LAWSONIA INTRACELLULARIS PROTEINS, AND RELATED METHODS AND MATERIALS
Isolated polynucleotide molecules contain a nucleotide sequence that encodes a L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein, a substantial portion of the sequences, or a homologous sequence. Related polypeptides, immunogenic compositions and assays are described.
The present invention relates to proteins derived from Lawsonia intracellularis and encompasses related proteins, nucleic acids, and immunogenic compositions. The immunogenic compositions are particularly useful in prevention of L. intracellularis infections in susceptible animals, such as pigs. The proteins, fragments, and nucleic acids can also be employed as diagnostic agents.
BACKGROUND OF THE INVENTIONCommercially raised pigs are sensitive to a wide spectrum of intestinal diseases or syndromes that are collectively referred to as porcine proliferative enteropathy (PPE). These diseases include intestinal adenomatosis complex (Barker I. K. et al., 1985, In “Pathology of Domestic Animals,” 3rd Edition, Vol. 2 p. 1-237, eds. K. V. F. Jubb et al. (Academic Press: Orlando)), porcine intestinal adenomatosis (PIA), necrotic enteritis (Rowland A. C. et al., 1976, Veterinary Record 97:-178-180), proliferative haemorrhagic enteropathy (Love, R. J. et al., 1977, Veterinary Record 100: 473), regional ileitis (Jonsson, L. et al., 1976, Acta Veterinaria Scandinavica 17: 223-232), haemorrhagic bowel syndrome (O'Neil, I. P. A., 1970, Veterinary Record 87: 742-747), porcine proliferative enteritis and Campylobacter spp—induced enteritis (Straw, B. E., 1990, Journal of American Veterinary Medical Association 197: 355-357).
One major type of PPE is non-haemorrhagic and is manifested by porcine intestinal adenomatosis (PIA). This form of PPE frequently causes growth retardation and mild diarrhea. Another important type of PPE is haemorrhagic. It is often fatal, and is manifested by proliferative haemorrhagic enteropathy (PHE) wherein the distal small intestine lumen becomes engorged with blood.
While PPE in pigs is commercially most important, PPE is also a problem in the raising of hamsters (Stills, H. F., 1991; Infection and Immunology 59: 3227-3236), ferrets (Fox et al., 1989, Veterinary Pathology 26: 515-517), guinea pigs (Elwell et al., 1981, Veterinary Pathology 18: 136-139), rabbits (Schodeb et al., 1990, Veterinary Pathology 27: 73-80) and certain birds (Mason et al, 1998).
The organism that causes PPE is the Campylobacter-like bacterium “L. intracellularis” (McOrist S et al, 1995, International Journal Of Systematic Bacteriology 45: 820-825). This organism is also known as Ileal symbiont intracellularis (Stills, 1991, supra). PPE-like diseases in pigs may also be caused by other species of Campylobacter (Gebhart et al., 1983, American Journal of Veterinary Research 44: 361-367).
L. intracellularis is located in the cytoplasm of villi and intestinal crypt cells of infected animals, where it causes structural irregularities and enterocyte proliferation. Abscesses form as the villi and intestinal crypts become branched and fill with inflammatory cells.
Current control of PPE relies on the use of antibacterial compounds. There is, however, a need for alternative means of controlling L. intracellularis infection.
International Patent Application No. PCT/AU96/00767 describes L. intracellularis polypeptides and immunogenic compositions that are useful as vaccines. There is, however, a need for additional compositions that confer resistance to L. intracellularis infection.
SUMMARY OF THE INVENTIONThe present invention relates to an isolated polynucleotide molecule comprising a nucleotide sequence that is selected from the group consisting of:
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- a) a nucleotide sequence encoding L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein;
- b) a nucleotide sequence that is a substantial part of the nucleotide sequence encoding the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein; and
- c) a nucleotide sequence that is homologous to the nucleotide sequence of a) or b).
In another aspect, the invention relates to a recombinant vector comprising these polynucleotide molecules, including those encoding a carrier or fusion partner such that expression of the recombinant vector results in a fusion protein comprising the carrier or fusion partner fused to a protein or polypeptide encoded by the nucleotide sequences described above. The invention also encompasses transformed host cells comprising these recombinant vectors and polypeptides produced by such transformed host cells.
In another aspect, the present invention relates to an isolated polypeptide that is selected from the group consisting of:
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- (a) L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein;
- (b) a polypeptide having an amino acid sequence that is homologous to that of the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein;
- (c) a polypeptide consisting of a substantial portion of the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein or of the polypeptide having an amino acid sequence that is homologous to that of the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein;
- (d) a fusion protein comprising the protein or polypeptide of (a), (b) or (c) fused to another protein or polypeptide; and
- (e) an analog or derivative of the protein or polypeptide of (a), (b), (c) or (d).
The present invention further provides a polynucleotide molecule comprising a nucleotide sequence of greater than 20 nucleotides having promoter activity and found within SEQ ID NO: 2 from about nt 2691 to about nt 2890.
The present invention further relates to a method of preparing any of these polypeptides, comprising culturing host cells transformed with a recombinant expression vector and recovering the expressed polypeptide from the cell culture. The vector comprises a polynucleotide molecule comprising a nucleotide sequence encoding any of the polypeptides, the nucleotide sequence being in operative association with one or more regulatory elements. Culturing is conducted under conditions conducive to expression of the polypeptide.
In yet another aspect, the invention relates to an isolated antibody that specifically reacts with any of the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 proteins or polypeptides described above.
The invention also relates to an immunizing composition that comprises an immunologically effective amount of a protein, polypeptide, antibody, or polynucleotide of the invention in combination with a pharmaceutically acceptable carrier. The present invention encompasses a method of immunizing a PPE susceptible animal against L. intracellularis infection that comprises administering to the animal the immunizing composition.
The invention also relates to a kit for immunizing a PPE susceptible animal against a disease condition caused or exacerbated by L. intracellularis that comprises a container having therein an immunologically effective amount of one of the proteins, polypeptides, antibodies, or polynucleotides described above. The invention also relates to a kit for detecting the presence of L. intracellularis, an L. intracellularis specific amino acid or nucleotide sequence, or an anti-L. intracellularis antibody, comprising a container that has therein a protein, polypeptide, polynucleotide, or antibody of the invention.
BRIEF DESCRIPTION OF DRAWINGS
All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.
Polynucleotide MoleculesAn isolated polynucleotide molecule of the present invention can have a nucleotide sequence derived from any species or strain of Lawsonia, but is preferably from the species intracellularis. Pathogenic strains or species of Lawsonia for use in practicing the present invention can be isolated from organs, tissues or body fluids of infected animals using isolation techniques as described below.
As used herein, the terms “polynucleotide molecule,” “polynucleotide sequence,” “coding sequence,” “open-reading frame (ORF),” and the like, are intended to refer to both DNA and RNA molecules, which can either be single-stranded or double-stranded, and that can include one or more prokaryotic sequences, cDNA sequences, genomic DNA sequences including exons and introns, and chemically synthesized DNA and RNA sequences, and both sense and corresponding anti-sense strands. As used herein, the term “ORF” refers to the minimal nucleotide sequence required to encode a Lawsonia HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein, without any intervening termination codons.
Production and manipulation of the polynucleotide molecules and oligonucleotide molecules disclosed herein are within the skill in the art and can be carried out according to recombinant techniques described, among other places, in Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Ausubel et al., 1989, Current Protocols In Molecular Biology, Greene Publishing Associates & Wiley Interscience, NY; Sambrook et al., 1989, Molecular Cloninq: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis et al. (eds), 1995, PCR Strategies, Academic Press, Inc., San Diego; and Erlich (ed), 1992, PCR Technology, Oxford University Press, New York and all revisions of these references.
References herein to the nucleotide sequences shown in SEQ ID NOS: 1 AND 2, and to substantial portions thereof, are intended to also refer to the corresponding nucleotide sequences and substantial portions thereof, respectively, as present in the following plasmids contained in E. coli Top10 cells deposited by Pfizer Inc. at Central Research, Eastern Point Road, Groton, Conn., 06340 with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md., 20852:
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- pER432 containing the ponA gene and accorded ATCC accession number PTA-635, deposited on Sep. 9, 1999;
- pER434 containing the htrA gene and accorded ATCC accession number PTA-636, deposited on Sep. 9, 1999;
- pER436 containing the hypC gene and accorded ATCC accession number PTA-637, deposited on Sep. 9, 1999;
- pER438 containing the ycfW and abc1 genes and accorded ATCC accession number PTA-638, deposited on Sep. 9, 1999;
- pER440 containing the omp100 gene and accorded ATCC accession number PTA-639, deposited on Sep. 9, 1999; and
- pT068 containing the lysS and ycfW genes and accorded ATCC accession number PTA-2232, deposited on Jul. 14, 2000.
In addition, references herein to the amino acid sequences shown in SEQ ID NOS:3-9, and SEQ ID NO: 102, and to substantial portions and peptide fragments thereof, are intended to also refer to the corresponding amino acid sequences, and substantial portions and peptide fragments thereof, respectively, encoded by the corresponding protein encoding nucleotide sequences present in the plasmids listed above, unless otherwise indicated.
HtrA-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the HtrA protein from L. intracellularis. In a preferred embodiment, the HtrA protein has the amino acid sequence of SEQ ID NO: 7. In a further preferred embodiment, the isolated HtrA-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 2 from about nt 2891 to about nt 4315, which is the nucleotide sequence of the open reading frame (ORF) of the htrA gene, and the nucleotide sequence of the HtrA-encoding ORF of plasmid pER434 (ATCC accession number PTA-636).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is homologous to the nucleotide sequence of a HtrA-encoding polynucleotide molecule of the present invention. The term “homologous” when used to refer to a HtrA-related polynucleotide molecule means a polynucleotide molecule having a nucleotide sequence: (a) that encodes the same protein as one of the aforementioned HtrA-encoding polynucleotide molecules of the present invention, but that includes one or more silent changes to the nucleotide sequence according to the degeneracy of the genetic code; or (b) that hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis HtrA protein under at least moderately stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.2×SSC/0.1% SDS at 42° C. (see Ausubel et al. (eds.), 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3), and that is useful in practicing the present invention. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis HtrA protein under highly stringent conditions, i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% SDS, 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel et al, 1989, above), and is useful in practicing the present invention. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the HtrA encoding ORF of SEQ ID NO: 2, which is from about nt 2891 to about nt 4315. As noted above, reference to homologous polynucleotide molecules herein is also intended to refer to the complements of such molecules.
As used herein, a polynucleotide molecule is “useful in practicing the present invention” where the polynucleotide molecule can be used to amplify a Lawsonia-specific polynucleotide molecule using a standard amplification technique, such as the polymerase chain reaction, or as a diagnostic reagent to detect the presence of a Lawsonia-specific polynucleotide in a fluid or tissue sample from a Lawsonia-infected animal, or where the polynucleotide molecule encodes a polypeptide that is useful in practicing the invention, as described below.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a HtrA-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules that have been described from bacteria such as E. coli, S. typhimurium, C. jejuni, H. influenzae, B. melitensis, B. abortus, C. trachomatis, Y. enterocolitia, Rickettsia, B. burgdorferi, and B. subtilis. The L. intracellularis HtrA protein encoded by SEQ ID NO: 2 has 39.6% identity of amino acid sequence with the B. abortus HtrA protein. The L. intracellularis protein is 474 residues in length and the B. abortus protein is 513 residues in length. The L. intracellularis protein is 35.4% identical to that of H. influenzae.
The homologous nucleotide sequence of the molecule of the invention preferably comprises a sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 2, which is from about nt 2891 to about nt 4315, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis HtrA protein. In another embodiment the sequence is more than 70%, in another embodiment the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis HtrA protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis HtrA protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 2 which is from about nt 2891 to about nt 4315.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is homologous to the L. intracellularis HtrA protein. As used herein to refer to polypeptides that are homologous to the L. intracellularis HtrA protein, the term “homologous” refers to a polypeptide otherwise having the amino acid sequence of the L. intracellularis HtrA protein, but in which one or more amino acid residues has been substituted with a different amino acid residue, where the resulting polypeptide is useful in practicing the present invention. Conservative amino acid substitutions are well-known in the art. Rules for making such substitutions include those described by Dayhof, M. D., 1978, Nat. Biomed. Res. Found., Washington, D.C., Vol. 5, Sup. 3, among others. More specifically, conservative amino acid substitutions are those that generally take place within a family of amino acids that are related in acidity, polarity, or bulkiness of their side chains. Genetically encoded amino acids are generally divided into four groups: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) non-polar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Phenylalanine, tryptophan and tyrosine are also jointly classified as aromatic amino acids. One or more replacements within any particular group, e.g., of a leucine with an isoleucine or valine, or of an aspartate with a glutamate, or of a threonine with a serine, or of any other amino acid residue with a structurally related amino acid residue, e.g., an amino acid residue with similar acidity, polarity, bulkiness of side chain, or with similarity in some combination thereof, will generally have an insignificant effect on the function or immunogenicity of the polypeptide. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 7.
In another embodiment, the polynucleotide encodes an isolated polypeptide consisting of the L. intracellularis HtrA protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the HtrA sequence of SEQ ID NO: 7.
As used herein, a polypeptide is “useful in practicing the present invention” where the polypeptide can be used as a diagnostic reagent to detect the presence of Lawsonia-specific antibodies in a blood, serum, or other biological fluid sample from an animal that has developed an immune response to Lawsonia. The polypeptide is also useful if it can be used to induce an immune response in an animal against Lawsonia.
The present invention further provides a polynucleotide molecule consisting of a substantial portion of any of the aforementioned Lawsonia HtrA-related polynucleotide molecules of the present invention. As used herein, a “substantial portion” of a HtrA-related polynucleotide molecule means a polynucleotide molecule consisting of less than the complete nucleotide sequence of the HtrA-related polynucleotide molecule, but comprising at least about 5%, more preferably at least about 10%, and even more preferably at least about 20%, and most preferably at least about 50% of the nucleotide sequence of the HtrA-related polynucleotide molecule, and that is useful in practicing the present invention. Such polynucleotide molecules include, for example polynucleotide molecules encoding peptide fragments of the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned HtrA-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the HtrA ORF or gene in situ in L. intracellularis, and include the nucleotide sequences shown in SEQ ID NO: 2 from about nt 2691 to about nt 2890 and from about nt 4316 to about nt 4580, or substantial portions thereof.
PonA-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the PonA protein from L. intracellularis. In a preferred embodiment, the PonA protein has the amino acid sequence of SEQ ID NO: 6. In a further preferred embodiment, the isolated PonA-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 2 from about nt 252 to about nt 2690 (the nucleotide sequence of the open reading frame (ORF) of the PonA gene) and the nucleotid sequence of the PonA-encoding ORF of plasmid pER432 (ATCC accession number PTA-635).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is “homologous” to the nucleotide sequence of a PonA-encoding polynucleotide molecule of the present invention, as that term is correspondingly defined above with respect to HtrA related polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis PonA protein under highly stringent conditions. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence of SEQ ID NO: 2 from about nt 252 to about nt 2690.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a PonA-encoding polynucleotide molecule of the present invention do not include known polynucleotide molecules encoding PonA proteins of Neisseria flavescens, N. gonorrhoeae, and N. meningitidis.
The homologous nucleotide sequence of the molecule of the invention preferably comprises a sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 2, which is from about nt 252 to about nt 2690, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis PonA protein. In another embodiment, the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis PonA protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis PonA protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 2.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is “homologous” to the L. intracellularis PonA protein, as that term is correspondingly described with respect to the HtrA prot in above. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 6.
In another embodiment, the polynucleotide encodes an isolated polypeptide consisting of the L. intracellularis PonA protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the PonA sequence of SEQ ID NO: 6.
The present invention further provides a polynucleotide molecule consisting of a “substantial portion” of any of the aforementioned Lawsonia PonA-related polynucleotide molecules of the present invention, as that term is correspondingly described above with respect to the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned PonA-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the ponA ORF or gene in situ in L. intracellularis, and include the nucleotide sequences shown in SEQ ID NO: 2 from about nt 126 to about nt 251 and from about nt 2691 to about nt 2890, or substantial portions thereof.
HypC-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the HypC protein from L. intracellularis. In a preferred embodiment, the HypC protein has the amino acid sequence of SEQ ID NO: 8. In a further preferred embodiment, the isolated HypC-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 2 from about nt 4581 to about nt 4829, and the nucleotide sequence of the HypC-encoding ORF of plasmid pER436 (ATCC accession number PTA-637).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is “homologous” to the nucleotide sequence of a HypC-encoding polynucleotide molecule of the present invention, as that term is correspondingly defined above with respect to HtrA related polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis HypC protein under highly stringent conditions. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the ORF of SEQ ID NO: 2 from about nt 4581 to about nt 4829.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a HypC-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules encoding HypC or HypD proteins of Desulfovibrio gigas and Rizobium leguminosarum.
The homologous nucleotide sequence of the molecule of the invention preferably comprises a sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 2, which is from about nt 4581 to about nt 4829, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis HypC protein. In another embodiment, the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis HypC protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis HypC protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis HypC protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 2.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is “homologous” to the L. intracellularis HypC protein, as that term is correspondingly described with respect to the HtrA protein above. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 8.
In another embodiment, the polynucleotide encodes an isolated polypeptide consisting of the L. intracellularis HypC protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the HypC sequence of SEQ ID NO: 8.
The present invention further provides a polynucleotide molecule consisting of a “substantial portion” of any of the aforementioned Lawsonia HypC-related polynucleotide molecules of the present invention, as that term is correspondingly described above with respect to the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned HypC-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the hypC ORF or gene in situ in L. intracellularis, and include the nucleotide sequences shown in SEQ ID NO: 2 from about nt 4316 to about nt 4580 and from about nt 4830 to about nt 4911, or substantial portions thereof.
LysS-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding a LysS protein from L. intracellularis. In a preferred embodiment, the LysS protein has the amino acid sequence of SEQ ID NO: 102. In a further preferred embodiment, the isolated LysS-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 from about nt 165 to about nt 1745 of the nucleotide sequence of the lysS gene, and the nucleotide sequence of the LysS-encoding ORF of plasmid pT068 (ATCC accession number PTA-2232).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is “homologous” to the nucleotide sequence of a LysS-encoding polynucleotide molecule of the present invention, as that term is correspondingly defined above with respect to HtrA related polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis LysS protein under highly stringent conditions. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence of SEQ ID NO: 1 from about nt 165 to about nt 1745.
The homologous nucleotide sequence of the molecule of the invention preferably comprises a sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 1 from about nt 165 to about nt 1745, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis LysS protein. In another embodiment, the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis LysS protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis LysS protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis LysS protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 1.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is “homologous” to the L. intracellularis LysS protein, as that term is correspondingly described with respect to the HtrA protein above. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 102.
In another embodiment, the polynucleotide encodes an isolated polypeptid consisting of the L. intracellularis LysS protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the LysS sequence of SEQ ID NO: 102.
The present invention further provides a polynucleotide molecule consisting of a “substantial portion” of any of the aforementioned Lawsonia lysS-related polynucleotide molecules of the present invention, as that term is correspondingly described above with respect to the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned lysS-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the lysS ORF or gene in situ in L. intracellularis.
YcfW-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the YcfW protein from L. intracellularis. In a preferred embodiment, the YcfW protein has the amino acid sequence of SEQ ID NO: 3. In a further preferred embodiment, the isolated YcfW-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 from about nt 1745 to about nt 3028 of the nucleotide sequence of the YcfW gene, and the nucleotide sequence of the YcfW-encoding ORF of plasmids pER438 (ATCC accession number PTA-638) and pT068 (ATCC accession number PTA-2232). The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is “homologous” to the nucleotide sequence of a YcfW-encoding polynucleotide molecule of the present invention, as that term is correspondingly defined above with respect to HtrA related polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis YcfW protein under highly stringent conditions. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence of SEQ ID NO: 1 from about nt 1745 to about nt 3028.
The homologous nucleotide sequence of the molecule of the invention preferably comprises a sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 1 from about nt 1745 to about nt 3028, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis YcfW protein. In another embodiment, the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis YcfW protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis YcfW protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis YcfW protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 1.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is “homologous” to the L. intracellularis YcfW protein, as that term is correspondingly described with respect to the HtrA protein above. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 3.
In another embodiment, the polynucleotide encodes an isolated polypeptide consisting of the L. intracellularis YcfW protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the YcfW sequence of SEQ ID NO: 3.
The present invention further provides a polynucleotide molecule consisting of a “substantial portion” of any of the aforementioned Lawsonia YcfW-related polynucleotide molecules of the present invention, as that term is correspondingly described above with respect to the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned YcfW-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the ycfW ORF or gene in situ in L. intracellularis.
ABC1-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the ABC1 protein from L. intracellularis. In a preferred embodiment, the ABC1 protein has the amino acid sequence of SEQ ID NO: 4. In a further preferred embodiment, the isolated ABC1-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 from about nt 3031 to about nt 3738 (the nucleotide sequence of the open reading frame (ORF) of the ABC1 gene) and the nucleotide sequence of the ABC1-encoding ORF of plasmid pER438 (ATCC accession number PTA-638).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is “homologous” to the nucleotide sequence of an ABC1-encoding polynucleotide molecule of the present invention, as that term is correspondingly defined above with respect to HtrA related polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis ABC1 protein under highly stringent conditions. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the ORF of SEQ ID NO: 1, which is from about nt 3031 to about nt 3738.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a ABC1-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules encoding ABC1 proteins of Neisseria flavescens, N. gonorrhoeae, and N. meningitidis.
The nucleotide sequence of the molecule of the invention preferably comprises a sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 1 from about nt 3031 to about nt 3738, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis ABC1 protein. In another embodiment, the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis ABC1 protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis ABC1 protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis ABC1 protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 1.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is “homologous” to the L. intracellularis ABC1 protein, as that term is correspondingly described with respect to the HtrA protein above. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 1.
In another embodiment, the polynucleotide encodes an isolated polypeptide consisting of the L. intracellularis ABC1 protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the ABC1 sequence of SEQ ID NO: 4.
The present invention further provides a polynucleotide molecule consisting of a “substantial portion” of any of the aforementioned Lawsonia ABC1-related polynucleotide molecules of the present invention, as that term is correspondingly described above with respect to the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned ABC1-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the abc1 ORF or gene in situ in L. intracellularis, and include the flanking nucleotide sequences shown in SEQ ID NO: 1.
Omp100-Related Polynucleotide MoleculesThe present invention provides an isolated polynucleotide molecule comprising a nucleotide sequence encoding the Omp100 protein from L. intracellularis. In a preferred embodiment, the Omp100 prot in has the amino acid sequence of SEQ ID NO: 5. In a further preferred embodiment, the isolated Omp100-encoding polynucleotide molecule of the present invention comprises a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 1 from about nt 3695 to about nt 6385 (the nucleotide sequence of the open reading frame (ORF) of the Omp100 gene), and the nucleotide sequence of the Omp100-encoding ORF of plasmid pER440 (ATCC accession number PTA-639).
The present invention further provides an isolated polynucleotide molecule having a nucleotide sequence that is “homologous” to the nucleotide sequence of a Omp100-encoding polynucleotide molecule of the present invention, as that term is correspondingly defined above with respect to HtrA related polynucleotide molecules. In a preferred embodiment, the homologous polynucleotide molecule hybridizes to the complement of a polynucleotide molecule having a nucleotide sequence that encodes the amino acid sequence of the L. intracellularis Omp100 protein under highly stringent conditions. In a more preferred embodiment, the homologous polynucleotide molecule hybridizes under highly stringent conditions to the complement of a polynucleotide molecule consisting of a nucleotide sequence selected from the group consisting of the ORF of SEQ ID NO: 1, which is from about nt 3695 to about nt 6385.
Polynucleotide molecules of the present invention having nucleotide sequences that are homologous to the nucleotide sequence of a Omp100-encoding polynucleotide molecule of the present invention do not include polynucleotide molecules encoding any of the following proteins listed in the GenBank database: YaeT (Accn. U70214 or AE000127) of E. coli; Oma90 (Accn. AF120927) of Shigella flexneri, Omp85 (Accn. AF021245) of Neisseria meningitidis, D15 (Accn. U60834) of Haemophilus influenzae (D15), and Oma87 (Accn. U60439) of Pasteurella multocida.
The nucleotide sequence of the molecule of the invention preferably comprises a homologous sequence that has more than 50%, more preferably more than about 90%, even more preferably more than about 95%, and most preferably more than about 99% sequence identity to the molecule of SEQ ID NO: 1 from about nt 3695 to about nt 6385, wherein sequence identity is determined by use of the BLASTN algorithm (GenBank, National Center for Biotechnology Information).
In another embodiment, the polynucleotide has a homologous sequence that is more than about 50% of the length of the nucleotide sequence encoding the L. intracellularis Omp100 protein. In another embodiment, the sequence is more than 90%, and in another embodiment more than about 98%, of the length of the nucleotide sequence encoding the L. intracellularis Omp100 protein. In yet another embodiment, the isolated polynucleotide that has a homologous sequence is equal in length to the sequence encoding the L. intracellularis Omp100 protein.
In yet another embodiment, the nucleotide sequence that is homologous to the L. intracellularis Omp100 protein encoding sequence has between 1 and 50, more preferably between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 5.
The present invention further provides an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a polypeptide that is “homologous” to the L. intracellularis Omp100 protein, as that term is correspondingly described with respect to the HtrA protein above. In a preferred embodiment, the homologous polypeptide has at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% sequence identity, and most preferably at least 95% sequence identity to SEQ ID NO: 5.
In another embodiment, the polynucleotide encodes an isolated polypeptide consisting of the L. intracellularis Omp100 protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the polynucleotide encodes an isolated polypeptide having between 1 and 5 amino acids conservatively substituted for the Omp100 sequence of SEQ ID NO: 5.
The present invention further provides a polynucleotide molecule consisting of a “substantial portion” of any of the aforementioned Lawsonia Omp100-related polynucleotide molecules of the present invention, as that term is correspondingly described above with respect to the HtrA protein.
In addition to the nucleotide sequences of any of the aforementioned Omp100-related polynucleotide molecules, polynucleotide molecules of the present invention can further comprise, or alternatively may consist of, nucleotide sequences selected from those that naturally flank the omp100 ORF or gene in situ in L. intracellularis, and include the nucleotide sequences shown in SEQ ID NO: 1.
Promoter SequencesThe present invention also relates to a polynucleotide molecule comprising a nucleotide sequence greater than 20 nucleotides having promoter activity and found within SEQ ID NO: 2 from about nt 2691 to about nt 2890, or its complement. As further discussed below in the Examples, it has been determined that this region of the Lawsonia sequence contains a temperature responsive promoter for the htrA gene. In a preferred embodiment, the polynucleotide comprises the sequence of about nt 2797 to nt 2829.
The present invention also relates to oligonucleotides having promoter activity that hybridize under moderately stringent, and more preferably under highly stringent conditions, to the complement of the nucleotide sequence greater than 20 nucleotides having promoter activity and found within SEQ ID NO: 2 from about nt 2691 to about nt 2890. Preferably the oligonucleotide having promoter activity hybridizes under moderately stringent or highly stringent conditions to the complement of the polynucleotide comprising the sequence from about nt 2797 to nt 2829. In another embodiment, the invention encompasses an oligonucleotide having promoter activity having between 1 and 25, and most preferably between 1 and 5 nucleotides inserted, deleted, or substituted with respect to the sequence of SEQ ID NO: 2 which is from about nt 2691 to about nt 2890.
The functional sequences having promoter activity of the present invention are useful for a variety of purposes including for controlling the recombinant expression of any of the genes of the present invention, or of other genes or coding sequences, in host cells of L. intracellularis or in host cells of any other species of Lawsonia, or in any other appropriate host cell. Such other genes or coding sequences can either be native or heterologous to the recombinant host cell. The promoter sequence can be fused to the particular gene or coding sequence using standard recombinant techniques as known in the art so that the promoter sequence is in operative association therewith, as “operative association” is defined below. By using the promoter, recombinant expression systems can, for example, be constructed and used to screen for compounds and transcriptional factors that can modulate the expression of the genes of Lawsonia or other bacteria. In addition, such promoter constructs can be used to express heterologous polypeptides in Lawsonia, E. coli, or other appropriate host cells.
Oligonucleotide MoleculesThe present invention further provides oligonucleotide molecules that hybridize to any one of the aforementioned polynucleotide molecules of the present invention, or that hybridize to a polynucleotide molecule having a nucleotide sequence that is the complement of any one of the aforementioned polynucleotide molecules of the present invention. Such oligonucleotide molecules are preferably at least about 10 nucleotides in length, and more preferably from about 15 to about 30 nucleotides in length, and hybridize to one or more of the aforementioned polynucleotide molecules under highly stringent conditions, i.e., washing in 6×SSC/0.5% sodium pyrophosphate at about 37° C. for ˜14-base oligos, at about 48° C. for ˜17-base oligos, at about 55° C. for ˜20-base oligos, and at about 60° C. for ˜23-base oligos. Other hybridization conditions for longer oligonucleotide molecules of the present invention can be determined by the skilled artisan using standard techniques. In a preferred embodiment, an oligonucleotide molecule of the present invention is complementary to a portion of at least one of the aforementioned polynucleotide molecules of the present invention.
The oligonucleotide molecules of the present invention are useful for a variety of purposes, including as primers in amplification of a Lawsonia-specific polynucleotide molecule for use, e.g., in differential disease diagnosis, or to act as antisense molecules useful in gene regulation. Suitably designed primers can also be used to detect the presence of Lawsonia-specific polynucleotide molecules in a sample of animal tissue or fluid, including brain tissue, lung tissue, intestinal tissue, placental tissue, blood, cerebrospinal fluid, feces, mucous, urine, amniotic fluid, etc. The oligonucleotide molecule specifically reacts with the Lawsonia organism; this is generally accomplished by employing a sequence of sufficient length. The production of a specific amplification product can support a diagnosis of Lawsonia infection, while lack of an amplified product can point to a lack of infection. Methods for conducting amplifications, such as the polymerase chain reaction (PCR), are described, among other places, in Innis et al. (eds), 1995, above; and Erlich (ed), 1992, above. Other amplification techniques known in the art, e.g., the ligase chain reaction, can alternatively be used. The sequences of the polynucleotide molecules disclosed herein can also be used to design primers for use in isolating homologous genes from other species or strains of Lawsonia or other bacterial cells.
Specific though non-limiting embodiments of oligonucleotide molecules useful in practicing the present invention include oligonucleotide molecules selected from the group consisting of SEQ ID NOS:10-101 and the complements thereof.
Recombinant Expression Systems Cloning and Expression VectorsThe present invention further encompasses methods and compositions for cloning and expressing any of the polynucleotide molecules of the present invention, including cloning vectors, expression vectors, transformed host cells comprising any of said vectors, and novel strains or cell lines derived therefrom. In a preferred embodiment, the present invention provides a recombinant vector comprising a polynucleotide molecule having a nucleotide sequence encoding the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein of L. intracellularis. In specific embodiments, the present invention provides plasmid pER432 containing the ponA gene (ATCC accession number PTA-635), plasmid pER434 containing the htrA gene (ATCC accession number PTA-636), plasmid pER436 containing the hypC gene (ATCC accession number PTA-637), plasmid pT068 containing the lysS and ycfW genes (ATCC accession number PTA-2232), plasmid pER438 containing the ycfW and abc1 genes (ATCC accession number PTA-638), and plasmid pER440 containing the Omp100 gene (ATCC accession number PTA-639). The invention also encompasses recombinant vectors and transformed cells employed to obtain polypeptides of the invention.
Recombinant vectors of the present invention, particularly expression vectors, are preferably constructed so that the coding sequence for the polynucleotide molecule of the invention is in operative association with one or more regulatory elements necessary for transcription and translation of the coding sequence to produce a polypeptide. As used herein, the term “regulatory element” includes but is not limited to nucleotide sequences that encode inducible and non-inducible promoters, enhancers, operators, ribosome-binding sites, and other elements known in the art that serve to drive and/or regulate expression of polynucleotide coding sequences. Also, as used herein, the coding sequence is in “operative association” with one or more regulatory elements where the regulatory elements effectively regulate and allow for the transcription of the coding sequence or the translation of its mRNA, or both.
Methods are well-known in the art for constructing recombinant vectors containing particular coding sequences in operative association with appropriate regulatory elements, and these can be used to practice the present invention. These methods include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination. See, e.g., the techniques described in Maniatis et al., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.
A variety of expression vectors are known in the art which can be utilized to express the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 coding sequences of the present invention, including recombinant bacteriophage DNA, plasmid DNA, and cosmid DNA expression vectors containing the particular coding sequences. Typical prokaryotic expression vector plasmids that can be engineered to contain a polynucleotide molecule of the present invention include pUC8, pUC9, pBR322 and pBR329 (Biorad Laboratories, Richmond, Calif.), pPL and pKK223 (Pharmacia, Piscataway, N.J.), pQE50 (Qiagen, Chatsworth, Calif.), and pGEM-T EASY (Promega, Madison, Wis.), among many others. Typical eukaryotic expression vectors that can be engineered to contain a polynucleotide molecule of the present invention include an ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.), cytomegalovirus promoter-enhancer-based systems (Promega, Madison, Wis.; Stratagene, La Jolla, Calif.; Invitrogen), and baculovirus-based expression systems (Promega), among others.
The regulatory elements of these and other vectors can vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements can be used. For instance, when cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells, e.g., mouse metallothionein promoter, or from viruses that grow in these cells, e.g., vaccinia virus 7.5K promoter or Moloney murine sarcoma virus long terminal repeat, can be used. Promoters obtained by recombinant DNA or synthetic techniques can also be used to provide for transcription of the inserted sequence. In addition, expression from certain promoters can be elevated in the presence of particular inducers, e.g., zinc and cadmium ions for metallothionein promoters. Non-limiting examples of transcriptional regulatory regions or promoters include for bacteria, the β-gal promoter, the T7 promoter, the TAC promoter, λ left and right promoters, trp and lac promoters, trp-lac fusion promoters, etc.; for yeast, glycolytic enzyme promoters, such as ADH-I and -II promoters, GPK promoter, PGI promoter, TRP promoter, etc.; and for mammalian cells, SV40 early and late promoters, adenovirus major late promoters, among others. The present invention further provides a polynucleotide molecule comprising the nucleotide sequence of the promoter of the htrA gene of L. intracellularis, which can be used to express any of the coding sequences of the present invention in Lawsonia, E. coli, or other suitable hosts.
Specific initiation signals are also required for sufficient translation of inserted coding sequences. These signals typically include an ATG initiation codon and adjacent sequences. In cases where the polynucleotide molecule of the present invention including its own initiation codon and adjacent sequences are inserted into the appropriate expression vector, no additional translation control signals may be needed. However, in cases where only a portion of a coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, may be required. These exogenous translational control signals and initiation codons can be obtained from a variety of sources, both natural and synthetic. Furthermore, the initiation codon must be in phase with the reading frame of the coding regions to ensure in-frame translation of the entire insert.
Expression vectors can also be constructed that will express a fusion protein comprising a protein or polypeptide of the present invention. Such fusion proteins can be used, e.g., to raise antisera against a Lawsonia protein, to study the biochemical properties of the Lawsonia protein, to engineer a Lawsonia protein exhibiting different immunological or functional properties, or to aid in the identification or purification, or to improve the stability, of a recombinantly-expressed Lawsonia protein. Possible fusion protein expression vectors include but are not limited to vectors incorporating sequences that encode β-galactosidase and trpE fusions, maltose-binding protein fusions (pMal series; New England Biolabs), glutathione-S-transferase fusions (pGEX series; Pharmacia), polyhistidine fusions (pET series; Novagen Inc., Madison, Wis.), and thioredoxin fusions (pTrxFus; Invitrogen, Carlsbad, Calif.). Methods are well-known in the art for constructing expression vectors encoding these and other fusion proteins.
The fusion protein can be useful to aid in purification of the expressed protein. In non-limiting embodiments, e.g., a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100-maltose-binding fusion protein can be purified using amylose resin; a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100-glutathione-S-transferase fusion protein can be purified using glutathione-agarose beads; and a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100-polyhistidine fusion protein can be purified using divalent nickel resin. Alternatively, antibodies against a carrier protein or peptide can be used for affinity chromatography purification of the fusion protein. For example, a nucleotide sequence coding for the target epitope of a monoclonal antibody can be engineered into the expression vector in operative association with the regulatory elements and situated so that the expressed epitope is fused to a Lawsonia protein of the present invention. In a non-limiting embodiment, a nucleotide sequence coding for the FLAG™ epitope tag (International Biotechnologies Inc.), which is a hydrophilic marker peptide, can be inserted by standard techniques into the expression vector at a point corresponding to the amino or carboxyl terminus of the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein. The expressed HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein-FLAG™ epitope fusion product can then be detected and affinity-purified using commercially available anti-FLAG™ antibodies.
The expression vector can also be engineered to contain polylinker sequences that encode specific protease cleavage sites so that the expressed Lawsonia protein can be released from the carrier region or fusion partner by treatment with a specific protease. For example, the fusion protein vector can include a nucleotide sequence encoding a thrombin or factor Xa cleavage site, among others.
A signal sequence upstream from and in the same reading frame with the Lawsonia coding sequence can be engineered into the expression vector by known methods to direct the trafficking and secretion of the expressed protein. Non-limiting examples of signal sequences include those from α-factor, immunoglobulins, outer membrane proteins, penicillinase, and T-cell receptors, among others.
To aid in the selection of host cells transformed or transfected with a recombinant vector of the present invention, the vector can be engineered to further comprise a coding sequence for a reporter gene product or other selectable marker. Such a coding sequence is preferably in operative association with the regulatory elements, as described above. Reporter genes that are useful in practicing the invention are well-known in the art and include those encoding chloramphenicol acetyltransferase (CAT), green fluorescent protein, firefly luciferase, and human growth hormone, among others. Nucleotide sequences encoding selectable markers are well-known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode thymidine kinase activity, or resistance to methotrexate, ampicillin, kanamycin, chloramphenicol, zeocin, pyrimethamine, aminoglycosides, or hygromycin, among others.
Transformation of Host CellsThe present invention further provides transformed host cells comprising a polynucleotide molecule or recombinant vector of the present invention, and cell lines derived therefrom. Host cells useful in practicing the invention can be eukaryotic or prokaryotic cells. Such transformed host cells include but are not limited to microorganisms, such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA vectors, or yeast transformed with a recombinant vector, or animal cells, such as insect cells infected with a recombinant virus vector, e.g., baculovirus, or mammalian cells infected with a recombinant virus vector, e.g., adenovirus or vaccinia virus, among others. For example, a strain of E. coli can be used, such as, e.g., the DH5α strain available from the ATCC, Rockville, Md., USA (Accession No. 31343), or from GIBCO BRL, Gaithersburg, Md. Eukaryotic host cells include yeast cells, although mammalian cells, e.g., from a mouse, hamster, cow, monkey, or human cell line, among others, can also be utilized effectively. Examples of eukaryotic host cells that can be used to express a recombinant protein of the invention include Chinese hamster ovary (CHO) cells (e.g., ATCC Accession No. CCL-61), NIH Swiss mouse embryo cells NIH/3T3 (e.g., ATCC Accession No. CRL-1658), and Madin-Darby bovine kidney (MDBK) cells (ATCC Accession No. CCL-22). Transfected cells can express the polynucleotide of the invention by chromosomal integration, or episomally.
The recombinant vector of the invention is preferably transformed or transfected into one or more host cells of a substantially homogeneous culture of cells. The vector is generally introduced into host cells in accordance with known techniques, such as, e.g., by protoplast transformation, calcium phosphate precipitation, calcium chloride treatment, microinjection, electroporation, transfection by contact with a recombined virus, liposome-mediated transfection, DEAE-dextran transfection, transduction, conjugation, or microprojectile bombardment, among others. Selection of transformants can be conducted by standard procedures, such as by selecting for cells expressing a selectable marker, e.g., antibiotic resistance, associated with the recombinant expression vector.
Once an expression vector is introduced into the host cell, the integration and maintenance of the polynucleotide molecule of the present invention, either in the host cell genome or episomally, can be confirmed by standard techniques, e.g., by Southern hybridization analysis, restriction enzyme analysis, PCR analysis including reverse transcriptase PCR (rt-PCR), or by immunological assay to detect the expected protein product. Host cells containing and/or expressing a polynucleotide molecule of the present invention can be identified by any of at least four general approaches that are well-known in the art, including: (i) DNA-DNA, DNA-RNA, or RNA-antisense RNA hybridization; (ii) detecting the presence of “marker” gene functions; (iii) assessing the level of transcription as measured by the expression of specific mRNA transcripts in the host cell; or (iv) detecting the presence of mature polypeptide product, e.g., by immunoassay, as known in the art.
Expression and Purification of Recombinant PolypeptidesOnce a polynucleotide molecule of the present invention has been stably introduced into an appropriate host cell, the transformed host cell is clonally propagated, and the resulting cells are grown under conditions conducive to the maximum production of the encoded polypeptide. Such conditions typically include growing transformed cells to high density. Where the expression vector comprises an inducible promoter, appropriate induction conditions such as, e.g., temperature shift, exhaustion of nutrients, addition of gratuitous inducers (e.g., analogs of carbohydrates, such as isopropyl-β-D-thiogalactopyranoside (IPTG)), accumulation of excess metabolic by-products, or the like, are employed as needed to induce expression.
Where the polypeptide is retained inside the host cells, the cells are harvested and lysed, and the product is substantially purified or isolated from the lysate under extraction conditions known in the art to minimize protein degradation such as, e.g., at 4° C., or in the presence of protease inhibitors, or both. Where the polypeptide is secreted from the host cells, the exhausted nutrient medium can simply be collected and the polypeptide substantially purified or isolated therefrom.
The polypeptide can be substantially purified or isolated from cell lysates or culture medium, as necessary, using standard methods, including but not limited to one or more of the following methods: ammonium sulfate precipitation, size fractionation, ion exchange chromatography, HPLC, density centrifugation, and affinity chromatography. If the polypeptide lacks biological activity, it can be detected as based, e.g., on size, or reactivity with a polypeptide-specific antibody, or by the presence of a fusion tag. For use in practicing the present invention, the polypeptide can be in an unpurified state as secreted into the culture fluid or as present in a cell lysate, but is preferably substantially purified or isolated therefrom. As used herein, a polypeptide is “substantially purified” where the polypeptide constitutes at least about 20 wt % of the protein in a particular preparation. Also, as used herein, a polypeptide is “isolated” where the polypeptide constitutes at least about 80 wt % of the protein in a particular preparation. In another embodiment of the invention, the protein is present in a preparation in at least about a 1000× higher concentration than its natural counterpart is normally found in a preparation of L. intracellularis cell lysate.
PolypeptidesThus, the present invention encompasses a substantially purified or isolated polypeptide encoded by a polynucleotide of the present invention. In a non-limiting embodiment, the polypeptide is a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 L. intracellularis protein. In a preferred embodiment, the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, and Omp100 proteins have the amino acid sequences of SEQ ID NOS: 3-8 or SEQ ID NO: 102. In another embodiment, the polypeptides are substantially free of other Lawsonia proteins.
The present invention further provides polypeptides that are homologous to any of the aforementioned L. intracellularis proteins, as the term “homologous” is defined above for polypeptides. Polypeptides of the present invention that are homologous to the proteins of the invention do not include polypeptides having the amino acid sequences of non-Lawsonia proteins described herein. The polypeptide of the invention, in one embodiment, has more than 70%, preferably more than about 90%, and most preferably more than about 95% amino acid sequence identity to the Lawsonia proteins, wherein sequence identity is determined by use of the BLASTP algorithm (GenBank, NCBI).
In another embodiment, the polypeptide consists of the L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein having between 1 and 10, and more preferably between 1 and 5, amino acids inserted, deleted, or substituted, including combinations thereof. In a more particular example of this embodiment, the isolated polypeptide has between 1 and 5 amino acids conservatively substituted in the amino acid sequence of the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein.
The present invention further provides polypeptides consisting of a substantial portion of any one of the aforementioned polypeptides of the present invention. As used herein, a “substantial portion” of a polypeptide of the present invention, or “peptide fragment,” means a polypeptide consisting of less than the complete amino acid sequence of the corresponding full-length polypeptide, but comprising at least about 5%, more preferably at least about 20%, even more preferably at least about 50%, and most preferably at least about 95% of the amino acid sequence thereof, and that is useful in practicing the present invention. Particularly preferred are peptide fragments that are immunogenic, i.e., capable of inducing an immune response which results in production of antibodies that react specifically against the corresponding full-length Lawsonia polypeptide.
In another embodiment, the polypeptide of the invention comprises an epitope of HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein that is specifically reactive with anti-Lawsonia antibodies. The epitope is preferably more than 8, more preferably more than 12, and most preferably, more than 20 amino acids of the protein sequence.
The present invention further provides fusion proteins comprising any of the polypeptides of the invention fused to a carrier or fusion partner as known in the art.
The present invention further provides a method of preparing any of the polypeptides described above, comprising culturing a host cell transformed with a recombinant expression vector, said recombinant expression vector comprising a polynucleotide molecule comprising a nucleotide sequence encoding the particular polypeptide, which polynucleotide molecule is in operative association with one or more regulatory elements, under conditions conducive to the expression of the polypeptide, and recovering the expressed polypeptide from the cell culture.
Use Of PolypeptidesOnce a polypeptide of the present invention of sufficient purity has been obtained, it can be characterized by standard methods, including by SDS-PAGE, size exclusion chromatography, amino acid sequence analysis, immunological activity, biological activity, etc. The polypeptide can be further characterized using hydrophilicity analysis (see, e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78: 3824), or analogous software algorithms, to identify hydrophobic and hydrophilic regions. Structural analysis can be carried out to identify regions of the polypeptide that assume specific secondary structures. Biophysical methods such as X-ray crystallography (Engstrom, 1974, Biochem. Exp. Biol. 11: 7-13), computer modeling (Fletterick and Zoller (eds), 1986, in: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and nuclear magnetic resonance (NMR) can be used to map and study potential sites of interaction between the polypeptide and other putative interacting proteins/receptors/molecules. Information obtained from these studies can be used to design deletion mutants and vaccine compositions, and to design or select therapeutic or pharmacologic compounds that can specifically block the biological function of the polypeptide in vivo.
Polypeptides of the present invention are useful for a variety of purposes, including as components of vaccine compositions to protect PPE susceptible animals against PPE; or as diagnostic reagents, e.g., using standard techniques such as ELISA assays, to screen for Lawsonia-specific antibodies in blood or serum samples from animals; or as antigens to raise polyclonal or monoclonal antibodies, as described below, which antibodies are useful as diagnostic reagents, e.g., using standard techniques such as Western blot assays, to screen for Lawsonia-specific proteins in cell, tissue or fluid samples from an animal.
Analogs and Derivatives of PolypeptidesA polypeptide of the present invention can be modified at the protein level to improve or otherwise after its biological or immunological characteristics. One or more chemical modifications of the polypeptide can be carried out using known techniques to prepare analogs therefrom, including but not limited to any of the following: substitution of one or more L-amino acids of the polypeptide with corresponding D-amino acids, amino acid analogs, or amino acid mimics, so as to produce, e.g., carbazates or tertiary centers; or specific chemical modification, such as, e.g., proteolytic cleavage with trypsin, chymotrypsin, papain or V8 protease, or treatment with NaBH4 or cyanogen bromide, or acetylation, formylation, oxidation or reduction, etc. Alternatively or additionally, polypeptides of the present invention can be modified by genetic recombination techniques.
A polypeptide of the present invention can be derivatized by conjugation thereto of one or more chemical groups, including but not limited to acetyl groups, sulfur bridging groups, glycosyl groups, lipids, and phosphates, and/or by conjugation to a second polypeptide of the present invention, or to another protein, such as, e.g., serum albumin, keyhole limpet hemocyanin, or commercially activated BSA, or to a polyamino acid (e.g., polylysine), or to a polysaccharide, (e.g., sepharose, agarose, or modified or unmodified celluloses), among others. Such conjugation is preferably by covalent linkage at amino acid side chains and/or at the N-terminus or C-terminus of the polypeptide. Methods for carrying out such conjugation reactions are well-known in the field of protein chemistry.
Derivatives useful in practicing the claimed invention also include those in which a water-soluble polymer such as, e.g., polyethylene glycol, is conjugated to a polypeptide of the present invention, or to an analog or derivative thereof, thereby providing additional desirable properties while retaining, at least in part, the immunogenicity of the polypeptide. These additional desirable properties include, e.g., increased solubility in aqueous solutions, increased stability in storage, increased resistance to proteolytic dehydration, and increased in vivo half-life. Water-soluble polymers suitable for conjugation to a polypeptide of the present invention include but are not limited to polyethylene glycol homopolymers, polypropylene glycol homopolymers, copolymers of ethylene glycol with propylene glycol, wherein said homopolymers and copolymers are unsubstituted or substituted at one end with an alkyl group, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, polyvinyl ethyl ethers, and α,β-poly[2-hydroxyethyl]-DL-aspartamide. Polyethylene glycol is particularly preferred. Methods for making water-soluble polymer conjugates of polypeptides are known in the art and are described in, among other places, U.S. Pat. No. 3,788,948; U.S. Pat. No. 3,960,830; U.S. Pat. No. 4,002,531; U.S. Pat. No. 4,055,635; U.S. Pat. No. 4,179,337; U.S. Pat. No. 4,261,973; U.S. Pat. No. 4,412,989; U.S. Pat. No. 4,414,147; U.S. Pat. No. 4,415,665; U.S. Pat. No. 4,609,546; U.S. Pat. No. 4,732,863; U.S. Pat. No. 4,745,180; European Patent (EP) 152,847; EP 98,110; and Japanese Patent 5,792,435, which patents are incorporated herein by reference.
AntibodiesThe present invention further provides isolated antibodies directed against a polypeptide of the present invention. In a preferred embodiment, antibodies can be raised against a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein from L. intracellularis using known methods. Various host animals selected from pigs, cows, horses, rabbits, goats, sheep, or mice, can be immunized with a partially or substantially purified, or isolated, L. intracellularis protein, or with a homolog, fusion protein, substantial portion, analog or derivative thereof, as these are described above. An adjuvant, such as described below, can be used to enhance antibody production.
Polyclonal antibodies can be obtained and isolated from the serum of an immunized animal and tested for specificity against the antigen using standard techniques. Alternatively, monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (Nature, 1975, 256: 495-497); the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4: 72; Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Alternatively, techniques described for the production of single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted to produce L. intracellularis antigen-specific single chain antibodies. These publications are incorporated herein by reference.
Antibody fragments that contain specific binding sites for a polypeptide of the present invention are also encompassed within the present invention, and can be generated by known techniques. Such fragments include but are not limited to F(ab′)2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab′)2 fragments. Alternatively, Fab expression libraries can be constructed (Huse et al., 1989, Science 246: 1275-1281) to allow rapid identification of Fab fragments having the desired specificity to the L. intracellularis protein.
Techniques for the production and isolation of monoclonal antibodies and antibody fragments are well-known in the art, and are additionally described, among other places, in Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, and in J. W. Goding, 1986, Monoclonal Antibodies: Principles and Practice, Academic Press, London, which are incorporated herein by reference.
Targeted Mutation of Lawsonia GenesBased on the disclosure of the polynucleotide molecules of the present invention, genetic constructs can be prepared for use in disabling or otherwise mutating a Lawsonia htrA, ponA, lysS, ycfW, hypC, abc1, or omp100 gene (which gene is hereinafter referred to as the “Lawsonia gene”). The Lawsonia gene can be mutated using an appropriately designed genetic construct. For example, the Lawsonia gene can be mutated using a genetic construct of the present invention that functions to: (a) delete all or a portion of the coding sequence or regulatory sequence of the Lawsonia gene; or (b) replace all or a portion of the coding sequence or regulatory sequence of the Lawsonia gene with a different nucleotide sequence; or (c) insert into the coding sequence or regulatory sequence of the Lawsonia gene one or more nucleotides, or an oligonucleotide molecule, or polynucleotide molecule, which can comprise a nucleotide sequence from Lawsonia or from a heterologous source; or (d) carry out some combination of (a), (b) and (c). Alternately, constructs can be employed to alter the expression of the Lawsonia gene or the stability of its encoded protein.
Lawsonia cells in which a Lawsonia gene has been mutated are, for example, useful in practicing the present invention where mutating the gene reduces the pathogenicity of the Lawsonia cells carrying the mutated gene compared to cells of the same strain of Lawsonia where the gene has not been so mutated, and where such Lawsonia cells carrying the disabled gene can be used in a vaccine composition, particularly in a modified live vaccine, to induce or contribute to the induction of, a protective response in an animal against PPE. In a preferred embodiment, the mutation serves to partially or completely disable the Lawsonia gene, or partially or completely disable the protein encoded by the Lawsonia gene. In this context, a Lawsonia gene or protein is considered to be partially or completely disabled if either no protein product is made (for example, the gene is deleted), or a protein product is made that can no longer carry out its normal biological function or can no longer be transported to its normal cellular location, or a product is made that carries out its normal biological function but at a significantly reduced rate. Lawsonia cells in which the Lawsonia gene has been mutated are also useful to increase expression of that gene or the stability of its encoded protein. Mutations are particularly useful that result in a detectable decrease in the pathogenicity of cells of a pathogenic strain of Lawsonia. The invention also encompasses cells expressing proteins and polypeptides of the invention where such cells are constitutive mutants.
In a non-limiting embodiment, a genetic construct of the present invention is used to mutate a wild-type Lawsonia gene by replacement of the coding sequence of the wild-type gene, or a promoter or other regulatory region thereof, or a portion thereof, with a different nucleotide sequence such as, e.g., a mutated coding sequence or mutated regulatory region, or portion thereof. Mutated Lawsonia gene sequences for use in such a genetic construct can be produced by any of a variety of known methods, including by use of error-prone PCR, or by cassette mutagenesis. For example, oligonucleotide-directed mutagenesis can be employed to alter the coding sequence or promoter sequence of a wild-type Lawsonia gene in a defined way, e.g., to introduce a frame-shift or a termination codon at a specific point within the sequence. Alternatively or additionally, a mutated nucleotide sequence for use in the genetic construct of the present invention can be prepared by insertion or deletion of the coding sequence or promoter sequence of one or more nucleotides, oligonucleotide molecules or polynucleotide molecules, or by replacement of a portion of the coding sequence or promoter sequence with one or more different nucleotides, oligonucleotide molecules or polynucleotide molecules. Such oligonucleotide molecules or polynucleotide molecules can be obtained from any naturally occurring source or can be synthetic. The inserted or deleted sequence can serve simply to disrupt the reading frame of the Lawsonia gene, or can further encode a heterologous gene product such as a selectable marker.
Alternatively or additionally, random mutagenesis can be used to produce a mutated Lawsonia gene sequence for use in a genetic construct of the present invention. Random mutagenesis can be carried out by any suitable techniques such as, e.g., by exposing cells carrying a Lawsonia gene to ultraviolet radiation or x-rays, or to chemical mutagens such as N-methyl-N′-nitrosoguanidine, ethyl methane sulfonate, nitrous acid or nitrogen mustards, and then selecting for cells carrying a mutation in the particular gene. See, e.g., Ausubel, 1989, above, for a review of mutagenesis techniques.
Mutations to produce modified Lawsonia cells that are useful in practicing the present invention can occur anywhere in the Lawsonia gene, including in the ORF, or in the promoter or other regulatory region, or in any other sequences that naturally comprise the gene or ORF, or that alter expression of the gene or the stability of its encoded protein. Such Lawsonia cells include mutants in which a modified form of the protein normally encoded by the Lawsonia gene is produced, or in which no protein normally encoded by the Lawsonia gene is produced, and can be null, conditional, constitutive, or leaky mutants.
Alternatively, a genetic construct of the present invention can comprise nucleotide sequences that naturally flank the Lawsonia gene or ORF in situ, with only a portion or no nucleotide sequences from the coding region of the gene itself. Such a genetic construct would be useful, e.g., to delete the entire Lawsonia gene or ORF.
In one embodiment, a genetic construct of the present invention comprises a polynucleotide molecule that can be used to disable a Lawsonia gene, comprising: (a) a polynucleotide molecule having a nucleotide sequence that is otherwise the same as a nucleotide sequence encoding a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein from L. intracellularis, but which nucleotide sequence further comprises one or more disabling mutations; or (b) a polynucleotide molecule comprising a nucleotide sequence that naturally flanks the ORF of a Lawsonia gene in situ. Once transformed into cells of a strain of Lawsonia, the polynucleotide molecule of the genetic construct is specifically targeted to the particular Lawsonia gene by homologous recombination, and thereby either replaces the gene or portion thereof or inserts into the gene. As a result of this recombination event, the Lawsonia gene otherwise native to that particular strain of Lawsonia is disabled.
In another embodiment, a genetic construct employs a mutation that alters expression, e.g., by constitutively expressing or overexpressing the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein. Such a mutation can be useful, for example, to weaken the host cells. The construct can also employ a mutation that increases stability of the protein to, e.g., attenuate the host cell.
For targeted gene mutation through homologous recombination, the genetic construct is preferably a plasmid, either circular or linearized, comprising a mutated nucleotide sequence as described above. In a non-limiting embodiment, at least about 200 nucleotides of the mutated sequence are used to specifically direct the genetic construct of the present invention to the particular targeted Lawsonia gene for homologous recombination, although shorter lengths of nucleotides can also be effective. In addition, the plasmid preferably comprises an additional nucleotide sequence encoding a reporter gene product or other selectable marker that is constructed so that it will insert into the Lawsonia genome in operative association with the regulatory element sequences of the native Lawsonia gene to be disrupted. Reporter genes that can be used in practicing the invention are well-known in the art and include those encoding CAT, green fluorescent protein, and β-galactosidase, among others. Nucleotide sequences encoding selectable markers are also well-known in the art, and include those that encode gene products conferring resistance to antibiotics or anti-metabolites, or that supply an auxotrophic requirement. Examples of such sequences include those that encode pyrimethamine resistance, or neomycin phosphotransferase (which confers resistance to aminoglycosides), or hygromycin phosphotransferase (which confers resistance to hygromycin).
Methods that can be used for creating the genetic constructs of the present invention are well-known in the art, and include in vitro recombinant techniques, synthetic techniques, and in vivo genetic recombination, as described, among other places, in Maniatis et al., 1989, above; Ausubel et al., 1989, above; Sambrook et al., 1989, above; Innis et al., 1995, above; and Erlich, 1992, above.
Lawsonia cells can be transformed or transfected with a genetic construct of the present invention in accordance with known techniques, such as, e.g., by electroporation. Selection of transformants can be carried out using standard techniques, such as by selecting for cells expressing a selectable marker associated with the construct. Identification of transformants in which a successful recombination event has occurred and the particular target gene has been altered can be carried out by genetic analysis, such as by Southern blot analysis, or by Northern analysis to detect a lack of mRNA transcripts encoding the particular protein, or cells lacking the particular protein, as determined, e.g., by immunological analysis, by the appearance of a novel phenotype, such as reduced pathogenicity, by PCR assay, or by some combination thereof.
In a further non-limiting embodiment, the genetic construct of the present invention can additionally comprise a different gene or coding region from Lawsonia or from a different pathogen that infects the animal, which gene or coding region encodes an antigen useful to induce, or contribute to the induction of, a separate and distinct protective immune response in the animal upon vaccination with the modified live Lawsonia cells of the present invention. This additional gene or coding region can be further engineered to contain a signal sequence that leads to secretion of the encoded antigen from the modified live Lawsonia cell, thereby allowing for the antigen to be displayed to the immune system of the vaccinated animal.
The present invention thus provides modified live Lawsonia cells in which the htrA, ponA, hypC, lysS, ycfW, abc1, or omp100 gene has been mutated. In addition, the present invention provides a method of preparing modified live Lawsonia cells, comprising: (a) transforming cells of Lawsonia with a genetic construct of the invention; (b) selecting transformed cells in which the htrA, ponA, hypC, lysS, ycfW, abc1, or omp100 gene has been mutated by the genetic construct; and (c) selecting from among the cells of step (b) those cells that can be used in a vaccine to protect a PPE susceptible animal against PPE. The invention also encompasses killed cell compositions prepared from such modified Lawsonia cells.
Culturing Lawsonia BacteriaLawsonia bacterium for use in the present invention can be cultured and maintained in vitro using methods described e.g. by Joens et al., 1997, Am. J. Vet. Res. 58: 1125-1131; Lawson et al., 1993, Journal of Clinical Microbiology 31: 1136-1142; and McOrist et al., 1995, supra.
Anti-Lawsonia VaccinesThe present invention further provides a vaccine against PPE, comprising an immunologically effective amount of a protein or polypeptide of the present invention, and a pharmaceutically acceptable carrier. In a preferred embodiment, the vaccine comprises a HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 L. intracellularis protein.
The present invention further provides a vaccine against PPE, comprising an immunologically effective amount of one or more polynucleotide molecules of the present invention, and a pharmaceutically acceptable carrier. In a preferred embodiment, the vaccine comprises a polynucleotide molecule having a nucleotide sequence encoding L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100.
The present invention further provides a vaccine against PPE, comprising an immunologically effective amount of modified Lawsonia bacteria of the present invention, and a pharmaceutically acceptable carrier. In one embodiment, the modified Lawsonia cells for use in the vaccine of the present invention are live L. intracellularis bacteria which express a HtrA−, PonA−, HypC−, LysS−, YcfW−, ABC1−, or Omp100− phenotype. Alternatively, the vaccine of the present invention can comprise any of such modified Lawsonia cells of the present invention that have been inactivated. Inactivation of modified Lawsonia cells can be carried out using any techniques known in the art, including by chemical treatment, such as with binary ethylenimine (BEI), or beta-propiolactone, or formaldehyde, or by freeze-thawing or heat treatment, or by homogenization of cells, or by a combination of these types of techniques. Vaccines prepared from homogenized, modified Lawsonia cells can consist of either the entire unfractionated cell homogenate, or an immunologically effective subfraction thereof.
As used herein, the term “immunologically effective amount” refers to that amount of antigen, e.g., protein, polypeptide, polynucleotide molecule, or modified cells, capable of inducing a protective response against PPE when administered to a member of a PPE susceptible animal species after either a single administration, or after multiple administrations.
The phrase “capable of inducing a protective response” is used broadly herein to include the induction or enhancement of any immune-based response in the animal in response to vaccination, including either an antibody or cell-mediated immune response, or both, that serves to protect the vaccinated animal against PPE. The terms “protective response” and “protect” as used herein to refer not only to the absolute prevention of PPE or absolute prevention of infection by Lawsonia, but also to any detectable reduction in the degree or rate of infection by such a pathogen, or any detectable reduction in the severity of the disease or any symptom or condition resulting from infection by the pathogen, including, e.g., any detectable reduction in the rate of formation, or in the absolute number, of lesions formed in one or more tissues, or the transmission of infection to other animals, in the vaccinated animal as compared to an unvaccinated infected animal of the same species.
In a further preferred embodiment, the vaccine of the present invention is a combination vaccine for protecting a PPE susceptible animal against PPE and, optionally, one or more other diseases or pathological conditions that can afflict the animal, which combination vaccine comprises an immunologically effective amount of a first component comprising a polypeptide, polynucleotide molecule, or modified Lawsonia cells of the present invention; an immunologically effective amount of a second component that is different from the first component, and that is capable of inducing, or contributing to the induction of, a protective response against a disease or pathological condition that can afflict the PPE susceptible animal; and a pharmaceutically acceptable carrier.
The second component of the combination vaccine is selected based on its ability to induce, or contribute to the induction of, a protective response against either PPE or another disease or pathological condition that can afflict members of the relevant species, as known in the art. Any antigenic component that is useful in a vaccine composition in the particular species can be used as the second component of the combination vaccine. Such antigenic components include but are not limited to those that provide protection against pathogens selected from the group consisting of Leptospira spp., Campylobacter spp., Staphylococcus aureus, Streptococcus agalactiae, Streptococcus suis, Mycoplasma spp., Klebsiella spp., Salmonella spp., rotavirus, coronavirus, rabies, Pasteurella hemolytica, Pasteurella multocida, Clostridia spp., Tetanus toxoid, E. coli, Cryptosporidium spp., Eimeria spp., Trichomonas spp., Serpulina (Brachyspira) hyodysenteriae, Actinobacillus pleuropneumoniae, Actinobacillus suis, Salmonella cholerasuis, Erysipelothrix rhusiopathiae, Leptospira sp., Staphylococcus hyicus, Haemophilus parasuis, Bordetella bronchiseptica, Mycoplasma hyopneumoniae, porcine reproductive and respiratory syndrome virus, swine influence virus, porcine immunodeficiency virus, transmissible gastroenteritis virus, porcine parvovirus, encophalomyocarditis virus, coronavirus, pseudorabies virus, circovirus and other eukaryotic parasites.
In one embodiment, the combination vaccine of the present invention comprises a combination of two or more components selected from the group consisting of an immunologically effective amount of a protein or polypeptide of the present invention, an immunologically effective amount of a polynucleotide molecule of the present invention, and an immunologically effective amount of modified Lawsonia cells of the present invention.
The vaccines of the present invention can further comprise one or more additional immunomodulatory components including, e.g., an adjuvant or cytokine, as described below.
The present invention further provides a method of preparing a vaccine against PPE, comprising combining an immunologically effective amount of a L. intracellularis protein or polypeptide, or polynucleotide molecule, or modified Lawsonia cells, of the present invention, with a pharmaceutically acceptable carrier, in a form suitable for administration to a PPE susceptible animal. In a preferred embodiment, the protein is L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100, the polynucleotide molecule preferably comprises a nucleotide sequence encoding L. intracellularis HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 protein and the modified Lawsonia bacteria has an HtrA−, PonA−, HypC−, LysS−, YcfW−, ABC1−, or Omp100− phenotype.
A vaccine comprising modified live Lawsonia cells of the present invention can be prepared using an aliquot of culture fluid containing said Lawsonia cells, either free in the medium or residing in mammalian host cells, or both, and can be administered directly or in concentrated form to the PPE susceptible animal. Alternatively, modified live Lawsonia cells can be combined with a pharmaceutically acceptable carrier, with or without an immunomodulatory agent, selected from those known in the art and appropriate to the chosen route of administration, preferably where at least some degree of viability of the modified live Lawsonia cells in the vaccine composition is maintained.
Vaccine compositions of the present invention can be formulated following accepted convention to include pharmaceutically acceptable carriers, such as standard buffers, stabilizers, diluents, preservatives, and/or solubilizers, and can also be formulated to facilitate sustained release. Diluents include water, saline, dextrose, ethanol, glycerol, and the like. Additives for isotonicity include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin, among others. Suitable other vaccine vehicles and additives, including those that are particularly useful in formulating modified live vaccines, are known or will be apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Science, 18th ed., 1990, Mack Publishing, which is incorporated herein by reference.
The vaccine of the present invention can further comprise one or more additional immunomodulatory components such as, e.g., an adjuvant or cytokine, among others. Non-limiting examples of adjuvants that can be used in the vaccine of the present invention include the RIBI adjuvant system (Ribi Inc., Hamilton, Mont.), alum, mineral gels such as aluminum hydroxide gel, oil-in-water emulsions, water-in-oil emulsions such as, e.g., Freund's complete and incomplete adjuvants, Block co polymer (CytRx, Atlanta Ga.), QS-21 (Cambridge Biotech Inc., Cambridge Mass.), SAF-M (Chiron, Emeryville Calif.), AMPHIGEN adjuvant, saponin, Quil A or other saponin fraction, monophosphoryl lipid A, Avridine lipid-amine adjuvant, and protein adjuvants such as Vibrio cholera toxin and E. coli labile toxin. Specific non-limiting examples of oil-in-water emulsions useful in the vaccine of the invention include modified SEAM62 and SEAM ½ formulations. Modified SEAM62 is an oil-in-water emulsion containing 5% (v/v) squalene (Sigma), 1% (v/v) SPAN® 85 detergent (ICI Surfactants), 0.7% (v/v) TWEEN® 80 detergent (ICI Surfactants), 2.5% (v/v) ethanol, 200 μg/ml Quil A, 100 μg/ml cholesterol, and 0.5% (v/v) lecithin. Modified SEAM ½ is an oil-in-water emulsion comprising 5% (v/v) squalene, 1%. (v/v) SPAN® 85 detergent, 0.7% (v/v) Tween 80 detergent, 2.5% (v/v) ethanol, 100 μg/ml Quil A, and 50 μg/ml cholesterol. Other immunomodulatory agents that can be included in the vaccine include, e.g., one or more interleukins, interferons, or other known cytokines. Where the vaccine comprises modified live Lawsonia cells, the adjuvant is preferably selected based on the ability of the resulting vaccine formulation to maintain at least some degree of viability of the modified live Lawsonia cells.
Where the vaccine composition comprises a polynucleotide molecule, the polynucleotide molecule can either be DNA or RNA, although DNA is preferred, and is preferably administered to a PPE susceptible animal to be protected against PPE in an expression vector construct, such as a recombinant plasmid or viral vector, as known in the art. Examples of recombinant viral vectors include recombinant adenovirus vectors and recombinant retrovirus vectors. The vaccine formulation can also comprise a non-viral DNA vector, such as a DNA plasmid-based vector. The polynucleotide molecule may be associated with lipids to form, e.g., DNA-lipid complexes, such as liposomes or cochleates. See, e.g., International Patent Publication WO 93/24640.
An expression vector useful as a vaccinal agent in a DNA vaccine preferably comprises a nucleotide sequence encoding one or more antigenic Lawsonia proteins, or a substantial portion of such a nucleotide sequence, in operative association with one or more transcriptional regulatory elements required for expression of the Lawsonia coding sequence in a eukaryotic cell, such as, e.g., a promoter sequence, as known in the art. In a preferred embodiment, the regulatory element is a strong viral promoter such as, e.g., a viral promoter from RSV or CMV. Such an expression vector also preferably includes a bacterial origin of replication and a prokaryotic selectable marker gene for cloning purposes, and a polyadenylation sequence to ensure appropriate termination of the expressed mRNA. A signal sequence may also be included to direct cellular secretion of the expressed protein.
The requirements for expression vectors useful as vaccinal agents in DNA vaccines are further described in U.S. Pat. No. 5,703,055, U.S. Pat. No. 5,580,859, U.S. Pat. No. 5,589,466, International Patent Publication WO 98/35562, and in various scientific publications, including Ramsay et al., 1997, Immunol. Cell Biol. 75: 360-363; Davis, 1997, Cur. Opinion Biotech. 8: 635-640; Maniackan et al., 1997, Critical Rev. Immunol. 17: 139-154; Robinson, 1997, Vaccine 15(8):785-787; Lai and Bennett, 1998, Critical Rev. Immunol. 18: 449-484; and Vogel and Sarver, 1995, Clin. Microbiol. Rev. 8(3):406-410, among others.
Where the vaccine composition comprises modified live Lawsonia cells, the vaccine can be stored cold, frozen, or lyophilized. Where the vaccine composition instead comprises a protein, polypeptide, polynucleotide molecule, or inactivated modified Lawsonia cells of the present invention, the vaccine may be stored cold, frozen, or in lyophilized form to be rehydrated prior to administration using an appropriate diluent.
The vaccine of the present invention can optionally be formulated for sustained release of the antigen. Examples of such sustained release formulations include antigen in combination with composites of biocompatible polymers, such as, e.g., poly(lactic acid), poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid, collagen and the like. The structure, selection and use of degradable polymers in drug delivery vehicles have been reviewed in several publications, including A. Domb et al., 1992, Polymers for Advanced Technologies 3: 279-292, which is incorporated herein by reference. Additional guidance in selecting and using polymers in pharmaceutical formulations can be found in the text by M. Chasin and R. Langer (eds), 1990, “Biodegradable Polymers as Drug Delivery Systems” in: Drugs and the Pharmaceutical Sciences, Vol. 45, M. Dekker, NY, which is also incorporated herein by reference. Alternatively, or additionally, the antigen can be microencapsulated to improve administration and efficacy. Methods for microencapsulating antigens are well-known in the art, and include techniques described, e.g., in U.S. Pat. No. 3,137,631; U.S. Pat. No. 3,959,457; U.S. Pat. No. 4,205,060; U.S. Pat. No. 4,606,940; U.S. Pat. No. 4,744,933; U.S. Pat. No. 5,132,117; and International Patent Publication WO 95/28227, all of which are incorporated herein by reference.
Liposomes can also be used to provide for the sustained release of antigen. Details concerning how to make and use liposomal formulations can be found in, among other places, U.S. Pat. No. 4,016,100; U.S. Pat. No. 4,452,747; U.S. Pat. No. 4,921,706; U.S. Pat. No. 4,927,637; U.S. Pat. No. 4,944,948; U.S. Pat. No. 5,008,050; and U.S. Pat. No. 5,009,956, all of which are incorporated herein by reference.
The present invention further provides a method of vaccinating a PPE susceptible animal against PPE, comprising administering to the animal an immunogenically effective amount of a vaccine of the present invention. The vaccine is preferably administered parenterally, e.g., either by subcutaneous or intramuscular injection. However, the vaccine can also be administered by intraperitoneal or intravenous injection, or by other routes, including, e.g., orally, intranasally, rectally, vaginally, intra-ocularly, or by a combination of routes, and also by delayed release devices as known in the art. The skilled artisan can determine optimal routes of vaccine administration, and recognize acceptable formulations for the vaccine composition according to the chosen route of administration.
An effective dosage can be determined by conventional means, starting with a low dose of antigen, and then increasing the dosage while monitoring its effects. Numerous factors may be taken into consideration when determining an optimal dose per animal. Primary among these is the species, size, age and general condition of the animal, the presence of other drugs in the animal, the virulence of a particular strain of Lawsonia against which the animal is being vaccinated, and the like. The actual dosage is preferably chosen after consideration of the results from other animal studies.
The dose amount of a protein or polypeptide of the present invention in a vaccine of the present invention preferably ranges from about 1 μg to about 10 mg, more preferably from about 50 μg to about 1 mg, and most preferably from about 100 μg to about 0.5 mg. The dose amount of a Lawsonia polynucleotide molecule of the present invention in a vaccine of the present invention preferably ranges from about 50 μg to about 1 mg. The dose amount of modified Lawsonia cells of the present invention in a vaccine of the present invention preferably ranges from about 1×103 to about 1×108 cells/ml, and more preferably from about 1×105 to about 1×107 cells/ml. A suitable dosage size ranges from about 0.1 ml to about 10 ml, and more preferably from about 1 ml to about 5 ml. The dose amounts of these antigens are also applicable to combination vaccines of the present invention. Where the second component of the combination vaccine is an antigen other than a Lawsonia protein, polypeptide, polynucleotide or modified cell of the present invention, the dose amount of the second component for use in the combination vaccine can be determined from prior vaccine applications of that second component, as known in the art.
The vaccine of the present invention is useful to protect animals, especially pigs, against PPE. The vaccine can be administered to any suitable animals, including, without limitation, hamsters, ferrets, guinea pigs, deer, and bovine, equine, and avian species. The vaccine of the invention can be administered at any time during the life of a particular animal depending upon several factors including, e.g., the timing of an outbreak of PPE among other animals, etc. The vaccine can be administered to animals of weaning age or younger, or to more mature animals. Effective protection may require only a primary vaccination, or one or more booster vaccinations may also be needed. One method of detecting whether adequate immune protection has been achieved is to determine seroconversion and antibody titer in the animal after vaccination. The timing of vaccination and the number of boosters, if any, is preferably determined by a veterinarian based on analysis of all relevant factors, some of which are described above.
In one embodiment, a protein or polypeptide of the invention, e.g., HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 L. intracellularis protein, or combinations thereof, is administered in a formulation containing 100 μg of polypeptide, and 25 μg of E. coli labile toxin as adjuvant, in 1 ml of buffered solution. The formulation is, for example, administered intramuscularly to pigs at between 5 and 7 days of age, and readministered 14 days later.
The present invention further provides a kit for vaccinating a PPE susceptible animal against PPE, comprising a container having an immunologically effective amount of a polypeptide, polynucleotide molecule, or modified Lawsonia cells of the present invention, or a combination thereof. The kit can optionally comprise a second container having a pharmaceutically acceptable carrier or diluent. In a preferred embodiment, the polypeptide is the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 L. intracellularis protein; the polynucleotide molecule preferably has a nucleotide sequence that encodes the HtrA, PonA, HypC, LysS, YcfW, ABC1, or Omp100 L. intracellularis protein and the modified Lawsonia cells preferably are live or inactivated cells that express an HtrA−, PonA−, HypC−, LysS−, YcfW−, ABC1−, or Omp100− phenotype.
The invention also relates to a kit for detecting the presence of L. intracellularis, an L. intracellularis specific amino acid or nucleotide sequence, or an anti-L. intracellularis antibody, that contains a protein, polypeptide, polynucleotide, or antibody of the invention. The kit can also contain means for detecting the protein, polypeptide, polynucleotide, or antibody of the invention including, for example, an enzyme, fluorescent, or radioactive label attached to the protein, polypeptide, polynucleotide, or antibody, or attached to a moiety that binds to the protein, polypeptide, polynucleotide, or antibody.
The following examples are illustrative only, and not intended to limit the scope of the present invention.
EXAMPLES Example 1 Molecular Cloning of L. intracellularis Chromosomal Region A Isolation of DNA and Construction of DNA LibrariesTemplate DNA was purified from pig intestinal mucosa isolated from the ileum of pigs experimentally infected with L. intracellularis. DNA purification from homogenized intestinal mucosa was performed according to (1) the method of Nollau et al. (Nollau et al., 1996, BioTechniques 20: 784-788) or (2) phenol extraction and sodium acetate-ethanol precipitation of DNA. To facilitate cloning of L. intracellularis gene sequences, several genomic libraries were constructed. These libraries were specifically modified by ligation of a known sequence (Vectorette II™, Genosys Biotechnologies, Inc., The Woodlands, Tex.) to the 5′ and 3′ ends of restricted DNA fragments. Vectorette™ libraries were constructed by separately digesting aliquots of L. intracellularis-infected pig mucosal DNA extract with restriction endonucleases HindIII, EcoRI, DraI or HpaI at 37° overnight. The reaction was then spiked with additional fresh restriction enzyme and adjusted to 2 mM ATP, 2 mM DTT final concentration. Vectorette™ tailing was carried out by addition of T4 DNA Ligase (400 U) plus 3 pMol of the appropriate compatible Vectorette™ linker (HindIII Vectorette™: HindIII digested DNA; EcoRI: EcoRI digested DNA; Blunt: DraI, HpaI digested DNA). The mixture was incubated for three cycles at 20°, 60 min; and 37°, 30 min to complete the tailing reaction then adjusted to 200 μl with water and stored at −20°.
Molecular Cloning of Genomic Region A Encoding LysS, YcfW, ABC1, and Omp100 Proteins
Identification of genomic Region A (shown in
For polymerase chain amplification of a fragment of the omp100 gene, oligonucleotides ER159 (SEQ ID NO: 37), ER161 (SEQ ID NO: 38), and ER162 (SEQ ID NO: 39) were used in combination with a Vectorette™ specific primer (ER70) (SEQ ID NO: 33) in 50 μl reactions containing 1×PCR Buffer II (Perkin Elmer), 1.5 mM MgCl2, 200 μM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq™ Gold (Perkin Elmer) thermostable polymerase. Multiple single reactions were performed with 4 μl of the Vectorette™ libraries as DNA template. Amplification was carried out as follows: denaturation (95° 9 min); 40 cycles of denaturation (95° 30 sec), annealing (65° 30 sec), and polymerization (72° 2.5 min); followed by a final extension at 72° for 7 minutes.
The amplified products were visualized by separation on a 1.2% agarose gel (Sigma). An approximately 2.5 kb product resulted from amplification of the HpaI Vectorette™ library when either ER159 or ER162 were used in combination with the Vectorette™-specific primer, ER70. This fragment represented a 1.9 kb region immediately upstream of the L. intracellularis gene encoding AmiB. The PCR product was purified following agarose gel electrophoresis using JETsorb™ kit (GENOMED, Inc., Research Triangle Park, NC) and cloned into pCR2.1 Topo (Invitrogen, Carlsbad, Calif.) to generate plasmid pER393. The insert was partially sequenced using vector specific primers. The sequence obtained was analyzed by the BLASTx algorithm (Altschul et al., 1990, J. Mol. Biol. 215: 403-10) and shown to partially encode a protein with similarity to an approximately 85 kDa protein in the GenBank database. The reported proteins from Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida were Omp85, protective surface antigen D15, and Oma87, respectively.
Based on the newly identified sequence of this partial gene fragment (encoding about the C-terminal ⅔ of the Omp100 protein) specific primers ER174 (SEQ ID NO: 46) and ER175 (SEQ ID NO: 47) were designed to obtain additional 5′ flanking sequences by a second round of screening the Vectorette™ libraries by PCR amplification. Oligonucleotides ER174 (SEQ ID NO: 46) and ER175 (SEQ ID NO: 47) were used in combination with primer ER70 (SEQ ID NO: 33) in 50 μl reactions containing 1×PCR Buffer II (Perkin Elmer), 1.5 mM MgCl2, 200 μM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq Gold (Perkin Elmer) thermostable polymerase. Multiple single reactions were performed with 2 μl of the Vectorette™ EcoRI and DraI libraries as DNA template. Amplification was carried out as follows: denaturation (95° 9 min); 35 cycles of denaturation (95° 30 sec), annealing (62° 30 sec), and polymerization (72° 2.5 min); followed by a final extension at 72° for 7 minutes.
Screening of the EcoRI and DraI Vectorette™ libraries by PCR (employing either ER174 or ER175 in combination with ER70) resulted in successful amplification of an approximately 1.4 kb fragment from the EcoRI Vectorette™ library. The PCR product was purified following agarose gel electrophoresis using JETsorb™ kit and cloned into pCR2.1 Topo to generate plasmid pER394. Sequence analysis of the insert termini within pER394 using ER175 and a vector specific primer confirmed this fragment was contiguous (e.g. overlapped) with the fragment insert within pER393 and allowed determination of the 5′ end of the omp100 gene. This analysis also indicated the presence of an additional partial ORF having homology to the ATP-binding cassette (ABC) superfamily of transporter proteins. Accordingly, the encoded partial protein was designated ABC1.
Based on the newly identified nucleotide sequence of this partial gene fragment (encoding about the C-terminal ½ of the ABC1 protein) specific primer ER188 (SEQ ID NO: 55) was designed to obtain additional 5′ flanking sequence by a third round of screening the Vectorette™ libraries by PCR amplification. Oligonucleotide ER188 (SEQ ID NO: 55) was used in combination with primer ER70 (SEQ ID NO: 33) in 50 μl reactions containing 1×PCR Buffer II, 1.5 mM MgCl2, 200 μM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq™ Gold thermostable polymerase. Multiple single reactions were performed with 4 μl of the Vectorette™ HindIII, DraI, and HpaI libraries as DNA template. Amplification was carried out as follows: denaturation (95° 9 min); 30 cycles of denaturation (95° 30 sec), annealing (60° 30 sec), and polymerization (72° 2.5 min); followed by a final extension at 72° for 7 minutes.
Screening of the HindIII, DraI, and HpaI Vectorette™ libraries by PCR (employing ER188 in combination with ER70) resulted in successful amplification of an approximately 0.8 kb fragment from the HindIII Vectorette™ library. The PCR product was purified following agarose gel electrophoresis using JETsorb™ kit and cloned into pCR2.1 Topo to generate plasmid pER395. Sequence analysis of the insert termini within pER395 using ER188 and vector specific primers confirmed this fragment was contiguous (e.g. overlapped) with the fragment insert within pER394 and allowed determination of the 5′ end of the abc1 gene. An additional partial ORF was identified upstream of the abc1 gene. The encoded protein was designated YcfW based on its homology with the conserved protein, YcfW, found in numerous bacteria.
The region encoding the remaining portion of the ycfW ORF was obtained by screening a Lambda ZAP Express™ phage library created by partial Tsp509I digestion of L. intracellularis genomic DNA. The phage library was plated onto XL1-Blue MRF′ E. coli cells in the presence of 10 mM MgSO4, IPTG, and X-Gal. Clear plaques were picked and phage inserts were amplified using the Expand Long Template PCR System™ as recommended by the supplier (Boehringer Mannheim, Indianapolis, Ind.). The T3 and T7 phage specific primers were used in PCR conditions consisting of denaturation (94° 2 min); 25 cycles of denaturation (94° 10 sec), annealing (50° 30 sec), and polymerization (68° 6 min); followed by a final extension at 68° for 7 min. Resulting PCR products were end-sequenced using the T3 and T7 primers and compared to genes in the GeneBank database by BLASTx analysis. One phage, designated clone A21, contained an approximately 6.1 kb insert encompassing 2.8 kb which overlapped the previously identified ycfW, ABC1, and omp100 DNA sequence. Accordingly this clone was used to determine the DNA sequence encoding the N-terminus of the YcfW protein. An additional ORF was identified upstream of the ycfW gene. This gene encoded a protein which shares homology with several lysyl-tRNA synthetases and was designated lysS.
The preliminary nucleotide sequence for the omp100 and C-terminal portion of the abc1 genes was obtained by sequencing the inserts within pER393 and pER394. Preliminary nucleotide sequence encoding the C-terminal 141 amino acid portion of YcfW and amino-terminal portion of ABC1 was obtained by sequencing the insert within pER395. Preliminary nucleotide sequence encoding the lysS and N-terminal portion of the ycfW gene was obtained by sequencing the PCR product representing the insert contained in phage clone A21. The primers employed for preliminary sequencing included the vector-specific M13 forward, M13 reverse, phage T3 and T7 primers in addition to ER159 (SEQ ID NO: 37), ER169 (SEQ ID NO: 41), ER170 (SEQ ID NO: 42), ER176 (SEQ ID NO: 48), and ER177 (SEQ ID NO: 49) for pER393; ER175 (SEQ ID NO: 47), ER185 (SEQ ID NO: 52), ER186 (SEQ ID NO: 53), and ER187 (SEQ ID NO: 54) for pER394; ER188 (SEQ ID NO: 55) for pER395; and ER246 (SEQ ID NO: 97), ER254 (SEQ ID NO: 98), ER255 (SEQ ID NO: 99), ER256 (SEQ ID NO: 100), and ER257 (SEQ ID NO: 101) for phage clone A21.
Specific PCR Amplification of Subgenomic DNA Fragments Encompassing L. intracellularis Region A
Results of the cloning and preliminary sequencing from the genomic fragments contained in plasmids pER393, pER394, pER395 and phage clone A21 were used to design oligonucleotide primers for the specific amplification of overlapping subgenomic fragments directly from L. intracellularis-infected pig mucosal DNA extracts. DNA extraction was carried out according to the methods described above. This approach was preferred based on the desire to eliminate introduction of sequencing artifacts due to possible mutations arising during the cloning of gene fragments in E. coli. Oligonucleotides ER246 (SEQ ID NO: 97) and ER254 (SEQ ID NO: 98), which flank the lysS and N-terminal ¾ of ycfW; oligonucleotides ER229 (SEQ ID NO: 73) and ER206 (SEQ ID NO: 66), which flank the abc1 gene; and ER231 (SEQ ID NO: 75) and ER232 (SEQ ID NO: 76), which flank the omp100 gene, were used to specifically amplify this region from the mucosal DNA extract. The lysS gene was amplified in a PCR reaction containing 2 μl mucosal DNA extract as template, 1× PC2 buffer (Ab Peptides, Inc.), 200 μM each dNTP, 50 pMol each primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable polymerases in a 50 μl final sample volume. Conditions for amplification consisted of denaturation at 94° for 5 minutes followed by 30 cycles of denaturation (94° 1 minute), annealing (55° 30 seconds), and polymerization (72° 3 minuntes). A final extension at 72° for 7 minutes completed the amplification of the targeted 2.6 kb region. The abc1 gene was amplified in triplicate PCR reactions containing 1 μl mucosal DNA extract as template, 1×PC2 buffer, 200 μM each dNTP, 50 pMol each primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable polymerases in a 50 μl final sample volume. Conditions for amplification consisted of denaturation at 95° for 5 min followed by 33 cycles of denaturation (94° 1 min), annealing (58° 30 sec), and polymerization (72° 80 sec). A final extension at 72° for 10 minutes completed the amplification of the targeted gene region. The omp100 gene was amplified in quadruplicate PCR reactions containing 2 μl mucosal DNA extract as template, 1× PC2 buffer, 200 μM each dNTP, 50 pMol each primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable polymerases in a 50 μl final sample volume. Conditions for amplification consisted of denaturation at 94° for 5 min followed by 35 cycles of denaturation (94° 30 sec), annealing (60° 30 sec), and polymerization (72° 3 min). A final extension at 72° for 7 minutes completed the amplification of the targeted gene region. Following amplification, each of the samples were pooled if appropriate and the specific product was purified by agarose gel electrophoresis prior to direct sequence analysis using DyeDeoxy™ termination reactions on an ABI automated DNA sequencer (Lark Technologies, Inc., Houston, Tex.).
Synthetic oligonucleotide primers were used to sequence both DNA strands of the amplified products from L. intracellularis. The primers used for sequence analysis included: AP58.1 (SEQ ID NO: 26), AP58.2 (SEQ ID NO: 27), AP59.1 (SEQ ID NO: 28), AP59.2 (SEQ ID NO: 29), AP59.3 (SEQ ID NO: 30), AP59.4 (SEQ ID NO: 31), AP59.5 (SEQ ID NO: 32), ER159 (SEQ ID NO: 37), ER169 (SEQ ID NO: 41), ER170 (SEQ ID NO: 42), ER175 (SEQ ID NO: 47), ER176 (SEQ ID NO: 48), ER177 (SEQ ID NO: 49), ER185 (SEQ ID NO: 52), ER186 (SEQ ID NO: 53), ER187 (SEQ ID NO: 54), ER188 (SEQ ID NO: 55), ER205 (SEQ ID NO: 65), ER206 (SEQ ID NO: 66), ER217 (SEQ ID NO: 71), ER229 (SEQ ID NO: 73), ER230 (SEQ ID NO: 74), RA138 (SEQ ID NO: 79), RA139 (SEQ ID NO: 80), RA140 (SEQ ID NO: 81), AP182.1 (SEQ ID NO: 83), AP182.2 (SEQ ID NO: 84), AP182.3 (SEQ ID NO: 85), AP182.4 (SEQ ID NO: 86), AP182.5 (SEQ ID NO: 87), AP182.6 (SEQ ID NO: 88), AP182.7 (SEQ ID NO: 89), AP182.8 (SEQ ID NO: 90), AP182.9 (SEQ ID NO: 91), AP182.10 (SEQ ID NO: 92), AP182.11 (SEQ ID NO: 93), AP182.12 (SEQ ID NO: 94), AP182.13 (SEQ ID NO: 95), AP182.14 (SEQ ID NO: 96), ER246 (SEQ ID NO: 97), ER254 (SEQ ID NO: 98), ER255 (SEQ ID NO: 99), ER256 (SEQ ID NO: 100), and ER257 (SEQ ID NO: 101).
The nucleotide sequence of the L. intracellularis genomic Region A is listed in SEQ ID NO: 1. The deduced amino acid sequences of the encoded LysS, YcfW, ABC1, and Omp100 proteins within this region are presented in SEQ ID NO: 102, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.
Molecular Analysis of the L. intracellularis Genes and Gene Products Specified by Region A
The L. intracellularis chromosomal Region A identified upstream of the amiB gene encodes proteins designated LysS, YcfW, ABC1, and Omp100 (
Similarity of L. intracellularis LysS Protein to lysyl-tRNA Synthetases
The deduced amino acid sequence of the LysS protein (SEQ ID NO: 102) was compared to other proteins reported in GeneBank by the BLASTp algorithm (Altschul, S. F et al., 1997, Nucleic Acids Res. 25: 3389-3402) and aligned by the CLUSTAL W algorithm (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680). As shown in
Similarity of L. intracellularis YcfW and ABC1 Proteins to Other Hypothetical Proteins
The YcfW protein shares limited homology with a family of conserved hypothetical proteins approximately 40-45 kDa in size. Members of this family are reported to be transmembrane or integral membrane proteins. A structural prediction comparison of representative proteins from this family was carried out using TMPred (EMBnet—European Molecular Biology Network; http://www.ch.embnet.org/index.html). The TMpred program makes a prediction of membrane-spanning regions and their orientation. The algorithm is based on the statistical analysis of TMbase, a database of naturally occurring transmembrane proteins. (Hofmann & Stoffel, 1993, Biol. Chem. Hoppe-Seyler 347: 166). This analysis indicates that homologs within this protein family have three strong transmembrane domains clustered near the C-terminus of the protein. We have noted an extremely well conserved domain at the very carboxyl-terminal four amino acids (LRYE) of representatives from this family. The observation that the C-terminal region contains multiple transmembrane domains while the extreme C-terminus is highly conserved suggests a universal functional requirement associated with this region of the YcfW family of homologous proteins. The L. intracellularis YcfW protein presented in SEQ ID NO: 3 also contains three C-terminal transmembrane domains in addition to the extreme C-terminal amino acids (LRYE). In addition to the three carboxyl domains above, TMPred analysis indicates that residues 19-44 of the YcfW protein are likely to form a transmembrane region. The amino terminus of YcfW was also examined by the PSORT (Ver. 6.4) computer algorithm using networks trained on known secretion signal sequences. This analysis indicates that residues 29-45 are likely to form a transmembrane region (P. Klein et al., 1985, Biochim. Biophys. Acta, 815: 468) which is predicted to act as an uncleavable signal sequence (D. J. McGeoch, Virus Research, 3: 271, 1985 and G. von Heijne, Nucl. Acids Res., 14: 4683, 1986). As shown in
The deduced amino acid sequence of the ABC1 protein (SEQ ID NO: 4) was compared to other known proteins reported in GenBank by the BLASTp algorithm. An especially well conserved region (GASGSGKS) was identified near the amino terminus of ABC1. This region corresponds to the nucleotide-binding motif A (P-loop) present in ABC-type transporters. The ABC-typ proteins consist of a very large superfamily of proteins which have a wide variety of cellular functions. The majority of these proteins are classified as ABC-type proteins based on regional homology within the nucleotide-binding motif and are generally thought to be involved in cellular transport functions.
Similarity of L. intracellularis Omp100 Protein to 85 kDa Proteins
Examination of the amino terminus of Omp100 indicates that amino acids 1-25 are hydrophobic and positively charged which is characteristic of signal sequences (von Heijm, 1985, J. Mol. Biol. 184: 99-105). The SignalP (Ver. 1.1) computer algorithm (Nielsen, H., et. al., 1997, Prot. Engineering, 10: 1-6; http://www.cbs.dtv.dk/services/signalP/), using networks trained on known signal sequences, predicted the most likely cleavage site between amino acids 25 and 26. Thus amino acids 1-25 are predicted to be removed from Omp100 during the outer membrane localization process. The Omp100 C-terminal amino acid is predicted to be a phenylalanine residue, a feature consistent with the correct localization of outer membrane proteins (Struyve, M., 1991, J. Mol. Biol. 218: 141-148).
The deduced amino acid sequence of the Omp100 protein (SEQ ID NO: 5) was compared to other known proteins reported in GenBank by the BLASTp algorithm (Altschul, S. F et al., 1997, Nucleic Acids Res. 25: 3389-3402) and aligned by the CLUSTAL W algorithm (Thompson, J. D. et al., 1994, Nucleic Acids Res. 22: 4673-4680). As shown in
Molecular Cloning of Genomic Region B Encoding PonA, HtrA, HypC, and ORF1 Proteins.
Identification of genomic Region B (shown in
For polymerase chain amplification of a fragment of the ponA gene, oligonucleotides ER142 (SEQ ID NO: 34), ER153 (SEQ ID NO: 35), and ER158 (SEQ ID NO: 36) were used in combination with a Vectorette™ specific primer (ER70) (SEQ ID NO: 33) in 50 μl reactions containing 1× PCR Buffer II, 1.5 mM MgCl2, 200 μM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq™ Gold thermostable polymerase. Multiple single reactions were performed with 4 μl of the Vectorette™ libraries as DNA template. Amplification annealing temperatures, extension times, and number of cycles varied between experiments and were carried out over the following ranges: denaturation (95° 9 min); 35-40 cycles of denaturation (95° 30 sec), annealing (50-60° 30 sec), and polymerization (72° 2.5-3 min); followed by a final extension at 72° for 7 minutes.
The amplified products were visualized by separation on a 1.2% agarose gel. An approximately 1.2 kb product resulted from amplification of the DraI Vectorette™ library when ER158 (SEQ ID NO: 36) was used in combination with the Vectorette™-specific primer, ER70. Conditions leading to specific amplification of this product included denaturation (95° 9 min); 40 cycles of denaturation (95° 30 sec), annealing (60° 30 sec), and polymerization (72° 2.5 min); followed by a final extension at 72° for 7 minutes. This fragment represented a 1.4 kb region immediately downstream of the L. intracellularis gene encoding FlgE. The PCR product was purified following agarose gel electrophoresis using a JETsorb™ kit and cloned into pCR2.1 Topo to generate plasmid pER390. The insert was partially sequenced using ER70 and ER158 primers. The sequence obtained was analyzed by the BLASTx algorithm (Altschul, S. F. et al., 1990) and shown to encode a polypeptide with similarity to the amino terminal one half of penicillin-binding proteins in the GenBank database.
Based on the newly identified sequence of this partial gene, primer ER163 (SEQ ID NO: 40) was designed to obtain additional 3′ flanking sequences by a second round of screening the Vectorette™ libraries. Oligonucleotide ER163 (SEQ ID NO: 40) was used in combination with primer ER70 (SEQ ID NO: 33) in 50 μl reactions containing 1× PCR Buffer II, 1.5 mM MgCl2, 200 μM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq Gold thermostable polymerase. Multiple single reactions were performed with 2 μl of the Vectorette™ HindIII, EcoRI and HpaI libraries as DNA template. Amplification was carried out as follows: denaturation (95° 9 min); 30 cycles of denaturation (95° 30 sec), annealing (62° 30 sec), and polymerization (72° 1.5 min); followed by a final extension at 72° for 7 minutes.
A 2.7 kb fragment was amplified from the HindIII Vectorette™ library. The PCR product was purified following agarose gel electrophoresis using JETsorb™ kit and cloned into pCR2.1 Topo to generate plasmid pER392. Sequence analysis of the cloned insert termini using vector specific primers confirmed this fragment was contiguous with the fragment insert within pER390. This analysis also indicated the presence of an additional partial ORF corresponding to approximately the NH2-terminal 400 residues of the HtrA protein family of serine proteases. Accordingly, the encoded partial protein was designated HtrA.
A third round of genomic walking was carried out to identify additional sequence within the htrA ORF. Specific primer ER173 (SEQ ID NO: 45) was designed based on the known sequence near the 3′ end of the insert within pER392. Oligonucleotide ER173 (SEQ ID NO: 45) was used in combination with primer ER70 (SEQ ID NO: 33) in 50 μl reactions as above. Multiple single reactions were performed with 2 μl of the Vectorette™ DraI and HpaI libraries as DNA template. Amplification (denaturation (95° 9 min); 35 cycles of denaturation (95° 30 sec), annealing (62° 30 sec), and polymerization (72° 2.5 min); followed by a final extension at 72° for 7 minutes) resulted in production of a 0.3 kb fragment from the DraI library. The PCR product was purified following agarose gel electrophoresis using a JETsorb™ kit, cloned into pCR2.1 Topo, and the insert sequenced on both strands using vector specific primers. Sequence and BLASTx analysis indicated that the htrA ORF remained open through the 3′ end of the cloned fragment and that an additional 10 amino acids would be expected to remain before the end of the encoded HtrA protein.
A final round of genomic walking was carried out to identify the remainder of the htrA ORF and 3′ flanking region. Specific primer ER189 (SEQ ID NO: 56) was designed based on the known sequence near the 3′ end of the htrA ORF. Oligonucleotide ER189 (SEQ ID NO: 56) was used in combination with primer ER70 (SEQ ID NO: 33) in 50 μl reactions as above. Multiple single reactions were performed with 4 μl of the Vectorette™ HindIII, EcoRI, and HpaI libraries as DNA template. Amplification was carried out as follows: denaturation (95° 9 min); 30 cycles of denaturation (95° 30 sec), annealing (60° 30 sec), and polymerization (72° 2.5 min); followed by a final extension at 72° for 7 minutes. Amplification resulted in production of an approximately 1 kb fragment from the EcoRI library. The PCR product was purified following agarose gel electrophoresis using a JETsorb™ kit and cloned into pCR2.1 Topo to generate pER396. Sequence analysis of the insert termini within pER396 using vector specific primers allowed determination of the 3′ end of the htrA gene. An additional small ORF was identified downstream of the htrA gene. The encoded protein was designated HypC based on its homology with the HypC protein found in other bacteria. Further downstream from hypC is an additional partial ORF, designated orf1, which is encoded by the opposite DNA strand. This truncated 177 amino acid polypeptide was designated ORF1.
The preliminary nucleotide sequence for the ponA, htrA, hypC, and C-terminal portion of the orf1 genes was obtained by sequencing the inserts within pER390, pER392 and pER396. The primers employed for preliminary sequencing included the vector-specific M13 forward and M13 revers primers in addition to ER193 (SEQ ID NO: 59) and ER194 (SEQ ID NO: 60) for pER390; ER171 (SEQ ID NO: 43), ER172 (SEQ ID NO: 44), ER178 (SEQ ID NO: 50), ER179 (SEQ ID NO: 51), ER190 (SEQ ID NO: 57), and ER191 (SEQ ID NO: 58) for pER392; and ER195 (SEQ ID NO: 61) and ER196 (SEQ ID NO: 62) for pER396.
Specific PCR Amplification of Subgenomic DNA Fragments Encompassing L. intracellularis Region B
Results of the cloning and preliminary sequencing from the genomic fragments contained in plasmids pER390, pER392, and pER396 were used to design oligonucleotide primers for the specific amplification of overlapping subgenomic fragments directly from L. intracellularis-infected pig mucosal DNA extracts (methods described above for DNA extraction were employed). This approach was preferred based on the desire to eliminate introduction of sequencing artifacts due to possible mutations arising during the cloning of gene fragments in E. coli. Oligonucleotides ER228 (SEQ ID NO: 72) and ER190 (SEQ ID NO: 57), which flank the ponA gene; oligonucleotides ER207 (SEQ ID NO: 67) and RA134 (SEQ ID NO: 78), which flank the htrA gene; and oligonucleotides ER189 (SEQ ID NO: 56) and ER196 (SEQ ID NO: 62), which flank the hypC gene were used to specifically amplify this region from the mucosal DNA extract. The endpoints of these fragments overlap thereby allowing specific amplification of subgenomic DNA fragments which are contiguous followed by subsequent confirmation by comparing the terminal nucleotide sequences present in each unique, overlapping DNA fragment. Accordingly, the final sequence represents the complete L. intracellularis genomic Region B.
The overlapping ponA, htrA, and hypC gene regions were amplified in triplicate PCR reactions each containing 1 μl mucosal DNA extract as template, 1× PC2 buffer (Ab Peptides, Inc.), 200 μM each dNTP, 50 pMol each primer, 7.5 U KlenTaq1 and 0.15 U cloned Pfu thermostable polymerases in a 50 μl final sample volume. Conditions for amplification of ponA consisted of denaturation at 95° for 5 min followed by 33 cycles of denaturation (95° 30 sec), annealing (62° 30 sec), and polymerization (72° 3 min). Conditions for amplification of htrA consisted of denaturation at 94° for 5 min followed by 33 cycles of denaturation (95° 30 sec), annealing (58° 30 sec), and polymerization (72° 3 min). Conditions for amplification of hypC consisted of denaturation at 94° for 5 min followed by 33 cycles of denaturation (95° 30 sec), annealing (62° 30 sec), and polymerization (72° 80 sec). A final extension at 72° for 7 minutes completed the amplification of each of the targeted gene regions. Following amplification, each of the samples were pooled separately and the specific product was purified by agarose gel electrophoresis prior to direct sequence analysis using DyeDeoxy™ termination reactions on an ABI automated DNA sequencer (Lark Technologies, Inc., Houston, Tex.).
Synthetic oligonucleotide primers were used to sequence both DNA strands of the amplified products from L. intracellularis. The primers used for sequence analysis included: AP55.1 (SEQ ID NO: 10), AP55.2 (SEQ ID NO: 11), AP55.3 (SEQ ID NO: 12), AP55.4 (SEQ ID NO: 13), AP55.5 (SEQ ID NO: 14), AP55.6 (SEQ ID NO: 15), AP55.7 (SEQ ID NO: 16), AP55.8 (SEQ ID NO: 17), AP55.9 (SEQ ID NO: 18), AP55.10 (SEQ ID NO: 19), AP55.11 (SEQ ID NO: 20), AP56.1 (SEQ ID NO: 21), AP56.2 (SEQ ID NO: 22), AP56.3 (SEQ ID NO: 23), AP57.1 (SEQ ID NO: 24), AP57.2 (SEQ ID NO: 25), ER158 (SEQ ID NO: 36), ER163 (SEQ ID NO: 40), ER171 (SEQ ID NO: 43), ER172 (SEQ ID NO: 44), ER173 (SEQ ID NO: 45), ER178 (SEQ ID NO: 50), ER179 (SEQ ID NO: 51), ER189 (SEQ ID NO: 56), ER190 (SEQ ID NO: 57), ER191 (SEQ ID NO: 58), ER193 (SEQ ID NO: 59), ER194 (SEQ ID NO: 60), ER195 (SEQ ID NO: 61), ER196 (SEQ ID NO: 62), ER203 (SEQ ID NO: 63), ER204 (SEQ ID NO: 64), ER207 (SEQ ID NO: 67), ER208 (SEQ ID NO: 68), ER213 (SEQ ID NO: 69), ER228 (SEQ ID NO: 72), RA133 (SEQ ID NO: 77), RA134 (SEQ ID NO: 78), and RA171 (SEQ ID NO: 82).
The nucleotide sequence of the L. intracellularis genomic Region B is listed in SEQ ID NO: 2. The deduced amino acid sequences of the encoded PonA, HtrA, HypC, and ORF1 proteins within this region are presented in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and SEQ ID NO: 9, respectively.
Molecular Analysis of the L. intracellularis Genes and Gene Products Specified by Region B
The L. intracellularis chromosomal Region B identified downstream of the flgE gene encodes proteins designated PonA, HtrA, HypC, and a partial “ORF1” protein (
Similarity of L. intracellularis HypC prot into Hydrogenase Maturation Proteins
The HypC protein shares homology with the hyp/hup family of hydrogenase maturation proteins. Hydrogenase, which catalyzes the reversible oxidation of molecular hydrogen, is involved in many relevant anaerobic processes where hydrogen is oxidized or produced (Adams, M. W. W., et al., 1980, Biochem. Biophys. Acta 594: 105-176). The HypC protein is required for the maturation of catalytically active hydrogenase isozymes in E coli. The precise role of HypC in this process is unknown but hydrogenase maturation involves nickel insertion, protein folding, C-terminal proteolytic processing, membrane integration, and reductive activation (Lutz, S., et al., 1991, Mol. Microbiol. 5: 123-135; Przybyla, A. E., et al., 1992, FEMS Microbiol. Rev. 88: 109-136). The HypC protein is 41% identical to the Desulfovibrio gigas 82 amino acid HynD protein (Accn. AJ223669, as shown in
Similarity of L. intracellularis PonA Protein to Penicillin Binding Proteins The ponA ORF encodes a deduced 812 amino acid protein, having a theoretical molecular weight of about 90,263 daltons. An alternative in-frame methionine codon is present which encodes a slightly smaller 804 amino acid protein having a theoretical molecular weight of about 89,313 daftons. Similar in-frame methionine codons have been identified in other characterized ponA ORF's. For example, PonA homologs from Neisseria flavescens (Accn. AF087677), N. gonorrhoeae (Accn. U72876), and N. meningitidis (Accn. U80933) contain amino-terminal in-frame methionine codons separated by 8, 6, and 6 codons, respectively. As with L. intracellularis, the neisserial ponA genes are preceded by undiscernable ribosome binding sites thus further complicating prediction of the true initiating methionine. N-terminal sequencing of the N. gonorrhoeae FA19 PonA protein indicated the second methionine was the preferred start site in this strain (Ropp et al., 1997, J. Bacteriol. 179: 2783-2787). The upstream methionine codon was used as the putative initiation site for the encoded PonA protein from L. intracellularis.
A structural prediction of the PonA protein was carried out using TMPred. The TMpred program makes a prediction of membrane-spanning regions and their orientation (K. Hofmann & W. Stoffel, 1993. TMbase—A database of membrane spanning proteins segments. Biol. Chem. Hoppe-Seyler 347: 166). This analysis indicates that PonA has a strong transmembrane domain at the NH2-terminus of the protein. The amino terminus of PonA was examined by the PSORT (Ver. 6.4) computer algorithm using networks trained on known signal sequences. This analysis indicates that residues 16-32 are likely to form a transmembrane region (P. Klein et al., 1985, Biochim. Biophys. Acta, 815: 468) which is predicted to act as an uncleavable signal sequence (D. J. McGeoch, Virus Research, 3: 271, 1985 and G. von Heijne, Nucl. Acids Res., 14: 4683, 1986). Thus the amino terminus of PonA is predicted to anchor the protein to the bacterial inner membrane, which is similar to the method of localization of other penicillin-binding proteins.
The PonA protein shares homology with Class A high-molecular-mass penicillin-binding proteins (PBP's) identified in other bacteria. Penicillin-binding proteins are bacterial cytoplasmic membrane proteins involved in the final steps of peptidoglycan synthesis. The Class A proteins generally exhibit two types of enzymatic activities: the glycosyltransferase, which polymerizes the glycan strand and the transpeptidase, which cross-links these strands by their peptide side chains. These reactions are catalyzed either on the outer surface of the cytoplasmic membrane or further outside and the major fraction of the proteins involved in peptidoglycan synthesis is therefore localized in the periplasm. The deduced amino acid sequence of the PonA protein (SEQ ID NO: 6) was compared to other known proteins reported in GenBank by the BLASTp algorithm (Altschul, S. F et al., 1997, supra) and aligned by the CLUSTAL W algorithm (Thompson et al., 1994, supra). As shown in
Similarity of L. intracellularis HtrA Protein to Periplasmic Serine Protease Proteins
Examination of the amino terminus of HtrA indicates that amino acids 1-26 are hydrophobic and positively charged which is characteristic of signal sequences (von Heijm, 1985, J. Mol. Biol. 184: 99-105). The PSORT computer algorithm (Nakai, K., 1991, PROTEINS: Structure, Function, and Genetics 11: 95-110), using networks trained on known signal sequences indicates that residues 1-26 likely function as a typical signal sequence and predicts the most likely cleavage site between amino acids 26 and 27. Thus amino acids 1-26 are predicted to be removed from HtrA during the maturation process.
The deduced amino acid sequence of the HtrA protein (SEQ ID NO: 7) was compared to other known proteins reported in GenBank by the BLASTp algorithm (Altschul, S. F et al., 1997, above) and aligned by the CLUSTAL W algorithm (Thompson, J. D. et al., 1994, supra). This analysis indicated HtrA belongs to the large HtrA/DegP family of periplasmic serine proteases. The reported proteins include those identified from E. coli, Salmonella typhimurium, Camplylbacter jejuni, Haemophilus influenzae, Brucella melitensis, Brucella abortus, Chlamydia trachomatis, Yersinia enterocolitica, Borrelia burgdorferi, and Bacillus subtilis, among others. In some instances the HtrA homolog is referred to as a heat shock protein and has been shown by deletion analysis to be required for bacterial survival at elevated temperatures or for survival of intracellular pathogens. In other cases an HtrA homolog is not induced by temperature but is expressed in response to other physiological stress. Several HtrA homologs have been shown to possess serine protease activity and in a number of cases is important for bacterial virulence and/or intracellular survival, for example resistance to high temperature, hydrogen peroxide, oxidative and osmotic stress.
Alignment of the L. intracellularis HtrA protein with its most similar relative from Pseudomonas aeruginosa (Accn. #U32853) indicates the two proteins share 40% identical amino acid residues (as shown in
Analysis of the htrA Promoter Region and Induction in Response to Temperature
The gene arrangement for L. intracellularis Region A and Region B differ with regard to the extent of intergenic spacing between the encoded proteins. Unlike Region A the ORF's within Region B are more distantly separated. For example, the flgE, ponA, htrA, and hypC genes are separated by approximately 125, 200, and 265 nucleotides between the respective open reading frames. The 200 bp region immediately upstream of htrA was examined in more detail to find a promoter region, particularly since several HtrA protein homologs have been shown to be induced in response to a number of different environmental signals including temperature, oxidative, and osmotic stress. Examination of the nucleotide sequence of SEQ ID NO: 2 upstream of htrA indicated a promoter located about nucleotide 2797-2802 (TTGATA; −35 region) and nucleotide 2824-2829 (TATMT; -10 region). These two hexamers are separated by a 21 nucleotide space and share near perfect homology to consensus sigma 70 type promoters. Other promoter elements may exist in this region which control htrA expression in response to various environmental signals. Plasmid pER434, which contains the htrA ORF and htrA promoter region imparts a temperature-dependent phenotype to E. coli host cells grown at either 30° C. or 37° C. Thus, the region upstream of htrA can be recognized as a likely functional promoter in response to temperature. It should therefore be possible to use the htrA promoter to operably control expression of heterologous proteins in E. coli and other organisms in response to temperature. The presence of other promoter elements that control expression in response to other environmental signals would allow those other signals to be used to control expression.
Example 3 Preparation of Plasmids and Deposit MaterialsPlasmids containing DNA fragments encompassing L. intracellularis Region A Plasmids were prepared containing the L. intracellularis genomic region representing the lysS, ycfW, abc1, and omp100 genes. A 2.6 kb fragment encompassing the lysS gene and a portion of the ycfW gene was amplified using primers ER246 (SEQ ID NO: 97) and ER254 (SEQ ID NO: 98) while a 0.87 kb fragment encompassing a portion of the ycfW gene and complete abc1 gene fragment was amplified using primers ER229 (SEQ ID NO: 73) and ER206 (SEQ ID NO: 66). These fragments were amplified as described in Example 1 under “Specific PCR amplification of subgenomic DNA fragments encompassing L. intracellularis Region A”. The 2.6 kb and 0.87 kb DNA fragments were isolated by extraction with spin chromatography (QIAquick™) and inserted into the TA cloning site of pCR2.1 Topo. Single sequence extension reactions utilizing vector-specific sequencing primers confirmed the endpoints of the cloned fragments, and revealed that the genes encoding LysS and YcfW in plasmid pT068 and YcfW and ABC1 in plasmid pER438 were in the opposite orientation relative to the lactose promoter.
A 2.97 kb DNA fragment containing the omp100 gene was amplified by PCR employing specific 5′ and 3′ primers ER187 (SEQ ID NO: 54) and ER170 (SEQ ID NO: 42). PCR reactions were carried out in triplicate and contained 1 μl DNA extract as template, 1× PCR Buffer II, 1.5 mM MgCl2, 200 μM each deoxy-NTP, 50 pMol each primer, and 2.5 U AmpliTaq Gold thermostable polymerase in a 50 μl final sample volume. Conditions for amplification consisted of denaturation at 95° for 9 min followed by 33 cycles of denaturation (95° 30 sec), annealing (62° 30 sec), and polymerization (72° 3 min). A final extension at 72° for 7 minutes completed the amplification of the target gene region. Following amplification, each of the triplicate samples were pooled and the specific product was isolated by extraction with spin chromatography (QIAquick™) and inserted into the TA cloning site of pCR2.1 Topo in the opposite orientation relative to the lactose promoter. This plasmid construct was designated pER440.
Plasmids pER438 and pER440 were introduced into E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.). The resulting strains, designated Pz438 and Pz440, were deposited with the ATCC (10801 University Blvd, Manassas, Va., 20110, USA) on Sep. 9, 1999 and assigned accession numbers PTA-638 and PTA-640 respectively. Plasmid pT068 was introduced into E. coli TOP10 cells and the resulting strain was deposited with the ATCC on Jul. 14, 2000 and assigned accession number PTA-2232.
Plasmids Containing DNA Fragments Encompassing L. intracellularis Region B
Plasmids were prepared containing the L. intracellularis genomic region representing the ponA, htrA, and hypC genes. The ponA, htrA, and hypC gene fragments were amplified as described above in Example 2, in the section entitled “Specific PCR amplification of subgenomic DNA fragments encompassing L. intracellularis Region B” using primers ER228 (SEQ ID NO: 72) and ER190 (SEQ ID NO: 57), which flank the ponA gene; primers ER207 (SEQ ID NO: 67) and RA134 (SEQ ID NO: 78), which flank the htrA gene; and primers ER189 (SEQ ID NO: 56) and ER196 (SEQ ID NO: 62), which flank the hypC gene. The resulting 2.98 kb fragment containing ponA was purified following agarose gel electrophoresis using a JETsorb™ kit and cloned into pCR2.1 Topo to generate plasmid pER432. The resulting 1.72 kb fragment containing htrA was isolated by extraction with spin chromotography (QIAquick™) and inserted into the TA cloning site of pCR2.1 Topo to generate plasmid pER434. The resulting 0.98 kb fragment containing hypC and additional flanking nucleotides encoding the C-terminal 94 amino acids of ORF1 was isolated by extraction with spin chromotography (QIAquick™) and inserted into the TA cloning site of pCR2.1 Topo to generate plasmid pER436. Single sequence extension reactions utilizing vector-specific sequencing primers confirmed the endpoints of the cloned fragments, and revealed that the genes encoding PonA and HypC were in the opposite orientation relative to the lactose promoter. The HtrA gene was cloned in the same orientation relative to the lactose promoter and cells containing such plasmids exhibited an unstable phenotype at 37° C. which was relieved when growth was maintained at 30° C.
Plasmids pER432, pER434 and pER436 were introduced into E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.). The resulting strains, designated Pz432, Pz434, and Pz436 were deposited with the ATCC (10801 University Blvd, Manassas, Va., 20110, USA) on Sep. 9, 1999 and assigned the accession numbers PTA-635, PTA-636, and PTA-637, respectively.
Example 4 Expression of Recombinant HtrA and Omp100 Proteins in E. coliPlasmid Expression Vectors
The expression vector used for production of recombinant HtrA and Omp100 was pET-28b (+) (Novagen, Inc., Madison, Wis.). The coding sequences for the HtrA and Omp100 proteins were amplified from L. intracellularis-infected pig mucosal DNA extract. The PCR products were purified following agarose gel electrophoresis using a JETsorb™ kit and cloned into pCR2.1 Topo to generate plasmids pRL001 (HtrA) and pER415 (Omp100). Specific PCR primers used to amplify the HtrA ORF included ER208 (SEQ ID NO: 68) and RA133 (SEQ ID NO: 77). Primer ER208 was designed to introduce an NdeI site (CATATG) while deleting the HtrA signal sequence. The HtrA insert present in pRL001 was subcloned into pET-28b (+) following digestion with NdeI and EcoRI. The resulting expression plasmid, designated pER405, was sequenced at both 5′ and 3′ ends of the inserted fragment and confirmed to encode an in-frame fusion with the vector encoded 6× His leader. Therefore the predicted amino terminal sequence of the encoded protein consisted of the sequence MGSSHHHHHHSSGLVPRGSHM encoded by the vector followed immediately by the sequence ALPNFVP beginning at Alanine-27 of the HtrA open reading frame.
Specific PCR primers used to amplify the Omp100 ORF included ER216 (SEQ ID NO: 70) and RA138 (SEQ ID NO: 79). Primer ER216 was designed to introduce an Nco/site (CCATGG) while deleting the Omp100 signal sequence. In addition, ER216 specified a leader peptide, termed a “protective peptide” which protects recombinant proteins from proteolytic degradation, based on information from Sung et al., 1986, Proc. Natl. Acad. Sci. USA 83: 561-565; Sung et al., 1987, Meth. Enzymol. 153: 385-389; and U.S. Pat. No. 5,460,954, which references are incorporated herein by reference. The protective peptide consisting of the amino acid sequence MGTTTTTTSL was encoded by the 5′ proximal nucleotide sequence of ER216. The Omp100 insert present in pER415 was subcloned into pET-28b (+) following digestion with NcoI and EcoRI. The resulting expression plasmid, designated pRL029, was sequenced at both 5′ and 3′ ends of the inserted fragment and confirmed to encode an in-frame fusion with the protective peptide leader. Therefore the predicted amino terminal sequence of the encoded protein consisted of the sequence MGTTTTTTSL specified by the 5′ proximal nucleotide sequence of ER216 followed immediately by the sequence ASKDDPSIV beginning at Alanine-26 of the Omp100 open reading frame.
Expression of Recombinant Proteins
The pET-28b (+) based expression vectors pER405 and pRL029, encoding recombinant HtrA and Omp100, respectively, were introduced into the expression host E. coli BL21 (DE3). This expression host has the genotype F, ompT hsdSB (rB−mB−) gal dcm (DE3) (Novagen, Inc.) which allows high level transcription of cloned genes driven by the IPTG-inducible phage T7 promoter. The E. coli transformants were propagated in SB#2 medium (2.4% yeast extract, 1.2% tryptone, 0.5% K2HPO4, 0.25% KH2PO4, 0.014% MgSO4) containing 50 μg/ml kanamycin sulfate in a 5 L BioFlow™ 3000 fermentor (New Brunswick Scientific, Edison, N.J.) at 30-37° C. until A625 was 2.5-30.1. Recombinant protein expression was obtained following induction with 1 mM IPTG for 1-4.5 h.
Wet cells of E. coli expressing recombinant HtrA were lysed by homogenization at 10,000 psi (2 passes) followed by centrifugation. The pellet, which contained HtrA, was washed with 2× RIPA/TET which was in a 5: 4 ratio. 2× RIPA is 20 mM Tris (pH 7.4), 0.3 M NaCl, 2.0% sodium deoxycholate, and 2% (v/v) Igepal CA-630™ (Sigma). TET is 0.1 M Tris (pH 8.0), 50 mM EDTA, and 2% (v/v) Triton X-100. The washed pellet was then solubilized in 8 M Urea, 10 mM Tris, 0.1 M NaH2PO4, pH 7.0. The solubilized protein was diluted 2 fold in 8 M Urea, 10 mM Tris, 0.1 M NaH2PO4, pH 7.0 and applied onto a Ni NTA column (QIAGEN, Santa Clarita, Calif.). The desired protein was eluted off this column by reduction of pH into 8 M Urea, 10 mM Tris, 0.1 M NaH2PO4, pH 4.5. The final pooled fractions were dialyzed against 4 M Guanidine HCl, 50 mM Tris, pH 6.5 and then step dialyzed to 2 M Guanidine HCl, 25 mM Tris, pH 6.5. The final product was filtered by 0.22 μM filtration. The protein concentration was 0.56 mg/ml with an estimated visual purity of 70% by SDS-PAGE.
Wet cells of E. coli expressing recombinant Omp100 were lysed with lysozyme and sonication in the presence of Benzonase™ (Benzonase™ (EM Industries Inc, Hawthorne, N.Y.)), to facilitate DNA degradation, followed by centrifugation. The pellet, which contained Omp100, was washed twice with 2 M Urea, 50 mM Tris, 10 mM EDTA, 25 mM DTT, 1% Zwittergent 3-14. The pellet was resuspended with 6 M Urea, 50 mM Tris (pH 8.0) followed by centrifugation. The pellet was washed with 2× RIPA/TET which was in a 5:4 ratio and the washed pellet was then solubilized in 8 M Urea, 50 mM Tris (pH 8.0). 25 mM DTT was added to the solubilized protein and further diluted 2:1 with 8 M Urea, 25 mM DTT, 50 mM Tris (pH 8.0). The diluted solubilized protein was applied onto a Q-Sepharose column equilibrated with 8 M Urea, 25 mM DTT, 50 mM Tris (pH 8.0). Recombinant Omp100 was eluted in a linear gradient of 0-1 M NaCl in 8 M Urea, 25 mM DTT, 50 mM Tris (pH 8.0). The pooled fractions were dialyzed against 6 M Guanidine HCl, 10 mM DTT, 50 mM Tris (pH 8.0) and then step dialyzed to 4 M Guanidine HCl, 6.7 mM DTT, 33.3 mM Tris (pH 8.0). The final product was filtered by 0.22 μM filtration and frozen at −70° C. The purified Omp100 protein was then thawed and centrifuged (16,000 rpm, 60 min) and the supernatant was subjected to 0.22 μM filtration again to remove insoluble particles and aggregates. The protein concentration was 1.08 mg/ml with an estimated visual purity of 80% by SDS-PAGE.
Claims
1-10. (canceled)
11. An isolated L. intracellularis HypC protein consisting of SEQ ID No: 8.
12-13. (canceled)
14. An isolated polypeptide comprising an epitope of a HypC protein consisting of SEQ ID No: 8. that is specifically reactive with anti-Lawsonia antibodies.
15-18. (canceled)
19. An immunogenic composition comprising an immunogenically effective amount of the polypeptide of claim 11 in combination with a pharmaceutically acceptable carrier.
20. (canceled)
Type: Application
Filed: May 30, 2003
Publication Date: Jun 30, 2005
Inventor: Everett Rosey (Preston, CT)
Application Number: 10/449,648
