCVLVTLAACFGGLGASHINDSGFWIVTKYLGLSVADGLK	430
Query:	438	TWTVNQTLIAFIGFALSALLFAIV	461
		TWTV  T++ F GF ++  ++A++
Sbjct:	431	TWTVLTTILGFTGFLITWCVWAVI	454

Based on this analysis, including the identification of the presence of a putative leader sequence (double-underlined) and several putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 71

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 593>:

1   ..GATTTCGGCA TATCGCCCGT GTATCTTTGG GTTGCCGCCG
    CGTTCAAACA
51  TTTGCTGTCG CCGTGGGCTG CCGACTCATA CGATGTCGCA
    CGCTTTGCAG
101 GCGTATTTTT TGCCGTTATC GGACTGACTT CCTGCGGCTT
    TGCCGGTTTC
151 AACTTTTTGG GCAGACACCA CGGGCGCAC. GTCGTCCTGA
    TTCTCATCGG
201 CTGTATCGGG CTGATTCCAG TTGCCCATTT CCTCAACCCC
    GCTGCCGCCG
251 CCTTTGCCGC CGCCGGACTG GTGCTGCACG GTTATTCTTT
    GGCTCGCCGG
301 CGCGTGATTG CCGCCTCTTT TCTGCTCGGT ACGGGCTGGA
    CGCTGATGTC
351 GTTGGCAGCA GCTTATCCGG CAGCATTTGC CCTGATGCTG
    CCCTTGCCCG
401 TACTGATGTT TTTCCGTCCG ..

This corresponds to the amino acid sequence <SEQ ID 594; ORF141>:

1   ..DFGISPVYLW VAAAFKHLLS PWAADSYDVA RFAGVFFAVI
    GLTSCGFAGF
51  NFLGRHHGRX VVLILIGCIG LIPVAHFLNP AAAAFAAAGL
    VLHGYSLARR
101 RVIAASFLLG TGWTLMSLAA AYPAAFALML PLPVLMFFRP
    ..

Further work revealed the complete nucleotide sequence <SEQ ID 595>:

1    ATGCTGACCT ATACCCCGCC CGATGCCCGC CCGCCCGCCA
     AAACCCACGA
51   AAAGCCGTGG CTGCTGCTGT TGATGGCGTT TGCCTGGTTG
     TGGCCCGGCG
101  TGTTTTCCCA CGATTTGTGG AATCCTGACG AACCTGCCGT
     CTATACCGCC
151  GTCGAAGCAC TGGCAGGCAG CCCCACCCCC TTGGTTGCCC
     ATCTGTTCGG
201  TCAAACCGAT TTCGGCATAC CGCCCGTGTA TCTTTGGGTT
     GCCGCCGCGT
251  TCAAACATTT GCTGTCGCCG TGGGCTGCCG ACTCATACGA
     TGCCGCACGC
301  TTTGCAGGCG TATTTTTTGC CGTTATCGGA CTGACTTCCT
     GCGGCTTTGC
351  CGGTTTCAAC TTTTTGGGCA GACACCACGG GCGCAgCGTC
     GTCCTGATTC
401  TCATCGGCTG TATCGGGCTG ATTCCAGTTG CCCATTTCCT
     CAACCCCGCT
451  GCCGCCGCCT TTGCCGCCGC CGGACTGGTG CTGCACGGTT
     ATTCTTTGGC
501  TCGCCGGCGC GTGATTGCCG CCTCTTTTCT GCTCGGTACG
     GGCTGGACGC
551  TGATGTCGTT GGCAGCAGCT TATCCGGCAG CATTTGCCCT
     GATGCTGCCC
601  TTGCCCGTAC TGATGTTTTT CCGTCCGTGG CAAAGCAGGC
     GTTTGATGTT
651  GACGGCAGTC GCCTCACTTG CCTTTGCCCT GCCGCTTATG
     ACCGTTTACC
701  CGCTGCTCTT GGCAAAAACG CAGCCCGCGC TGTTCGCGCA
     ATGGCTCGAC
751  TATCACGTTT TCGGTACGTT CGGCGGCGTG CGGCACGTTC
     AGACGGCATT
801  CAGTTTGTTT TACTATCTGA AAAACCTGCT TTGGTTTGCA
     TTGCCCGCGC
851  TGCCGCTGGC GGTTTGGACG GTTTGCCGCA CGCGCCTGTT
     TTCGACCGAC
901  TGGGGGATTT TGGGCGTCGT CTGGATGCTT GCCGTTTTGG
     TGCTGCTTGC
951  CGTCAATCCG CAGCGTTTTC AGGATAACCT CGTCTGGCTG
     CTTCCGCCGC
1001 TTGCCCTGTT CGGCGCGGCG CAACTGGACA GCCTGAGGCG
     CGGCGCGGCG
1051 GCGTTTGTCA ACTGGTTCGG CATTATGGCG TTCGGACTGT
     TTGCCGTGTT
1101 CCTGTGGACG GGCTTTTTCG CCATGAATTA CGGCTGGCCC
     GCCAAGCTTG
1151 CCGAACGCGC CGCCTATTTC AGCCCGTATT ATGTTCCTGA
     TATCGATCCC
1201 ATTCCGATGG CGGTTGCCGT ACTGTTCACA CCCTTGTGGC
     TGTGGGCGAT
1251 TACCCGGAAA AACATACGCG GCAGGCAGGC GGTTACCAAC
     TGGGCGGCAG
1301 GCGTTACCCT GACCTGGGCT TTGCTGATGA CGCTGTTCCT
     GCCGTGGCTG
1351 GACGCGGCGA AAAGCCACGC GCCGGTCGTC CGGAGTATGG
     AGGCATCGCT
1401 TTCCCCGGAA TTGAAACGGG AGCTTTCAGA CGGCATCGAG
     TGTATCGGCA
1451 TAGGCGGCGG CGACCTGCAC ACGCGGATTG TTTGGACGCA
     GTACGGCACA
1501 TTGCCGCACC GCGTCGGCGA TGTACAATGC CGCTACCGCA
     TCGTCCTCCT
1551 GCCCCAAAAT GCGGATGCGC CGCAAGGCTG GCAGACGGTT
     TGGCAGGGTG
1601 CGCGTCCGCG CAACAAAGAC AGTAAGTTCG CACTGATACG
     GAAAATCGGG
1651 GAAAATATAT AA

This corresponds to the amino acid sequence <SEQ ID 596; ORF141-1>:

1   MLTYTPPDAR PPAKTHEKPW LLLLMAFAWL WPGVFSHDLW
    NPDEPAVYTA
51  VEALAGSPTP LVAHLFGQTD FGIPPVYLWV AAAFKHLLSP
    WAADSYDAAR
101 FAGVFFAVIG LTSCGFAGFN FLGRHHGRSV VLILIGCIGL
    IPVAHFLNPA
151 AAAFAAAGLV LHGYSLARRR VIAASFLLGT GWTLMSLAAA
    YPAAFALMLP
201 LPVLMFFRPW QSRRLMLTAV ASLAFALPLM TVYPLLLAKT
    QPALFAQWLD
251 YHVFGTFGGV RHVQTAFSLF YYLKNLLWFA LPALPLAVWT
    VCRTRLFSTD
301 WGILGVVWML AVLVLLAVNP QRFQDNLVWL LPPLALFGAA
    QLDSLRRGAA
351 AFVNWFGIMA FGLFAVFLWT GFFAMNYGWP AKLAERAAYF
    SPYYVPDIDP
401 IPMAVAVLFT PLWLWAITRK NIRGRQAVTN WAAGVTLTWA
    LLMTLFLPWL
451 DAAKSHAPVV RSMEASLSPE LKRELSDGIE CIGIGGGDLH
    TRIVWTQYGT
501 LPHRVGDVQC RYRIVLLPQN ADAPQGWQTV WQGARPRNKD
    SKFALIRKIG
551 ENI*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF141 shows 95.0% identity over a 140aa overlap with an ORF (ORF141a) from strain A of N. meningitidis:

The complete length ORF141a nucleotide sequence <SEQ ID 597> is:

1    ATGCTGACCT ATACCCCGCC CGATGCCCGC CCGCCCGCCA
     AAACCCACGA
51   AAAGCCGTGG CTGTTGCTGT TGATGGCGTT TGCCTGGTTG
     TGGCCCGGCG
101  TGTTTTCCCA CGATTTGTGG AATCCTGACG AACCTGCCGT
     CTATACCGCC
151  GTCGAAGCAC TGGCAGGCAG CCCCACCCCT TTGGTTGCCC
     ATCTGTTCGG
201  TCAAATCGAT TTCGGCATAC CGCCCGTGTA TCTTTGGGTT
     GCCGCCGCGT
251  TCAAACATTT GCTGTCGCCG TGGGCTGCCG ACCCGTATGA
     TGCCGCACGC
301  TTTGCCGGCG TGTTTTTCGC CGTTGTCGGA CTGACTTCCT
     GCGGCTTTGC
351  CGGTTTCAAC TTTTTGGGCA GACACCACGG GCGCAGCGTC
     GTCCTGATTC
401  TCATCGGCTG TATCGGGCTG ATTCCGACCG TACACTTTCT
     CAACCCCGCT
451  GCCGCCGCCT TTGCCGCCGC CGGACTGGTG CTGCACGGTT
     ATTCTTTGGC
501  TCGCCGGCGC GTGATTGCCG CCTCTTTTCT GCTCGGTACG
     GGTTGGACGC
551  TGATGTCGTT GGCAGCAGCT TATCCGGCGG CATTTGCCCT
     GATGCTGCCC
601  CTGCCCGTGC TGATGTTTTT CCGTCCGTGG CAAAGCAGGC
     GTTTGATGTT
651  GACGGCAGTC GCCTCGCTTG CCTTTGCCCT GCCGCTTATG
     ACCGTTTACC
701  CGCTGCTCTT GGCAAAAACG CAGCCCGCGC TGTTCGCGCA
     ATGGCTCGAC
751  GATCACGTTT TCGGTACGTT CGGCGGCGTG CGGCACATTC
     AGACGGCATT
801  CAGTTTGTTT TACTATCTGA AAAACCTGCT TTGGTTTGCA
     TTGCCTGCGC
851  TGCCGCTGGC GGTTTGGACG GTTTGCCGCA CGCGCCTGTT
     TTCGACCGAC
901  TGGGGGATTT TGGGCGTCGT CTGGATGCTT GCCGTTTTGG
     TGCTGCTTGC
951  CGTCAATCCG CAGCGTTTTC AGGATAACCT CGTCTGGCTG
     CTTCCGCCGC
1001 TTGCCCTGTT CGGCGCGGCG CAACTGGACA GCCTGAGACG
     CGGCGCGGCG
1051 GCGTTTGTCA ACTGGTTCGG CATTATGGCG TTCGGACTGT
     TTGCCGTGTT
1101 CCTGTGGACG GGCTTTTTCG CCATGAATTA CGGCTGGCCC
     GCCAAGCTTG
1151 CCGAACGCGC CGCCTATTTC AGCCCGTATT ATGTTCCTGA
     TATCGATCCC
1201 ATTCCGATGG CGGTTGCCGT ACTGTTCACA CCCTTGTGGC
     TGTGGGCGAT
1251 TACCCGCAAA AACATACGCG GCAGGCAGGC GGTTACCAAC
     TGGGCGGCAG
1301 GCGTTACCCT GACCTGGGCT TTGCTGATGA CGCTGTTCCT
     GCCGTGGCTG
1351 GACGCGGCGA AAAGCCACGC GCCCGTCGTC CGGAGTATGG
     AGGCATCGCT
1401 TTCCCCGGAA TTAAAACGGG AGCTTTCAGA CGGCATCGAG
     TGTATCGACA
1451 TAGGCGGCGG CGACCTACAC ACGCGGATTG TTTGGACGCA
     GTACGGCACA
1501 TTGCCGCACC GCGTCGGCGA TGTACAATGC CGCTACCGCA
     TCGTCCGCTT
1551 GCCCCAAAAC GCGGATGCGC CGCAAGGCTG GCAGACGGTC
     TGGCAGGGTG
1601 CGCGCCCGCG CAACAAAGAC AGTAAGTTCG CACTGATACG
     GAAAACCGGG
1651 GAAAATATAT TAAAAACAAC AGATTGA

This encodes a protein having amino acid sequence <SEQ ID 598>:

1   MLTYTPPDAR PPAKTHEKPW LLLLMAFAWL WPGVFSHDLW
    NPDEPAVYTA
51  VEALAGSPTP LVAHLFGQID FGIPPVYLWV AAAFKHLLSP
    WAADPYDAAR
101 FAGVFFAVVG LTSCGFAGFN FLGRHHGRSV VLILIGCIGL
    IPTVHFLNPA
151 AAAFAAAGLV LHGYSLARRR VIAASFLLGT GWTLMSLAAA
    YPAAFALMLP
201 LPVLMFFRPW QSRRLMLTAV ASLAFALPLM TVYPLLLAKT
    QPALFAQWLD
251 DHVFGTFGGV RHIQTAFSLF YYLKNLLWFA LPALPLAVWT
    VCRTRLFSTD
301 WGILGVVWML AVLVLLAVNP QRFQDNLVWL LPPLALFGAA
    QLDSLRRGAA
351 AFVNWFGIMA FGLFAVFLWT GFFAMNYGWP AKLAERAAYF
    SPYYVPDIDP
401 IPMAVAVLFT PLWLWAITRK NIRGRQAVTN WAAGVTLTWA
    LLMTLFLPWL
451 DAAKSHAPVV RSMEASLSPE LKRELSDGIE CIDIGGGDLH
    TRIVWTQYGT
501 LPHRVGDVQC RYRIVRLPQN ADAPQGWQTV WQGARPRNKD
    SKFALIRKTG
551 ENILKTTD*

ORF141a and ORF141-1 show 98.2% identity in 553 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF141 shows 95% identity over a 140aa overlap with a predicted ORF (ORF141ng) from N. gonorrhoeae:

An ORF141ng nucleotide sequence <SEQ ID 599> was predicted to encode a protein having amino acid sequence <SEQ ID 600>:

1   MPSEAVSARP LCEYLLHLAI RPFLLTLMLT YTPPDARPPA
    KTHEKPWLLL
51  LMAFAWLWPG VFSHDLWNPA EPAVYTAVEA LAGSPTPLVA
    HLFGQTDFGI
101 PPVYLWVAAA FKHLLSPWAA HPYDAARFAG VFFAVIGLTS
    CGFAGFNFLG
151 RHHGRSVVLI HIGCIGLIPV AHFFNPAAAA FAAAGLVLHG
    YSLARRRVIA
201 ASFLLGTGWT LMSLAAAYPA AFALMLPLPV LMFFRPWQSR
    RLMLTAVASL
251 AFALPLMTVY PLLLAKTQPA LFAQWLNYHV FGTFGGVRHI
    QRAFSLFHYL
301 KNLLWFAPPG LPLAVWTVCR TRLFSTDWGI LGIVWMLAVL
    VLLAFNPQRF
351 QDNLVWLLPP LALFGAAQLD SLRRGAAAFV NWFGIMAFGL
    FAVFLWTGFF
401 AMNYGWPAKL AERAAYFSPY YVPDIDPIPM AVAVLFTPLW
    LWAITRKNIR
451 GRQAVTNWAA GVTLTWALLM TLFLPWLDAA KSHAPVVRSM
    EASFSPELKR
501 ELSDGIECIG IGGGDLHTRI VWTQYGTLPH RVGDVRCRYR
    IVRLPQNADA
551 PQGWQTVWQG ARPRNKDSKF ALIRKIGENI LKTTD*

Further work revealed the following gonococcal DNA sequence <SEQ ID 601>:

1    ATGCTGACCT ATACCCCGCC CGATGCCCGC CCGCCCGCCA
     AAACCCACGA
51   AAAACCGTGG CTGCTGCTGT TGATGGCGTT TGCCTGGCTG
     TGGCCCGGCG
101  TGTTTTCCCA CGATTTGTGG AATCCTGCCG AACCTGCCGT
     CTATACCGCC
151  GTCGAAGCAC TGGCAGGCAG CCCCACCCCC TTGGTTGCCC
     ATCTGTTCGG
201  TCAAACCGAT TTCGGCATAC CGCCCGTGTA TCTTTGGGTT
     GCCGCCGCAT
251  TCAAACATTT GCTGTCGCCG TGGGCAGCCG ACCCGTATGA
     TGCCGCACGC
301  TTTGCAGGCG TATTTTTTGC CGTTATCGGA CTGACTTCTT
     GCGGCTTTGC
351  CGGTTTCAAC TTTTTGGGCA GACACCACGG GCGCAGCGTT
     GTTTTAATCC
401  ATATCGGCTG TATCGGGCTG ATTCCGGTTG CCCATTTCCT
     CAATCCcgcc
451  gccgccgcct tTGCCGCCGC CGGACTGGTG CTGCacggct
     actcgctgGC
501  ACGCCGGCGC GTGATtgccg cctctTtccT GCTCGGTACG
     GGTTGGACGT
551  TGATGTCGCT GGCGGCAGCT TATCCGGCGG CGTTTGCGCT
     GATGCTGCCC
601  CTGCCCGTGC TGATGTTTTT CCGTCCGTGG CAAAGCAGGC
     GTTTGATGTT
651  GACGGCAGTC GCCTCGCTTG CCTTTGCCCT GCCGCTTATG
     ACCGTTTACC
701  CGCTGCTCtt gGCAAAAACG CAGCCCGCGC TGTTTGCGCA
     ATGGCTCAAC
751  TATCACGTTT TCGGTACGTt cggcgGCGTG CGGCAcaTTC
     AGAggGCatT
801  Cagtttgttt cactatctgA AAaatctgct ttggttcgca
     ccgcccgggC
851  TGCCGCTGGC GGTTTGGACG GTTTGCCGCA CACGCCTGTT
     TTCGACCGAC
901  TGGGGGATTT TGGGCATTGT CTGGATGCTT GCCGTTTTGG
     TGCTGCTCGC
951  CTTTAATCCG CAGCGTTTTC AAGACAACCT CGTCTGGCTG
     CTGCCGCCGC
1001 TTGCCCTGTT CGGCGCGGCG CAACTGGACA GCCTGAGGCG
     CGGCGCGGCG
1051 GCTTTTGTCA ACTGGTTCGG CATTATGGCG TTCGGGCTGT
     TTGCCGTGTT
1101 CCTGTGGACG GGCTTTTTCG CCATGAATTA CGGCTGGCCC
     GCCAAGCTTG
1151 CCGAACGCGC CGCCTACTTC AGCCCGTATT ACGTTCCCGA
     CATCGATCCC
1201 ATTCCGATGG CGGTTGCCGT ACTGTTCACA CCCTTGTGGC
     TGTGGGCGAT
1251 TACCCGGAAA AACATACGCG GCAGGCAGGC GGTTACCAAC
     TGGGCGGCAG
1301 GCGTTACCCT GACCTGGGCT TTGCTGATGA CGCTGTTCCT
     GCCGTGGCTG
1351 GACGCGGCGA AAAGCCACGC GCCCGTCGTC CGGAGTATGG
     AGGCATCGTT
1401 TTCCCCGGAA TTAAAACGGG AGCTTTCAGA CGGCATCGAG
     TGTATCGGCA
1451 TAGGCGGCGG CGACCTGCAC ACGCGGATTG TTTGGACGCA
     GTACGGCACA
1501 TTGCCGCACC GCGTCGGCGA TGTCCGTTGC CGCTACCGTA
     TCGTCCGCCT
1551 GCCCCAAAAC GCGGATGCGC CGCAAGGCTG GCAGACGGTC
     TGGCAGGGTG
1601 CGCGCCCGCG CAACAAAGAC AGTAAGTTTG CACTGATACG
     GAAAATCGGG
1651 GAAAATATAT TAAAAACAAC AGATTGA

This corresponds to the amino acid sequence <SEQ ID 602; ORF141ng-1>:

1   MLTYTPPDAR PPAKTHEKPW LLLLMAFAWL WPGVFSHDLW
    NPAEPAVYTA
51  VEALAGSPTP LVAHLFGQTD FGIPPVYLWV AAAFKHLLSP
    WAADPYDAAR
101 FAGVFFAVIG LTSCGFAGFN FLGRHHGRSV VLIHIGCIGL
    IPVAHFLNPA
151 AAAFAAAGLV LHGYSLARRR VIAASFLLGT GWTLMSLAAA
    YPAAFALMLP
201 LPVLMFFRPW QSRRLMLTAV ASLAFALPLM TVYPLLLAKT
    QPALFAQWLN
251 YHVFGTFGGV RHIQRAFSLF HYLKNLLWFA PPGLPLAVWT
    VCRTRLFSTD
301 WGILGIVWML AVLVLLAFNP QRFQDNLVWL LPPLALFGAA
    QLDSLRRGAA
351 AFVNWFGIMA FGLFAVFLWT GFFAMNYGWP AKLAERAAYF
    SPYYVPDIDP
401 IPMAVAVLFT PLWLWAITRK NIRGRQAVTN WAAGVTLTWA
    LLMTLFLPWL
451 DAAKSHAPVV RSMEASFSPE LKRELSDGIE CIGIGGGDLH
    TRIVWTQYGT
501 LPHRVGDVRC RYRIVRLPQN ADAPQGWQTV WQGARPRNKD
    SKFALIRKIG
551 ENILKTTD*

ORF141ng-1 and ORF141-1 show 97.5% identity in 553 aa overlap:

Based on the presence of several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 72

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 603>:

1   ..CAATCCGCCA AATGGTTATC GGGCCAAACT CTAGTCGGCA
    CAGCAATTGG
51  GATACGCGGG CAGATAAAGC TTGGCGGCAA CCTGCATTAC
    GATATATTTA
101 CCGGCCGCGC ATTGAAAAAG CCCGAATTTT TCCAATCAAG
    GAAATGGGCA
151 AGCGGTTTTC AGGTAGGCTA TACGTTTTAA

This corresponds to the amino acid sequence <SEQ ID 604; ORF142>:

1  ..QSAKWLSGQT LVGTAIGIRG QIKLGGNLHY DIFTGRALKK
   PEFFQSRKWA
51 SGFQVGYTF*

Further work revealed the complete nucleotide sequence <SEQ ID 605>:

1    ATGGATAATT CGGGTAGTGA GGCGACAGGA AAATACCAAG
     GAAATATCAC
51   TTTCTCTGCC GACAATCCTT TGGGACTGAG TGATATGTTC
     TATGTAAATT
101  ATGGACGTTC GATTGGCGGT ACGCCCGATG AGGAAAGTTT
     TGACGGCCAT
151  CGCAAAGAAG GCGGATCAAA CAATTACGCC GTACATTATT
     CAGCCCCTTT
201  CGGTAAATGG ACATGGGCAT TCAATCACAA TGGCTACCGT
     TACCATCAGG
251  CAGTTTCCGG ATTATCGGAA GTCTATGACT ATAATGGAAA
     AAGTTACAAT
301  ACTGATTTCG GCTTCAACCG CCTGTTGTAT CGTGATGCCA
     AACGCAAAAC
351  CTATCTCGGT GTAAAACTGT GGATGAGGGA AACAAAAAGT
     TACATTGATG
401  ATGCCGAACT GACTGTACAA CGGCGTAAAA CTGCGGGTTG
     GTTGGCAGAA
451  CTTTCCCACA AAGAATATAT CGGTCGCAGT ACGGCAGATT
     TTAAGTTGAA
501  ATATAAACGC GGCACCGGCA TGAAAGATGC TCTGCGCGCG
     CCTGAAGAAG
551  CCTTTGGCGA AGGCACGTCA CGTATGAAAA TTTGGACGGC
     ATCGGCTGAT
601  GTAAATACTC CTTTTCAAAT CGGTAAACAG CTATTTGCCT
     ATGACACATC
651  CGTTCATGCA CAATGGAACA AAACCCCGCT AACATCGCAA
     GACAAACTGG
701  CTATCGGCGG ACACCACACC GTACGTGGCT TCGACGGTGA
     AATGAGTTTG
751  TCTGCCGAGC GGGGATGGTA TTGGCGCAAC GATTTGAGCT
     GGCAATTTAA
801  ACCAGGCCAT CAGCTTTATC TTGGGGCTGA TGTAGGACAT
     GTTTCAGGAC
851  AATCCGCCAA ATGGTTATCG GGCCAAACTC TAGTCGGCAC
     AGCAATTGGG
901  ATACGCGGGC AGATAAAGCT TGGCGGCAAC CTGCATTACG
     ATATATTTAC
951  CGGCCGCGCA TTGAAAAAGC CCGAATTTTT CCAATCAAGG
     AAATGGGCAA
1001 GCGGTTTTCA GGTAGGCTAT ACGTTTTAA

This corresponds to the amino acid sequence <SEQ ID 606; ORF142-1>:

1   MDNSGSEATG KYQGNITFSA DNPLGLSDMF YVNYGRSIGG
    TPDEESFDGH
51  RKEGGSNNYA VHYSAPFGKW TWAFNHNGYR YHQAVSGLSE
    VYDYNGKSYN
101 TDFGFNRLLY RDAKRKTYLG VKLWMRETKS YIDDAELTVQ
    RRKTAGWLAE
151 LSHKEYIGRS TADFKLKYKR GTGMKDALRA PEEAFGEGTS
    RMKIWTASAD
201 VNTPFQIGKQ LFAYDTSVHA QWNKTPLTSQ DKLAIGGHHT
    VRGFDGEMSL
251 SAERGWYWRN DLSWQFKPGH QLYLGADVGH VSGQSAKWLS
    GQTLVGTAIG
301 IRGQIKLGGN LHYDIFTGRA LKKPEFFQSR KWASGFQVGY
    TF*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. gonorrhoeae

ORF142 shows 88.1% identity over a 59aa overlap with a predicted ORF (ORF142ng) from N. gonorrhoeae:

The complete length ORF142ng nucleotide sequence <SEQ ID 607> is:

1    ATGGATAATT CGGGTAGTGA GGCGACAGGA AAATACCAAG
     GAAATATCAC
51   TTTCTCTGCC GACAATCCTT TTGGACTGAG TGATATGTTC
     TATGTAAATT
101  ATGGACGTTC AATTGGCGGT ACGCCCGATG AGGAAAATTT
     TGACGGCCAT
151  CGCAAAGAAG GCGGATCAAA CAATTACGCC GTACATTATT
     CAGCCCCTTT
201  CGGTAAATGG ACATGGGCAT TCAATCACAA TGGCTACCGT
     TACCATCAGG
251  CGGTTTCCGG ATTATCGGAA GTCTATGACT ATAATGGAAA
     AAGTTACAAC
301  ACTGATTTCG GCTTCAACCG CCTGTTGTAT CGTGATGCCA
     AACGCAAAAC
351  CTATCTCAGT GTAAAACTGT GGACGAGGGA AACAAAAAGT
     TACATTGATG
401  ATGCCGAACT GACTGTACAA CGGCGTAAAA CCACAGGTTG
     GTTGGCAGAA
451  CTTTCCCACA AAGGATATAT CGGTCGCAGT ACGGCAGATT
     TTAAGTTGAA
501  ATATAAACAC GGCACCGGCA TGAAAGATGC TCTGCGCGCG
     CCTGAAGAAG
551  CCTTTGGCGA AGGCACGTCA CGTATGAAAA TTTGGACGGC
     ATCGGCTGAT
601  GTAAATACTC CTTTTCAAAT CGGTAAACAG CTATTTGCCT
     ATGACACATC
651  CGTTCATGCA CAATGGAACA AAACCCCGCT AACATCGCAA
     GACAAACTGG
701  CTATCGGCGG ACACCACACC GTACGTGGCT TCGACGGTGA
     AATGAGTTTG
751  CCTGCCGAGC GGGGATGGTA TTGGCGCAAC GATTTGAGCT
     GGCAATTTAA
801  ACCAGGCCAT CAGCTTTATC TTGGGGCTGA TGTAGGACAT
     GTTTCAGGAC
851  AATCCGCCAA ATGGTTATCG GGCCAAACTC TAGCCGGCAC
     AGCAATTGGG
901  ATACGCGGGC AGATAAAGCT TGGCGGCAAC CTGCATTACG
     ATATATTTAC
951  CGGCCGTGCA TTGAAAAAGC CCGAATATTT TCAGACGAAG
     AAATGGGTAA
1001 CGGGGTTTCA GGTGGGTTAT TCGTTTTGA

This encodes a protein having amino acid sequence <SEQ ID 608>:

1   MDNSGSEATG KYQGNITFSA DNPFGLSDMF YVNYGRSIGG
    TPDEENFDGH
51  RKEGGSNNYA VHYSAPFGKW TWAFNHNGYR YHQAVSGLSE
    VYDYNGKSYN
101 TDFGFNRLLY RDAKRKTYLS VKLWTRETKS YIDDAELTVQ
    RRKTTGWLAE
151 LSHKGYIGRS TADFKLKYKH GTGMKDALRA PEEAFGEGTS
    RMKIWTASAD
201 VNTPFQIGKQ LFAYDTSVHA QWNKTPLTSQ DKLAIGGHHT
    VRGFDGEMSL
251 PAERGWYWRN DLSWQFKPGH QLYLGADVGH VSGQSAKWLS
    GQTLAGTAIG
301 IRGQIKLGGN LHYDIFTGRA LKKPEYFQTK KWVTGFQVGY
    SF*

The underlined sequence (aromatic-Xaa-aromatic amino acid motif) is usually found at the C-terminal end of outer membrane proteins.

ORF142ng and ORF142-1 show 95.6% identity over 342aa overlap:

In addition, ORF142ng is homologous to the HecB protein of E. chrysanthemi:

gi|1772622 (L39897) HecB [Erwinia chrysanthemi] Length = 558
Score = 119 bits (295), Expect = 3e−26
Identities = 88/346 (25%), Positives = 151/346 (43%),
Gaps = 22/346 (6%)
Query:	2	DNSGSEATGKYQGNITFSADNPFGLSDMFYVNYGRSIGGTPDEENFDGHRKEGGSNNYAV	61
		DNSG ++TG+ Q N + + DN FGL+D ++++ G S   +    + D    + G
Sbjct:	230	DNSGQKSTGEEQLNGSLALDNVFGLADQWFISAGHS---SRFATSHDAESLQAG------	280
Query:	62	HYSAPFGKWTWAFNHNGYRYHQAVSGLSEVYDYNGKSYNTDFGFNRLLYRDAKRKTYLSV	121
		+S P+G W   +N++  RY          +   G S    F  +R+++RD   KT ++
Sbjct:	281	-FSMPYGYWNLGYNYSQSRYRNTFINRDFPWHSTGDSDTHRFSLSRVVFRDGTMKTAIAG	339
Query:	122	KLWTRETKSYIDDAELTVQRRKTTGWLAELSHKGYIGRSTADFKLKYKHGTGMKDALRAP	181
		R   +Y++ + L    RK +     ++H   +    A F   Y  G     +
Sbjct:	340	TFSQRTGNNYLNGSLLPSSSRKLSSVSLGVNHSQKLWGGLATFNPTYNRGVRWLGSETDT	399
Query:	182	EEAFGEGTSRMKIWTASADVNTPFQIGKQLFAYDTSVHAQWNKTPLTSQDKLAIGGHHTV	241
		+++  E  +    WT SA    P         Y  S++ Q++   L   ++L +GG  ++
Sbjct:	400	DKSADEPRAEFNKWTLSASYYHPV---TDSITYLGSLYGQYSARALYGSEQLTLGGESSI	456
Query:	242	RGFDGEMSLPAERGWYWRNDLSWQFKP----GHQLYLGA-DVGHVSGQSAKWLSGQTLAG	296
		RGF  E      RG YWRN+L+WQ       G+  ++ A D GH+        +  +L G
Sbjct:	457	RGF-REQYTSGNRGAYWRNELNWQAWQLPVLGNVTFMAAVDGGHLYNHKQDNSTAASLWG	515
Query:	297	TAIGIRGQIKLGGNLHYDIFTGRALKKPEYFQTKKWVTGFQVGYSF	342
		A+G+    +    L   +  G  +  P + Q    V G++VG SF
Sbjct:	516	GAVGMTVASRW---LSQQVTVGWPISYPAWLQPDTMVVGYRVGLSF	558

On the basis of this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 73

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 609>:

1   ATGCGGACGA AATGGTCAGC AGTGAGAAGC TGCTTACTTG
    GgCGGACACC
51  GCCGACATCG ATACCGCTTT GAACCTGTTG TACCGTTTGC
    AAAAACTCGA
101 ATTCCTCTAT GGCGATGAAA ACGGTCATTC AGACGGCATC
    AATTTGwCGG
151 ACGAGCAATT GCCGTTGCTG ATGGAACAAT TGTCCGGCAG
    CGGTAAGGCG
201 TTATTGGTCG ATCGGAACGG TCTGTATCTT GCCAACGCCA
    ATTTCCATCA
251 TGAGGCGGCG GAAGAGTTGG GGTTGTTGGC GGCAGAAGTC
    GCACAGATGG
301 AAAAGAAATA CCGGCTGCTG ATTAAGAACA AC..

This corresponds to the amino acid sequence <SEQ ID 610; ORF143>:

1   MRTKWSAVRS CTWADTADID TALNLLYRLQ KLEFLYGDEN
    GHSDGINLXD
51  EQLPLLMEQL SGSGKALLVD RNGLYLANAN FHHEAAEELG
    LLAAEVAQME
101 KKYRLLIKNN ..

Further work revealed the complete nucleotide sequence <SEQ ID 611>:

1   ATGGAATCAA CACTTTCACT ACAAGCAAAT TTATATCCCC
    GCCTGACTCC
51  TGCCGGTGCA TTTTATGCCG TATCCAGCGA TGCCCCCAGT
    GCCGGTAAAA
101 CTTTGTTGCA CAGCCTGTTG AAAGCAGATG CGGACGAAAT
    GGTCAGCAGT
151 GAGAAGCTGC TTACTTGGGC GGACACCGCC GACATCGATA
    CCGCTTTGAA
201 CCTGTTGTAC CGTTTGCAAA AACTCGAATT CCTCTATGGC
    GATGAAAACG
251 GTCATTCAGA CGGCATCAAT TTGTCGGACG AGCAATTGCC
    GTTGCTGATG
301 GAACAATTGT CCGGCAGCGG TAAGGCGTTA TTGGTCGATC
    GGAACGGTCT
351 GTATCTTGCC AACGCCAATT TCCATCATGA GGCGGCGGAA
    GAGTTGGGGT
401 TGTTGGCGGC AGAAGTCGCA CAGATGGAAA AGAAATACCG
    GCTGCTGATT
451 AAGAACAACC TGTATATCAA CAATAACGCT TGGGGCGTTT
    GCGATCCTTC
501 CGGTCAGAGC GAATTGACAT TTTTCCCATT GTATATCGGT
    TCAACCAAAT
551 TTATTTTGGT TATCGGCGGC ATTCCCGATT TGGGCAAAGA
    GGCATTTGTT
601 ACTTTGGTAA GGATTTTATA CCGCCGTTAC AGCAACCGCG
    TGTAA

This corresponds to the amino acid sequence <SEQ ID 612; ORF143-1>:

1   MESTLSLQAN LYPRLTPAGA FYAVSSDAPS AGKTLLHSLL
    KADADEMVSS
51  EKLLTWADTA DIDTALNLLY RLQKLEFLYG DENGHSDGIN
    LSDEQLPLLM
101 EQLSGSGKAL LVDRNGLYLA NANFHHEAAE ELGLLAAEVA
    QMEKKYRLLI
151 KNNLYINNNA WGVCDPSGQS ELTFFPLYIG STKFILVIGG
    IPDLGKEAFV
201 TLVRILYRRY SNRV*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF143 shows 92.4% identity over a 105aa overlap with an ORF (ORF143a) from strain A of N. meningitidis:

The complete length ORF143a nucleotide sequence <SEQ ID 613> is:

1   ATGGAATCAA CANTTTCACT ACAAGCAAAT TTATATCNCC
    GCCTGACTCC
51  TGCCGGTGCA TTTTATGCCG TATCCAGCGA TGNCCCCAGT
    GCCGGTAAAA
101 CTTTGTTGCA CAGCCTGTTG AAAGCGGATG CGGACGAAAT
    GGTNAGCAGT
151 GAGAAGCTGC TTACCTGGGC GGANACCGCC GACATCGATA
    CCGCTTTGAA
201 CCTGTTGTAC CGTTTGCAAA AACTCGAATT CCTCTATGGC
    GATGAAAACG
251 GTCATTCAGA CGGCATCAAT TTGTCGGACG AGCAATTGCC
    GTTGCTGATG
301 GAACAATTGT CCGGCAGCGG TAAGGCGTTA TTGGTCGATC
    GGAACGGTCT
351 GTATCTTGCC AACGCCAATT TCCATCATGA GGCGGCGGAA
    GAGTTGGGGT
401 TGTTGGCGGC AGAAGTCGCA CAGATGGAAA AGAAATACCG
    GCTGCNNATT
451 AAGAACAACC TGTATATCAA CAATAACGCT TGGGGCGTTT
    GCGATCCTTC
501 CGGTCAGAGC GAATTGACAT TTTTCCCATT GTATATCGGT
    TCAACCAAAT
551 TTATTTTGGT TATCGGCGGC ATTCCCGATT TGGGCAAAGA
    GGCATTTGTT
601 ACTTTGGTAA GGATNTTATA CCNCCNGTTA CAGCAACCGC
    GTGTAAAACT
651 TGGGAGAGAG GANGGGTTAT GCAGCAATTA TTGA

This encodes a protein having amino acid sequence <SEQ ID 614>:

1   MESTXSLQAN LYXRLTPAGA FYAVSSDXPS AGKTLLHSLL
    KADADEMVSS
51  EKLLTWAXTA DIDTALNLLY RLQKLEFLYG DENGHSDGIN
    LSDEQLPLLM
101 EQLSGSGKAL LVDRNGLYLA NANFHHEAAE ELGLLAAEVA
    QMEKKYRLXI
151 KNNLYINNNA WGVCDPSGQS ELTFFPLYIG STKFILVIGG
    IPDLGKEAFV
201 TLVRXLYXXL QQPRVKLGRE XGLCSNY*

ORF143a and ORF143-1 show 97.1% identity in 207 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF143 shows 95.5% identity over a 110aa overlap with a predicted ORF (ORF143ng) from N. gonorrhoeae:

An ORF143ng nucleotide sequence <SEQ ID 615> was predicted to encode a protein having amino acid sequence <SEQ ID 616>:

1   MRTKWSAVRS CSRADTADID TALNLLYRLQ KLEFLYGDEN
    GHSDGINLSD
51  EQLPLLMEQL SGSGKALLVD RNGLYLANAN FHHESAEELG
    LLAAEVAQME
101 KKYRLLIRNN LYINNNAWGV CDPSGQSELT FFPLYIGSTK
    FILVIAGIPD
151 LSKGGICYFG KDFIPPLQQP RVKLGTGGIM RQLLISILED
    LNNTSTDIIA
201 SAVISTDGLP MATMLPSHLN SDRVGAISAT LLALGSRSVQ
    ELACGELEQV
251 MIKGKSGYIL LSQAGKDAVL VLVAKETGRL GLILLDAKRA
    ARHIAEAI*

Further work revealed the following gonococcal DNA sequence <SEQ ID 617>:

1   ATGGAATCAA CACTTTCACT ACAAGCGAAT TTATATCCCT
    GCCTGACTCC
51  TGCCGGTGCA TTTTATGCCG TATCCAGCGA TGCCCCCAGT
    GCCGGTAAAA
101 CTTTGTTGCG CAGCCTGTTG AAAGCGGATG CGGACGAAGT
    GGTCAGCAGT
151 GAGAAGCTGC TCGCGGCGGA CACCGCCGAC ATCGATACCG
    CTTTGAACCT
201 GTTGTACCGT TTGCAAAAAC TCGAATTCCT CTATGGCGAT
    GAAAACGGTC
251 ATTCAGACGG CATCAATTTG TCGGACGAGC AATTGCCGTT
    GCTGATGGAA
301 CAATTGTCCG GCAGCGGTAA GGCATTATTG GTCGATCGGA
    ACGGTCTGTA
351 TCTTGCCAAC GCCAATTTCC ATCATGAGTC GGCGGAAGAG
    TTGGGGTTGT
401 TGGCGGCAGA AGTCGCACAG ATGGAAAAGA AATACCGGCT
    GCTGATTAGG
451 AACAACCTGT ATATCAACAA TAACGCTTGG GGCGTTTGCG
    ATCCTTCCGG
501 TCAGAGCGAA TTGACATTTT TCCCATTGTA TATCGGTTCA
    ACCAAATTTA
551 TTTTGGTTAT CGCCGGCATT CCCGATTTGA GCAAAGAGGC
    ATTTGTTACT
601 TTGGTAAGGA TTTTATACCG CCGTTACAGC AACCGCGTGT
    AA

This corresponds to the amino acid sequence <SEQ ID 618; ORF143ng-1>:

1   MESTLSLQAN LYPCLTPAGA FYAVSSDAPS AGKTLLRSLL
    KADADEVVSS
51  EKLLAADTAD IDTALNLLYR LQKLEFLYGD ENGHSDGINL
    SDEQLPLLME
101 QLSGSGKALL VDRNGLYLAN ANFHHESAEE LGLLAAEVAQ
    MEKKYRLLIR
151 NNLYINNNAW GVCDPSGQSE LTFFPLYIGS TKFILVIAGI
    PDLSKEAFVT
201 LVRILYRRYS NRV*

ORF143ng-1 and ORF143-1 show 95.8% identity in 214 aa overlap:

Based on the presence of the putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 74

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 619>:

1   ATGACCTTTT TACAACGTTT GCAAGGTTTG GCAGACAATA
    AAATCTGTGC
51  GTTTGCATGG TTCGTCGTCC GCCGCTTTGA TGAAGAACGC
    GTACCGCAGr
101 CGGCGGCAAG CATGACGTTT ACGACGCTGC TGGCACTCGT
    CCCCGTGCTG
151 ACCGTGATGG TGGCGGTCGC TTCGATTTTC CCCGTGTTCG
    ACCGCTGGTC
201 GGATTCGTTC GTCTCCTTCG TCAACCAAAC CATTGTGCCG
    CA.GGCGCGG
251 ACATGGTGTT CGACTATATC AATGCGTTCC GCGAGCAGGC
    GAACCGGCTG
301 ACGGCAATCG GCAGCGTGAT GCTGGTCGTT ACCTCGCTGA
    TGCTGATTCG
351 GACGATAGAC AATACGTTCA ACCGCATCTG GaCGGGTCAA
    wTyCCAGCGT
401 CCGTGGATG..

This corresponds to the amino acid sequence <SEQ ID 620; ORF144>:

1   MTFLQRLQGL ADNKICAFAW FVVRRFDEER VPQXAASMTF
    TTLLALVPVL
51  TVMVAVASIF PVFDRWSDSF VSFVNQTIVP XGADMVFDYI
    NAFREQANRL
101 TAIGSVMLVV TSLMLIRTID NTFNRIWRVX XQRPWM...

Further work revealed the complete nucleotide sequence <SEQ ID 621>:

1    ATGACCTTTT TACAACGTTT GCAAGGTTTG GCAGACAATA
     AAATCTGTGC
51   GTTTGCATGG TTCGTCGTCC GCCGCTTTGA TGAAGAACGC
     GTACCGCAGG
101  CGGCGGCAAG CATGACGTTT ACGACGCTGC TGGCACTCGT
     CCCCGTGCTG
151  ACCGTGATGG TGGCGGTCGC TTCGATTTTC CCCGTGTTCG
     ACCGCTGGTC
201  GGATTCGTTC GTCTCCTTCG TCAACCAAAC CATTGTGCCG
     CAGGGCGCGG
251  ACATGGTGTT CGACTATATC AATGCGTTCC GCGAGCAGGC
     GAACCGGCTG
301  ACGGCAATCG GCAGCGTGAT GCTGGTCGTT ACCTCGCTGA
     TGCTGATTCG
351  GACGATAGAC AATACGTTCA ACCGCATCTG GCGGGTCAAT
     TCCCAGCGTC
401  CGTGGATGAT GCAGTTTCTC GTCTATTGGG CTTTACTGAC
     GTTCGGGCCG
451  CTGTCTTTGG GCGTGGGCAT TTCCTTTATG GTCGGCTCGG
     TACAGGATGC
501  CGCGCTTGCC TCAGGTGCGC CGCAGTGGTC GGGCGCGTTG
     CGAACGGCGG
551  CGACGCTGAC CTTCATGACG CTTTTGCTGT GGGGGCTGTA
     CCGCTTCGTG
601  CCAAACCGCT TCGTTCCCGC GCGGCAGGCG TTTGTCGGGG
     CTTTGGCAAC
651  AGCGTTTTGT CTGGAAACCG CGCGCTCCCT CTTCACTTGG
     TATATGGGCA
701  ATTTCGACGG CTACCGCTCG ATTTACGGCG CGTTTGCCGC
     CGTGCCGTTT
751  TTTCTGTTGT GGCTGAACCT GTTGTGGACG CTGGTCTTGG
     GCGGCGCGGT
801  GCTGACTTCT TCACTCTCCT ACTGGCAGGG AGAAGCGTTC
     CGCAGGGGCT
851  TCGACTCGCG CGGACGGTTT GACGACGTGT TGAAAATCCT
     GCTGCTTCTG
901  GATGCGGCGC AAAAAGAAGG CAAAGCCTTG CCTGTTCAGG
     AGTTCAGACG
951  GCATATCAAT ATGGGCTACG ACGAGTTGGG CGAGCTTTTG
     GAAAAGCTGG
1001 CGCGGCACGG CTACATCTAT TCCGGCAGAC AGGGTTGGGT
     GTTGAAAACG
1051 GGGGCGGATT CGATTGAGTT GAACGAACTC TTCAAGCTCT
     TCGTTTACCG
1101 TCCGTTGCCT GTGGAAAGGG ATCATGTGAA CCAAGCTGTC
     GATGCGGTAA
1151 TGACACCGTG TTTGCAGACT TTGAACATGA CGCTGGCAGA
     GTTTGACGCT
1201 CAGGCGAAAA AACGGCAGTA G

This corresponds to, the amino acid sequence <SEQ ID 622; ORF144-1>:

1   MTFLQRLQGL ADNKICAFAW FVVRRFDEER VPQAAASMTF
    TTLLALVPVL
51  TVMVAVASIF PVFDRWSDSF VSFVNQTIVP QGADMVFDYI
    NAFREQANRL
101 TAIGSVMLVV TSLMLIRTID NTFNRIWRVN SQRPWMMQFL
    VYWALLTFGP
151 LSLGVGISFM VGSVQDAALA SGAPQWSGAL RTAATLTFMT
    LLLWGLYRFV
201 PNRFVPARQA FVGALATAFC LETARSLFTW YMGNFDGYRS
    IYGAFAAVPF
251 FLLWLNLLWT LVLGGAVLTS SLSYWQGEAF RRGFDSRGRF
    DDVLKILLLL
301 DAAQKEGKAL PVQEFRRHIN MGYDELGELL EKLARHGYIY
    SGRQGWVLKT
351 GADSIELNEL FKLFVYRPLP VERDHVNQAV DAVMTPCLQT
    LNMTLAEFDA
401 QAKKRQ*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF144 shows 96.3% identity over a 136aa overlap with an ORF (ORF144a) from strain A of N. meningitidis:

The complete length ORF144a nucleotide sequence <SEQ ID 623> is:

1    ATGACCTTTT TACAACGTTT GCAAGGTTTG GCAGACAATA
     AAATCTGTGC
51   GTTTGCATGG TTCGTCGTCC GCCGCTTTGA TGAAGAACGC
     GTACCGCAGG
101  CGGCGGCAAG CATGACGTTT ACGACACTGC TGGCACTCGT
     CCCCGTGCTG
151  ACCGTGATGG TGGCGGTCGC TTCGATTTTC CCCGTGTTCG
     ACCGNTGGTC
201  GGATTCGTTC GTCTCCTTCG TCAACCAAAC CATTGTGCCG
     CAGGGCGCGG
251  ACATGGTNTT CGACTATATC AATGCGTTCC GCGAGCAGGC
     GAACCGGCTG
301  ACGGCAATCG GCAGCGTGAT GCTGGTCGTT ACCTCGCNGA
     TGCTGATTCG
351  GACGATAGAC AATACGTTCA ACCGCATCTG GCGGGTCAAT
     TCCCAGCGTC
401  CGTGGATGAT GCAGTTTCTC GTCTATTGGG CTTTACTGAC
     GTTCGGGCCG
451  CTGTCTTTGG GCGTGGGCAT TTCCTTTATN GTCGGCTCGG
     TACAGGATGC
501  CGCGCTTGCC TCAGGTGCGC CGCAGTGGTC GGGCGCGTTG
     CGAACGGCGG
551  CGACGCTGAN CTTCATGACG CTTTTGCTGT GGGGGCTGTA
     CCGCTNCGTG
601  CCAAACCGCT TCGTTCCCGC GCGGCANGCG TTTGTCGGGG
     CTTTGGCAAC
651  AGCGTTCTGT CTGGAAACCG CGCGTTCCCT CTTTACTTGG
     TATATGGGCA
701  ATTTCGACGG CTACCGCTCG ATTTACGGNG CGTTTGCCGC
     CGTGCCGTTT
751  TTTCTGTTGT GGCTGAACCT GTTGTGGACG CTGGTCTTGG
     GCGGCGCGGT
801  GCTGACTTCT TCACTCTCCT ACTGGCAGGG AGAAGCGTTC
     CGCAGGGNCT
851  TCGACTCGCG CGGACGGTTT GACGACGTGT TGAAAATCCT
     GCTGCTTCTG
901  GATGCGGCGC AAAAAGAAGG CNAAGCCTTG CCTGTTCAGG
     AGTTCAGACG
951  GCATATCAAT ATGGGCTACG ACGAGTTGGG CGAGCTTTTG
     GAAAAGCTGG
1001 CGCGGCACGG CTACATCTAT TCCGGCAGAC AGGGTTGGGT
     GTTGAAAACG
1051 GGGGCGGATT CGATTGAGTT GAACGAACTC TTCAAGCTCT
     TCGTTTACCG
1101 TCCGTTGCCT GTGGAAAGGG ATCATGTGAA CCAAGCTGTC
     GATGCGGTAA
1151 TGATGCCGTG TTTGCAGACT TTGAACATGA CGCTGGCAGA
     GTTTGACGCT
1201 CAGGCGAAAA AACAGCAGCA ATCTTGA

This encodes a protein having amino acid sequence <SEQ ID 624>:

1   MTFLQRLQGL ADNKICAFAW FVVRRFDEER VPQAAASMTF
    TTLLALVPVL
51  TVMVAVASIF PVFDRWSDSF VSFVNQTIVP QGADMVFDYI
    NAFREQANRL
101 TAIGSVMLVV TSXMLIRTID NTFNRIWRVN SQRPWMMQFL
    VYWALLTFGP
151 LSLGVGISFX VGSVQDAALA SGAPQWSGAL RTAATLXFMT
    LLLWGLYRXV
201 PNRFVPARXA FVGALATAFC LETARSLFTW YMGNFDGYRS
    IYGAFAAVPF
251 FLLWLNLLWT LVLGGAVLTS SLSYWQGEAF RRXFDSRGRF
    DDVLKILLLL
301 DAAQKEGXAL PVQEFRRHIN MGYDELGELL EKLARHGYIY
    SGRQGWVLKT
351 GADSIELNEL FKLFVYRPLP VERDHVNQAV DAVMMPCLQT
    LNMTLAEFDA
401 QAKKQQQS*

ORF144a and ORF144-1 show 97.8% identity in 406 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF144 shows 91.2% identity over a 136aa overlap with a predicted ORF (ORF144ng) from N. gonorrhoeae:

The complete length ORF144ng nucleotide sequence <SEQ ID 625> is predicted to encode a protein having amino acid sequence <SEQ ID 626>:

1   MTFLQCWQGS ADNKICAFAW FVIRRFSEER VPQAAASMTF
    TTLLALVPVL
51  TVMVAVASIF PVFDRWSDSF VSFVNQTIVP QGADMVFDYI
    DAFRDQANRL
101 TAIGSVMLVV TSLMLIRTID NAFNRIWRVN TQRPWMMQFL
    VYWALLTFGP
151 LSLGVGISFM VGSVQDSVLS SGAQQWADAL KTAARLAFMT
    LLLWGLYRFV
201 PNRFVPARQA FVGALITAFC LETARFLFTW YMGNFDGYRS
    IYGAFAAVPF
251 FLLWLNLLWT LVLGGAVLTS SLSYWQGEAF RRGFDSRGRF
    DDVLKILLLL
301 DAAQKEGRTL SVQEFRRHIN MGYDELGELL EKLARYGYIY
    SGRQGWVLKT
351 GADSIELSEL FKLFVYRPLP VERDHVNQAV DAVMTPCLQT
    LNMTLAEFDA
401 QAKKQQQS*

Further work revealed the following gonococcal DNA sequence <SEQ ID 627>:

1    ATGACCTTTT TACAACGTTG GCAAGGTTTG GCGGACAATA
     AAATCTGTGC
51   ATTTGCATGG TTCGTCATCC GCCGTTTCAG TGAAGAGCGC
     GTACCGCAGG
101  CAGCGGCGAG CATGACGTTT ACGACACTGC TGGCACTCGT
     CCCCGTACTG
151  ACCGTAATGG TCGCGGTCGC TTCGATTTTC CCCGTGTTCG
     ACCGCTGGTC
201  GGATTCGTTC GTCTCCTTCG TCAACCAAAC CATTGTGCCG
     CAGGGCGCGG
251  ATATGGTGTT CGACTATATC GACGCATTCC GCGATCAGGC
     AAACCGGCTG
301  ACCGCCATCG GCAGCGTGAT GCTGGTCGTA ACCTCGCTGA
     TGCTGATTCG
351  GACGATAGAC AATGCGTTCA ACCGCATCTG GCGGGTTAAC
     ACGCAACGCC
401  CCTGGATGAT GCAGTTCCTC GTTTATTGGG CGTTGCTGAC
     TTTCGGGCCT
451  TTGTCTTTGG GTGTGGGCAT TTCCTTTATG GTCGGGTCGG
     TTCAAGACTC
501  CGTACTCTCC TCCGGAGCGC AACAATGGGC GGACGCGTTG
     AAGACGGCGG
551  CAAGGCTGGC TTTCATGACG CTTTTGCTGT GGGGGCTGTA
     CCGCTTCGTG
601  CCCAACCGCT TCGTGCCCGC CCGGCAGGCG TTTGTCGGAG
     CTTTGATTAC
651  GGCATTCTGC CTGGAGACGG CACGTTTCCT GTTCACCTGG
     TATATGGGCA
701  ATTTCGACGG CTACCGCTCG ATTTACGGCG CATTTGCCGC
     CGTGCCGTTT
751  TTCCTGCTGT GGTTAAACCT GCTGTGGACG CTGGTCTTGG
     GCGGGGCGGT
801  GCTGACTTCG TCGCTGTCTT ATTGGCAGGG CGAGGCCTTC
     CGCAGGGGAT
851  TCGACTCGCG CGGACGGTTT GACGACGTGT TGAAAATCCT
     GCTGCTTCTG
901  GATGCGGCGC AAAAAGAAGG CCGAACCCTG TCCGTTCAGG
     AGTTCAGACG
951  GCATATCAAT ATGGGTTACG ATGAATTGGG CGAGCTTTTG
     GAAAAGCTGG
1001 CGCGGTACGG CTATATCTAT TCCGGCAGAC AGGGCTGGGT
     TTTGAAAACG
1051 GGGGCGGATT CGATTGAGTT GAGCGAACTC TTCAAGCTCT
     TCGTGTACCG
1101 CCCGTTGCct gtggaAAGGG ATCATGTGAA CCAAGCTGtc
     gaTGCGGTAA
1151 TGAcgccgtG TTTGCAGACT TTGAACATGA CGCTGGCGGA
     GTTTGACGCT
1201 CAGgcgAAAA AACAGCAGCA GTCTTGA

This encodes a variant of ORF144ng, having the amino acid sequence <SEQ ID 628; ORF144ng-1>:

1   MTFLQCWQGL ADNKICAFAW FVIRRFSEER VPQAAASMTF
    TTLLALVPVL
51  TVMVAVASIF PVFDRWSDSF VSFVNQTIVP QGADMVFDYI
    DAFRDQANRL
101 TAIGSVMLVV TSLMLIRTID NAFNRIWRVN TQRPWMMQFL
    VYWALLTFGP
151 LSLGVGISFM VGSVQDSVLS SGAQQWADAL KTAARLAFMT
    LLLWGLYRFV
201 PNRFVPARQA FVGALITAFC LETARFLFTW YMGNFDGYRS
    IYGAFAAVPF
251 FLLWLNLLWT LVLGGAVLTS SLSYWQGEAF RRGFDSRGRF
    DDVLKILLLL
301 DAAQKEGRTL SVQEFRRHIN MGYDELGELL EKLARYGYIY
    SGRQGWVLKT
351 GADSIELSEL FKLFVYRPLP VERDHVNQAV DAVMTPCLQT
    LNMTLAEFDA
401 QAKKQQQS*

ORF144ng-1 and ORF144-1 show 94.1% identity in 406 aa overlap:

On this basis of this analysis, including the identification of several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 75

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 629>:

1   ..AGACACGCCC GCCGCATCCG CATCGACACC GCCATCAACC
    CCGAACTGGA
51  AGCCCTCGCC GAACACCTCC ACTACCAATG GCAGGGCTTC
    CTCTGGCTCA
101 GCACCGATAT GCGTCAGGAA ATTTCCGCCC TCGTCATCCT
    GCTGCAACGC
151 ACCCGCCGCA AATGGCTGGA TGCCCACGAA CGCCAACACC
    TGCGCCAAAG
201 CCTGCTTGAA ACACGGGAAC ACGGCTGA

This corresponds to the amino acid sequence <SEQ ID 630; ORF146>:

1  ..RHARRIRIDT AINPELEALA EHLHYQWQGF LWLSTDMRQE
   ISALVILLQR
51 TRRKWLDAHE RQHLRQSLLE TREHG*

Further work revealed the complete nucleotide sequence <SEQ ID 631>:

1    ATGAACACCT CGCAACGCAA CCGCCTCGTC AGCCGCTGGC
     TCAACTCCTA
51   CGAACGCTAC CGCTACCGCC GCCTCATCCA CGCCGTCCGG
     CTCGGCGGGG
101  CCGTCCTGTT CGCCACCGCC TCCGCCCGGC TGCTCCACCT
     CCAACACGGC
151  GAGTGGATAG GGATGACCGT CTTCGTCGTC CTCGGCATGC
     TCCAGTTTCA
201  AGGGGCGATT TACTCCAAGG CGGTGGAACG TATGCTCGGC
     ACGGTCATCG
251  GGCTGGGCGC GGGTTTGGGC GTTTTATGGC TGAACCAGCA
     TTATTTCCAC
301  GGCAACCTCC TCTTCTACCT CACCGTCGGC ACGGCAAGCG
     CACTGGCCGG
351  CTGGGCGGCG GTCGGCAAAA ACGGCTACGT CCCTATGCTG
     GCAGGGCTGA
401  CGATGTGTAT GCTCATCGGC GACAACGGCA GCGAATGGCT
     CGACAGCGGA
451  CTCATGCGCG CCATGAACGT CCTCATCGGC GCGGCCATCG
     CCATCGCCGC
501  CGCCAAACTG CTGCCGCTGA AATCCACACT GATGTGGCGT
     TTCATGCTTG
551  CCGACAACCT GGCCGACTGC AGCAAAATGA TTGCCGAAAT
     CAGCAACGGC
601  AGGCGCATGA CCCGCGAACG CCTCGAGGAG AACATGGCGA
     AAATGCGCCA
651  AATCAACGCA CGCATGGTCA AAAGCCGCAG CCATCTCGCC
     GCCACATCGG
701  GCGAAAGCCG CATCAGCCCC GCCATGATGG AAGCCATGCA
     GCACGCCCAC
751  CGTAAAATCG TCAACACCAC CGAGCTGCTC CTGACCACCG
     CCGCCAAGCT
801  GCAATCTCCC AAACTCAACG GCAGCGAAAT CCGGCTGCTT
     GACCGCCACT
851  TCACACTGCT CCAAACCGAC CTGCAACAAA CCGTCGCCCT
     TATCAACGGC
901  AGACACGCCC GCCGCATCCG CATCGACACC GCCATCAACC
     CCGAACTGGA
951  AGCCCTCGCC GAACACCTCC ACTACCAATG GCAGGGCTTC
     CTCTGGCTCA
1001 GCACCAATAT GCGTCAGGAA ATTTCCGCCC TCGTCATCCT
     GCTGCAACGC
1051 ACCCGCCGCA AATGGCTGGA TGCCCACGAA CGCCAACACC
     TGCGCCAAAG
1101 CCTGCTTGAA ACACGGGAAC ACGGCTGA

This corresponds to the amino acid sequence <SEQ ID 632; ORF146-1>:

1   MNTSQRNRLV SRWLNSYERY RYRRLIHAVR LGGAVLFATA
    SARLLHLQHG
51  EWIGMTVFVV LGMLQFQGAI YSKAVERMLG TVIGLGAGLG
    VLWLNQHYFH
101 GNLLFYLTVG TASALAGWAA VGKNGYVPML AGLTMCMLIG
    DNGSEWLDSG
151 LMRAMNVLIG AAIAIAAAKL LPLKSTLMWR FMLADNLADC
    SKMIAEISNG
201 RRMTRERLEE NMAKMRQINA RMVKSRSHLA ATSGESRISP
    AMMEAMQHAH
251 RKIVNTTELL LTTAAKLQSP KLNGSEIRLL DRHFTLLQTD
    LQQTVALING
301 RHARRIRIDT AINPELEALA EHLHYQWQGF LWLSTNMRQE
    ISALVILLQR
351 TRRKWLDAHE RQHLRQSLLE TREHG*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF146 shows 98.6% identity over a 74aa overlap with an ORF (ORF146a) from strain A of N. meningitidis:

The complete length ORF146a nucleotide sequence <SEQ ID 633> is:

1    ATGAACACCT CGCAACGCAA CCGCCTCGTC AGCCGCTGGC
     TCAACTCCTA
51   CGAACGCTAC CGCTACCGCC GCCTCATCCA CGCCGTCCGG
     CTCGGCGGGG
101  CCGTCCTGTT CGCCACCGCC TCCGCCCGGC TGCTCCACCT
     CCAACACGGC
151  GAGTGGATAG GGATGACCGT CTTCGTCGTC CTCGGCATGC
     TCCAGTTTCA
201  AGGGGCGATT TACTCCAAGG CGGTGGAACG TATGCTCGGC
     ACGGTCATCG
251  GGCTGGGCGC GGGTTTGGGC GTTTTATGGC TGAACCAGCA
     TTATTTCCAC
301  GGCAACCTCC TCTTCTACCT CACCGTCGGC ACGGCAAGCG
     CACTGGCCGG
351  CTGGGCGGCG GTCGGCAAAA ACGGCTACGT CCCTATGCTG
     GCGGGGCTGA
401  CGATGTGCAT GCTCATCGGC GACAACGGCA GCGAATGGTT
     CGACAGCGGC
451  CTGATGCGCG CGATGAACGT CCTCATCGGC GCGGCCATCG
     CCATCGCCGC
501  CGCCAAACTG CTGCCGCTGA AATCCACACT GATGTGGCGT
     TTCATGCTTG
551  CCGACAACCT GACCGACTGC AGCAAAATGA TTGCCGAAAT
     CAGCAACGGC
601  AGGCGCATGA CCCGCGAACG CCTCGAAGAG AACATGGCGA
     AAATGCGCCA
651  AATCAACGCA CGCATGGTCA AAAGCCGCAG CCACCTCGCC
     GCCACATCGG
701  GCGAAAGCCG CATCAGCCCC GCCATGATGG AAGCCATGCA
     GCACGCCCAC
751  CGTAAAATTG TCAACACCAC CGAGCTGCTC CTGACCACCG
     CCGCCAAGCT
801  GCAATCTCCC AAACTCAACG GCAGCGAAAT CCGGCTGCTT
     GACCGCCACT
851  TCACACTGCT CCAAACCGAC CTGCAACAAA CCGTCGCCCT
     TATCAACGGC
901  AGACACGCCC GCCGCATCCG CATCGACACC GCCATCAACC
     CCGAACTGGA
951  AGCCCTCGCC GAACACCTCC ACTACCAATG GCAGGGCTTC
     CTCTGGCTCA
1001 GCACCAATAT GCGTCAGGAA ATTTCCGCCC TCGTCATCCT
     GCTGCAACGC
1051 ACCCGCCGCA AATGGCTGGA TGCCCACGAA CGCCAACACC
     TGCGCCAAAG
1101 CCTGCTTGAA ACACGGGAAC ACAGTTGA

This encodes a protein having amino acid sequence <SEQ ID 634>:

1   MNTSQRNRLV SRWLNSYERY RYRRLIHAVR LGGAVLFATA
    SARLLHLQHG
51  EWIGMTVFVV LGMLQFQGAI YSKAVERMLG TVIGLGAGLG
    VLWLNQHYFH
101 GNLLFYLTVG TASALAGWAA VGKNGYVPML AGLTMCMLIG
    DNGSEWFDSG
151 LMRAMNVLIG AAIAIAAAKL LPLKSTLMWR FMLADNLTDC
    SKMIAEISNG
201 RRMTRERLEE NMAKMRQINA RMVKSRSHLA ATSGESRISP
    AMMEAMQHAH
251 RKIVNTTELL LTTAAKLQSP KLNGSEIRLL DRHFTLLQTD
    LQQTVALING
301 RHARRIRIDT AINPELEALA EHLHYQWQGF LWLSTNMRQE
    ISALVILLQR
351 TRRKWLDAHE RQHLRQSLLE TREHS*

ORF146a and ORF146-1 show 99.5% identity in 374 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF146 shows 97.3% identity over a 75aa overlap with a predicted ORF (ORF146ng) from N. gonorrhoeae:

An ORF146ng nucleotide sequence <SEQ ID 635> was predicted to encode a protein having amino, acid sequence <SEQ ID 636>:

1   MSGVRFPSPA PIPSTDPPSG SLCFFTFPLQ TASDMNSSQR
    KRLSGRWLNS
51  YERYRHRRLI HAVRLGGTVL FATALARLLH LQHGEWIGMT
    VFVVLGMLQF
101 QGAIYSNAVE RMLGTVIGLG AGLGVLWLNQ HYFHGNLLFY
    LTIGTASALA
151 GWAAVGKNGY VPMLAGLTMC MLIGDNGSEW LDSGLMRAMN
    VLIGAAIAIA
201 AAKLLPLKST LMWRFMLADN LADCSKMIAE ISNGRRMTRE
    RLEQNMVKMR
251 QINARMVKSR SHLAATSGES RISPSMMEAM QHAHRKIVNT
    TELLLTTAAK
301 LQSPKLNGSE IRLLDRHFTL LQTDLQQTAA LINGRHARRI
    RIDTAINPEL
351 EALAEHLHYQ WQGFLWLSTN MRQEISALVI PLQRTRRKWL
    DAHERQHLRQ
401 SLLETREHG*

Further work revealed the following gonococcal DNA sequence <SEQ ID 637>:

1    ATGAACTCCT CGCAACGCAA ACGCCTTTCC GgccGCTGGC
     TCAACTCCTA
51   CGAACGCTac cGCCaccGCC GCCTCATACA TGCCGTGCGG
     CTCGGCggaa
101  ccgtCCTGTT CGCCACCGCA CTCGCCCGgc tACTCCACCT
     CCAacacggc
151  gAATGGATAG GGAtgaCCGT CTTCGTCGTC CTCGGCATGC
     TCCAGTTCCA
201  AGGCgcgatt tActccaacg cggtgGAacg taTGctcggt
     acggtcatcg
251  ggctgGGCGC GGGTTTGGgc gTTTTATGGC TGAACCAGCA
     TTAtttccac
301  ggcaacCTcc tcttctacct gaccatcggc acggcaagcg
     cactggccgg
351  ctGGGCGGCG GTCGGCAAAA acggctacgt ccctatgctg
     GCGGGGctgA
401  CGATGTGCAT gctcatcggc gACAACGGCA GCGAATGGCT
     CGACAGCGGC
451  CTGATGCGCG CGATGAACGT CCTCATCGGC GCCGCCATCG
     CCATTGCCGC
501  CGCCAAACTG CTGCCGCTGA AATCCACACT GATGTGGCGT
     TTCATGCTTG
551  CCGACAACCT GGCCGACTGC AGCAAAATGA TTGCCGAAAT
     CAGCAACGGC
601  AGGCGTATGA CGCGCGAACG TTTGGAGCAG AATATGGTCA
     AAATGCGCCA
651  AATCAACGCA CGCATGGTCA AAAGCCGCAG CCACCTCGCC
     GCCACATCGG
701  GCGAAAGCCG CATCAGCCCC TCCATGATGG AAGCCATGCA
     GCACGCCCAC
751  CGCAAAATCG TCAACACCAC CGAGCTGCTC CTGACCACCG
     CCGCCAAGCT
801  GCAATCTCCC AAACTCAACG GCAGCGAAAT CCGGCTGCTC
     GACCGCCACT
851  TCACACTGCT CCAAACCGAC CTGCAACAAA CCGCCGCCCT
     CATCAACGGC
901  AGACACGCCC GCCGCATCCG CATCGACACC GCCATCAACC
     CCGAACTGGA
951  AGCCCTCGCC GAACACCTCC ACTACCAATG GCAGGGCTTC
     CTCTGGCTCA
1001 GCACCAATAT GCGTCAGGAA ATTTCCGCCC TCGTCATCCT
     GCTGCAACGC
1051 ACCCGCCGCA AATGGCTGGA TGCCCACGAA CGCCAACACC
     TGCGCCAAAG
1101 CCTGCTTGAA ACACGGGAAC ACGGCTGA

This corresponds to the amino acid sequence <SEQ ID 638; ORF146ng-1>:

1   MNSSQRKRLS GRWLNSYERY RHRRLIHAVR LGGTVLFATA
    LARLLHLQHG
51  EWIGMTVFVV LGMLQFQGAI YSNAVERMLG TVIGLGAGLG
    VLWLNQHYFH
101 GNLLFYLTIG TASALAGWAA VGKNGYVPML AGLTMCMLIG
    DNGSEWLDSG
151 LMRAMNVLIG AAIAIAAAKL LPLKSTLMWR FMLADNLADC
    SKMIAEISNG
201 RRMTRERLEQ NMVKMRQINA RMVKSRSHLA ATSGESRISP
    SMMEAMQHAH
251 RKIVNTTELL LTTAAKLQSP KLNGSEIRLL DRHFTLLQTD
    LQQTAALING
301 RHARRIRIDT AINPELEALA EHLHYQWQGF LWLSTNMRQE
    ISALVILLQR
351 TRRKWLDAHE RQHLRQSLLE TREHG*

ORF146ng-1 and ORF146-1 show 96.5% identity in 375 aa overlap

Furthermore, ORF146ng-1 shows homology with a hypothetical E. coli protein:

sp|P33011|YEEA_ECOLI HYPOTHETICAL 40.0 KD PROTEIN IN
COBU-SBMC INTERGENIC REGION
>gi|1736674|gnl|PID|d1016553 (D90838) ORF_ID: o348#20;
similar to [SwissProt Accession Number P33011] [Escherichia coli]
>gi|1736682|gnl|PID|d1016560 (D90839) ORF_ID: o348#20;
similar to [SwissProt Accession Number P33011] [Escherichia coli]
>gi|1788318 (AE000292) f352; 100% identical to fragment YEEA_ECOLI
SW: P33011 but has 203 additional C-terminal residues [Escherichia coli]
Length = 352 Score = 109 bits (271), Expect = 2e−23
Identities = 89/347 (25%), Positives = 150/347 (42%), Gaps = 21/347 (6%)
Query:	20	YRHRRLIHAVRLGGTVLFATALARLLHLQHGEWIGMTVFVVLGMLQFQGAIYSNAVERML	79
		YRH R++H  R+    L    + RL  +    W  +T+ V++G + F G +   A ER+
Sbjct:	15	YRHYRIVHGTRVALAFLLTFLIIRLFTIPESTWPLVTMVVIMGPISFWGNVVPRAFERIG	74
Query:	80	GTVIGLGAGLGVLWLNQHYFHGNLLFYLTIGTASALAGWAAVGKNGYVPMLAGLTMCMLI	139
		GTV+G   GL  L L         L  +    A  L GW A+GK  Y  +L G+T+ +++
Sbjct:	75	GTVLGSILGLIALQLE---LISLPLMLVWCAAAMFLCGWLALGKKPYQGLLIGVTLAIVV	131
Query:	140	GDNGSEWLDSGLMRAMNVLIGXXXXXXXXKLLPLKSTLMWRFMLADNLADCSKMIAEISN	199
		G    E +D+ L R+ +V++G         + P ++ + WR  LA +L + +++     +
Sbjct:	132	GSPTGE-IDTALWRSGDVILGSLLAMLFTGIWPQRAFIHWRIQLAKSLTEYNRVYQSAFS	190
Query:	200	GRRMTRERLEQNMVKMRQINARMVKSRSHLAATSGESRISPSMMEAMQHAHRKIVNXXXX	259
		+ R RLE ++ K+       VK R  +A  S E+RI  S+ E +Q  +R +V
Sbjct:	191	PNLLERPRLESHLQKLL---TDAVKMRGLIAPASKETRIPKSIYEGIQTINRNLVCMLEL	247
Query:	260	XXXXXXXXQSPK---LNGSEIRLLDRHFXXXXXXXXXXAALINGRHARRIRIDTAINPEL	316
		+      LN  ++R  D              AL  G           +N  +
Sbjct:	248	QINAYWATRPSHFVLLNAQKLR--DTQHMMQQILLSLVHALYEGNPQPVFANTEKLNDAV	305
Query:	317	EALAEHL--HYQWQ-------GFLWLSTNMRQEISALVILLQRTRRK	354
		E L + L  H+  +       G++WL+     ++  L  L+ R  RK
Sbjct:	306	EELRQLLNNHHDLKVVETPIYGYVWLNMETAHQLELLSNLICRALRK	352

On the basis of this analysis, including the identification of several transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 76

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 639>

1   ..GCCGAAGACA CGCGCGTTAC CGCACAGCTT TTGAGCGCGT
    ACGGCATTCA
51  GGGCAAACTC GTCAGTGTGC GCGAACACAA CGAACGGCAG
    ATGGCGGACA
101 AGATTGTCGG CTATCTTTCA GACGGCATGG TTGTGGCACA
    GGTTTCCGAT
151 GCGGGTACGC CGGCCGTGTG CGACCCGGGC GCGAAACTCG
    CCCGCCGCGT
201 GCGTGAGGCC GGGTTTAAAG TCGTTCCCGT CGTGGGCGCA
    AC.GCGGTGA
251 TGGCGGCTTT GAGCGTGGCC GGTGTGGAAG GATCCGATTT
    TTATTTCAAC
301 GGTTTTGTAC CGCCGAAATC GGGAGAACGC AGGAAACTGT
    TTGCCAAATG
351 GGTGCGGGCG GCGTTTCCTA TCGTCATGTT TGAAACGCCG
    CACCGCATCG
401 GTGCAGCGCT TGCCGATATG GCGGAACTGT TCCCCGAACG
    CCGATTAATG
451 CTGGCGCGCG AAATTACGAA AACGTTTGAA ACGTTCTTAA
    GCGGCACGGT
501 TGGGGAAATT CAGACGGCAT TGTCTGCCGA CGGCGACCAA
    TCGCGCGGCG
551 AGATGGTGTT GGTGCTTTAT CCGGCGCAGG ATGAAAAACA
    CGAAGGCTTG
601 TCCGAGTCCG CGCAAAACAT CATGAAAATC CTCACAGCCG
    AGCTGCCGAC
651 CAAACAGGCG GCGGAGCTTG CTGCCAAAAT CACGGGCGAG
    GGAAAGAAAG
701 CTTTGTACGA T..

This corresponds to the amino acid sequence <SEQ ID 640; ORF147>:

1   ..AEDTRVTAQL LSAYGIQGKL VSVREHNERQ MADKIVGYLS
    DGMVVAQVSD
51  AGTPAVCDPG AKLARRVREA GFKVVPVVGA XAVMAALSVA
    GVEGSDFYFN
101 GFVPPKSGER RKLFAKWVRA AFPIVMFETP HRIGAALADM
    AELFPERRLM
151 LAREITKTFE TFLSGTVGEI QTALSADGDQ SRGEMVLVLY
    PAQDEKHEGL
201 SESAQNIMKI LTAELPTKQA AELAAKITGE GKKALYD..

Further work revealed the complete nucleotide sequence <SEQ ID 641>:

1   ATGTTTCAGA AACATTTGCA GAAAGCCTCC GACAGCGTCG
    TCGGAGGGAC
51  ATTATACGTG GTTGCCACGC CCATCGGCAA TTTGGCGGAC
    ATTACCCTGC
101 GCGCTTTGGC GGTATTGCAA AAGGCGGACA TCATCTGTGC
    CGAAGACACG
151 CGCGTTACCG CACAGCTTTT GAGCGCGTAC GGCATTCAGG
    GCAAACTCGT
201 CAGTGTGCGC GAACACAACG AACGGCAGAT GGCGGACAAG
    ATTGTCGGCT
251 ATCTTTCAGA CGGCATGGTT GTGGCACAGG TTTCCGATGC
    GGGTACGCCG
301 GCCGTGTGCG ACCCGGGCGC GAAACTCGCC CGCCGCGTGC
    GTGAGGCCGG
351 GTTTAAAGTC GTTCCCGTCG TGGGCGCAAG CGCGGTGATG
    GCGGCTTTGA
401 GCGTGGCCGG TGTGGAAGGA TCCGATTTTT ATTTCAACGG
    TTTTGTACCG
451 CCGAAATCGG GAGAACGCAG GAAACTGTTT GCCAAATGGG
    TGCGGGCGGC
501 GTTTCCTATC GTCATGTTTG AAACGCCGCA CCGCATCGGT
    GCGACGCTTG
551 CCGATATGGC GGAACTGTTC CCCGAACGCC GATTAATGCT
    GGCGCGCGAA
601 ATTACGAAAA CGTTTGAAAC GTTCTTAAGC GGCACGGTTG
    GGGAAATTCA
651 GACGGCATTG TCTGCCGACG GCAACCAATC GCGCGGCGAG
    ATGGTGTTGG
701 TGCTTTATCC GGCGCAGGAT GAAAAACACG AAGGCTTGTC
    CGAGTCCGCG
751 CAAAACATCA TGAAAATCCT CACAGCCGAG CTGCCGACCA
    AACAGGCGGC
801 GGAGCTTGCT GCCAAAATCA CGGGCGAGGG AAAGAAAGCT
    TTGTACGATC
851 TGGCTCTGTC TTGGAAAAAC AAATAG

This corresponds to the amino acid sequence <SEQ ID 642; ORF147-1>:

1   MFQKHLQKAS DSVVGGTLYV VATPIGNLAD ITLRALAVLQ
    KADIICAEDT
51  RVTAQLLSAY GIQGKLVSVR EHNERQMADK IVGYLSDGMV
    VAQVSDAGTP
101 AVCDPGAKLA RRVREAGFKV VPVVGASAVM AALSVAGVEG
    SDFYFNGFVP
151 PKSGERRKLF AKWVRAAFPI VMFETPHRIG ATLADMAELF
    PERRLMLARE
201 ITKTFETFLS GTVGEIQTAL SADGNQSRGE MVLVLYPAQD
    EKHEGLSESA
251 QNIMKILTAE LPTKQAAELA AKITGEGKKA LYDLALSWKN
    K*

Computer analysis of this amino acid sequence gave the following results:

Homology with Hypothetical Protein ORF286 of E. coli (Accession Number U 18997)

ORF147 and E. coli ORF286 protein show 36% aa identity in 237aa overlap:

Orf147:	1	AEDTRVTAQLLSAYGIQGKLVSVREHNERQMADKIVGYLSDGMVVAQVSDAGTPAVCDPG	60
		AEDTR T  LL  +GI  +L ++ +HNE+Q A+ ++  L +G  +A VSDAGTP + DPG
Orf286:	43	AEDTRHTGLLLQHFGINARLFALHDHNEQQKAETLLAKLQEGQNIALVSDAGTPLINDPG	102
Orf147:	61	AKLARRVREXXXXXXXXXXXXXXXXXXXXXXXEGSDFYFNGFVPPKSGERRKLFAKWVRA	120
		L R  RE                           F + GF+P KS  RR
Orf286:	103	YHLVRTCREAGIRVVPLPGPCAAITALSAAGLPSDRFCYEGFLPAKSKGRRDALKAIEAE	162
Orf147:	121	AFPIVMFETPHRIGAALADMAELFPERR-LMLAREITKTFETFLSGTVGEIQTALSADGD	179
		++ +E+ HR+  +L D+  +  E R ++LARE+TKT+ET     VGE+   +  D +
Orf286:	163	PRTLIFYESTHRLLDSLEDIVAVLGESRYVVLARELTKTWETIHGAPVGELLAWVKEDEN	222
Orf147:	180	QSRGEMVLVLYPAQDEKHEGLSESAQNIMKILTAELPTKQAAELAAKITGEGKKALY	236
		+ +GEMVL++      + E L   A   + +L AELP K+AA LAA+I G  K ALY
Orf286:	223	RRKGEMVLIV-EGHKAQEEDLPADALRTLALLQAELPLKKAAALAAEIHGVKKNALY	278

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF147 shows 96.6% identity over a 237aa overlap with ORF75a from strain A of N. meningitidis:

ORF147a is identical to ORF75a, which includes aa 56-292 of ORF75.

Homology with a Predicted ORF from N. gonorrhoeae

ORF147 shows 94.1% identity over a 237aa overlap with a predicted ORF (ORF147ng) from N. gonorrhoeae:

An ORF147ng nucleotide sequence <SEQ ID 643> was predicted to encode a protein having amino acid sequence <SEQ ID 644>:

1   MSVFQTAFFM FQKHLQKASD SVVGGTLYVV ATPIGNLADI
    TLRALAVLQK
51  ADIICAEDTR VTAQLLSAYG IQGRLVSVRE HNERQMADKV
    IGFLSDGLVV
101 AQVSDAGTPA VCDPGAKLAR RVREAGFKVV PVVGASAVMA
    ALSVAGVAES
151 DFYFNGFVPP KSGERRKLFA KWVRAAFPVV MFETPHRIGA
    TLADMAELFP
201 ERRLMLAREI TKTFETFLSG TVGEIQTALA ADGNQSRGEM
    VLVLYPAQDE
251 KHEGLSESAQ NAMKILAAEL PTKQAAELAA KITGEGKKAL
    YDLALSWKNK
301 *

Further work revealed the following gonococcal DNA sequence <SEQ ID 645>:

1   ATGTTTCAGA AACACTTGCA GAAAGCCTCC GACAGCGTCG
    TCGGAGGGAC
51  ATTATACGTG GTTGCCACGC CCATCGGCAA TTTGGCAGAC
    ATTACCCTGC
101 GCGCTTTGGC GGTATTGCAA AAGGCGGACA TCATTTGTGC
    CGAAGACACG
151 CGCGTTACTG CGCAGCTTTT GAGCGCGTAC GGCATTCAGG
    GCAGGTTGGT
201 CAGTGTGCGC GAACACAACG AGCGGCAGAT GGCGGACAAG
    GTAATCGGTT
251 TCCTTTCAGA CGGCCTGGTT GTGGCGCAGG TTTCCGATGC
    GGGTACGCCG
301 GCCGTGTGCG ACCCGGGCGC GAAACTCGCC CGCCGCGTGC
    GCGAAGCAGG
351 GTTCAAAGTC GTTCCCGTCG TGGGCGCAAG CGCGGTAATG
    GCGGCGTTGA
401 GTGTGGCCGG TGTGGCGGAA TCCGATTTTT ATTTCAACGG
    TTTTGTACCG
451 CCGAAATCGG GCGAACGTAG GAAATTGTTT GCCAAATGGG
    TGCGGGCGGC
501 ATTTCCTGTC GTCATGTTTG AAACGCCGCA CCGAATCGGG
    GCAACGCTTG
551 CCGATATGGC GGAATTGTTC CCCGAACGCC GTCTGATGCT
    GGCGCGCGAA
601 ATCACGAAAA CGTTTGAAAC GTTCTTAAGC GGCACGGTTG
    GGGAAATTCA
651 GACGGCATTG GCGGCGGACG GCAACCAATC GCGCGGCGAG
    ATGGTGTTGG
701 TGCTTTATCC GGCGCAGGAT GAAAAACACG AAGGCTTGTC
    CGAGTCTGCG
751 CAAAATGCGA TGAAAATCCT TGCGGCCGAG CTGCCGACCA
    AGCAGGCGGC
801 GGAGCTTGCC GCCAAGATTA CAGGTGAGGG CAAAAAGGCT
    TTGTACGATT
851 TGGCACTGTC GTGGAAAAAC AAATGA

This corresponds to the amino acid sequence <SEQ ID 646; ORF147ng-1>:

1   MFQKHLQKAS DSVVGGTLYV VATPIGNLAD ITLRALAVLQ
    KADIICAEDT
51  RVTAQLLSAY GIQGRLVSVR EHNERQMADK VIGFLSDGLV
    VAQVSDAGTP
101 AVCDPGAKLA RRVREAGFKV VPVVGASAVM AALSVAGVAE
    SDFYFNGFVP
151 PKSGERRKLF AKWVRAAFPV VMFETPHRIG ATLADMAELF
    PERRLMLARE
201 ITKTFETFLS GTVGEIQTAL AADGNQSRGE MVLVLYPAQD
    EKHEGLSESA
251 QNAMKILAAE LPTKQAAELA AKITGEGKKA LYDLALSWKN
    K*

ORF147ng shows homology to a hypothetical E. coli protein:

sp|P45528|YRAL_ECOLI HYPOTHETICAL 31.3 KD PROTEIN IN AGAI-MTR
INTERGENIC REGION (F286)
>gi|606086 (U18997) ORF_f286 [Escherichia coli]
>gi|1789535 (AE000395) hypothetical 31.3 kD protein in agai-mtr intergenic region
[Escherichia coli] Length = 286
Score = 218 bits (550), Expect = 3e−56
Identities = 128/284 (45%), Positives = 171/284 (60%), Gaps = 4/284 (1%)
Query:	4	KHLQKASDSVVGGTLYVVATPIGNLADITLRALAVLQKADIICAEDTRVTAQLLSAYGIQ	63
		K  Q A +S   G LY+V TPIGNLADIT RAL VLQ  D+I AEDTR T  LL  +GI
Sbjct:	2	KQHQSADNSQ--GQLYIVPTPIGNLADITQRALEVLQAVDLIAAEDTRHTGLLLQHFGIN	59
Query:	64	GRLVSVREHNERQMADKVIGFLSDGLVVAQVSDAGTPAVCDPGAKLARRVREAGFKVVPV	123
		RL ++ +HNE+Q A+ ++  L +G  +A VSDAGTP + DPG  L R  REAG +VVP+
Sbjct:	60	ARLFALHDHNEQQKAETLLAKLQEGQNIALVSDAGTPLINDPGYHLVRTCREAGIRVVPL	119
Query:	124	VGASAVMAALSVAGVAESDFYFNGFVPPKSGERRKLFAKWVRAAFPVVMFETPHRIGATL	183
		G  A + ALS AG+    F + GF+P KS  RR            ++ +E+ HR+  +L
Sbjct:	120	PGPCAAITALSAAGLPSDRFCYEGFLPAKSKGRRDALKAIEAEPRTLIFYESTHRLLDSL	179
Query:	184	ADMAELFPERR-LMLAREITKTFETFLSGTVGEIQTALAADGNQSRGEMVLVLYPAQDEK	242
		D+  +  E R ++LARE+TKT+ET     VGE+   +  D N+ +GEMVL++      +
Sbjct:	180	EDIVAVLGESRYVVLARELTKTWETIHGAPVGELLAWVKEDENRRKGEMVLIV-EGHKAQ	238
Query:	243	HEGLSESAQNAMKILAAELPTKQAAELAAKITGEGKKALYDLAL	286
		E L   A   + +L AELP K+AA LAA+I G  K ALY  AL
Sbjct:	239	EEDLPADALRTLALLQAELPLKKAAALAAEIHGVKKNALYKYAL	282

Based on the computer analysis and the presence of a putative transmembrane domain in the gonococcal protein, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 77

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 647>

This corresponds to the amino acid sequence <SEQ ID 648; ORF1>:

Further sequencing analysis revealed the complete nucleotide sequence <SEQ ID 649>:

1    ATGAAAACAA CCGACAAACG GACAACCGAA ACACACCGCA
     AAGCCCCGAA
51   AACCGGCCGC ATCCGCTTCT CGCCTGCTTA CTTAGCCATA
     TGCCTGTCGT
101  TCGGCATTCT TCCCCAAGCC TGGGCGGGAC ACACTTATTT
     CGGCATCAAC
151  TACCAATACT ATCGCGACTT TGCCGAAAAT AAAGGCAAGT
     TTGCAGTCGG
201  GGCGAAAGAT ATTGAGGTTT ACAACAAAAA AGGGGAGTTG
     GTCGGCAAAT
251  CAATGACAAA AGCCCCGATG ATTGATTTTT CTGTGGTGTC
     GCGTAACGGC
301  GTGGCGGCAT TGGTGGGCGA TCAATATATT GTGAGCGTGG
     CACATAACGG
351  CGGCTATAAC AACGTTGATT TTGGTGCGGA AGGAAGAAAT
     CCCGATCAAC
401  ATCGTTTTAC TTATAAAATT GTGAAACGGA ATAATTATAA
     AGCAGGGACT
451  AAAGGCCATC CTTATGGCGG CGATTATCAT ATGCCGCGTT
     TGCATAAATT
501  TGTCACAGAT GCAGAACCTG TTGAAATGAC CAGTTATATG
     GATGGGCGGA
551  AATATATCGA TCAAAATAAT TACCCTGACC GTGTTCGTAT
     TGGGGCAGGC
601  AGGCAATATT GGCGATCTGA TGAAGATGAG CCCAATAACC
     GCGAAAGTTC
651  ATATCATATT GCAAGTGCGT ATTCTTGGCT CGTTGGTGGC
     AATACCTTTG
701  CACAAAATGG ATCAGGTGGT GGCACAGTCA ACTTAGGTAG
     TGAAAAAATT
751  AAACATAGCC CATATGGTTT TTTACCAACA GGAGGCTCAT
     TTGGCGACAG
801  TGGCTCACCA ATGTTTATCT ATGATGCCCA AAAGCAAAAG
     TGGTTAATTA
851  ATGGGGTATT GCAAACGGGC AACCCCTATA TAGGAAAAAG
     CAATGGCTTC
901  CAGCTGGTTC GTAAAGATTG GTTCTATGAT GAAATCTTTG
     CTGGAGATAC
951  CCATTCAGTA TTCTACGAAC CACGTCAAAA TGGGAAATAC
     TCTTTTAACG
1001 ACGATAATAA TGGCACAGGA AAAATCAATG CCAAACATGA
     ACACAATTCT
1051 CTGCCTAATA GATTAAAAAC ACGAACCGTT CAATTGTTTA
     ATGTTTCTTT
1101 ATCCGAGACA GCAAGAGAAC CTGTTTATCA TGCTGCAGGT
     GGTGTCAACA
1151 GTTATCGACC CAGACTGAAT AATGGAGAAA ATATTTCCTT
     TATTGACGAA
1201 GGAAAAGGCG AATTGATACT TACCAGCAAC ATCAATCAAG
     GTGCTGGAGG
1251 ATTATATTTC CAAGGAGATT TTACGGTCTC GCCTGAAAAT
     AACGAAACTT
1301 GGCAAGGCGC GGGCGTTCAT ATCAGTGAAG ACAGTACCGT
     TACTTGGAAA
1351 GTAAACGGCG TGGCAAACGA CCGCCTGTCC AAAATCGGCA
     AAGGCACGCT
1401 GCACGTTCAA GCCAAAGGGG AAAACCAAGG CTCGATCAGC
     GTGGGCGACG
1451 GTACAGTCAT TTTGGATCAG CAGGCAGACG ATAAAGGCAA
     AAAACAAGCC
1501 TTTAGTGAAA TCGGCTTGGT CAGCGGCAGG GGTACGGTGC
     AACTGAATGC
1551 CGATAATCAG TTCAACCCCG ACAAACTCTA TTTCGGCTTT
     CGCGGCGGAC
1601 GTTTGGATTT AAACGGGCAT TCGCTTTCGT TCCACCGTAT
     TCAAAATACC
1651 GATGAAGGGG CGATGATTGT CAACCACAAT CAAGACAAAG
     AATCCACCGT
1701 TACCATTACA GGCAATAAAG ATATTGCTAC AACCGGCAAT
     AACAACAGCT
1751 TGGATAGCAA AAAAGAAATT GCCTACAACG GTTGGTTTGG
     CGAGAAAGAT
1801 ACGACCAAAA CGAACGGGCG GCTCAACCTT GTTTACCAGC
     CCGCCGCAGA
1851 AGACCGCACC CTGCTGCTTT CCGGCGGAAC AAATTTAAAC
     GGCAACATCA
1901 CGCAAACAAA CGGCAAACTG TTTTTCAGCG GCAGACCAAC
     ACCGCACGCC
1951 TACAATCATT TAAACGACCA TTGGTCGCAA AAAGAGGGCA
     TTCCTCGCGG
2001 GGAAATCGTG TGGGACAACG ACTGGATCAA CCGCACATTT
     AAAGCGGAAA
2051 ACTTCCAAAT TAAAGGCGGA CAGGCGGTGG TTTCCCGCAA
     TGTTGCCAAA
2101 GTGAAAGGCG ATTGGCATTT GAGCAATCAC GCCCAAGCAG
     TTTTTGGTGT
2151 CGCACCGCAT CAAAGCCACA CAATCTGTAC ACGTTCGGAC
     TGGACGGGTC
2201 TGACAAATTG TGTCGAAAAA ACCATTACCG ACGATAAAGT
     GATTGCTTCA
2251 TTGACTAAGA CCGACATCAG CGGCAATGTC GATCTTGCCG
     ATCACGCTCA
2301 TTTAAATCTC ACAGGGCTTG CCACACTCAA CGGCAATCTT
     AGTGCAAATG
2351 GCGATACACG TTATACAGTC AGCCACAACG CCACCCAAAA
     CGGCAACCTT
2401 AGCCTCGTGG GCAATGCCCA AGCAACATTT AATCAAGCCA
     CATTAAACGG
2451 CAACACATCG GCTTCGGGCA ATGCTTCATT TAATCTAAGC
     GACCACGCCG
2501 TACAAAACGG CAGTCTGACG CTTTCCGGCA ACGCTAAGGC
     AAACGTAAGC
2551 CATTCCGCAC TCAACGGTAA TGTCTCCCTA GCCGATAAGG
     CAGTATTCCA
2601 TTTTGAAAGC AGCCGCTTTA CCGGACAAAT CAGCGGCGGC
     AAGGATACGG
2651 CATTACACTT AAAAGACAGC GAATGGACGC TGCCGTCAGG
     CACGGAATTA
2701 GGCAATTTAA ACCTTGACAA CGCCACCATT ACACTCAATT
     CCGCCTATCG
2751 CCACGATGCG GCAGGGGCGC AAACCGGCAG TGCGACAGAT
     GCGCCGCGCC
2801 GCCGTTCGCG CCGTTCGCGC CGTTCCCTAT TATCCGTTAC
     ACCGCCAACT
2851 TCGGTAGAAT CCCGTTTCAA CACGCTGACG GTAAACGGCA
     AATTGAACGG
2901 TCAGGGAACA TTCCGCTTTA TGTCGGAACT CTTCGGCTAC
     CGCAGCGACA
2951 AATTGAAGCT GGCGGAAAGT TCCGAAGGCA CTTACACCTT
     GGCGGTCAAC
3001 AATACCGGCA ACGAACCTGC AAGCCTCGAA CAATTGACGG
     TAGTGGAAGG
3051 AAAAGACAAC AAACCGCTGT CCGAAAACCT TAATTTCACC
     CTGCAAAACG
3101 AACACGTCGA TGCCGGCGCG TGGCGTTACC AACTCATCCG
     CAAAGACGGC
3151 GAGTTCCGCC TGCATAATCC GGTCAAAGAA CAAGAGCTTT
     CCGACAAACT
3201 CGGCAAGGCA GAAGCCAAAA AACAGGCGGA AAAAGACAAC
     GCGCAAAGCC
3251 TTGACGCGCT GATTGCGGCC GGGCGCGATG CCGTCGAAAA
     GACAGAAAGC
3301 GTTGCCGAAC CGGCCCGGCA GGCAGGCGGG GAAAATGTCG
     GCATTATGCA
3351 GGCGGAGGAA GAGAAAAAAC GGGTGCAGGC GGATAAAGAC
     ACCGCCTTGG
3401 CGAAACAGCG CGAAGCGGAA ACCCGGCCGG CTACCACCGC
     CTTCCCCCGC
3451 GCCCGCCGCG CCCGCCGGGA TTTGCCGCAA CTGCAACCCC
     AACCGCAGCC
3501 CCAACCGCAG CGCGACCTGA TCAGCCGTTA TGCCAATAGC
     GGTTTGAGTG
3551 AATTTTCCGC CACGCTCAAC AGCGTTTTCG CCGTACAGGA
     CGAATTAGAC
3601 CGCGTATTTG CCGAAGACCG CCGCAACGCC GTTTGGACAA
     GCGGCATCCG
3651 GGACACCAAA CACTACCGTT CGCAAGATTT CCGCGCCTAC
     CGCCAACAAA
3701 CCGACCTGCG CCAAATCGGT ATGCAGAAAA ACCTCGGCAG
     CGGGCGCGTC
3751 GGCATCCTGT TTTCGCACAA CCGGACCGAA AACACCTTCG
     ACGACGGCAT
3801 CGGCAACTCG GCACGGCTTG CCCACGGCGC CGTTTTCGGG
     CAATACGGCA
3851 TCGACAGGTT CTACATCGGC ATCAGCGCGG GCGCGGGTTT
     TAGCAGCGGC
3901 AGCCTTTCAG ACGGCATCGG AGGCAAAATC CGCCGCCGCG
     TGCTGCATTA
3951 CGGCATTCAG GCACGATACC GCGCCGGTTT CGGCGGATTC
     GGCATCGAAC
4001 CGCACATCGG CGCAACGCGC TATTTCGTCC AAAAAGCGGA
     TTACCGCTAC
4051 GAAAACGTCA ATATCGCCAC CCCCGGCCTT GCATTCAACC
     GCTACCGCGC
4101 GGGCATTAAG GCAGATTATT CATTCAAACC GGCGCAACAC
     ATTTCCATCA
4151 CGCCTTATTT GAGCCTGTCC TATACCGATG CCGCTTCGGG
     CAAAGTCCGA
4201 ACACGCGTCA ATACCGCCGT ATTGGCTCAG GATTTCGGCA
     AAACCCGCAG
4251 TGCGGAATGG GGCGTAAACG CCGAAATCAA AGGTTTCACG
     CTGTCCCTCC
4301 ACGCTGCCGC CGCCAAAGGC CCGCAACTGG AAGCGCAACA
     CAGCGCGGGC
4351 ATCAAATTAG GCTACCGCTG GTAA

This corresponds to the amino acid sequence <SEQ ID 650; ORF1-1>:

1    MKTTDKRTTE THRKAPKTGR IRFSPAYLAI CLSFGILPQA
     WAGHTYFGIN
51   YQYYRDFAEN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM
     IDFSVVSRNG
101  VAALVGDQYI VSVAHNGGYN NVDFGAEGRN PDQHRFTYKI
     VKRNNYKAGT
151  KGHPYGGDYH MPRLHKFVTD AEPVEMTSYM DGRKYIDQNN
     YPDRVRIGAG
201  RQYWRSDEDE PNNRESSYHI ASAYSWLVGG NTFAQNGSGG
     GTVNLGSEKI
251  KHSPYGFLPT GGSFGDSGSP MFIYDAQKQK WLINGVLQTG
     NPYIGKSNGF
301  QLVRKDWFYD EIFAGDTHSV FYEPRQNGKY SFNDDNNGTG
     KINAKHEHNS
351  LPNRLKTRTV QLFNVSLSET AREPVYHAAG GVNSYRPRLN
     NGENISFIDE
401  GKGELILTSN INQGAGGLYF QGDFTVSPEN NETWQGAGVH
     ISEDSTVTWK
451  VNGVANDRLS KIGKGTLHVQ AKGENQGSIS VGDGTVILDQ
     QADDKGKKQA
501  FSEIGLVSGR GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH
     SLSFHRIQNT
551  DEGAMIVNHN QDKESTVTIT GNKDIATTGN NNSLDSKKEI
     AYNGWFGEKD
601  TTKTNGRLNL VYQPAAEDRT LLLSGGTNLN GNITQTNGKL
     FFSGRPTPHA
651  YNHLNDHWSQ KEGIPRGEIV WDNDWINRTF KAENFQIKGG
     QAVVSRNVAK
701  VKGDWHLSNH AQAVFGVAPH QSHTICTRSD WTGLTNCVEK
     TITDDKVIAS
751  LTKTDISGNV DLADHAHLNL TGLATLNGNL SANGDTRYTV
     SHNATQNGNL
801  SLVGNAQATF NQATLNGNTS ASGNASFNLS DHAVQNGSLT
     LSGNAKANVS
851  HSALNGNVSL ADKAVFHFES SRFTGQISGG KDTALHLKDS
     EWTLPSGTEL
901  GNLNLDNATI TLNSAYRHDA AGAQTGSATD APRRRSRRSR
     RSLLSVTPPT
951  SVESRFNTLT VNGKLNGQGT FRFMSELFGY RSDKLKLAES
     SEGTYTLAVN
1001 NTGNEPASLE QLTVVEGKDN KPLSENLNFT LQNEHVDAGA
     WRYQLIRKDG
1051 EFRLHNPVKE QELSDKLGKA EAKKQAEKDN AQSLDALIAA
     GRDAVEKTES
1101 VAEPARQAGG ENVGIMQAEE EKKRVQADKD TALAKQREAE
     TRPATTAFPR
1151 ARRARRDLPQ LQPQPQPQPQ RDLISRYANS GLSEFSATLN
     SVFAVQDELD
1201 RVFAEDRRNA VWTSGIRDTK HYRSQDFRAY RQQTDLRQIG
     MQKNLGSGRV
1251 GILFSHNRTE NTFDDGIGNS ARLAHGAVFG QYGIDRFYIG
     ISAGAGFSSG
1301 SLSDGIGGKI RRRVLHYGIQ ARYRAGFGGF GIEPHIGATR
     YFVQKADYRY
1351 ENVNIATPGL AFNRYRAGIK ADYSFKPAQH ISITPYLSLS
     YTDAASGKVR
1401 TRVNTAVLAQ DFGKTRSAEW GVNAEIKGFT LSLHAAAAKG
     PQLEAQHSAG
1451 IKLGYRW*

Computer analysis of these sequences gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF1 shows 57.8% identity over a 1456aa overlap with an ORF (ORF1a) from strain A of N. meningitidis:

The complete length ORF1a nucleotide sequence <SEQ ID 651> is:

1    ATGAAAACAA CCGACAAACG GACAACCGAA ACACACCGCA
     AAGCCCCGAA
51   AACCGGCCGC ATCCGCTTCT CGCCTGCTTA CTTAGCCATA
     TGCCTGTCGT
101  TCGGCATTCT TCCCCAAGCT TGGGCGGGAC ACACTTATTT
     CGGCATCAAC
151  TACCAATACT ATCGCGACTT TGCCGAAAAT AAAGGCAAGT
     TTGCAGTCGG
201  GGCGAAAGAT ATTGAGGTNT ACAACAAAAA AGGGGAGTTG
     GTCGGCAAAT
251  CAATGACAAA AGCCCCGATG ATTGATTTTT CTGTGGTGTC
     GCGTAACGGC
301  GTGGCGGCAT TGGTGGGCGA TCAATATATT GTGAGCGTGG
     CACATAACGG
351  CGGCTATAAC AACGTTGATT TTGGTGCGGA AGGAAGNAAT
     CCCGATCAGC
401  ACCGTTTTTC TTACCAAATT GTGAAAAGAA ATAATTATAA
     GCCTGACAAT
451  TCACACCCTT ACAACGGCGA TTANCATATG CCGCGTTTGC
     ATAAATTTGT
501  CACAGATGCA GAACCTGTCG AAATGACGAG TGACATGAGG
     GGGAATACCT
551  ATTCCGATAA AGAAAAATAT CCCGAGCGTG TCCGCATCGG
     CTCAGGACAC
601  CACTATTGGC GTTATGATGA TGACAAACAC GGCGATTTAT
     CCTACTCCGG
651  CGCATGGTTA ATTGGCGGCA ATACACATAT GCAGGGTTGG
     GGAAATAATG
701  GCGTANTTAG TTTGAGCGGC GATGTGCGCC ATGCCAACGA
     CTATGGCCCT
751  ATGCCGATTG CAGGTGCGGC AGGCGACAGC GGTTCGCCAA
     TGTTTATTTA
801  TGACAAAACA AACAATAAAT GGCTGCTCAA CGGAGTTTTA
     CAAACCGGCT
851  ACCCTTATTC CGGCAGGGAA AACGGTTTCC AGCTGATACG
     CAAAGATTGG
901  TTCTACGATG ACATTTACAG AGGCGATACA CATACCGTCT
     NTTTTGAACC
951  GCGCAGTAAC GGACATTTTT CCTTTACATC CAACAACAAC
     GGTACGGGTA
1001 CGGTAACAGA AACCAACGAA AAGGTNTCCA ATCCAAAGCT
     TAAAGTACAG
1051 ACAGTCCGAC TGTTTGACGA ATCTTTGAAT GAAACTGATA
     AAGAACCAGT
1101 TTACGCGGCA GGGGGTGTTA ATCAGTACCG TCCAAGGTTA
     AACAACGGTG
1151 AAAACCTTTC TTTTATCGAT TACGGCAACG GCAAACTCAT
     CTTATCAAAC
1201 AACATCAACC AAGGCGCGGG CGGTTTGTAT TTTGAAGGTG
     ATTTTACGGT
1251 CTCGCCTGAA AACAACGAAA CGTGGCAAGG CGCGGGCGTT
     CATATCAGTG
1301 AAGACAGTAC CGTTACTTGG AAAGTAAACG GCGTGGCAAA
     CGACCGCCTG
1351 TCCAAAATCG GCAAAGGCAC GCTGCACGTT CAAGCCAAAG
     GGGAAAACCA
1401 AGGCTCGATC AGCGTGGGCG ACGGTACAGT CATTTTGGAT
     CAGCAGGCAG
1451 ACGATAAAGG CAAAAAACAA GCCTTTAGTG AAATCGGCTT
     GNTCAGCGGC
1501 AGGGGTACGG TGCAACTGAA TGCCGATAAT CAGTTCAACC
     CCGACAAACT
1551 CTATTTCGGC TTTCGCGGCG GACGTTTGGA TTTAAACGGG
     CATTCGCTTT
1601 CGTTCCACCG TATTCAAAAT ACCGATGAAG GGGCGATGAT
     TGNCNATCAT
1651 AATGCCACAA CAACATCCAC CGTTACCATT ACAGGGAATG
     AAAGTATTAC
1701 ACAACCGAGT GGTAAGAATA TCAATAGACT TAATTACAGC
     AAAGAAATTG
1751 CCTACAACGG TTGGTTTGGC GAGAAAGATA CGACCAAAAC
     GAACGGGCGG
1801 CTCAACCTTG TTTACCAGCC CGCCGCAGAA GACCGCACCC
     NGCTGCTTTC
1851 CGGCGGAACA AATTTAAACG GCAACATCAC GCAAACAAAC
     GGCAAACTGT
1901 TTTTCAGCGG CAGACCGACA CCGCACGCCT ACAATCATTT
     AGGAAGCGGG
1951 TGGTCAAAAA TGGAAGGTAT CCCACAAGGA GAAATCGTGT
     GGGACAACGA
2001 CTGGATCNAC CGCACGTTTA AAGCGGAAAA TTTCCATATT
     CAGGGCGGGC
2051 AGGCGGTGAT TTCCCGCAAT GTTGCCAAAG TGGAAGGCGA
     TTGNCATTTG
2101 AGCAATCACG CCCAAGCAGT TTTTGGTGTC GCACCGCATC
     AAAGCCATAC
2151 AATCTGTACA CGTTCGGACT GGACNGGTCT GACAAATTGT
     GTCGAANAAA
2201 NCATTACCGA CGATAAAGTG ATTGCTTCAT TGACTAAGAC
     NGACNTNAGC
2251 GGCANTGTNA GNCTNNCCNA TNACGNTNNT TNAAANCTCN
     CNGGGCNTGC
2301 NNCACTNAAN GGCAATCTTA GTGCAAATGG CGATACACGT
     TATACAGTCA
2351 GCCACAACGC CACCCAAAAC GGCAACCTTA GCCTCGTGGG
     CAATGCCCAA
2401 GCAACATTTA ATCAAGCCAC ATTAAACGGC AACNCATCGG
     NTTCGGGCAA
2451 TGCTTCATTT AATCTAAGCA ACAACGCCGC ACAAAACGGC
     AGTCTGACGC
2501 TTTCCGACAA CGCTAAGGCA AACGTAAGCC ATTCCGCACT
     CAACGGCAAT
2551 GTCTCCCTAG CCGATAAGGC AGTATTCCAT TTTGAAAACA
     GCCGCTTTAC
2601 CGGACAACTC AGCGGCAGCA AGGANACAGC ATTACACTTA
     AAAGACAGCG
2651 AATGGACGCT GCCGTCAGGC ACGGAATTAG GCAATTTAAA
     CCTTGACAAC
2701 GCCACCATTA CACTCAATTC CGCCTATCGC CACGATGCTG
     CAGGCGCGCA
2751 AACCGGCAGN GTGTCAGACA CGCCGCGCCG CCGTTCGCGC
     CGTTCCCTAT
2801 TATCCGTTAC ACCGCCAACT TCGGTAGAAT CCCGTTTCAA
     CACGCTGACG
2851 GTAAACGGCA AATTGAACNG TCAAGGAACA TTCCGCTTTA
     TGTCGGAACT
2901 CTTCGGCTAC CGAAGCGACA AATTGAAGCT GGCGGAAAGT
     TCCGAAGGNA
2951 CTTACACCTT GGCGGTCAAC AATACCGGCA ACGAACCCGT
     AAGCCTCGAT
3001 CAATTGACGG TAGTGGAAGG GAAAGACAAC AAACCGCTGT
     CCGAAAACCT
3051 TAATTTCACC CTGCAAAACG AACACGTCGA TGCCGGCGCG
     TGGCGTTACC
3101 AACTCATCCG CAAAGACGGC GAGTTCCGCC TGCATAATCC
     GGTCAAAGAA
3151 CAAGAGCTTT CCGACAAACT CGGCAAGGCA GAAGCCAAAA
     AACAGGCGGA
3201 AAAAGACAAC GCGCAAAGCC TTGACGCGCT GATTGCGGCC
     GGGCGCGATG
3251 CCGCCGAAAA GACAGAAAGC GTTGCCGAAC CGGCCCGGCN
     GGCAGGCGGG
3301 GAAAATGTCG GCATTATGCA GGCGGAGGAA GAGAAAAAAC
     GGGTGCAGGC
3351 GGATAAAGAC AGCGCNTTGG CGAAACAGCG CGAAGCGGAA
     ACCCGGCCGG
3401 NTACCACCGC CTTCCCCCGC GCCCGCNGCG CCCGCCGGGA
     TTTGCCGCAA
3451 CCGCAGCCCC AACCGCAACC TCAACCCCAA CCGCAGCGCG
     ACCTGATNAG
3501 CCGTTATGCC AATAGCGGTT TGAGTGAATT TTCCGCCACG
     CTCAACAGCG
3551 TTTTCGCCGT ACAGGACGAA TTGGACCGCG TGTTTGCCGA
     AGACCGCCGC
3601 AACGCNGTTT GGACAAGCNG CATCCGGNAC ACCAAACACT
     ACCGTTCGCA
3651 AGATTTCCGC GCCTACCGCC AACAAACCGA CCTGCGCCAA
     ATCGGTATGC
3701 AGAAAAACCT CGGCAGCGGG CGCGTCGGCA TCCTGTTTTC
     GCACAACCGG
3751 ACCGAAAACA NCTTCGACGA CGGCATCGGC AACTCGGCAC
     GGCTTGCCCA
3801 CGGCGCCGTT TTCGGGCAAT ACGGCATCGG CAGGTTCGAC
     ATCGGCATCA
3851 GCACGGGCGC GGGTTTTAGC AGCGGCANTC TNTCAGACGG
     CATCGGAGGC
3901 AAAATCCGCC GCCGCGTGCT GCATTACGGC ATTCAGGCAC
     GATACCGCGC
3951 CGGTTTCGGC GGATTCGGCA TCGAACCGTA CATCGGCGCA
     ACGCGCTATT
4001 TCGTCCAAAA AGCGGATTAC CGCTACGAAA ACGTCAATAT
     CGCCACCCCC
4051 GGTCTTGCGT TCAACCGNTA CCGNGCGGGC ATTAAGGCAG
     ATTATTCATT
4101 CAAACCGGCG CAACACATNT CCATCACNCC TTATTTNAGC
     CTGTCCTATA
4151 CCGATGCCGC TTCGGGCAAA GTCCGAACAC GCGTCAATAC
     CGCNGTATTG
4201 GCTCAGGATT TCGGCAAAAC CCGCAGTGCG GAATGGGGCG
     TAAACGCCGA
4251 AATCAAAGGT TTCACGCTGT CCNTCCACGC TGCCGCCGCC
     AAAGGNCCGC
4301 AACTGGAAGC GCAACACAGC GCGGGCATCA AATTAGGCTA
     CCGCTGGTAA

This encodes a protein having amino acid sequence <SEQ ID 652>:

1    MKTTDKRTTE THRKAPKTGR IRFSPAYLAI CLSFGILPQA
     WAGHTYFGIN
51   YQYYRDFAEN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM
     IDFSVVSRNG
101  VAALVGDQYI VSVAHNGGYN NVDFGAEGXN PDQHRFSYQI
     VKRNNYKPDN
151  SHPYNGDXHM PRLHKFVTDA EPVEMTSDMR GNTYSDKEKY
     PERVRIGSGH
201  HYWRYDDDKH GDLSYSGAWL IGGNTHMQGW GNNGVXSLSG
     DVRHANDYGP
251  MPIAGAAGDS GSPMFIYDKT NNKWLLNGVL QTGYPYSGRE
     NGFQLIRKDW
301  FYDDIYRGDT HTVXFEPRSN GHFSFTSNNN GTGTVTETNE
     KVSNPKLKVQ
351  TVRLFDESLN ETDKEPVYAA GGVNQYRPRL NNGENLSFID
     YGNGKLILSN
401  NINQGAGGLY FEGDFTVSPE NNETWQGAGV HISEDSTVTW
     KVNGVANDRL
451  SKIGKGTLHV QAKGENQGSI SVGDGTVILD QQADDKGKKQ
     AFSEIGLXSG
501  RGTVQLNADN QFNPDKLYFG FRGGRLDLNG HSLSFHRIQN
     TDEGAMIXXH
551  NATTTSTVTI TGNESITQPS GKNINRLNYS KEIAYNGWFG
     EKDTTKTNGR
601  LNLVYQPAAE DRTXLLSGGT NLNGNITQTN GKLFFSGRPT
     PHAYNHLGSG
651  WSKMEGIPQG EIVWDNDWIX RTFKAENFHI QGGQAVISRN
     VAKVEGDXHL
701  SNHAQAVFGV APHQSHTICT RSDWTGLTNC VEXXITDDKV
     IASLTKTDXS
751  GXVXLXXXXX XXLXGXAXLX GNLSANGDTR YTVSHNATQN
     GNLSLVGNAQ
801  ATFNQATLNG NXSXSGNASF NLSNNAAQNG SLTLSDNAKA
     NVSHSALNGN
851  VSLADKAVFH FENSRFTGQL SGSKXTALHL KDSEWTLPSG
     TELGNLNLDN
901  ATITLNSAYR HDAAGAQTGX VSDTPRRRSR RSLLSVTPPT
     SVESRFNTLT
951  VNGKLNXQGT FRFMSELFGY RSDKLKLAES SEGTYTLAVN
     NTGNEPVSLD
1001 QLTVVEGKDN KPLSENLNFT LQNEHVDAGA WRYQLIRKDG
     EFRLHNPVKE
1051 QELSDKLGKA EAKKQAEKDN AQSLDALIAA GRDAAEKTES
     VAEPARXAGG
1101 ENVGIMQAEE EKKRVQADKD SALAKQREAE TRPXTTAFPR
     ARXARRDLPQ
1151 PQPQPQPQPQ PQRDLXSRYA NSGLSEFSAT LNSVFAVQDE
     LDRVFAEDRR
1201 NAVWTSXIRX TKHYRSQDFR AYRQQTDLRQ IGMQKNLGSG
     RVGILFSHNR
1251 TENXFDDGIG NSARLAHGAV FGQYGIGRFD IGISTGAGFS
     SGXLSDGIGG
1301 KIRRRVLHYG IQARYRAGFG GFGIEPYIGA TRYFVQKADY
     RYENVNIATP
1351 GLAFNRYRAG IKADYSFKPA QHXSITPYXS LSYTDAASGK
     VRTRVNTAVL
1401 AQDFGKTRSA EWGVNAEIKG FTLSXHAAAA KGPQLEAQHS
     AGIKLGYRW*

A transmembrane region is underlined.

ORF1-1 shows 86.3% identity over a 1462aa overlap with ORF1a:

Homology with Adhesion and Penetration Protein Hap Precursor of H. influenzae (Accession Number P45387)

Amino acids 23-423 of ORF1 show 59% aa identity with hap protein in 450aa overlap:

orf1	23	FXAAYLAICLSFGILPQAWAGHTYFGINYQYYRDFAENKGKFAVGAKDIEVYNKKGELVG	82
		F   +L  C+S GI  QAWAGHTYFGI+YQYYRDFAENKGKF VGAK+IEVYNK+G+LVG
hap	6	FRLNFLTACVSLGIASQAWAGHTYFGIDYQYYRDFAENKGKFTVGAKNIEVYNKEGQLVG	65
orf1	83	KSMTKAPMIDFSVVSRNGVAALVGVQYIVSVAHNGGYNNVDFGAEGXNIXDQXRXTYKIV	142
		SMTKAPMIDFSVVSRNGVAALVG QYIVSVAHNGGYN+VDFGAEG N  DQ R TY+IV
hap	66	TSMTKAPMIDFSVVSRNGVAALVGDQYIVSVAHNGGYNDVDFGAEGRN-PDQHRFTYQIV	124
orf1	143	KRNNYKAGTKGHPYGGDYHMPRLHKXVTDAEPVEMTSYMDGRKYIDQNNYPDRVRIGAGR	202
		KRNNY+A  + HPY GDYHMPRLHK VT+AEPV MT+ MDG+ Y D+ NYP+RVRIG+GR
hap	125	KRNNYQAWERKHPYDGDYHMPRLHKFVTEAEPVGMTTNMDGKVYADRENYPERVRIGSGR	184
orf1	203	QYWRSDEDEPNNRESSYHIA----------------------------------------	222
		QYWR+D+DE  N  SSY+++
hap	185	QYWRTDKDEETNVHSSYYVSGAYRYLTAGNTHTQSGNGNGTVNLSGNVVSPNHYGPLPTG	244
orf1	223	-----SGSPMFIYDAQKQKWLINGVLQTGNPYIGKSNGFQLVRKDWFYDEIFAGDTHSVF	277
		SGSPMFIYDA+K++WLIN VLQTG+P+ G+ NGFQL+R++WFY+E+ A DT SVF
hap	245	GSKGDSGSPMFIYDAKKKQWLINAVLQTGHPFFGRGNGFQLIREEWFYNEVLAVDTPSVF	304
orf1	278	--YEPRQNGKYSFNDDNNGTGKIN-AKHEHNSLPNRLKTRTVQLFNVSLSETAREPVYHA	334
		Y P  NG YSF  +N+GTGK+   +   +    + +  TV+LFN SL++TA+E V  A
hap	305	QRYIPPINGHYSFVSNNDGTGKLTLTRPSKDGSKAKSEVGTVKLFNPSLNQTAKEHV-KA	363
orf1	335	AGGVNSYRPRLNNGENISFIDEGKGELILTSNINQGAGGLYFQGDFTV-SPENNETWQGA	393
		A G N Y+PR+  G+NI   D+GKG L + +NINQGAGGLYF+G+F V   +NN TWQGA
hap	364	AAGYNIYQPRMEYGKNIYLGDQGKGTLTIENNINQGAGGLYFEGNFVVKGKQNNITWQGA	423
orf1	394	GVHISEDSTVTWKVNGVANDRLSKIGKGTL	423
		GV I +D+TV WKV+   NDRLSKIG GTL
hap	424	GVSIGQDATVEWKVHNPENDRLSKIGIGTL	453

Amino acids 715-1011 of ORF1 show 50% aa identity with hap protein in 258aa overlap:

Orf1	41	DTRYTVSHNATQ-NGNXSLVXNAQATFNQ-ATLNGNTSASGNASFNLSDHAVQNGSLTLS	98
		DT+   S   TQ NG+ +L  NA    +  A LNGN +   ++ F LS++A Q G++ LS
hap	733	DTKVINSIPITQINGSINLTNNATVNIHGLAKLNGNVTLIDHSQFTLSNNATQTGNIKLS	792
orf1	99	GNAKANVSHSALNGNVSLADKAVFHFESSRFTGQISGGKDTALHLKDSEWTLPSGXELGN	158
		+A A V+++ LNGNV L D A F  ++S F  QI G KDT + L+++ WT+PS   L N
hap	793	NHANATVNNATLNGNVHLTDSAQFSLKNSHFWHQIQGDKDTTVTLENATWTMPSDTTLQN	852
orf1	159	LNLDNATITLNSAYRHDAAGAQTGSATDAPXXXXXXXXXXLLXVTPPTSVESRFNTLTVN	218
		L L+N+T+TLNSAY        + S+ +AP          L   T PTS E RFNTLTVN
hap	853	LTLNNSTVTLNSAY--------SASSNNAPRHRRS-----LETETTPTSAEHRFNTLTVN	899
orf1	219	GKLNGQGTFRFMSELFGYRSDKLKLAESSEGTYTLAVNNTGNEPASLEQLTVVEGKDNKP	278
		GKL+GQGTF+F S LFGY+SDKLKL+  +EG YTL+V NTG EP +LEQLT++E  DNKP
hap	900	GKLSGQGTFQFTSSLFGYKSDKLKLSNDAEGDYTLSVRNTGKEPVTLEQLTLIESLDNKP	959
orf1	279	LSENLNFTLQNEHVDAGA	296
		LS+ L FTL+N+HVDAGA
hap	960	LSDKLKFTLENDHVDAGA	977

Amino acids 1192-1450 of ORF1 show 41% aa identity with hap protein in 259aa overlap:

Orf1	1	LDRVFAEDRRNAVWTSGIRDTKHYRSQDFRAYRQQTDLRQIGMQKNLGSGRVGILFSHNR	60
		LDR+F +  ++AVWT+  +D + Y S  FRAY+Q+T+LRQIG+QK L +GR+G +FSH+R
hap	1135	LDRLFVDQAQSAVWTNIAQDKRRYDSDAFRAYQQKTNLRQIGVQKALANGRIGAVFSHSR	1194
orf1	61	TENTFDDGIGNSARLAHGAVFGQYGIDRFYXXXXXXXXXXXXXXXXXIGXKXRRRVLHYG	120
		++NTFD+ + N A L   + F QY                          K  R+ ++YG
hap	1195	SDNTFDEQVKNHATLTMMSGFAQYQWGDLQFGVNVGTGISASKMAEEQSRKIHRKAINYG	1254
orf1	121	IQARYRAGFGGFGIEPHIGATRYFVQKADYRYENVNIATPGLAFNRYRAGIKADYSFKPA	180
		+ A Y+   G  GI+P+ G  RYF+++ +Y+ E V + TP LAFNRY AGI+ DY+F P
hap	1255	VNASYQFRLGQLGIQPYFGVNRYFIERENYQSEEVRVKTPSLAFNRYNAGIRVDYTFTPT	1314
orf1	181	QHISITPYLSLSYTDAASGKVRTRVNTAVLAQDFGKTRSAEWGVNAEIKGFTLSLHAAAA	240
		+IS+ PY  ++Y D ++  V+T VN  VL Q FG+    E G+ AEI  F +S   + +
hap	1315	DNISVKPYFFVNYVDVSNANVQTTVNLTVLQQPFGRYWQKEVGLKAEILHFQISAFISKS	1374
orf1	241	KGPQLEAQHSAGIKLGYRW	259
		+G QL  Q + G+KLGYRW
hap	1375	QGSQLGKQQNVGVKLGYRW	1393

Homology with a Predicted ORF from N. gonorrhoeae

The blocks of ORF1 show 83.5%, 88.3%, and 97.7% identities in 467, 298, and 259 aa overlap, respectively with a predicted ORF (ORF1ng) from N. gonorrhoeae:

The complete length ORF1ng nucleotide sequence was identified <SEQ ID 653>:

1    ATGAAAACAA CCGACAAACG GACAACCGAA ACACACCGCA
     AAGCCCCTAA
51   AACCGGCCGC ATCCGCTTCT CGCCCGCTTA CTTAGCCATA
     TGCCTGTCGT
101  TCGGCATTCT GCCCCAAGCC CGGGCGGGAC ACACTTATTT
     CGGCATCAAC
151  TACCAATACT ATCGCGACTT TGCCGAAAAT AAAGGCAAGT
     TTGCAGTCGG
201  GGCGAAAGAT ATTGAGGTTT ACAACAAAAA AGGGGAGTTG
     GTCGGCAAAT
251  CGATGACGAA AGCCCCGATG ATTGATTTTT CTGTGGTATC
     GCGTAACGGC
301  GTGGCGGCAT TGGCGGGCGA TCAATATATT GTGAGCGTGG
     CACATAACGG
351  CGGCTATAAC AATGTTGATT TTGGTGCGGA GGGAAGCAAT
     CCCGATCAGC
401  ACCGCTTTTC TTACCAAATT GTGAAAAGAA ATAATTATAA
     AGCAGGGACT
451  AACGGCCATC CTTATGGCGG CGATTATCAT ATGCCGCGTT
     TGCACAAATT
501  TGTCACAGAT GCAGAACCTG TTGAGATGAC CAGTTATATG
     GATGGGTGGA
551  AATACGCTGA TTTAAATAAA TACCCTGATC GTGTTCGAAT
     CGGAGCAGGC
601  AGACAATATT GGCGGTCTGA TGAAGACGAA CCCAATAACC
     GCGAAAGTTC
651  ATATCATATT GCAAGCGCAT ATTCTTGGCT CGTCGGTGGC
     AATACCTTTG
701  CACAAAATGG ATCAGGTGGT GGCACAGTCA ACTTAGGTAG
     CGAAAAAATT
751  AAACATAGCC CATATGGTTT TTTACCAACA GGAGGCTCAT
     TTGGCGACAG
801  TGGCTCACCA ATGTTTATCT ATGATGCCCA AAAGCAAAAG
     TGGTTAATTA
851  ATGGGGTATT GCAAACAGGC AACCCCTATA TAGGAAAAAG
     CAATGGCTTC
901  CAGCTAGTTC GTAAAGATTG GTTCTATGAT GAAATCTTTG
     CTGGAGATAC
951  CCATTCAGTA TTCTACGAAC CACATCAAAA TGGGAAATAC
     TTTTTTAACG
1001 ACAATAATAA TGGCGCAGGA AAAATCGATG CCAAACATAA
     ACACTATTCT
1051 CTACCTTATA GATTAAAAAC ACGAACCGTT CAATTGTTTA
     ATGTTTCTTT
1101 ATCCGAGACA GCAAGAGAAC CTGTTTATCA TGCTGCAGGT
     GGGGTCAACA
1151 GTTATCGACC CAGACTGAAT AATGGAGAAA ATATTTCCTT
     TATTGACAAA
1201 GGAAAAGGTG AATTGATACT TACCAGCAAC ATCAACCAAG
     GCGCGGGCGG
1251 TTTGTATTTT GAGGGTAATT TTACGGTCTC GCCTAAAAAC
     AACGAAACGT
1301 GGCAAGGCGC GGGCGTTCAT ATCAGTGATG GCAGTACCGT
     TACTTGGAAA
1351 GTAAACGGCG TGGCAAACGA CCGCCTGTCC AAAATCGGCA
     AAGGCACGCT
1401 GCTGGTTCAA GCCAAAGGGG AAAACCAAGG CTCGGTCAGC
     GTGGGCGACG
1451 GTAAAGTCAT CTTAGATCAG CAGGCGGACG ATCAAGGCAA
     AAAACAAGCC
1501 TTTAGTGAAA TCGGCTTGGT CAGCGGCAGG GGGACGGTGC
     AACTGAATGC
1551 CGATAATCAG TTCAACCCCG ACAAACTCTA TTTCGGCTTT
     CGCGGCGGAC
1601 GTTTGGATTT GAACGGGCAT TCGCTTTCGT TCCACCGCAT
     TCAAAATACC
1651 GATGAAGGGG CGATGATTGT CAACCACAAT CAAGACAAAG
     AATCCACCGT
1701 TACCATTACA GGCAATAAAG ATATTACTAC AACCGGCAAT
     AACAACAACT
1751 TGGATAGCAA AAAAGAAATT GCCTACAACG GTTGGTTTGG
     CGAGAAAGAT
1801 GCAACCAAAA CGAACGGGCG GCTCAATCTG AATTACCAAC
     CGGAAGAAGC
1851 GGATCGCACT TTACTGCTTT CCGGCGGAAC AAATTTAAAC
     GGCAATATCA
1901 CGCAAACAAA CGGCAAACTG TTTTTCAGCG GCAGACCGAC
     ACCGCACGCC
1951 TACAATCATT TAGGAAGCGG GTGGTCAAAA ATGGAAGGTA
     TCCCACAAGG
2001 AGAAATCGTG TGGGACAACG ATTGGATCGA CCGCACATTT
     AAAGCGGAAA
2051 ACTTCCATAT TCAGGGCGGA CAAGCGGTGG TTTCCCGCAA
     TGTTGCCAAA
2101 GTGGAAGGCG ATTGGCATTT AAGCAATCAC GCCCAAGCAG
     TTTTCGGTGT
2151 CGCACCGCAT CAAAGCCACA CAATCTGTAC ACGTTCGGAC
     TGGACGGGTC
2201 TGACAAGTTG TACCGAAAAA ACCATTACCG ACGATAAAGT
     GATTGCTTCA
2251 TTGAGCAAGA CCGACATCAG AGGCAATGTC AGCCTTGCCG
     ATCACGCTCA
2301 TTTAAATCTC ACAGGACTTG CCACACTCAA CGGCAATCTT
     AGTGCAGGCG
2351 GAGACACGCA CTATACGGTT ACGCGCAACG CCACCCAAAA
     CGGCAACCTC
2401 AGCCTCGTGG GCAATGCCCA AGCAACATTT AATCAAGCCA
     CATTAAACGG
2451 CAACACATCG GCTTCGGACA ATGCTTCATT TAATCTAAGC
     AACAACGCCG
2501 TACAAAACGG CAGTCTGACG CTTTCCGACA ACGCTAAGGC
     AAACGTAAGC
2551 CATTCCGCAC TCAACGGCAA TGTCTCCCTA GCCGATAAGG
     CAGTATTCCA
2601 TTTTGAAAAC AGCCGCTTTA CCGGAAAAAT CAGCGGCGGC
     AAGGATACGG
2651 CATTACACTT AAAAGACAGC GAATGGACGC TGCCGTCGGG
     CACGGAATTA
2701 GGCAATTTAA ACCTTGACAA CGCCACCATT ACACTCAATT
     CCGCCTATCG
2751 ACACGATGCG GCAGGCGCGC AAACCGGCAG TGCGGCAGAT
     GCGCCGCGCC
2801 GCCGTTCGCG CCGTTCCCTA TTATCCGTTA CGCCGCCAAC
     TTCGGCAGAA
2851 TCCCGTTTCA ACACGCTGAC GGTAAACGGC AAATTGAACG
     GTCAGGGAAC
2901 ATTCCGCTTT ATGTCGGAAC TCTTCGGCTA CCGCAGCGGC
     AAATTGAAGC
2951 TGGCGGAAAG TTCCGAAGGC ACTTACACCT TGGCTGTCAA
     CAATACCGGC
3001 AACGAACCCG TAAGTCTCGA GCAATTGACG GTAGTGGAAG
     GAAAAGACAA
3051 CACACCGCTG TCCGAAAATC TTAATTTCAC CCTGCaaaAc
     gaacacgtcg
3101 atgccggcgc atggCGTTAT CAGCTTATCC gcaaagacgG
     CGAGTTCCgc
3151 CTGCATAATC CGGTCAAAGA ACAAGAGCTT TCCGACAAAC
     TCGGCAAGgc
3201 gggagaaACA GAggccgccT TGACGGCAAA ACAGGCacaA
     CTTGCCGCCA
3251 AAcaacaggc ggaaaAAGAC AACgcgcaaa gccttgAcgc
     gctgattgcg
3301 gCcgggcgca atgccaccga AAAGGCAgaa agtgttgccg
     aaccgGCCCG
3351 GCAGGCAGGC GGGGAAAAtg ccgGCATTAT GCAGGCGGAG
     GAAGAGAAAA
3401 AACGGGTGCA GGCGGATAAA GACACCGCCT TGGCGAAACA
     GCGCGAAGCG
3451 GAAACCCGGC CGGCTACCAC CGCCTTCCCC CGCGCCCGCC
     GCGCCCGCCG
3501 GGATTTGCCG CAACCGCAGC CCCAACCGCA ACCCCAACCG
     CAGCGCGACC
3551 TGATCAGCCG TTATGCCAAT AGCGGTTTGA GTGAATTTTC
     CGCCACGCTC
3601 AACAGCGTTT TCGCCGTACA GGACGAATTG GACCGCGTGT
     TTGCCGAAGA
3651 CCGCCGCAAC GCCGTTTGGA CAAGCGGCAT CCGGGACACC
     AAACACTACC
3701 GTTCGCAAGA TTTCCGCGCC TACCGCCAAC AAACCGACCT
     GCGCCAAATC
3751 GGTATGCAGA AAAACCTCGG CAGCGGGCGC GTCGGCATCC
     TGTTTTCGCA
3801 CAACCGGACC GGAAACACCT TCGACGACGG CATCGGCAAC
     TCGGCACGGC
3851 TTGCCCACGG TGCCGTTTTC GGGCAATACG GCATCGGCAG
     GTTCGACATC
3901 GGCATCAGCG CGGGCGCGGG TTTTAGTAGC GGCAGCCTTT
     CAGACGGCAT
3951 CAGAGGCAAA ATCCGCCGCC GCGTGCTGCA TTACGGCATT
     CAGGCAAGAT
4001 ACCGCGCAGG TTTCGGCGGA TTCGGCATCG AACCGCACAT
     CGGCGCAACG
4051 CGCTATTTCG TCCAAAAAGC GGATTACCGA TACGAAAACG
     TCAATATCGC
4101 CACCCCGGGC CTTGCATTCA ACCGCTACCG CGCGGGCATT
     AAGGCAGATT
4151 ATTCATTCAA ACCGGCGCAA CACATTTCCA TCACGCCTTA
     TTTGAGCCTG
4201 TCCTATACCG ATGCCGCTTC CGGCAAAGTC CGAACGCGCG
     TCAATACCGC
4251 CGTATTGGCG CAGGATTTCG GCAAAACCCG CAGTGCGGAA
     TGGGGCGTAA
4301 ACGCCGAAAT CAAAGGTTTC ACGCTGTCCC TCCACGCTGC
     CGCCGCCAAG
4351 GGGCCGCAAT TGGAAGCGCA GCACAGCGCG GGCATCAAAT
     TAGGCTACCG
4401 CTGGTAA

This is predicted to encode a protein having amino acid sequence <SEQ ID 654>:

1    MKTTDKRTTE THRKAPKTGR IRFSPAYLAI CLSFGILPQA
     RAGHTYFGIN
51   YQYYRDFAEN KGKFAVGAKD IEVYNKKGEL VGKSMTKAPM
     IDFSVVSRNG
101  VAALAGDQYI VSVAHNGGYN NVDFGAEGSN PDQHRFSYQI
     VKRNNYKAGT
151  NGHPYGGDYH MPRLHKFVTD AEPVEMTSYM DGWKYADLNK
     YPDRVRIGAG
201  RQYWRSDEDE PNNRESSYHI ASAYSWLVGG NTFAQNGSGG
     GTVNLGSEKI
251  KHSPYGFLPT GGSFGDSGSP MFIYDAQKQK WLINGVLQTG
     NPYIGKSNGF
301  QLVRKDWFYD EIFAGDTHSV FYEPHQNGKY FFNDNNNGAG
     KIDAKHKHYS
351  LPYRLKTRTV QLFNVSLSET AREPVYHAAG GVNSYRPRLN
     NGENISFIDK
401  GKGELILTSN INQGAGGLYF EGNFTVSPKN NETWQGAGVH
     ISDGSTVTWK
451  VNGVANDRLS KIGKGTLLVQ AKGENQGSVS VGDGKVILDQ
     QADDQGKKQA
501  FSEIGLVSGR GTVQLNADNQ FNPDKLYFGF RGGRLDLNGH
     SLSFHRIQNT
551  DEGAMIVNHN QDKESTVTIT GNKDITTTGN NNNLDSKKEI
     AYNGWFGEKD
601  ATKTNGGLNL NYPPEEADRT LLLSGGTNLN GNITQTNGKL
     FFSGRPTPHA
651  YNHLGSGWSK MEGIPQGEIV WDNDWIDRTF KAENFHIQGG
     QAVVSRNVAK
701  VEGDWHLSNH AQAVFGVAPH QSHTICTRSD WTGLTSCTEK
     TITDDKVIAS
751  LSKTDVRGNV SLADHAHLNL TGLATFNGNL VQAETRTIRL
     RANATQNGNL
801  SLVGNAQATF NQATLNGNTS ASDNASFNLS NNAVQNGSLT
     LSDNAKANVS
851  HSALNGNVSL ADKAVFHFEN SRFTGKISGG KDTALHLKDS
     EWTLPSGTEL
901  GNLNLDNATI TLNSAYRHDA AGAQTGSAAD APRRRSRRSL
     LSVTPPTSAE
951  SRFNTLTVNG KLNGQGTFRF MSELFGYRSG KLKLAESSEG
     TYTLAVNNTG
1001 NEPVSLEQLT VVEGKDNTPL SENLNFTLQN EHVDAGAWRY
     QLIRKDGEFR
1051 LHNPVKEQEL SDKLGKAGET EAALTAKQAQ LAAKQQAEKD
     NAQSLDALIA
1101 AGRNATEKAE SVAEPARQAG GENAGIMQAE EEKKRVQADK
     DTALAKQREA
1151 ETRPATTAFP RARRARRDLP QPQPQPQPQP QRDLISRYAN
     SGLSEFSATL
1201 NSVFAVQDEL DRVFAEDRRN AVWTSGIRDT KHYRSQDFRA
     YRQQTDLRQI
1251 GMQKNLGSGR VGILFSHNRT GNTFDDGIGN SARLAHGAVF
     GQYGIGRFDI
1301 GISAGAGFSS GSLSDGIRGK IRRRVLHYGI QARYRAGFGG
     FGIEPHIGAT
1351 RYFVQKADYR YENVNIATPG LAFNRYRAGI KADYSFKPAQ
     HISITPYLSL
1401 SYTDAASGKV RTRVNTAVLA QDFGKTRSAE WGVNAEIKGF
     TLSLHAAAAK
1451 GPQLEAQHSA GIKLGYRW*

Underlined and double-underlined sequences represent the active site of a serine protease (trypsin family) and an ATP/GTP-binding site motif A (P-loop).

ORF1-1 and ORF1ng show 93.7% identity in 1471 aa overlap:

In addition, ORF1ng shows 55.7% identity with hap protein (P45387) over a 1455aa overlap:

Based on this analysis, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 78

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 655>:

1   ..AAGGTGTGGC AATTTGTCGA AGA.CCGCTG CGTGCCGTCG
    TGCCTGCCGA
51  CAGTTTTGAA CCGACCGCGC AAAAATTGAA CCTGTTTAAG
    GCGGGTGCGG
101 CAACCATTTT GTTTTATGAA GATCAAAATG TCGTCAAAGG
    TTTGCAGGAG
151 CAGTTCCCTG CTTATGCCGC TAACTTCCCC GTTTGGGCGg
    ATCAGGCAAA
201 CGCGATGGTG CAGTATGCCG TTTGGACGAC ACTTGCCGCG
    GTCGGCGTAG
251 GTGCAAACCT GCAACATTAC AATCCCTTGC CCGATGCGGC
    GATTGCCAAA
301 GCGTGGAATA TCCCCGAAAA CTGGTTGTTG CGCGCACAAA
    TGGTTATCGG
351 CGGTATTGAA GGGGCGGCAG GTGAAAAGAC CTTTGAACCC
    GTTGCAGAAC
401 GTTTGAAAGT GTTCGGCGCA TAA

This corresponds to the amino acid sequence <SEQ ID 656; ORF6>:

1   . . . KVWQFVEXPL RAWPADSFE PTAQKLNLFK AGAATILFYE  DQNVVKGLQE
51  QFPAYAANFP VWADQANAMV QYAVWTTLAA VGVGANLQHY NPLPDAAIAK
101 AWNIPENWLL RAQMVIGGIE GAAGEKTFEP VAERLKVFGA *

Further sequence analysis revealed a further partial DNA sequence <SEQ ID 657>:

1   . . . CTGCGTGCCG TCGTGCCTGC CGACAGTTTT GAACCGACCG CGCAAAAATT
51  GAACCTGTTT AAGGCGGGTG CGGCAACCAT TTTGTTTTAT GAAGATCAAA
101 ATGTCGTCAA AGGTTTGCAG GAGCAGTTCC CTGCTTATGC CGCTAACTTC
151 CCCGTTTGGG CGGATCAGGC AAACGCGATG GTGCAGTATG CCGTTTGGAC
201 GACACTTGCC GCGGTCGGCG TAGGTGCAAA CCTGCAACAT TACAATCCCT
251 TGCCCGATGC GGCGATTGCC AAAGCGTGGA ATATCCCCGA AAACTGGTTG
301 TTGCGCGCAC AAATGGTTAT CGGCGGTATT GAAGGGGCGG CAGGTGAAAA
351 GACCTTTGAA CCCGTTGCAG AACGTTTGAA AGTGTTCGGC GCATAA

This corresponds to the amino acid sequence <SEQ ID 658; ORF6-1>:

1   . . . LRAVVPADSF EPTAQKLNLF KAGAATILFY EDQNVVKGLQ EQFPAYAANF
51  PVWADQANAM VQYAVWTTLA AVGVGANLQH YNPLPDAAIA KAWNIPENWL
101 LRAQMVIGGI EGAAGEKTFE PVAERLKVFG A*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF6 shows 98.6% identity over a 140aa overlap with an ORF (ORF6a) from strain A of N. meningitidis:

The complete length ORF6a nucleotide sequence <SEQ ID 659> is:

1   ATGACCCGTC AATCTCTGCA ACAGGCTGCC GAAAGCCGCC GTTCCATTTA
51  TTCGTTAAAT AAAAATCTGC CCGTCGGCAA AGATGAAATC GTCCAAATCG
101 TCGAACACGC CGTTTTGCAC ACACCTTCTT CGTTCAATTC CCAATCTGCC
151 CGTGTGGTCG TGCTGTTTGG CGAAGAGCAT GATAAGGTGT GGCAATTTGT
201 CGAAGACGCG CTGCGTGCCG TCGTGCCTGC CGACAGTTTT GAACCGACCG
251 CGCAAAAATT GAACCTGTTT AAGGCGGGTG CGGCAACTAT TTTGTTTTAT
301 GAAGATCAAA ATGTCGTCAA AGGTTTGCAG GAGCAGTTCC CTGCTTATGC
351 CGCCAACTTT CCCGTTTGGG CGGACCAGGC GAACGCGATG GTGCAGTATG
401 CCGTTTGGAC GACACTTGCC GCGGTCGGCG TAGGTGCAAA CCTGCAACAT
451 TACAATCCCT TGCCCGATGC GGCGATTGCC AAAGCGTGGA ATATCCCCGA
501 AAACTGGTTG TTGCGCGCAC AAATGGTTAT CGGCGGTATT GAAGGGGCGG
551 CAGGTGAAAA GACCTTTGAA CCAGTTGCAG AACGTTTGAA AGTGTTCGGC
601 GCATAA

This is predicted to encode a protein having amino acid sequence <SEQ ID 660>:

1   MTRQSLQQAA ESRRSIYSLN KNLPVGKDEI VQIVEHAVLH TPSSFNSQSA
51  RVVVLFGEEH DKVWQFVEDA LRAVVPADSF EPTAQKLNLF KAGAATILFY
101 EDQNVVKGLQ EQFPAYAANF PVWADQANAM VQYAVWTTLA AVGVGANLQH
151 YNPLPDAAIA KAWNIPENWL LRAQMVIGGI EGAAGEKTFE PVAERLKVFG
201 A*

ORF6a and ORF6-1 show 100.0% identity in 131 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF6 shows 95.7% identity over a 140aa overlap with a predicted ORF (ORF6ng) from N. gonorrhoeae:

The complete length ORF6ng nucleotide sequence <SEQ ID 661> was identified as:

1   ATGGCCGTTG CGTCAAATGT CAGCTTGGAT ATGTCCAATC CTACGGTGTT
51  ACGCATGGGA TTACCCTTAT ATATTGCGTC CCTAAGAAGG GGCGCAATAT
101 ATAAGGTGTG GCAATTTGTC GAAGACGCGC TGCGTGCCGT CGTGCCTGCC
151 GACAGTTTTG AACCGACCGC GCAAAAATTG AAGCTGTTTA AGGCGGGCGC
201 GGCAACCATT TTGTTTTATG AAGATCAAAA TGTCGTCAAA GGTTTGCAGG
251 AGCAGTTCCC TGCTTATGCC GCCAACTTTC CCGTTTGGGC GGACCAGGCG
301 AACGCTATGG TACAGTATGC CGTCTGGACG ACACTTGCCG CGGTCGGTGC
351 AGGTGCAAAT CTGCAACATT ACAACCCCTT GCCCGATGTG GCGATTGCTA
401 AAGCGTGGAA TATTCCCGAA AACTGGCTGT TGCGCGCGCA AATGGTTATC
451 GGTGGTATTG AAGGGGcggc aggtgaaaaa gtctttgaac CCGTTGCgga
501 acgtttgAAA GTGTTCGGCG CATAA

This encodes a protein having amino acid sequence <SEQ ID 662>:

1   MAVASNVSLD MSNPTVLRMG LPLYIASLRR GAIYKVWQFV EDALRAVVPA
51  DSFEPTAQKL KLFKAGAATI LFYEDQNVVK GLQEQFPAYA ANFPVWADQA
101 NAMVQYAVWT TLAAVGAGAN LQHYNPLPDV AIAKAWNIPE NWLLRAQMVI
151 GGIEGAAGEK VFEPVAERLK VFGA*

ORF6ng and ORF6-1 show 96.9% identity in 131 aa overlap:

It is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 79

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 663>

1   . . . GGCTACAACT ACCTGTTCGC GCGCGGCAGC CGCATCGCCA ACTACCAAAT
51  CAACGGCATC CCCGTTGCCG ACGCGCTGGC CGATACGGGt CAATGCCAAC
101 ACCGCCGCCT ATGAGCGCGT AGAAGTCGTG CGCGGCGTGG CGGGGCTGCT
151 GGACGGCACG GGCGAGCCTT CCGCCACCGT CAATCTGGTG CGCAAACGCC
201 TGACCCGCAA GCCATTGTTT GAAGTCCGCG CCGAAGCgGG CAACCGcAAA
251 CATTTCGGGC TGGACGCGGA CGTATCGGGC AGCCTGAACA CCGAAG.crC
301 rCTGCGCgGC CGCCTGGTTT CCAcCTTCGG ACGCGGCGAC TCGTGGCGGC
351 GGCGCGAACG CAGCCGskAT GCCGAACTCT ACGGCATTTT GGAATACGAC
401 ATCGCACCGC AAACCCGCGT CCACGCArGC ATGGACTACC AGCAGGCGAA
451 AGAAACCGCC GACGCGCCGC TCAGcTACGC CGTGTACGAC AGCCAAGGTT
501 ATGCCACCGC CTTCGGCCCG AAAGACAACC CCGCCACAAA TTGGGCGAAC
551 AGCCACCACC GTGCGCTCAA CCTGTTCGCC GGCATCGAAC ACCGCTTCAA
601 CCAAGACTGG AAACTCAAAG CCGAATACGA CTAC . . .

This corresponds to the amino acid sequence <SEQ ID 664; ORF23>:

1   . . . GYNYLFARGS RIANYQINGI PVADALADTG NANTAAYERV EVVRGVAGLL
51  DGTGEPSATV NLVRKRLTRK PLFEVRAEAG NRKHFGLDAD VSGSLNTEXX
101 LRGRLVSTFG RGDSWRRRER SRXAELYGIL EYDIAPQTRV HAXMDYQQAK
151 ETADAPLSYA VYDSQGYATA FGPKDNPATN WANSHHRALN LFAGIEHRFN
201 QDWKLKAEYD Y . . .

Further work revealed the complete nucleotide sequence <SEQ ID 665>:

1    ATGACACGCT TCAAATATTC CCTGCTGTTT GCCGCCCTGT TGCCCGTGTA
51   CGCGCAGGCC GATGTTTCTG TTTCAGACGA CCCCAAACCG CAGGAAAGCA
101  CTGAATTGCC GACCATCACC GTTACCGCCG ACCGCACCGC GAGTTCCAAC
151  GACGGCTACA CTGTTTCCGG CACGCACACC CCGCTCGGGC TGCCCATGAC
201  CCTGCGCGAA ATCCCGCAGA GCGTCAGCGT CATCACATCG CAACAAATGC
251  GCGACCAAAA CATCAAAACG CTCGACCGCG CCCTGTTGCA GGCGACCGGC
301  ACCAGCCGCC AGATTTACGG CTCCGACCGC GCGGGCTACA ACTACCTGTT
351  CGCGCGCGGC AGCCGCATCG CCAACTACCA AATCAACGGC ATCCCCGTTG
401  CCGACGCGCT GGCCGATACG GGCAATGCCA ACACCGCCGC CTATGAGCGC
451  GTAGAAGTCG TGCGCGGCGT GGCGGGGCTG CTGGACGGCA CGGGCGAGCC
501  TTCCGCCACC GTCAATCTGG TGCGCAAACG CCTGACCCGC AAGCCATTGT
551  TTGAAGTCCG CGCCGAAGCG GGCAACCGCA AACATTTCGG GCTGGACGCG
601  GACGTATCGG GCAGCCTGAA CACCGAAGGC ACGCTGCGCG GCCGCCTGGT
651  TTCCACCTTC GGACGCGGCG ACTCGTGGCG GCGGCGCGAA CGCAGCCGCG
701  ATGCCGAACT CTACGGCATT TTGGAATACG ACATCGCACC GCAAACCCGC
751  GTCCACGCAG GCATGGACTA CCAGCAGGCG AAAGAAACCG CCGACGCGCC
801  GCTCAGCTAC GCCGTGTACG ACAGCCAAGG TTATGCCACC GCCTTCGGCC
851  CGAAAGACAA CCCCGCCACA AATTGGGCGA ACAGCCGCCA CCGTGCGCTC
901  AACCTGTTCG CCGGCATCGA ACACCGCTTC AACCAAGACT GGAAACTCAA
951  AGCCGAATAC GACTACACCC GCAGCCGCTT CCGCCAGCCC TACGGCGTAG
1001 CAGGCGTGCT TTCCATCGAC CACAACACCG CCGCCACCGA CCTGATTCCC
1051 GGTTATTGGC ACGCCGACCC GCGCACCCAC AGCGCCAGCG TGTCATTGAT
1101 CGGCAAATAC CGCCTGTTCG GCCGCGAACA CGATTTAATC GCGGGTATCA
1151 ACGGTTACAA ATACGCCAGC AACAAATACG GCGAACGCAG CATCATCCCC
1201 AACGCCATTC CCAACGCCTA CGAATTTTCC CGCACGGGTG CCTACCCGCA
1251 GCCTGCATCG TTTGCCCAAA CCATCCCGCA ATACGGCACC AGGCGGCAAA
1301 TCGGCGGCTA TCTCGCCACC CGTTTCCGCG CCGCCGACAA CCTTTCGCTG
1351 ATTTTGGGCG GACGATACAC CCGTTACCGC ACCGGCAGCT ACGACAGCCG
1401 CACACAAGGC ATGACCTATG TGTCCGCCAA CCGTTTCACC CCCTACACAG
1451 GCATCGTGTT CGACCTGACC GGCAACCTGT CTCTTTACGG CTCGTACAGC
1501 AGCCTGTTCG TCCCGCAATC GCAAAAAGAC GAACACGGCA GCTACCTGAA
1551 ACCCGTAACC GGCAACAATC TGGAAGCCGG CATCAAAGGC GAATGGCTTG
1601 AAGGCCGTCT GAACGCATCC GCCGCCGTGT ACCGCGCCCG TAAAAACAAC
1651 CTCGCCACCG CAGCAGGACG CGACCCGAGC GGCAACACCT ACTACCGCGC
1701 CGCCAACCAA GCCAAAACCC ACGGCTGGGA AATCGAAGTC GGCGGCCGCA
1751 TCACGCCCGA ATGGCAGATA CAGGCAGGTT ACAGCCAAAG CAAAACCCGC
1801 GACCAAGACG GCAGCCGCCT GAACCCCGAC AGCGTACCCG AACGCAGCTT
1851 CAAACTCTTC ACTGCCTACC ACTTTGCCCC CGAAGCCCCC AGCGGCTGGA
1901 CCATCGGCGC AGGCGTGCGC TGGCAGAGCG AAACCCACAC CGACCCTGCC
1951 ACGCTCCGCA TCCCCAACCC CGCCGCCAAA GCCCGCGCCG CCGACAACAG
2001 CCGCCAAAAA GCCTACGCCG TCGCCGACAT CATGGCGCGT TACCGCTTCA
2051 ATCCGCGCGC CGAACTGTCG CTGAACGTGG ACAATCTGTT CAACAAACAC
2101 TACCGCACCC AGCCCGACCG CCACAGCTAC GGCGCACTGC GGACAGTGAA
2151 CGCGGCGTTT ACCTATCGGT TTAAATAA

This corresponds to the amino acid sequence <SEQ ID 666; ORF23-1>:

1   MTRFKYSLLF AALLPVYAQA DVSVSDDPKP QESTELPTIT VTADRTASSN
51  DGYTVSGTHT PLGLPMTLRE IPQSVSVITS QQMRDQNIKT LDRALLQATG
101 TSRQIYGSDR AGYNYLFARG SRIANYQING IPVADALADT GNANTAAYER
151 VEVVRGVAGL LDGTGEPSAT VNLVRKRLTR KPLFEVRAEA GNRKHFGLDA
201 DVSGSLNTEG TLRGRLVSTF GRGDSWRRRE RSRDAELYGI LEYDIAPQTR
251 VHAGMDYQQA KETADAPLSY AVYDSQGYAT AFGPKDNPAT NWANSRHRAL
301 NLFAGIEHRF NQDWKLKAEY DYTRSRFRQP YGVAGVLSID HNTAATDLIP
351 GYWHADPRTH SASVSLIGKY RLFGREHDLI AGINGYKYAS NKYGERSIIP
401 NAIPNAYEFS RTGAYPQPAS FAQTIPQYGT RRQIGGYLAT RFRAADNLSL
451 ILGGRYTRYR TGSYDSRTQG MTYVSANRFT PYTGIVFDLT GNLSLYGSYS
501 SLFVPQSQKD EHGSYLKPVT GNNLEAGIKG EWLEGRLNAS AAVYRARKNN
551 LATAAGRDPS GNTYYRAANQ AKTHGWEIEV GGRITPEWQI QAGYSQSKTR
601 DQDGSRLNPD SVPERSFKLF TAYHFAPEAP SGWTIGAGVR WQSETHTDPA
651 TLRIPNPAAK ARAADNSRQK AYAVADIMAR YRFNPRAELS LNVDNLFNKH
701 YRTQPDRHSY GALRTVNAAF TYRFK*

Computer analysis of this amino acid sequence gave the following results:

Homology with the Ferric-Pseudobactin Receptor PupB of Pseudomonas putida (Accession Number P38047)

ORF23 and PupB protein show 32% aa identity in 205aa overlap:

Orf23	6	FARGSRIANYQINGIPVADALADTGNANTAAYERVEVVRGVAGLLDGTGEPSATVNLVRK	65
		++RG  I NY+++G+P +   L D  + + A ++RVE+VRG  GL+ G G PSAT+NL+RK
PupB	215	WSRGFAIQNYEVDGVPTSTRL-DNYSQSMAMFDRVEIVRGATGLISGMGNPSATINLIRK	273
Orf23	66	RLTRKPLFEVRAEAGNRKHFGLDADVSGSLNTEXXLRGRLVSTFXXXXXXXXXXXXXXAE	125
		R T +    +  EAGN   +G   DVSG L     +RGR V+ +
PupB	274	RPTAEAQASITGEAGNWDRYGTGFDVSGPLTETGNIRGRFVADYKTEKAWIDRYNQQSQL	333
Orf23	126	LYGILEYDIAPQTRVHAXMDYQQAKETADAPLSYAVYD--SQGYATAFGPKDNPATNWAN	183
		+YGI E+D++  T+      Y   +    D+PL   +    S G  T      N A +W+
PupB	334	MYGITEFDLSEDTLLTVGFSY--LRSDIDSPLRSGLPTRFSTGERTNLKRSLNAAPDWSY	391
Orf23	184	SHHRALNLFAGIEHRFNQDWKLKAE	208
		+ H   + F  IE +    W  K E
PupB	392	NDHEQTSFFTSIEQQLGNGWSGKIE	416

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF23 shows 95.7% identity over a 211aa overlap with an ORF (ORF23a) from strain A of N. meningitidis:

The complete length ORF23a nucleotide sequence <SEQ ID 667> is:

1    ATGACACGCT TCAAATATTC CCTGCTGTTT GCCGCCCTGT TGCCCGTGTA
51   CGCGCAGGCC GATGTTTCTG TTTCAGACGA CCCAAAACCG CAGGAAAGCA
101  CTGAATTGCC GACCATCACC GTTACCGCCG ACCGCACCGC GAGTTCCAAC
151  GACGGCTACA CTGTTTCCGG CACGCACACC CCGCTCGGGC TGCCCATGAC
201  CCTGCGCGAA ATCCCGCAGA GCGTCAGCGT CATCACATCG CAACAAATGC
251  GCGACCAAAA CATCAAAGCG CTCGACCGCG CCCTGTTGCA GGCGACCGGC
301  ACCAGCCGCC AGATTTACGG CTCCGACCGC GCGGGCTACA ACTACCTGTT
351  CGCGCGCGGC AGCCGCATCG CCAACTACCA AATCAACGGC ATCCCCGTTG
401  CCGACGCGCT GGCCGATACG GGCAATGCCA ACACCGCCGC CTATGAGCGC
451  GTAGAAGTCG TGCGCGGCGT GGCGGGGCTG CTGGACGGCA CGGGCGAGCC
501  TTCCGCCACC GTCAATCTGG TGCGCAAACG CCCGACCCGC AAGCCATTGT
551  TTGAAGTCCG CGCCGAAGCG GGCAACCGCA AACATTTCGG GCTGGGCGCG
601  GACGTATCGG GCAGCCTGAA TGCCGAAGGC ACGCTGCGCG GCCGCCTGGT
651  TTCCACCTTC GGACGCGGCG ACTCGTGGCG GCAGCGCGAA CGCAGCCGCG
701  ATGCCGAACT CTACGGCATT TTGGAATACG ACATCGCACC GCAAACCCGC
751  GTCCACGCAG GCATGGACTA CCAGCAGGCG AAAGAAACCG CCGACGCGCC
801  GCTCAGCTAC GCCGTGTACG ACAGCCAAGG TTATGCCACC GCCTTCGGCC
851  CGAAAGACAA CCCCGCCACA AATTGGGCGA ACAGCCGCCA CCGTGCGCTC
901  AACCTGTTCG CCGGCATCGA ACACCGCTTC AACCAAGACT GGAAACTCAA
951  AGCCGAATAC GACTACACCC GCAGCCGCTT CCGCCAGCCC TACGGCGTAG
1001 CAGGCGTGCT TTCCATCGAC CACAACACCG CCGCCACCGA CCTGATTCCC
1051 GGTTATTGGC ACGCCGACCC GCGCACCCAC AGCGCCAGCG TGTCATTAAT
1101 CGGCAAATAC CGCCTGTTCG GCCGCGAACA CGATTTAATC GCGGGTATCA
1151 ACGGTTACAA ATACGCCAGC AACAAATACG GCGAACGCAG CATCATCCCC
1201 AACGCCATTC CCAACGCCTA CGAATTTTCC CGCACGGGTG CCTACCCGCA
1251 GCCTGCATCG TTTGCCCAAA CCATCCCGCA ATACGGCACC AGGCGGCAAA
1301 TCGGCGGCTA TCTCGCCACC CGTTTCCGCG CCGCCGACAA CCTTTCGCTG
1351 ATACTCGGCG GCAGATACAG CCGTTACCGC ACCGGCAGCT ACGACAGCCG
1401 CACACAAGGC ATGACCTATG TGTCCGCCAA CCGTTTCACC CCCTACACAG
1451 GCATCGTGTT CGACCTGACC GGCAACCTGT CGCTTTACGG CTCGTACAGC
1501 AGCCTGTTCG TCCCGCAATC GCAAAAAGAC GAACACGGCA GCTACCTGAA
1551 ACCCGTAACC GGCAACAATC TGGAAGCCGG CATCAAAGGC GAATGGCTTG
1601 AAGGCCGTCT GAACGCATCC GCCGCCGTGT ACCGCGCCCG TAAAAACAAC
1651 CTCGCCACCG CAGCAGGACG CGACCCGAGC GGCAACACCT ACTACCGCGC
1701 CGCCAACCAA GCCAAAACCC ACGGCTGGGA AATCGAAGTC GGCGGCCGCA
1751 TCACGCCCGA ATGGCAGATA CAGGCAGGTT ACAGCCAAGG CAAAACCCGC
1801 GACCAAGACG GCAGCCGCCT GAACCCCGAC AGCGTACCCG AACGCAGCTT
1851 CAAACTCTTC ACTGCCTACC ACTTTGCCCC CGAAGCCCCC AGCGGCTGGA
1901 CCATCGGCGC AGGCGTGCGC TGGCAGAGCG AAACCCACAC CGACCCTGCC
1951 ACGCTCCGCA TCCCCAACCC CGCCGCCAAA GCCCGCGCCG CCGACAACAG
2001 CCGCCAAAAA GCCTACGCCG TCGCCGACAT CATGGCGCGT TACCGCTTCA
2051 ATCCGCGCGC CGAACTGTCG CTGAACGTGG ACAATCTGTT CAACAAACAC
2101 TACCGCACCC AGCCCGACCG CCACAGCTAC GGCGCACTGC GGACAGTGAA
2151 CGCGGCGTTT ACCTATCGGT TTAAATAA

This encodes a protein having amino acid sequence <SEQ ID 668>:

1   MTRFKYSLLF AALLPVYAQA DVSVSDDPKP QESTELPTIT VTADRTASSN
51  DGYTVSGTHT PLGLPMTLRE IPQSVSVITS QQMRDQNIKA LDRALLQATG
101 TSRQIYGSDR AGYNYLFARG SRIANYQING IPVADALADT GNANTAAYER
151 VEVVRGVAGL LDGTGEPSAT VNLVRKRPTR KPLFEVRAEA GNRKHFGLGA
201 DVSGSLNAEG TLRGRLVSTF GRGDSWRQRE RSRDAELYGI LEYDIAPQTR
251 VHAGMDYQQA KETADAPLSY AVYDSQGYAT AFGPKDNPAT NWANSRHRAL
301 NLFAGIEHRF NQDWKLKAEY DYTRSRFRQP YGVAGVLSID HNTAATDLIP
351 GYWHADPRTH SASVSLIGKY RLFGREHDLI AGINGYKYAS NKYGERSIIP
401 NAIPNAYEFS RTGAYPQPAS FAQTIPQYGT RRQIGGYLAT RFRAADNLSL
451 ILGGRYSRYR TGSYDSRTQG MTYVSANRFT PYTGIVFDLT GNLSLYGSYS
501 SLFVPQSQKD EHGSYLKPVT GNNLEAGIKG EWLEGRLNAS AAVYRARKNN
551 LATAAGRDPS GNTYYRAANQ AKTHGWEIEV GGRITPEWQI QAGYSQSKTR
601 DQDGSRLNPD SVPERSFKLF TAYHFAPEAP SGWTIGAGVR WQSETHTDPA
651 TLRIPNPAAK ARAADNSRQK AYAVADIMAR YRFNPRAELS LNVDNLFNKH
701 YRTQPDRHSY GALRTVNAAF TYRFK*

ORF23a and ORF23-1 show 99.2% identity in 725 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF23 shows 93.4% identity over a 211 as overlap with a predicted ORF (ORF23.ng) from N. gonorrhoeae:

The ORF23ng nucleotide sequence <SEQ ID 669> is predicted to encode a protein comprising amino acid sequence <SEQ ID 670>:

1   SAVDACRIPG YNYLFARGSR IANYQINGIP VADALADTGN ANTAAYERVE
51  VVRGVAGLPD GTGEPSATVN LVRKHPTRKP LFEVRAEAGN RKHFGLGADV
101 SGSLNAEGTL RGRLVSTFGR GDSWRQLERS RDAELYGILE YDIAPQTRVH
151 AGMDYQQAKE TADAPLSYAV YDSQGYATAF GPKDNPATNW SNSRNRALNL
201 FAGIEHRFNQ DWKLKAEYDY TRSRFRQPYG VAGVLSIDHS TAATDLIPGY
251 WHADPRTHSA SMSLTGKYRL FGREHDLIAG INGYKYASNK YGERSIIPNA
301 IPNAYEFSRT GAYPQPSSFA QTIPQYDTRR QIGGYLATRF RAADNLSLIL
351 GGRYSRYRAG SYNSRTQGMT YVSANRFTPY TGIVFDLTGN LSLYGSYSSL
401 FVPQLQKDEH GSYLKPVTGN NLEADIKGEW LEGRLNASAA VYRARKNNLA
451 TAAGRDQSGN TYYRAANQAK THGWEIEVGG RITPEWQIQA GYSQSKPRDQ
501 DGSRLNPDSV PERSFKLFTA YHLAPEAPSG RTIGAGVRRQ GETHTDPAAL
551 RIPNPAAKAR AVANSRQKAY AVADIMARYR FNPRTELSLN VDNLFNKHYR
601 TQPDRHSYGA LRTVNAAFTY RFK*

Further work revealed the complete nucleotide sequence <SEQ ID 671>:

1    ATGACACGCT TCAAATACTC CCTGCTTTTT GCCGCCCTGC
     TACCCGTGTA
51   CGCGCAGGCC GATGTTTCTG TTTCAGACGA CCCCAAACCG
     CAGGAAAGCA
101  CCGAATTGCC GACCATCACC GTTACCGCCG ACCGCACCGC
     GAGTTCCAAC
151  GACGGCTACA CCGTTTCCGG CACGCACACC CCGTTCGGGC
     TGCCCATGAC
201  CCTGCGCGAA ATCCCGCAGA GCGTCAGCGT CATCACATCG
     CAACAAATGC
251  GCGACCAAAA CATCAAAACG CTCGACCGCG CCCTGTTGCA
     GGCGACCGGC
301  ACCAGCCGCC AGATTTACGG CTCCGACCGC GCGGGCTACA
     ACTACCTGTT
351  CGCGCGCGGC AGCCGCATCG CCAACTACCA AATCAACGGC
     ATCCCCGTTG
401  CCGACGCGCT GGCCGATACG GGCAATGCCA ACACCGCCGC
     CTATGAGCGC
451  GTAGAAGTCG TGCGCGGCGT GGCGGGGCTG CCGGACGGCA
     CGGGCGAGCC
501  TTCTGCCACC GTCAATCTGG TACGCAAACA CCCGACCCGC
     AAGCCATTGT
551  TTGAAGTCCG CGCCGAAGCC GGCAACCGCA AACATTTCGG
     GCTGGGCGCG
601  GACGTATCGG GCAGCCTGAA CGCCGAAGGC ACGCTGCGCG
     GCCGCCTGGT
651  TTCCACCTTC GGACGCGGCG ACTCGTGGCG GCAGCTCGAA
     CGCAGCCGCG
701  ATGCCGAACT CTACGGCATT TTGGAATACG ACATCGCACC
     GCAAACCCGC
751  GTCCACGCAG GCATGGACTA CCAGCAGGCG AAAGAAACCG
     CAGACGCGCC
801  GCTCAGCTAC GCCGTGTACG ACAGCCAAGG TTATGCCACC
     GCCTTCGGCC
851  CAAAAGACAA CCCCGCCACA AATTGGTCGA ACAGCCGCAA
     CCGTGCGCTC
901  AACCTGTTCG CCGGCATAGA ACACCGCTTC AACCAAGACT
     GGAAACTCAA
951  AGCCGAATAC GACTACACCC GTAGCCGCTT CCGCCAGCCC
     TACGGTGTGG
1001 CAGGCGTACT TTCCATCGAC CACAGCACTG CCGCCACCGA
     CCTGATTCCC
1051 GGTTATTGGC ACGCcgatcc GCGCACCCAC AGCGCCAGCA
     TGTCATTGAC
1101 CGGCAAATAC CgcctGTTCG GCCGCGAGCA CGATTTAATC
     GCGGGTATCA
1151 ACGGCTACAA ATACGCCAGC AACAAATACG GCGAACGCAG
     CATCATTCCC
1201 AACGCCATTC CCAACGCCTA CGAATTTTCC CGCACGGGCG
     CCTATCCGCA
1251 GCCATCATCG TTTGCCCAAA CCATCCCGCA ATACGACACC
     AGGCGGCAAA
1301 TCGGCGGCTA TCTCGCCACC CGTTTCCGCG CCGCCGACAA
     CCTTTCGCTG
1351 ATACTCGGCG GCAGATACAG CCGCTACCGC GCAGGCAGCT
     ACAACAGCCG
1401 CACACAAGGC ATGACCTATG TGTCCGCCAA CCGTTTCACC
     CCCTACACAG
1451 GCATCGTGTT CGATCTGACC GGCAACCTGT CGCTTTACGG
     CTCGTACAGC
1501 AGCCTGTTCG TCCCGCAATT GCAAAAAGAC GAACACGGCA
     GCTACCTGAA
1551 ACCCGTAACC GGCAACAATC TGGAAGCCGA CATCAAAGGC
     GAATGGCTTG
1601 AAGGGCGTCT GAACGCATCC GCCGCCGTGT ACCGCGCCCG
     TAAAAACAAC
1651 CTCGCCACCG CAGCAGGACG CGACCAGAGC GGCAACACCT
     ACTATCGCGC
1701 CGCCAACCAA GCCAAAACCC ACGGCTGGGA AATCGAAGTC
     GGCGGCCGCA
1751 TCACGCCCGA ATGGCAGATA CAGGCAGGCT ACAGCCAAAG
     CAAACCCCGC
1801 GACCAAGACG GCAGCCGCCT GAACCCCGAC AGCGTAcCCG
     AACGCAGCTT
1851 CAAACTCTTC ACCGCCTACC ACTTAGCCCC CGAAGCCCCC
     AGCGGCCGGA
1901 CCATcggTGC GGGTGTGCGC CGGCAGGGCG AAACCCACAC
     CGACCCAGCC
1951 GCGCTCCGCA TCCCCAACCC CGCCGCCAAA GCCCGCGCCG
     TCGCCAACAG
2001 CCGCCAGAAA GCCTACGCCG TCGCCGACAT CATGGCGCGT
     TACCGCTTCA
2051 ATCCGCGCAC CGAACTGTCG CTGAACGTGG ACAACCTGTT
     CAACAAACAC
2101 TACCGCACCC AGCCCGACCG CCACAGCTAC GGCGCACTGC
     GGACAGTGAA
2151 CGCGGCGTTT ACCTATCGGT TTAAATAA

This corresponds to the amino acid sequence <SEQ ID 672; ORF23ng-1>:

1   MTRFKYSLLF AALLPVYAQA DVSVSDDPKP QESTELPTIT
    VTADRTASSN
51  DGYTVSGTHT PFGLPMTLRE IPQSVSVITS QQMRDQNIKT
    LDRALLQATG
101 TSRQIYGSDR AGYNYLFARG SRIANYQING IPVADALADT
    GNANTAAYER
151 VEVVRGVAGL PDGTGEPSAT VNLVRKHPTR KPLFEVRAEA
    GNRKHFGLGA
201 DVSGSLNAEG TLRGRLVSTF GRGDSWRQLE RSRDAELYGI
    LEYDIAPQTR
251 VHAGMDYQQA KETADAPLSY AVYDSQGYAT AFGPKDNPAT
    NWSNSRNRAL
301 NLFAGIEHRF NQDWKLKAEY DYTRSRFRQP YGVAGVLSID
    HSTAATDLIP
351 GYWHADPRTH SASMSLTGKY RLFGREHDLI AGINGYKYAS
    NKYGERSIIP
401 NAIPNAYEFS RTGAYPQPSS FAQTIPQYDT RRQIGGYLAT
    RFRAADNLSL
451 ILGGRYSRYR AGSYNSRTQG MTYVSANRFT PYTGIVFDLT
    GNLSLYGSYS
501 SLFVPQLQKD EHGSYLKPVT GNNLEADIKG EWLEGRLNAS
    AAVYRARKNN
551 LATAAGRDQS GNTYYRAANQ AKTHGWEIEV GGRITPEWQI
    QAGYSQSKPR
601 DQDGSRLNPD SVPERSFKLF TAYHLAPEAP SGRTIGAGVR
    RQGETHTDPA
651 ALRIPNPAAK ARAVANSRQK AYAVADIMAR YRFNPRTELS
    LNVDNLFNKH
701 YRTQPDRHSY GALRTVNAAF TYRFK*

ORF23ng-1 and ORF23-1 show 95.9% identity in 725 aa overlap:

In addition, ORF23ng-1 shows significant homology with an OMP from E. coli:

sp|P16869|FHUE_ECOLI OUTER-MEMBRANE RECEPTOR FOR FE(III)-COPROGEN,
FE(III)-FERRIOXAMINE B AND FE(III)-RHODOTRULIC ACID PRECURSOR
>gi|1651542|gnl|PID|d1015403
(D90745) Outer membrane protein FhuE precursor [Escherichia coli]
>gi|1651545|gnl|PID|d1015405 (D90746) Outer membrane protein
FhuE precursor [Escherichia coli] >gi|1787344 (AE000210)
outer-membrane receptor for Fe(III)-
coprogen, Fe(III)-ferrioxamine B and Fe(III)-rhodotrulic acid precursor
[Escherichia coli] Length = 729
Score = 332 bits (843), Expect = 3e−90
Identities = 228/717 (31%), Positives = 350/717 (48%),
Gaps = 60/717 (8%)
Query:	38	TITVTADRTASSN--DGYTVSGTHTPFGLPMTLREIPQSVSVITSQQMRDQNIKTLDRAL	95
		T+ V    TA  +  + Y+V+ T     + MT R+IPQSV++++ Q+M DQ ++TL   +
Sbjct:	43	TVIVEGSATAPDDGENDYSVTSTSAGTKMQMTQRDIPQSVTIVSQQRMEDQQLQTLGEVM	102
Query:	96	LQATGTSRQIYGSDRAGYNYLFARGSRIANYQINGIP--------VADALADTGNANTAA	147
		G S+    SDRA Y   ++RG +I NY ++GIP        + DAL+D      A
Sbjct:	103	ENTLGISKSQADSDRALY---YSRGFQIDNYMVDGIPTYFESRWNLGDALSDM-----AL	154
Query:	148	YERVEVVRGVAGLPDGTGEPSATVNLVRKHPTRKPLF-EVRAEAGNRKHFGLGADVSGSL	206
		+ERVEVVRG  GL  GTG PSA +N+VRKH T +    +V AE G+       AD+   L
Sbjct:	155	FERVEVVRGATGLMTGTGNPSAAINMVRKHATSREFKGDVSAEYGSWNKERYVADLQSPL	214
Query:	207	NAEGTLRGRLVSTFGRGDSWRQLERSRDAELYGILEYDIAPQTRVHAGMDYQQAKETADA	266
		+G +R R+V  +   DSW     S      GI++ D+   T + AG +YQ+    +
Sbjct:	215	TEDGKIRARIVGGYQNNDSWLDRYNSEKTFFSGIVDADLGDLTTLSAGYEYQRIDVNSPT	274
Query:	267	PLSYAVYDSQGYATAFGPKDNPATNWSNSRNRALNLFAGIEHRFNQDWKLKAEYDYTRSR	326
		+++ G + ++    + A +W+ +      +F  ++ +F   W+      ++
Sbjct:	275	WGGLPRWNTDGSSNSYDRARSTAPDWAYNDKEINKVFMTLKQQFADTWQATLNATHSEVE	334
Query:	327	F--RQPYGVAGVLSIDHSTAA--TDLIPGY-------WHADPRTHSA-SMSLTGKYRLFG	374
		F  +  Y  A V   D       ++  PG+       W++  R   A  +   G Y LFG
Sbjct:	335	FDSKMMYVDAYVNKADGMLVGPYSNYGPGFDYVGGTGWNSGKRKVDALDLFADGSYELFG	394
Query:	375	REHDLIAGINGYKYASNKYGER--SIIPNAIPNAYEFSRTGAYPQPSSFAQTIPQYDTRR	432
		R+H+L+ G   Y   +N+Y     +I P+ I + Y F+  G +PQ     Q++ Q DT
Sbjct:	395	RQHNLMFG-GSYSKQNNRYFSSWANIFPDEIGSFYNFN--GNFPQTDWSPQSLAQDDTTH	451
Query:	433	QIGGYLATRFRAADNLSLILGGRYSRYRAGSYNSRTQGMTY-VSANRFTPYTGIVFDXXX	491
		Y ATR   AD L LILG RY+ +R  +       +TY +  N  TPY G+VFD
Sbjct:	452	MKSLYAATRVTLADPLHLILGARYTNWRVDT-------LTYSMEKNHTTPYAGLVFDIND	504
Query:	492	XXXXXXXXXXXFVPQLQKDEHGSYLKPVTGNNLEADIKGEWLEGRLNASAAVYRARKNNL	551
		F PQ  +D  G YL P+TGNN E  +K +W+  RL  + A++R  ++N+
Sbjct:	505	NWSTYASYTSIFQPQNDRDSSGKYLAPITGNNYELGLKSDWMNSRLTTTLAIFRIEQDNV	564
Query:	552	ATAAGR---DQSGNTYYRAANQAKTHGWEIEVGGRITPEWQIQAGYSQSKPRDQDGSRLN	608
		A + G      +G T Y+A +   + G E E+ G IT  WQ+  G ++    D +G+ +N
Sbjct:	565	AQSTGTPIPGSNGETAYKAVDGTVSKGVEFELNGAITDNWQLTFGATRYIAEDNEGNAVN	624
Query:	609	PDSVPERSFKLFTAYHLAPEAPSGRTIGAGVRRQGETHTDPAALRIPNPAAKARAVANSR	668
		P ++P  + K+FT+Y L P  P   T+G GV  Q   +TD        P    RA
Sbjct:	625	P-NLPRTTVKMFTSYRL-PVMPE-LTVGGGVNWQNRVYTDTV-----TPYGTFRA----E	672
Query:	669	QKAYAVADIMARYRFNPRTELSLNVDNLFNKHYRTQPDRH-SYGALRTVNAAFTYRF	724
		Q +YA+ D+  RY+      L  NV+NLF+K Y T  +    YG  R  +   TY+F
Sbjct:	673	QGSYALVDLFTRYQVTKNFSLQGNVNNLFDKTYDTNVEGSIVYGTPRNFSITGTYQF	729

Based on this analysis, it was predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF23-1 (77.5 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 15A shows the results of affinity purification of the His-fusion protein, and FIG. 15B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot (FIG. 15C) and for ELISA (positive result). These experiments confirm that ORF23-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 80

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 673>:

1   ATGCGCACGG CAGTGGTTTT GCTGTTGATC ATGCCGATGG
    CGGCTTCGTC
51  GGCAATGATG CCGGAAATGG TGTGCGCGGG CGTGTCGCCG
    GGAACGGCAA
101 TCATATCCAA GCCGACCGAA CAAACGGCGG TCATGGCTTC
    GAGTTTGTCC
151 AGCGTCAgcA CGCCTGCTTC GGCGgcGgCa ATCATACCTT
    CGTCTTCGGA
201 AACGGGGATA AACGcGCCAC TCAAACCCCC GACCGCGCTG
    GAAGCCATCA
251 TGCCGCCTTT TTTCACGGCA TCGTTCAGCA ATGCCAAAGC
    TGCTGTTGTG
301 CCGTGCGTAC CGCAGACGCT CAAGCCCATT TnTTCAAGAA
    TGCGTGCCAC
351 TnAGTCGCCG ACGGGG..

This corresponds to the amino acid sequence <SEQ ID 674; ORF24>:

1   MRTAVVLLLI MPMAASSAMM PEMVCAGVSP GTAIISKPTE
    QTAVMASSLS
51  SVSTPASAAA IIPSSSETGI NAPLKPPTAL EAIMPPFFTA
    SFSNAKAAVV
101 PCVPQTLKPI XSRMRATXSP TG..

Further work revealed the complete nucleotide sequence <SEQ ID 675>:

1   ATGCGCACGG CAGTGGTTTT GCTGTTGATC ATGCCGATGG
    CGGCTTCGTC
51  GGCAATGATG CCGGAAATGG TGTGCGCGGG CGTGTCGCCG
    GGAACGGCAA
101 TCATATCCAA GCCGACCGAA CAAACGGCGG TCATGGCTTC
    GAGTTTGTCC
151 AGCGTCAGCA CGCCTGCTTC GGCGGCGGCA ATCATACCTT
    CGTCTTCGGA
201 AACGGGGATA AACGCGCCAC TCAAACCCCC GACCGCGCTG
    GAAGCCATCA
251 TGCCGCCTTT TTTCACGGCA TCGTTCAGCA ATGCCAAAGC
    TGCTGTTGTG
301 CCGTGCGTAC CGCAGACGCT CAAGCCCATT TCTTCAAGAA
    TGCGTGCCAC
351 TGAGTCGCCG ACGGCGGGGG TCGGCGCCAG CGACAAGTCG
    AGAATACCAA
401 ACGGGATATT CAGCATTTTT GAGGCTTCGC GGCCGATGAG
    TTCGCCCACG
451 CGGGTAATTT TGAAAGCAGT TTTCTTCACT ACTTCCGCAA
    CTTCGGTCAA
501 TGTCGTTGCA TCTGAATTTT CCAACGCGGC TTTTACGACA
    CCTGGGCCGG
551 ATACGCCGAC ATTGATAACG GCATCCGCTT CGCCCGAACC
    ATGAAACGCG
601 CCCGCCATAA ACGGGTTGTC TTCCACCGCG TTGCAGAACA
    CGACAATTTT
651 AGCGCAGCCG AAACCTTCGG GCGTGATTTC CGCCGTGCGT
    TTGACGGTTT
701 CGCCCGCCAG CTTGACCGCA TCCATATTGA TACCGGCACG
    CGTACTGCCG
751 ATATTGATGG AGCTGCACAC AATATCGGTA GTCTTCATCG
    CTTCGGGAAT
801 GGAGCGGATT AACACCTCAT CCGAAGGCGA CATCCCTTTT
    TGCACCAACG
851 CGGAAAAACC GCCGATAAAA GACACACCGA TGGCTTTGGC
    AGCTTTATCC
901 AAAGTTTGCG CCACGCTGAC GTAA

This corresponds to the amino acid sequence <SEQ ID 676; ORF24-1>:

1   MRTAVVLLLI MPMAASSAMM PEMVCAGVSP GTAIISKPTE
    QTAVMASSLS
51  SVSTPASAAA IIPSSSETGI NAPLKPPTAL EAIMPPFFTA
    SFSNAKAAVV
101 PCVPQTLKPI SSRMRATESP TAGVGASDKS RIPNGIFSIF
    EASRPMSSPT
151 RVILKAVFFT TSATSVNVVA SEFSNAAFTT PGPDTPTLIT
    ASASPEP*NA
201 PAINGLSSTA LQNTTILAQP KPSGVISAVR LTVSPASLTA
    SILIPARVLP
251 ILMELHTISV VFIASGMERI NTSSEGDIPF CTNAEKPPIK
    DTPMALAALS
301 KVCATLT*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF24 shows 96.4% identity over a 307 aa overlap with an ORF (ORF24a) from strain A of N. meningitidis:

The complete length ORF24a nucleotide sequence <SEQ ID 677> is:

1   ATGCGCACGG CAGTGGTTTT GCTGTTGATC ATGCCGATGG
    CGGCTTCGTC
51  GGCAATGATG CCGGAAATGG TGTGCGCGGG TGTGTCGCCG
    GGAACGGCAA
101 TCATATCCAA NCCGACCGAA CAAACGGCGG TCATCGCTTC
    GAGTTTATCC
151 AACGTCAGCA CGCCTGCTTC GGCGGCGGCA ATCATACCTT
    CGTCTTCGGA
201 NACGGGGATA AACGCGCCAC TCAAACCGCC AACCGCGCTC
    GAAGCCATCA
251 TGCCGCCCTT TTTCACGGCA TCGTTCAGCA ATGCCAAAGC
    TGCTGTTGTG
301 CCGTGCGTAC CGCAGACGCT CAAACCCATT TCTTCAAGAA
    TGCGCGCCAC
351 CGAGTCGCCG ACGGCAGGGG TCGGTGCCAG CGACAAGTCG
    AGAATACCAA
401 ACGGGATATT CAGCATTTTT GAGGCTTCGC GGCCGATGAG
    TTCGCCCACG
451 CGGGTAATTT TGAAGGCGGT TTTCTTCACA ACTTCGGCAA
    CTTCGGTCAA
501 TGTCGTTGCA TCCGAATTTT CCAACGCGGC TTTTACGACA
    CCCGGGCCGG
551 ATACGCCGAC ATTAATCACA GCATCCGCTT CGCCTGAGCC
    GTGAAACGCG
601 CCCGCCATAN ACGGGTTGTC TTCCNCCGCG TTGCAGAACA
    CGACGATTTT
651 GGCGCAGCCG AAACCTTCTA GTGTGATTTC ANCCGTGCGT
    TTGATGGTTT
701 CGCCCGCCAG TCTGACCGCG TCCATATTGA TACCGGCGCG
    CGTACTGCCG
751 ATATTGATGG AGCTGCACAC GATATCAGTA GTCTTCATCG
    CTTCGGGAAT
801 GGAACGGATN AACACCTCGT CAGAAGGCGA CATACCTTTT
    TGCACCAGCG
851 CGGAAAAGCC GCCAATAAAA GACACGCCGA TGGCTTTGGC
    AGCCTTATCC
901 AAAGTTTGCG CCACGCTGAC GTAA

This encodes a protein having amino acid sequence <SEQ ID 678>:

1   MRTAVVLLLI MPMAASSAMM PEMVCAGVSP GTAIISXPTE
    QTAVIASSLS
51  NVSTPASAAA IIPSSSXTGI NAPLKPPTAL EAIMPPFFTA
    SFSNAKAAVV
101 PCVPQTLKPI SSRMRATESP TAGVGASDKS RIPNGIFSIF
    EASRPMSSPT
151 RVILKAVFFT TSATSVNVVA SEFSNAAFTT PGPDTPTLIT
    ASASPEP*NA
201 PAIXGLSSXA LQNTTILAQP KPSSVISXVR LMVSPASLTA
    SILIPARVLP
251 ILMELHTISV VFIASGMERX NTSSEGDIPF CTSAEKPPIK
    DTPMALAALS
301 KVCATLT*

It should be noted that this protein includes a stop codon at position 198.

ORF24a and ORF24-1 show 96.4% identity in 307 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF24 shows 96.7% identity over a 121 aa overlap with a predicted ORF (ORF24ng) from N. gonorrhoeae:

The complete length ORF24ng nucleotide sequence <SEQ ID 679> is:

1   ATGCGCACGG CGGTGGTTTT GCTGTTGATC ATGCCGATGG
    CGGCTTCGTC
51  GGCGATGATG CCGGAAATGG TGTGCGCGGG CGTGTCGCCG
    GGAACGGCAA
101 TCATGTCCAA ACCAACGGAG CAGACGGCGG TCATGGCTTC
    GAGTTTGTCC
151 AGCGTCAACA CGCCTGCCTC GGCGGCGGCA ATCATACCTT
    CGTCTTCGGA
201 AACGGGGATA AACGCGCCGC TCAAACCGCC GACCGCGCTG
    GAAGCCATCA
251 TGCCGCCCTT TTTCACGGCA TCGTTCAGCA ATGCCAAAGC
    TGCTGTTGTG
301 CCGTGCGTAC CGCAGACGCT CAAGCCCATT TCTTCAAGAA
    TGCGCGCCAC
351 CGAGTCGCCG ACGGCGGGGG TCGGTGCCAG CGACAAATCG
    AGAATGCCGA
401 ACGGGATATT CAGCATTTTT GAGGCTTCGC GACCGATGAG
    TTCGCCCACG
451 CGGGTGATTT TGAAAGCGGT TTTCTTCACG ACTTCGGCGA
    CCTCGGTCAG
501 GCTGACCGCG TCCGAATTTT CCAGCGCGGC TTTGACCACG
    CCTGGACCGG
551 ATACGCCGAC ATTAATCACA GCATCCGCTT CGCCCGAGCC
    GTGGAACGCA
601 CCCGCCATAA ACGGATTGTC TTCCACCGCG TTGCAGAACA
    CGACGATTTT
651 GGCGCAGCCG AAACCTTCGG GTGTGATTTC AGCCGTGCGT
    TTGATGGTTT
701 CGCCTGCCAG CTTGACCGCA TCCATATTGA TACCGGCACG
    CGTGCTGCCG
751 ATATTGATGG AGCTGCACAC GATATCGGTA GTTTTCATCG
    CTTCGGGAAC
801 GGAACGGATC AACACCTCAT CCGAAGGCGA CATACCTTTT
    TGCACCAGCG
851 CGGAAAAGCC GCCGATAAAG GACACGCCGA TGGCTTTGGC
    TGCCTTGTCC
901 AAAGTCTGCG CCACGCTGAC ATAA

This encodes a protein having amino acid sequence <SEQ ID 680>:

1   MRTAVVLLLI MPMAASSAMM PEMVCAGVSP GTAIMSKPTE
    QTAVMASSLS
51  SVNTPASAAA IIPSSSETGI NAPLKPPTAL EAIMPPFFTA
    SFSNAKAAVV
101 PCVPQTLKPI SSRMRATESP TAGVGASDKS RMPNGIFSIF
    EASRPMSSPT
151 RVILKAVFFT TSATSVRLTA SEFSSAALTT PGPDTPTLIT
    ASASPEPWNA
201 PAINGLSSTA LQNTTILAQP KPSGVISAVR LMVSPASLTA
    SILIPARVLP
251 ILMELHTISV VFIASGTERI NTSSEGDIPF CTSAEKPPIK
    DTPMALAALS
301 KVCATLT*

ORF24ng and ORF24-1 show 96.1% identity in 307 aa overlap:

Based on this analysis, including the presence of a putative leader sequence (first 18 aa—double-underlined) and putative transmembrane domains (single-underlined) in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 81

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 681>:

1   ..ACCGACGTGC AAAAAGAGTT GGTCGGCGAA CAACGCAAGT
    GGGCGCAGGA
51  AAAAATCAGC AACTGCCGAC AAGCCGCCGC GCAGGCAGAC
    CGGCAGGAAT
101 ACGCCGAATA CCTCAAGCTG CAATGCGACA CGCGGATGAC
    GCGCGAACGG
151 ATACAGTATC TTCGCGGCTA TTCCATCGAT TAG

This corresponds to the amino acid sequence <SEQ ID 682; ORF25>:

• • 1 . . . TDVQKELVGE QRKWAQEKIS NCRQAAAQAD RQEYAEYLKL QCDTRMTRER
  • 51 IQYLRGYSID *

Further work revealed the complete nucleotide sequence <SEQ ID 683>:

1    ATGTATCGGA AACTCATTGC GCTGCCGTTT GCCCTGCTGC
     TTGCCGCTTG
51   CGGCAGGGAA GAACCGCCCA AGGCATTGGA ATGCGCCAAC
     CCCGCCGTGT
101  TGCAAGGCAT ACGCGGCAAT ATTCAGGAAA CGCTCACGCA
     GGAAGCGCGT
151  TCTTTCGCGC GCGAAGACGG CAGGCAGTTT GTCGATGCCG
     ACAAAATTAT
201  CGCCGCCGCC TACGGTTTGG CGTTTTCTTT GGAACACGCT
     TCGGAAACGC
251  AGGAAGGCGG GCGCACGTTC TGTATCGCCG ATTTGAACAT
     TACCGTGCCG
301  TCTGAAACGC TTGCCGATGC CAAGGCAAAC AGCCCCCTGT
     TGTACGGGGA
351  AACTGCTTTG TCGGATATTG TGCGGCAGAA GACGGGCGGC
     AATGTCGAGT
401  TTAAAGACGG CGTATTGACG GCAGCCGTCC GCTTCCTGCC
     CGTCAAAGAC
451  GGTCAGACGG CATTTGTCGA CAACACGGTC GGTATGGCGG
     CGCAAACGCT
501  GTCTGCCGCG CTGCTGCCTT ACGGCGTGAA GAGCATCGTG
     ATGATAGACG
551  GCAAGGCGGT GAAAAAAGAA GACGCGGTCA GGATTTTGAG
     CGGAAAAGCC
601  CGTGAAGAAG AACCGTCCAA ACCCACGCCC GAAGACATTT
     TGGAACACAA
651  TGCCGCCGGC GGCGATGCGG GCGTACCCCA AGCCGCAGAA
     GGCGCGCCCG
701  AACCGGAAAT CCTGCATCCT GACGACGGCG AGCGTGCCGA
     TACCGTTACC
751  GTATCACGGG GCGAAGTGGA AGAGGCGCGC GTACAAAACC
     AGCGTGCGGA
801  ATCCGAAATT ACCAAACTTT GGGGAGGACT CGATACCGAC
     GTGCAAAAAG
851  AGTTGGTCGG CGAACAACGC AAGTGGGCGC AGGAAAAAAT
     CAGCAACTGC
901  CGACAAGCCG CCGCGCAGGC AGACCGGCAG GAATACGCCG
     AATACCTCAA
951  GCTGCAATGC GACACGCGGA TGACGCGCGA ACGGATACAG
     TATCTTCGCG
1001 GCTATTCCAT CGATTAG

This corresponds to the amino acid sequence <SEQ ID 684; ORF25-1>:

1   MYRKLIALPF ALLLAACGRE EPPKALECAN PAVLQGIRGN
    IQETLTQEAR
51  SFAREDGRQF VDADKIIAAA YGLAFSLEHA SETQEGGRTF
    CIADLNITVP
101 SETLADAKAN SPLLYGETAL SDIVRQKTGG NVEFKDGVLT
    AAVRFLPVKD
151 GQTAFVDNTV GMAAQTLSAA LLPYGVKSIV MIDGKAVKKE
    DAVRILSGKA
201 REEEPSKPTP EDILEHNAAG GDAGVPQAAE GAPEPEILHP
    DDGERADTVT
251 VSRGEVEEAR VQNQRAESEI TKLWGGLDTD VQKELVGEQR
    KWAQEKISNC
301 RQAAAQADRQ EYAEYLKLQC DTRMTRERIQ YLRGYSID*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF25 shows 98.3% identity over a 60aa overlap with an ORF (ORF25a) from strain A of N. meningitidis:

The complete length ORF25a nucleotide sequence <SEQ ID 685> is:

1    ATGTATCGGA AACTCATTGC GCTGCCGTTT GCCCTGCTGC
     TTGCCGCTTG
51   CGGCAGGGAA GAACCGCCCA AGGCATTGGA ATGCGCCAAC
     CCCGCCGTGT
101  TGCAANGCAT ACGCNGCAAT ATTCAGGAAA CGCTCACGCA
     GGAAGCGCGT
151  TCTTTCGCGC GCGAAGACNG CANGCAGTTT GTCGATGCCG
     ACNAAATTAT
201  CGCCGCCGCC TANGNTNNGN NGNTNTCTTT GGAACACGCT
     TCGGAAACGC
251  AGGAAGGCGG GCGCACGTTC TGTNTCGCCG ATTTGAACAT
     TACCGTGCCG
301  TCTGAAACGC TTGCCGATGC CAAGGCAAAC AGCCCCCTGC
     TGTACGGGGA
351  AACCGCTTTG TCGGATATTG TGCGGCAGAA GACGGGCGGC
     AATGTCGAGT
401  TTAAAGACGG CGTATTGACG GCAGCCGTCC GCTTCCTACC
     CGTCAAAGAC
451  GGTCAGANGG CATTTGTCGA CAACACGGTC GGTATGGCGG
     CGCAAACGCT
501  GTCTGCCGCG TTGCTGCCTT ACGGCGTGAA GAGCATCGTG
     ATGATAGACG
551  GCAAGGCGGT AAAAAAAGAA GACGCGGTCA GGATTNTGAG
     CNGANAAGCC
601  CGTGAANAAG AACCGTCCAA ANCCNNGCCC GAAGACATTT
     TGGAACATAA
651  TGCCGCCGGA GGGGATGCAG ACGTACCCCA AGCCGGAGAA
     GACGCGCCCG
701  AACCGGAAAT CCTGCATCCT GACGACGGCG AGCGTGCCGA
     TACCGTTACC
751  GTATCACGGG GCGAAGTGGA AGAGGCGCGN GTACAAAACC
     AGCGTGCGGA
801  ATCCGAAATT ACCAAACTTT GGGGAGGACT CGATACCGAC
     GTGCAAAAAG
851  AGTTGGTCGG CGAANAACGC AAGTGGGCGC AGGAAAAAAT
     CAGCAACTGC
901  CGACAAGCCG CCGCGCAGGC AGACCGGCAG GAATACGCCG
     AATACCTCAA
951  GCTGCAATGC GACACGCGGA TGACGCGCGA ACGGATACAG
     TATCTTCGCG
1001 GCTATTCCAT CGATTAG

This encodes a protein having amino acid sequence <SEQ ID 686>:

1   MYRKLIALPF ALLLAACGRE EPPKALECAN PAVLQXIRXN
    IQETLTQEAR
51  SFAREDXXQF VDADXIIAAA XXXXXSLEHA SETQEGGRTF
    CXADLNITVP
101 SETLADAKAN SPLLYGETAL SDIVRQKTGG NVEFKDGVLT
    AAVRFLPVKD
151 GQXAFVDNTV GMAAQTLSAA LLPYGVKSIV MIDGKAVKKE
    DAVRIXSXXA
201 REXEPSKXXP EDILEHNAAG GDADVPQAGE DAPEPEILHP
    DDGERADTVT
251 VSRGEVEEAR VQNQRAESEI TKLWGGLDTD VQKELVGEXR
    KWAQEKISNC
301 RQAAAQADRQ EYAEYLKLQC DTRMTRERIQ YLRGYSID*

ORF25a and ORF25-1 show 93.5% identity in 338 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF25 shows 100% identity over a 60aa overlap with a predicted ORF (ORF25ng) from N. gonorrhoeae:

The complete length ORF25ng nucleotide sequence <SEQ ID 687> is:

1    ATGTATCGGA AACTCATTGC GCTGCCGTTT GCCCTGCTGC
     TTGCAGCGTG
51   CGGCAGGGAA GAACCGCCCA AGGCGTTGGA ATGCGCCAAC
     CCCGCCGTGT
101  TGCAGGACAT ACGCGGCAGT ATTCAGGAAA CGCTCACGCA
     GGAAGCGCGT
151  TCTTTCGCGC GCGAAGACGG CAGGCAGTTT GTCGATGCCG
     ACAAAATTAT
201  CGCCGCCGCC TACGGTTTGG CGTTTTCTTT GGAACACGCT
     TCGGAAACGC
251  AGGAAGGCGG GCGCACGTTC TGTATCGCCG ATTTGAACAT
     TACCGTGCCG
301  TCTGAAACGC TTGCCGATGC CGAGGCAAAC AGCCCCCTGC
     TGTATGGGGA
351  AACGTCTTTG GCAGACATCG TGCAGCAGAA GACGGGCGGC
     AATGTCGAGT
401  TTAAAGACGG CGTATTGACG GCAGCCGTCC GCTTCCTGCC
     CGCCAAAGAC
451  GCTCGGACGG CATTTATCGA CAACACGGTC GGTATGGCGA
     CGCAAACGCT
501  GTCTGCCGCG TTGCTGCCTT ACGGCGTGAA GAGCATCGTG
     ATGATAGACG
551  GCAAGGCGGT GACAAAAGAA GACGCGGTCA GGGTTTTGAG
     CGGCAAAGCC
601  CGTGAAGAAG AACCGTCCAA ACCCACCCCC GAAGACATTT
     TGGAACACAA
651  TGCCGCCGGC GGCGATGCGG GCGTACCCCA AGCCGCAGAA
     GGCGCACCCG
701  AACCCGAAAT CCTGCATCCC GACGACGTCG AGCGTGCCGA
     TACCGTTACC
751  GTATCACGGG GCGAAGTGGA AGAGGCGCGC GTACAAAACC
     AACGTGCGGA
801  ATCCGAAATT ACCAAACTTT GGGGAGGACT CGATACCGAC
     GTGCAAAAAG
851  AGTTGGTCGG CGAACAGCGC AAGTGGGCGC AGGAAAAAAT
     CAGcaactgc
901  cgACAAGCCG CCGCGCAGGC AGACCGGCAG GAATACGCCG
     AATACCTCAA
951  GCTCCAATGC GACACGCGGA TGACGCGCGA ACggaTACAG
     TATCTTCGCG
1001 GCTATTCCAT CGATTAG

This encodes a protein having amino acid sequence <SEQ ID 688>:

1   MYRKLIALPF ALLLAACGRE EPPKALECAN PAVLQDIRGS
    IQETLTQEAR
51  SFAREDGRQF VDADKIIAAA YGLAFSLEHA SETQEGGRTF
    CIADLNITVP
101 SETLADAEAN SPLLYGETSL ADIVQQKTGG NVEFKDGVLT
    AAVRFLPAKD
151 ARTAFIDNTV GMATQTLSAA LLPYGVKSIV MIDGKAVTKE
    DAVRVLSGKA
201 REEEPSKPTP EDILEHNAAG GDAGVPQAAE GAPEPEILHP
    DDVERADTVT
251 VSRGEVEEAR VQNQRAESEI TKLWGGLDTD VQKELVGEQR
    KWAQEKISNC
301 RQAAAQADRQ EYAEYLKLQC DTRMTRERIQ YLRGYSID*

ORF25ng and ORF25-1 show 95.9% identity in 338 aa overlap:

Based on this analysis, including the presence of a predicted prokaryotic membrane lipoprotein lipid attchment site (underlined) in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF25-1 (37 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 16A shows the results of affinity purification of the GST-fusion protein, and FIG. 16B shows the results of expression of the His-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot (FIG. 16C), ELISA (positive result), and FACS analysis (FIG. 16D). These experiments confirm that ORF25-1 is a surface-exposed protein, and that it is a useful immunogen.

FIG. 16E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF25-1.

Example 82

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 689>

1    ATGCAGCTGA TCGACTATTC ACATTCATTT TTCTCGGTTG
     TGCCACCCTT
51   TTTGGCACTG GCACTTGCCG TCATTACCCG CCGCGTACTG
     CTGTCTTTAG
101  GCATCGGTAT TCTGGwysGC GTTGCCTTTT TGGTCGGCGG
     CAACCCCGTC
151  GACGGTCTGA CACACCTGAA AGACATGGTC GTCGGCTTGG
     CTTGGTCAGA
201  CGsyGATTGG TCGCTGGGCA AACCAAAAAT CTTGGTTTTC
     CkGATACTTT
251  TGGGTATTTT TACTTCCCTG CTGACCTACT CCGGCAGCAA
     T.........
     //
851  .......... .......... .......... ........AC
     TTCGCTGGTA
901  TTCGGCGGCA CTTGCGGCGT CTTTGCCGTC GTTCTCTGCA
     CGCTCGGCAC
951  GATTAAAACC GCCGACTATC CCAAAGCCGT TTGGCAGGGT
     GCGAAATCTA
1001 TGTTCGGCGC AATCGCCATT TTAATCCTCG CTTGGCTCAT
     CAGTACGGTT
1051 GTCGGCGAAA TGCACACCGG CGATTACCTC TCCACACTGG
     TTGCGGGCAA
1101 CATCCATCCC GGCTTCCTGC CCGTCATCCT CTTCCTGCTC
     GCCAGCGTGA
1151 TGGCGTTTGC CACAGGCACA AGCTGGGGGA CGTTCGGCAT
     TATGCTGCCG
1201 ATTGCCGCCG CCATGGCGGT CAAAGTCGAA CCCGCGCTGA
     TTATCCCGTG
1251 TATGTCCGCA GTAATGGCGG GGGCGGTATG CGGCGACCAC
     TGCTCGCCCA
1301 TTTCCGACAC GACCATCCTG TCGTCCACCG GCGCGCGCTG
     CAACCACATC
1351 GACCACGTTA CCTCGCAACT GCCTTACGCC TTAACCGTTG
     CCGCCGCCGC
1401 CGCATCGGGC TACCTCGCAT TGGGTCTGAC AAAATCCGCG
     CTGTTGGGCT
1451 TTGGCACGAC AGGCATTGTA TTGGCGGTGC TGATTTTTCT
     GTTGAAAGAT
1501 AAAAAA..

This corresponds to the amino acid sequence <SEQ ID 690; ORF26>:

1   MQLIDYSHSF FSVVPPFLAL ALAVITRRVL LSLGIGILXX
    VAFLVGGNPV
51  DGLTHLKDMV VGLAWSDXDW SLGKPKILVF XILLGIFTSL
    LTYSGSN...
    //
251 .......... .......... .......... ..........
    ......TSLV
301 FGGTCGVFAV VLCTLGTIKT ADYPKAVWQG AKSMFGAIAI
    LILAWLISTV
351 VGEMHTGDYL STLVAGNIHP GFLPVILFLL ASVMAFATGT
    SWGTFGIMLP
401 IAAAMAVKVE PALIIPCMSA VMAGAVCGDH CSPISDTTIL
    SSTGARCNHI
451 DHVTSQLPYA LTVAAAAASG YLALGLTKSA LLGFGTTGIV
    LAVLIFLLKD
501 KK..

Further work revealed the complete nucleotide sequence <SEQ ID 691>:

1    ATGCAGCTGA TCGACTATTC ACATTCATTT TTCTCGGTTG
     TGCCACCCTT
51   TTTGGCACTG GCACTTGCCG TCATTACCCG CCGCGTACTG
     CTGTCTTTAG
101  GCATCGGTAT TCTGGTCGGC GTTGCCTTTT TGGTCGGCGG
     CAACCCCGTC
151  GACGGTCTGA CACACCTGAA AGACATGGTC GTCGGCTTGG
     CTTGGTCAGA
201  CGGCGATTGG TCGCTGGGCA AACCAAAAAT CTTGGTTTTC
     CTGATACTTT
251  TGGGTATTTT TACTTCCCTG CTGACCTACT CCGGCAGCAA
     TCAGGCGTTT
301  GCCGACTGGG CAAAACGGCA CATTAAAAAC CGGCGCGGCG
     CGAAAATGCT
351  GACCGCCTGC CTCGTGTTCG TAACCTTTAT CGACGACTAT
     TTCCACAGTC
401  TCGCCGTCGG TGCGATTGCC CGCCCCGTTA CCGACAAGTT
     TAAAGTTTCC
451  CGCACCAAAC TCGCCTACAT CCTCGACTCC ACTGCCGCTC
     CTATGTGCGT
501  GCTGATGCCC GTTTCAAGCT GGGGCGCGTC GATTATCGCC
     ACGCTTGCCG
551  GACTGCTCGT TACCTACAAA ATCACCGAAT ACACGCCGAT
     GGGGACGTTT
601  GTCGCCATGA GCCTGATGAA CTATTACGCA CTGTTTGCCC
     TGATTATGGT
651  GTTCGTCGTC GCATGGTTTT CCTTCGACAT CGGCTCGATG
     GCACGTTTCG
701  AACAAGCCGC GTTGAACGAA GCCCACGATG AAACTGCCGT
     TTCAGACGCT
751  ACCAAAGGTC GTGTTTACGC ACTGATTATT CCCGTTTTGG
     CCTTAATCGC
801  CTCAACGGTT TCCGCCATGA TCTACACCGG CGCGCAGGCA
     AGCGAAACCT
851  TCAGCATTTT GGGGGCATTT GAAAACACGG ACGTAAACAC
     TTCGCTGGTA
901  TTCGGCGGCA CTTGCGGCGT CCTTGCCGTC GTTCTCTGCA
     CGCTCGGCAC
951  GATTAAAACC GCCGACTATC CCAAAGCCGT TTGGCAGGGT
     GCGAAATCTA
1001 TGTTCGGCGC AATCGCCATT TTAATCCTCG CTTGGCTCAT
     CAGTACGGTT
1051 GTCGGCGAAA TGCACACCGG CGATTACCTC TCCACACTGG
     TTGCGGGCAA
1101 CATCCATCCC GGCTTCCTGC CCGTCATCCT CTTCCTGCTC
     GCCAGCGTGA
1151 TGGCGTTTGC CACAGGCACA AGCTGGGGGA CGTTCGGCAT
     TATGCTGCCG
1201 ATTGCCGCCG CCATGGCGGT CAAAGTCGAA CCCGCGCTGA
     TTATCCCGTG
1251 TATGTCCGCA GTAATGGCGG GGGCGGTATG CGGCGACCAC
     TGCTCGCCCA
1301 TTTCCGACAC GACCATCCTG TCGTCCACCG GCGCGCGCTG
     CAACCACATC
1351 GACCACGTTA CCTCGCAACT GCCTTACGCC TTAACCGTTG
     CCGCCGCCGC
1401 CGCATCGGGC TACCTCGCAT TGGGTCTGAC AAAATCCGCG
     CTGTTGGGCT
1451 TTGGCACGAC AGGCATTGTA TTGGCGGTGC TGATTTTTCT
     GTTGAAAGAT
1501 AAAAAACGCG CCAACGCCTG A

This corresponds to the amino acid sequence <SEQ ID 692; ORF26-1>:

1   MQLIDYSHSF FSVVPPFLAL ALAVITRRVL LSLGIGILVG
    VAFLVGGNPV
51  DGLTHLKDMV VGLAWSDGDW SLGKPKILVF LILLGIFTSL
    LTYSGSNQAF
101 ADWAKRHIKN RRGAKMLTAC LVFVTFIDDY FHSLAVGAIA
    RPVTDKFKVS
151 RTKLAYILDS TAAPMCVLMP VSSWGASIIA TLAGLLVTYK
    ITEYTPMGTF
201 VAMSLMNYYA LFALIMVFVV AWFSFDIGSM ARFEQAALNE
    AHDETAVSDA
251 TKGRVYALII PVLALIASTV SAMIYTGAQA SETFSILGAF
    ENTDVNTSLV
301 FGGTCGVLAV VLCTLGTIKT ADYPKAVWQG AKSMFGAIAI
    LILAWLISTV
351 VGEMHTGDYL STLVAGNIHP GFLPVILFLL ASVMAFATGT
    SWGTFGIMLP
401 IAAAMAVKVE PALIIPCMSA VMAGAVCGDH CSPISDTTIL
    SSTGARCNHI
451 DHVTSQLPYA LTVAAAAASG YLALGLTKSA LLGFGTTGIV
    LAVLIFLLKD
501 KKRANA*

Computer analysis of this amino acid sequence gave the following results:

Homology with the Hypothetical Transmembrane Protein HI1586 of H. influenzae (Accession Number P44263)

ORF26 and HI1586 show 53% and 49% amino acid identity in 97 and 221 aa overlap at the N-terminus and C-terminus, respectively:

Orf26	1	MQLIDYSHSFFSVVPPFLALALAVITRRVXXXXXXXXXXXVAFLVGGNPVDGLTHLKDMV	60
		M+LID+S S +S+VP  LA+ LA+ TRRV              L          +L   V
HI1586	14	MELIDFSSSVWSIVPALLAIILAIATRRVLVSLSAGIIIGSLMLSDWQIGSAFNYLVKNV	73
Orf26	61	VGLAWSDXDWSLGKPKILVFXILLGIFTSLLTYSGSN	97
		V L ++D + +     I++F +LLG+ T+LLT SGSN
HI1586	74	VSLVYADGEIN-SNMNIVLFLLLLGVLTALLTVSGSN	109
		//
Orf26	86	IFTSLLTYSGS--NTSLVFGGTCGVFAVVLCTL--GTIKTADYPKAVWQGAKSMFGXXXX	141
		+F+ L T+  +   TSLV GG C +    L  +    +   +Y ++   G KSM G
HI1586	299	VFSVLGTFENTVVGTSLVVGGFCSIIISTLLIILDRQVSVPEYVRSWIVGIKSMSGAIAI	358
Orf26	142	XXXXXXXSTVVGEMHTGDYLSTLVAGNIHPGFLPVILFLLASVMAFATGTSWGTFGIMLP	201
		+ +VG+M TG YLS+LV+GNI   FLPVILF+L + MAF+TGTSWGTFGIMLP
HI1586	359	LFFAWTINKIVGDMQTGKYLSSLVSGNIPMQFLPVILFVLGAAMAFSTGTSWGTFGIMLP	418
Orf26	202	IAAAMAVKVEPALIIPCMSAVMAGAVCGDHCSPISDTTILSSTGARCNHIDHVTSQXXXX	261
		IAAAMA    P L++PC+SAVMAGAVCGDHCSP+SDTTILSSTGA+CNHIDHVT+Q
HI1586	419	IAAAMAANAAPELLLPCLSAVMAGAVCGDHCSPVSDTTILSSTGAKCNHIDHVTTQLPYA	478
Orf26	262	XXXXXXXXXXXXXXXXXKSALLGFGTTGIVLAVLIFLLKDK	302
		S L GF  T + L V+IF +K +
HI1586	479	ATVATATSIGYIVVGFTYSGLAGFAATAVSLIVIIFAVKKR	519

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF26 shows 58.2% identity over a 502aa overlap with an ORF (ORF26a) from strain A of N. meningitidis:

The complete length ORF26a nucleotide sequence <SEQ ID 693> is:

1    ATGCAGCTGA TCGACTATTC ACATTCATTT TTCTCGGTTG
     TGCCACCCTT
51   TTTGGCACTG GCACTTGCCG TCATTACCCG CCGCGTACTG
     CTGTCTTTAG
101  GCATCGGTAT TCTGGTCGGC GTTGCCTTTT TGGTCGGCGG
     CAACCCCGTC
151  GACGGTCTGA CACACCTGAA AGACATGGTC GTCGGCTTGG
     CTTGGTCAGA
201  CGGCGATTGG TCGCTGGGCA AACCAAAANT CTTGGTTTTC
     CTGATACTTT
251  TGGGTATTTT TACTTCCCTG CTGACCTACT CCGGCAGCAA
     TCAGGCGTTT
301  GCCGACTGGG CAAAACGGCA CATTAAAAAC CGGCGCGGCG
     CGAAAATGCT
351  GACCGCCTGC CTCGTGTTCG TAACCTTTAT CGACGACTAT
     TTCCACAGTC
401  TCGCCGTCGG TGCGNTTGCC CGCCCCGTTA CCGACAAGTT
     TAAAGTTTCC
451  CGCGCCAAAC TCGCCTACAT CCTCGACTCC ACTGCCGCGC
     CTATGTGCGT
501  GCTGATGCCC GTTTCAAGCT GGGGCGCGTC GATTATCGCC
     ACGCTTGCCG
551  GACTGCTCGT TACCTACAAA ATCACCGAAT ACACGCCGAT
     GGGGACGTTT
601  GTCGCCATGA GCCTGATGAA CTATTACGCA CTGTTTGCCC
     TGATTATGGT
651  GTTCGTCGTC GCATGGTTCT CCTTCGACAT CGGCTCGATG
     GCACGTTTCG
701  AACAAGCCGC GTTGAACGAA GCCCACGATG AAACTGCCGT
     TTCAGACGGC
751  AGCTGGGGCA GGGTTTACGC ATTGATTATT CCCGTTTTGG
     CCTTAATCGC
801  CTCAACGGTT TCCGCCATGA TCTACACCGG TGCACAGGCA
     AGCGAAACCT
851  TCAGCATTTT GGGTGCATTT GAAAATACGG ACGTGAACAC
     TTCGCTGGTA
901  TTCGGCGGCA CTTGCGGCGT GCTTGCCGTC GTCCTCTGCA
     CGCTCGGCAC
951  GATTAAAATC GCCGATTATC CCAAAGCCGT TTGGCAGGGT
     GCGAAATCCA
1001 TGTTCGGCGC AATCGCCATT TTAATCCTTG CCTGGCTCAT
     CAGTACGGTT
1051 GTCGGCGAAA TGCACACAGG CGACTACCTC TCCACGCTGG
     TTGCGGGCAA
1101 CATCCATCCC GGCTTCCTGN CCGTCATCCT TTTCCTGCTC
     GCCAGCGTGA
1151 TGGCGTTTGC CACAGGCACA AGCTGGGGGA CGTTCGGCAT
     CATGCTGCCG
1201 ATTGCCGCCG CCATGGCGGT CAAAGTCGAT CCCTCACTGA
     TTATCCCGTG
1251 TATGTCCGCC GTGATGGCGG GGGCGGTATG CGGCGACCAC
     TGCTCGCCCA
1301 TTTCCGACAC GACCATCCTG TCGTCCACCG GCGCGCGCTG
     CAACCACATC
1351 GACCACGTTA CNTCGCAACT GCCTTACGCC TTAACCGTTG
     CCGCCGCCGC
1401 CGCATCGGGN TACCTCGCAT TGGGTCTGAC AAAATCCGCG
     CTGTTGGGTT
1451 TTGGCANGAC AGGCATTGTA TTGGCGGTGC TGATTTTTCT
     GTTGAAAGAT
1501 AAAAAACGCG CCAACGCCTG A

This encodes a protein having amino acid sequence <SEQ ID 694>:

1   MQLIDYSHSF FSVVPPFLAL ALAVITRRVL LSLGIGILVG
    VAFLVGGNPV
51  DGLTHLKDMV VGLAWSDGDW SLGKPKXLVF LILLGIFTSL
    LTYSGSNQAF
101 ADWAKRHIKN RRGAKMLTAC LVFVTFIDDY FHSLAVGAXA
    RPVTDKFKVS
151 RAKLAYILDS TAAPMCVLMP VSSWGASIIA TLAGLLVTYK
    ITEYTPMGTF
201 VAMSLMNYYA LFALIMVFVV AWFSFDIGSM ARFEQAALNE
    AHDETAVSDG
251 SWGRVYALII PVLALIASTV SAMIYTGAQA SETFSILGAF
    ENTDVNTSLV
301 FGGTCGVLAV VLCTLGTIKI ADYPKAVWQG AKSMFGAIAI
    LILAWLISTV
351 VGEMHTGDYL STLVAGNIHP GFLXVILFLL ASVMAFATGT
    SWGTFGIMLP
401 IAAAMAVKVD PSLIIPCMSA VMAGAVCGDH CSPISDTTIL
    SSTGARCNHI
451 DHVTSQLPYA LTVAAAAASG YLALGLTKSA LLGFGXTGIV
    LAVLIFLLKD
501 KKRANA*

ORF26a and ORF26-1 show 97.8% identity in 506 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF26 shows 94.8% and 99% identity in 97 and 206 aa overlap at the N-terminus and C-terminus, respectively, with a predicted ORF (ORF26ng) from N. gonorrhoeae:

The complete length ORF26ng nucleotide sequence <SEQ ID 695> is:

1    ATGCAGCTGA TTGACTATTC ACATTCATTT TTCTCGGTTG
     TGCCACCCTT
51   TTTGGCACTG GCACTTGCCG TCATTACCCG CCGCGTACTG
     CTGTCTTTAG
101  GCATCGGTAT TTTGGTCGGC GTTGCCTTTT TGGTCGGCGG
     CAACCCCGTC
151  GACGGTCTGA CACACCTGAA AGACATGGTC GTCGGCTTGG
     CTTGGGCAGA
201  CGGCGATTGG TCGCTGGGCA AACCAAAAAT CTTGGTTTTC
     CTGATACTTT
251  TGGGCATTTT CACTTCACTG CTGACCTACT CCGGCAGCAA
     TCAGGCGTTT
301  GCCGACTGGG CAAAACGGCA CATTAAAAAC CGGTGCGGCG
     CGAAAATGCT
351  GACCGCCTGC CTCGTGTTCG TAACCTTTAT CGACGACTAT
     TTCCACAGCC
401  TCGCCGTCGG TGCGATTGCC CGCCCCGTTA CCGACAAGTT
     TAAAGTTTCC
451  CGCGCCAAAC TCGCCTACAT CCTCGACTCC ACTGCCTCGC
     CCATGTGCGT
501  GCTGATGCCC GTTTCAAGCT GGGGCGCGTC GATTATCGCC
     ACGCTTGCCG
551  GATTGCTCGT TACCTACAAA ATTACCGAAT ACACGCCGAT
     GGGGACGTTT
601  GTCGCCATGA GCCTGATGAA CTATTACGCG CTGTTTGCCC
     TGATTATGGT
651  ATTCGTCGTC GCATGGTTCT CCTTCGACAT CGGCTCGAtg
     gCGCGTTTCG
701  AACAGGCTGC GTTGAACGAA gcccaggacg aaaccgccgc
     tTCAGACgCT
751  ACCAAAGGTC GTGTTTACGC ATTGATTATT CCCGTTTTGG
     CCTTAATCGC
801  CTCAACGGTT TCCGCCATGA TCTACACCGG CGCGCAGGCA
     AGCGAAACCT
851  TCAGCATTTT GGGGGCATTT GAAAATACCG ACGTAAACAC
     TTCGCTGGTA
901  TTCGGCGGCA CTTGCGGCGT GCTTGCCGTC GTCCTCTGCA
     CGTTCGGCAC
951  GATTAAAACC GCCGATTATC CCAAAGCCGT GTGGCAGGGT
     GCGAAATCCA
1001 TGTTCGGCGC AATCGCCATT TTAATCCTCG CCTGGCTCAT
     CAGTACGGTT
1051 GTCGGCGAAA TGCACACGGG CGACTACCTC TCCACGCTGG
     TTGCGGGCAA
1101 CATCCATCCC GGCTTCCTGC CCGTCATCCT CTTCCTGCTC
     GCCAGCGTGA
1151 TGGCGTTTGC CACAGGCACA AGCTGGGGGA CGTTCGGCAT
     TATGCTGCCG
1201 ATTGCCGCCG CCATGGCGGT CAAAGTCGAA CCCGCGCTGA
     TTAtcccGTG
1251 TATGTCCGCA GTAATGGCGG GGGCGGTATG CGGCGACCAC
     TGTTCGCCCA
1301 TCTCCGACAC GACCATCCTG TCGTCCACCG GCGCGCGCTG
     CAACCACATC
1351 GACCACGTTA CCTCGCAACT GCCTTATGCC CTGACGGTTG
     CCGCCGCCGC
1401 CGCATCGGGC TACCTCGCAT TGGGTCTGAC AAAATCCGCG
     CTGTTGGGCT
1451 TTGGCACGAC CGGTATTGTA TTGGCGGTGC TGATTTTTCT
     GTTGAAAGAT
1501 AAAAAACGCG CCGACGTTTG A

This encodes a protein having amino acid sequence <SEQ ID 696>:

1   MQLIDYSHSF FSVVPPFLAL ALAVITRRVL LSLGIGILVG
    VAFLVGGNPV
51  DGLTHLKDMV VGLAWADGDW SLGKPKILVF LILLGIFTSL
    LTYSGSNQAF
101 ADWAKRHIKN RCGAKMLTAC LVFVTFIDDY FHSLAVGAIA
    RPVTDKFKVS
151 RAKLAYILDS TASPMCVLMP VSSWGASIIA TLAGLLVTYK
    ITEYTPMGTF
201 VAMSLMNYYA LFALIMVFVV AWFSFDIGSM ARFEQAALNE
    AQDETAASDA
251 TKGRVYALII PVLALIASTV SAMIYTGAQA SETFSILGAF
    ENTDVNTSLV
301 FGGTCGVLAV VLCTFGTIKT ADYPKAVWQG AKSMFGAIAI
    LILAWLISTV
351 VGEMHTGDYL STLVAGNIHP GFLPVILFLL ASVMAFATGT
    SWGTFGIMLP
401 IAAAMAVKVE PALIIPCMSA VMAGAVCGDH CSPISDTTIL
    SSTGARCNHI
451 DHVTSQLPYA LTVAAAAASG YLALGLTKSA LLGFGTTGIV
    LAVLIFLLKD
501 KKRADV*

ORF26ng and ORF26-1 show 98.4% identity in 505 aa overlap:

In addition, ORF26 ng shows significant homology to a hypothetical H. influenzae protein:

sp|P44263|YF86_HAEIN HYPOTHETICAL PROTEIN HI1586 >gi|1074850|pir||C64037
hypothetical
protein HI1586 - Haemophilus influenzae (strain Rd KW20) >gi|1574427
(U32832) H. influenzae predicted coding region HI1586 [Haemophilus
influenzae] Length = 519
Score = 538 bits (1370), Expect = e−152
Identities = 280/507 (55%), Positives = 346/507 (68%), Gaps = 7/507 (1%)
Query:	1	MQLIDYSHSFFSVVPPFLALALAVITRRXXXXXXXXXXXXXAFLVGGNPVDGLTHLKDMV	60
		M+LID+S S +S+VP  LA+ LA+ TRR               L          +L   V
Sbjct:	14	MELIDFSSSVWSIVPALLAIILAIATRRVLVSLSAGIIIGSLMLSDWQIGSAFNYLVKNV	73
Query:	61	VGLAWADGDWSLGKPKILVFLILLGIFTSLLTYSGSNQAFADWAKRHIKNRCGAKMLTAC	120
		V L +ADG+ +     I++FL+LLG+ T+LLT SGSN+AFA+WA+  IK R GAK+L A
Sbjct:	74	VSLVYADGEIN-SNMNIVLFLLLLGVLTALLTVSGSNRAFAEWAQSRIKGRRGAKLLAAS	132
Query:	121	LVFVTFIDDYFHSLAVGAIARPVTDKFKVSRAKLAYILDSTASPMCVLMPVSSWGASIIA	180
		LVFVTFIDDYFHSLAVGAIARPVTD+FKVSRAKLAYILDSTA+PMCV+MPVSSWGA II
Sbjct:	133	LVFVTFIDDYFHSLAVGAIARPVTDRFKVSRAKLAYILDSTAAPMCVMMPVSSWGAYIIT	192
Query:	181	TLAGLLVTYKITEYTPMGTFVAMSLMNYYALFALIMVFVVAWFSFDIGSMARFEQAALNE	240
		+ GLL TY ITEYTP+G FVAMS MN+YA+F++IMVF VA+FSFDI SM R E+ AL
Sbjct:	193	LIGGLLATYSITEYTPIGAFVAMSSMNFYAIFSIIMVFFVAYFSFDIASMVRHEKLALKN	252
Query:	241	AQDETAASDATKGRVYALIIPVLALIASTVSAMIYTGAQA----SETFSILGAFENTDVN	296
		+D+      TKG+V  LI+P+L LI +TVS MIYTGA+A     + FS+LG FENT V
Sbjct:	253	TEDQLEEETGTKGQVRNLILPILVLIIATVSMMIYTGAEALAADGKVFSVLGTFENTVVG	312
Query:	297	TSLVFGGTCGVL--AVVLCTFGTIKTADYPKAVWQGAKSMFGXXXXXXXXXXXSTVVGEM	354
		TSLV GG C ++   +++     +   +Y ++   G KSM G           + +VG+M
Sbjct:	313	TSLVVGGFCSIIISTLLIILDRQVSVPEYVRSWIVGIKSMSGAIAILFFAWTINKIVGDM	372
Query:	355	HTGDYLSTLVAGNIHPGFLPVILFLLASVMAFATGTSWGTFGIMLPIAAAMAVKVEPALI	414
		TG YLS+LV+GNI   FLPVILF+L + MAF+TGTSWGTFGIMLPIAAAMA    P L+
Sbjct:	373	QTGKYLSSLVSGNIPMQFLPVILFVLGAAMAFSTGTSWGTFGIMLPIAAAMAANAAPELL	432
Query:	415	IPCMSAVMAGAVCGDHCSPISDTTILSSTGARCNHIDHVTSQXXXXXXXXXXXXXXXXXX	474
		+PC+SAVMAGAVCGDHCSP+SDTTILSSTGA+CNHIDHVT+Q
Sbjct:	433	LPCLSAVMAGAVCGDHCSPVSDTTILSSTGAKCNHIDHVTTQLPYAATVATATSIGYIVV	492
Query:	475	XXXKSALLGFGTTGIVLAVLIFLLKDK	501
		S L GF  T + L V+IF +K +
Sbjct:	493	GFTYSGLAGFAATAVSLIVIIFAVKKR	519

Based on this analysis, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 83

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 697>:

1   ..AAGCAATGGT ATGCCGACGN .AGTATCAAG ACGGAAATGG
    TTATGGTCAA
51  CGATGAGCCT GCCAAAATTC TGACTTGGGA TGAAAGCGGC
    CGATTACTCT
101 CGGAACTGTC TATCCGCCAC CATCAACGCA ACGGGGTGGT
    TTTGGAGTGG
151 TATGAAGATG GTTCTAAAAA GAGCGAAGT. GTTTATCAGG
    ATGACAAGTT
201 GGTCAGGAAA ACCCAGTGGG ATAAGGATGG TTATTTAATC
    GAACCCTGA

This corresponds to the amino acid sequence <SEQ ID 698; ORF27>:

1  ..KQWYADXSIK TEMVMVNDEP AKILTWDESG RLLSELSIRH
   HQRNGVVLEW
51 YEDGSKKSEX VYQDDKLVRK TQWDKDGYLI EP*

Further work revealed the complete nucleotide sequence <SEQ ID 699>:

1   ATGAAAAAAT TATCTCGGAT TGTATTTTCA ACTGTCCTGT
    TGGGTTTTTC
51  GGCCGCTTTG CCGGCGCAGA CCTATTCTGT TTATTTTAAT
    CAGAACGGAA
101 AGCTGACGGC GACGATGTCT TCTGCCGCTT ATATCAGGCA
    ATATAGTGTG
151 GTGGCGGGTA TTGCGCACGC GCAGGATTTT TATTATCCGT
    CGATGAAGAA
201 ATATTCTGAA CCTTATATCG TTGCTTCAAC GCAAATCAAA
    TCTTTTGTGC
251 CTACCCTGCA AAACGGTATG TTGATTTTGT GGCATTTTAA
    TGGTCAGAAA
301 AAAATGGCGG GGGGCTTCAG CAAGGGTAAG CCGGACGGGG
    AGTGGGTCAA
351 CTGGTATCCG AACGGTAAAA AATCTGCCGT TATGCCTTAT
    AAAAATGGCT
401 TGAGTGAGGG TACGGGATAC CGCTATTACC GTAACGGCGG
    CAAGGAAAGC
451 GAAATCCAGT TTAAGCAAAA TAAGGCAAAC GGCGTATGGA
    AGCAATGGTA
501 TGCCGACGGC AGTATCAAGA CGGAAATGGT TATGGTCAAC
    GATGAGCCTG
551 CCAAAATTCT GACTTGGGAT GAAAGCGGCC GATTACTCTC
    GGAACTGTCT
601 ATCCGCCACC ATCAACGCAA CGGGGTGGTT TTGGAGTGGT
    ATGAAGATGG
651 TTCTAAAAAG AGCGAAGCTG TTTATCAGGA TGACAAGTTG
    GTCAGGAAAA
701 CCCAGTGGGA TAAGGATGGT TATTTAATCG AACCCTGA

This corresponds to the amino acid sequence <SEQ ID 700; ORF27-1>:

1   MKKLSRIVFS TVLLGFSAAL PAQTYSVYFN QNGKLTATMS
    SAAYIRQYSV
51  VAGIAHAQDF YYPSMKKYSE PYIVASTQIK SFVPTLQNGM
    LILWHFNGQK
101 KMAGGFSKGK PDGEWVNWYP NGKKSAVMPY KNGLSEGTGY
    RYYRNGGKES
151 EIQFKQNKAN GVWKQWYADG SIKTEMVMVN DEPAKILTWD
    ESGRLLSELS
201 IRHHQRNGVV LEWYEDGSKK SEAVYQDDKL VRKTQWDKDG
    YLIEP*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF27 shows 91.5% identity over a 82aa overlap with an ORF (ORF27a) from strain A of N. meningitidis:

The complete length ORF27a nucleotide sequence <SEQ ID 701> is:

1   ATGAAAAAAT TATCTCGGAT TGTATTTTCA ACTGTCCTGT
    TGGGTTTTTC
51  GGCCGCTTTG CCGGCGCAGA NCTATTCTGT TTATTTTAAT
    CAGAACGGGA
101 AACTGACGGC GACGNTGTCT TCTGCCGCNT ATATCAGGCA
    ATATAGTGTG
151 GCGGAGGGTA TTGCGCACGC GCAGGANTTT TANTATCCGT
    CGATGAAGAA
201 ATATTCCGAA CCTTATATCG TTGCTTCAAC GCAAATCAAA
    TCTTTTGTGC
251 CTACCCTGCA AAACGGTATG TTGATTTTGT GGCATTTTAA
    NGGTCAGAAA
301 AAAATGGCNG GGGGCTTCAG CAAGGGTAAG CCGGACGGGG
    AGTGGGTCAA
351 CTGGTATCCG AACGGTAAAA AATCTGCCGT TATGCCTTAT
    AAAAATGGTT
401 TGAGTGAAGG TACGGGGTNN CGCTATTACC GTAACGGCGG
    CAAGGAAAGC
451 GAAATCCAGT TTAAACAGAA TAAGGCAAAC GGCGTATGGA
    AGCAATGGTA
501 TGCCGACGGC AATATCAAAA CGGAAATGGT TATGGTCAAT
    GATGAGCCTG
551 CCAAAATTCT GACATGGGAT GAAAGCGGTC GATTACTCTC
    GGAACTGTCT
601 ATCCATCATC ATNAACGTAA TGGAGTAGTC TTAGAGTGGT
    ATGAAGATGG
651 TTCTAAAAAG ANTGAAGCTG TTTATCAGGA TGATAAGTTG
    GTCAGGAAAA
701 CCCAGTGGGA TAANGATGGT TATTTAATCG AACCCTGA

This encodes a protein having amino acid sequence <SEQ ID 702>:

1   MKKLSRIVFS TVLLGFSAAL PAQXYSVYFN QNGKLTATXS
    SAAYIRQYSV
51  AEGIAHAQXF XYPSMKKYSE PYIVASTQIK SFVPTLQNGM
    LILWHFXGQK
101 KMAGGFSKGK PDGEWVNWYP NGKKSAVMPY KNGLSEGTGX
    RYYRNGGKES
151 EIQFKQNKAN GVWKQWYADG NIKTEMVMVN DEPAKILTWD
    ESGRLLSELS
201 IHHHXRNGVV LEWYEDGSKK XEAVYQDDKL VRKTQWDXDG
    YLIEP*

ORF27a and ORF27-1 show 94.7% identity in 245 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF27 shows 96.3% identity over 82 aa overlap with a predicted ORF (ORF27ng) from N. gonorrhoeae:

The complete length ORF27ng nucleotide sequence <SEQ ID 703> is:

1   ATGAAGAAAT TATCTCGGAT TGTATTTTCA ATCGTACTGT
    TGGGTTTTTC
51  GGCCGCTTTG CCGGCGCAGA CCTATTCTGT TTATTTTAAT
    CAGAACGGGA
101 AACTGACGGC GACGATGTCT TCTGCCGCTT ATATCAGGCA
    ATATAGTGTG
151 GCGGCGGGTA TCGCACACGC GCAGGATTTT TATTATCCGT
    CGATGAAGAA
201 ATATTCCGAA CCTTATATCG TTGCTTCAAC GCAAATCAAA
    TCTTTTGTGC
251 CTACCCTGCA AAACGGTATG TTGATTTTGT GGCATTTTAA
    TGGTCAGAAA
301 AAAATGGCGG GGGGCTTCAG CAAGGGTAAG CCGGACGGGG
    AATGGGTCAA
351 CTGGTATCCG AACGGTAAAA AATCTGCGGT TATGCCTTAT
    AAAAATGGCT
401 TGAGTGAGGG TACGGGATAC CGTTATTACC GTAACGGCGG
    CAAGGAAAGC
451 GAAATCCAGT TTAAGCAAAA TAAGGCGAAC GGCGTATGGA
    AGCAATGGTA
501 TGCCGATGGA AGTATCAAGA CGGAAATGGT TATGGTCAAC
    GATGAGCCTG
551 CCAAAATTCT GACTTGGGAT GAAAGCGGCC GATTACTTTC
    GGAACTGTCT
601 ATCCGCCACC ATAAACGCAA CGGGGTGGTT TTGGAGTGGT
    ATGAAGATGG
651 TTCTAAAAAG AGCGAGGCTG TTTATCAGGA TGACAAGTTG
    GTCAGGAAAA
701 CCCAATGGGA TAAGGATGGT TATTTAATCG AACCCTGA

This encodes a protein having amino acid sequence <SEQ ID 704>:

1   MKKLSRIVFS IVLLGFSAAL PAQTYSVYFN QNGKLTATMS
    SAAYIRQYSV
51  AAGIAHAQDF YYPSMKKYSE PYIVASTQIK SFVPTLQNGM
    LILWHFNGQK
101 KMAGGFSKGK PDGEWVNWYP NGKKSAVMPY KNGLSEGTGY
    RYYRNGGKES
151 EIQFKQNKAN GVWKQWYADG SIKTEMVMVN DEPAKILTWD
    ESGRLLSELS
201 IRHHKRNGVV LEWYEDGSKK SEAVYQDDKL VRKTQWDKDG
    YLIEP*

ORF27ng and ORF27-1 show 98.8% identity in 245 aa overlap:

Based on this analysis, including the putative leader sequence in the gonococcal protein, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF27-1 (24.5 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 17A shows the results of affinity purification of the GST-fusion protein, and FIG. 17B shows the results of expression of the His-fusion in E. coli. Purified GST-fusion protein was used to immunise mice, whose sera were used for ELISA, which gave a positive result, confirming that ORF27-1 is a surface-exposed protein and a useful immunogen.

Example 84

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 705>:

1   ATGAAATTTA CCAAGCACCC CGTCTGGGCA ATGGCGTTCC
    GCCCATTTTA
51  TTCGCTGGCG GCTCTGTACG GCGCATTGTC CGTATTGCTG
    TGGGGTTTCG
101 GCTACACGGG AACGCACkAG CTGTCCGGTT TCTATTGGCA
    CGCGCATGAg
151 ATGATTTGGG GTTATGCCGG ACTGGTCGTC ATCGCCTTCC
    TGCTGACCGC
201 CGTCGCCACT TGGACGGGGC AGCCGCCCAC GCGGGGCGGC
    GTaTCTGGTC
251 GGCTTGACTA TCTTTTGGCT GGCTGCGCGG ATTGCCGCCT
    TTATCCCGGG
301 TTGGGGTGCG TCGGCAAGCG GCATACTCGG TACGCTGTTT
    TTCTGGTACG
351 GCGCGGTGTG CATGGCTTTG CCCGTTATCC GTTCGCAGAA
    TCAACGCAAC
401 TATGTTgCCG TGTTCGCGCT GTTCGTCTTG GGCGGCACGC
    ATGCGGCGTT
451 CCACGTCCAG CTGCACAACG GCAACCTAGG CGGACTCTTG
    AGCGGATTGC
501 AGTCGGGCTT GGTGATG

This corresponds to the amino acid sequence <SEQ ID 706; ORF47>:

1   MKFTKHPVWA MAFRPFYSLA ALYGALSVLL WGFGYTGTHX
    LSGFYWHAHE
51  MIWGYAGLVV IAFLLTAVAT WTGQPPTRGG VLVGLTIFWL
    AARIAAFIPG
101 WGASASGILG TLFFWYGAVC MALPVIRSQN QRNYVAVFAL
    FVLGGTHAAF
151 HVQLHNGNLG GLLSGLQSGL VM

Further work revealed the complete nucleotide sequence <SEQ ID 707>:

1    ATGAAATTTA CCAAGCACCC CGTCTGGGCA ATGGCGTTCC
     GCCCATTTTA
51   TTCGCTGGCG GCTCTGTACG GCGCATTGTC CGTATTGCTG
     TGGGGTTTCG
101  GCTACACGGG AACGCACGAG CTGTCCGGTT TCTATTGGCA
     CGCGCATGAG
151  ATGATTTGGG GTTATGCCGG ACTGGTCGTC ATCGCCTTCC
     TGCTGACCGC
201  CGTCGCCACT TGGACGGGGC AGCCGCCCAC GCGGGGCGGC
     GTTCTGGTCG
251  GCTTGACTAT CTTTTGGCTG GCTGCGCGGA TTGCCGCCTT
     TATCCCGGGT
301  TGGGGTGCGT CGGCAAGCGG CATACTCGGT ACGCTGTTTT
     TCTGGTACGG
351  CGCGGTGTGC ATGGCTTTGC CCGTTATCCG TTCGCAGAAT
     CAACGCAACT
401  ATGTTGCCGT GTTCGCGCTG TTCGTCTTGG GCGGCACGCA
     TGCGGCGTTC
451  CACGTCCAGC TGCACAACGG CAACCTAGGC GGACTCTTGA
     GCGGATTGCA
501  GTCGGGCTTG GTGATGGTGT CGGGTTTTAT CGGTCTGATT
     GGTACGCGGA
551  TTATTTCGTT TTTTACGTCC AAACGCTTGA ATGTGCCGCA
     GATTCCCAGT
601  CCGAAATGGG TGGCGCAGGC TTCGCTGTGG CTGCCCATGC
     TGACTGCCAT
651  GCTGATGGCG CACGGTGTGT TGGCTTGGCT GTCTGCCGTT
     TTTGCCTTTG
701  CGGCAGGTGT GATTTTTACC GTGCAGGTGT ACCGCTGGTG
     GTATAAACCC
751  GTGTTGAAAG AGCCGATGCT GTGGATTCTG TTTGCCGGCT
     ATCTGTTTAC
801  CGGATTGGGG CTGATTGCGG TCGGCGCGTC TTATTTCAAA
     CCCGCTTTCC
851  TCAATCTGGG TGTGCATCTG ATCGGGGTCG GCGGTATCGG
     CGTGCTGACT
901  TTGGGCATGA TGGCGCGTAC CGCGCTTGGT CATACGGGCA
     ATCCGATTTA
951  TCCGCCGCCC AAAGCCGTTC CCGTTGCGTT TTGGCTGATG
     ATGGCGGCAA
1001 CCGCCGTCCG TATGGTTGCC GTATTTTCTT CCGGCACTGC
     CTACACGCAC
1051 AGCATCCGCA CCTCTTCGGT TTTGTTTGCA CTCGCGCTTT
     TGGTGTATGC
1101 GTGGAAGTAT ATTCCTTGGC TGATTCGTCC GCGTTCGGAC
     GGCAGGCCCG
1151 GTTGA

This corresponds to the amino acid sequence <SEQ ID 708; ORF47-1>:

1   MKFTKHPVWA MAFRPFYSLA ALYGALSVLL WGFGYTGTHE
    LSGFYWHAHE
51  MIWGYAGLVV IAFLLTAVAT WTGQPPTRGG VLVGLTIFWL
    AARIAAFIPG
101 WGASASGILG TLFFWYGAVC MALPVIRSQN QRNYVAVFAL
    FVLGGTHAAF
151 HVQLHNGNLG GLLSGLQSGL VMVSGFIGLI GTRIISFFTS
    KRLNVPQIPS
201 PKWVAQASLW LPMLTAMLMA HGVLAWLSAV FAFAAGVIFT
    VQVYRWWYKP
251 VLKEPMLWIL FAGYLFTGLG LIAVGASYFK PAFLNLGVHL
    IGVGGIGVLT
301 LGMMARTALG HTGNPIYPPP KAVPVAFWLM MAATAVRMVA
    VFSSGTAYTH
351 SIRTSSVLFA LALLVYAWKY IPWLIRPRSD GRPG*

Computer analysis of this amino acid sequence predicts a leader peptide and also gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF47 shows 99.4% identity over a 172aa overlap with an ORF (ORF47a) from strain A of N. meningitidis:

The complete length ORF47a nucleotide sequence <SEQ ID 709> is:

1    ATGAAATTTA CCAAGCACCC CGTTTGGGCA ATGGCGTTCC
     GCCCGTTTTA
51   TTCACTGGCG GCTCTGTACG GCGCATTGTC CGTATTGCTG
     TGGGGTTTCG
101  GCTACACGGG AACGCACGAG CTGTCCGGTT TCTATTGGCA
     CGCGCATGAG
151  ATGATTTGGG GTTATGCCGG ACTGGTCGTC ATCGCCTTCC
     TGCTGACCGC
201  CGTCGCCACT TGGACGGGGC AGCCGCCCAC GCGGGGCGGC
     GTTCTGGTCG
251  GCTTGACTAT CTTTTGGCTG GCTGCGCGGA TTGCCGCCTT
     TATCCCGGGT
301  TGGGGTGCGT CGGCAAGCGG CATACTCGGT ACGCTGTTTT
     TCTGGTACGG
351  CGCGGTGTGC ATGGCTTTGC CCGTTATCCG TTCGCAGAAT
     CAACGCAATT
401  ATGTTGCCGT GTTCGCGCTG TTCGTCTTGG GCGGTACGCA
     CGCGGCGTTC
451  CACGTCCAGC TGCACAACGG CAACCTAGGC GGACTCTTGA
     GCGGATTGCA
501  GTCGGGCTTG GTGATGGTGT CGGGTTTTAT CGGTCTGATT
     GGTACGCGGA
551  TTATTTCGTT TTTTACGTCC AAACGGTTGA ATGTGCCGCA
     GATTCCCAGT
601  CCGAAATGGG TGGCGCAGGC TTCGCTGTGG CTGCCCATGC
     TGACCGCCAT
651  GCTGATGGCG CACGGCGTGA TGCCTTGGCT GTCGGCGGCT
     TTCGCGTTTG
701  CGGCAGGTGT GATTTTTACC GTGCAGGTGT ACCGCTGGTG
     GTATAAGCCT
751  GTGTTGAAAG AGCCGATGCT GTGGATTCTG TTTGCCGGCT
     ATCTGTTTAC
801  CGGATTGGGG CTGATTGCGG TCGGCGCGTC TTATTTCAAA
     CCCGCTTTCC
851  TCAATCTGGG TGTGCATCTG ATCGGGGTCG GCGGTATCGG
     CGTGCTGACT
901  TTGGGCATGA TGGCGCGTAC CGCGCTCGGT CATACGGGCA
     ATCCGATTTA
951  TCCGCCGCCC AAAGCCGTTC CCGTTGCGTT TTGGCTGATG
     ATGGCGGCAA
1001 CCGCCGTCCG TATGGTTGCC GTATTTTCTT CCGGCACTGC
     CTACACGCAC
1051 AGCATACGCA CCTCTTCGGT TTTGTTTGCA CTCGCGCTTT
     TGGTGTATGC
1101 GTGGAAGTAT ATTCCTTGGC TGATTCGTCC GCGTTCGGAC
     GGCAGGCCCG
1151 GTTGA

This encodes a protein having amino acid sequence <SEQ ID 710>:

1   MKFTKHPVWA MAFRPFYSLA ALYGALSVLL WGFGYTGTHE
    LSGFYWHAHE
51  MIWGYAGLVV IAFLLTAVAT WTGQPPTRGG VLVGLTIFWL
    AARIAAFIPG
101 WGASASGILG TLFFWYGAVC MALPVIRSQN QRNYVAVFAL
    FVLGGTHAAF
151 HVQLHNGNLG GLLSGLQSGL VMVSGFIGLI GTRIISFFTS
    KRLNVPQIPS
201 PKWVAQASLW LPMLTAMLMA HGVMPWLSAA FAFAAGVIFT
    VQVYRWWYKP
251 VLKEPMLWIL FAGYLFTGLG LIAVGASYFK PAFLNLGVHL
    IGVGGIGVLT
301 LGMMARTALG HTGNPIYPPP KAVPVAFWLM MAATAVRMVA
    VFSSGTAYTH
351 SIRTSSVLFA LALLVYAWKY IPWLIRPRSD GRPG*

ORF47a and ORF47-1 show 99.2% identity in 384 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF47 shows 97.1% identity over 172 aa overlap with a predicted ORF (ORF47ng) from N. gonorrhoeae:

The ORF47ng nucleotide sequence <SEQ ID 711> is predicted to encode a protein comprising amino acid sequence <SEQ ID 712>:

1   MKFTKHPVWA MAFRPFYSLA ALYGALSVLL WGFGYTGTHE
    LSGFYWHAHE
51  MIWGYAGLVV IAFLLTAVAT WTGQPPTRGG VLVGLTAFWL
    AARIAAFIPG
101 WGAAASGILG TLFFWYGAVC MALPVIRSQN RRNYVAVFAI
    FVLGGTHAAF
151 HVQLHNGNLG GLLSGLQSGL VMVWGFIGLI GMKIISFFTS
    KRLKLPQIPS
201 PKWVAHASLW LPMLNAILMA HRVMPWLSAA FPFAAGVIFT
    VQVYAGGITP
251 IEETSCGSVA GICYRLGNSS G

The predicted leader peptide and transmembrane domains are identical (except for an Ile/Ala substitution at residue 87 and an Leu/Ile substitution at position 140) to sequences in the meningococcal protein (see also Pseudomonas stutzeri orf396, accession number e246540):

TM segments in ORF47ng
INTEGRAL	Likelihood = −5.63	Transmembrane	52 - 68
INTEGRAL	Likelihood = −3.88	Transmembrane	169 - 185
INTEGRAL	Likelihood = −3.08	Transmembrane	82 - 98
INTEGRAL	Likelihood = −1.91	Transmembrane	134 - 150
INTEGRAL	Likelihood = −1.44	Transmembrane	107 - 123
INTEGRAL	Likelihood = −1.38	Transmembrane	227 - 243

Further work revealed the complete gonococcal DNA sequence <SEQ ID 713>:

1    ATGAAATTTA CCAAACATCC CGTCTGGGCA ATGGCGTTCC
     GCCCGTTTTA
51   TTCACTGGCG GCACTGTACG GCGCATTGTC CGTATTGCTG
     TGGGGTTTCG
101  GCTACACGGG AACGCACGAG CTGTCCGGTT TCTATTGGCA
     CGCGCATGAG
151  ATGATTTGGG GTTATGCCGG TCTCGTCGTC ATCGCCTTCC
     TGCTGACCGC
201  CGTCGCCACT TGGACGGGAC AGCCGCCCAC GAGGGGCGGC
     GTTCTGGTCG
251  GCTTGACCGC CTTTTGGCTG GCTGCGCGGA TTGCCGCCTT
     TATCCCGGGT
301  TGGGGTGCGG CGGCAAGCGG CATACTCGGT ACGCTGTTTT
     TCTGGTACGG
351  CGCGGTGTGC ATGGCTTTGC CCGTTATCCG TtcgCAAAAC
     CGGCGCAACT
401  ATGtcgCCGT ATTCGCAATA TTTGTGCTGG GCGGTACGCA
     TGCGgcgTTC
451  CACGtccAgc tGCACAACGG CAACCTAGGC GGACTCTTGA
     GCGGATTGCA
501  GTCGGGCCTG GTTATGGTGT CGGGCTTTAT CGGCCTGATT
     GGGATGAGGA
551  TTATTTCGTT TTTTACGTCC AAACGGTTGA ACGTGCCGCA
     GATTCCCAGT
601  CCGAAATGGG TGGCGCAGGC TTCGCTGTGG CTACCCATGC
     TGACCGCCAT
651  ACTGATGGCG CACGGCGTGA TGCCTTGGCT GTCGGCGGCT
     TTCGCGTTTG
701  CGGCGGGCGT GATTTTTACC GTACAGGTGT ACCGCTGGTG
     GTATAAACCC
751  GTATTGAAAG AACCGATGCT GTGGATTCTG TTTGCCGGCT
     ATCTGTTTAC
801  CGGATTGGGG CTGATTGCGG TCGGCGCGTC TTATTTCAAA
     CCTGCCTTCC
851  TCAATCTGGG CGTACATCTG ATCGGGGTCG GCGGTATCGG
     CGTGCTGACT
901  TTGGGCATGA TGGCGCGTAC CGCGCTCGGT CATACGGGCA
     ATTCGATTTA
951  TCCGCCGCCC AAAGCCGTTC CCGTTGCGTT TTGGCTGATG
     ATGGCGGCAA
1001 CCGCCGTCCG TATGGTTGCC GTATTTTCTT CCGGCACTGC
     CTACACGCAC
1051 AGCATCCGCA CGTCTTCGGT TTTGTTTGCA CTCGCGCTGC
     TGGTGTATGC
1101 GTGGAAATAC ATTCCGTGGC TGATCCGTCC GCGTTCGGAC
     GGCAGGCCCG
1151 GTTGA

This encodes a protein having amino acid sequence <SEQ ID 714; ORF47ng-1>:

1   MKFTKHPVWA MAFRPFYSLA ALYGALSVLL WGFGYTGTHE
    LSGFYWHAHE
51  MIWGYAGLVV IAFLLTAVAT WTGQPPTRGG VLVGLTAFWL
    AARIAAFIPG
101 WGAAASGILG TLFFWYGAVC MALPVIRSQN RRNYVAVFAI
    FVLGGTHAAF
151 HVQLHNGNLG GLLSGLQSGL VMVSGFIGLI GMRIISFFTS
    KRLNVPQIPS
201 PKWVAQASLW LPMLTAILMA HGVMPWLSAA FAFAAGVIFT
    VQVYRWWYKP
251 VLKEPMLWIL FAGYLFTGLG LIAVGASYFK PAFLNLGVHL
    IGVGGIGVLT
301 LGMMARTALG HTGNSIYPPP KAVPVAFWLM MAATAVRMVA
    VFSSGTAYTH
351 SIRTSSVLFA LALLVYAWKY IPWLIRPRSD GRPG*

ORF47ng-1 and ORF47-1 show 97.4% identity in 384 aa overlap:

Furthermore, ORF47ng-1 shows significant homology to an ORF from Pseudomonas stutzeri:

gnl|PID|e246540 (Z73914) ORF396 protein [Pseudomonas stutzeri]
Length = 396 Score = 155 bits (389), Expect = 5e−37
Identities = 121/391 (30%), Positives = 169/391 (42%), Gaps = 21/391 (5%)
Query:	7	PVWAMAFRPFYSLAALYGALSVLLWGFGYTGTHELSGFY-------WHAHEMIWGYAGLV	59
		P+W +AFRPF+   +LY  L++ LW   +TG     GF        WH HEM++G+A  +
Sbjct:	14	PIWRLAFRPFFLAGSLYALLAIPLWVAAWTGLWP--GFQPTGGWLAWHRHEMLFGFAMAI	71
Query:	60	VIAFLLTAVATWTGQPPTRGGVLVGLTAFWLAARIAAFIPGWGAAASGILGTLFFWYGAV	119
		V  FLLTAV TWTGQ    G  LVGL A WLAAR+  ++ G  AA    L  LF
Sbjct:	72	VAGFLLTAVQTWTGQTAPSGNRLVGLAAVWLAARL-GWLFGLPAAWLAPLDLLFLVALVW	130
Query:	120	CMALPVIRSQNRRNYVAVFAIFVLGGTHAAFXXXXXXXXXXXXXXXXXXXXXMVSGFIGL	179
		MA  +   + +RNY  V  + ++ G                          +V+  + L
Sbjct:	131	MMAQMLWAVRQKRNYPIVVVLSLMLGADVLILTGLLQGNDALQRQGVLAGLWLVAALMAL	190
Query:	180	IGMRIISFFTSKRLNVPQIPSP-KWVAQASLWLPMLTAILMAHGV----MPWLSAAFAFA	234
		IG R+I FFT + L       P  W+  A L    + A+L A GV     P L   F  A
Sbjct:	191	IGGRVIPFFTQRGLGKVDAVKPWVWLDVALLVGTGVIALLHAFGVAMRPQPLLGLLFV-A	249
Query:	235	AGVIFTVQVYRWWYKPVLKEPMLWILFAGYLFTGLGLIAVGASYF-KPAFXXXXXXXXXX	293
		GV   +++ RW+ K + K  +LW L    L+  +    +   +F   A
Sbjct:	250	IGVGHLLRLMRWYDKGIWKVGLLWSLHVAMLWLVVAAFGLALWHFGLLAQSSPSLHALSV	309
Query:	294	XXXXXXXXXMMARTALGHTGNSIYPPPKAVPVAFWLXXXXXXXXXXXXFSSGTAYTHSIR	353
		M+AR  LGHTG  +  P   +  AF L            F S       +
Sbjct:	310	GSMSGLILAMIARVTLGHTGRPLQLPAGIIG-AFVL---FNLGTAARVFLSVAWPVGGLW	365
Query:	354	TSSVLFALALLVYAWKYIPWLIRPRSDGRPG	384
		++V + LA  +Y W+Y P L+  R DG PG
Sbjct:	366	LAAVCWTLAFALYVWRYAPMLVAARVDGHPG	396

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 85

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 715>:

1   ..ATGCCGTCTG AAGGTTCAGA CGGCmTCGGT GyCGGGGAAy
    CAGAAGyGGT
51  AGCGCATGCC CAATGAGACT TCGTGGGTTT TGAAGCGGGT
    GTTTTCCAAG
101 CGTCCCCAGT TGTGGTAACG GTATCCGGTG TCyAArGTCA
    GCTTGGGyGT
151 GATGTCGAAa CCGACACCGG CGATGACACC AAGACCyAmG
    CTGCTGATrC
201 TGTkGCTTTC GTGATAGGsA GGTTTGyTGG kmksAsyTTG
    TAyrATwkkG
251 CCTssCwsTG kAGmGCCkTk CkyTGGTkkA swGrwArTAG
    TCGTGGTTTy
301 TkTTyyCACC GAATGAACyT GATGTTTAAC GTGTCCGTAG
    GCGACGCGCG
351 CGCCGATATA GGGTTTGAAT TTATCGTTGA GTTTGAAATC
    GTAAATGGCG
401 GACAAGCCGA GAGAAGAAAC GGCGTGGAAG CTGCCGTTTC
    CCTGATGTTT
451 TGTTTGGGTT TCTTTGTAGT TGTTGTTTAT CTCTTCAGTA
    ACTTTTTTAG
501 TAGAAGAATT ACTTTCTTTC CATTTTCTGT AACTGGCATA
    ATCTGCCGCT
551 ATTCTCCAGC CGCCGAAATC ..

This corresponds to the amino acid sequence <SEQ ID 716; ORF67>:

1   ..MPSEGSDGXG XGEXEXVAHA QXDFVGFEAG VFQASPVVVT
    VSGVXXQLGX
51  DVETDTGDDT KTXAADXVAF VIGRFXGXXL YXXAXXXXAX
    XWXXXXSRGF
101 XXHRMNLMFN VSVGDARADI GFEFIVEFEI VNGGQAERRN
    GVEAAVSLMF
151 CLGFFVVVVY LFSNFFSRRI TFFPFSVTGI ICRYSPAAEI
    ..

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. gonorrhoeae

ORF67 shows 51.8% identity over 199 aa overlap with a predicted ORF (ORF67ng) from N. gonorrhoeae:

The ORF67ng nucleotide sequence <SEQ ID 717> is predicted to encode a protein comprising amino acid sequence <SEQ ID 718>:

1   MPSETVGSIV NVGVDESVGF SPPFPSIQHF YRFHRIHRIR
    LFRPPGPMQL
51  NRHSHGSGNL GRGVWATVLS DKFPCGQVRI PACAGMTNFE
    IAVLSGMTVR
101 VFYCARPAPV NGGRLKMPSE GSDGIGIGES EAVAHAQRGF
    VGFEAGVFQA
151 SPVVVAVAGV QGQAGRDVYA HARHRAEAQA AAAVAFLIGV
    FLRMSVRINR
201 NCCVSITRVG GKSTCYFFSR IDAVSDVSVG DARTDIGFEF
    VVEFEIVNGG
251 QAERRNGVEC AVFLMFRLLV FYVKLVAAKS FIILSFQLFY
    VHGIFIVVPF
301 PVTGIIRGDA PAAEVVADRH PGVDGMRTDV SEIIAYRAYF
    VFAWSGWFRI
351 IVGNAFGGVG *

Based on the presence of a several putative transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 86

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 719>

1   ATGTTTGCTT TTTTAGAAGC CTTTTTTGTC GAATACGGTT
    ATGCGGCTGT
51  TTTTTTTGTA TTGGTCATCT GCGGTTTCGG CGTGCCGATT
    CCCGAGGATT
101 TGACCTTGGT AACAGGCGGC GTGATTTCGG GTATGGGTTA
    TACCAATCCG
151 CATATTATGT TTGCAGTCGG TATGCTCGGC GTATTGGTCG
    GGGACGGCAT
201 CATGTTCGCC GCCGGACGAA TTTGGGGGCA GArArTCCTA
    rGGTTCArAC
251 CTATTGCGsG CATCATGACG CCGrAACGTT ATGAGCAGGT
    TCAGGAAAAA
301 TTCGACAAAT ACGGTAACTG GGTCTTATTT GTCGCCCGTT
    TCCTGCCCGG
351 TTTGAGAACG GCCGTATTTG TTACAGCCGG TATCAGCCGC
    AAGGTTTCAT
401 ACTTGCGTTT TATCATTATG GATGGACTGG CCGCA...

This corresponds to the amino acid sequence <SEQ ID 720; ORF78>:

1   MFAFLEAFFV EYGYAAVFFV LVICGFGVPI PEDLTLVTGG
    VISGMGYTNP
51  HIMFAVGMLG VLVGDGIMFA AGRIWGQXXL XFXPIAXIMT
    PXRYEQVQEK
101 FDKYGNWVLF VARFLPGLRT AVFVTAGISR KVSYLRFIIM
    DGLAA...

Further work revealed the complete nucleotide sequence <SEQ ID 721>:

1   ATGTTTGCTT TTTTAGAAGC CTTTTTTGTC GAATACGGTT
    ATGCGGCTGT
51  TTTTTTTGTA TTGGTCATCT GCGGTTTCGG CGTGCCGATT
    CCCGAGGATT
101 TGACCTTGGT AACAGGCGGC GTGATTTCGG GTATGGGTTA
    TACCAATCCG
151 CATATTATGT TTGCAGTCGG TATGCTCGGC GTATTGGTCG
    GGGACGGCAT
201 CATGTTCGCC GCCGGACGAA TTTGGGGGCA GAAAATCCTA
    AGGTTCAAAC
251 CTATTGCGCG CATCATGACG CCGAAACGTT ATGAGCAGGT
    TCAGGAAAAA
301 TTCGACAAAT ACGGTAACTG GGTCTTATTT GTCGCCCGTT
    TCCTGCCCGG
351 TTTGAGAACG GCCGTATTTG TTACAGCCGG TATCAGCCGC
    AAGGTTTCAT
401 ACTTGCGTTT TATCATTATG GATGGACTGG CCGCACTGAT
    TTCCGTCCCT
451 ATTTGGATTT ATCTGGGCGA ATACGGTGCG CACAACATCG
    ATTGGCTGAT
501 GGCGAAAATG CACAGCCTGC AATCGGGTAT TTTTGTTATC
    TTGGGTATAG
551 GTGCGACCGT TGTCGCTTGG ATTTGGTGGA AAAAACGCCA
    ACGTATCCAG
601 TTTTACCGCA GCAAATTGAA AGAAAAGCGG GCGCAACGCA
    AAGCCGCCAA
651 GGCAGCCAAA AAAGCCGCGC AAAGCAAACA ATAA

This corresponds to the amino acid sequence <SEQ ID 722; ORF78-1>:

1   MFAFLEAFFV EYGYAAVFFV LVICGFGVPI PEDLTLVTGG
    VISGMGYTNP
51  HIMFAVGMLG VLVGDGIMFA AGRIWGQKIL RFKPIARIMT
    PKRYEQVQEK
101 FDKYGNWVLF VARFLPGLRT AVFVTAGISR KVSYLRFIIM
    DGLAALISVP
151 IWIYLGEYGA HNIDWLMAKM HSLQSGIFVI LGIGATVVAW
    IWWKKRQRIQ
201 FYRSKLKEKR AQRKAAKAAK KAAQSKQ*

Computer analysis of this amino acid sequence predicts several transmembrane domains, and also gave the following results:

Homology with the dedA Homologue of H. influenzae (Accession Number P45280)

ORF78 and the dedA homologue show 58% aa identity in 144aa overlap:

Orf78:	4	FLEAFFVEYGYAAVFFVLVICGFGVPIPEDLTLVTGGVISGM--GYTNPHIMFAVGMLGV	61
		FL  FF EYGY AV FVL+ICGFGVPIPED+TLV+GGVI+G+     N H+M  V M+GV
DedA:	20	FLIGFFTEYGYWAVLFVLIICGFGVPIPEDITLVSGGVIAGLYPENVNSHLMLLVSMIGV	79
Orf78:	62	LVGDGIMFAAGRIWGQXXLXFXPIAXIMTPKRYEQVQEKFDKYGNWVLFVARFLPGLRTA	121
		L GD  M+  GRI+G   L F PI  I+T  R   V+EKF +YGN VLFVARFLPGLR
DedA:	80	LAGDSCMYWLGRIYGTKILRFRPIRRIVTLQRLRMVREKFSQYGNRVLFVARFLPGLRAP	139
Orf78:	122	VFVTAGISRKVSYLRFIIMDGLAA	145
		+++ +GI+R+VSY+RF+++D  AA
DedA:	140	IYMVSGITRRVSYVRFVLIDFCAA	163

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF78 shows 93.8% identity over a 145aa overlap with an ORF (ORF78a) from strain A of N. meningitidis:

The complete length ORF78a nucleotide sequence <SEQ ID 723> is:

1   ATGTTTGCCC TTTTGGAAGC CTTTTTTGTC GAATACGGCT
    ATGCGGCCGT
51  GTTTTTCGTT TTGGTCATCT GCGGTTTCGG CGTGCCGATT
    CCCGAGGATT
101 TGACCTTGGT AACAGGCGGC GTGATTTCGG GTATGGGTTA
    TACCAATCCG
151 CATATTATGT TTGCAGTCGG TATGCTCGGC GTATTGGTCG
    GGGACGGCAT
201 CATGTTCGCC GCCGGACGCA TCTGGGGGCA GAAAATCCTC
    AAGTTCAAAC
251 CGATTGCGCG CATCATGACG CCGAAACGTT ACGCACAGGT
    TCAGGAAAAA
301 TTCGACAAAT ACGGCAACTG GGTGTTATTT GTCGCTCGTT
    TCCTGCCCGG
351 TTTGCGGACT GCCGTTTTCG TTACCGCCGG CATCAGCCGC
    AAAGTATCGT
401 ATCTGCGCTT TCTGATTATG GACGGGCTTG CCGCGCTGAT
    TTCCGTGCCC
451 GTTTGGATTT ACTTGGGCGA GTACGGCGCG CACAACATCG
    ATTGGCTGAT
501 GGCGAAAATG CACAGCCTGC AATCCGGCAT CTTCATCGCA
    TTGGGCGTGC
551 TGGCGGCGGC GCTGGCGTGG TTCTGGTGGC GCAAACGCCG
    ACATTATCAG
601 CTTTACCGCG CACAATTGAG CGAAAAACGC GCCAAACGCA
    AGGCGGAAAA
651 GGCAGCGAAA AAAGCGGCAC AGAAGCAGCA GTAA

This encodes a protein having amino acid sequence <SEQ ID 724>:

1   MFALLEAFFV EYGYAAVFFV LVICGFGVPI PEDLTLVTGG
    VISGMGYTNP
51  HIMFAVGMLG VLVGDGIMFA AGRIWGQKIL KFKPIARIMT
    PKRYAQVQEK
101 FDKYGNWVLF VARFLPGLRT AVFVTAGISR KVSYLRFLIM
    DGLAALISVP
151 VWIYLGEYGA HNIDWLMAKM HSLQSGIFIA LGVLAAALAW
    FWWRKRRHYQ
201 LYRAQLSEKR AKRKAEKAAK KAAQKQQ*

ORF78a and ORF78-1 show 89.0% identity in 227 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF78 shows 97.4% identity over 38 aa overlap with a predicted ORF (ORF78ng) from N. gonorrhoeae:

The ORF78ng nucleotide sequence <SEQ ID 725> is predicted to encode a protein comprising amino acid sequence <SEQ ID 726>:

1   ..YPVLFVARFL PGLRTAVFVT AGISRKVSYL RFLIMDGLAA
    LISVPVWIYL
51  GEYGAHNIDW LMAKMHSLQS GIFIALGVLA AALAWFWWRK
    RRHYQLYRAQ
101 LSEKRAKRKA EKAAKKAAQK QQ*

Further work revealed the complete gonococcal nucleotide sequence <SEQ ID 727>:

1   atgtttgccc tttTggaagc CTTTTTTGTC GAAtacggCt
    atgcGGCCGT
51  GTTTTTCGTT TTGGTCATCT GCGGTTTCGG CGTGCCGATT
    CCCGAAGATT
101 TGACCTTGGT AACGGGCGGC GTGATTTCGG GTATGGGTTA
    TACCAATCCG
151 CATATTATGT TTGCGGTCGG TATGCTCGGC GTGTTGGCGG
    GCGACGGCGT
201 GATGTTTGCC GCCGGACGCA TCTGGGGGCA GAAAATCCTC
    AAGTTCAAAC
251 CGATTGCGCG CATCATGACG CCGAAACGTT ACGCGCAGGT
    TCAGGAAAAA
301 TTCGACAAAT ACGGCAACTG GGTTCTGTTT GTCGCCCGTT
    TCCTGCCGGG
351 TTTGCGGACT GCCGTTTTCG TTACCGCCGG CATCAGCCGC
    AAAGTATCGT
401 ATCTGCGCTT TCTGATTATG GACGGGCTGG CCGCGCTGAT
    TTCCGTGCCC
451 GTTTGGATTT ACTTGGGCGA GTACGGCGCG CACAACATCG
    ATTGGCTGAT
501 GGCGAAAATG CACAGCCTGC AATCGGGCAT CTTCATCGCA
    TTGGGCGTGC
551 TGGCGGCGGC GCTGGCGTGG TTCTGGTGGC GCAAACGCCG
    ACATTATCAG
601 CTTTACCGCG CACAATTGAG CGAAAAACGC GCCAAACGCA
    AGGCGGAAAA
651 GGCAGCGAAA AAAGCGGCAC AGAAGCAGCA GTAa

This corresponds to the amino acid sequence <SEQ ID 728; ORF78ng-1>:

1   MFALLEAFFV EYGYAAVFFV LVICGFGVPI PEDLTLVTGG
    VISGMGYTNP
51  HIMFAVGMLG VLAGDGVMFA AGRIWGQKIL KFKPIARIMT
    PKRYAQVQEK
101 FDKYGNWVLF VARFLPGLRT AVFVTAGISR KVSYLRFLIM
    DGLAALISVP
151 VWIYLGEYGA HNIDWLMAKM HSLQSGIFIA LGVLAAALAW
    FWWRKRRHYQ
201 LYRAQLSEKR AKRKAEKAAK KAAQKQQ*

ORF78ng-1 and ORF78-1 show 88.1% identity in 227 aa overlap:

Furthermore, orf78ng-1 shows homology to the dedA protein from H. influenzae:

sp|P45280|YG29_HAEIN HYPOTHETICAL PROTEIN HI1629 >gi|1073983|pir||D64133
dedA protein (dedA) homolog - Haemophilus influenzae (strain Rd KW20)
>gi|1574476 (U32836) dedA protein (dedA) [Haemophilus influenzae]
Length = 212 Score = 223 bits (563), Expect = 7e−58
Identities = 108/182 (59%), Positives = 140/182 (76%), Gaps = 2/182 (1%)
Query:	5	LEAFFVEYGYAAVFFVLVICGFGVPIPEDLTLVTGGVISGM--GYTNPHIMFAVGMLGVL	62
		L  FF EYGY AV FVL+ICGFGVPIPED+TLV+GGVI+G+     N H+M  V M+GVL
Sbjct:	21	LIGFFTEYGYWAVLFVLIICGFGVPIPEDITLVSGGVIAGLYPENVNSHLMLLVSMIGVL	80
Query:	63	AGDGVMFAAGRIWGQKILKFKPIARIMTPKRYAQVQEKFDKYGNWVLFVARFLPGLRTAV	122
		AGD  M+  GRI+G KIL+F+PI RI+T +R   V+EKF +YGN VLFVARFLPGLR  +
Sbjct:	81	AGDSCMYWLGRIYGTKILRFRPIRRIVTLQRLRMVREKFSQYGNRVLFVARFLPGLRAPI	140
Query:	123	FVTAGISRKVSYLRFLIMDGLAALISVPVWIYLGEYGAHNIDWLMAKMHSLQSGIFIALG	182
		++ +GI+R+VSY+RF+++D  AA+ISVP+WIYLGE GA N+DWL  ++   Q  I+I +G
Sbjct:	141	YMVSGITRRVSYVRFVLIDFCAAIISVPIWIYLGELGAKNLDWLHTQIQKGQIVIYIFIG	200
Query:	183	VL	184
		L
Sbjct:	201	YL	202

Based on this analysis, including the presence of putative transmembrane domains, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 87

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 729>:

1   ATGAAAAAAT TATTGGCGGC CGTGATGATG GCAGGTTTGG
    CAGGCGCGGT
51  TTCCGCCGCC GGAGTCCACG TTGAGGACGG CTGGGCGCGC
    ACCACCGTCG
101 AAGGTATGAA AATAGGCGGC GCGTTCATGA AAATCCACAA
    CGACGAAGCC
151 AAACAAGACT TTTTGCTCGG CGGAAGCAGC CCCGTTGCCG
    ACCGCGTCGA
201 AGTGCATACC CACATCAACG ACAACGGCGT GATGCGGATG
    CGCGAAGTCG
251 AAGGCGGCGT GCCTTTGGAA GCGAAATCCG TTACCGAACT
    CAAACCCGGC
301 AGCTATCATG TGATGTTTAT GGGTTTGAAA AAACAATTAA
    AAGAGGGCGA
351 TAAAATTCCC GTTACCCTGA AATTTAAAAA CGCCAAAGCG
    CAAACCGTCC
401 AACTGGAAGT CAAAATCGCG CCGATGCCGG CAATGAACCA
    C...

This corresponds to the amino acid sequence <SEQ ID 730; ORF79>:

1   MKKLLAAVMM AGLAGAVSAA GVHVEDGWAR TTVEGMKIGG
    AFMKIHNDEA
51  KQDFLLGGSS PVADRVEVHT HINDNGVMRM REVEGGVPLE
    AKSVTELKPG
101 SYHVMFMGLK KQLKEGDKIP VTLKFKNAKA QTVQLEVKIA
    PMPAMNH..

Further work revealed the complete nucleotide sequence <SEQ ID 731>:

1   ATGAAAAAAT TATTGGCGGC CGTGATGATG GCAGGTTTGG
    CAGGCGCGGT
51  TTCCGCCGCC GGAGTCCACG TTGAGGACGG CTGGGCGCGC
    ACCACCGTCG
101 AAGGTATGAA AATAGGCGGC GCGTTCATGA AAATCCACAA
    CGACGAAGCC
151 AAACAAGACT TTTTGCTCGG CGGAAGCAGC CCCGTTGCCG
    ACCGCGTCGA
201 AGTGCATACC CACATCAACG ACAACGGCGT GATGCGGATG
    CGCGAAGTCG
251 AAGGCGGCGT GCCTTTGGAA GCGAAATCCG TTACCGAACT
    CAAACCCGGC
301 AGCTATCATG TGATGTTTAT GGGTTTGAAA AAACAATTAA
    AAGAGGGCGA
351 TAAAATTCCC GTTACCCTGA AATTTAAAAA CGCCAAAGCG
    CAAACCGTCC
401 AACTGGAAGT CAAAATCGCG CCGATGCCGG CAATGAACCA
    CGGTCATCAC
451 CACGGCGAAG CGCATCAGCA CTAA

This corresponds to the amino acid sequence <SEQ ID 732; ORF79-1>:

1   MKKLLAAVMM AGLAGAVSAA GVHVEDGWAR TTVEGMKIGG
    AFMKIHNDEA
51  KQDFLLGGSS PVADRVEVHT HINDNGVMRM REVEGGVPLE
    AKSVTELKPG
101 SYHVMFMGLK KQLKEGDKIP VTLKFKNAKA QTVQLEVKIA
    PMPAMNHGHH
151 HGEAHQH*

Computer analysis of this amino acid sequence revealed a putative leader peptide and also gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF79 shows 94.6% identity over a 147aa overlap with an ORF (ORF79a) from strain A of N. meningitidis:

The complete length ORF79a nucleotide sequence <SEQ ID 733> is:

1   ATGAAANAAC TATTGGCAGC CGTGATGATG GCAGGTTTGG
    CAGGCGCGGT
51  TTCCGCCGCC GGAATCCACG TTGAGGACGG CTGGGCGCGC
    ACCACCGTCG
101 AAGGTATGAA AATGGGCGGC GCGTTCATGA AAATCCACAA
    CGACGAAGCC
151 AAACAAGACT TTTTGCTCGG CGGAAGCAGC CCTGTTGCCG
    ACCGCGTCGA
201 AGTGCATACC CATATCAATG ATAACGGTGT GATGCGGATG
    CGCGAAGTCG
251 AAGGCGGCGT GCCTTTGGAG GCGAAATCCG TTACCGAACT
    CAAACCCGGC
301 AGCTATCATG TCATGTTTAT GGGTNTGAAA AAACAATTAA
    AAGANGGCGA
351 CAAGATTCCC GTTACCCTGA AATTTAAAAA CGCCAAAGCA
    CAAACCGTCC
401 AACTGGAAGT CAAAACCGCG CCGATGTCGG CAATGGACCA
    CGGTCATCAC
451 CACGGCGAAG CGCATCAGCA CTAA

This encodes a protein having amino acid sequence <SEQ ID 734>:

1   MKXLLAAVMM AGLAGAVSAA GIHVEDGWAR TTVEGMKMGG
    AFMKIHNDEA
51  KQDFLLGGSS PVADRVEVHT HINDNGVMRM REVEGGVPLE
    AKSVTELKPG
101 SYHVMFMGXK KQLKXGDKIP VTLKFKNAKA QTVQLEVKTA
    PMSAMDHGHH
151 HGEAHQH*

ORF79a and ORF79-1 show 94.9% identity in 157 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF79 shows 96.1% identity over 76 aa overlap with a predicted ORF (ORF79ng) from N. gonorrhoeae:

An ORF79ng nucleotide sequence <SEQ ID 735> was predicted to encode a protein comprising amino acid sequence <SEQ ID 736>:

1  ..INDNGVMRMR EVKGGVPLEA KSVTELKPGS YHVMFMGLKK
   QLKEGDKIPV
51 TLKFKNAKAQ TVQLEVKTAP MSAMNHGHHH GEAHQH*

Further work revealed the complete gonococcal DNA sequence <SEQ ID 737>:

1   ATGAAAAAAT TATTGGCAGC CGTGATGATG GCAGGTTTGG
    CAGGCGCGGT
51  TTccgccgCc GGagTccAtG TCGAggACGG CTGGGCGCGc
    accaCTGtcg
101 aaggtATgaa aatggGCGGC GCgttCATga aaATCCACAA
    CGACGaaGcc
151 atacaaGACt ttgtgcTCgg CGGaagcatg cccgttgccg
    accgcGTCGA
201 AGTGCAtaca cacATCAACG ACAACGGCGT GATGCGTATG
    CGCGAAGTCA
251 AAGGCGGCGT GCCTTTGGAG GCGAAATCCG TTACCGAACT
    CAAACCCGGC
301 AGCTATCACG TGATGTTTAT GGGTTTGAAA AAACAACTGA
    AAGAGGGCGA
351 CAAGATTCCC GTTACCCTGA AATTTAAAAA CGCCAAAGCG
    CAAACCGTCC
401 AACTGGAAGT CAAAACCGCG CCGATGTCGG CAATGAACCA
    CGGTCATCAC
451 CACGGCGAAG CGCATCAGCA CTAA

This corresponds to the amino acid sequence <SEQ ID 738; ORF79ng-1>:

1   MKKLLAAVMM AGLAGAVSAA GVHVEDGWAR TTVEGMKMGG
    AFMKIHNDEA
51  IQDFVLGGSM PVADRVEVHT HINDNGVMRM REVKGGVPLE
    AKSVTELKPG
101 SYHVMFMGLK KQLKEGDKIP VTLKFKNAKA QTVQLEVKTA
    PMSAMNHGHH
151 HGEAHQH*

ORF79ng-1 and ORF79-1 show 95.5% identity in 157 aa overlap:

Furthermore, ORF79ng-1 shows significant homology to a protein from Aquifex aeolicus:

gi|2983695 (AE000731) putative protein [Aquifex aeolicus] Length = 151
Score = 63.6 bits (152), Expect = 6e−10
Identities = 38/114 (33%), Positives = 58/114 (50%), Gaps = 1/114 (0%)
Query:	24	VEDGWARTTVEGMKMGGAFMKIHNDEAIQDFVLGGSMPVADRVEVHTHINDNGVMRMREV	83
		V+  W      G       M I N+    D+++G    +A RVE+H  + +N V +M
Sbjct:	27	VKHPWVMEPPPGPNTTMMGMIIVNEGDEPDYLIGAKTDIAQRVELHKTVIENDVAKMVPQ	86
Query:	84	KGGVPLEAKSVTELKPGSYHVMFMGLKKQLKEGDKIPVTLKFKNAKAQTVQLEV	137
		+  + +  K   E K   YHVM +GLKK++KEGDK+ V L F+ +   TV+  V
Sbjct:	87	ER-IEIPPKGKVEFKHHGYHVMIIGLKKRIKEGDKVKVELIFEKSGKITVEAPV	139

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF79-1 (15.6 kDa) was cloned in the pET vector and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 18A shows the results of affinity purification of the His-fusion protein. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA (positive result) and FACS analysis (FIG. 18B) These experiments confirm that ORF79-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 88

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 739>:

1   ATGACGGTAA CTGCGGCCGA AGGCGGCAAA GCTGCCAAGG
    CGTTAAAAAA
51  ATATCTGATT ACGGGCATTT TGGTCTGGCT GCCGATTGCG
    GTAACGGTTT
101 GGGTGGTTTC CTATATCGTT TCCGCGTCCG ATCAGCTCGT
    CAACCTGCTG
151 CCGAAGCAAT GGCGGCCGCA ATATGTTTTG GGGTTTAATA
    TCCCGGGGCT
201 GGGCGTTATC GTTGCCATTG CCGTATTGTT TGTAACCGGA
    TTGTTTGCCG
251 CCAACGTATT GGGTCGGCAG ATCCTCGCCG CGTGGGACAG
    CCTGTTGGGG
301 CGGATTCCGG TTGTGAAAtC CATCTATTCG AGTGTGAAAA
    AAGTATCCGA
351 ATacgTGCTG TCCGACAGCA GCCGTTCGTT TAAAACGCCG
    GTACTCGTGC
401 CGTTTCCCCA GCCCGGTATT TGGACGATyG CTTTCGTGTC
    AGGGCAGGTG
451 TCGAATGCGG TTAAGGCCGC ATTGCCGAAs GACGGCGATT
    ATCTTTCCGT
501 GTATGTTCCG ACCACGCCGA ATCCGACCGG CGGTTACTAT
    ATTATGGTAA
551 AGAAAAGCGA TGTGCGCGAA CTCGATATGA GCGTGGACGA
    AsCATTGAAA
601 TATGTGATTT CGCTGGGTAT GGTCATCCCT GACGACCTGC
    CCGTCAAAAC
651 ATTGGCAsGA CCTATGCCGT CTGAAAAGGC GGATTTGCCC
    GAACAACAAT
701 AA

This corresponds to the amino acid sequence <SEQ ID 740; ORF98>:

1   MTVTAAEGGK AAKALKKYLI TGILVWLPIA VTVWVVSYIV
    SASDQLVNLL
51  PKQWRPQYVL GFNIPGLGVI VAIAVLFVTG LFAANVLGRQ
    ILAAWDSLLG
101 RIPVVKSIYS SVKKVSEYVL SDSSRSFKTP VLVPFPQPGI
    WTIAFVSGQV
151 SNAVKAALPX DGDYLSVYVP TTPNPTGGYY IMVKKSDVRE
    LDMSVDEXLK
201 YVISLGMVIP DDLPVKTLAX PMPSEKADLP EQQ*

Further work revealed the complete nucleotide sequence <SEQ ID 741>:

1   ATGACGGAAC nTGCGGCCGA AGGCGGCAAA GCTGCCAArG
    CGTTAAAAAA
51  ATATCTGATT ACGGGCATTT TGGTCTGGCT GCCGATTGCG
    GTAACGGTTT
101 GGGTGGTTTC CTATATCGTT TCCGCGTCCG ATCAGCTCGT
    CAACCTGCTG
151 CCGAAGCAAT GGCGGCCGCA ATATGTTTTG GGGTTTAATA
    TCCCGGGGCT
201 GGGCGTTATC GTTGCCATTG CCGTATTGTT TGTAACCGGA
    TTGTTTGCCG
251 CCAACGTATT GGGTCGGCAG ATCCTCGCCG CGTGGGACAG
    CCTGTTGGGG
301 CGGATTCCGG TTGTGAAATC CATCTATTCG AGTGTGAAAA
    AAGTATCCGA
351 ATCGCTGCTG TCCGACAGCA GCCGTTCGTT TAAAACGCCG
    GTACTCGTGC
401 CGTTTCCCCA GCCCGGTATT TGGACGATTG CTTTCGTGTC
    AGGGCAGGTG
451 TCGAATGCGG TTAAGGCCGC ATTGCCGAAG GACGGCGATT
    ATCTTTCCGT
501 GTATGTTCCG ACCACGCCGA ATCCGACCGG CGGTTACTAT
    ATTATGGTAA
551 AGAAAAGCGA TGTGCGCGAA CTCGATATGA GCGTGGACGA
    AGCATTGAAA
601 TATGTGATTT CGCTGGGTAT GGTCATCCCT GACGACCTGC
    CCGTCAAAAC
651 ATTGGCAGGA CCTATGCCGT CTGAAAAGGC GGATTTGCCC
    GAACAACAAT
701 AA

This corresponds to the amino acid sequence <SEQ ID 742; ORF98-1>:

1   MTEXAAEGGK AAKALKKYLI TGILVWLPIA VTVWVVSYIV
    SASDQLVNLL
51  PKQWRPQYVL GFNIPGLGVI VAIAVLFVTG LFAANVLGRQ
    ILAAWDSLLG
101 RIPVVKSIYS SVKKVSESLL SDSSRSFKTP VLVPFPQPGI
    WTIAFVSGQV
151 SNAVKAALPK DGDYLSVYVP TTPNPTGGYY IMVKKSDVRE
    LDMSVDEALK
201 YVISLGMVIP DDLPVKTLAG PMPSEKADLP EQQ*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF98 shows 96.1% identity over a 233aa overlap with an ORF (ORF98a) from strain A of N. meningitidis:

The complete length ORF98a nucleotide sequence <SEQ ID 743> is:

1   ATGACGGAAC CTGCGGCCGA AGGCGGCAAA GCTGCCAAGG
    CGTTAAAAAA
51  ATATCTGATT ACGGGCATTT TGGTCTGGCT GCCGATTGCG
    GTAACGGTTT
101 GGGTGGTTTC CTATATCGTT TCCGCGTCCG ATCAGCTCGT
    CAACCTGCTG
151 CCGAAGCAAT GGCGGCCGCA ATATGTTTTG GGGTTTAATA
    TCCCGGGGCT
201 GGGCGTTATC GTTGCCATTG CCGTATTGTT TGTAACCGGA
    TTATTTGCCG
251 CAAACGTATT GGGCCGGCAG ATTCTTGCCG CGTGGGACAG
    CTTGTTGGGG
301 CGGATTCCGG TTGTGAAGTC CATCTATTCG AGTGTGAAAA
    AAGTATCCGA
351 NTCGTTGCTG TCCGACAGCA GCCGTTCGTT TAAAACACCA
    GTACTCGTGC
401 CGTTTCCCCA ATCGGGTATT TGGACAATCG CATTCGTGTC
    CGGTCAGGTG
451 TCGAATGCGG TTAAGGCCGC ATTGCCGAAG GACGGCGATT
    ATCTTTCCGT
501 GTATGTTCCG ACCACGCCGA ATCCGACCGG CGGTTACTAT
    ATTATGGTAA
551 AGAAAAGCGA TGTGCGCGAA CTCGATATGA GCGTGGACGA
    AGCGTTGAAA
601 TATGTGATTT CGCTGGGTAT GGTCATCCCT GACGACCTGC
    CCGTCAAAAC
651 ATTGGCAGGA CCTATGCCGT CTGAAAAGGC GGATTTGCCC
    GAACAACAAT
701 AA

This encodes a protein having amino acid sequence <SEQ ID 744>:

1   MTEPAAEGGK AAKALKKYLI TGILVWLPIA VTVWVVSYIV
    SASDQLVNLL
51  PKQWRPQYVL GFNIPGLGVI VAIAVLFVTG LFAANVLGRQ
    ILAAWDSLLG
101 RIPVVKSIYS SVKKVSXSLL SDSSRSFKTP VLVPFPQSGI
    WTIAFVSGQV
151 SNAVKAALPK DGDYLSVYVP TTPNPTGGYY IMVKKSDVRE
    LDMSVDEALK
201 YVISLGMVIP DDLPVKTLAG PMPSEKADLP EQQ*

ORF98a and ORF98-1 show 98.7% identity in 233 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF98 shows 95.3% identity over a 233 aa overlap with a predicted ORF (ORF98ng) from N. gonorrhoeae:

The complete length ORF98ng nucleotide sequence <SEQ ID 745> is predicted to encode a protein having amino acid sequence <SEQ ID 746>:

1   MTEPAAEGGK AAKALKKYLI TGILVWLPIA VTVWVVSYIV
    SASDQLVNLL
51  PKQWRPQYVL GFNIPGLGVI VAIAVLFVTG LFAANVLGRQ
    ILAAWDSLLX
101 RIPVVKSIYS SVKKVSESLL SDSSRSFKTP VLVPFPQSGI
    WTIAFVSGQV
151 SNAVKAALPQ DGDYLSVYVP TTPNPTGGYY IMVKKSDVRE
    LDMSVDEALK
201 YVISLGMVIP DDLPVKTLAG PMPPEKAELP EQQ*

Further work revealed the complete nucleotide sequence <SEQ ID 747>:

1   ATGACGGAAC CTGCGGCCGA AGGCGGCAAA GCTGCCAAGG
    CGTTAAAAAA
51  ATATCTGATT ACAGGCATTT TGGTCTGGCT GCCGATTGCG
    GTAACGGTTT
101 GGGTGGTTTC CTATATCGTT TCCGCGTCCG ACCAGCTTGT
    CAACCTGCTG
151 CCGAAGCAAT GGCGGCCGCA ATATGTTTTG GGGTTTAATA
    TCCCCGGGCT
201 CGGCGTTATT GTTGCCATTG CCGTATTGTT TGTAACCGGA
    TTATTTGCCG
251 CAAACGTGTT GGGCCGGCAG ATTCTTGCCG CGTGGGACAG
    CCTGTTgggg
301 cggaTTCCGG TTGTCAAATC CATCTATTCG AGTGTGAAAA
    AAGTATCCGA
351 ATCGCTGCTG TCCGACAGCA GCCGTTCGTT TAAAACGCCG
    GTACTCGTGC
401 CGTTTCCCCA ATCGGGTATT TGGACAATCG CATTCGTGTC
    CGGTCAGGTG
451 TCGAATGCGG TTAAGGCCGC ATTGCCGCAG GATGGCGATT
    ATCTTTCCGT
501 GTATGTCCCG ACCACGCCCA ACCCGACCGG CGGTTACTAT
    ATTATGGTAA
551 AGAAAAGCGA TGTGCGCGAA CTCGATATGA GCGTGGACGA
    AGCGTTGAAA
601 TATGTGATTT CGCTGGGTAT GGTCATCCCT GACGACCTGC
    CCGTCAAAAC
651 ATTGGCAGGA CCTATGCCGC CTGAAAAGGC GGAGTTGCCC
    GAACAACAAT
701 AA

This corresponds to the amino acid sequence <SEQ ID 748; ORF98ng-1>:

1   MTEPAAEGGK AAKALKKYLI TGILVWLPIA VTVWVVSYIV
    SASDQLVNLL
51  PKQWRPQYVL GFNIPGLGVI VAIAVLFVTG LFAANVLGRQ
    ILAAWDSLLG
101 RIPVVKSIYS SVKKVSESLL SDSSRSFKTP VLVPFPQSGI
    WTIAFVSGQV
151 SNAVKAALPQ DGDYLSVYVP TTPNPTGGYY IMVKKSDVRE
    LDMSVDEALK
201 YVISLGMVIP DDLPVKTLAG PMPPEKAELP EQQ*

ORF98ng-1 and ORF98-1 show 97.9% identity in 233 aa overlap:

Based on this analysis, including the fact that the putative transmembrane domains in the gonococcal protein are identical to the sequences in the meningococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 89

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 749>:

1    ATgAAAACGG TAGTCTGGAT TGTCGTCCTG TTTGCCGCCG
     CCGTCGGACT
51   GGCGCTGGCT TCGGGCATTT ACACCGGCGA CGTGTATATC
     GTACTCGGAC
101  AGACCATGCT CAGAATCAAC CTGCACGCCT TTGTGTTAGG
     TTCGCTGATT
151  GCCGTCGTGG TGTGGTATTT CTTGTTTAAA TTCATTATCG
     GsGgTACTCA
201  ATATCCCCGA AAAGATGCAG CGTTTCGGTT CGGCnCGTAA
     AGGCCkCAAG
251  ssCGsGCTTG CCTTGAACAA GGCGGGTTTG GCGTATTTTG
     AAGGGCGTTT
301  TGAAAAGGCG GAACTAGAAG CCTCACGCGT GTTGGTCAAC
     AAAGtAGGCC
351  GaGAGACAAC CGGACTTTGG CATTGATGCT GrGCGCGCAC
     GCCGCCGGAC
401  AGATGGAAAA CATCGAssTG CGCGACCGTT ATCTTGCGGA
     AATCGCCAAA
451  CTGCCGGAAA AACAGCAGCT TTCCCGTTAT CTTTTGTTGG
     CGGAATCGGC
501  GTTGAACCGG CGCGATTACG AAGCGGCGGA AGCCAATCTT
     CATGCGGCGG
551  CGAAGATGAA TGCCAACCTT ACGCGCCTCG TGCGTCTGCA
     .ATTCGTTAC
601  GCTTTCGACA GGGGCGACGC GTTGCAGGTT CTGGCAAAAA
     CCGAAAAACT
651  TTCCAAGGCG GGCGCGTTGG GCAAATCGGA AATGGAACGG
     TATCAAAATT
701  GGGCATATCC GTCGCCAGCT GGCGGATGCT GCCGATGCCG
     CCGCTTTGAA
751  AACCTGCCTG AAGCGGATTC CCGACAGCCT CAAAAACGGG
     GAATTGAGCG
801  TATCGGTTGC GGAAAAGTAC GAACGTTTGG GACTGTATGC
     CGATGCGGTC
851  AAATGGGTCA AACAGCATTA TCCGCAsAAC CGCCGCCCCG
     AGCTTTTGGA
901  AGCCTTTGTC GAAAGCGTGC GCTTTTTGGG CGAGCGCGAA
     CAGCAGAAAG
951  CCATCGATTT TGCCGATGCT TGGCTGAAAG AACAGCCCGA
     TAACGCGCTT
1001 CTGCTGATGT ATCTCGGTCG GCTCGCCTTC GGCCGCAAAC
     TTTGGGGCAA
1051 GGCAAAAGGC TACCTTGAAG CGAGCATTGC ATTAAAGCCG
     AGTATTTCCG
1101 CGCGTTTGGT TCTAACAAAG GTTTTCGACG AAATCGGAGA
     ACCGCAGAAG
1151 GCGGAGGCGC AC...

This corresponds to the amino acid sequence <SEQ ID 750; ORF100>:

1   MKTVVWIVVL FAAAVGLALA SGIYTGDVYI VLGQTMLRIN
    LHAFVLGSLI
51  AVVVWYFLFK FIIGVLNIPE KMQRFGSARK GXKXXLALNK
    AGLAYFEGRF
101 EKAELEASRV LVNKVGRDNR TLALMLXAHA AGQMENIXXR
    DRYLAEIAKL
151 PEKQQLSRYL LLAESALNRR DYEAAEANLH AAAKMNANLT
    RLVRLXIRYA
201 FDRGDALQVL AKTEKLSKAG ALGKSEMERY QNWAYRRQLA
    DAADAAALKT
251 CLKRIPDSLK NGELSVSVAE KYERLGLYAD AVKWVKQHYP
    XNRRPELLEA
301 FVESVRFLGE REQQKAIDFA DAWLKEQPDN ALLLMYLGRL
    AFGRKLWGKA
351 KGYLEASIAL KPSISARLVL TKVFDEIGEP QKAEAH...

Further work revealed the complete nucleotide sequence <SEQ ID 751>:

1    ATGAAAACGG TAGTCTGGAT TGTCGTCCTG TTTGCCGCCG
     CCGTCGGACT
51   GGCGCTGGCT TCGGGCATTT ACACCGGCGA CGTGTATATC
     GTACTCGGAC
101  AGACCATGCT CAGAATCAAC CTGCACGCCT TTGTGTTAGG
     TTCGCTGATT
151  GCCGTCGTGG TGTGGTATTT CTTGTTTAAA TTCATTATCG
     GCGTACTCAA
201  TATCCCCGAA AAGATGCAGC GTTTCGGTTC GGCGCGTAAA
     GGCCGCAAGG
251  CCGCGCTTGC CTTGAACAAG GCGGGTTTGG CGTATTTTGA
     AGGGCGTTTT
301  GAAAAGGCGG AACTAGAAGC CTCACGCGTG TTGGTCAACA
     AAGAGGCCGG
351  AGACAACCGG ACTTTGGCAT TGATGCTGGG CGCGCACGCC
     GCCGGACAGA
401  TGGAAAACAT CGAGCTGCGC GACCGTTATC TTGCGGAAAT
     CGCCAAACTG
451  CCGGAAAAAC AGCAGCTTTC CCGTTATCTT TTGTTGGCGG
     AATCGGCGTT
501  GAACCGGCGC GATTACGAAG CGGCGGAAGC CAATCTTCAT
     GCGGCGGCGA
551  AGATGAATGC CAACCTTACG CGCCTCGTGC GTCTGCAACT
     TCGTTACGCT
601  TTCGACAGGG GCGACGCGTT GCAGGTTCTG GCAAAAACCG
     AAAAACTTTC
651  CAAGGCGGGC GCGTTGGGCA AATCGGAAAT GGAACGGTAT
     CAAAATTGGG
701  CATACCGCCG CCAGCTGGCG GATGCTGCCG ATGCCGCCGC
     TTTGAAAACC
751  TGCCTGAAGC GGATTCCCGA CAGCCTCAAA AACGGGGAAT
     TGAGCGTATC
801  GGTTGCGGAA AAGTACGAAC GTTTGGGACT GTATGCCGAT
     GCGGTCAAAT
851  GGGTCAAACA GCATTATCCG CACAACCGCC GCCCCGAGCT
     TTTGGAAGCC
901  TTTGTCGAAA GCGTGCGCTT TTTGGGCGAG CGCGAACAGC
     AGAAAGCCAT
951  CGATTTTGCC GATGCTTGGC TGAAAGAACA GCCCGATAAC
     GCGCTTCTGC
1001 TGATGTATCT CGGTCGGCTC GCCTACGGCC GCAAACTTTG
     GGGCAAGGCA
1051 AAAGGCTACC TTGAAGCGAG CATTGCATTA AAGCCGAGTA
     TTTCCGCGCG
1101 TTTGGTTCTA GCAAAGGTTT TCGACGAAAT CGGAGAACCG
     CAGAAGGCGG
1151 AGGCGCAGCG CAACTTGGTT TTGGAAGCCG TCTCCGATGA
     CGAACGTCAC
1201 GCAGCGTTAG AGCAGCATAG CTGA

This corresponds to the amino acid sequence <SEQ ID 752; ORF100-1>:

1   MKTVVWIVVL FAAAVGLALA SGIYTGDVYI VLGQTMLRIN
    LHAFVLGSLI
51  AVVVWYFLFK FIIGVLNIPE KMQRFGSARK GRKAALALNK
    AGLAYFEGRF
101 EKAELEASRV LVNKEAGDNR TLALMLGAHA AGQMENIELR
    DRYLAEIAKL
151 PEKQQLSRYL LLAESALNRR DYEAAEANLH AAAKMNANLT
    RLVRLQLRYA
201 FDRGDALQVL AKTEKLSKAG ALGKSEMERY QNWAYRRQLA
    DAADAAALKT
251 CLKRIPDSLK NGELSVSVAE KYERLGLYAD AVKWVKQHYP
    HNRRPELLEA
301 FVESVRFLGE REQQKAIDFA DAWLKEQPDN ALLLMYLGRL
    AYGRKLWGKA
351 KGYLEASIAL KPSISARLVL AKVFDEIGEP QKAEAQRNLV
    LEAVSDDERH
401 AALEQHS*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF100 shows 93.5% identity over a 386aa overlap with an ORF (ORF100a) from strain A of N. meningitidis:

The complete length ORF100a nucleotide sequence <SEQ ID 753> is:

1    ATGAAAACGG TAGTCTGGAT TGTCGTCCTG TTTGCCGCCG
     CNNTCGGGCT
51   GGCATTGGCG TCGGGCATTN ACACCGGCGA CGTGTATATC
     GTACTCGGAC
101  AGACCATGCT CAGAATCAAC CTGCACGCCT TTGTGTTAGG
     TTCGCTGATT
151  GCCGTCGTGG TGTGGTATTT CCTGTTCAAA TTCATCATCG
     GCGTACTCAA
201  TANCCCCGAA AAGATGCAGC GTTTCGGTTC GGCGCGTAAA
     GGCCGCAAGG
251  CCGCGCTTGC TTTGAACAAG GCGGGTTTGG CGTATTTTGA
     AGGGCGTTTT
301  GAAAAGGCGG AACTTGAAGC CTCGCGCGTA TTGGGAAACA
     AAGAGGCGGG
351  GGATAACCGG ACTTTGGCAT TGATGTTGGG CGCACATGCC
     GCCGGGCAGA
401  TGGAAAACAT CGAGCTGCGC GACCGTTATC TTGCGGAAAT
     CGCCAAACTG
451  CCGGAAAAGC AGCAGCTTTC CCGTTATCTT TTGTTGGCGG
     AATCGGCGTT
501  GAACCGGCGC GATTACGAAG CGGCGGAAGC CAATCTTCAT
     GCGGCGGCGA
551  AGATGAATGC CAACCTTACG CGCCTCGTGC GTCTGCAACT
     TCGTTACGCT
601  TTCGACAGGG GCGACGCGTT GCAGGTTCTG GCAAAAACCG
     AAAAANTTTC
651  CAAGGCGGGC GCGTNGGGCA AATCGGAAAT GGAACGGTAT
     CAAAATTGGG
701  CATACCGCCG CCAGCTGNCG GATGCTGCCG ATGCCGCCGC
     TTTGAAAACC
751  TGCCTGAAGC GGATTCCCGA CAGCCTCAAA AACGGGGAAT
     TGAGCGTATC
801  GGTTGCGGAA AAGTACGAAC GTTTGGGACT GTATGCCGAT
     GCGGTCAAAT
851  GGGTCAAACA GCATTATCCG CACAACCGCC GACCCGAACT
     TTTGGAAGCN
901  TTTGTCGAAA GCGTGCGCTT TTTGGGCGAA CGCGATCAGC
     AGAAAGCCAT
951  CGATTTTGCC GATGCTTGGC TGAAAGAACA GCCCGATAAT
     GCGCTTCTGC
1001 TGANGTATCT CGGTCGGCTC GCCTACGGCC GCAAACTTTG
     GGGCAAGGCA
1051 AAAGGCTACC TTGAAGCGAG CATTGCATTA AAGCCGAGTA
     TTTCCGCGCG
1101 TTTGGTTCTG GCAAAGGTTT TTGACGAAAC CGGAGAACCG
     CAGAAGGCGG
1151 AGGCGCAGCG CAACTTGGTT TTGGCAAGCG TTGCCGAGGA
     AAACCGNCCT
1201 TCCGCCGAAA CCCATTGA

This encodes a protein having amino acid sequence <SEQ ID 754>:

1   MKTVVWIVVL FAAAXGLALA SGIXTGDVYI VLGQTMLRIN
    LHAFVLGSLI
51  AVVVWYFLFK FIIGVLNXPE KMQRFGSARK GRKAALALNK
    AGLAYFEGRF
101 EKAELEASRV LGNKEAGDNR TLALMLGAHA AGQMENIELR
    DRYLAEIAKL
151 PEKQQLSRYL LLAESALNRR DYEAAEANLH AAAKMNANLT
    RLVRLQLRYA
201 FDRGDALQVL AKTEKXSKAG AXGKSEMERY QNWAYRRQLX
    DAADAAALKT
251 CLKRIPDSLK NGELSVSVAE KYERLGLYAD AVKWVKQHYP
    HNRRPELLEA
301 FVESVRFLGE RDQQKAIDFA DAWLKEQPDN ALLLXYLGRL
    AYGRKLWGKA
351 KGYLEASIAL KPSISARLVL AKVFDETGEP QKAEAQRNLV
    LASVAEENRP
401 SAETH*

ORF100a and ORF100-1 show 95.1% identity in 406 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF100 shows 93.3% identity over a 386 aa overlap with a predicted ORF (ORF100ng) from N. gonorrhoeae:

The complete length ORF100ng nucleotide sequence <SEQ ID 755> is:

1    ATGAAAACGG TAGTCTGGAT TGTTGTCCTG TTTGCCGCCG
     CCGTCGGACT
51   GGCGCTGGCT TCGGGCATTT ACACCGGCGA CGTGTATATC
     GTACTCGGAC
101  AGACCATGCT CAGAATCAAC CTGCACGCCT TTGTGTTAGG
     TTCGCTGATT
151  GCCGTCGTGG TGTGGTATTT CCTGTTTAAA TTCATCATCG
     GCGTACTCAA
201  TATCCCCGAA AATATGCGGC GTTCCGGTTC GGCGCGGAAA
     GGCCGCAAGG
251  CCGCGCTTGC CTTGAATAAG GCGGGTTTGG CGTATTTCGA
     AGGGCGTTTT
301  GAAAAGGCGG AACTCGAAGC CTCTCGAGTG TTGGGCAACA
     AAGAGGCCGG
351  AGACAACCGG ACTTTGGCAT TGATGCTGGG CGCGCACGCG
     GCAGGACAGA
401  TGGAAAATAT CGAGCTGCGC GACCGTTATC TTGCGGAAAT
     CGCCAAACTG
451  CCGGAAAAAC AGCAGCTTTC CCGCTATCTT CTGCTGGCGG
     AATCGGCGTT
501  AAACCGGCGC GATTACGAAG CGGCGGAAGC CAATCTTCAT
     GCGGCGGCGA
551  AGATGAATGC CAACCTTACG CGCCTCGTGC GTCTGCAACT
     TCGTTACGCC
601  TTCGATCGGG GCGATGCGTT GCAGGTTCTG GCAAAAaccG
     AAAAACTTTC
651  CAAGGCGGGC GCGTTGGGCA AATCGGAAAT GGAACGGTAT
     CAAAATTGGG
701  CATACCGCCG CCAGATGGCG GATGCTGCCG ATGCCGCCGC
     TTTGAAAACC
751  TGCCTGAAGC GGATTCCCGA CAGCCTCAAA AACGGGGAAT
     TGagcGTATC
801  GGTTGCGGAA AAGTACGAAC GTTTGGGACT GTATGCCGAT
     GCGGTCAAAT
851  GGGTCAAACA GCATTATCCG CACAACCGCC GCCCCGAGCT
     TTTGGAAGCC
901  TTTGTCGAAA GCGTGCGCTT TTTGGGCGAG CGCGAACAGC
     AGAAAGCCAT
951  CGATTTTGCC GATTCTTGGC TGAAAGAACA GCCCGATAAC
     GCGCTTCTGC
1001 TGATGTATCT CGGCCGGCTC GCCTACGGCC GCAAACTTTG
     GGGTAAGGCA
1051 AAAGGCTACC TTGAAGCGAG TATTGCACTG AAGCCGAGTA
     TTCCGGCGCG
1101 TTTGGTGTTG GCAAAGGTTT TTGACGAAAC CGCACAGTCG
     CAAAAAGCCG
1151 AAGCACAGCG CAACTTGGTT TTGGCAAGCG TTGCCGGGGA
     AAACCGCCCT
1201 TCCGCCGAAA CCCGTTGA

This encodes a protein having amino acid sequence <SEQ ID 756>:

1   MKTVVWIVVL FAAAVGLALA SGIYTGDVYI VLGQTMLRIN
    LHAFVLGSLI
51  AVVVWYFLFK FIIGVLNIPE NMRRSGSARK GRKAALALNK
    AGLAYFEGRF
101 EKAELEASRV LGNKEAGDNR TLALMLGAHA AGQMENIELR
    DRYLAEIAKL
151 PEKQQLSRYL LLAESALNRR DYEAAEANLH AAAKMNANLT
    RLVRLQLRYA
201 FDRGDALQVL AKTEKLSKAG ALGKSEMERY QNWAYRRQMA
    DAADAAALKT
251 CLKRIPDSLK NGELSVSVAE KYERLGLYAD AVKWVKQHYP
    HNRRPELLEA
301 FVESVRFLGE REQQKAIDFA DSWLKEQPDN ALLLMYLGRL
    AYGRKLWGKA
351 KGYLEASIAL KPSIPARLVL AKVFDETAQS QKAEAQRNLV
    LASVAGENRP
401 SAETR*

ORF100ng and ORF100-1 show 95.3% identity in 402 aa overlap:

Based on this analysis, including the presence of a putative leader sequence, a putative transmembrane domain, and a RGD motif, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 90

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 757>

1   ATGATGTTTT CTTGGTTCAA GCTGTTTCAC TTGTTTTTTG
    TCATTTCGTG
51  GTTTGCAGGG CTGTTTTACC TGCCGAGGAT TTTCGTCAAT
    ATGGCGATGA
101 TTGATGTGCC GCGCGGCAAT CCCGAGTATG TGCGTCTGTC
    GGGCATGGCG
151 GTGCGGCTGT ACCGTTTTAT GTCGCCGTTG GGCTTCGGCG
    CGGTCGTGTT
201 CGGCGCGGCG ATACCGTTTG CCGCCGGCTG GTGGGGCAGC
    GGCTGGGTAC
251 ACGTCAAACT GTGTTTGGGC TTGATGCTCT TGGCTTACCA
    GTTGTATTGC
301 GGCGTGCTGC TGCGCCGTTT TCAGGATTAC AGCAATGCTT
    TTTCACACCG
351 CTGGTACCGC GTGTTCAACG AAATCCCCGT GCTGCTGATG
    GTTGCCGCGC
401 TGTATsTGGT CGTGTTCAAA CCGTTTTGA

This corresponds to the amino acid sequence <SEQ ID 758; ORF102>:

1   MMFSWFKLFH LFFVISWFAG LFYLPRIFVN MAMIDVPRGN
    PEYVRLSGMA
51  VRLYRFMSPL GFGAVVFGAA IPFAAGWWGS GWVHVKLCLG
    LMLLAYQLYC
101 GVLLRRFQDY SNAFSHRWYR VFNEIPVLLM VAALYXVVFK
    PF*

Further work revealed the complete nucleotide sequence <SEQ ID 759>:

1   ATGATGTTTT CTTGGTTCAA GCTGTTTCAC TTGTTTTTTG
    TCATTTCGTG
51  GTTTGCAGGG CTGTTTTACC TGCCGAGGAT TTTCGTCAAT
    ATGGCGATGA
101 TTGATGTGCC GCGCGGCAAT CCCGAGTATG TGCGTCTGTC
    GGGCATGGCG
151 GTGCGGCTGT ACCGTTTTAT GTCGCCGTTG GGCTTCGGCG
    CGGTCGTGTT
201 CGGCGCGGCG ATACCGTTTG CCGCCGGCTG GTGGGGCAGC
    GGCTGGGTAC
251 ACGTCAAACT GTGTTTGGGC TTGATGCTCT TGGCTTACCA
    GTTGTATTGC
301 GGCGTGCTGC TGCGCCGTTT TCAGGATTAC AGCAATGCTT
    TTTCACACCG
351 CTGGTACCGC GTGTTCAACG AAATCCCCGT GCTGCTGATG
    GTTGCCGCGC
401 TGTATCTGGT CGTGTTCAAA CCGTTTTGA

This corresponds to the amino acid sequence <SEQ ID 760; ORF102-1>:

1   MMFSWFKLFH LFFVISWFAG LFYLPRIFVN MAMIDVPRGN
    PEYVRLSGMA
51  VRLYRFMSPL GFGAVVFGAA IPFAAGWWGS GWVHVKLCLG
    LMLLAYQLYC
101 GVLLRRFQDY SNAFSHRWYR VFNEIPVLLM VAALYLVVFK
    PF*

Computer analysis of this amino acid sequence gave the following results:

Homology with HP1484 Hypothetical Integral Membrane Protein of H. pylori (Accession Number AE000647)

ORF102 and HP1484 show 33% aa identity in 143aa overlap:

orf102	3	FSWFKLFHLFFVISWFAGLFYLPRIFVNMAMIDVPRGNPEYVRLSGMAVRLYRFMSPLGF	62
		F W K FH+  VISW A LFYLPR+FV  A     +     V++     +LY F++
HP1484	8	FLWVKAFHVIAVISWMAALFYLPRLFVYHAENAHKKEFVGVVQIQEK--KLYSFIASPAM	65
orf102	63	GAVVFGAAIPFAAG---WWGSGWVHVKLCLGLMLLAYQLYCGVLLRRFQDYSNAFSHRWY	119
		G  +    +        +   GW+H KL L ++LLAY  YC   +R  +      + R+Y
HP1484	66	GFTLITGILMLLIEPTLFKSGGWLHAKLALVVLLLAYHFYCKKCMRELEKDPTRRNARFY	125
orf102	120	RVFNEIPXXXXXXXXXXXXFKPF	142
		RVFNE P             KPF
HP1484	126	RVFNEAPTILMILIVILVVVKPF	148

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF102 shows 99.3% identity over a 142aa overlap with an ORF (ORF102a) from strain A of N. meningitidis:

The complete length ORF102a nucleotide sequence <SEQ ID 761> is:

1   ATGATGTTTT CTTGGTTCAA GCTGTTTCAC TTGTTTTTTG
    TCATTTCGTG
51  GTTTGCAGGG CTGTTTTACC TGCCGAGGAT TTTCGTCAAT
    ATGGCGATGA
101 TTGATGTGCC GCGCGGCAAT CCCGAGTATG TGCGTCTGTC
    GGGCATGGCG
151 GTGCGGCTGT ACCGTTTTAT GTCGCCGTTG GGCTTCGGCG
    CGGTCGTGTT
201 CGGCGCGGCG ATACCGTTTG CCGCCGGCTG GTGGGGCAGC
    GGCTGGGTAC
251 ACGTCAAACT GTGTTTGGGC TTGATGCTCT TGGCTTACCA
    GTTGTATTGC
301 GGCGTGCTGC TGCGCCGTTT TCAGGATTAC AGCAATGCTT
    TTTCACACCG
351 CTGGTACCGC GTGTTCAACG AAATCCCCGT GCTGCTGATG
    GTTGCCGCGC
401 TGTATCTGGT CGTGTTCAAA CCGTTTTGA

This encodes a protein having amino acid sequence <SEQ ID 762>:

1   MMFSWFKLFH LFFVISWFAG LFYLPRIFVN MAMIDVPRGN
    PEYVRLSGMA
51  VRLYRFMSPL GFGAVVFGAA IPFAAGWWGS GWVHVKLCLG
    LMLLAYQLYC
101 GVLLRRFQDY SNAFSHRWYR VFNEIPVLLM VAALYLVVFK
    PF*

ORF102a and ORF102-1 show complete identity in 142 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF102 shows 97.9% identity over a 142 aa overlap with a predicted ORF (ORF102ng) from N. gonorrhoeae:

The complete length ORF102ng nucleotide sequence <SEQ ID 763> is:

1   ATGATGTTTT CTTGGTTCAA GCTGTTTCAC TTGTTTTTTG
    TCATTTCGTG
51  GTTTGCAGGG CTGTTTTACC TGCCGAGGAT TTTCGTCAAT
    ATGGCGATGA
101 TTGATGCGCC GCGCGGCAAT CCCGAGTATG TGCGCCTGTC
    GGGGATGGCG
151 GTGCGGTTGT ACCGTTTTAT GTCGCCTTTG GGTTTCGGCG
    CGGTCGTGTT
201 CGGCGCGGCG ATACCGTTTG CCGCcggccg GTGGGGCagc
    ggctggGTTC
251 ACGTCAAACT GTGTTTGGGC TTGATGCTCT TGGCTTATCA
    GTTGTATTGC
301 GGCGTGCTGC TGCGCCGTTT TCAGGATTAC AGCAATGCTT
    TTTCACACCG
351 CTGGTACCGC GTGTTCAAcg aAATCCCCGT GCTGCTGATG
    GTTGCCGCGC
401 TGTATCTGGT CGTGTTCAAA CCGTTTTGA

This encodes a protein having amino acid sequence <SEQ ID 764>:

1   MMFSWFKLFH LFFVISWFAG LFYLPRIFVN MAMIDAPRGN
    PEYVRLSGMA
51  VRLYRFMSPL GFGAVVFGAA IPFAAGRWGS GWVHVKLCLG
    LMLLAYQLYC
101 GVLLRRFQDY SNAFSHRWYR VFNEIPVLLM VAALYLVVFK
    PF*

ORF102ng and ORF102-1 show 98.6% identity in 142 aa overlap:

In addition, ORF102ng shows significant homology to a membrane protein from H. pylori:

gi|2314656 (AE000647) conserved hypothetical integral membrane protein
[Helicobacter pylori] Length = 148
Score = 79.2 bits (192), Expect = 1e−14
Identities = 50/147 (34%), Positives = 68/147 (46%), Gaps = 13/147 (8%)
Query:	3	FSWFKLFHLFFVISWFAGLFYLPRIFVNMAMIDAPRGNPEYVRLSGMAVRLYRFMSPLGF	62
		F W K FH+  VISW A LFYLPR+FV  A     +     V++     +LY F++
Sbjct:	8	FLWVKAFHVIAVISWMAALFYLPRLFVYHAENAHKKEFVGVVQIQEK--KLYSFIASPAM	65
Query:	63	GAVVFGAAIP-------FAAGRWGSGWVHVKLCLGLMLLAYQLYCGVLLRRFQDYSNAFS	115
		G  +    +        F +G    GW+H KL L ++LLAY  YC   +R  +      +
Sbjct:	66	GFTLITGILMLLIEPTLFKSG----GWLHAKLALVVLLLAYHFYCKKCMRELEKDPTRRN	121
Query:	116	HRWYRVFNEIPXXXXXXXXXXXXFKPF	142
		R+YRVFNE P             KPF
Sbjct:	122	ARFYRVFNEAPTILMILIVILVVVKPF	148

Based on this analysis, it is predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 91

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 765>:

1  ATGGCAAAAA TGATGAAATG GGCGGCTGTT GCGGCGGTCG CGGCGGCAGC
51  GGTTTGGGGC GGATGGTCTT AACTGAAGCC CGAGCCGCAC GTGCTTGATA
101  TTACGGAAAC GGTCAGGCGC GGC // .....
//.. ATTTCGTTTA CGATTTTGTC CGAACCGGAT ACGCCGATTA AGGCGAAGCT
51  CGACAGCGTC GACCCCGGGC TGACCACGAT GTCGTCGGGC GGTTACAACA
101  GCAGTACGGA TACGGCTTCC AATGCGGTCT ACTATTATGC CCGTTCGTTT
151  GTGCCGAATC CGGACGGCAA ACTCGCCACG GGGATGACGA CGCAGAATAC
201  GGTTGAAATC GACGGCGTGA AAAATGTGCT GATTATTCCG TCGCTGACCG
251  TGAAAAATCG CGGCGGCAAG GCGTTTGTGC GCGTGTTGGG TGCGGACGGC
301  AAGGCGGCGG AACGCGAAAT CCGGACCGGT ATGAGAGACA GTATGAATAC
351  CGAAGTAAAA AGCGGGTTGA AAGAGGGGGA CAAAGTGGTC ATCTCCGAAA
401  TAACCGCCGC CGAGCAACAG GAAAGCGGCG AACGCGCCCT AGGCGGCCCG
451  CCGCGCCGAT AA

This corresponds to the amino acid sequence <SEQ ID 766; ORF85>:

1   MAKMMKWAAV AAVAAAAVWG GWS.LKPEPH VLDITETVRR
    G.........
51  .......... .......... .......... ..........
    ..........
101 .......... .......... .......... ..........
    ..........
151 .......... .......... .......... ..........
    ..........
201 .......... .......... .......... .........I
    SFTILSEPDT
251 PIKAKLDSVD PGLTTMSSGG YNSSTDTASN AVYYYARSFV
    PNPDGKLATG
301 MTTQNTVEID GVKNVLIIPS LTVKNRGGKA FVRVLGADGK
    AAEREIRTGM
351 RDSMNTEVKS GLKEGDKVVI SEITAAEQQE SGERALGGPP
    RR*

Further work revealed the further partial nucleotide sequence <SEQ ID 767>:

1    ..GTATCGGTCG GCGCGCAGGC ATCGGGGCAG ATTAAGATAC
     TTTATGTCAA
51   ACTCGGGCAA CAGGTTAAAA AGGGCGATTT GATTGCGGAA
     ATCAATTCGA
101  CCTCGCAGAC CAATACGCTC AATACGGAAA AATCCAAGTT
     GGAAACGTAT
151  CAGGCGAAGC TGGTGTCGGC ACAGATTGCA TTGGGCAGCG
     CGGAGAAGAA
201  ATATAAGCGT CAGGCGGCGT TATGGAAGGA AAACGCGACT
     TCCAAAGAGG
251  ATTTGGAAAG CGCGCAGGAT GCGTTTGCCG CCGCCAAAGC
     CAATGTTGCC
301  GAGCTGAAGG CTTTAATCAG ACAGAGCAAA ATTTCCATCA
     ATACCGCCGA
351  GTCGGAATTG GGCTACACGC GCATTACCGC AACGATGGAC
     GGCACGGTGG
401  TGGCGATTCT CGTGGAAGAG GGGCAGACTG TGAACGCGGC
     GCAGTCTACG
451  CCGACGATTG TCCAATTGGC GAATCTGGAT ATGATGTTGA
     ACAAAATGCA
501  GATTGCCGAG GGCGATATTA CCAAGGTGAA GGCGGGGCAG
     GATATTTCGT
551  TTACGATTTT GTCCGAACCG GATACGCCGA TTAAGGCGAA
     GCTCGACAGC
601  GTCGACCCCG GGCTGACCAC GATGTCGTCG GGCGGTTACA
     ACAGCAGTAC
651  GGATACGGCT TCCAATGCGG TCTACTATTA TGCCCGTTCG
     TTTGTGCCGA
701  ATCCGGACGG CAAACTCGCC ACGGGGATGA CGACGCAGAA
     TACGGTTGAA
751  ATCGACGGCG TGAAAAATGT GCTGATTATT CCGTCGCTGA
     CCGTGAAAAA
801  TCGCGGCGGC AAGGCGTTTG TGCGCGTGTT GGGTGCGGAC
     GGCAAGGCGG
851  CGGAACGCGA AATCCGGACC GGTATGAGAG ACAGTATGAA
     TACCGAAGTA
901  AAAAGCGGGT TGAAAGAGGG GGACAAAGTG GTCATCTCCG
     AAATAACCGC
951  CGCCGAGCAA CAGGAAAGCG GCGAACGCGC CCTAGGCGGC
     CCGCCGCGCC
1001 GATAA

This corresponds to the amino acid sequence <SEQ ID 768; ORF85-1>:

1   ..VSVGAQASGQ IKILYVKLGQ QVKKGDLIAE INSTSQTNTL
    NTEKSKLETY
51  QAKLVSAQIA LGSAEKKYKR QAALWKENAT SKEDLESAQD
    AFAAAKANVA
101 ELKALIRQSK ISINTAESEL GYTRITATMD GTVVAILVEE
    GQTVNAAQST
151 PTIVQLANLD MMLNKMQIAE GDITKVKAGQ DISFTILSEP
    DTPIKAKLDS
201 VDPGLTTMSS GGYNSSTDTA SNAVYYYARS FVPNPDGKLA
    TGMTTQNTVE
251 IDGVKNVLII PSLTVKNRGG KAFVRVLGAD GKAAEREIRT
    GMRDSMNTEV
301 KSGLKEGDKV VISEITAAEQ QESGERALGG PPRR*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF85 shows 87.8% identity over a 41 aa overlap and 99.3% identity over a 153aa overlap with an ORF (ORF85a) from strain A of N. meningitidis:

The complete length ORF85a nucleotide sequence <SEQ ID 769> is:

1    ATGGCAAAAA TGATGAAATG GGCGGCTGTT GCGGCGGTCG
     CGGCGGCAGC
51   GGTTTGGGGC GGATGGTCTT ATCTGAAGCC CGAGCCGCAG
     GCTGCTTATA
101  TTACGGAAAC GGTCAGGCGC GGCGACATCA GCCGGACGGT
     TTCTGCAACA
151  GGGGAGATTT CGCCGTCCAA CCTGGTATCG GTCGGCGCGC
     AGGCATCGGG
201  GCAGATTAAG AAACTTTATG TCAAACTCGG GCAACAGGTT
     AAAAAGGGCG
251  ATTTGATTGC GGAAATCAAT TCGACCTCGC AGACCAATAC
     GCTCAATACG
301  GAAAAATCCA AATTGGAAAC GTATCAGGCG AAGCTGGTGT
     CGGCACAGAT
351  TGCATTGGGC AGCGCGGAGA AGAAATATAA GCGTCAGGCG
     GCGTTGTGGA
401  AGGATGATGC GACCGCTAAA GAAGATTTGG AAAGCGCACA
     GGATGCGCTT
451  GCCGCCGCCA AAGCCAATGT TGCCGAGCTG AAGGCTCTAA
     TCAGACAGAG
501  CAAAATTTCC ATCAATACCG CCGAGTCGGA ATTGGGCTAC
     ACGCGCATTA
551  CCGCAACGAT GGACGGCACG GTGGTGGCGA TTCTCGTGGA
     AGAGGGGCAG
601  ACTGTGAACG CGGCGCAGTC TACGCCGACG ATTGTCCAAT
     TGGCGAATCT
651  GGATATGATG TTGAACAAAA TGCAGATTGC CGAGGGCGAT
     ATTACCAAGG
701  TGAAGGCGGG GCAGGATATT TCGTTTACGA TTTTGTCCGA
     ACCGGATACG
751  CCGATTAAGG CGAAGCTCGA CAGCGTCGAC CCCGGGCTGA
     CCACGATGTC
801  GTCGGGCGGC TACAACAGCA GTACGGATAC GGCTTCCAAT
     GCGGTCTACT
851  ATTATGCCCG TTCGTTTGTG CCGAATCCGG ACGGCAAACT
     CGCCACGGGG
901  ATGACGACGC AGAATACGGT TGAAATCGAC GGTGTGAAAA
     ATGTGCTGAT
951  TATTCCGTCG CTGACCGTGA AAAATCGCGG CGGCAGGGCG
     TTTGTGCGCG
1001 TGTTGGGTGC AGACGGCAAG GCGGCGGAAC GCGAAATCCG
     GACCGGTATG
1051 AGAGACAGTA TGAATACCGA AGTAAAAAGC GGGTTGAAAG
     AGGGGGACAA
1101 AGTGGTCATC TCCGAAATAA CCGCCGCCGA GCAGCAGGAA
     AGCGGCGAAC
1151 GCGCCCTAGG CGGCCCGCCG CGCCGATAA

This encodes a protein having amino acid sequence <SEQ ID 770>:

1   MAKMMKWAAV AAVAAAAVWG GWSYLKPEPQ AAYITETVRR
    GDISRTVSAT
51  GEISPSNLVS VGAQASGQIK KLYVKLGQQV KKGDLIAEIN
    STSQTNTLNT
101 EKSKLETYQA KLVSAQIALG SAEKKYKRQA ALWKDDATAK
    EDLESAQDAL
151 AAAKANVAEL KALIRQSKIS INTAESELGY TRITATMDGT
    VVAILVEEGQ
201 TVNAAQSTPT IVQLANLDMM LNKMQIAEGD ITKVKAGQDI
    SFTILSEPDT
251 PIKAKLDSVD PGLTTMSSGG YNSSTDTASN AVYYYARSFV
    PNPDGKLATG
301 MTTQNTVEID GVKNVLIIPS LTVKNRGGRA FVRVLGADGK
    AAEREIRTGM
351 RDSMNTEVKS GLKEGDKVVI SEITAAEQQE SGERALGGPP
    RR*

ORF85a and ORF85-1 show 98.2% identity in 334 aa overlap:

FIG. 19D shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF85a.

Homology with a Predicted ORF from N. gonorrhoeae

ORF85 shows a high degree of identity with a predicted ORF (ORF85ng) from N. gonorrhoeae:

The complete length ORF85ng nucleotide sequence <SEQ ID 771> is:

1    ATGGCAAAAA TGATGAAATG GGCGGCTGTT GCGGCGGTCG
     CGGCGGCaac
51   GGTTTGGGGC GGATGGTCTT ATCTGAAGCC CGAACCGCAG
     GCTGCTTATA
101  TTACGGAaac ggTCAGGCGC GGCGATATCA GCCGGACGGT
     TTCCGCGACG
151  GgcgAGATTT CGCCGTCCAA CCTGGTATCG GTCGGCGCGC
     AGGCTTCGGG
201  GCAGATTAAA AAGCTTTATG TCAAACTCGG GCAACAGGTC
     AAAAAGGGCG
251  ATTTGATTGC GGAAATCAAT TCGACCACGC AGACCAACAC
     GATCGATATG
301  GAAAAATCCA AATTGGAAAC GTATCAGGCG AAGCTGGTGT
     CGGCACAGAT
351  TGCATTGGGC AGCGCGGAGA AGAAATATAA GCGTCAGGCG
     GCGTTGTGGA
401  AGGATGATGC GACCTCTAAA GAAGATTTGG AAAGCGCGCA
     GGATGCGCTT
451  GCCGCCGCCA AAGCCAATGT TGCCGAGTTG AAGGCTTTAA
     TCAGACAGAG
501  CAAAATTTCC ATCAATACCG CCGAGTCGGA TTTGGGCTAC
     ACGCGCATTA
551  CCGCGACGAT GGACGGCACG GTGGTGGCGA TTCCCGTGGA
     AGAGGGGCAG
601  ACTGTGAACG CGGCGCAGTC TACGCCGACG ATTGTCCAAT
     TGGCGAATCT
651  GGATATGATG TTGAACAAAA TGCAGATTGC CGAGGGCGAT
     ATTACCAAGG
701  TGAAGGCGGG GCAGGATATT TCGTTTACGA TTTTGTCCGA
     ACCGGATACG
751  CCGATTAAGG CGAAGCTCGA CAGCGTCGAC CCCGGGCTGA
     CCACGATGTC
801  GTCGGGCGGC TACAACAGCA GTACGGATAC GGCTTCCAAT
     GCGGTCTATT
851  ATTATGCCCG TTCGTTTGTG CCGAATCCGG ACGGCAAACT
     CGCCACGGGG
901  ATGACGACGC AGAATACGGT TGAAATCGAC GGTGTGAAAA
     ATGTGTTGCT
951  TATTCCGTCG CTGACCGTGA AAAATCGCGG CGGCAAGGCG
     TTCGTACGCG
1001 TGTTGGGTGC GGACGGCAAG GCAGTGGAAC GCGAAATCCG
     GACCGGTATG
1051 AAAGACAGTA TGAATACCGA AGTGAAAAGC GGGTTGAAAG
     AGGGGGACAA
1101 AGTGGTCATC TCCGAAATAA CCGCCGCCGA GCAGCAGGAA
     AGCGGCGAAC
1151 GCGCCCTAGG CGGCCCGCCG CGCCGATAA

This encodes a protein having amino acid sequence <SEQ ID 772>:

1   MAKMMKWAAV AAVAAAAVWG GWSYLKPEPQ AAYITEAVRR
    GDISRTVSAT
51  GEISPSNLVS VGAQASGQIK KLYVKLGQQV KKGDLIAEIN
    STTQTNTIDM
101 EKSKLETYQA KLVSAQIALG SAEKKYKRQA ALWKDDATSK
    EDLESAQDAL
151 AAAKANVAEL KALIRQSKIS INTAESDLGY TRITATMDGT
    VVAIPVEEGQ
201 TVNAAQSTPT IVQLANLDMM LNKMQIAEGD ITKVKAGQDI
    SFTILSEPDT
251 PIKAKLDSVD PGLTTMSSGG YNSSTDTASN AVYYYARSFV
    PNPDGKLATG
301 MTTQNTVEID GVKNVLLIPS LTVKNRGGKA FVRVLGADGK
    AVEREIRTGM
351 KDSMNTEVKS GLKEGDKVVI SEITAAEQQE SGERALGGPP
    RR*

ORF85ng and ORF85-1 show 96.1% identity in 334 aa overlap:

In addition, ORF85ng shows significant homology to an E. coli membrane fusion protein:

gi|1787104 (AE000189) o380; 27% identical (27 gaps) to 332 residues from
membrane fusion protein precursor, MTRC_NEIGO SW: P43505 (412 aa)
[Escherichia coli] Length = 380
Score = 193 bits (485), Expect = 2e−48
Identities = 120/345 (34%), Positives = 182/345 (51%), Gaps = 13/345 (3%)
Query:	29	PQAAYITETVRRGDISRTVSATGEISPSNLVSVGAQASGQIKKLYVKLGQQVKKGDLIAE	88
		P   Y T  VR GD+ ++V ATG++     V VGAQ SGQ+K L V +G +VKK  L+
Sbjct:	41	PVPTYQTLIVRPGDLQQSVLATGKLDALRKVDVGAQVSGQLKTLSVAIGDKVKKDQLLGV	100
Query:	89	INSTTQTNTIDMEKSKLETYQAKLVSAQIALGSAEKKYKRQAALWKDDATSKEXXXXXXX	148
		I+     N I   ++ L   +A+   A+  L  A   Y RQ  L +  A S++
Sbjct:	101	IDPEQAENQIKEVEATLMELRAQRQQAEAELKLARVTYSRQQRLAQTKAVSQQDLDTAAT	160
Query:	149	XXXXXXXXXXXXXXXIRQSKISINTAESDLGYTRITATMDGTVVAIPVEEGQTVNAAQST	208
		I++++ S++TA+++L YTRI A M G V  I   +GQTV AAQ
Sbjct:	161	EMAVKQAQIGTIDAQIKRNQASLDTAKTNLDYTRIVAPMAGEVTQITTLQGQTVIAAQQA	220
Query:	209	PTIVQLANLDMMLNKMQIAEGDITKVKAGQDISFTILSEPDTPIKAKLDSVDPGLTTMSS	268
		P I+ LA++  ML K Q++E D+  +K GQ   FT+L +P T  + ++  V P
Sbjct:	221	PNILTLADMSAMLVKAQVSEADVIHLKPGQKAWFTVLGDPLTRYEGQIKDVLP-------	273
Query:	269	GGYNSSTDTASNAVYYYARSFVPNPDGKLATGMTTQNTVEIDGVKNVLLIPSLTVKNRGG	328
		+ +  ++A++YYAR  VPNP+G L   MT Q  +++  VKNVL IP   + +  G
Sbjct:	274	-----TPEKVNDAIFYYARFEVPNPNGLLRLDMTAQVHIQLTDVKNVLTIPLSALGDPVG	328
Query:	329	KAFVRV-LGADGKAVEREIRTGMKDSMNTEVKSGLKEGDKVVISE	372
		+V L  +G+  ERE+  G ++  + E+  GL+ GD+VVI E
Sbjct:	329	DNRYKVKLLRNGETREREVTIGARNDTDVEIVKGLEAGDEVVIGE	373

Based on this analysis, it was predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF85-1 (40.4 kDa) was cloned in the pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 19A shows the results of affinity purification of the GST-fusion protein. Purified GST-fusion protein was used to immunise mice, whose sera were used for Western blot (FIG. 19B), FACS analysis (FIG. 19C), and ELISA (positive result). These experiments confirm that ORF85-1 is a surface-exposed protein, and that it is a useful immunogen.

Example 92

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 773>:

1   ..ATTCCCGCCA CGATGACATT TGAACGCAGC GGCAATGCTT
    ACAAAATCGT
51  TTCGACGATT AAAGTGCCGC TATACAATAT CCGTTTCGAG
    TCCGGCGGTA
101 CGGTTGTCGG CAATACCCTG CACCCTACCT ACTATAGAGA
    CATACGCAGG
151 GGCAAACTGT ATGCGGAAgc CAAATTCGCC GACgGcAGCG
    TAACTTACGG
201 CAAAGCGGGC GAGAGCAAAA CCGAGCAAAG CCCCAAGGCT
    ATGGATTTGT
251 TCACGCTTGC CTGGCAGTTG GCGGCAAATG ACGCGAAACT
    CCCCCCGGGG
301 CTGAAAATCA CCAACGGCAA AAAACTTTAT TCCGTCGGCG
    GTTTGAATAA
351 GGCGGGTACA GGAAAATACA GCATAGGCGG CGTGGAAACC
    GAAGTCGTCA
401 AATATCGGGT GCGGCGCGGC GACGATGCGG TAATGTATTT
    cTTCGCACCG
451 TCCCTGAACA ATATTCCGGC ACAAATCGGC TATACCGACG
    ACGGCAAAAC
501 CTATACGCTG AAACTCAAAT CGGTGCAGAT CAACGGCCAG
    GCAGCCAAAC
551 CGTAA

This corresponds to the amino acid sequence <SEQ ID 774; ORF120>:

1   ..IPATMTFERS GNAYKIVSTI KVPLYNIRFE SGGTVVGNTL
    HPTYYRDIRR
51  GKLYAEAKFA DGSVTYGKAG ESKTEQSPKA MDLFTLAWQL
    AANDAKLPPG
101 LKITNGKKLY SVGGLNKAGT GKYSIGGVET EVVKYRVRRG
    DDAVMYFFAP
151 SLNNIPAQIG YTDDGKTYTL KLKSVQINGQ AAKP*

Further work revealed the complete nucleotide sequence <SEQ ID 775>:

1   ATGATGAAGA CTTTTAAAAA TATATTTTCC GCCGCCATTT
    TGTCCGCCGC
51  CCTGCCGTGC GCGTATGCGG CAGGGCTGCC CCAATCCGCC
    GTGCTGCACT
101 ATTCCGGCAG CTACGGCATT CCCGCCACGA TGACATTTGA
    ACGCAGCGGC
151 AATGCTTACA AAATCGTTTC GACGATTAAA GTGCCGCTAT
    ACAATATCCG
201 TTTCGAGTCC GGCGGTACGG TTGTCGGCAA TACCCTGCAC
    CCTACCTACT
251 ATAGAGACAT ACGCAGGGGC AAACTGTATG CGGAAGCCAA
    ATTCGCCGAC
301 GGCAGCGTAA CTTACGGCAA AGCGGGCGAG AGCAAAACCG
    AGCAAAGCCC
351 CAAGGCTATG GATTTGTTCA CGCTTGCCTG GCAGTTGGCG
    GCAAATGACG
401 CGAAACTCCC CCCGGGGCTG AAAATCACCA ACGGCAAAAA
    ACTTTATTCC
451 GTCGGCGGTT TGAATAAGGC GGGTACAGGA AAATACAGCA
    TAGGCGGCGT
501 GGAAACCGAA GTCGTCAAAT ATCGGGTGCG GCGCGGCGAC
    GATGCGGTAA
551 TGTATTTCTT CGCACCGTCC CTGAACAATA TTCCGGCACA
    AATCGGCTAT
601 ACCGACGACG GCAAAACCTA TACGCTGAAA CTCAAATCGG
    TGCAGATCAA
651 CGGCCAGGCA GCCAAACCGT AA

This corresponds to the amino acid sequence <SEQ ID 776; ORF120-1>:

1   MMKTFKNIFS AAILSAALPC AYAAGLPQSA VLHYSGSYGI
    PATMTFERSG
51  NAYKIVSTIK VPLYNIRFES GGTVVGNTLH PTYYRDIRRG
    KLYAEAKFAD
101 GSVTYGKAGE SKTEQSPKAM DLFTLAWQLA ANDAKLPPGL
    KITNGKKLYS
151 VGGLNKAGTG KYSIGGVETE VVKYRVRRGD DAVMYFFAPS
    LNNIPAQIGY
201 TDDGKTYTLK LKSVQINGQA AKP*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF120 shows 92.4% identity over a 184aa overlap with an ORF (ORF120a) from strain A of N. meningitidis:

The complete length ORF120a nucleotide sequence <SEQ ID 777> is:

1   ATGATGAAGA CTTTTAAAAA TATATTTTCC GCCGCCATTT
    TGTCCGCCGC
51  CCTGCCGTGC GCGTATGCGG CAGGGCTGCC CNAATCCGCC
    GTGCTGCACT
101 ATTCCGGCAG CTACGGCATT CCCGCCACNA NNANNTNNGN
    ACNNNGNGNC
151 AATGCTTNCA AAATCGTTTC GACGATTAAA GTGCCGCTAT
    ACAATATCCG
201 TTTCGAGTCC GGCGGTACGG TTGTCGGCAA TACCCTGCAC
    CCTACCTACT
251 ATAGAGACAT ACGCAGGGGC AAACTGTATG CGGAAGCCAA
    ATTCGCCGAC
301 GGCAGCGTAA CCTACGGCAA AGCGGNNNNN ANCNNNNNNG
    NGCAAAGCCC
351 CAAGGCTATG GATTTGTTCA CGCTTGCNTG GCAGTTGGCG
    GCAAATGACG
401 CGAAACTCCC CCCGGGGCTG AAAATCACCA ACGGCAAAAA
    ACTTTATTCC
451 GTCGGCGGTT TGAATAAGGC GGGTACAGGA AAATACAGCA
    TAGGCGGCGT
501 GGAAACCGAA GTCGTCAAAT ATCGGGTGCG GCGCGGCGAC
    GATGCGGTAA
551 TGTATTTCTT CGCACCGTCC CTGAACAATA TTCCGGCACA
    AATCGGCTAT
601 ACCGACGACG GCAAAACCTA TACGCTGAAA CTCAAATCGG
    TGCAGATCAA
651 CGGCCAGGCA GCCAAACCGT AA

This encodes a protein having amino acid sequence <SEQ ID 778>:

1   MMKTFKNIFS AAILSAALPC AYAAGLPXSA VLHYSGSYGI
    PATXXXXXXX
51  NAXKIVSTIK VPLYNIRFES GGTVVGNTLH PTYYRDIRRG
    KLYAEAKFAD
101 GSVTYGKAXX XXXXQSPKAM DLFTLAWQLA ANDAKLPPGL
    KITNGKKLYS
151 VGGLNKAGTG KYSIGGVETE VVKYRVRRGD DAVMYFFAPS
    LNNIPAQIGY
201 TDDGKTYTLK LKSVQINGQA AKP*

ORF120a and ORF120-1 show 93.3% identity in 223 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF120 shows 97.8% identity over 184 aa overlap with a predicted ORF (ORF120ng) from N. gonorrhoeae:

The complete length ORF120ng nucleotide sequence <SEQ ID 779> is:

1   ATGATGAAGA CTTTTAAAAA TATATTTTCC GCCGCCATTT
    TGTCCGCCGC
51  CCTGCCGTGC GCGTATGCGG CAAGGCTACC CCAATCCGCC
    GTGCTGCACT
101 ATTCCGGCAG CTACGGCATT CCCGCCACGA TGACATTTGA
    ACGCAGCGGC
151 AATGCTTACA AAATCGTTTC GACGATTAAA GTGCCGCTAT
    ACAATATCCG
201 TTTCGAATCC GGCGGTACGG TTGTCGGCAA TACCCTGCAC
    CCTGCCTACT
251 ATAAAGACAT ACGCAGGGGC AAACTGTATG CGGAAGCCAA
    ATTCGCCGAC
301 GGCAGCGTAA CCTACGGCAA AGCGGGCGAG AGCAAAACCG
    AGCAAAGCCC
351 CAAGGCTATG GATTTGTTCA CGCTTGCCTG GCAGTTGGCG
    GCAAATGACG
401 CGAAACTCCC CCCGGGTCTG AAAATCACCA ACGGCAAAAA
    ACTTTATTCC
451 GTCGGCGGCC TGAATAAGGC GGGTACGGGA AAATACAGCA
    TaggCGGCGT
501 GGAAACCGAA GTCGTCAAAT ATCGGGTGCG GCGCGGCGAC
    GATACGGTAA
551 CGTATTTCTT CGCACCGTCC CTGAACAATA TTCCGGCACA
    AATCGGCTAT
601 ACCGACGACG GCAAAACCTA TACGCTGAAG CTCAAATCGG
    TGCAGATCAA
651 CGGACAGGCC GCCAAACCGT AA

This encodes a protein having amino acid sequence <SEQ ID 780>:

1   MMKTFKNIFS AAILSAALPC AYAARLPQSA VLHYSGSYGI
    PATMTFERSG
51  NAYKIVSTIK VPLYNIRFES GGTVVGNTLH PAYYKDIRRG
    KLYAEAKFAD
101 GSVTYGKAGE SKTEQSPKAM DLFTLAWQLA ANDAKLPPGL
    KITNGKKLYS
151 VGGLNKAGTG KYSIGGVETE VVKYRVRRGD DTVTYFFAPS
    LNNIPAQIGY
201 TDDGKTYTLK LKSVQINGQA AKP*

In comparison with ORF120-1, ORF120ng shows 97.8% identity in 223 aa overlap:

This analysis, including the presence of a putative leader sequence in the gonococcal protein suggests that the proteins from N. meningitidis and N. gonorrhoeae, and, their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 93

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 781>:

1   ATGTATCGGA GGAAAGGGCG GGGCATCAAG CCGTGGATGG
    GTGCCGGTGC
51  .GCGTTTGCC GCCTTGGTCT GGCTGGTTTT CGCGCTCGGC
    GATACTTTGA
101 CTCCGTTTGC GGTTGCGGCG GTGCTGGCGT ATGTATTGGA
    CCCTTTGGTC
151 GAATGGTTGC AGAAAAAGGG TTTGAACCGT GCATCCGCTT
    CGATGTCTGT
201 GATGGTGTTT TCCTTGATTT TGTTGTTGGC ATTATTGTTG
    ATTATCGTCC
251 CTATGCTGGT CGGGCAGTTC AACAATTTGG CATCGCGCCT
    GCCCCAATTA
301 ATCGGTTTTA TGCAGAACAC GCTGCTGCCG TGGTTGAAAA
    ATACAATCGG
351 CGGATATGTG GAAATCGATC AGGCATCTAT TATTGCGTGG
    CTTCAGGCGC
401 ATACGGGAGA GTTGAGCAAC GCGCTTAAGG CGTGGTTTCC
    CGTTTTGATG
451 AGGCAGGGCG GCAATATT..

This corresponds to the amino acid sequence <SEQ ID 782; ORF121>:

1   MYRRKGRGIK PWMGAGXAFA ALVWLVFALG DTLTPFAVAA
    VLAYVLDPLV
51  EWLQKKGLNR ASASMSVMVF SLILLLALLL IIVPMLVGQF
    NNLASRLPQL
101 IGFMQNTLLP WLKNTIGGYV EIDQASIIAW LQAHTGELSN
    ALKAWFPVLM
151 RQGGNI..

Further work revealed the complete nucleotide sequence <SEQ ID 783>:

1    ATGTATCGGA GGAAAGGGCG GGGCATCAAG CCGTGGATGG
     GTGCCGGTGC
51   GGCGTTTGCC GCCTTGGTCT GGCTGGTTTT CGCGCTCGGC
     GATACTTTGA
101  CTCCGTTTGC GGTTGCGGCG GTGCTGGCGT ATGTATTGGA
     CCCTTTGGTC
151  GAATGGTTGC AGAAAAAGGG TTTGAACCGT GCATCCGCTT
     CGATGTCTGT
201  GATGGTGTTT TCCTTGATTT TGTTGTTGGC ATTATTGTTG
     ATTATCGTCC
251  CTATGCTGGT CGGGCAGTTC AACAATTTGG CATCGCGCCT
     GCCCCAATTA
301  ATCGGTTTTA TGCAGAACAC GCTGCTGCCG TGGTTGAAAA
     ATACAATCGG
351  CGGATATGTG GAAATCGATC AGGCATCTAT TATTGCGTGG
     CTTCAGGCGC
401  ATACGGGAGA GTTGAGCAAC GCGCTTAAGG CGTGGTTTCC
     CGTTTTGATG
451  AGGCAGGGCG GCAATATTGT CAGCAGTATC GGCAACCTGC
     TGCTGCTTCC
501  CTTGCTGCTT TACTATTTCC TGCTGGATTG GCAGCGGTGG
     TCGTGCGGCA
551  TTGCCAAACT GGTTCCGAgG CGTTTTGCCG GTGCTTATAC
     GCGCATTACA
601  GGCAATTTGA ACGAGGTATT GGGCGAATTT TTGCGCGGGC
     AGCTTCTGGT
651  AATGCTGATT ATGGGCTTGG TTTACGGTTT GGGATTGGTG
     CTGGTCGGGC
701  TGGATTCGGG GTTTGCCATC GGTATGCTTG CCGGTATTTT
     GGTGTTTGTC
751  CCTTATCTCG GGGCGTTTAC GGGATTGCTG CTTGCCACCG
     TCGCCGCCTT
801  GCTCCAGTTC GGTTCGTGGA ACGGCATCCT ATCGGTTTGG
     GCGGTTTTTG
851  CCGTAGGACA GTTTCTCGAA AGTTTTTTCA TTACGCCGAA
     AATCGTGGGA
901  GACCGTATCG GGCTGTCGCC GTTTTGGGTT ATCTTTTCGC
     TGATGGCGTT
951  CGGGCAGCTG ATGGGCTTTG TCGGAATGTT GGCGGGATTG
     CCTTTGGCCG
1001 CCGTAACCTT GGTCTTGCTT CGCGAGGGCG TGCAGAAATA
     TTTTGCCGGC
1051 AGTTTTTACC GGGGCAGGTA G

This corresponds to the amino acid sequence <SEQ ID 784; ORF121-1>:

1   MYRRKGRGIK PWMGAGAAFA ALVWLVFALG DTLTPFAVAA
    VLAYVLDPLV
51  EWLQKKGLNR ASASMSVMVF SLILLLALLL IIVPMLVGQF
    NNLASRLPQL
101 IGFMQNTLLP WLKNTIGGYV EIDQASIIAW LQAHTGELSN
    ALKAWFPVLM
151 RQGGNIVSSI GNLLLLPLLL YYFLLDWQRW SCGIAKLVPR
    RFAGAYTRIT
201 GNLNEVLGEF LRGQLLVMLI MGLVYGLGLV LVGLDSGFAI
    GMLAGILVFV
251 PYLGAFTGLL LATVAALLQF GSWNGILSVW AVFAVGQFLE
    SFFITPKIVG
301 DRIGLSPFWV IFSLMAFGQL MGFVGMLAGL PLAAVTLVLL
    REGVQKYFAG
351 SFYRGR*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF121 shows 98.7% identity over a 156aa overlap with an ORF (ORF121a) from strain A of N. meningitidis:

The complete length ORF121a nucleotide sequence <SEQ ID 785> is:

1    ATGTATCGGA GGAAAGGGCG GGGCATCAAG CCGTGGATGG
     ATGCCGGTGC
51   GGCGTTTGCC GCCTTGGTCT GGCTGGTTTT CGCGCTCGGC
     GATACTTTGA
101  CTCCGTTTGC GGTTGCGGCG GTGCTGGCGT ATGTATTGGA
     CCCTTTGGTC
151  GAATGGTTGC AGAAAAAGGG TTTGAACCGT GCATCCGCTT
     CGATGTCTGT
201  GATGGTGTTT TCCTTGATTT TGTTGTTGGC ATTATTGTTG
     ATTATTGTCC
251  CTATGCTGGT CGGGCAGTTC AACAATTTGG CATCGCGCCT
     GCCCCAATTA
301  ATCGGTTTTA TGCAGAACAC GCTGCTGCCG TGGTTGAAAA
     ATACAATCGG
351  CGGATATGTG GAAATCGATC AGGCATCTAT TATTGCGTGG
     CTTCAGGCGC
401  ATACGGGCGA GTTGAGCAAC GCGCTTAAGG CGTGGTTTCC
     CGTTTTGATG
451  AGGCAGGGCG GCAATATTGT CAGCAGTATC GGCAACCTGC
     TGCTGCTTCC
501  CTTGCTGCTT TACTATTTCC TGCTGGATTG GCAGCGGTGG
     TCGTGCGGCA
551  TTGCCAAACT GGTTCCGAGG CGTTTTGCCG GTGCTTATAC
     GCGCATTACA
601  GGCAATTTGA ACGAGGTATT GGGCGAATTT TTGCGCGGGC
     AGCTTCTGGT
651  GATGCTGATT ATGGGTTTGG TTTACGGCTT GGGGTTGGTG
     CTGGTCGGGC
701  TGGATTCGGG GTTTGCAATC GGTATGGTTG CCGGTATTTT
     GGTTTTTGTT
751  CCCTATTTGG GCGCGTTTAC AGGACTGCTG CTGGCAACCG
     TCGCCGCCTT
801  GCTCCAGTTC GGTTCGTGGA ACGGCATCTT GGCTGTTTGG
     GCGGTTTTTG
851  CCGTAGGACA GTTTCTCGAA AGTTTTTTCA TTACGCCGAA
     AATCGTGGGA
901  GACCGTATCG GCCTGTCGCC GTTTTGGGTT ATCTTTTCGC
     TGATGGCGTT
951  CGGGCAGCTG ATGGGCTTTG TCGGAATGTT GGCCGGATTG
     CCTTTGGCCG
1001 CCGTAACCTT GGTCTTGCTT CGCGAGGGCG TGCAGAAATA
     TTTTGCCGGC
1051 AGTTTTTACC GGGGCAGGTA G

This encodes a protein having amino acid sequence <SEQ ID 786>:

1   MYRRKGRGIK PWMDAGAAFA ALVWLVFALG DTLTPFAVAA
    VLAYVLDPLV
51  EWLQKKGLNR ASASMSVMVF SLILLLALLL IIVPMLVGQF
    NNLASRLPQL
101 IGFMQNTLLP WLKNTIGGYV EIDQASIIAW LQAHTGELSN
    ALKAWFPVLM
151 RQGGNIVSSI GNLLLLPLLL YYFLLDWQRW SCGIAKLVPR
    RFAGAYTRIT
201 GNLNEVLGEF LRGQLLVMLI MGLVYGLGLV LVGLDSGFAI
    GMVAGILVFV
251 PYLGAFTGLL LATVAALLQF GSWNGILAVW AVFAVGQFLE
    SFFITPKIVG
301 DRIGLSPFWV IFSLMAFGQL MGFVGMLAGL PLAAVTLVLL
    REGVQKYFAG
351 SFYRGR*

ORF121a and ORF121-1 show 99.2% identity in 356 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF121 shows 97.4% identity over a 156 aa overlap with a predicted ORF (ORF121ng) from N. gonorrhoeae:

An ORF121ng nucleotide sequence <SEQ ID 787> was predicted to encode a protein having amino acid sequence <SEQ ID 788>:

1   MYRRKGRGIK PWMGAGAAFA ALVWLVYALG DTLTPFAVAA
    VLAYVLDPLV
51  EWLQKKGLNR ASASMSVMVF SLILLLALLL IIVPMLVGQF
    NNLASRLPQL
101 IGFMQNTLLP WLKNTIGGYV EIDQASIIAW FQAHTGELSN
    ALKAWFPVLM
151 KQGGNIVSTI GNLLLPPLLL YYFLLDWHRW SCGIPKLVPR
    RFAGAYTRIT
201 GNLNKVWGKF LRGQLLGETE RGAVVCRVGR ECWEGGGARS
    RPSDDGWPRW
251 GGG*

Further work revealed the following gonoccocal DNA sequence <SEQ ID 789>:

1    ATGTATCGGA GAAAAGGACG GGGCATCAAG CCGTGGATGG
     GTGCCGGCGC
51   GGCGTTTGCC GCCTTGGTCT GGCTGGTTTA CGCGCTCGGC
     GATACTTTGA
101  CTCCGTTTGC GGTTGCGGCG GTGCTGGCGT ATGTGTTGGA
     CCCTTTGGTC
151  GAATGGTTGC AGAAAAAGGG TTTGAACCGT GCATCCGCTT
     CGATGTCTGT
201  GATGGTGTTT TCCTTGATTT TGTTGTTGGC ATTATTGTTG
     ATTATTGTCC
251  CTATGCTGGT CGGGCAGTTC AATAATTTGG CATCTCGCCT
     GCCCCAATTA
301  ATCGGTTTTA TGCAGAACAC GCTGCTGCCG TGGTTGAAAA
     ATACAATCGG
351  CGGATATGTG GAAATCGATC AGGCATCTAT TATTGCGTGG
     TTTCAGGCGC
401  ATACGGGCGA GTTGAGCAAC GCGCTTAAGG CGTGGTTTCC
     CGTTTTGATG
451  AAACAGGGCG GCAATATTGT CAGCAGTATC GGCAACCTGC
     TGCTGCCGCC
501  CTTGCTGCTT TACTATTTCC TGCTGGATTG GCAGCGGTGG
     TCGTGCGGCA
551  TCGCCAAACT GGTTCCGAGG CGTTTTGCCG GTGCTTATAC
     GCGCATTACG
601  GGTAATTTGA ACGAGGTATT GGGCGAATTT TTGCGCGGTC
     AGCTTCTGGT
651  GATGCTGATT ATGGGCTTGG TTTACGGTTT GGGATTGATG
     CTAGTCGGAC
701  TGGATTCGGG ATTTGCCATC GGTATGGTTG CCGGTATTTT
     GGTGTTTGTC
751  CCCTATTTGG GTGCGTTTAC GGGATTGCTG CTTGCCACTG
     TTGCAGCCTT
801  GCTCCAGTTC GGTTCGTGGA ACGGAATCTT GGCTGTTTGG
     GCGGTTTTTG
851  CCGTCGGTCA GTTTCTCGAA AGTTTTTTCA TTACGCCGAA
     AATTGTAGGA
901  GACCGTATCG GCCTGTCGCC GTTTTGGGTT ATCTTTTCGC
     TGATGGCGTT
951  CGGAGAGCTG ATGGGCTTTG TCGGAATGTT GGCCGGATTG
     CCTTTGGCCG
1001 CCGTAACCTT GGTCTTGCTT CGCGAGGGCG CGCAGAAATA
     TTTTGCCGGC
1051 AGTTTTTACC GGGGCAGGTA G

This corresponds to the amino acid sequence <SEQ ID 790; ORF121ng-1>:

1   MYRRKGRGIK PWMGAGAAFA ALVWLVYALG DTLTPFAVAA
    VLAYVLDPLV
51  EWLQKKGLNR ASASMSVMVF SLILLLALLL IIVPMLVGQF
    NNLASRLPQL
101 IGFMQNTLLP WLKNTIGGYV EIDQASIIAW FQAHTGELSN
    ALKAWFPVLM
151 KQGGNIVSSI GNLLLPPLLL YYFLLDWQRW SCGIAKLVPR
    RFAGAYTRIT
201 GNLNEVLGEF LRGQLLVMLI MGLVYGLGLM LVGLDSGFAI
    GMVAGILVFV
251 PYLGAFTGLL LATVAALLQF GSWNGILAVW AVFAVGQFLE
    SFFITPKIVG
301 DRIGLSPFWV IFSLMAFGEL MGFVGMLAGL PLAAVTLVLL
    REGAQKYFAG
351 SFYRGR*

ORF121ng-1 and ORF121-1 show 97.5% identity in 356 aa overlap:

In addition, ORF121ng-1 shows homology to a permease from H. influenzae:

sp|P43969|PERM_HAEIN PUTATIVE PERMEASE PERM HOMOLOG Length = 349
Score = 69.9 bits (168), Expect = 2e−11
Identities = 67/317 (21%), Positives = 120/317 (37%), Gaps = 7/317 (2%)
Query:	26	VYALGDTLTPFAVAAVLAYVLDPLVEWL-QKKGLNRASASMSVMVFSXXXXXXXXXXXVP	84
		+Y  GD + P  +A VL+Y+L+  + +L Q     R  A++ +               VP
Sbjct:	32	IYFFGDLIAPLLIALVLSYLLEIPINFLNQYLKCPRMLATILIFGSFIGLAAVFFLVLVP	91
Query:	85	MLVGQFNNLASRLPQLIGFMQNTLLPWLKNTIGGYVE-IDQASIIAWFQAHTGELSNALK	143
		ML  Q  +L S LP +     N    WL N    Y E ID + + + F +   ++    +
Sbjct:	92	MLWNQTISLLSDLPAMF----NKSNEWLLNLPKNYPELIDYSMVDSIFNSVREKILGFGE	147
Query:	144	AWFPVLMKQGGNIVSSIGNXXXXXXXXXXXXXDWQRWSCGIAKLVPRRFAGAYTRITGNL	203
		+   + +    N+VS                 D      G+++ +P+    A+ R    +
Sbjct:	148	SAVKLSLASIMNLVSLGIYAFLVPLMMFFMLKDKSELLQGVSRFLPKNRNLAFXRWK-EM	206
Query:	204	NEVLGEFLRGQXXXXXXXXXXXXXXXXXXXXDSGFAIGMVAGILVFVPYXXXXXXXXXXX	263
		+ +  ++ G+                    +    +    G+ V VPY
Sbjct:	207	QQQISNYINGKLLEILIVTLITYIIFLIFGLNYPLLLAFAVGLSVLVPYIGAVIVTIPVA	266
Query:	264	XXXXXQFGSWNGILAVWAVFAVGQFLESFFITPKIVGDRIGLSPFWVIFSLMAFGELMGF	323
		QFG       +   FAV Q L+   + P +  + + L P  +I S++ FG L GF
Sbjct:	267	LVALFQFGISPTFWYIIIAFAVSQLLDGNLLVPYLFSEAVNLHPLIIIISVLIFGGLWGF	326
Query:	324	VGMLAGLPLAAVTLVLL	340
		G+   +PLA +   ++
Sbjct:	327	WGVFFAIPLATLVKAVI	343

Based on this analysis, including the presence of a putative leader sequence and transmembrane domains in the two proteins, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 94

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 791>:

1   ..ACTGCTTTTT CGGCGGCGCT GCGCTTGAGT CCATCATGAC
    TCGTCATATT
51  TTTGTCCTTT GGGAAACCGT ATCAACAAAC AGCCGCCATC
    TTAACATTTT
101 TTTGCACGTC CTGCCCGCCG CGTTCAAATG CGTACCAGCA
    ATACCGCCGC
151 CTGCGCCTCT ATGCCTTCCA TCCGCCCGAG ATAGCCGAGT
    TTTTCGTTGG
201 TTTTGCCTTT GATGTTGACG CACGAAATGT CTATGCCCAA
    ATCGGCGGCG
251 ATGTTGGCAC GCATTTGCGG AATGTGCGGC GCGAGTGTGG
    GTTTCTGTGC
301 AATCACGGTC GTATCGACAT TGACCGCCTG CCAACCCTGC
    GCCTGAACGC
351 TTTGATACGC CGCACGCAAA AGGACGCGGC TGTCCGCATC
    TTTGAACTCT
401 GCGGCGGTGT CGGGGAAATG GCTGCCGATA TCGCCCAAAC
    CTGCCGCACC
451 GAGCAGCGCG TCGGTAACGG CGTGCAGCAG CGCATCGGCA
    TCGGAGTGTC
501 CGAGCAGCCC TTTTTCAAAT GGGATTTCAA CTCCGCCAAG
    TATCAG..

This corresponds to the amino acid sequence <SEQ ID 792; ORF122>:

1   ..TAFSAALRLS PSXLVIFLSF GKPYQQTAAI LTFFCTSCPP
    RSNAYQQYRR
51  LRLYAFHPPE IAEFFVGFAF DVDARNVYAQ IGGDVGTHLR
    NVRRECGFLC
101 NHGRIDIDRL PTLRLNALIR RTQKDAAVRI FELCGGVGEM
    AADIAQTCRT
151 EQRVGNGVQQ RIGIGVSEQP FFKWDFNSAK YQ..

Further work revealed the complete nucleotide sequence <SEQ ID 793>:

1   ATATCGTACT GGGCAAGCAG TTCGCCGGAT TTTTTGGAAG
    TAGATACCGC
51  GCCTTTGATT TTTTTGCCGC TCTTACCCAA GGCTTCGATG
    AAAAAGTTGA
101 TGGTCGAGCC GGTACCGATG CCGATATATT CATTTTCGGG
    TACGAATTCG
151 ACTGCTTTTT CGGCGGCGAT GCGCTTGAGT TCGTCTTGTG
    TCGTCATATT
201 TTTGTCCTTT GGGAAACCGT ATCAACAAAC AGCCGCCATC
    TTAACATTTT
251 TTTGCACGTC CTGCCCGCCG CGTTCAAATG CGTACCAGCA
    ATACCGCCGC
301 CTGCGCCTCT ATGCCTTCCA TCCGCCCGAG ATAGCCGAGT
    TTTTCGTTGG
351 TTTTGCCTTT GATGTTGACG CACGAAATGT CTATGCCCAA
    ATCGGCGGCG
401 ATGTTGGCAC GCATTTGCGG AATGTGCGGC GCGAGTTTGG
    GTTTCTGTGC
451 AATCACGGTC GTATCGACAT TGACCGCCTG CCAACCCTGC
    GCCTGAACGC
501 TTTGATACGC CGCACGCAAA AGGACGCGGC TGTCCGCATC
    TTTGAACTCT
551 GCGGCGGTGT CGGGGAAATG GCTGCCGATA TCGCCCAAAC
    CTGCCGCACC
601 GAGCAGCGCG TCGGTAACGG CGTGCAGCAG CGCATCGGCA
    TCGGAGTGTC
651 CGAGCAGCCC TTTTTCAAAT GGGATTTCAA CTCCGCCAAG
    TATCAGCTTT
701 CTGCCTTCGG TCAGTTGGTG GACATCGTAG CCCTGTCCGA
    TACGGATGTT
751 CGTCATCGTT TGTGTTCCTG A

This corresponds to the amino acid sequence <SEQ ID 794; ORF122-1>:

1   ISYWASSSPD FLEVDTAPLI FLPLLPKASM KKLMVEPVPM
    PIYSFSGTNS
51  TAFSAAMRLS SSCVVIFLSF GKPYQQTAAI LTFFCTSCPP
    RSNAYQQYRR
101 LRLYAFHPPE IAEFFVGFAF DVDARNVYAQ IGGDVGTHLR
    NVRREFGFLC
151 NHGRIDIDRL PTLRLNALIR RTQKDAAVRI FELCGGVGEM
    AADIAQTCRT
201 EQRVGNGVQQ RIGIGVSEQP FFKWDFNSAK YQLSAFGQLV
    DIVALSDTDV
251 RHALCS*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF122 shows 94.0% identity over a 182aa overlap with an ORF (ORF122a) from strain A of N. meningitidis:

The complete length ORF122a nucleotide sequence <SEQ ID 795> is:

1   ATATCATATT GGGCAAGCAG TTCACTGGAT TTTTTGGAAG
    TAGATACCGC
51  GCCTTTGATT TTTTTGCCGC TCTTACCCAA GGCTTCGATG
    AAAAAGTTGA
101 TGGTCGAACC GGTACCGATG CCGATGTATT CGTTTTCGGG
    TACGAATTCG
151 ACTGCNTTTT CGGCGGCGAT GCGCTTGAGT TCGTCTTGTG
    TCGTCATATT
201 TTTGTCCTTT GGGAAACCGT ATCAACAAAC AGCCGCCATC
    TTAACATTTT
251 TTNNNACGTC CTGCCCGCCG CGTTCAAATC CTTACCAGCA
    ATACCGCCGC
301 CTGCGACTCT ATGCCTTCCA TGCGCCCGAG ATAACCGAGT
    TTTTCGTTGG
351 TTTTGCCTTT GANGTTGACG CACGAAATGT CTATGCCCAA
    ATCGGCGGCG
401 ATGTTGGCAC GCATTTGCGG AATATGCGGC GCGAGTTTGG
    GTTTCTGTGC
451 AATCACGGTC GTATCGACAT TGACCGCCTG CCAACCCTGC
    GCCTGAACGC
501 TTTGATACGC CGCACGCAAA AGGACGCGGC TGTCCGCATC
    TTTGAACTCT
551 GCGGCGGTGT CGGGGAAATG GCTGCCGATA TCGCCCAAAC
    CTGCCGCACC
601 GAGCAGCGCG TCGGTAACGG CGTGCAGCAG CGCATCGGCA
    TCGGAGTGTC
651 CGAGCAGCCC TTTTTCAAAT GGGATTTCAA CTCCGCCAAG
    TATCAGCTTT
701 CTGCCTTCGG TCAGTTGGTG GACATCGTAG CCCTGTCCGA
    TACGGATGTT
751 CGTCATCGTT TGTGTTCCTG A

This encodes a protein having amino acid sequence <SEQ ID 796>:

1   ISYWASSSLD FLEVDTAPLI FLPLLPKASM KKLMVEPVPM
    PMYSFSGTNS
51  TAFSAAMRLS SSCVVIFLSF GKPYQQTAAI LTFFXTSCPP
    RSNPYQQYRR
101 LRLYAFHAPE ITEFFVGFAF XVDARNVYAQ IGGDVGTHLR
    NMRREFGFLC
151 NHGRIDIDRL PTLRLNALIR RTQKDAAVRI FELCGGVGEM
    AADIAQTCRT
201 EQRVGNGVQQ RIGIGVSEQP FFKWDFNSAK YQLSAFGQLV
    DIVALSDTDV
251 RHRLCS*

ORF122a and ORF122-1 show 96.9% identity in 256 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF122 shows 89.6% identity over a 182 aa overlap with a predicted ORF (ORF122ng) from N. gonorrhoeae:

The complete length ORF122ng nucleotide sequence <SEQ ID 797> is:

1   ATGTCGTACC GGGCAAGCAG TTCGCCGGAT TTTTTGGAGG
    TTGAAACCGC
51  GCCTTTGATT TTTTTACCGC TTTTGCCCAA GGCTTCGATG
    AAGAAATTGa
101 tgGTCGAACC GgtaCCGATG CCGATGTATT CGTTTTCGGG
    TACGAATTCG
151 ACTGCTTTTT CGGCGGCGAT GCGCttgAgt TCgtcttgcg
    TcgTCATATT
201 TTTAtccttt gGGAAaccct atcaAcaAAc agccgccatC
    TTAACATTTT
251 TTTGCACGtc ctggccgccg cgttcaAATc cgtaccaGca
    ataccgccgc
301 ctgcgcctCT AtgcCTTCCA TCCGCCCGAG ATAGCCGAGT
    TTTTCGTTGG
351 TTTTGCCTTT GATatTGACG CACGAAATAT CGatacCCAa
    atcggcgGCG
401 ATGTTGGCAC GCATTTGCGG AATGTGCGGT GCGAGTTTGG
    GTTTCTGTGC
451 AATCACGGTC GTATCGACAT TGACCACCTG CCAACCCTGC
    GCCTGAACGC
501 TTTGATACGC CGCACGCAAA AGGACGCGGC TGTCCGCATC
    TTTGAACTCT
551 GCGGCGGTGT CGGGAAAATG GCTGCCGATG TCGCCCAAAC
    CTGCCGCACC
601 GAGCAGCgcg tcggtaaCGG CGTGCAGCAG cgcgTcgGCA
    TCCGAATGCC
651 CGAGCAGCCC TTTTTCAAAT GGGATTTCAA CTCCGCCAAG
    TATCAGCTTT
701 CTGCCTTCGG TCAATTGGTG GACATCGTAG CCCTGTCCGA
    TACGGATATT
751 CGTCATCGTT TGTGTTCCTG A

This encodes a protein having amino acid sequence <SEQ ID 798>:

1   MSYRASSSPD FLEVETAPLI FLPLLPKASM KKLMVEPVPM
    PMYSFSGTNS
51  TAFSAAMRLS SSCVVIFLSF GKPYQQTAAI LTFFCTSWPP
    RSNPYQQYRR
101 LRLYAFHPPE IAEFFVGFAF DIDARNIDTQ IGGDVGTHLR
    NVRCEFGFLC
151 NHGRIDIDHL PTLRLNALIR RTQKDAAVRI FELCGGVGKM
    AADVAQTCRT
201 EQRVGNGVQQ RVGIRMPEQP FFKWDFNSAK YQLSAFGQLV
    DIVALSDTDI
251 RHRLCS*

ORF122ng and ORF122-1 show 92.6% identity in 256 aa overlap:

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 95

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 799>:

1   ..GCCGGCGCGA GTGCGAACAA CATTTCCGCG CGTTTTGCGG
    AAACACCCGT
51  CGCTGTCAGC GTTACCCTGA TCGGCACGGT ACTTGCCGTC
    ATGCTGCCCG
101 TTACCGAATA TGAAAACTTC CTGCTGCTTA TCGGCTCGGT
    ATTTGCGCCG
151 ATGGGGCGGA TTTTGATTGC CGACTTTTTC GTCTTGAAAC
    GGCGTGA

This corresponds to the amino acid sequence <SEQ ID 800; ORF125>:

1  ..AGASANNISA RFAETPVAVS VTLIGTVLAV MLPVTEYENF
   LLLIGSVFAP
51 MGGFDCRLFR LETA*

Further work revealed the complete nucleotide sequence <SEQ ID 801>:

1    ATGTCGGGCA ATGCCTCCTC TCCTTCATCT TCCTCCGCCA
     TCGGGCTGAT
51   TTGGTTCGGC GCGGCGGTAT CGATTGCCGA AATCAGCACG
     GGTACGCTGC
101  TTGCGCCTTT GGGCTGGCAG CGCGGTCTGG CGGCTCTACT
     TTTGGGTCAT
151  GCCGTCGGCG GCGCGCTGTT TTTTGCGGCG GCGTATATCG
     GCGCACTGAC
201  CGGACGCAGC TCGATGGAAA GCGTGCGCCT GTCGTTCGGC
     AAACGCGGTT
251  CAGTGCTGTT TTCCGTGGCG AATATGCTGC AACTGGCCGG
     CTGGACGGCG
301  GTGATGATTT ACGCCGGCGC AACGGTCAGC TCCGCTTTGG
     GCAAAGTGTT
351  GTGGGACGGC GAATCTTTTG TCTGGTGGGC ATTGGCAAAC
     GGCGCGCTGA
401  TTGTGCTGTG GCTGGTTTTC GGCGCACGCA AAACAGGCGG
     GCTGAAAACC
451  GTTTCGATGC TGCTGATGCT GTTGGCGGTT CTGTGGCTGA
     GTGCCGAAGT
501  CTTTTCCACG GCAGGCAGCA CCGCCGCACA GGTTTCAGAC
     GGCATGAGTT
551  TCGGAACGGC AGTCGAGCTG TCCGCCGTGA TGCCGCTTTC
     CTGGCTGCCG
601  CTTGCCGCCG ACTACACGCG CCACGCGCGC CGCCCGTTTG
     CGGCAACCCT
651  GACGGCAACG CTCGCCTACA CGCTGACCGG CTGCTGGATG
     TATGCCTTGG
701  GTTTGGCAGC GGCGTTGTTC ACCGGAGAAA CCGACGTGGC
     AAAAATCCTG
751  CTGGGCGCAG GTTTGGGTGC GGCAGGCATT TTGGCGGTCG
     TCCTCTCCAC
801  CGTTACCACA ACGTTTCTCG ATGCCTATTC CGCCGGCGCG
     AGTGCGAACA
851  ACATTTCCGC GCGTTTTGCG GAAACACCCG TCGCTGTCGG
     CGTTACCCTG
901  ATCGGCACGG TACTTGCCGT CATGCTGCCC GTTACCGAAT
     ATGAAAACTT
951  CCTGCTGCTT ATCGGCTCGG TATTTGCGCC GATGGCGGCG
     GTTTTGATTG
1001 CCGACTTTTT CGTCTTGAAA CGGCGTGAGG AGATTGAAGG
     CTTTGACTTT
1051 GCCGGACTGG TTCTGTGGCT TGCGGGCTTC ATCCTCTACC
     GCTTCCTGCT
1101 CTCGTCCGGC TGGGAAAGCA GCATCGGTCT GACCGCCCCC
     GTAATGTCTG
1151 CCGTTGCCAT TGCCACCGTA TCGGTACGCC TTTTCTTTAA
     AAAAACCCAA
1201 TCTTTACAAA GGAACCCGTC ATGA

This corresponds to the amino acid sequence <SEQ ID 802; ORF125-1>:

1   MSGNASSPSS SSAIGLIWFG AAVSIAEIST GTLLAPLGWQ
    RGLAALLLGH
51  AVGGALFFAA AYIGALTGRS SMESVRLSFG KRGSVLFSVA
    NMLQLAGWTA
101 VMIYAGATVS SALGKVLWDG ESFVWWALAN GALIVLWLVF
    GARKTGGLKT
151 VSMLLMLLAV LWLSAEVFST AGSTAAQVSD GMSFGTAVEL
    SAVMPLSWLP
201 LAADYTRHAR RPFAATLTAT LAYTLTGCWM YALGLAAALF
    TGETDVAKIL
251 LGAGLGAAGI LAVVLSTVTT TFLDAYSAGA SANNISARFA
    ETPVAVGVTL
301 IGTVLAVMLP VTEYENFLLL IGSVFAPMAA VLIADFFVLK
    RREEIEGFDF
351 AGLVLWLAGF ILYRFLLSSG WESSIGLTAP VMSAVAIATV
    SVRLFFKKTQ
401 SLQRNPS*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF125 shows 76.5% identity over a 51 as overlap with an ORF (ORF125a) from strain A of N. meningitidis:

The ORF125a partial nucleotide sequence <SEQ ID 803> is:

1    ATGTCGGGCA ATGCCTCCTC TCNTTCATCT TCCGCCGCCA
     TCGGGCTGAT
51   TTGGTTCGGC GCGGCGGTAT CGATTGCCGA AATCAGCACG
     GGTACACTGC
101  TTGCGCCTTT GGGCTGGCAG CGCGGTCTGG CNGCTCTGCT
     TTTGGGTCAT
151  GCCGTCGGCG GCGCGCTGTT TTTTGCGGCG GCGTATATCG
     GCGCACTGAC
201  CGGACNCANC TCGATGGAAA GCGTGCGCCT GTCGTTCGGC
     AAACGCGGTT
251  CAGTGCTGTT TTCCGTGGCG AATATGCTGC AACTGGCCGG
     CTGGACGGCG
301  GTGATGATTT ACGCCGGCGC AACGGTCAGC TCCGCTTTGG
     GCAAAGTGTT
351  GTGGGACGGC GAATCTTTTG TCTGGTGGGC ATTGGCAAAC
     GGCGCGCTGA
401  TTGTGCTGTG GCTGGTTTTC GGCGCACGCA AAACAGGCGG
     GCTGAAAACC
451  GTTTCGATGC TGCTGATGCT GTTGGCGGTT CTGTGGCTGA
     GTGCCGAANT
501  NTTTTCCACG GCAGGCAGCA CCGCCGCANN GGTNNCAGAC
     GGCATGAGTT
551  TCGGAACGGC AGTCGAGCTG TCCGCCGTNA TGCCGCTTTC
     TTGGCTGCCG
601  CTGGCCGCCG ACTACACGCG CCACGCGCGC CGCCCGTTTG
     CGGCAACCCT
651  GACGGCAACG CTCGCCTACA CGCTGACCGG CTGCTGGATG
     TATGCCTTGG
701  GTTTGGCAGC GGCGTTGTTC ACCGGAGAAA CCGACGTGGC
     AAAAATCCTG
751  CTGGGCGCAG GTTTGGGTGC GGCAGGCATT TTGGCGGTCG
     TCCTGTCGAC
801  CGTTACCACC ACTTTTCTCG ATGCNTACTC CGCCGGCGTA
     AGTGCCAACA
851  ATATTTCCGC CAAACTTTCG GAAATACCNA TCGCCGTTGC
     CGTCGCCGTT
901  GTCGGCACAC TGCTTGCCGT CCTCCTGCCC GTTACCGAAT
     ATGAAAACTT
951  CCTGCTGCTT ATCGGCTCGG TATTTGCGCC GATGGCGGCG
     GTTTTGATTG
1001 CCGACTTTTT CGTCTTGAAA CGGCGTGAGG AGATTGAAGG
     C..

This encodes a protein having the partial amino acid sequence <SEQ ID 804>:

1   MSGNASSXSS SAAIGLIWFG AAVSIAEIST GTLLAPLGWQ
    RGLAALLLGH
51  AVGGALFFAA AYIGALTGXX SMESVRLSFG KRGSVLFSVA
    NMLQLAGWTA
101 VMIYAGATVS SALGKVLWDG ESFVWWALAN GALIVLWLVF
    GARKTGGLKT
151 VSMLLMLLAV LWLSAEXFST AGSTAAXVXD GMSFGTAVEL
    SAVMPLSWLP
201 LAADYTRHAR RPFAATLTAT LAYTLTGCWM YALGLAAALF
    TGETDVAKIL
251 LGAGLGAAGI LAVVLSTVTT TFLDAYSAGV SANNISAKLS
    EIPIAVAVAV
301 VGTLLAVLLP VTEYENFLLL IGSVFAPMAA VLIADFFVLK
    RREEIEG..

ORF125a and ORF125-1 show 94.5% identity in 347 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF125 shows 86.2% identity over a 65aa overlap with a predicted ORF (ORF125ng) from N. gonorrhoeae:

An ORF125ng nucleotide sequence <SEQ ID 805> was predicted to encode a protein having amino acid sequence <SEQ ID 806>:

1   MSGNASSPSS SAAIGLVWFG AAVSIAEIST GTLLAPLGWQ
    RGLAALLLGH
51  AVGGALFFAA AYIGALTGRS SMESVRLSFG KCGSVLFSVA
    NMLQLAGWTA
101 VMIYVGATVS SALGKVLWDG ESFVWWALAN GALIVLWLVF
    GARRTGGLKT
151 VSMLLMLLAV LWLSVEVFAS SGTNAAPAVS DGMTFGTAVE
    LSAVMPLSWL
201 PLAADYTRQA RRPFAATLTA TLAYTLTGCW MYALGLAAAL
    FTGETDVAKI
251 LLGAGLGITG ILAVVLSTVT TTFLDTYSAG ASANNISARF
    AEIPVAVGVT
301 LIRTVLAVML PVTEYKNFLL LIRSVFGPMA GGFDCRLFCL
    KTA*

Further work revealed the following gonococcal DNA sequence <SEQ ID 807>:

1    ATGTCGGGCA ATGCCTCCTC TCCTTCATCT TCCGCCGCCA
     TCGGGCTGGT
51   TTGGTTCGGC GCGGCGGTAT CGATTGCCGA AATCAGCACG
     GGTACGCTGC
101  TCGCCCCCTT GGGCTGGCAG CGCGGTCTGG CGGCCCTGCT
     TTTGGGTCAT
151  GCCGTCGGCG GCGCGCTGTT TTTTGCGGCG GCGTATATCG
     GCGCACTGAC
201  CGGACGCAGC TCGATGGAAA GTGTGCGCCT GTCGTTCGGC
     AAATGCGGTT
251  CAGTGCTGTT TTCCGTGGCG AATATGCTGC AACTGGCCGG
     CTGGACGGCG
301  GTGATGATTT ACGTCGGCGC AACGGTCAGC TCCGCTTTGG
     GCAAAGTGTT
351  GTGGGACGGC GAATCCTTTG TCTGGTGGGC ATTGGCAAAC
     GGCGCACTGA
401  TCGTGCTGTG GCTGGTTTTC GGCGCACGCA GAACGGGCGG
     GCTGAAAACC
451  GTTTCGATGC TGCTGATGCT GCTTGCCGTG TTGTGGTTGA
     GCGTCGAAGT
501  GTTCGCTTCG TCCGGCACAA ACGCCGCGCC CGCCGTTTCA
     GACGGCATGA
551  CCTTCGGAAC GGCAGTCGAA CTGTCCGCCG TCATGCCGCT
     TTCCTGGCTG
601  CCGCTGGCCG CCGACTACAC GCGCCAAGCA CGCCGCCCGT
     TTGCGGCAAC
651  CCTGACGGCA ACGCTCGCCT ATACGCTGAC GGGCTGCTGG
     ATGTATGCCT
701  TGGGTTTGGC GGCGGCTCTG TTTACCGGAG AAACCGACGT
     GGCGAAAATC
751  CTGTTGGGCG CGGGCTTGGG CATAACGGGC ATTCTGGCAG
     TCGTCCTCTC
801  CACCGTTACC ACAACGTTTC TCGATACCTA TTCCGCCGGC
     GCGAGTGCGA
851  ACAACATTTC CGCGCGTTTT GCGGAAATAC CCGTCGCTGT
     CGGCGTTACC
901  CTGATCGGCA CGGTGCTTGC CGTCATGCTG CCCGTTACCG
     AATATAAAAA
951  CTTCCTGCTG CTTATCGGCT CGGTATTTGC GCCGATGGCG
     GCGGTTTTGA
1001 TTGCCGACTT TTTCGTCTTA AAACGGCGTG AGGAGATTGA
     AGGCTTTGAC
1051 TTTGCCGGAC TGGTTCTGTG GCTGGCAGGC TTCATCCTCT
     ACCGCTTCCT
1101 GCTCTCGTCC GGTTGGGAAA GCAGCATCGG TCTGACCGCC
     CCCGTAATGT
1151 CTGCCGTTGC CATTGCCACC GTATCGGTAC GCCTTTTCTT
     TAAAAAAACC
1201 CAATCTTTAC AAAGGAACCC GTCATGA

This corresponds to the amino acid sequence <SEQ ID 808; ORF125ng-1>:

1   MSGNASSPSS SAAIGLVWFG AAVSIAEIST GTLLAPLGWQ
    RGLAALLLGH
51  AVGGALFFAA AYIGALTGRS SMESVRLSFG KCGSVLFSVA
    NMLQLAGWTA
101 VMIYVGATVS SALGKVLWDG ESFVWWALAN GALIVLWLVF
    GARRTGGLKT
151 VSMLLMLLAV LWLSVEVFAS SGTNAAPAVS DGMTFGTAVE
    LSAVMPLSWL
201 PLAADYTRQA RRPFAATLTA TLAYTLTGCW MYALGLAAAL
    FTGETDVAKI
251 LLGAGLGITG ILAVVLSTVT TTFLDTYSAG ASANNISARF
    AEIPVAVGVT
301 LIGTVLAVML PVTEYKNFLL LIGSVFAPMA AVLIADFFVL
    KRREEIEGFD
351 FAGLVLWLAG FILYRFLLSS GWESSIGLTA PVMSAVAIAT
    VSVRLFFKKT
401 QSLQRNPS*

ORF125ng-1 and ORF125-1 show 95.1% identity in 408 aa overlap:

Based on this analysis, including the presence of putative leader sequence and transmembrane domains in the gonococcal protein, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 96

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 809>:

1   ATGACCCGTA TCGCCATCCT CGGCGGCGGC CTCTCGGGAA
    GGCTGACCGC
51  GTTGCAGCTT GCAGAACAAG GTTATCAGAT TGCACTTTTC
    GATAAAAGCT
101 GCCGCCGGGG CGAACACGCC GCCGCCTATG TAGCCGCCGC
    CATGCTCGCG
151 CCTGCAGCGG A.ACGGTCGA AGCCACGCCC GAAGTGGTCA
    GGCTGGGCAG
201 GCAGAGCATC CCGCTTTGGC GCGGCATCCG ATGCCGTCTG
    AACACGCACA
251 CGATGATGCA GGAAAACGGC AGCCTGATTG TATGGCACGG
    GCAGGACAAG
301 CCATTATCCA GCGAGTTCGT CCGCCATCTC AAACGCGGCG
    GCGT.ACGGA
351 TGACGAAATC GTCCGTTGGC GCGCCGACGA CATCGCCGAA
    CGCGAACCGC
401 AACTCGGCGG ACGTTTTTAA GACGGCATCT ACCTGCCGAC
    CGAAGC.CAG
451 CTCGACGGGC GGCAATTATA GTCTGCACTT GCCGACGCTT
    TGGACGAACT
501 GAACGTCCCC TGCCATTGGG AACACGAATG CGTCCCCGAA
    GCCTGCAAG..

This corresponds to the amino acid sequence <SEQ ID 810; ORF126>:

1   MTRIAILGGG LSGRLTALQL AEQGYQIALF DKSCRRGEHA
    AAYVAAAMLA
51  PAAXTVEATP EVVRLGRQSI PLWRGIRCRL NTHTMMQENG
    SLIVWHGQDK
101 PLSSEFVRHL KRGGXTDDEI VRWRADDIAE REPQLGGRFX
    DGIYLPTEXQ
151 LDGRQLXSAL ADALDELNVP CHWEHECVPE ACK...

Further work revealed the complete nucleotide sequence <SEQ ID 811>:

1    ATGACCCGTA TCGCCATCCT CGGCGGCGGC CTCTCGGGAA
     GGCTGACCGC
51   GTTGCAGCTT GCAGAACAAG GTTATCAGAT TGCACTTTTC
     GATAAAGGCT
101  GCCGCCGGGG CGAACACGCC GCCGCCTATG TTGCCGCCGC
     CATGCTCGCG
151  CCTGCGGCGG AAGCGGTCGA AGCCACGCCC GAAGTGGTCA
     GGCTGGGCAG
201  GCAGAGCATC CCGCTTTGGC GCGGCATCCG ATGCCGTCTG
     AACACGCACA
251  CGATGATGCA GGAAAACGGC AGCCTGATTG TGTGGCACGG
     GCAGGACAAG
301  CCATTATCCA GCGAGTTCGT CCGCCATCTC AAACGCGGCG
     GCGTAGCGGA
351  TGACGAAATC GTCCGTTGGC GCGCCGACGA CATCGCCGAA
     CGCGAACCGC
401  AACTCGGCGG ACGTTTTTCA GACGGCATCT ACCTGCCGAC
     CGAAGGCCAG
451  CTCGACGGGC GGCAAATATT GTCTGCACTT GCCGACGCTT
     TGGACGAACT
501  GAACGTCCCC TGCCATTGGG AACACGAATG CGTCCCCGAA
     GGCCTGCAAG
551  CCCAATACGA CTGGCTGATC GACTGCCGCG GCTACGGCGC
     AAAAACCGCG
601  TGGAACCAAT CCCCCGAGCA CACCAGCACC CTGCGCGGCA
     TACGCGGCGA
651  AGTGGCGCGG GTTTACACAC CCGAAATCAC GCTCAACCGC
     CCCGTGCGTC
701  TGCTCCATCC GCGTTATCCG CTCTACATCG CCCCGAAAGA
     AAACCACGTC
751  TTCGTCATCG GCGCGACCCA AATCGAAAGC GAAAGCCAAG
     CCCCCGCCAG
801  CGTGCGTTCA GGGTTGGAAC TCTTGTCCGC ACTCTATGCC
     ATCCACCCCG
851  CCTTCGGCGA AGCCGACATC CTCGAAATCG CCACCGGCCT
     GCGCCCCACG
901  CTCAACCACC ACAACCCCGA AATCCGTTAC AACCGCGCCC
     GACGCCTGAT
951  TGAAATCAAC GGCCTTTTCC GCCACGGTTT CATGATCTCC
     CCCGCCGTAA
1001 CCGCCGCCGC CGCCAGATTG GCAGTGGCAC TGTTTGACGG
     AAAAGACGCG
1051 CCCGAACGCG ATAAAGAAAG CGGTTTGGCG TATATCCGAA
     GACAAGATTA
1101 A

This corresponds to the amino acid sequence <SEQ ID 812; ORF126-1>:

1   MTRIAILGGG LSGRLTALQL AEQGYQIALF DKGCRRGEHA
    AAYVAAAMLA
51  PAAEAVEATP EVVRLGRQSI PLWRGIRCRL NTHTMMQENG
    SLIVWHGQDK
101 PLSSEFVRHL KRGGVADDEI VRWRADDIAE REPQLGGRFS
    DGIYLPTEGQ
151 LDGRQILSAL ADALDELNVP CHWEHECVPE GLQAQYDWLI
    DCRGYGAKTA
201 WNQSPEHTST LRGIRGEVAR VYTPEITLNR PVRLLHPRYP
    LYIAPKENHV
251 FVIGATQIES ESQAPASVRS GLELLSALYA IHPAFGEADI
    LEIATGLRPT
301 LNHHNPEIRY NRARRLIEIN GLFRHGFMIS PAVTAAAARL
    AVALFDGKDA
351 PERDKESGLA YIRRQD*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF126 shows 90.0% identity over a 180aa overlap with an ORF (ORF126a) from strain A of N. meningitidis:

The complete length ORF126a nucleotide sequence <SEQ ID 813> is:

1    ATGACCCGTA TCGCCATCCT CGGCGGCGGC CTCTCNGGAA
     GGCTGACCGC
51   ACTGCAGCTT GCAGAACAAG GTTATCAGAT TGCACTTTTC
     GATAAAGGCT
101  GCCGCCGGGG CGAACACGCC GCCGCCTATG TTGCCGCCGC
     CATGCTCGCG
151  CCTGCGGCGG AAGCGGTCGA AGCCACGCCT GAAGTGGTCA
     GGCTGGGCAG
201  GCAGANCATC CCGCTTTGGC GCGGCATCCG ATGCCATCTG
     AAAACGCCTG
251  CCATGATGCA NGAAAACGGC AGCCTGATTG TGTGGCACGG
     GCAGGACAAA
301  CCTTTATCCA ACGAGTTCGT CCGCCATCTC AAACGCGGCG
     GCGTAGCGGA
351  TGACNAAATC GTCCGTTGGC GCGCCGACGA CATCGCCGAA
     CGCGAACCGC
401  AACTCGGCGG ACGTTTTTCA GACGGCATCT ACCTGCCGAC
     CGAAGGCCAG
451  CTCGACGGGC GGCAAATATT GTCTGCACTT GCCGACGCTT
     TGGACGAACT
501  GAACGTCCCC TGCCATTGGG AACACGAATG TGCCCCCGAA
     GACTTGCAAG
551  CCCAATACGA CTGGCTGATC GACTGCCGCG GCTACGGCGC
     AAAAACCGCG
601  TGGAACCAAT CCCCCGANNA NACCAGCACC CTGCGCGGCA
     TACGCGGCGA
651  AGTGGCGCGG GTTTACACAC CCGAAATCAC GCTCAACCGC
     CCCGTGCGCC
701  TGCTACACCC GCGCTATCCG CTNTACATCG CCCCGAAAGA
     AAACCNCGTC
751  TTCGTCATCG GCGCGACCCA AATCGAAAGC GAAAGCCAAG
     CACCTGCCAG
801  CGTGCGTTCC GGGCTGGAAC TCTTATCCGC ACTCTATGCC
     GTCCACCCCG
851  CCTTCGGCGA AGCCGACATC CTCGAAATCG CCACCGGCCT
     GCGCCCCACG
901  CTCAATCACC ACAACCCCGA AATCCGTTAC AACCGCGCCC
     GACGCCTGAT
951  TGAAATCAAC GGCCTTTTCC GCCACGGTTT CATGATCTCC
     CCCGCCGTAA
1001 CCGCCGCCGC CGTCAGATTG GCAGTGGCAC TGTTTGACGG
     AAAAGANGCG
1051 CCCGAACGCG ATGAAGAAAG CGGTTTGGCG TATATCCGAA
     GACAAGATTA
1101 A

This encodes a protein having amino acid sequence <SEQ ID 814>:

1   MTRIAILGGG LSGRLTALQL AEQGYQIALF DKGCRRGEHA
    AAYVAAAMLA
51  PAAEAVEATP EVVRLGRQXI PLWRGIRCHL KTPAMMXENG
    SLIVWHGQDK
101 PLSNEFVRHL KRGGVADDXI VRWRADDIAE REPQLGGRFS
    DGIYLPTEGQ
151 LDGRQILSAL ADALDELNVP CHWEHECAPE DLQAQYDWLI
    DCRGYGAKTA
201 WNQSPXXTST LRGIRGEVAR VYTPEITLNR PVRLLHPRYP
    LYIAPKENXV
251 FVIGATQIES ESQAPASVRS GLELLSALYA VHPAFGEADI
    LEIATGLRPT
301 LNHHNPEIRY NRARRLIEIN GLFRHGFMIS PAVTAAAVRL
    AVALFDGKXA
351 PERDEESGLA YIRRQD*

ORF126a and ORF126-1 show 95.4% identity in 366 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF126 shows 90% identity over a 180 aa overlap with a predicted ORF (ORF126ng) from N. gonorrhoeae:

An ORF126ng nucleotide sequence <SEQ ID 815> was predicted to encode a protein having amino acid sequence <SEQ ID 816>:

1   MTRIAVLGGG LSGRLTALQL AEQGYQIELF DKGTRQGEHA
    AAYVAAAMLA
51  PAAEAVEATP EVIRLGRQSI PLWRGIRCRL NTLTMMQENG
    SLIVWHGQDK
101 PLSSEFVRHL KRGGVADDEI VRWRADEIAE REPQLGGRFS
    DGIYLPTEGQ
151 LDGRQILSAL ADALDELNVP CHWEHECAPQ DLQAQYDWVI
    DCRGYGAKTA
201 WNQSPEHTST LRGIRGEVRG FTRPKSRSTA PCACCTRAIR
    STSPRKKTTS
251 SSSARPKSKA KAKPPPAYVP GWNSYPRSMP STPPSAKPTS
    SKWRPGLRPT
301 LNHHNPEIRY SRERRLIEIN GLFRHGFMIS PAVTAAAVRL
    AVALFDGKDA
351 PERDEESGLA YIGRQD*

Further work revealed the following gonococcal DNA sequence <SEQ ID 817>:

1    ATGACCCGTA TCGCCGTCCT CGGAGGCGGC CTTTCCGGAA
     GGCTGACCGC
51   ATTGCAGCTT GCAGAACAAG GTTATCAGAT TGAACTTTTC
     GACAAGGGCA
101  CCCGCCAAGG CGAACACGCC GCCGCCTATG TTGCCGCCGC
     GATGCTCGCG
151  CCTGCGGCGG AAGCGGTCGA GGCAACGCCC GAAGTCATCA
     GGCTGGGCAG
201  GCAGAGCATT CCGCTTTGGC GCGGCATCCG ATGCCGTCTG
     AACACGCTCA
251  CGATGATGCA GGAAAACGGC AGCCTGATTG TGTGGCACGG
     GCAGGACAAG
301  CCATTATCCA GCGAGTTCGT CCGCCATCTC AAACGCGGCG
     GCGTAGCGGA
351  TGACGAAATC GTCCGTTGGC GCGCCGATGA AATCGCCGAA
     CGCGAACCGC
401  AACTCGGCGG ACGTTTTTCA GACGGCATCT ACCTGCCGAC
     CGAAGGCCAG
451  CTCGACGGGC GGCAAATATT GTCTGCACTT GCCGACGCTT
     TGGACGAACT
501  GAACGTCCCT TGCCATTGGG AACACGAATG CGCCCCCCAA
     GACCTGCAAG
551  CCCAATACGA CTGGGTAATC GACTGCCGGG GCTACGGCGC
     GAAAACCGCG
601  TGGAACCAAT CCCCCGAGCA CACCAGCACC TTGCGCGGCA
     TACGCGGCGA
651  AGTGGCGCGG GTTTACACGC CCGAAATCAC GCTCAACCGC
     CCCGTGCGCC
701  TGCTGCACCC GCGCTATCCG CTCTACATCG CCCCGAAAGA
     AAACCACGTC
751  TTCGTCATCG GCGCGACCCA AATCGAAAGC GAAAGCCAAG
     CCCCCGCCAG
801  CGTACGTTCC GGGCTGGAAC TCTTATCCGC GCTCTATGCC
     GTCCACCCCG
851  CCTTCGGCGA AGCCGACATC CTCGAAATCG CCGCCGGCCT
     GCGCCCCACG
901  CTCAACCACC ACAACCCCGA AATCCGCTAC AGCCGCGAAC
     GCCGCCTCAT
951  CGAAATCAAC GGCCTTTTCC GGCACGGCTT TATGATTTCC
     CCCGCCGTAA
1001 CCGCCGCCGC CGTCAGATTG GCAGTGGCAC TGTTTGACGG
     AAAAGACGCG
1051 CCCGAACGTG ATGAAGAAAG CGGTTTGGCG TATATCGGAA
     GACAAGATTA
1101 A

This corresponds to the amino acid sequence <SEQ ID 818; ORF126ng-1>:

1   MTRIAVLGGG LSGRLTALQL AEQGYQIELF DKGTRQGEHA
    AAYVAAAMLA
51  PAAEAVEATP EVIRLGRQSI PLWRGIRCRL NTLTMMQENG
    SLIVWHGQDK
101 PLSSEFVRHL KRGGVADDEI VRWRADEIAE REPQLGGRFS
    DGIYLPTEGQ
151 LDGRQILSAL ADALDELNVP CHWEHECAPQ DLQAQYDWVI
    DCRGYGAKTA
201 WNQSPEHTST LRGIRGEVAR VYTPEITLNR PVRLLHPRYP
    LYIAPKENHV
251 FVIGATQIES ESQAPASVRS GLELLSALYA VHPAFGEADI
    LEIAAGLRPT
301 LNHHNPEIRY SRERRLIEIN GLFRHGFMIS PAVTAAAVRL
    AVALFDGKDA
351 PERDEESGLA YIGRQD*

ORF126ng-1 and ORF126-1 show 95.1% identity in 366 aa overlap:

Furthermore, ORF126ng-1 shows homology to a putative Rhizobium oxidase flavoprotein:

gi|2627327 (AF004408) putative amino acid oxidase
flavoprotein [Rhizobium etli]
Length = 327
Score = 169 bits (423), Expect = 3e−41
Identities = 112/329 (34%), Positives = 163/329 (49%), Gaps = 25/329 (7%)
Query:	3	RIAVLGGGLSGRLTALQLAEQGYQIELFDKGTRQGEHXXXXXXXXXXXXXXXXXXXXXXX	62
		RI V G G++G   A QL   G+++ L ++    G
Sbjct:	2	RILVNGAGVAGLTVAWQLYRHGFRVTLAERAGTVGA-GASGFAGGMLAPWCERESAEEPV	60
Query:	63	IRLGRQSIPLWRGIRCRLNTLTMMQENGSLIVWHGQDKPLSSEFVRHLKRGGVADDEIVR	122
		+ LGR +   W            +   G+L+V  G+D      F R    G    DE+
Sbjct:	61	LTLGRLAADWWEAA-----LPGHVHRRGTLVVAGGRDTGELDRFSRRTS-GWEWLDEVA-	113
Query:	123	WRADEIAEREPQLGGRFSDGIYLPTEGQLDGRQILSALADALDELNVPCHWEHECAPQDL	182
		IA  EP L GRF   ++   E  LD RQ L+ALA  L++  +           +
Sbjct:	114	-----IAALEPDLAGRFRRALFFRQEAHLDPRQALAALAAGLEDARMRLTLG---VVGES	165
Query:	183	QAQYDWVIDCRGYGAKTAWNQSPEHTSTLRGIRGEVARVYTPEITLNRPVRLLHPRYPLY	242
		+D V+DC G                LRG+RGE+  V T E++L+RPVRLLHPR+P+Y
Sbjct:	166	DVDHDRVVDCTGAA-------QIGRLPGLRGVRGEMLCVETTEVSLSRPVRLLHPRHPIY	218
Query:	243	IAPKENHVFVIGATQIESESQAPASVRSGLELLSALYAVHPAFGEADILEIAAGLRPTLN	302
		I P++ + F++GAT IES+   P + RS +ELL+A YA+HPAFGEA + E  AG+RP
Sbjct:	219	IVPRDKNRFMVGATMIESDDGGPITARSLMELLNAAYAMHPAFGEARVTETGAGVRPAYP	278
Query:	303	HHNPEIRYSRERRLIEINGLFRHGFMISP	331
		+ P  R ++E R + +NGL+RHGF+++P
Sbjct:	279	DNLP--RVTQEGRTLHVNGLYRHGFLLAP	305

This analysis suggests that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 97

The following DNA sequence, believed to be complete, was identified in N. meningitidis <SEQ ID 819>:

1   ATGACTGATA ATCGGGGGTT TACGCTGGTT GAATTAATAT
    CAGTGGTCTT
51  GATATTGTCT GTACTTGCTT TAATTGTTTA TCCGAGCTAT
    CGCAATTATG
101 TTGAGAAAGC AAAGATAAAT GCAGTGCGGG CAGCCTTGTT
    AGAAAATGCA
151 CATTTTATGG AAAAGTTTTA TCTGCAGAAT GGGAGGTTTA
    AACAAACATC
201 TACCAAGTGG CCAAGTTTGC CGATTAAAGA GGCAGAAGGC
    TTTTGTATCC
251 GTTTGAATGG AATCGtCGCG CGGG..GCTT TAGACAGTAA
    ATTCATGTTG
301 AAGGCGGTAG CCATAGATAA AGATAAAAAT CCTTTTATTA
    TTAAGATGAA
351 TGAAAATCTA GTAACCTTTA aTTTGCAAGA AGTCCGCCAG
    TTCGTGTAGT
401 GACGGGCTGG ATTATTTTAA AGGAAATGAT AAGGACTGCA
    AGTTACTTAA
451 GTAG

This corresponds to the amino acid sequence <SEQ ID 820; ORF127>:

1   MTDNRGFTLV ELISVVLILS VLALIVYPSY RNYVEKAKIN
    AVRAALLENA
51  HFMEKFYLQN GRFKQTSTKW PSLPIKEAEG FCIRLNGIVA
    RXALDSKFML
101 KAVAIDKDKN PFIIKMNENL VTFICKKSAS SCSDGLDYFK
    GNDKDCKLLK
151 *

Further work revealed the following DNA sequence <SEQ ID 821>:

1   ATGACTGATA ATCGGGGGTT TACGCTGGTT GAATTAATAT
    CAGTGGTCTT
51  GATATTGTCT GTACTTGCTT TAATTGTTTA TCCGAGCTAT
    CGCAATTATG
101 TTGAGAAAGC AAAGATAAAT GCAGTGCGGG CAGCCTTGTT
    AGAAAATGCA
151 CATTTTATGG AAAAGTTTTA TCTGCAGAAT GGGAGGTTTA
    AACAAACATC
201 TACCAAGTGG CCAAGTTTGC CGATTAAAGA GGCAGAAGGC
    TTTTGTATCC
251 GTTTGAATGG AATCGCGCGC GGGGCTTTAG ACAGTAAATT
    CATGTTGAAG
301 GCGGTAGCCA TAGATAAAGA TAAAAATCCT TTTATTATTA
    AGATGAATGA
351 AAATCTAGTA ACCTTTATTT GCAAGAAGTC CGCCAGTTCG
    TGTAGTGACG
401 GGCTGGATTA TTTTAAAGGA AATGATAAGG ACTGCAAGTT
    ACTTAAGTAG

This corresponds to the amino acid sequence <SEQ ID 822; ORF127-1>:

1   MTDNRGFTLV ELISVVLILS VLALIVYPSY RNYVEKAKIN
    AVRAALLENA
51  HFMEKFYLQN GRFKQTSTKW PSLPIKEAEG FCIRLNGIAR
    GALDSKFMLK
101 AVAIDKDKNP FIIKMNENLV TFICKKSASS CSDGLDYFKG
    NDKDCKLLK*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF127 shows 98.0% identity over a 150aa overlap with an ORF (ORF127a) from strain A of N. meningitidis:

The complete length ORF127a nucleotide sequence <SEQ ID 823> is:

1   ATGACTGATA ATCGGGGGTT TACGCTGGTT GAATTAATAT
    CAGTGGTCTT
51  GATATTGTCT GTACTTGCTT TAATTGTTTA TCCGAGCTAT
    CGCAATTATG
101 TTGAGAAAGC AAAGATAAAT ACAGTGCGGG CAGCCTTGTT
    AGAAAATGCA
151 CATTTTATGG AAAAGTTTTA TCTGCAGAAT GGGAGATTTA
    AACAAACATC
201 TACCAAATGG CCAAGTTTGC CGATTAAAGA GGCAGAAGGC
    TTTTGTATCC
251 GTTTGAATGG AATCGCGCGC GGGGCCTTAG ACAGTAAATT
    CATGTTGAAG
301 GCGGTAGCCA TAGATAAAGA TAAAAATCCT TTTATTATTA
    AGATGAATGA
351 AAATCTAGTA ACCTTTATTT GCAAGAAGTC CGCCAGTTCG
    TGTAGTGACG
401 GGCTGGATTA TTTTAAAGGA AATGATAAGG ACTGCAAGTT
    ACTTAAGTAG

This encodes a protein having amino acid sequence <SEQ ID 824>:

1   MTDNRGFTLV ELISVVLILS VLALIVYPSY RNYVEKAKIN
    TVRAALLENA
51  HFMEKFYLQN GRFKQTSTKW PSLPIKEAEG FCIRLNGIAR
    GALDSKFMLK
101 AVAIDKDKNP FIIKMNENLV TFICKKSASS CSDGLDYFKG
    NDKDCKLLK*

ORF127a and ORF127-1 show 99.3% identity in 149 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF127 shows 97.3% identity over a 150 aa overlap with a predicted ORF (ORF127ng) from N. gonorrhoeae:

The complete length ORF127ng nucleotide sequence <SEQ ID 825> is:

1   ATGACTGATA ATCGGGGGTT TACACTGGTT GAATTAATAT
    CAGTGGTCTT
51  GATATTGTCT GTACTTGCTT TAATTGTTTA TCCGAGCTAT
    CGCAATTATG
101 TTGAGAAAGC AAAGATAAAT GCAGTGCGGG CAGCCTTGTT
    AGAAAATGCA
151 CATTTTATGG AAAAGTTTTA TCTGCAGAAT GGGAGATTTA
    AACAAACATC
201 TACCAAATGG CCAAGTTTGC CGATTAAAGA GGCAGAAGGC
    TTTTGTATCC
251 GTTTGAATGG AATCGCGCGC GGGGCTTTAG ACAGTAAATT
    CATGTTGAAG
301 GCGGTAGCCA TAGATAAAGA TAAAAATCCT TTTATTATTA
    AGATGAATGA
351 AAATCTAGTA ACCTTTATTT GCAAGAAGTC CGCCAGTTCG
    TGTAGTGACG
401 GGCTGGATTA TTTTAAAGGA AATGATAAGG ACTGCAAGTT
    ACTTAAGTAG

This encodes a protein having amino acid sequence <SEQ ID 826>:

1   MTDNRGFTLV ELISVVLILS VLALIVYPSY RNYVEKAKIN
    AVRAAFLENA
51  HFMEKFYLQN GRFKQTSTKW PSLPIKEAEG FCIRLNGIAR
    GALDSKFMLK
101 AVAIDKDKNP FIIKMNENLV TFICKKSASS CSDRLDYFKG
    NDKDCKLLK*

ORF127ng and ORF127-1 show 100.0% identity in 149 aa overlap:

This analysis, including the fact that the predicted transmembrane domain is shared by the meningococcal and gonococcal proteins, suggests that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 98

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 827>

1   ..GTGTCGCTGG CTTCGGTGAT TGCCTCTCAA ATCTTCCTTT
    ACGAAGATTT
51  CAACCAAATG CGGAAAACCC GTGGAGCTAT CTGCGGTTTT
    CTTGTCCAAT
101 ATTTATCTGG GGTTTCAGCA GGGGTATTTC GATTTGAGTG
    CCGACGAGAA
151 CCCCGTACTG CATATCTGGT CTTTGGCAGT AGAGGAACAG
    TATTACCTCC
201 TGTATCCCCT TTTGCTGATA TTTTGCTGCA AAAAAACCAA
    ATCGCTACGG
251 GTGCTGCGTA ACATCAGCAT CATCCTGTTT TTGATTTTGA
    CTGCCTCATC
301 GTTTTTGCCA AGCGGGTTTT ATACCGACAT CCTCAACCAA
    CCCAATACTT
351 ATTACCTTTC GACACTGAGG TTTCCCGAGC TGTTGGCAGG
    TTCGCTGCTG
401 GCGGTTTACG GGCAAACGCA AAACGGCAGA CGGCAAACAG
    CAAATGGAAA
451 ACGGCAGTTG CTTTCATCAC TCTGCTTCGG CGCATTGCTT
    GCCTGCCTGT
501 TCGTGATTGA CAAACACAAT CCGTTTATCC CGGGAATGAC
    CCTGCTCCTT
551 CCCTGCCTGC TGACGGCACT GCTTATCCGG AGTATGCAAT
    ACGGGACACT
601 TCCGACCCGC ATCCTGTCGG CAAGCCCCAT CGTATTTGTC
    GGCAAAATCT
651 CTTATTCCCT ATACCTGTAC CATTGGATTT TTATTGCTTT
    CGCTCCGCTC
701 ATTAGAGGCG GGAAACAGCT CGGACTGCCT GCCG..

This corresponds to the amino acid sequence <SEQ ID 828; ORF128>:

1   ..VSLASVIASQ IFLYEDFNQM RKTVELSAVF LSNIYLGFQQ
    GYFDLSADEN
51  PVLHIWSLAV EEQYYLLYPL LLIFCCKKTK SLRVLRNISI
    ILFLILTASS
101 FLPSGFYTDI LNQPNTYYLS TLRFPELLAG SLLAVYGQTQ
    NGRRQTANGK
151 RQLLSSLCFG ALLACLFVID KHNPFIPGMT LLLPCLLTAL
    LIRSMQYGTL
201 PTRILSASPI VFVGKISYSL YLYHWIFIAF APLIRGGKQL
    GLPA..

Further work revealed the complete nucleotide sequence <SEQ ID 829>:

1    ATGCAAGCTG TCCGATACAG ACCGGAAATT GACGGATTGC
     GGGCCGTCGC
51   CGTGCTATCC GTCATGATTT TCCACCTGAA TAACCGCTGG
     CTGCCCGGAG
101  GATTCCTGGG GGTGGACATT TTCTTTGTCA TCTCAGGATT
     CCTCATTACC
151  GGCATCATTC TTTCTGAAAT ACAGAACGGT TCTTTTTCTT
     TCCGGGATTT
201  TTATACCCGC AGGATTAAGC GGATTTATCC TGCCTTTATT
     GCGGCCGTGT
251  CGCTGGCTTC GGTGATTGCC TCTCAAATCT TCCTTTACGA
     AGATTTCAAC
301  CAAATGCGGA AAACCGTGGA GCTTTCTGCG GTTTTCTTGT
     CCAATATTTA
351  TCTGGGGTTT CAGCAGGGGT ATTTCGATTT GAGTGCCGAC
     GAGAACCCCG
401  TACTGCATAT CTGGTCTTTG GCAGTAGAGG AACAGTATTA
     CCTCCTGTAT
451  CCCCTTTTGC TGATATTTTG CTGCAAAAAA ACCAAATCGC
     TACGGGTGCT
501  GCGTAACATC AGCATCATCC TGTTTTTGAT TTTGACTGCC
     TCATCGTTTT
551  TGCCAAGCGG GTTTTATACC GACATCCTCA ACCAACCCAA
     TACTTATTAC
601  CTTTCGACAC TGAGGTTTCC CGAGCTGTTG GCAGGTTCGC
     TGCTGGCGGT
651  TTACGGGCAA ACGCAAAACG GCAGACGGCA AACAGCAAAT
     GGAAAACGGC
701  AGTTGCTTTC ATCACTCTGC TTCGGCGCAT TGCTTGCCTG
     CCTGTTCGTG
751  ATTGACAAAC ACAATCCGTT TATCCCGGGA ATGACCCTGC
     TCCTTCCCTG
801  CCTGCTGACG GCACTGCTTA TCCGGAGTAT GCAATACGGG
     ACACTTCCGA
851  CCCGCATCCT GTCGGCAAGC CCCATCGTAT TTGTCGGCAA
     AATCTCTTAT
901  TCCCTATACC TGTACCATTG GATTTTTATT GCTTTCGCCC
     ATTACATTAC
951  AGGCGACAAA CAGCTCGGAC TGCCTGCCGT ATCGGCGGTT
     GCCGCGTTGA
1001 CGGCCGGATT TTCCCTGTTG AGTTATTATT TGATTGAACA
     GCCGCTTAGA
1051 AAACGGAAGA TGACCTTCAA AAAGGCATTT TTCTGCCTCT
     ATCTCGCCCC
1101 GTCCCTGATA CTTGTCGGTT ACAACCTGTA CGCAAGGGGG
     ATATTGAAAC
1151 AGGAACACCT CCGCCCGTTG CCCGGCGCGC CCCTTGCTGC
     GGAAAATCAT
1201 TTTCCGGAAA CCGTCCTGAC CCTCGGCGAC TCGCACGCCG
     GACACCTGAG
1251 GGGGTTTCTG GATTATGTCG GCAGCCGGGA AGGGTGGAAA
     GCCAAAATCC
1301 TGTCCCTCGA TTCGGAGTGT TTGGTTTGGG TAGATGAGAA
     GCTGGCAGAC
1351 AACCCGTTAT GTCGAAAATA CCGGGATGAA GTTGAAAAAG
     CCGAAGCCGT
1401 TTTCATTGCC CAATTCTATG ATTTGAGGAT GGGCGGCCAG
     CCTGTGCCGA
1451 GATTTGAAGC GCAATCCTTC CTAATACCCG GGTTCCCAGC
     CCGATTCAGG
1501 GAAACCGTCA AAAGGATAGC CGCCGTCAAA CCCGTCTATG
     TTTTTGCAAA
1551 CAACACATCA ATCAGCCGTT CGCCCCTGAG GGAGGAAAAA
     TTGAAAAGAT
1601 TTGCCGCAAA CCAATATCTC CGCCCCATTC AGGCTATGGG
     CGACATCGGC
1651 AAGAGCAATC AGGCGGTCTT TGATTTGATT AAAGATATTC
     CCAATGTGCA
1701 TTGGGTGGAC GCACAAAAAT ACCTGCCCAA AAACACGGTC
     GAAATATACG
1751 GCCGCTATCT TTACGGCGAC CAAGACCACC TGACCTATTT
     CGGTTCTTAT
1801 TATATGGGGC GGGAATTCCA CAAACACGAA CGCCTGCTTA
     AATCTTCCCA
1851 CGGCGGCGCA TTGCAGTAG

This corresponds to the amino acid sequence <SEQ ID 830; ORF128-1>:

1   MQAVRYRPEI DGLRAVAVLS VMIFHLNNRW LPGGFLGVDI
    FFVISGFLIT
51  GIILSEIQNG SFSFRDFYTR RIKRIYPAFI AAVSLASVIA
    SQIFLYEDFN
101 QMRKTVELSA VFLSNIYLGF QQGYFDLSAD ENPVLHIWSL
    AVEEQYYLLY
151 PLLLIFCCKK TKSLRVLRNI SIILFLILTA SSFLPSGFYT
    DILNQPNTYY
201 LSTLRFPELL AGSLLAVYGQ TQNGRRQTAN GKRQLLSSLC
    FGALLACLFV
251 IDKHNPFIPG MTLLLPCLLT ALLIRSMQYG TLPTRILSAS
    PIVFVGKISY
301 SLYLYHWIFI AFAHYITGDK QLGLPAVSAV AALTAGFSLL
    SYYLIEQPLR
351 KRKMTFKKAF FCLYLAPSLI LVGYNLYARG ILKQEHLRPL
    PGAPLAAENH
401 FPETVLTLGD SHAGHLRGFL DYVGSREGWK AKILSLDSEC
    LVWVDEKLAD
451 NPLCRKYRDE VEKAEAVFIA QFYDLRMGGQ PVPRFEAQSF
    LIPGFPARFR
501 ETVKRIAAVK PVYVFANNTS ISRSPLREEK LKRFAANQYL
    RPIQAMGDIG
551 KSNQAVFDLI KDIPNVHWVD AQKYLPKNTV EIYGRYLYGD
    QDHLTYFGSY
601 YMGREFHKHE RLLKSSHGGA LQ*

Computer analysis of this amino acid sequence gave the following results:

Homology with Hypothetical Integral Membrane Protein H10392 of H. influenzae (Accession Number U32723)

ORF128 and HI0392 show 52% aa identity in 180aa overlap:

Orf128:	1	VSLASVIASQIFLYEDFNQMRKTVELSAVFLSNIYLGFQQGYFDLSADENPVLHIWSLAV	60
		++L S IAS IF+Y DFN++RKT+EL+  FLSN YLG  QGYFDLSA+ENPVLHIWSLAV
HI0392:	46	MALVSFIASAIFIYNDFNKLRKTIELAIAFLSNFYLGLTQGYFDLSANENPVLHIWSLAV	105
Orf128:	61	EEQXXXXXXXXXIFCCKKTKSLRVLRNISIILFLILTASSFLPSGFYTDILNQPNTYYLS	120
		E Q         I   KK + ++VL  I++ILF IL A+SF+ + FY ++L+QPN YYLS
HI0392:	106	EGQYYLIFPLILILAYKKFREVKVLFIITLILFFILLATSFVSANFYKEVLHQPNIYYLS	165
Orf128:	121	TLRFPELLAGSLLAVYGQTQNGRRQTANGKRQLLSSLCFGALLACLFVIDKHNPFIPGMT	180
		LRFPELL GSLLA+Y    N + Q +     +L+ L    L +CLF+++ +  FIPG+T
HI0392:	166	NLRFPELLVGSLLAIYHNLSN-KVQLSKQVNNILAILSTLLLFSCLFLMNNNIAFIPGIT	224

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF128 shows 98.0% identity over a 244aa overlap with an ORF (ORF128a) from strain A of N. meningitidis:

The complete length ORF128a nucleotide sequence <SEQ ID 831> is:

1    ATGCAAGCTG TCCGATACAG ACCGGAAATT GACGGATTGC
     GGGCCGTCGC
51   CGTGCTATCC GTCATGATTT TCCACCTGAA TAACCGCTGG
     CTGCCCGGAG
101  GATTCCTGGG GGTGGACATT TTCTTTGTCA TCTCAGGATT
     CCTCATTACC
151  GGCATCATTC TTTCTGAAAT ACAGAACGGT TCTTTTTCTT
     TCCGGGATTT
201  TTATACCCGC AGGATTAAGC GGATTTATCC TGCTTTTATT
     GCGGCCGTGT
251  CGCTGGCTTC GGTGATTGCC TCTCAAATCT TCCTTTACGA
     AGATTTCAAC
301  CAAATGCGGA AAACCGTGGA GCTTTCTGCG GTTTTCTTGT
     CCAATATTTA
351  TCTGGGGTTT CAGCAGGGGT ATTTCGATTT GAGTGCCGAC
     GAGAACCCCG
401  TACTGCATAT CTGGTCTTTG GCAGTAGAGG AACAGTATTA
     CCTCCTGTAT
451  CCTCTTTTGC TGATATTTTG CTGCAAAAAA ACAAAATCGC
     TACGGGTGCT
501  GCGTAACATC AGCATCATCC TATTTCTGAT TTTGACTGCC
     ACATCGTTTT
551  TGCCAAGCGG GTTTTATACC GATATTCTCA ACCAACCCAA
     TACTTATTAC
601  CTTTCGACAC TGAGGTTTCC CGAGCTGTTG GCAGGTTCGC
     TGCTGGCGGT
651  TTACGGGCAA ACGCAAAACG GCAGACGGCA AACAGCAAAT
     GGAAAACGGC
701  AGTTGCTTTC ATCACTCTGC TTCGGCGCAT TGCTTGCCTG
     CCTGTTCGTG
751  ATTGACAAAC ACAATCCGTT TATCCCGGGA ATGACCCTGC
     TCCTTCCCTG
801  CCTGCTGACG GCACTGCTTA TCCGGAGTAT GCAATACGGG
     ACACTTCCGA
851  CCCGCATCCT GTCGGCAAGC CCCATCGTAT TTGTCGGCAA
     AATCTCTTAT
901  TCCCTATACC TGTACCATTG GATTTTTATT GCTTTCGCCC
     ATTACATTAC
951  AGGCGACAAA CAGCTCGGAC TGCCTGCCGT ATCGGCGGTT
     GCCGCGTTGA
1001 CGGCCGGATT TTCCCTGTTG AGTTATTATT TGATTGAACA
     GCCGCTTAGA
1051 AAACGGAAGA TGACCTTCAA AAAGGCATTT TTCTGCCTCT
     ATCTCGCCCC
1101 GTCCCTGATA CTTGTCGGTT ACAACCTGTA CGCAAGGGGG
     ATATTGAAAC
1151 AGGAACACCT CCGCCCGTTG CCCGGCGCGC CCCTTGCTGC
     GGAAAATCAT
1201 TTTCCGGAAA CCGTCCTGAC CCTCGGCGAC TCGCACGCCG
     GACACCTGCG
1251 GGGGTTTCTG GATTATGTCG GCAGCCGGGA AGGGTGGAAA
     GCCAAAATCC
1301 TGTCCCTCGA TTCGGAGTGT TTGGTTTGGG TAGATGAGAA
     GCTGGCAGAC
1351 AACCCGTTAT GTCGAAAATA CCGGGATGAA GTTGAAAAAG
     CCGAAGCCGT
1401 TTTCATTGCC CAATTCTATG ATTTGAGGAT GGGCGGCCAG
     CCCGTGCCGA
1451 GATTTGAAGC GCAATCCTTC CTAATACCCG GGTTCCCAGC
     CCGATTCAGG
1501 GAAACCGTCA AAAGGATAGC CGCCGTCAAA CCCGTCTATG
     TTTTTGCAAA
1551 CAACACATCA ATCAGCCGTT CGCCCCTGAG GGAGGAAAAA
     TTGAAAAGAT
1601 TTGCCGCAAA CCAATATCTC CGCCCCATTC AGGCTATGGG
     CGACATCGGC
1651 AAGAGCAATC AGGCGGTCTT TGATTTGATT AAAGATATTC
     CCAATGTGCA
1701 TTGGGTGGAC GCACAAAAAT ACCTGCCCAA AAACACGGTC
     GAAATATACG
1751 GCCGCTATCT TTACGGCGAC CAAGACCACC TGACCTATTT
     CGGTTCTTAT
1801 TATATGGGGC GGGAATTTCA CAAACACGAA CGCCTGCTTA
     AATCTTCTCG
1851 CGACGGCGCA TTGCAGTAG

This encodes a protein having amino acid sequence <SEQ ID 832>:

1   MQAVRYRPEI DGLRAVAVLS VMIFHLNNRW LPGGFLGVDI
    FFVISGFLIT
51  GIILSEIQNG SFSFRDFYTR RIKRIYPAFI AAVSLASVIA
    SQIFLYEDFN
101 QMRKTVELSA VFLSNIYLGF QQGYFDLSAD ENPVLHIWSL
    AVEEQYYLLY
151 PLLLIFCCKK TKSLRVLRNI SIILFLILTA TSFLPSGFYT
    DILNQPNTYY
201 LSTLRFPELL AGSLLAVYGQ TQNGRRQTAN GKRQLLSSLC
    FGALLACLFV
251 IDKHNPFIPG MTLLLPCLLT ALLIRSMQYG TLPTRILSAS
    PIVFVGKISY
301 SLYLYHWIFI AFAHYITGDK QLGLPAVSAV AALTAGFSLL
    SYYLIEQPLR
351 KRKMTFKKAF FCLYLAPSLI LVGYNLYARG ILKQEHLRPL
    PGAPLAAENH
401 FPETVLTLGD SHAGHLRGFL DYVGSREGWK AKILSLDSEC
    LVWVDEKLAD
451 NPLCRKYRDE VEKAEAVFIA QFYDLRMGGQ PVPRFEAQSF
    LIPGFPARFR
501 ETVKRIAAVK PVYVFANNTS ISRSPLREEK LKRFAANQYL
    RPIQAMGDIG
551 KSNQAVFDLI KDIPNVHWVD AQKYLPKNTV EIYGRYLYGD
    QDHLTYFGSY
601 YMGREFHKHE RLLKSSRDGA LQ*

ORF128a and ORF128-1 show 99.5% identity in 622 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF128 shows 93.4% identity over 244 aa overlap with a predicted ORF (ORF128ng) from N. gonorrhoeae:

The complete length ORF128ng nucleotide sequence <SEQ ID 833> is:

1    ATGCAAGCTG TCCGATACAG GCCTGAAATT GACGGATTGC
     GGGCCGTCGC
51   CGTGCTATCC GTCATTATTT TCCACCTGAA TAACCGCTGG
     CTGCCCGGAG
101  GATTCCTGGG GGTGGACATT TTCTTTGTCA TCTCGGGATT
     CCTCATTACC
151  AACATCATTC TTTCTGAAAT ACAGAACGGT TCTTTTTCTT
     TCCGGGATTT
201  TTATACCCGC AGGATTAAGC GGATTTATCC TGCTTTTATT
     GCGGCCGTGT
251  CCCTGGCTTC GGTGATTGCT TCTCAAATCT TCCTTTACGA
     AGATTTCAAC
301  CAAATGAGGA AAACCATAGA GCTTTCTACG GTTTTTTTGT
     CCAATATTTA
351  TTTGGGGTTC CGATTGGGGT ATTTCGATTT GAGTGCCGAC
     GAGAACCCCG
401  TACTGCATAT CTGGTCTTTG GCGGTAGAGG AACAGTATTA
     CCTCCTGTAT
451  CCTCTTTTGC TGATATTCTG TTACAAAAAA ACCAAATCAC
     TACGGGTGCT
501  GCGTAATATC AGCATCATCC TGTTTCTGAT TTTGACCGCA
     TCATCGTTTT
551  TGCCGGCCGG GTTTTATACC GACATCCTCA ACCAACCcaa
     TACTTATTAC
601  CTTTCGACAC TGAGGTTTCC CGAGCTGTTG GTGGGTTCGC
     TGTTGGCGGT
651  TTACGGGCAA ACGCAAAACG GCAGACGGCA AACAGAAAAT
     GGAAAACGGC
701  AGTTGCTTTC ATTACTCTGT TTCGGCGCat tgCTTGTCTG
     CCTGTTCGTG
751  ATCGACAAAC ACGATCCGTT TATCCCGGGA ATAACCCTGC
     TCCTTCCCTG
801  CCTGCTGACG GCGCTGCTTA TCCGGAGTAT GCAATACGGG
     ACACTTCCGA
851  CCCGCATCCT GTCGGCAAGC CCCATCGTAT TTGTCGGCAA
     AATCTCTTAT
901  TCCCTATACC TGTACCATTG GATTTTTATT GCCTTCGCCC
     ATTACATTAC
951  AGGCGACAAA CAGCTCGGAC TGCCTGCCGT ATCGGCGGTT
     GCCGCGTTGA
1001 CGGCCGGATT TTCCCTGTTG AGCTATTATT TGATTGAACA
     GCCGCTTAGA
1051 AAACGGAAGA TGACCTTCAA AAAGGCATTT TTCTGCCTTT
     ATCTCGCCCC
1101 GTCCCTGATG CTTGTCGGTT ACAACCTGTA TTCAAGAGGG
     ATATTGAAAC
1151 AGGAACACCT CCGCCCGCTG CCCGGCACGC CCGTTGCTGC
     GGAAAATAAT
1201 TTTCCGGAAA CCGTCTTGAC CCTCGGCGAC TCGCACGCCG
     GACACCTGCG
1251 GGGGTTTCTG GATTATGTCG GCGGCAGGGA AGGGTGGAAA
     GCTAAAATCC
1301 TGTCCCTCGA TTCGGAGTGT TTGGTTTGGG TGGATGAGAA
     GCTGGCAGAC
1351 AACCCGTTGT GCCGAAAATA CCGGGATGAA GTTGAAAAAG
     CCGAAGCTGT
1401 TTTCATTGCC CAATTCTATG ATTTGAGGAT GGGCGGCCAG
     CCCGTGCCGA
1451 GATTTGAAGC GCAATCCTTC CTGATACCCG GGTTCAAAGC
     CCGATTCAGG
1501 GAAACCGTCA AGAGGATAGC CGCCGTCAAA CCTGTATATG
     TTTTTGCAAA
1551 CAATACATCA ATCAGCCGTT CTCCCTTGAG GGAGGAAAAA
     TTGAAAAGAT
1601 TTGCTATAAA CCAATACCTC CGGCCTATTC GGGCTATGGG
     CGACATCGGC
1651 AAGAGCAATC AGGCGGTCTT TGATTTGGTT AAAGATATTC
     CCAATGTGCA
1701 TTGGGTGGAC GCACAAAAAT ACCTGCCCAA AAACACGGTC
     GAAATACACG
1751 GACGCTATCT TTACGGCGAC CAAGACCACC TGACCTATTT
     CGGTTCTTAT
1801 TATATGGGGC GGGAATTTCA CAAACACGAA CGCCTGCTCA
     AGCATTCCCG
1851 AGGCGGCGCA TTGCAGTAG

This encodes a protein having amino acid sequence <SEQ ID 834>:

1   MQAVRYRPEI DGLRAVAVLS VIIFHLNNRW LPGGFLGVDI
    FFVISGFLIT
51  NIILSEIQNG SFSFRDFYTR RIKRIYPAFI AAVSLASVIA
    SQIFLYEDFN
101 QMRKTIELST VFLSNIYLGF RLGYFDLSAD ENPVLHIWSL
    AVEEQYYLLY
151 PLLLIFCYKK TKSLRVLRNI SIILFLILTA SSFLPAGFYT
    DILNQPNTYY
201 LSTLRFPELL VGSLLAVYGQ TQNGRRQTEN GKRQLLSLLC
    FGALLVCLFV
251 IDKHDPFIPG ITLLLPCLLT ALLIRSMQYG TLPTRILSAS
    PIVFVGKISY
301 SLYLYHWIFI AFAHYITGDK QLGLPAVSAV AALTAGFSLL
    SYYLIEQPLR
351 KRKMTFKKAF FCLYLAPSLM LVGYNLYSRG ILKQEHLRPL
    PGTPVAAENN
401 FPETVLTLGD SHAGHLRGFL DYVGGREGWK AKILSLDSEC
    LVWVDEKLAD
451 NPLCRKYRDE VEKAEAVFIA QFYDLRMGGQ PVPRFEAQSF
    LIPGFKARFR
501 ETVKRIAAVK PVYVFANNTS ISRSPLREEK LKRFAINQYL
    RPIRAMGDIG
551 KSNQAVFDLV KDIPNVHWVD AQKYLPKNTV EIHGRYLYGD
    QDHLTYFGSY
601 YMGREFHKHE RLLKHSRGGA LQ*

ORF128ng and ORF128-1 show 95.7% identity in 622 aa overlap:

In addition, ORF218ng shows homology to a hypothetical H. influenzae protein:

sp|P43993|Y392_HAEIN HYPOTHETICAL PROTEIN HI0392 >gi|1074385|pir||B64007
hypothetical protein HI0392 - Haemophilus influenzae (strain Rd KW20)
>gi|1573364 (U32723) H. influenzae predicted coding region HI0392
[Haemophilus influenzae] Length = 245
Score = 239 bits (604), Expect = 3e−62
Identities = 124/225 (55%), Positives = 152/225 (67%), Gaps = 1/225 (0%)
Query:	38	VDIFFVISGFLITNIILSEIQNGSFSFRDFYTRRIKRIYPXXXXXXXXXXXXXXXXFLYE	97
		+DIFFVISGFLIT II++EIQ  SFS + FYTRRIKRIYP                F+Y
Sbjct:	1	MDIFFVISGFLITGIIITEIQQNSFSLKQFYTRRIKRIYPAFITVMALVSFIASAIFIYN	60
Query:	98	DFNQMRKTIELSTVFLSNIYLGFRLGYFDLSADENPVLHIWSLAVEEQXXXXXXXXXIFC	157
		DFN++RKTIEL+  FLSN YLG   GYFDLSA+ENPVLHIWSLAVE Q         I
Sbjct:	61	DFNKLRKTIELAIAFLSNFYLGLTQGYFDLSANENPVLHIWSLAVEGQYYLIFPLILILA	120
Query:	158	YKKTKSLRVLRNISIILFLILTASSFLPAGFYTDILNQPNTYYLSTLRFPELLVGSLLAV	217
		YKK + ++VL  I++ILF IL A+SF+ A FY ++L+QPN YYLS LRFPELLVGSLLA+
Sbjct:	121	YKKFREVKVLFIITLILFFILLATSFVSANFYKEVLHQPNIYYLSNLRFPELLVGSLLAI	180
Query:	218	YGQTQNGRRQTENGKRQLLSLLCFGALLVCLFVIDKHDPFIPGIT	262
		Y    N + Q       +L++L    L  CLF+++ +  FIPGIT
Sbjct:	181	YHNLSN-KVQLSKQVNNILAILSTLLLFSCLFLMNNNIAFIPGIT	224

This analysis, including the identification of several putative transmembrane domains, suggests that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 99

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 835>:

1   ..ATTATTTACG AATACCGCTG GATGTTTCTT TACGGCGCAC
    TGACGACCTT
51  GGGGCTGACG GTCGTGGCAA C.GCGGGCGG TTCGGTATTG
    GGTCTGTTGT
101 TGGCGTTGGC GCGCCTGATT CACTTGGAAA AAGCCGGTGC
    GCCGATGCGC
151 GTGCTGGCGT GGGCGTTGCG TAAAGTTTCG CTGCTGTATG
    TTACGCTGTT
201 CCGGGGTACG CCGCTGTTTG TGCAGATTGT GATTTGGGCG
    TATGTGTGGT
251 TTCCGTTTTT CGTC..

This corresponds to the amino acid sequence <SEQ ID 836; ORF129>:

1  ..IIYEYRWMFL YGALTTLGLT VVAXAGGSVL GLLLALARLI
   HLEKAGAPMR
51 VLAWALRKVS LLYVTLFRGT PLFVQIVIWA YVWFPFFV..

Further work revealed the complete nucleotide sequence <SEQ ID 837>:

1   ATGGATTTTC GTTTTGACAT TATTTACGAA TACCGCTGGA
    TGTTTCTTTA
51  CGGCGCACTG ACGACCTTGG GGCTGACGGT CGTGGCAACG
    GCGGGCGGTT
101 CGGTATTGGG TCTGTTGTTG GCGTTGGCGC GCCTGATTCA
    CTTGGAAAAA
151 GCCGGTGCGC CGATGCGCGT GCTGGCGTGG GCGTTGCGTA
    AAGTTTCGCT
201 GCTGTATGTT ACGCTGTTCC GGGGTACGCC GCTGTTTGTG
    CAGATTGTGA
251 TTTGGGCGTA TGTGTGGTTT CCGTTTTTCG TCCATCCTTC
    AGACGGCATT
301 TTGGTCAGCG GCGAGGCGGC AATCGCGCTG CGTCGCGGAT
    ACGGGCCGCT
351 GATTGCCGGT TCTTTGGCAC TGATCGCCAA CTCGGGGGCG
    TATATCTGTG
401 AGATTTTCCG CGCGGGCATC CAGTCTATAG ACAAAGGACA
    GATGGAGGCG
451 GCGCGTTCTT TGGGGCTGAC CTATCCGCAG GCGATGCGCT
    ATGTGATTCT
501 GCCGCAGGCA TTGCGCCGCA TGCTGCCGCC TTTGGCGAGC
    GAGTTCATCA
551 CGCTCTTGAA AGACAGCTCG CTGCTGTCGG TCATTGCTGT
    GGCGGAGTTG
601 GCGTATGTTC AGAATACGAT TACGGGCCGG TATTCGGTTT
    ATGAAGAACC
651 GCTTTACACC GTCGCCCTGA TTTATCTGTT GATGACGACT
    TTCTTAGGCT
701 GGATATTCCT GCGTTTGGAA AAACGTTACA ATCCGCAACA
    CCGCTGA

This corresponds to the amino acid sequence <SEQ ID 838; ORF129-1>:

1   MDFRFDIIYE YRWMFLYGAL TTLGLTVVAT AGGSVLGLLL
    ALARLIHLEK
51  AGAPMRVLAW ALRKVSLLYV TLFRGTPLFV QIVIWAYVWF
    PFFVHPSDGI
101 LVSGEAAIAL RRGYGPLIAG SLALIANSGA YICEIFRAGI
    QSIDKGQMEA
151 ARSLGLTYPQ AMRYVILPQA LRRMLPPLAS EFITLLKDSS
    LLSVIAVAEL
201 AYVQNTITGR YSVYEEPLYT VALIYLLMTT FLGWIFLRLE
    KRYNPQHR*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF129 shows 98.9% identity over a 88aa overlap with an ORF (ORF129a) from strain A of N. meningitidis:

The complete length ORF129a nucleotide sequence <SEQ ID 839> is:

1   ATGGATTTTC GTTTTGACAT TATTTACGAA TACCGCTGGA
    TGTTTCTTTA
51  CGGCGCACTG ACGACCTTGG GGCTGACGGT CGTGGCGACG
    GCGGGCGGTT
101 CGGTATTGGG TCTGTTGTTG GCGTTGGCGC GCCTGATTCA
    CTTGGAAAAA
151 GCCGGTGCGC CGATGCGCGT GCTGGCGTGG GCGTTGCGTA
    AGGTTTCGCT
201 GCTGTATGTT ACGCTGTTCC GGGGTACGCC GCTGTTTGTG
    CAGATTGTGA
251 TTTGGGCGTA TGTGTGGTTT CCGTTTTTCG TCCATCCTTC
    AGACGGCATT
301 TTGGTTAGCG GCGAGGCGGC AATCGCGCTG CGTCGCGGAT
    ACGGGCCGCT
351 GATTGCCGGT TCTTTGGCAC TGATCGCCAA CTCGGGGGCG
    TATATCTGTG
401 AGATTTTCCG CGCGGGCATC CAGTCTATAG ACAAAGGACA
    GATGGAGGCG
451 GCGCGTTCTT TGGGGCTGAC CTATCCGCAG GCGATGCGCT
    ATGTGATTCT
501 GCCGCAGGCA TTGCGCCGTA TGCTGCCGCC TTTGGCGAGC
    GAGTTCATCA
551 CGCTCTTGAA AGACAGCTCG CTGCTGTCGG TCATTGCTGT
    GGCGGAGTTG
601 GCGTATGTTC AGAATACGAT TACGGGCCGG TATTCGGTTT
    ATGAAGAACC
651 GCTTTACACC GTCGCCCTGA TTTATCTGTT GATGACGACT
    TTCTTAGGCT
701 GGATATTCCT GCGTTTGGAA AAACGTTACA ATCCGCAACA
    CCGCTGA

This encodes a protein having amino acid sequence <SEQ ID 840>:

1   MDFRFDIIYE YRWMFLYGAL TTLGLTVVAT AGGSVLGLLL
    ALARLIHLEK
51  AGAPMRVLAW ALRKVSLLYV TLFRGTPLFV QIVIWAYVWF
    PFFVHPSDGI
101 LVSGEAAIAL RRGYGPLIAG SLALIANSGA YICEIFRAGI
    QSIDKGQMEA
151 ARSLGLTYPQ AMRYVILPQA LRRMLPPLAS EFITLLKDSS
    LLSVIAVAEL
201 AYVQNTITGR YSVYEEPLYT VALIYLLMTT FLGWIFLRLE
    KRYNPQHR*

ORF129a and ORF129-1 show 100.0% identity in 248 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF129 shows 98.9% identity over a 88 aa overlap with a predicted ORF (ORF129ng) from N. gonorrhoeae:

An ORF129ng nucleotide sequence <SEQ ID 841> was predicted to encode a protein having amino acid sequence <SEQ ID 842>:

1   MDFRFDIIYE YRWMFLYGAL TTLGLTVVAT AGGSVLGLLL
    ALARLIHLEK
51  AGAPMRVLAW ALRKVSLLYV TLFRGTPLFV QIVIWAYVWF
    PFFVILHTAF
101 LGNAMRQSRR VPDKGRWIAG SLELNCQPRG RKTRGEFPPG
    ESNLGTEPRN
151 PLSMGQRRFP GCENWYPPQN FIKK*

Further work revealed the following gonococcal sequence <SEQ ID 843>:

1   ATGGATTTTc gtTTTGACAT TATTTAcgaA TACCGCTGGA
    TGTTTCTTTA
51  CGGCGCACTG Acgaccttgg ggctgacggt cgtggcgacg
    gCGGGCGGTT
101 CGGtattggG TCTGTTGTTG GCGTTGGCGC GCCTGATTCA
    CTTGGAAAAA
151 GCCGGTGCGC CGATGCGCGT GCTGGCGTGG GCGTTGCGTA
    AGGTTTCGCT
201 GCTGTACGTT ACCCTGTTCC GGGGTACGCC GCTGTTTGTG
    CAGATTGTGA
251 TTTGGGCGTA TGTGTGGTTT CCGTTTTTCG TCCATCCTTC
    AGACGGCATT
301 TTGGTCAGCG GCGAGGCGGC AATCGCGCTG CGTCGCGGAT
    ACGGGCCGCT
351 GATTGCCGGT TCTTTGGCAC TGATCGCCAA CTCGGGGGCG
    TATATCTGTG
401 AGATTTTCCG CGCGGGCATC CAGTCTATAG ACAAAGGACA
    GATGGAGGCG
451 GCGTGTTCTT TGGGACTGAC CTATCCGCAG GCGATGCGCT
    ATGTGATTCT
501 GCCGCAGGCA TTGCGCCGTA TGCTGCCGCC TTTGGCGAGC
    GAGTTCATCA
551 CGCTCTTGAA AGACAGCTCG CTGCTGTCGG TCATTGCTGT
    GGCGGAGTTG
601 GCGTATGTTC AGAATACGAT TACGGGCCGG TATTCGGTTT
    ATGAAGAACC
651 GCTTTACACC GCCGCCCTGA TTTATCTGTT GATGACGACT
    TTCTTAGGCT
701 GGATATTCCT GCGTTTGGAA AAACGTTACA ATCCGCAACA
    CCGCTGA

This corresponds to the amino acid sequence <SEQ ID 844; ORF129ng-1>:

1   MDFRFDIIYE YRWMFLYGAL TTLGLTVVAT AGGSVLGLLL
    ALARLIHLEK
51  AGAPMRVLAW ALRKVSLLYV TLFRGTPLFV QIVIWAYVWF
    PFFVHPSDGI
101 LVSGEAAIAL RRGYGPLIAG SLALIANSGA YICEIFRAGI
    QSIDKGQMEA
151 ARSLGLTYPQ AMRYVILPQA LRRMLPPLAS EFITLLKDSS
    LLSVIAVAEL
201 AYVQNTITGR YSVYEEPLYT VALIYLLMTT FLGWIFLRLE
    KRYNPQHR*

ORF129ng-1 and ORF129-1 show 99.2% identity in 248 aa overlap:

In addition, ORF129ng-1 is homologous to an ABC transporter from A. fulgidus:

2650409(AE001090) glutamine ABC transporter, permease protein (glnP)
[Archaeoglobus fulgidus] Length = 224
Score = 132 bits (329), Expect = 2e−30
Identities = 86/178 (48%), Positives = 103/178 (57%), Gaps = 18/178 (10%)
Query:	65	VSLLYVTLFRGTPLFVQIVIWAYVWFPFFVHPSDGILVSGEAAIALRRGYGPLIAGSLAL	124
		+S  YV + RGTPL VQI+I       +F  P+ GI +  E A            G +AL
Sbjct:	58	ISTAYVEVIRGTPLLVQILI------VYFGLPAIGINLQPEPA------------GIIAL	99
Query:	125	IANSGAYICEIFRAGIQSIDKGQMEAACSLGLTYPQAMRYVILPQALRRMLPPLASEFIT	184
		SGAYI EI RAGI+SI  GQMEAA SLG+TY QAMRYVI PQA R +LP L +EFI
Sbjct:	100	SICSGAYIAEIVRAGIESIPIGQMEAARSLGMTYLQAMRYVIFPQAFRNILPALGNEFIA	159
Query:	185	LLKDSSLLSVIAVAELAYVQNTITGRYSVYEEPLYTAALIYLLMTTFLGWIFLRLEKR	242
		LLKDSSLLSVI++ EL  V   I         P    AL YL+MT  L  +    +K+
Sbjct:	160	LLKDSSLLSVISIVELTRVGRQIVNTTFNAWTPFLGVALFYLMMTIPLSRLVAYSQKK	217

This analysis, including the identification of transmembrane domains in the two proteins, suggests that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 100

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 845>:

1   ..CTGAAAGAAT GCCGTCTGAA AGACCCTGTT TTTATTCCAA
    ATATCGTTTA
51  TAAGAACATC GCCATTACTT TCCTGCTCTT GCACGCCGCC
    GCCGAACTTT
101 GGCTGCCCGC GCAAACCGCC GGTTTTACCG CGCTCGCCGT
    CGGCTTCATC
151 CTGCTCGCCA AGCTGCGTGA gCTTCACCAT CACGAACTCT
    TACGTAAACA
201 cTACGTCCGC ACTTATTACy TGCTCCAACT CTTTGCCGCC
    GCAGgcTAgT
251 TTGTGGACAG GCGCGGCGwA ATTACAAAAC CTGCCCGCyT
    CCGCGCCCCT
301 GCACCTGATT ACCCTCGGCG GCATGATGGG CGGCGTGATG
    ATGGTGTGGc
351 TGACCGCCGG ACTGTGGCAC AGCGGCTTTA CCAAACTCGA
    CTACCCCAAA
401 CTCTGCCGCA TTGCCGTCCC CATCCTTTTC GCCGCCGCCG
    TCTCGCGCGC
451 TTTCTTGrTG AACGTGAACC CGrTATTTTT CATTACCGTT
    CCTGCGATTC
501 TGACCGCCGC CGTATTCGTA CTGTATCTTT TCrCGTTTAT
    ACCGATATTT
551 CGGGCGAATG CGTTTACAGA CGATCCGGAr TAr

This corresponds to the amino acid sequence <SEQ ID 846; ORF130>:

1   ..LKECRLKDPV FIPNIVYKNI AITFLLLHAA AELWLPAQTA
    GFTALAVGFI
51  LLAKLRELHH HELLRKHYVR TYYLLQLFAA AGSLWTGAAX
    LQNLPASAPL
101 HLITLGGMMG GVMMVWLTAG LWHSGFTKLD YPKLCRIAVP
    ILFAAAVSRA
151 FLXNVNPXFF ITVPAILTAA VFVLYLFXFI PIFRANAFTD
    DPE*

Further work revealed the complete nucleotide sequence <SEQ ID 847>:

1    ATGCGGCCGT TTTTCGTCGG CGCGGCGGTG CTTGCCATAC
     TCGGTGCGCT
51   GGTGTTTTTC ATCAACCCCG GTGCCATCGT CCTGCACCGC
     CAAATTTTCT
101  TGGAACTTAT GCTGCCGGCG GCATACGGCG GTTTTTTGAC
     TGCGGCTTTG
151  TTGGACTGGA CGGGTTTTTC GGGTAACCTG AAACCTGTCG
     CGACTTTGAT
201  GGCGGCATTA TTGCTCGCCG CATCCGCTAT ACTGCCCTTT
     TCGCCGCAAA
251  CTGCCTCGTT TTTCGTCGCC GCCTATTGGC TGGTGTTGCT
     GCTGTTCTGC
301  GCCCGGCTGA TTTGGCTAGA CCGAAACACC GACAACTTCG
     CCCTGCTAAT
351  GTTACTTGCC GCGTTCACTG TTTTTCAGAC GGCATATGCC
     GTCAGCGGCG
401  ATTTGAACCT GTTGCGCGCG CAAGTGCATC TAAATATGGC
     GGCGGTGATG
451  TTCGTATCCG TGCGCGTCAG TATTCTTTTG GGCGCGGAAG
     CCCTGAAAGA
501  ATGCCGTCTG AAAGACCCTG TTTTTATTCC AAATATCGTT
     TATAAAAACA
551  TCGCCATTAC TTTCCTGCTC TTGCACGCCG CCGCCGAACT
     TTGGCTGCCC
601  GCGCAAACCG CCGGTTTTAC CGCGCTCGCC GTCGGCTTCA
     TCCTGCTCGC
651  CAAGCTGCGT GAGCTTCACC ATCACGAACT CTTACGTAAA
     CACTACGTCC
701  GCACTTATTA CCTGCTCCAA CTCTTTGCCG CCGCAGGCTA
     TTTGTGGACA
751  GGCGCGGCGA AATTACAAAA CCTGCCCGCC TCCGCGCCCC
     TGCACCTGAT
801  TACCCTCGGC GGCATGATGG GCGGCGTGAT GATGGTGTGG
     CTGACCGCCG
851  GACTGTGGCA CAGCGGCTTT ACCAAACTCG ACTACCCCAA
     ACTCTGCCGC
901  ATTGCCGTCC CCATCCTTTT CGCCGCCGCC GTCTCGCGCG
     CTTTCTTGAT
951  GAACGTGAAC CCGATATTTT TCATTACCGT TCCTGCGATT
     CTGACCGCCG
1001 CCGTATTCGT ACTGTATCTT TTCACGTTTA TACCGATATT
     TCGGGCGAAT
1051 GCGTTTACAG ACGATCCGGA ATAA

This corresponds to the amino acid sequence <SEQ ID 848; ORF130-1>:

1   MRPFFVGAAV LAILGALVFF INPGAIVLHR QIFLELMLPA
    AYGGFLTAAL
51  LDWTGFSGNL KPVATLMAAL LLAASAILPF SPQTASFFVA
    AYWLVLLLFC
101 ARLIWLDRNT DNFALLMLLA AFTVFQTAYA VSGDLNLLRA
    QVHLNMAAVM
151 FVSVRVSILL GAEALKECRL KDPVFIPNIV YKNIAITFLL
    LHAAAELWLP
201 AQTAGFTALA VGFILLAKLR ELHHHELLRK HYVRTYYLLQ
    LFAAAGYLWT
251 GAAKLQNLPA SAPLHLITLG GMMGGVMMVW LTAGLWHSGF
    TKLDYPKLCR
301 IAVPILFAAA VSRAFLMNVN PIFFITVPAI LTAAVFVLYL
    FTFIPIFRAN
351 AFTDDPE*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF130 shows 94.3% identity over a 193aa overlap with an ORF (ORF130a) from strain A of N. meningitidis:

The complete length ORF130a nucleotide sequence <SEQ ID 849> is:

1    ATGCGGCCGT TTTTCGTCGG CGCGGCGGTG CTTGCCATAC
     TCGGTGCGCT
51   GGTGTTTTTC ATCAACCCCG GTGCCATCGT CCTGCACCGC
     CAAATTTTCT
101  TGGAACTTAT GCTGCCGGCG GCATACGGCG GTTTTTTGAC
     TGCGGCTTTG
151  TTGGACTGGA CGGGTTTTTC GGGTAACCTG AAACCTGTCG
     CGACTTTGAT
201  GGCGGCATTA TTGCTCGCCG CATCCGCTAT ACTGCCCTTT
     TCGCCGCAAA
251  CTGCCTCGTT TTTCGTCGCC GCCTATTGGC TGGTGTTGCT
     GCTGTTCTGC
301  GCCCGGCTGA TTTGGCTAGA CCGAAACACC GACAACTTCG
     CCCTGCTAAT
351  GTTACTTGCC GCGTTCACTG TTTTTCAGAC GGCATATGCC
     GTCAGCGGCG
401  ATTTGAACCT GTTGCGCGCG CAAGTGCATC TAAATATGGC
     GGCGGTGATG
451  TTCGTATCCG TGCGCGTCAG TATTCTTTTG GGCGCGGAAG
     CCCTGAAAGA
501  ATGCCGTCTG AAAGACCCAG TATTCATCCC CAATGTCGTC
     TATAAAAACA
551  TCGCCATTAC CTTCCTGCTC CTGCACGCCG CCGCCGAACT
     TTGGCTGCCT
601  GCGCAAACCG CCGGTTTTAC CTCGCTCGCC GTCGGCTTTA
     TCCTGCTTGC
651  CAAGCTGCGT GAGCTTCACC ATCACGAACT CCTGCGCAAA
     CACTACGTCC
701  GCACTTATTA CCTGCTCCAA CTCTTTGCCG CCGCAGGCTA
     TTTGTGGACA
751  GGCGCGGCGA AATTACAAAA CCTGCCCGCC TCCGCGCCCC
     TGCACCTGAT
801  TACCCTCGGT GGCATGATGG GCAGCGTGAT GATGGTGTGG
     CTGACTGCCG
851  GACTGTGGCA CAGCGGCTTT ACCAAGCTCG ACTACCCGAA
     ACTCTGCCGC
901  ATCGCCGTCC CCATCCTNTT CGCCGCCGCC GTTTCGCGCG
     CTGTTTTAAT
951  GAACGTAAAC CCGATATTCT TCATCACCGT CCCCGCAATT
     CTGACCGCCG
1001 CCGTGTTCGT GCTTTACCTG CTGACATTCG TACCGATCTT
     TCGGGCGAAC
1051 GCGTTTACAG ACGATCCGGA ATAA

This encodes a protein having amino acid sequence <SEQ ID 850>:

1   MRPFFVGAAV LAILGALVFF INPGAIVLHR QIFLELMLPA
    AYGGFLTAAL
51  LDWTGFSGNL KPVATLMAAL LLAASAILPF SPQTASFFVA
    AYWLVLLLFC
101 ARLIWLDRNT DNFALLMLLA AFTVFQTAYA VSGDLNLLRA
    QVHLNMAAVM
151 FVSVRVSILL GAEALKECRL KDPVFIPNVV YKNIAITFLL
    LHAAAELWLP
201 AQTAGFTSLA VGFILLAKLR ELHHHELLRK HYVRTYYLLQ
    LFAAAGYLWT
251 GAAKLQNLPA SAPLHLITLG GMMGSVMMVW LTAGLWHSGF
    TKLDYPKLCR
301 IAVPILFAAA VSRAVLMNVN PIFFITVPAI LTAAVFVLYL
    LTFVPIFRAN
351 AFTDDPE*

ORF130a and ORF130-1 show 98.3% identity in 357 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF130 shows 91.7% identity over a 193 aa overlap with a predicted ORF (ORF130ng) from N. gonorrhoeae:

An ORF130ng nucleotide sequence <SEQ ID 851> was predicted to encode a protein having amino acid sequence <SEQ ID 852>:

1   MNKFFTHPMR PFFVGAAVLA ILGALVFFHQ PRRYHPAPPN
    FLGTYAAGCI
51  RRFFDYRFVG PDGFFRQPET CRYFDGGVVA CCGCFIAVFT
    ATCRIFRRRL
101 LAGVAAVLRL ADLARRQHRT LRSVDVTAAF TVFQTAYAVS
    GDLNLLRAQV
151 HLNMAAVMFV SVRVSVLLGT ETLKECRLKD PVFIPNVIYK
    NIAITLLLHA
201 AAELWLPAQT AGFTALAVGF ILLAKLRELH HHELLRKHYV
    RTYYLLQLFA
251 AAGYLWTGAA KLQNLPASAP LHLITLGGMT GGVMMVWLTA
    GLWHSGFTKL
301 DYPKLCRIAV SILFASAVSR AVLMNVNPIF FITVPEILTA
    AVFMLYLLTF
351 VPIFRANAFT DDPE*

Further work revealed the following gonococcal DNA sequence <SEQ ID 853>:

1    ATGCGCCCGT TTTTCGTCGG TGCGGCAGTA CTTGCCATAC
     TCGGTGCGTT
51   GGTGTTTTTT ATCAACCCCG GCGCTATCAT CCTGCACCGC
     CAAATTTTCT
101  TGGAACTTAT GCTGCCGGCT GCATACGGCG GTTTTTTGAC
     TACCGCTTTG
151  TTGGACCGGA CGGGTTTTTC AGGCAACCTG AAACCTGCCG
     CTACTTTGAT
201  GGCGGTGTTG TTGCTTGTTG CGGCTGTTTT ATTGCCGTTT
     TTACCGCAAC
251  TTGCCGCATT TTTCGTCGCC GCCTATTGGC TGGTGTTGCT
     GCTGTTCTGC
301  GCCTGGCTGA TTTGGCTCGA CCGCAACACC GACAACTTCG
     CTCTGTTGAT
351  GTTACTTGCC GCATTTACCG TTTTTCAGAC GGCCTATGCC
     GTCAGCGGCG
401  ATTTGAACTT ACTGCGCGCG CAAGTGCATT TGAATATGGC
     GGCGGTCATG
451  TTCGTATCCG TCCGCGTCAG CGTCCTTTTG GGCACGGAAA
     CCCTGAAAGA
501  ATGCCGTCTG AAAGACCCCG TATTCATCCC CAACGTTATC
     TATAAAAACA
551  TCGCCATCAC CCTGCTGCTG CACGCCGCCG CCGAACTTTG
     GCTGCCCGCG
601  CAAACCGCCG GTTTTACTGC GCTTGCCGTC GGCTTCATCC
     TGCTCGCCAA
651  GCTGCGCGAA CTGCACCATC ACGAACTCTT ACGCAAACAC
     TACGTCCGCA
701  CTTATTACCT GCTCCAGCTC TTTGCCGCCG CAGGTTATCT
     GTGGACAGGC
751  GCGGCGAAAC TGCAAAACCT GCCCGCCTCC GCGCCCCTGC
     ACCTGATTAC
801  CCTCGGCGGC ATGACGGGTG GCGTGATGAT GGTGTGGCTG
     ACTGCCGGAC
851  TGTGGCACAG CGGCTTTACC AAACTCGACT ACCCGAAACT
     CTGCCGCATC
901  GCCGTCTCCA TCCTTTTCGC CTCCGCCGTT TCGCGCGCTG
     TTTTAATGAA
951  CGTGAATCCG ATATTCTTCA TCACCGTTCC CGAGATTCTG
     ACCGCCGCCG
1001 TGTTCATGCT TTACCTGCTG ACGTTCGTAC CGATTTTTCG
     AGCGAACGCG
1051 TTTACAGACG ATCCGGAATA A

This corresponds to the amino acid sequence <SEQ ID 854; ORF130ng-1>:

1   MRPFFVGAAV LAILGALVFF INPGAIILHR QIFLELMLPA
    AYGGFLTTAL
51  LDRTGFSGNL KPAATLMAVL LLVAAVLLPF LPQLAAFFVA
    AYWLVLLLFC
101 AWLIWLDRNT DNFALLMLLA AFTVFQTAYA VSGDLNLLRA
    QVHLNMAAVM
151 FVSVRVSVLL GTETLKECRL KDPVFIPNVI YKNIAITLLL
    HAAAELWLPA
201 QTAGFTALAV GFILLAKLRE LHHHELLRKH YVRTYYLLQL
    FAAAGYLWTG
251 AAKLQNLPAS APLHLITLGG MTGGVMMVWL TAGLWHSGFT
    KLDYPKLCRI
301 AVSILFASAV SRAVLMNVNP IFFITVPEIL TAAVFMLYLL
    TFVPIFRANA
351 FTDDPE*

ORF130ng-1 and ORF130-1 show 92.4% identity in 357 aa overlap:

Based on this analysis, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 101

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 855>:

1   ATGGAAATTC GGGCAATAAA ATATACGGCA ATGGCTGCGT
    TGCTTGCATT
51  TACGGTTGCA GGCTGCCGGC TGGCGGGGTG GTATGAGTGT
    TCGTCCCTCA
101 CCGGCTGGTG TAAGCCGAGA AAACCGGCTG CCATCGATTT
    TTGGGATATT
151 GGCGGCGAGA GTCCGCCGTC TTTAGGGGAC TACGAGATAC
    CGCTTTCAGA
201 CGGCAATAGT TCCGTCAGGG CAAACGAATA TGAATCCGCA
    CAACAATCTT
251 ACTTTTACAG GAAAATAGGG AAGTTTGAAG C.TGCGGGCT
    GGATTGGCGT
301 ACGCGTGACG GCAAACCTTT GATTGAGACG TTCAAACAGG
    GAGGATTTGA
351 CTGCTTGGAA AAG..

This corresponds to the amino acid sequence <SEQ ID 856; ORF131>:

1   MEIRAIKYTA MAALLAFTVA GCRLAGWYEC SSLTGWCKPR
    KPAAIDFWDI
51  GGESPPSLGD YEIPLSDGNS SVRANEYESA QQSYFYRKIG
    KFEXCGLDWR
101 TRDGKPLIET FKQGGFDCLE K..

Further work revealed the complete nucleotide sequence <SEQ ID 857>:

1   ATGGAAATTC GGGCAATAAA ATATACGGCA ATGGCTGCGT
    TGCTTGCATT
51  TACGGTTGCA GGCTGCCGGC TGGCGGGGTG GTATGAGTGT
    TCGTCCCTCA
101 CCGGCTGGTG TAAGCCGAGA AAACCGGCTG CCATCGATTT
    TTGGGATATT
151 GGCGGCGAGA GTCCGCCGTC TTTAGGGGAC TACGAGATAC
    CGCTTTCAGA
201 CGGCAATCGT TCCGTCAGGG CAAACGAATA TGAATCCGCA
    CAACAATCTT
251 ACTTTTACAG GAAAATAGGG AAGTTTGAAG CCTGCGGGCT
    GGATTGGCGT
301 ACGCGTGACG GCAAACCTTT GATTGAGACG TTCAAACAGG
    GAGGATTTGA
351 CTGCTTGGAA AAGCAGGGGT TGCGGCGCAA CGGTCTGTCC
    GAGCGCGTCC
401 GATGGTAA

This corresponds to the amino acid sequence <SEQ ID 858; ORF131-1>:

1   MEIRAIKYTA MAALLAFTVA GCRLAGWYEC SSLTGWCKPR
    KPAAIDFWDI
51  GGESPPSLGD YEIPLSDGNR SVRANEYESA QQSYFYRKIG
    KFEACGLDWR
101 TRDGKPLIET FKQGGFDCLE KQGLRRNGLS ERVRW*

Computer analysis of this amino acid sequence gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF131 shows 95.0% identity over a 121 aa overlap with an ORF (ORF131a) from strain A of N. meningitidis:

The complete length ORF131a nucleotide sequence <SEQ ID 859> is:

1   ATGGAAATTC GGGCAATAAA ATATACGGCA ATGGCTGCGT
    TGCTTGCATT
51  TACGGTTGCA GGCTGCCGGT TGGCAGGTTG GTATGAGTGT
    TCGTCCCTGT
101 CCGGCTGGTG TAAGCCGAGA AAACCTGCCG CCATCGATTT
    TTGGGATATT
151 GGCGGCGAGA GTCCTCCGTC TTTAGAGGAC TACGAGATAC
    CGCTTTCAGA
201 CGGCAATCGT TCCGTCAGGG CAAACGAATA TGAATCCGCA
    CAACAATCTT
251 ACTTTTACAG GAAAATAGGG AAGTTTGAAG CCTGCGGGTT
    GGATTGGCGT
301 ACGCGTGACG GCAAACCTTT GATTGAGACG TTCAAACAGG
    AAGGTTTTGA
351 TTGTTTGAAA AAGCAGGGGT TGCGGCGCAA CGGTCTGTCC
    GAGCGCGTCC
401 GATGGTAA

This encodes a protein having amino acid sequence <SEQ ID 860>:

1   MEIRAIKYTA MAALLAFTVA GCRLAGWYEC SSLSGWCKPR
    KPAAIDFWDI
51  GGESPPSLED YEIPLSDGNR SVRANEYESA QQSYFYRKIG
    KFEACGLDWR
101 TRDGKPLIET FKQEGFDCLK KQGLRRNGLS ERVRW*

ORF131a and ORF131-1 show 97.0% identity in 135 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF131 shows 89.3% identity over 121 aa overlap with a predicted ORF (ORF131ng) from N. gonorrhoeae:

A complete length ORF131ng nucleotide sequence <SEQ ID 861> was predicted to encode a protein having amino acid sequence <SEQ ID 862>:

1   MEIRVIKYTA TAALFAFTVA GCRLAGWYEC LSLSGWCKPR
    KPAAIDFWDI
51  GGESPLSLED YEIPLSDGNR SVRANEYESA QKSYFYRKIG
    KFEACGLDWR
101 TRDGKPLVER FKQEGFDCLE KQGLRRNGLS ERVRW*

Further work revealed the following gonococcal DNA sequence <SEQ ID 863>:

1   ATGGAAATTC GGGTAATAAA ATATACGGCA ACGGCTGCGT
    TGTTTGCATT
51  TACGGTTGCA GGCTGCCGGC TGGCGGGGTG GTATGAGTGT
    TCGTCCTTGT
101 CCGGCTGGTG TAAGCCGAGA AAACCTGCCG CCATCGATTT
    TTGGGATATT
151 GGCGGCGAGA GtccgctGTC TTTAGAGGAC TACGAGATAC
    CGCTTTCAGA
201 CGGCAATCGT TCCGTCAGGG CAAACGAATA TGAATCCGCG
    CAAAAATCTT
251 ACTTTTATAG GAAAATAGGG AAGTTTGAAG CCTGCGGGTT
    GGATTGGCGT
301 ACGCGTGACG GCAAACCTTT GGTTGAGAGG TTCAAACAGG
    AAGGTTTCGA
351 CTGTTTGGAA AAGCAGGGGT TGCGGCGCAA CGGCCTGTCC
    GAGCGCGTCC
401 GATGGTAA

This corresponds to the amino acid sequence <SEQ ID 864; ORF131ng-1>:

1   MEIRVIKYTA TAALFAFTVA GCRLAGWYEC SSLSGWCKPR
    KPAAIDFWDI
51  GGESPLSLED YEIPLSDGNR SVRANEYESA QKSYFYRKIG
    KFEACGLDWR
101 TRDGKPLVER FKQEGFDCLE KQGLRRNGLS ERVRW*

ORF131ng-1 and ORF131-1 show 92.6% identity in 135 aa overlap:

Based on the presence of a predicted prokaryotic membrane lipoprotein lipid attachment site, it is predicted that the proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 102

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 865>

1   ATGAAACACA TCCATATTAT CGGTATCGGC GGCACGTTTA
    TGGGCGGGCT
51  TGCCGCCATT GCCAAAGAAG CGGGGTTTGA AGTCAGCGGT
    TGCGACGCGA
101 AGATGTATCC GCCGATGAGC ACCCAGCTCG AAGCCTTGGG
    TATAGACGTG
151 TATGAAGGCT TCGATGCCGC TCAGTTGGAC GAATTTAAAG
    CCGACGTTTA
201 CGTTATCGGC AATGTCGCCA AGCGCGGGAT GGATGTGGTT
    GAAGCGATTT
251 TGAACCTCGG CCTGCCtTAT ATtTcCGGCC CGCAATGGCT
    GTCGGAAAAC
301 GTGCTGCACC ATCATTGGGT ACTCGGTGTG GCGGGGACgC
    ACGGCAAAAC
351 GACCACCGCC TCCATGCTCG CATGGGTCTT GGAATATgCC
    GGCCTCGCGC
401 CGGGCTTCCT TATtGGCGGC GTACC.GGAA AATttCGGCG
    TTTCCGCCCG
451 CCTGCCGCAA ACGCCGCGCC AAGACCCGAA CAGCCAATCG
    CCGTTTTTcG
501 TCATCGAAGC CGACGAATAC GACACCGCCT TTtTCGACAA
    ACGTTCTAAA
551 TtCGTGCATT ACCGTCCGCG TACCGCCGTG TTGAACAATC
    TGGAATTCGA
601 CCACGCCGAC ATCTTTGCCG ACTTGGGCGC GATACAGACc
    CAGTTCCACT
651 ACCTCGTGCG TACCGTGCCG TCTGAAGGCT TAATCGTCTG
    CAACGGACGG
701 CAGCAAAGCC TGCAAGATAC TTTGGACAAA GGCTGCTGGA
    CGCCGGTGGA
751 AAAATTCGGC ACGGAACACG GCTGGCA..

This corresponds to the amino acid sequence <SEQ ID 866; ORF132>:

1   MKHIHIIGIG GTFMGGLAAI AKEAGFEVSG CDAKMYPPMS
    TQLEALGIDV
51  YEGFDAAQLD EFKADVYVIG NVAKRGMDVV EAILNLGLPY
    ISGPQWLSEN
101 VLHHHWVLGV AGTHGKTTTA SMLAWVLEYA GLAPGFLIGG
    VXGKFRRFRP
151 PAANAAPRPE QPIAVFRHRS RRIRHRLFRQ TFXIRALPSA
    YRRVEQSGIR
201 PRRHLCRLGR DTDPVPLPRA YRAVXRLNRL QRTAAKPARY
    FGQRLLDAGG
251 KIRHGTRLA..

Further work revealed the complete nucleotide sequence <SEQ ID 867>:

1    ATGAAACACA TCCATATTAT CGGTATCGGC GGCACGTTTA
     TGGGCGGGCT
51   TGCCGCCATT GCCAAAGAAG CGGGGTTTGA AGTCAGCGGT
     TGCGACGCGA
101  AGATGTATCC GCCGATGAGC ACCCAGCTCG AAGCCTTGGG
     TATAGACGTG
151  TATGAAGGCT TCGATGCCGC TCAGTTGGAC GAATTTAAAG
     CCGACGTTTA
201  CGTTATCGGC AATGTCGCCA AGCGCGGGAT GGATGTGGTT
     GAAGCGATTT
251  TGAACCTCGG CCTGCCTTAT ATTTCCGGCC CGCAATGGCT
     GTCGGAAAAC
301  GTGCTGCACC ATCATTGGGT ACTCGGTGTG GCGGGGACGC
     ACGGCAAAAC
351  GACCACCGCC TCCATGCTCG CATGGGTCTT GGAATATGCC
     GGCCTCGCGC
401  CGGGCTTCCT TATTGGCGGC GTACCGGAAA ATTTCGGCGT
     TTCCGCCCGC
451  CTGCCGCAAA CGCCGCGCCA AGACCCGAAC AGCCAATCGC
     CGTTTTTCGT
501  CATCGAAGCC GACGAATACG ACACCGCCTT TTTCGACAAA
     CGTTCTAAAT
551  TCGTGCATTA CCGTCCGCGT ACCGCCGTGT TGAACAATCT
     GGAATTCGAC
601  CACGCCGACA TCTTTGCCGA CTTGGGCGCG ATACAGACCC
     AGTTCCACTA
651  CCTCGTGCGT ACCGTGCCGT CTGAAGGCTT AATCGTCTGC
     AACGGACGGC
701  AGCAAAGCCT GCAAGATACT TTGGACAAAG GCTGCTGGAC
     GCCGGTGGAA
751  AAATTCGGCA CGGAACACGG CTGGCAGGCC GGCGAAGCCA
     ATGCCGACGG
801  CTCGTTCGAC GTGTTGCTCG ACGGCAAAAC CGCCGGACGC
     GTCAAATGGG
851  ATTTGATGGG CAGGCACAAC CGCATGAACG CGCTCGCCGT
     CATTGCCGCC
901  GCGCGTCATG TCGGTGTCGA TATTCAGACC GCCTGCGAAG
     CCTTGGGCGC
951  GTTTAAAAAC GTCAAACGCC GGATGGAAAT CAAAGGCACG
     GCAAACGGCA
1001 TCACCGTTTA CGACGACTTC GCCCACCACC CGACCGCCAT
     CGAAACCACG
1051 ATTCAAGGTT TGCGCCAACG CGTCGGCGGC GCGCGCATCC
     TCGCCGTCCT
1101 CGAACCGCGT TCCAACACGA TGAAGCTGGG CACGATGAAG
     TCCGCCCTGC
1151 CTGTAAGCCT CAAAGAAGCC GACCAAGTGT TCTGCTACGC
     CGGCGGCGTG
1201 GACTGGGACG TCGCCGAAGC CCTCGCGCCT TTGGGCGGCA
     GGCTGAACGT
1251 CGGCAAAGAC TTCGATGCCT TCGTTGCCGA AATCGTGAAA
     AACGCCGAAG
1301 TAGGCGACCA TATTTTGGTG ATGAGCAACG GCGGTTTCGG
     CGGAATACAC
1351 GGAAAGCTGC TGGAAGCTTT GAGATAG

This corresponds to the amino acid sequence <SEQ ID 868; ORF132-1>:

1   MKHIHIIGIG GTFMGGLAAI AKEAGFEVSG CDAKMYPPMS
    TQLEALGIDV
51  YEGFDAAQLD EFKADVYVIG NVAKRGMDVV EAILNLGLPY
    ISGPQWLSEN
101 VLHHHWVLGV AGTHGKTTTA SMLAWVLEYA GLAPGFLIGG
    VPENFGVSAR
151 LPQTPRQDPN SQSPFFVIEA DEYDTAFFDK RSKFVHYRPR
    TAVLNNLEFD
201 HADIFADLGA IQTQFHYLVR TVPSEGLIVC NGRQQSLQDT
    LDKGCWTPVE
251 KFGTEHGWQA GEANADGSFD VLLDGKTAGR VKWDLMGRHN
    RMNALAVIAA
301 ARHVGVDIQT ACEALGAFKN VKRRMEIKGT ANGITVYDDF
    AHHPTAIETT
351 IQGLRQRVGG ARILAVLEPR SNTMKLGTMK SALPVSLKEA
    DQVFCYAGGV
401 DWDVAEALAP LGGRLNVGKD FDAFVAEIVK NAEVGDHILV
    MSNGGFGGIH
451 GKLLEALR*

Computer analysis of this amino acid sequence gave the following results:

Homology with the Hypothetical o457 Protein of E. coli (Accession Number U14003)

ORF132 and o457 show 58% aa identity in 140 aa overlap:

Orf132:	4	IHIIGIGGTFMGGLAAIAKEAGFEVSGCDAKMYPPMSTQLEALGIDVYEGFDAAQLDEFK	63
		IHI+GI GTFMGGLA +A++ G EV+G DA +YPPMST LE  GI++ +G+DA+QL+  +
o457:	3	IHILGICGTFMGGLAMLARQLGHEVTGSDANVYPPMSTLLEKQGIELIQGYDASQLEP-Q	61
Orf132:	64	ADVYVIGNVAKRGMDVVEAILNLGLPYISGPQWLSENVLHHHWVLGVAGTHGKTTTASML	123
		D+ +IGN   RG   VEA+L   +PY+SGPQWL + VL   WVL VAGTHGKTTTA M
o457:	62	PDLVIIGNAMTRGNPCVEAVLEKNIPYMSGPQWLHDFVLRDRWVLAVAGTHGKTTTAGMA	121
Orf132:	124	AWVLEYAGLAPGFLIGGVXG	143
		W+LE  G  PGF+IGGV G
o457:	122	TWILEQCGYKPGFVIGGVPG	141

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF132 shows 74.6% identity over a 189aa overlap with an ORF (ORF132a) from strain A of N. meningitidis:

The complete length ORF132a nucleotide sequence <SEQ ID 869> is:

1 ATGAAACACA TCCACATTAT CGGTATCGGC GGCACGTTTA TGGGTGGGAT
51 TGCCGCCATT GCCAAAGAAG CAGGGTTTGA ANTCAGCGGT TGCGATGCGA
101 AGATGTATCC GCCGATGAGC ACCCAGCTCG AAGCCTTGGG CATAGGCGTG
151 TATGAAGGCT TCGACACCGC GCAGTTGGAC GAATTTAAAG CCGACGTTTA
201 CGTTATCGGC AATGTCGCCA AGCGCGGGAT GGATGTGGTT GAAGCGATTT
251 TGAACCGTGG GCTGCCTTAT ATTTCCGGCC CGCAATGGCT GGCTGAAAAC
301 NTGCTGCACC ATCATTGGNN ACTCGGCGTG GCGGNGACGC ACGGCAAAAC
351 GACCACCGCG TCTATGCTCG CGTGGGTTTT GGAATATGCC GGACTCGCAC
401 CGGGCTTCNT TATCGGCGGC GTACCGGAAA ACTTCAGCGT TTCCGCCCGC
451 CTGCCGCAAA CGCCGCGCCA AGACCCGAAC AGCCAATCGC CGTTTTTCGT
501 CATTGAAGCC GACGAATACG ACACCGCGTT TTTCGACAAA CGCTCCAAAT
551 TCGTGCATTA CCGTCCGCGT ACCGCCGTGT TGAACAATCT GGAATTCGAC
601 CACGCCGACA TCTTCGCCGA TTTGGGCGCG ATACAGACCC AGTTCCACCA
651 CCTCGTGCGT ACCGTGCCGT CTGAAGGCCT CATCGTCTGC AACGGACGGC
701 AGCAAAGCCT GCAAGACACT TTGGACAAAG GCTGCTGGAC GCCGGTGGAA
751 AAATTCGGCA CGGAACACGG CTGGCAGGCC GGCGAAGCCA ATGCCGATGG
801 CTCGTTCGAC GTGTTGCTTG ACGGCAAAAA AGCCGGACAC GTCGCTTGGA
851 GTTTGATGGG CGGACACAAC CGCATGAACG CGCTCGCNGT CATCGCCGCC
901 GCGCGTCATG CCGGAGTNGA CATTCAGACG GCCTGCGAAG CCTTGAGCAC
951 GTTTAAAAAC GTCAAACGCC GCATGGAAAT CAAAGGCACG GCAAACGGTA
1001 TCACCGTTTA CGACGACTTC GCCCACCATC CGACCGCTAT CGAAACCACG
1051 ATTCAAGGTT TGCGCCAGCG CGTCGGCGGC GCGCGCATCC TCGCCGTCCT
1101 CGAACCGCGT TCCAATACGA TGAAGCTGGG TACGATGAAA GCCGCCCTGC
1151 CCGCAAGCCT CAAAGAAGCC GACCAAGTGT TCTGNTACGC CGGCGGCGCG
1201 GACTGGGACG TTGCCGAAGC CCTCGCGCCT TTGGGCGGCA GGCTGCACGT
1251 CGGCAAAGAC TTCGATGCCT TCGTTGCCGA AATCGTGAAA AACGCCGAAG
1301 CAGGCGACCA TATTTTGGTG ATGAGCAACG GCGGTTTCGG CGGAATACAC
1351 ACCAAACTGC TGGACGCTTT GAGATAG

This encodes a protein having-amino acid sequence <SEQ ID 870>:

1 MKHIHIIGIG GTFMGGIAAI AKEAGFEXSG CDAKMYPPMS TQLEALGIGV
51 YEGFDTAQLD EFKADVYVIG NVAKRGMDVV EAILNRGLPY ISGPQWLAEN
101 XLHHHWXLGV AXTHGKTTTA SMLAWVLEYA GLAPGFXIGG VPENFSVSAR
151 LPQTPRQDPN SQSPFFVIEA DEYDTAFFDK RSKFVHYRPR TAVLNNLEFD
201 HADIFADLGA IQTQFHHLVR TVPSEGLIVC NGRQQSLQDT LDKGCWTPVE
251 KFGTEHGWQA GEANADGSFD VLLDGKKAGH VAWSLMGGHN RMNALAVIAA
301 ARHAGVDIQT ACEALSTFKN VKRRMEIKGT ANGITVYDDF AHHPTAIETT
351 IQGLRQRVGG ARILAVLEPR SNTMKLGTMK AALPASLKEA DQVFXYAGGA
401 DWDVAEALAP LGGRLHVGKD FDAFVAEIVK NAEAGDHILV MSNGGFGGIH
451 TKLLDALR*

ORF132a and ORF132-1 show 93.9% identity in 458 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF132 shows 89.6% identity over 259 aa overlap with a predicted ORF (ORF132ng) from N. gonorrhoeae:

An ORF132ng nucleotide sequence <SEQ ID 871> was predicted to encode a protein having amino acid sequence <SEQ ID 872>:

1   MKHIHIIGIG GTFMGGIAAI AKEAGFKVSG CDAKMYPPMS
    TQLEALGIGV
51  HEGFDAAQLE EFQADIYVIG NVARRGMDVV EAILNRGLPY
    ISGPQWLAEN
101 VLHHHWVLGV AGTHGKTTTA SMLAWVLEYA GLAPGFLIGG
    VPGKFRRFRP
151 PTANAASRPE QQIAVFRHRS RRIRHRLFRQ TLQIRALSPA
    YRRVEQSGIR
201 PRRHLRRLGR DTDPVPPPRA HRTIRRPHRL QRTAAKPARY
    FGQRLLDAGG
251 KIRHRTRLAD W*

Further work revealed the following gonococcal DNA sequence <SEQ ID 873>:

1    ATGAAACACA TCCACATTAT CGGTATCGGC GGCACGTTTA
     TGGGCGGGAT
51   TGCCGCCATT GCCAAAGAAG CCGGGTTCAA AGTCAGCGGT
     TGCGACGCGA
101  AGATGTATCC GCCGATGAGC ACCCAGCTCG AAGCCTTGGG
     CATAGGCGTA
151  CACGAAGGCT TCGATGCCGC GCAGTTGGAA GAATTTCAAG
     CCGATATTTA
201  CGTCATCGGC AATGTCGCCA GGCGCGGGAT GGATGTGGTC
     GAGGCGATTT
251  TGAACCGTGG GCTGCCTTAT ATTTCCGGCC CGCAATGGCT
     GGCTGAAAac
301  GTGCtgcacc atcaTTGGgt ACTCGGCGTG GcagggaCGC
     ACGGcaaAac
351  gaccaCcGcg tCCATGCTCG CCTGGGTCTT GGAATATGCC
     GGACTCGCGC
401  CGGGCTTCCT CATCGGCGGt gtaccggaAA ATTTCGGCGT
     TTCCGCCCGC
451  CTACCGCAAA CGCCGCGTCA AGACCCGAAC AGCAAATCGC
     CGTTTTTCGT
501  CATCGAAGCC GACGAATACG ACACCGCCTT TTTCGACAAA
     CGCTCCAAAT
551  TCGTGCATTA TCGCCCGCGT ACCGCCGTGT TGAACAATCT
     GGAATTCGAC
601  CACGCCGACA TCTTCGCCGA CTTGGGCGCG ATACAGACCC
     AGTTCCACCA
651  CCTCGTGCGC ACCGTACCAT CCGAAGGCCT CATCGTCTGC
     AACGGACAGC
701  AGCAAAGCCT GCAAGATACT TTGGACAAAG GCTGCTGGAC
     GCCGGTGGAA
751  AAATTCGGCA CCGGACACGG CTGGCAGATT GGTGAAGTCA
     ATGCCGACGG
801  CTCGTTCGAC GTATTGCTTG ACGGCAAAAA AGCCGGACAC
     GTCGCATGGG
851  ATTTGATGGG CGGACACAAC CGCATGAACG CGCTCGCCGT
     CATCGCTGCC
901  GCACGCCATG CCGGAGTCGA TGTTCAGACG GCCTGCGAAG
     CCTTGGGTGC
951  GTTTAAAAAC GTCAAACGCC GCATGGAAAT CAAAGGCACG
     GCAAACGGCA
1001 TCACCGTTTA CGACGATTTC GCCCACCACC CGACCGCCAT
     CGAAACCACG
1051 ATTCAAGGTT TGCGCCAACG TGTCGGCGGC GCGCGCATCC
     TCGCCGTCCT
1101 CGAGCCGCGT TCCAACACCA TGAAACTCGG CACGATGAAG
     TCCGCCCTGC
1151 CCGCAAGCCT CAAAGAAGCC GACCAAGTGT TCTGCTACGC
     CGGCGGCGCG
1201 GACTGGGACG TTGCCGAAGC CCTCGCGCCT TTGGGCTGCA
     GGCTGCGCGT
1251 CGGTAAAGAT TTCGATACCT TCGTTGCCGA AATTGTGAAA
     AACGCCCGAA
1301 CCGGCGACCA TATTTTGGTG ATGAGCAACG GCGGTTTCGG
     CGGAATACAC
1351 ACCAAACTGC TGGACGCTTT GAGATAG

This corresponds to the amino acid sequence <SEQ ID 874; ORF132ng-1>:

1   MKHIHIIGIG GTFMGGIAAI AKEAGFKVSG CDAKMYPPMS
    TQLEALGIGV
51  HEGFDAAQLE EFQADIYVIG NVARRGMDVV EAILNRGLPY
    ISGPQWLAEN
101 VLHHHWVLGV AGTHGKTTTA SMLAWVLEYA GLAPGFLIGG
    VPENFGVSAR
151 LPQTPRQDPN SKSPFFVIEA DEYDTAFFDK RSKFVHYRPR
    TAVLNNLEFD
201 HADIFADLGA IQTQFHHLVR TVPSEGLIVC NGQQQSLQDT
    LDKGCWTPVE
251 KFGTGHGWQI GEVNADGSFD VLLDGKKAGH VAWDLMGGHN
    RMNALAVIAA
301 ARHAGVDVQT ACEALGAFKN VKRRMEIKGT ANGITVYDDF
    AHHPTAIETT
351 IQGLRQRVGG ARILAVLEPR SNTMKLGTMK SALPASLKEA
    DQVFCYAGGA
401 DWDVAEALAP LGCRLRVGKD FDTFVAEIVK NARTGDHILV
    MSNGGFGGIH
451 TKLLDALR*

ORF132ng-1 and ORF132-1 show 93.2% identity in 458 aa overlap:

In addition, ORF132ng-1 is homologous to a hypothetical E. coli protein:

pir||S556459 hypothetical protein o457 - Escherichia coli >gi|537075 (U14003)
ORF_o457 [Escherichia coli] >gi|1790660 (AE000494). hypothetical 48.5 kD protein
in fbp-pmba intergenic region [Escherichia coli] Length = 457
Score = 474 bits (1207), Expect = e−133
Identities = 249/439 (56%), Positives = 294/439 (66%), Gaps = 13/439 (2%)
Query:  22 KEAGFKVSGCDAKMYPPMSTQLEALGIGVHEGFDAAQLEEFQADIYVIGNVARRGMDVVE  81
++ G +V+G DA +YPPMST LE  GI + +G+DA+QLE  Q D+ +IGN   RG    VE
Sbjct:  21 RQLGHEVTGSDANVYPPMSTLLEKQGIELIQGYDASQLEP-OPDLVIIGNAMTRGNPCVE  79
Query:  82 AILNRGLPYISGPQWLAENVLHHHWVLGVAGTHGKTTTASMLAWVLEYAGLAPGFLIGGV 141
A+L  ++PY+SGPQWL  +VL   WVL VAGTHGKTTTA M  W+LE  G   PGF+IGGV
Sbjct:  80 AVLEKNIPYMSGPQWLHDFVLADRWVLAVAGTHGKTTTAGMATWILEQCGYKPGFVIGGV 139
Query: 142 PENFGVSARLPQTPRQDPNSKSPFFVIEADEYDTAFFDKRSKFVHYRPRTAVLNNLEFDH 201
P NF VSA L          +S FFVIEADEYD AFFDKRSKFVHY PRT +LNNLEFDH
Sbjct: 140 PGNFEVSAHL---------GESDFFVIEADEYDCAFFDKRSKFVHYCPRTLILNNLEFDH 190
Query: 202 ADIFADLGAIQTQFHHLVRTVPSEGLIVCNGQQQSLQDTLDKGCWTPVEKFGTGHGWQIG 261
ADIF DL AIQ QFHHLVR VP +G I+      +L+ T+  GCW+  E  G     WQ
Sbjct: 191 ADIFDDLKAIQKQFHHLVRIVPGOGRIIWPENDINLKQTMAMGCWSEQELVGEQGHWQAK 250
Query: 262 EVNADGS-FDVLLDGKKAGHVAWDLMGGHNRMNALAVIAAARHAGVDVQTACEALGAFKN 320
++  D S ++VLLDG+K G V W L+G HN  N L  IAAARH GV    A   ALG+FN
Sbjct: 251 KLTTDASEWEVLLDGEKVGEVKWSLVGEHNMHNGLMAIAAARHVGVAPADAANALGSFIN 310
Query: 321 VKRRMEIKGTANGITVYDDFAHHPTAIETTIQGLRQRVGG-ARILAVLEPRSNTMKLGTM 379
+RR+E++G ANG+TVYDDFAHHPTAI  T+  LR +VGG  ARI+AVLEPRSNTMK+G
Sbjct: 311 ARRRLELRGEANGVTVYDDFAHHPTAILATLAALRGKVGGTARIIAVLEPRSNTMKMGIC 370
Query: 380 KSALPASLKEADQVF-CYAGGADWDVAEALAPLGCRLRVGKDFDTFVAEIVKNARTGDHI 438
K  L  SL  AD+VF        W VAE             D DT    +VK A+ GDHI
Sbjct: 371 KDDLAPSLGRADEVFLLQPAHIPWQVAEVAEACVQPAHWSGDVDTLADMVVKTAQPGDHI 430
Query: 439 LVMSNGGFGGIHTKLLDAL                                          457
LVMSNGGFGGIH KLLD L
Sbjct: 931 LVMSNGGFGGIHQKLLDGL                                          999

Based on this analysis, it was predicted that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

ORF132-1 (26.4 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 20A shows the results of affinity purification of the His-fusion protein, and FIG. 20B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for FACS analysis (FIG. 20C) and ELISA (positive result). These experiments confirm that ORF132 is a surface-exposed protein, and that it is a useful immunogen.

Example 103

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 875>

1 ..CCGGGCTATT ACGGCTCGGA TGACGAATTT AAGCGGGCAT TCGGAGAAAA
51   CTCGCCGACA TmCAAGAAAC ATTGCAACCG GAGCTGCGGG ATTTATGAAC
101   CCGTATTGAA AAAATACGGC AAAAAGCGCG CCAACAACCA TTCGGTCAGC
151   ATTAGTGCGG ACTTCGGCGA TTATTTCATG CCGTTCGCCA GCTATTCGCG
201   CACACACCGT ATGCCCAACA TCCAAGAAAT GTATTTTTCC CAAATCGGCG
251   ACTCCGGCGT TCACACCGCC TTAAAACCAG AGCGCGCAAA CACTTGGCAA
301   TTTGGCTTCr ATACCTATAA AAAAGGATTG TTAAAACAAG ATGATACATT
351   AGGATTAAAA CTGGTCGGCT ACCGCAGCCG CATCGACAAC TACATCCACA
401   ACGTTTACGG GAAATGGTGG GATTTGAACG GGGATATTCC GAGCTGGGTC
451   AGCAGCACCG GGCTTGCCTA CACCATCCAA CATCGCrATT TCAwAGACAA
501   AGTGCATCAA nnnnnnnnnn nnnnnnnnnn nnnnTACGAT TATGGGCGTT
551   TTTTCACCAA CCTTTCTTAC GCCTATCAAA AAAGCACGCA ACCGACCAAC
601   TTCAGCGATG CGAGCGAATC GCCCAACAAT GCGTCCAAAG AAGACCAACT
651   CAAACAAGGT TATGGGTTGA GCAGGGTTTC CGCCCTGCCG CGAGATTACG
701   GACGTTTGGA AGTCGGTACG CGCTGGTTGG GCAACAAACT GACTTTGGGC
751   GGCGCGATGC GCTATTTCGG CAAGAGCATC CGCGCGACGG CTGAAGAACG
801   CTATATCGAC GGCACCAACG GGGGAAATAC CAGCAATTTC CGGCAACTGG
851   GCAAGCGTTC CATCAAACAA ACCGAAACTC TTGCCCGCCA GCCTTTGATT
901   TTwGATTTTa ACGCCGCTTA CGAGCCGAAG AAAAACCTTA TTTTCCGCGC
951   CGAAGTCAAA AATCTGTTCG ACAGGCGTTA TATCGATCCG CTCGATGCGG
1001   GCAATGATGC GGCAAC.GAG CGTTATTACA GCTCGTTCGA CCCGAAAGAC
1051   AAGGACrrAG ACGTAACGTG TAATGCTGAT AAAACGTTGT GCaACGGCAA
1101   ATACGGCGGC ACAAGCAAAA GCGTATTGAC CAATTTTGCA CGCGGACGCA
1151   CCTTTTTgAT GACGATGAGC TACAAGTTTT AA

This corresponds to the amino acid sequence <SEQ ID 876; ORF133>:

1   ..PGYYGSDDEF KRAFGENSPT XKKHCNRSCG IYEPVLKKYG
    KKRANNHSVS
51  ISADFGDYFM PFASYSRTHR MPNIQEMYFS QIGDSGVHTA
    LKPERANTWQ
101 FGFXTYKKGL LKQDDTLGLK LVGYRSRIDN YIHNVYGKWW
    DLNGDIPSWV
151 SSTGLAYTIQ HRXFXDKVHQ XXXXXXXXYD YGRFFTNLSY
    AYQKSTQPTN
201 FSDASESPNN ASKEDQLKQG YGLSRVSALP RDYGRLEVGT
    RWLGNKLTLG
251 GAMRYFGKSI RATAEERYID GTNGGNTSNF RQLGKRSIKQ
    TETLARQPLI
301 XDFNAAYEPK KNLIFRAEVK NLFDRRYIDP LDAGNDAAXE
    RYYSSFDPKD
351 KDXDVTCNAD KTLCNGKYGG TSKSVLTNFA RGRTFLMTMS
    YKF*

Further work revealed the further partial DNA sequence <SEQ ID 877>:

1    GAGGCGCAGA TACAGGTTTT GGAAGATGTG CACGTCAAGG
     CGAAGCGCGT
51   ACCGAAAGAC AAAAAAGTGT TTACCGATGC GCGTGCCGTA
     TCGACCCGTC
101  AGGATATATT CAAATCCAGC GAAAACCTCG ACAACATCGT
     ACGCAGCATC
151  CCCGGTGCGT TTACACAGCA AGATAAAAGC TCGGGCATTG
     TGTCTTTGAA
201  TATTCGCGGC GACAGCGGGT TCGGGCGGGT CAATACGATG
     GTGGACGGCA
251  TCACGCAGAC CTTTTATTCG ACTTCTACCG ATGCGGGCAG
     GGCAGGCGGT
301  TCATCTCAAT TCGGTGCATC TGTCGACAGC AATTTTATTG
     CCGGACTGGA
351  TGTCGTCAAA GGCAGCTTCA GCGGCTCGGC AGGCATCAAC
     AGCCTTGCCG
401  GTTCGGCGAA TCTGCGGACT TTAGGCGTGG ATGACGTCGT
     TCAGGGCAAT
451  AATACCTACG GCCTGCTGCT AAAAGGTCTG ACCGGCACCA
     ATTCAACCAA
501  AGGTAATGCG ATGGCGGCGA TAGGTGCGCG CAAATGGCTG
     GAAAGCGGAG
551  CATCTGTCGG TGTGCTTTAC GGGCACAGCA GGCGCAGCGT
     GGCGCAAAAT
601  TACCGCGTGG GCGGCGGCGG GCAGCACATC GGAAATTTTG
     GCGCGGAATA
651  TTTGGAACGG CGCAAGCAGC GATATTTTGT ACAAGAGGGT
     GCTTTGAAAT
701  TCAATTCCGA CAGCGGAAAA TGGGAGCGGG ATTTACAAAG
     GCAACAGTGG
751  AAATACAAGC CGTATAAAAA TTACAACAAC CAAGAACTAC
     AaAAATACAT
801  CGAAGAGCAT GACAAAAGCT GGCGGGAAAA CCTg.CaCCG
     CAATACGACA
851  TTACCCCCAT CGATCCGTCC AGCCTGAAGC AGCAGTCGGC
     AGGCAATCTG
901  TTTAAATTGG AATACGACGG CGTATTCAAT AAATACACGG
     CGCAATTTCG
951  CGATTTAAAC ACCAAAATCG GCAGCCGCAA AATCATCAAC
     CGCAATTATC
1001 AGTTCAATTA CGGTTTGTCT TTGAACCCGT ATACCAACCT
     CAATCTGACC
1051 GCAGCCTACA ATTCGGGCAG GCAGAAATAT CCGAAAGGGT
     CGAAGTTTAC
1101 AGGCTGGGGG CTTTTAAAGG ATTTTGAAAC CTACAACAAC
     GCGAAAATCC
1151 TCGACCTCAA CAACACCGCC ACCTTCCGGC TGCCCCGCGA
     AACCGAGTTG
1201 CAAACCACTT TGGGCTTCAA TTATTTCCAC AACGAATACG
     GCAAAAACCG
1251 CTTTCCTGAA GAATTGGGGC TGTTTTTCGA CGGTCCTGAT
     CAGGACAACG
1301 GGCTTTATTC CTATTTGGGG CGGTTTAAGG GCGATAAAGG
     GCTGCTGCCC
1351 CAAAAATCAA CCATTGTCCA ACCGGCCGGC AGCCAATATT
     TCAACACGTT
1401 CTACTTCGAT GCCGCGCTCA AAAAAGACAT TTACCGCTTA
     AACTACAGCA
1451 CCAATACCGT CGGCTACCGT TTCGGCGGCG AATATACGGG
     CTATTACGGC
1501 TCGGATGACG AATTTAAGCG GGCATTCGGA GAAAACTCGC
     CGACATACAA
1551 GAAACATTGC AACCGGAGCT GCGGGATTTA TGAACCCGTA
     TTGAAAAAAT
1601 ACGGCAAAAA GCGCGCCAAC AACCATTCGG TCAGCATTAG
     TGCGGACTTC
1651 GGCGATTATT TCATGCCGTT CGCCAGCTAT TCGCGCACAC
     ACCGTATGCC
1701 CAACATCCAA GAAATGTATT TTTCCCAAAT CGGCGACTCC
     GGCGTTCACA
1751 CCGCCTTAAA ACCAGAGCGC GCAAACACTT GGCAATTTGG
     CTTCAATACC
1801 TATAAAAAAG GATTGTTAAA ACAAGATGAT ACATTAGGAT
     TAAAACTGGT
1851 CGGCTACCGC AGCCGCATCG ACAACTACAT CCACAACGTT
     TACGGGAAAT
1901 GGTGGGATTT GAACGGGGAT ATTCCGAGCT GGGTCAGCAG
     CACCGGGCTT
1951 GCCTACACCA TCCAACATCG CAATTTCAAA GACAAAGTGC
     ACAAACACGG
2001 TTTTGAGTTG GAGCTGAATT ACGATTATGG GCGTTTTTTC
     ACCAACCTTT
2051 CTTACGCCTA TCAAAAAAGC ACGCAACCGA CCAACTTCAG
     CGATGCGAGC
2101 GAATCGCCCA ACAATGCGTC CAAAGAAGAC CAACTCAAAC
     AAGGTTATGG
2151 GTTGAGCAGG GTTTCCGCCC TGCCGCGAGA TTACGGACGT
     TTGGAAGTCG
2201 GTACGCGCTG GTTGGGCAAC AAACTGACTT TGGGCGGCGC
     GATGCGCTAT
2251 TTCGGCAAGA GCATCCGCGC GACGGCTGAA GAACGCTATA
     TCGACGGCAC
2301 CAACGGGGGA AATACCAGCA ATTTCCGGCA ACTGGGCAAG
     CGTTCCATCA
2351 AACAAACCGA AACTCTTGCC CGCCAGCCTT TGATTTTTGA
     TTTTTACGCC
2401 GCTTACGAGC CGAAGAAAAA CCTTATTTTC CGCGCCGAAG
     TCAAAAATCT
2451 GTTCGACAGG CGTTATATCG ATCCGCTCGA TGCGGGCAAT
     GATGCGGCAA
2501 CGCAGCGTTA TTACAGCTCG TTCGACCCGA AAGACAAGGA
     CGAAGACGTA
2551 ACGTGTAATG CTGATAAAAC GTTGTGCAAC GGCAAATACG
     GCGGCACAAG
2601 CAAAAGCGTA TTGACCAATT TTGCACGCGG ACGCACCTTT
     TTGATGACGA
2651 TGAGCTACAA GTTTTAA

This corresponds to the amino acid sequence <SEQ ID 878; ORF133-1>:

1   EAQIQVLEDV HVKAKRVPKD KKVFTDARAV STRQDIFKSS
    ENLDNIVRSI
51  PGAFTQQDKS SGIVSLNIRG DSGFGRVNTM VDGITQTFYS
    TSTDAGRAGG
101 SSQFGASVDS NFIAGLDVVK GSFSGSAGIN SLAGSANLRT
    LGVDDVVQGN
151 NTYGLLLKGL TGTNSTKGNA MAAIGARKWL ESGASVGVLY
    GHSRRSVAQN
201 YRVGGGGQHI GNFGAEYLER RKQRYFVQEG ALKFNSDSGK
    WERDLQRQQW
251 KYKPYKNYNN QELQKYIEEH DKSWRENLXP QYDITPIDPS
    SLKQQSAGNL
301 FKLEYDGVFN KYTAQFRDLN TKIGSRKIIN RNYQFNYGLS
    LNPYTNLNLT
351 AAYNSGRQKY PKGSKFTGWG LLKDFETYNN AKILDLNNTA
    TFRLPRETEL
401 QTTLGFNYFH NEYGKNRFPE ELGLFFDGPD QDNGLYSYLG
    RFKGDKGLLP
451 QKSTIVQPAG SQYFNTFYFD AALKKDIYRL NYSTNTVGYR
    FGGEYTGYYG
501 SDDEFKRAFG ENSPTYKKHC NRSCGIYEPV LKKYGKKRAN
    NHSVSISADF
551 GDYFMPFASY SRTHRMPNIQ EMYFSQIGDS GVHTALKPER
    ANTWQFGFNT
601 YKKGLLKQDD TLGLKLVGYR SRIDNYIHNV YGKWWDLNGD
    IPSWVSSTGL
651 AYTIQHRNFK DKVHKHGFEL ELNYDYGRFF TNLSYAYQKS
    TQPTNFSDAS
701 ESPNNASKED QLKQGYGLSR VSALPRDYGR LEVGTRWLGN
    KLTLGGAMRY
751 FGKSIRATAE ERYIDGTNGG NTSNFRQLGK RSIKQTETLA
    RQPLIFDFYA
801 AYEPKKNLIF RAEVKNLFDR RYIDPLDAGN DAATQRYYSS
    FDPKDKDEDV
851 TCNADKTLCN GKYGGTSKSV LTNFARGRTF LMTMSYKF*

Computer analysis of this amino acid sequence gave the following results:

Homology with the Probable TonB-Dependent Receptor HI121 of H. influenzae (Accession Number U32801)

ORF133 and HI121 show 57% aa identity in 363aa overlap:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF133 shows 90.8% identity over a 392aa overlap with an ORF (ORF133a) from strain A of N. meningitidis:

A partial ORF133a nucleotide sequence <SEQ ID 879> is:

1    AAAGACAAAA AAGTGTTTAC CGATGCGCGT GCCGTATCGA
     CCCGTCAGGA
51   TATATTCAAA TCCANCGAAA ACCTCGACAA CATCGTACGC
     ANCATCCCCG
101  GTGCGTTTAC ACANCAANAT AAAAGCTCGG GCNTTGTGTC
     TTTGAATATT
151  CGCNGCGACA GCGGGTTCGG GCGGGTCAAT ACNATGGTNG
     ACGGCATCAC
201  NCANACCTTT TATTCGACTT CTACCGATGC GGGCAGGGCA
     GGCGGTTCAT
251  CTCAATTCGG TGCATCTGTC GACAGCAATT TTATNGCCGG
     ACTGGATGTC
301  GTCAAAGGCA GCTTCAGCGG CTCGGCAGGC ATCAACAGCC
     TTGCCGGTTC
351  GGCGAATCTG CGGACTTTAN GCGTGGATGA TGTCGTTCAG
     GGCAATANTA
401  CNTACGGCCT GCTGCTAAAA GGTCTGACCG GCACCAATTC
     AACCAAAGGT
451  AATGCGATGG CGGCGATAGG TGCGCGCAAA TGGCTGGAAA
     GCGGAGCATC
501  TGTCGGTGTG CTTTACGGGC ACAGCAGGCG CAGCGTGGCG
     CAAAATTACC
551  GCGTGGGCGG CGGCGGGCAG CACATCGGAA ATTTTGGCGC
     GGAATATCTG
601  GAACGACGCA AGCAACGATA TTTTGAGCAA GAAGGCGGGT
     TGAAATTCAA
651  TTCCAACAGC GGAAAATGGG AGCGGGATTT CCAAAAGTCG
     TACTGGAAAA
701  CCAAGTGGTA TCAAAAATAC GATGCCCCCC AAGAACTGCA
     AAAATACATC
751  GAAGGTCATG ATAAAAGCTG GCGGGAAAAC CTGGCGCCGC
     AATACGACAT
801  CACCCCCATC GATCCGTCCA GCCTGAAGCN GCAGTCGGCA
     GGCAACCTGT
851  TTAAATTGGA ATACGACGGC GTATTCAATA AATACACGGC
     GCAATTTCGC
901  GATTTAAACA CCAAAATCGG CAGCCGCAAA ATCATCAACC
     GCAATTATCA
951  ATTCAATTAC GGTTTGTCTT TGAACCCGTA TACCAACCTC
     AATCTGACCG
1001 CAGCCTACAA TTCGGGCAGG CAGAAATATC CGAAAGGGTC
     GAAGTTTACA
1051 GGCTGGGGGC TTTTNAAAGA TTTTGAAACC TACAACAACG
     CAAAAATCCT
1101 CGACCTCANC AACACCTCCA CCTTCCGGCT GCCCCGTGAA
     ACCGAGTTGC
1151 AAACCACTTT GGGCTTCAAT TATTTCCACA ACGAATACGG
     CAAAAACCGC
1201 TTTCCTGAAG AATTGGGGCT GTTTTTCGAC GGTCCGGATC
     ANGACAACGG
1251 GCTTTATTCC TATTTGGGGC GGTTTAAGGG CGATAAAGGG
     CTGCTGCCCC
1301 AAAAATCAAC CATTGTCCAA CCGGCCGGCA GCCAATATTT
     CAACACGTTC
1351 TACTTCGATG CCGCGCTCAA AAAAGACATT TACCGCTTAA
     ACTACAGCAC
1401 CAATACCGTC GGCTACCGTT TCGGCGGCNA ATATACGGGC
     TATTACNGCT
1451 CGGATGACGA ATTTAAGCGG GCATTCGGAG AAAACTCGCC
     GACATACANG
1501 AAACATTGCA ACCAGAGCTG CGGAATTTAT GAACCCGTAT
     TGAAAAAATA
1551 CGGCAAAAAG CGCGCCAACA ACCATTCGGT CAGCATTAGT
     GCGGACTTCG
1601 GCGATTATTT CATGCCGTTC GCCAGCTATT CGCGCACACA
     CCGTATGCCC
1651 AACATCCAAG AAATGTATTT TTCCCAAATC GGCGACTCCG
     GCGTTCACAC
1701 CGCCTTAAAA CCAGAGCGCG CAAACACTTG GCAATTTGGC
     TTCAATACCT
1751 ATAAAAAAGG ATTGTTAAAA CAAGATGATA TATTAGGATT
     AAAACTGGTC
1801 GGCTACCGCA GCCGCATCGA CNACTACATC CACAACGTTT
     ACGGGAAATG
1851 GTGGGATTTG AACGGGAATA TTCCGAGCTG GGTCAGCAGC
     ACCGGGCTTG
1901 CCTACACCAT CCAACACCGC AATTTCAAAG ACAAAGTGCA
     CAAACACGGT
1951 TTTGAGTTGG AGCTGAATTA CGATTATNGG CGTTTTTTCA
     CCAACCTTTC
2001 TTACGCCTAT CAAAAAAGCA CGCAACCGAC CAACTTCAGC
     GATGCGAGCG
2051 AATCGCCCAA CAATGCGTCC AAAGAAGACC AACTCAAACA
     AGGTTATGGG
2101 TTGAGCAGGG TTTCCGCCCT GCCGCGAGAT TACGGACGTT
     TGGAAGTCGG
2151 TACGCGCTGG TTGGGCAACA AACTGACTTT GGGCGGCGCG
     ATGCGCTATT
2201 TCGGCAAGAG CATCCGCGCG ACGGCTGAAG AACGCTATAT
     CGACGNCACC
2251 AATGGGGNAN NTACCAGCAA TTTCCGGCAA CTGGGCAAGC
     GTTCCATCAN
2301 ACAAACCGAA ACCCTTGCCC GCCAGCCTTT GATTTTTGAT
     TTNTACGCCG
2351 CTTACGAGCC GAAGAAAAAN CTTATTTTCC GCGCCGAAGT
     CAAAAATCTG
2401 TTCGACAGGC GTTATATCGA TCCGCTCGAT GCGGGCAATG
     ATGCGGCAAC
2451 GCAGCGTTAT TACAGTTCGT TCGACCCGAA AGACAAGGAC
     GAAGAAGTAA
2501 CGTGTAATGA TGATAACACG TTATGCAACG GCAAATACGG
     CGGCACAAGC
2551 AAAAGCGTAT TGACCAATTT TGCACGCGGA CNCACCTTTT
     TGATAACGAT
2601 GAGCTACAAG TTTTAA

This encodes a protein having (partial) amino acid sequence <SEQ ID 880>:

1   KDKKVFTDAR AVSTRQDIFK SXENLDNIVR XIPGAFTXQX
    KSSGXVSLNI
51  RXDSGFGRVN TMVDGITXTF YSTSTDAGRA GGSSQFGASV
    DSNFXAGLDV
101 VKGSFSGSAG INSLAGSANL RTLXVDDVVQ GNXTYGLLLK
    GLTGTNSTKG
151 NAMAAIGARK WLESGASVGV LYGHSRRSVA QNYRVGGGGQ
    HIGNFGAEYL
201 ERRKQRYFEQ EGGLKFNSNS GKWERDFQKS YWKTKWYQKY
    DAPQELQKYI
251 EGHDKSWREN LAPQYDITPI DPSSLKXQSA GNLFKLEYDG
    VFNKYTAQFR
301 DLNTKIGSRK IINRNYQFNY GLSLNPYTNL NLTAAYNSGR
    QKYPKGSKFT
351 GWGLXKDFET YNNAKILDLX NTSTFRLPRE TELQTTLGFN
    YFHNEYGKNR
401 FPEELGLFFD GPDXDNGLYS YLGRFKGDKG LLPQKSTIVQ
    PAGSQYFNTF
451 YFDAALKKDI YRLNYSTNTV GYRFGGXYTG YYXSDDEFKR
    AFGENSPTYX
501 KHCNQSCGIY EPVLKKYGKK RANNHSVSIS ADFGDYFMPF
    ASYSRTHRMP
551 NIQEMYFSQI GDSGVHTALK PERANTWQFG FNTYKKGLLK
    QDDILGLKLV
601 GYRSRIDXYI HNVYGKWWDL NGNIPSWVSS TGLAYTIQHR
    NFKDKVHKHG
651 FELELNYDYX RFFTNLSYAY QKSTQPTNFS DASESPNNAS
    KEDQLKQGYG
701 LSRVSALPRD YGRLEVGTRW LGNKLTLGGA MRYFGKSIRA
    TAEERYIDXT
751 NGXXTSNFRQ LGKRSIXQTE TLARQPLIFD XYAAYEPKKX
    LIFRAEVKNL
801 FDRRYIDPLD AGNDAATQRY YSSFDPKDKD EEVTCNDDNT
    LCNGKYGGTS
851 KSVLTNFARG XTFLITMSYK F*

ORF133a and ORF133-1 show 94.3% identity in 871 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF133 shows 92.3% identity over 392 aa overlap with a predicted ORF (ORF133ng) from N. gonorrhoeae:

The complete length ORF133ng nucleotide sequence <SEQ ID 881> is predicted to encode a protein having amino acid sequence <SEQ ID 882>:

1   MRSSFRLKPI CFYLMGVMLY HHSYAEDAGR AGSEAQIQVL
    EDVHVKAKRV
51  PKDKKVFTDA RAVSTRQDVF KSGENLDNIV RSIPGAFTQQ
    DKSSGIVSLN
101 IRGDSGFGRV NTMVDGITQT FYSTSTDAGR AGGSSQFGAS
    VDSNFIAGLD
151 VVKGSFSGSA GINSLAGSAN LRTLGVDDVV QGNNTYGLLL
    KGLTGTNSTK
201 GNAMAAIGAR KWLESGASVG VLYGHSRRGV AQNYRVGGGG
    QHIGNFGEEY
251 LERRKQQYFV QEGGLKFNAG SGKWERDLQR QYWKTKWYKK
    YEDPQELQKY
301 IEEHDKSWRE NLAPQYDITP IDPSGLKQQS AGNLLNLEYD
    GVFNKYTAQF
351 RDLNTRIGSR KIINRNYQFN YGLSLNPYTN LNLTAAYNSG
    RQKYPKGAKF
401 TGWGLLKDFE TYNNAKILDL NNTATFRLPR ETELQTTLGF
    NYFHNEYGKN
451 RFPEELGLFF DGPDQDNGLY SYLGRFKGDK GLLPQKSTIV
    QPAGSQYFNT
501 FYFDAALKKD IYRLNYSTNA INYRFGGEYT GYYGSENEFK
    RAFGENSPAY
551 KEHCDPSCGL YEPVLKKYGK KRANNHSVSI SADFGDYFMP
    FAGYSRTHRM
601 PNIQEMYFSQ IGDSGVHTAL KPERANTWQF GFNTYKKGLL
    KQDDILGLKL
651 VGYRSRIDNY IHNVYGKWWD LNGDIPSWVG STGLAYTIRH
    RNFKDKVHKH
701 GFELELNYDY GRFFTNLSYA YQKSTQPTNF SDASESPNNA
    SKEDQLKQGY
751 GLSRVSALPR DYGRLEVGTR WLGNKLTLGG AMRYFGKSIR
    ATAEERYIDG
801 TNGGNTSNVR QLGKRSIKQT ETLARQPLIF DFYAAYEPKK
    NLIFRAEVKN
851 LFDRRYIDPL DAGNDAATQR YYSSFDPKDK DEDVTCNADK
    TLCNGKYGGT
901 SKSVLTNFAR GRTFLMTMSY KF*

A variant was also identified, being encoded by the gonococcal DNA sequence <SEQ ID 883>:

1 ATGAGATCTT CTTTCCGGTT GAAGCCGATT TGTTTTTATC TTATGGGTGT
51 TATGCTATAT CATCATAGTT ATGCCGAAGA TGCAGGGCGC GCGGGCAGCG
101 AGGCGCAGAT ACAGGTTTTG GAAGATGTGC ACGTCAAGGC GAAGCGCGTA
151 CCGAAAGACA AAAAAGTGTT TACCGATGCG CGTGCCGTAT CGACCCGTca
201 gGATGTGTTC AAATCCGGCG AAAACCTCGA CAACATCGTA CGCAGCATAC
251 CCGGTGCGTT TACACAGCAA GATAAAAGCT CGGGCATTGT GTCTTTGAAT
301 ATTCCCGGCG ACAGCGGGTT CGGGCGGGTC AATACGATGG TGGACGGCAT
351 CACGCAGACC TTTTATTCGA CTTCTACCGA TGCGGGCAGG GCAGGCGGTT
401 CATCTCAATT CGGTGCATCT GTCGACAGCA ATTTTATTGC CGGACTGGAT
451 GTCGTCAAAG GCAGCTTCAG CGGCTCGGCA GGCATCAACA GCCTTGCCGG
501 TTCGGCGAAT CTGCGGACTT TAGGCGTGGA TGACGTCGTT CAGGGCAATA
551 ATACCTACGG CCTGCTGCTA AAAGGTCTGA CCGGCACCAA TTCAACCAAA
601 GGTAATGCGA TGGCGGCGAT AGGTGCGCGC AAATGGCTGG AAAGCGGAGC
651 GTCTGTCGGT GTGCTTTACG GGCACAGCAG GCGCGGCGTG GCGCAAAATT
701 ACCGCGTGGG CGGCGGCGGG CAGCACATCG GAAATTTTGG TGAAGAATAT
751 CTGGAACGGC GCAAACAGCA ATATTTTGTA CAAGAGGGTG GTTTGAAATT
801 CAATGCCGGC AGCGGAAAAT GGGAACGGGA TTTGCAAAGG CAATACTGGA
851 AAACAAAGTG GTATAAAAAA TACGAAGACC CCCAAGAACT GCAAAAATAC
901 ATCGAAGAGC ATGATAAAAG CTGGCGGGAA AACCTGGCGC CGCAATACGA
951 CATCACCCCC ATCGATCCGT CCGGCCTGAA GCAGCAGTCG GCAGGCAATC
1001 TGTTTAAATT GGAATACGAC GGCGTATTCA ATAAATACAC GGCGCAATTT
1051 CGCGATTTAA ACACCAGAAT CGGCAGCCGC AAAATCATCA ACCGCAATTA
1101 TCAATTCAAT TACGGTTTGT CTTTGAACCC GTATACCAAC CTCAATCTGA
1151 CCGCAGCCTA CAATTCGGGC AGGCAGAAAT ATCCGAAAGG GGCGAAGTTT
1201 ACAGGCTGGG GGCTTTTAAA AGATTTTGAA ACCTACAACA ACGCGAAAAT
1251 CCTCGACCTC AACAACACCG CCACCTTCCG GCTGCCCCGC GAAACCGAGT
1301 TGCAAACCAC TTTGGGCTTC AATTATTTCC ACAACGAATA CGGCAAAAAC
1351 CGCTTTCCTG AAGAATTGGG GCTGTTTTTC GACGGTCCTG ATCAGGACAA
1401 CGGGCTTTAT TCCTATTTGG GGCGGTTTAA GGGCGATAAA GGGCTGTTGC
1451 CTCAAAAATC AACCATTGTC CAACCGGCCG GCAGCCAATA TTTCAACACG
1501 TTCTACTTCG ATGCCGCGCT CAAAAAAGAC ATTTACCGCT TAAACTACAG
1551 CACCAATGCA ATCAACTACC GTTTCGGCGG CGAATATACG GGCTATTACG
1601 GCTCGGAAAA CGAATTTAAG CGGGCATTCG GAGAAAACTC GCCGGCATAC
1651 AAGGAACATT GCGACCCGAG CTGCGGGCTT TATGAACCCG TATTGAAAAA
1701 ATACGGCAAA AAGCGCGCCA ACAACCATTC GGTCAGCATT AGTGCGGACT
1751 TCGGCGATTA TTTCATGCCG TTCGCCGGCT ATTCGCGCAC ACACCGTATG
1801 CCCAACATCC AAGAAATGTA TTTTTCCCAA ATCGGCGACT CCGGCGTTCA
1851 CACCGCCTTA AAACCAGAGC GCGCAAACAC TTGGCAATTT GGCTTCAATA
1901 CCTATAAAAA AGGATTGTTA AAACAAGATG ATATATTAGG ATTGAAACTG
1951 GTCGGCTACC GCAGCCGCAT TGACAACTAC ATCCACAACG TTTACGGGAA
2001 ATGGTGGGAT TTGAACGGGG ATATTCCGAG CTGGGTCGGC AGCACCGGGC
2051 TTGCCTACAC CATCCGACAC CGCAATTTCA AAGACAAAGT GCACAAACAC
2101 GGTTTTGAGC TGGAGCTGAA TTACGATTAT GGGCGTTTTT TCACCAACCT
2151 TTCTTACGCC TATCAAAAAA GCACGCAACC GACCAATTTC AGCGATGCGA
2201 GCGAATCGCC CAACAATGCC tccaaAGAAG ACCAACTCAA ACAAGGTTAT
2251 GGGCTGAGCA GGGTTTCCGC CCTGCCGCGA GATTACGGAC GTTTGGAAGT
2301 CGGTACGCGC TGGTTGGGCA ACAAACTGAC TTTGGGCGGC GCGAtgCGCT
2351 ATTTCGGCAA GAGCATCCGC GCGACGGCTG AAGAACGCTA TATCGACGGC
2401 ACCAACGGGG GAAATACCAG CAATGTCCGG CAACTGGGCA AGCGTTCCAT
2451 CAAACAAACC GAAACCCTTG CCCGACAGCC TTTGATTTTT GATTTTTACG
2501 CCGCTTACGA GCCGAAGAAA AACCTTATTT TCCGCGCCGA AGTCAAAAAC
2551 CTGTTCGACA GGCGTTATAT CGATCCGCTC GATGCGGGCA ATGATGCGGC
2601 AACGCAGCGT TATTACAGCT CGTTCGACCC GAAAGACAAG GACGAAGACG
2651 TAACGTGTAA TGCTGATAAA ACGTTGTGCA ACGGCAAATA CGGCGGCACA
2701 AGCAAAAGCG TATTGACCAA TTTCGCACGC GGACGCACCT TCTTGATGAC
2751 GATGAGCTAC AAGTTTTAA

This corresponds to the amino acid sequence <SEQ ID 884; ORF133ng-1>:

1 MRSSFRLKPI CFYLMGVMLY HHSYAEDAGR AGSEAQIQVL EDVHVKAKRV
51 PKDKKVFTDA RAVSTRQDVF KSGENLDNIV RSIPGAFTQQ DKSSGIVSLN
101 IRGDSGFGRV NTMVDGITQT FYSTSTDAGR AGGSSQFGAS VDSNFIAGLD
151 VVKGSFSGSA GINSLAGSAN LRTLGVDDVV QGNNTYGLLL KGLTGTNSTK
201 GNAMAAIGAR KWLESGASVG VLYGHSRRGV AQNYRVGGGG QHIGNFGEEY
251 LERRKQQYFV QEGGLKFNAG SGKWERDLQR QYWKTKWYKK YEDPQELQKY
301 IEEHDKSWRE NLAPQYDITP IDPSGLKQQS AGNLFKLEYD GVFNKYTAQF
351 RDLNTRIGSR KIINRNYQFN YGLSLNPYTN LNLTAAYNSG RQKYPKGAKF
401 TGWGLLKDFE TYNNAKILDL NNTATFRLPR ETELQTTLGF NYFHNEYGKN
451 RFPEELGLFF DGPDQDNGLY SYLGRFKGDK GLLPQKSTIV QPAGSQYFNT
501 FYFDAALKKD IYRLNYSTNA INYRFGGEYT GYYGSENEFK RAFGENSPAY
551 KEHCDPSCGL YEPVLKKYGK KRANNHSVSI SADFGDYFMP FAGYSRTHRM
601 PNIQEMYFSQ IGDSGVHTAL KPERANTWQF GFNTYKKGLL KQDDILGLKL
651 VGYRSRIDNY IHNVYGKWWD LNGDIPSWVG STGLAYTIRH RNFKDKVHKH
701 GFELELNYDY GRFFTNLSYA YQKSTQPTNF SDASESPNNA SKEDQLKQGY
751 GLSRVSALPR DYGRLEVGTR WLGNKLTLGG AMRYFGKSIR ATAEERYIDG
801 TNGGNTSNVR QLGKRSIKQT ETLARQPLIF DFYAAYEPKK NLIFRAEVKN
851 LFDRRYIDPL DAGNDAATQR YYSSFDPKDK DEDVTCNADK TLCNGKYGGT
901 SKSVLTNFAR GRTFLMTMSY KF*

ORF133ng-1 and ORF133-1 show 96.2% identity in 889 aa overlap:

In addition, ORF133ng-1 is homologous to a TonB-dependent receptor in H. influenzae:

sp|P45114|YC17_HAEIN PROBABLE TONB-DEPENDENT RECEPTOR HI1217 PRECURSOR
>gi|1075372|pir||G64110 transferrin binding protein 1 precursor (tbp1)
homolog - Haemophilus influenzae (strain Rd KW20) >gi|1574147 (U32801)
transferrin binding protein 1 precursor (tbp1) [Haemophilus influenzae]
Length = 913
Score = 930 bits (2377), Expect = 0.0
Identities = 476/921 (51%), Positives = 619/921 (66%), Gaps = 72/921 (7%)
Query:	38	QVLEDVHVKAKRVPKDKKVFTDARAVSTRQDVFKSGENLDNIVRSIPGAFTQQDKSSGIV	97
		+ L  + V  K +  DKK FT+A+A STR++VFK  + +D ++RSIPGAFTQQDK SG+V
Sbjct:	29	ETLGQIDVVEKVISNDKKPFTEAKAKSTRENVFKETQTIDQVIRSIPGAFTQQDKGSGVV	88
Query:	98	SLNIRGDSGFGRVNTMVDGITQTFYSTSTDAGRAGGSSQFGASVDSNFIAGLDVVKGSFS	157
		S+NIRG++G GRVNTMVDG+TQTFYST+ D+G++GGSSQFGA++D NFIAG+DV K +FS
Sbjct:	89	SVNIRGENGLGRVNTMVDGVTQTFYSTALDSGQSGGSSQFGAAIDPNFIAGVDVNKSNFS	148
Query:	158	GSAGINSLAGSANLRTLGVDDVVQXXXXXXXXXXXXXXXXXXXXXAMAAIGARKWLESGA	217
		G++GIN+LAGSAN RTLGV+DV+                       M     RKWL++G
Sbjct:	149	GASGINALAGSANFRTLGVNDVITDDKPFGIILKGMTGSNATKSNFMTMAAGRKWLDNGG	208
Query:	218	SVGVLYGHSRRGVAQNYRVGGGGQHIGNFGEEYLERRKQQYFVQEGGLKFNAGSGKWERD	277
		VGV+YG+S+R V+Q+YR+ GGG+ + + G++ L + K+ YF +  G   N   G+W  D
Sbjct:	209	YVGVVYGYSQREVSQDYRI-GGGERLASLGQDILAKEKEAYF-RNAGYILNP-EGQWTPD	265
Query:	278	LQRQYWK-----------TKWY--------------------KKYEDPQELQK---YIEE	303
		L +++W              +Y                    KK +D ++LQK    IEE
Sbjct:	266	LSKKHWSCNKPDYQKNGDCSYYRIGSAAKTRREILQELLTNGKKPKDIEKLQKGNDGIEE	325
Query:	304	HDKSWRENLAPQYDITPIDPSGLKQQSAGNLFKLEYDGVFNKYTAQFRDLNTRIGSRKII	363
		DKS+  N   QY + PI+P  L+ +S  +L K EY        AQ R L+ +IGSRKI
Sbjct:	326	TDKSFERN-KDQYSVAPIEPGSLQSRSRSHLLKFEYGDDHQNLGAQLRTLDNKIGSRKIE	384
Query:	364	NRNYQFNYGLSLNPYTNLNLTAAYNSGRQKYPKGAKFTGWGLLKDFETYNNAKILDLNNT	423
		NRNYQ NY  + N Y +LNL AA+N G+  YPKG  F GW +     T N A I+D+NN+
Sbjct:	385	NRNYQVNYNFNNNSYLDLNLMAAHNIGKTIYPKGGFFAGWQVADKLITKNVANIVDINNS	444
Query:	424	ATFRLPRETELQTTLGFNYFHNEYGKNRFPEELGLFFDGPDQDNGLYSY--LGRFKGDKG	481
		TF LP+E +L+TTLGFNYF NEY KNRFPEEL LF++    D GLYS+   GR+ G K
Sbjct:	445	HTFLLPKEIDLKTTLGFNYFTNEYSKNRFPEELSLFYNDASHDQGLYSHSKRGRYSGTKS	504
Query:	482	LLPQKSTIVQPAGSQYFNTFYFDAALKKDIYRLNYSTNAINYRFGGEYTGYYGSENEFKR	541
		LLPQ+S I+QP+G Q F T YFD AL K IY LNYS N  +Y F GEY GY
Sbjct:	505	LLPQRSVILQPSGKQKFKTVYFDTALSKGIYHLNYSVNFTHYAFNGEYVGY---------	555
Query:	542	AFGENSPAYKEHCDPSCGLYEPVLKKYGKKRANNHSVSISADFGDYFMPFAGYSRTHRMP	601
		EN+   +        + EP+L K G K+A NHS ++SA+  DYFMPF  YSRTHRMP
Sbjct:	556	---ENTAGQQ--------INEPILHKSGHKKAFNHSATLSAELSDYFMPFFTYSRTHRMP	604
Query:	602	NIQEMYFSQIGDSGVHTALKPERANTWQFGFNTYKKGLLKQDDILGLKLVGYRSRIDNYI	661
		NIQEM+FSQ+ ++GV+TALKPE+++T+Q GFNTYKKGL  QDD+LG+KLVGYRS I NYI
Sbjct:	605	NIQEMFFSQVSNAGVNTALKPEQSDTYQLGFNTYKKGLFTQDDVLGVKLVGYRSFIKNYI	664
Query:	662	HNVYGKWWDLNGDIPSWVGSTGLAYTIRHRNFKDKVHKHGFELELNYDYGRFFTNLSYAY	721
		HNVYG WW     +P+W  S G  YTI H+N+K  V K G ELE+NYD GRFF N+SYAY
Sbjct:	665	HNVYGVWW--RDGMPTWAESNGFKYTIAHQNYKPIVKKSGVELEINYDMGRFFANVSYAY	722
Query:	722	QKSTQPTNFSDASESPNNASKEDQLKQGYGLSRVSALPRDYGRLEVGTRWLGNKLTLGGA	781
		Q++ QPTN++DAS  PNNAS+ED LKQGYGLSRVS LP+DYGRLE+GTRW   KLTLG A
Sbjct:	723	QRTNQPTNYADASPRPNNASQEDILKQGYGLSRVSMLPKDYGRLELGTRWFDQKLTLGLA	782
Query:	782	MRYFGKSIRATAEERYIDGTNGGNTSNVRQLGKRSIKQTETLARQPLIFDFYAAYEPKKN	841
		RY+GKS RAT EE YI+G+     + +R+    ++K+TE + +QP+I D + +YEP K+
Sbjct:	783	ARYYGKSKRATIEEEYINGSR-FKKNTLRRENYYAVKKTEDIKKQPIILDLHVSYEPIKD	841
Query:	842	LIFRAEVKNLFDRRYIDPLDAGNDAATQRYYSSFDPKDKDEDVTCNADKTLCNGKYGGTS	901
		LI +AEV+NL D+RY+DPLDAGNDAA+QRYYSS      +  + C  D + C    GG+
Sbjct:	842	LIIKAEVQNLLDKRYVDPLDAGNDAASQRYYSSL-----NNSIECAQDSSAC----GGSD	892
Query:	902	KSVLTNFARGRTFLMTMSYKF	922
		K+VL NFARGRT++++++YKF
Sbjct:	893	KTVLYNFARGRTYILSLNYKF	913

The underlined motif in the gonococcal protein (also present in the meningococcal protein) is predicted to be an ATP/GTP-binding site motif A (P-loop), and the analysis suggests that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

Example 104

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 885>

1   ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG
    TTATGGCGGT
51  TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT
    GAAATCCTGT
101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG
    GGAAATGCTG
151 GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC
    TGATTCCCCT
201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT
    GCCGCCGGCA
251 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA
    AAAGCTGCTG
301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA
    CCGTCGCGCT
351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA
    AACATCAAAG
401 CCGCCGCCAT CAACGGCAAA ATCAGCACCG GCAATACCGG
    CCTTTGGCTG
451 AAAGAAAAAA ACAGCGTGAT CAATGTGCGC GAAATGTTGC
    CCGACCAT..

This corresponds to the amino acid sequence <SEQ ID 886; ORF112>:

1   MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG
    KGSYGIWEML
51  GYTALKMPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK
    ASGMSTKKLL
101 LILSQFGFIF AIATVALGEW VAPTLSQKAE NIKAAAINGK
    ISTGNTGLWL
151 KEKNSVINVR EMLPDH...

Further work revealed further partial nucleotide sequence <SEQ ID 887>:

1   ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG
    TTATGGCGGT
51  TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT
    GAAATCCTGT
101 ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG
    GGAAATGCTG
151 gGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC
    TGATTCCCCT
201 CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT
    GCCGCCGGCA
251 GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA
    AAAGCTGCTG
301 TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA
    CCGTCGCGCT
351 CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA
    AACATCAAAG
401 CCGCCGCCAT CAACGGCAAA ATCAGCACCG GCAATACCGG
    CCTTTGGCTG
451 AAAGAAAAAA ACAGCrTkAT CAATGTGCGC GAAATGTTGC
    CCGACCATAC
501 GCTTTTGGGC ATCAAAATTT GGGCGCGCAA CGATAAAAAC
    GAATTGGCAG
551 AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG
    CAGTTGGCAG
601 TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG
    TCGAGGTCTC
651 TATTGCGGCT GAAGAAAACT GGCCGATTTC CGTCAAACGC
    AACCTGATGG
701 ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA
    ACTGACCACC
751 TACATCCGCC ACCTCCAAAA CAACAGCCAA AACACCCGAA
    TCTACGCCAT
801 CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG
    GTGATGGCGC
851 TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG
    CAATATGGGC
901 TTAAAACTCT TCGGCGGCAT CTGTsTCGGA TTGCTGTTCC
    ACCTTGCCGG
951 ACGGCTCTTT GGGTTTACCA GCCAACTCGG...

This corresponds to the amino acid sequence <SEQ ID 888; ORF112-1>:

1   MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG
    KGSYGIWEML
51  GYTALKMPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK
    ASGMSTKKLL
101 LILSQFGFIF AIATVALGEW VAPTLSQKAE NIKAAAINGK
    ISTGNTGLWL
151 KEKNSXINVR EMLPDHTLLG IKIWARNDKN ELAEAVEADS
    AVLNSDGSWQ
201 LKNIRRSTLG EDKVEVSIAA EENWPISVKR NLMDVLLVKP
    DQMSVGELTT
251 YIRHLQNNSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT
    PQTTRHGNMG
301 LKLFGGICXG LLFHLAGRLF GFTSQL...

Computer analysis of this amino acid sequence predicts two transmembrane domains and gave the following results:

Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF112 shows 96.4% identity over a 166aa overlap with an ORF (ORF112a) from strain A of N. meningitidis:

The ORF112a nucleotide sequence <SEQ ID 889> is:

1    ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG
     TTATGGCGGT
51   TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT
     GAAATCCTGT
101  ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG
     GGAAATGNTG
151  GGNTACACCG CCCTCAAAAT GNCCGCCCGC GCCTACGAAC
     TGATGCCCCT
201  CGCCGTCCTT ATCGGCGGAC TGGTCTCTNT CAGCCAGCTT
     GCCGCCGGCA
251  GCGAACTGAN CGTCATCAAA GCCAGCGGCA TGAGCACCAA
     AAAGCTGCTG
301  TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA
     CCGTCGCGCT
351  CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA
     AACATCAAAG
401  CCGCGGCCAT CAACGGCAAA ATCAGTACCG GCAATACCGG
     CCTTTGGCTG
451  AAAGAAAAAA ACAGCATTAT CAATGTGCGC GAAATGTTGC
     CCGACCATAC
501  CCTGCTGGGC ATTAAAATCT GGGCCCGCAA CGATAAAAAC
     GAACTGGCAG
551  AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG
     CAGTTGGCAG
601  TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG
     TCGAGGTCTC
651  TATTGCGGCT GAAGAAAANT GGCCGATTTC CGTCAAACGC
     AACCTGATGG
701  ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA
     ACTGACCACC
751  TACATCCGCC ACCTCCAAAN NNACAGCCAA AACACCCGAA
     TCTACGCCAT
801  CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG
     GTGATGGCGC
851  TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG
     CAATATGGGC
901  TTAAAANTCT TCGGCGGCAT CTGTCTCGGA TTGCTGTTCC
     ACCTTGCCGG
951  NCGGCTCTTC NGGTTTACCA GCCAACTCTA CGGCATCCCG
     CCCTTCCTCG
1001 NCGGCGCACT ACCTACCATA GCCTTCGCCT TGCTCGCCGT
     TTGGCTGATA
1051 CGCAAACAGG AAAAACGCTA A

This encodes a protein having the amino acid sequence <SEQ ID 890>:

1   MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG
    KGSYGIWEMX
51  GYTALKMXAR AYELMPLAVL IGGLVSXSQL AAGSELXVIK
    ASGMSTKKLL
101 LILSQFGFIF AIATVALGEW VAPTLSQKAE NIKAAAINGK
    ISTGNTGLWL
151 KEKNSIINVR EMLPDHTLLG IKIWARNDKN ELAEAVEADS
    AVLNSDGSWQ
201 LKNIRRSTLG EDKVEVSIAA EEXWPISVKR NLMDVLLVKP
    DQMSVGELTT
251 YIRHLQXXSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT
    PQTTRHGNMG
301 LKXFGGICLG LLFHLAGRLF XFTSQLYGIP PFLXGALPTI
    AFALLAVWLI
351 RKQEKR*

ORF112a and ORF112-1 show 96.3% identity in 326 aa overlap:

Homology with a Predicted ORF from N. gonorrhoeae

ORF112 shows 95.8% identity over 166aa overlap with a predicted ORF (ORF112ng) from N. gonorrhoeae:

The complete length ORF112ng nucleotide sequence <SEQ ID 891> is:

1    ATGAACCTGA TTTCACGTTA CATCATCCGC CAAATGGCGG
     TTATGGCGGT
51   TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT
     GAAATCCTGT
101  ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG
     GGAAATGCTG
151  GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC
     TCATGCCCCT
201  CGCCGTCCTC ATCGGCGGAC TGGCCTCTCT CAGCCAGCTT
     GCCGCCGGCA
251  GCGAACTGGC CGTCATCAAA GCCAGCGGCA TGAGCACCAA
     AAAGCTGCTG
301  TTGATTCTGT CTCAGTTCGG TTTTATTTTT GCTATTGCCG
     CCGTCGCGCT
351  CGGCGAATGG GTTGCGCCCA CGCTGAGCCA AAAAGCCGAA
     AACATCAAag
401  cCGCCGCCAt taacggCAAA ATCAGCAccg gcAATACCGG
     CCTTTggcTG
451  AAAGAAAAAa ccAGCATTAT CAATGTGcGc GGAATGTTGC
     CCGACCATAC
501  GCTTTTGGGC ATCAAAATTT GGGCGCGCAA CGATAAAAAC
     GAATTGGCAG
551  AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG
     CAGCTGGCAG
601  TTGAAAAACA TCCGCCGCAG CATCATGGGT ACAGACAAAA
     TCGAAACATC
651  cgCCGCCGCC GAAGAAACTT gGCCGATTGC CGTCAGACGC
     AACCTGATGG
701  ACGTATTGCT CGTCAAGCCC GACCAAATGT CCGTCGGCGA
     GCTGACCACC
751  TACATCCGCC ACCTCCAAAA CAACAGCCAA AACACCCAAA
     TCTACGCCAT
801  CGCATGGTGG CGTAAACTCG TTTACCCCGT CGCCGCATGG
     GTCATGGCGC
851  TCGTTGCCTT CGCCTTTACG CCGCAAACCA CGCGCCACGG
     CAATATGGGC
901  TTAAAACTCT TCGGCGGCAT CTGTCTCGGA TTGCTGTTCC
     ACCTTGCCGG
951  CAGGCTCTTC GGGTTTACCA GCCAACTCTA CGGCACCCCA
     CCCTTCCTCG
1001 CCGGCGCACT GCCTACCATA GCCTTCGCCT TGCTCGCTGT
     TTGGCTGATA
1051 CGCAAACAGG AAAAACGTTG A

This encodes a protein having amino acid sequence <SEQ ID 892>:

1   MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG
    KGSYGIWEML
51  GYTALKMPAR AYELMPLAVL IGGLASLSQL AAGSELAVIK
    ASGMSTKKLL
101 LILSQFGFIF AIAAVALGEW VAPTLSQKAE NIKAAAINGK
    ISTGNTGLWL
151 KEKTSIINVR GMLPDHTLLG IKIWARNDKN ELAEAVEADS
    AVLNSDGSWQ
201 LKNIRRSIMG TDKIETSAAA EETWPIAVRR NLMDVLLVKP
    DQMSVGELTT
251 YIRHLQNNSQ NTQIYAIAWW RKLVYPVAAW VMALVAFAFT
    PQTTRHGNMG
301 LKLFGGICLG LLFHLAGRLF GFTSQLYGTP PFLAGALPTI
    AFALLAVWLI

• • 351 RKQEKR*

ORF112ng and ORF112-1 show 94.2% identity in 326 aa overlap:

This analysis suggests that these proteins from N. meningitidis and N. gonorrhoeae, and their epitopes, could be useful antigens for vaccines or diagnostics, or for raising antibodies.

It will be appreciated that the invention has been described by means of example only, and that modifications may be made whilst remaining within the spirit and scope of the invention.

TABLE I
PCR primers
ORF	Primer	Sequence	Restriction sites
ORF 1	Forward	CGCGGATCCGCTAGC-GGACACACTTATTTCGG	BamHI-NheI
	Reverse	CCCGCTCGAG-CCAGCGGTAGCCTAATT	XhoI
ORF 2	Forward	GCGGATCCCATATG-TTTGATTTCGGTTTGGG	BamHI-NdeI
	Reverse	CCCGCTCGAG-GACGGCATAACGGCG	XhoI
ORF 2-1	Forward	GCGGATCCCATATG-TTTGATTTCGGTTTGGG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TGATTTACGGACGCGCA	XhoI
ORF 4	Forward	GCGGATCCCATATG-TGCGGAGGTCAAAAAGAC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTGGCTGCGCCTTC	XhoI
ORF 5	Forward	GGAATTCCATATGGCCATGG-TGGAAGGCGCACAACC	NdeI-NcoI
	Forward	CGGGATCC-ATGGAAGGCGCACAAC	BamHI
	Reverse	CCCGCTCGAG-GACTGTGCAAAAACGG	XhoI
ORF 6	Forward	CGCGGATCCCATATG-ACCCGTCAATCTCTGCA	BamHI-NdeI
	Reverse	CCCGCTCGAG-TGCGCCGAACACTTTC	XhoI
ORF 7	Forward	CGCGGATCCGCTAGC-GCGCTGCTTTTTGTTCC	BamHI-NheI
	Reverse	CCCGCTCGAG-TTTCAAAATATATTTGCGGA	XhoI
ORF 8	Forward	GCGGATCCCATATG-GCTCAACTGCTTCGTAC	BamHI-NdeI
	Reverse	CCCGCTCGAG-AGCAGGCTTTGGCGC	XhoI
ORF 9	Forward	CGCGGATCCCATATG-CCGAAGGAAGTCGGAAA	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTCCGAGGTTTTCGGG	XhoI
ORF 10	Forward	GCGGATCCCATATG-GACACAAAAGAAATCCTC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TAATGGGAAACCTTGTTTT	XhoI
ORF 11	Forward	GCGGATCCCATATG-GCGGTCAACCTCTACG	BamHI-NdeI
	Reverse	CCCGCTCGAG-GGAAACGACTTCGCC	XhoI
ORF 13	Forward	CGCGGATCCCATATG-GCTCTGCTTTCCGCGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-AGGGTGTGTGATAATAAG	XhoI
ORF 15	Forward	GGAATTCCATATGGCCATGG-GCGGGACACTGACAG	NdeI-NcoI
	Forward	CGGGATCC-TGCGGGACACTGACAGG	BamHI
	Reverse	CCCGCTCGAG-AGGTTGGCCTTGTCTATG	XhoI
ORF 17	Forward	GGAATTCCATATGGCCATGG-TTGCCGGCCTGTTCG	NdeI-NcoI
	Forward	CGGGATCC-ATTGCCGGCCTGTTCG	BamHI
	Reverse	CCCGCTCGAG-AAGCAGGTTGTACAGC	XhoI
ORF 18	Forward	GCGGATCCCATATG-ATTTTGCTGCATTTGGAT	BamHI-NdeI
	Reverse	CCCGCTCGAG-TCTTCCAATTTCTGAAAGC	XhoI
ORF 19	Forward	GGAATTCCATATGGCCATGG -TCGCCAGTGTTTTTACC	NdeI-NcoI
	Forward	CGGGATCC-TTCGCCAGTGTTTTTACCG	BamHI
	Reverse	CCCGCTCGAG-GGTGTTTTTGAAGCTGCC	XhoI
ORF 20	Forward	GGAATTCCATATGGCCATGG -TCGGCGCGGGTATG	NdeI-NcoI
	Forward	CGGGATCC-TTCGGCGCGGGTATG	BamHI
	Reverse	CCCGCTCGAG-CGGCGAGCGAGAGCA	XhoI
ORF 22	Forward	GGAATTCCATATGGCCATGG-TGATTAAAATCAAAAAAGGTCT	NdeI-NcoI
	Forward	CGGGATCC-ATGATTAAAATCAAAAAAGGTCTAAACC	BamHI
	Reverse	CCCGCTCGAG-ATTATGATAGCGGCCC	XhoI
ORF 23	Forward	CGCGGATCCCATATG-GATGTTTCTGTTTCAGAC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTAAACCGATAGGTAAACG	XhoI
ORF 24	Forward	GGAATTCCATATGGCCATGG- TGATGCCGGAAATGGTG	NdeI-NcoI
	Forward	CGGGATCC-ATGATGCCGGAAATGGTG	BamHI
	Reverse	CCCGCTCGAG-TGTCAGCGTGGCGCA	XhoI
ORF 25	Forward	GCGGATCCCATATG-TATCGCAAACTGATTGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-ATCGATGGAATAGCCG	XhoI
ORF 26	Forward	GCGGATCCCATATG -CAGCTGATCGACTATTC	BamHI-NdeI
	Reverse	CCCGCTCGAG-GACATCGGCGCGTTTT	XhoI
ORF 27	Forward	GGAATTCCATATGGCCATGG-AGACCTATTCTGTTTA	NdeI-NcoI
	Forward	CGGGATCC- CAGACCTATTCTGTTTATTTTAATC	BamHI
	Reverse	CCCGCTCGAG-GGGTTCGATTAAATAACCAT	XhoI
ORF 28	Forward	GGAATTCCATATGGCCATGG-ACGGCTGTACGTTGATGT	NdeI-NcoI
	Forward	CGGGATCC-AACGGCTGTACGTTGATG	BamHI
	Reverse	CCCGCTCGAG-TTTGTCAGAGGAATTCGCG	XhoI
ORF 29	Forward	GCGGATCCCATATG -AACGGTTTGGATGCCCG	BamHI-NdeI
	Forward	CGCGGATCCGCTAGC-AACGGTTTGGATGCCCG	BamHI-NheI
	Reverse	CCCGCTCGAG-TTTGTCTAAGTTCCTGATATG	XhoI
ORF 32	Forward	CGCGGATCCCATATG-AATACTCCTCCTTTTG	BamHI-NdeI
	Reverse	CCCGCTCGAG-GCGTATTTTTTGATGCTTTG	XhoI
ORF 33	Forward	GCGGATCCCATATG -ATTGATAGGGATCGTATG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTGATCTTTCAAACGGCC	XhoI
ORF 35	Forward	GCGGATCCCATATG-TTCAGAGCTCAGCTT	BamHI-NdeI
	Forward	CGCGGATCCGCTAGC-TTCAGAGCTCAGCTT	BamHI-NheI
	Reverse	CCCGCTCGAG-AAACAGCCATTTGAGCGA	XhoI
ORF 37	Forward	GCGGATCCCATATG-GATGACGTATCGGATTTT	BamHI-NdeI
	Reverse	CCCGCTCGAG-ATAGCCCGCTTTCAGG	XhoI
ORF 58	Forward	CGCGGATCCGCTAGC-TCCGAACGCGAGTGGAT	BamHI-NheI
	Reverse	CCCGCTCGAG-AGCATTGTCCAAGGGGAC	XhoI
ORF 65	Forward	GGAATTCCATATGGCCATGG -TGCTGTATCTGAATCAAG	NdeI-NcoI
	Forward	CGGGATCC-TTGCTGTATCTGAATCAAGG	BamHI
	Reverse	CCCGCTCGAG-CCGCATCGGCAGACA	XhoI
ORF 66	Forward	GCGGATCCCATATG-TACGCATTTACCGCCG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TGGATTTTGCAGAGATGG	XhoI
ORF 72	Forward	CGCGGATCCCATATG- AATGCAGTAAAAATATCTGA	BamHI-NdeI
	Reverse	CCCGCTCGAG-GCCTGAGACCTTTGCAA	XhoI
ORF 73	Forward	GCGGATCCCATATG-AGATTTTTCGGTATCGG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTCATCTTTTTCATGTTCG	XhoI
ORF 75	Forward	GCGGATCCCATATG- TCTGTCTTTCAAACGGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTGTTTTTGCAAGACAG	XhoI
ORF 76	Forward	GATCAGCTAGCCATATG-AAACAGAAAAAAACCGC	NheI-NdeI
	Reverse	CGGGATCC-TTACGGTTTGACACCGTT	BamHI
ORF 79	Forward	CGCGGATCCCATATG-GTTTCCGCCGCCG	BamHI-NdeI
	Reverse	CCCGCTCGAG-GTGCTGATGCGCTTCG	XhoI
ORF 83	Forward	GCGGATCCCATATG-AAAACCCTGCTGCTGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-GCCGCCTTTGCGGC	XhoI
ORF 84	Forward	GCGGATCCCATATG-GCAGAGATCTGTTTG	BamHI-NdeI
	Reverse	CCCGCTCGAG-GTTTGCCGATCCGACCA	XhoI
ORF 85	Forward	CGCGGATCCCATATG- GCGGTTTGGGGCGGA	BamHI-NdeI
	Reverse	CCCGCTCGAG-TCGGCGCGGCGGGC	XhoI
ORF 89	Forward	GGAATTCCATATGGCCATGG-CCATACCTTCTTATCA	NdeI-NcoI
	Forward	CGGGATCC-GCCATACCTTCTTATCAGAG	BamHI
	Reverse	CCCGCTCGAG-TTTTTTGCGATTAGAAAAAGC	XhoI
ORF 97	Forward	GCGGATCCCATATG-CATCCTGCCAGCGAAC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTCGCCTACGGTTTTTTG	XhoI
ORF 98	Forward	GCGGATCCCATATG-ACGGTAACTGCGG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTGTTGTTCGGGCAAATC	XhoI
ORF 100	Forward	GCGGATCCCATATG-TCGGGCATTTACACCG	BamHI-NdeI
	Reverse	CCCGCTCGAG-ACGGGTTTCGGCGGAA	XhoI
ORF 101	Forward	GCGGATCCCATATG-ATTTATCAAAGAAACCTC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTTCCGCCTTTCAATGT	XhoI
ORF 102	Forward	GCGGATCCCATATG-GCAGGGCTGTTTTACC	BamHI-NdeI
	Reverse	CCCGCTCGAG-AAACGGTTTGAACACGAC	XhoI
ORF 103	Forward	GCGGATCCCATATG-AACCACGACATCAC	BamHI-NdeI
	Reverse	CCCGCTCGAG-CAGCCACAGGACGGC	XhoI
ORF 104	Forward	GCGGATCCCATATG-ACGTGGGGAACGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-GCGGCGTTTGAACGGC	XhoI
ORF 105	Forward	GCGGATCCCATATG-ACCAAATTTCAAACCCCTC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TAAACGAATGCCGTCCAG	XhoI
ORF 106	Forward	GCGGATCCCATATG-AGGATAACCGACGGCG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTGTTCCCGATGATGTT	XhoI
ORF 109	Forward	GCGGATCCCATATG-GAAGATTTATATATAATACTCG	BamHI-NdeI
	Reverse	CCCGCTCGAG-ATCAGCTTCGAACCGAAG	XhoI
ORF 110	Forward	AAAGAATTC-ATGAGTAAATCCCGTAGATCTCCC	EcoRI
	Reverse	AAACTGCAG-GGAAAACCACATCCGCACTCTGCC	PstI
ORF 111	Forward	AAAGAATTC-GCACCGCAAAAGGCAAAAACCGCA	EcoRI
	Reverse	AAACTGCAG-TCTGCGCGTTTTCGGGCAGGGTGG	PstI
ORF 113	Forward	AAAGAATTC-ATGAACAAAACCCTCTATCGTGTGATTTTCAACCG	EcoRI
	Reverse	AAACTGCAG-TTACGAATGCCTGCTTGCTCGACCGTACTG	PstI
ORF 115	Forward	AAAGAATTC-TTGCTTGTGCAAACAGAAAAAGACGG	EcoRI
	Reverse	AAAAAAGTCGAC-CTATTTTTTAGGGGCTTTTGCTTGTTTGAAAAGCCTGCC	SalI
ORF 119	Forward	AAAGAATTC-TACAACATGTATCAGGAAAACCAATACCG	EcoRI
	Reverse	AAACTGCAG-TTATGAAAACAGGCGCAGGGCGGTTTTGCC	PstI
ORF 120	Forward	AAAGAATTC-GCAAGGCTACCCCAATCCGCCGTG	EcoRI
	Reverse	AAACTGCAG-CGGTTTGGCTGCCTGGCCGTTGAT	PstI
ORF 121	Forward	AAAGAATTC-GCCTTGGTCTGGCTGGTTTTCGC	EcoRI
	Reverse	AAACTGCAG-TCATCCGCCACCCCACCTCGGCCATCCATC	PstI
ORF 122	Forward	AAAAAAGTCGAC-ATGTCTTACCGCGCAAGCAGTTCTCC	SalI
	Reverse	AAACTGCAG-TCAGGAACACAAACGATGACGAATATCCGTATC	PstI
ORF 125	Forward	AAAGAATTC-GCGCTGTTTTTTGCGGCGGCGTAT	EcoRI
	Reverse	AAACTGCAG-CGCCGTTTCAAGACGAAAAAGTCG	PstI
ORF 126	Forward	AAAGAATTC-GCGGAAACGGTCGAAG	EcoRI
	Reverse	AAACTGCAG-TTAATCTTGTCTTCCGATATAC	PstI
ORF 127	Forward	AAAGAATTC-ATGACTGATAATCGGGGGTTTACG	EcoRI
	Reverse	AAAAAAGTCGAC-CTTAAGTAACTTGCAGTCCTTATC	SalI
ORF 128	Forward	AAAGAATTC-ATGCAAGCTGTCCGCTACAGGCC	EcoRI
	Reverse	AAACTGCAG-CTATTGCAATGCGCCGCCGCGGGAATGTTTGAGCAGGCG	PstI
ORF 129	Forward	AAAGAATTC-ATGGATTTTCGTTTTGACATTATTTACGAATACCG	EcoRI
	Reverse	AAACTGCAG-TTATTTTTTGATGAAATTTTGGGGCGG	PstI
ORF 130	Forward	AAAGAATTC-GCAGTACTTGCCATTCTCGGTGCG	EcoRI
	Reverse	AAACTGCAG-CTCCGGATCGTCTGTAAACGCATT	PstI
ORF 131	Forward	GCGGATCCCATATG-GAAATTCGGGCAATAAAAT	BamHI-NdeI
	Reverse	CCCGCTCGAG-CCAGCGGACGCGTTC	XhoI
ORF 132	Forward	GCGGATCCCATATG-AAAGAAGCGGGGTTTG	BamHI-NdeI
	Reverse	CCCGCTCGAG-CCAATCTGCCAGCCGT	XhoI
ORF 133	Forward	CGCGGATCCCATATG-GAAGATGCAGGGCGCG	BamHI-NdeI
	Reverse	CCCGCTCGAG-AAACTTGTAGCTCATCGT	XhoI
ORF 134	Forward	GCGGATCCCATATG-TCTGTGCAAGCAGTATTG	BamHI-NdeI
	Reverse	CCCGCTCGAG-ATCCTGTGCCAATGCG	XhoI
ORF 135	Forward	GCGGATCCCATATG-CCGTCTGAAAAAGCTTT	BamHI-NdeI
	Reverse	CCCGCTCGAG-AAATACCGCTGAGGATG	XhoI
ORF 136	Forward	CGCGGATCCGCTAGC-ATGAAGCGGCGTATAGCC	BamHI-NheI
	Reverse	CCCGCTCGAG-TTCCGAATATTTGGAACTTTT	XhoI
ORF 137	Forward	CGCGGATCCCATATG-GGCACGGCGGGAAATA	BamHI-NdeI
	Reverse	CCCGCTCGAG-ATAACGGTATGCCGCC	XhoI
ORF 138	Forward	GCGGATCCCATATG-TTTCGTTTACAATTCAGGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-CGGCGTTTTATAGCGG	XhoI
ORF 139	Forward	GCGGATCCCATATG-GCTTTTTTGGCGGTAATG	BamHI-NdeI
	Reverse	CCCGCTCGAG-TAACGTTTCCGTGCGTTT	XhoI
ORF 140	Forward	GCGGATCCCATATG-TTGCCCACAGGCAGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-GACGATGGCAAACAGC	XhoI
ORF 141	Forward	GCGGATCCCATATG-CCGTCTGAAGCAGTCT	BamHI-NdeI
	Reverse	CCCGCTCGAG-ATCTGTTGTTTTTAAAATATT	XhoI
ORF 142	Forward	GCGGATCCCATATG-GATAATTCTGGTAGTGAAG	BamHI-NdeI
	Reverse	CCCGCTCGAG-AAACGTATAGCCTACCT	XhoI
ORF 143	Forward	GCGGATCCCATATG-GATACCGCTTTGAACCT	BamHI-NdeI
	Reverse	CCCGCTCGAG-AATGGCTTCCGCAATATG	XhoI
ORF 144	Forward	GCGGATCCCATATG-ACCTTTTTACAACGTTTGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-AGATTGTTGTTGTTTTTTCG	XhoI
ORF 147	Forward	GCGGATCCCATATG-TCTGTCTTTCAAACGGC	BamHI-NdeI
	Reverse	CCCGCTCGAG-TTTGTTTTTGCAAGACAG	XhoI
NB:
restriction sites are underlined
for ORFs 110-130, where the ORF itself carries an EcoRI site (eg. ORF 122), a SalI site was used in the forward primer instead. Similarly, where the ORF carries a PstI site (eg. ORFs 115 and 127), a SalI site was used in the reverse primer.

TABLE II
Summary of cloning, expression and purification
		PCR/	His-fusion	GST-fusion
	ORF	cloning	expression	expression	Purification
	orf 1	+	+	+	His-fusion
	orf 2	+	+	+	GST-fusion
	orf 2.1	+	n.d.	+	GST-fusion
	orf 4	+	+	+	His-fusion
	orf 5	+	n.d.	+	GST-fusion
	orf 6	+	+	+	GST-fusion
	orf 7	+	+	+	GST-fusion
	orf 8	+	n.d.	n.d.
	orf 9	+	+	+	GST-fusion
	orf 10	+	n.d.	n.d.
	orf 11	+	n.d.	n.d.
	orf 13	+	n.d.	+	GST-fusion
	orf 15	+	+	+	GST-fusion
	orf 17	+	n.d.	n.d.
	orf 18	+	n.d.	n.d.
	orf 19	+	n.d.	n.d.
	orf 20	+	n.d.	n.d.
	orf 22	+	+	+	GST-fusion
	orf 23	+	+	+	His-fusion
	orf 24	+	n.d.	n.d.
	orf 25	+	+	+	His-fusion
	orf 26	+	n.d.	n.d.
	orf 27	+	+	+	GST-fusion
	orf 28	+	+	+	GST-fusion
	orf 29	+	n.d.	n.d.
	orf 32	+	+	+	His-fusion
	orf 33	+	n.d.	n.d.
	orf 35	+	n.d.	n.d.
	orf 37	+	+	+	GST-fusion
	orf 58	+	n.d.	n.d.
	orf 65	+	n.d.	n.d.
	orf 66	+	n.d.	n.d.
	orf 72	+	+	n.d.	His-fusion
	orf 73	+	n.d.	+	n.d.
	orf 75	+	n.d.	n.d.
	orf 76	+	+	n.d.	His-fusion
	orf 79	+	+	n.d.	His-fusion
	orf 83	+	n.d.	+	n.d.
	orf 84	+	n.d.	n.d.
	orf 85	+	n.d.	+	GST-fusion
	orf 89	+	n.d.	+	GST-fusion
	orf 97	+	+	+	GST-fusion
	orf 98	+	n.d.	n.d.
	orf 100	+	n.d.	n.d.
	orf 101	+	n.d.	n.d.
	orf 102	+	n.d.	n.d.
	orf 103	+	n.d.	n.d.
	orf 104	+	n.d.	n.d.
	orf 105	+	n.d.	n.d.
	orf 106	+	+	+	His-fusion
	orf 109	+	n.d.	n.d.
	orf 110	+	n.d.	n.d.
	orf 111	+	+	n.d.	His-fusion
	orf 113	+	+	n.d.	His-fusion
	orf 115	n.d.	n.d.	n.d.
	orf 119	+	+	n.d.	His-fusion
	orf 120	+	+	n.d.	His-fusion
	orf 121	+	n.d.	n.d.
	orf 122	+	+	n.d.	His-fusion
	orf 125	+	+	n.d.	His-fusion
	orf 126	+	+	n.d.	His-fusion
	orf 127	+	+	n.d.	His-fusion
	orf 128	+	n.d.	n.d.
	orf 129	+	+	n.d.	His-fusion
	orf 130	+	n.d.	n.d.
	orf 131	+	+	+	n.d.
	orf 132	+	+	+	His-fusion
	orf 133	+	n.d.	+	GST-fusion
	orf 134	+	n.d.	n.d.
	orf 135	+	n.d.	n.d.
	orf 136	+	n.d.	n.d.
	orf 137	+	n.d.	+	GST-fusion
	orf 138	+	n.d.	+	GST-fusion
	orf 139	+	n.d.	n.d.
	orf 140	+	n.d.	n.d.
	orf 141	+	n.d.	n.d.
	orf 142	+	n.d.	n.d.
	orf 143	+	n.d.	n.d.
	orf 144	+	n.d.	+	n.d.
	orf 147	+	n.d.	n.d.

Claims

1: An isolated protein comprising:

(a) the amino acid sequence of SEQ ID NO: 654; or

(b) an amino acid sequence having 80% or greater sequence identity to the amino acid sequence of SEQ ID NO: 654; or

(b) a fragment of SEQ ID NO: 654 of at least 10 contiguous amino acids in length.

2: The isolated protein of claim 1 comprising (b).

3: The isolated protein of claim 2, wherein the amino acid sequence has 90% or greater sequence identity to the amino acid sequence of SEQ ID NO: 654.

4: The isolated protein of claim 2, wherein the amino acid sequence has 95% or greater sequence identity to the amino acid sequence of SEQ ID NO: 654.

5: The isolated protein of claim 1 comprising (c).

6: A composition comprising the protein of any one of claims 1-5 and an adjuvant.

7: The composition of claim 6 further comprising a pharmaceutically acceptable carrier.