EP0491859A1 - ANTIGENES CONTRE LA TREPONEMA HYODYSENTERIAE AYANT UNE MASSE MOLECULAIRE DE 39 kDa ET CODAGE D'ADN POUR LEUR PRODUCTION - Google Patents

ANTIGENES CONTRE LA TREPONEMA HYODYSENTERIAE AYANT UNE MASSE MOLECULAIRE DE 39 kDa ET CODAGE D'ADN POUR LEUR PRODUCTION

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Publication number
EP0491859A1
EP0491859A1 EP90914865A EP90914865A EP0491859A1 EP 0491859 A1 EP0491859 A1 EP 0491859A1 EP 90914865 A EP90914865 A EP 90914865A EP 90914865 A EP90914865 A EP 90914865A EP 0491859 A1 EP0491859 A1 EP 0491859A1
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European Patent Office
Prior art keywords
protein
hyo
gene
dna
antigen
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EP90914865A
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German (de)
English (en)
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EP0491859A4 (en
Inventor
Jeffrey Gabe
Elizabeth Dragon
Michael Mccaman
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ML Technology Ventures LP
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ML Technology Ventures LP
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Publication of EP0491859A1 publication Critical patent/EP0491859A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Gram-negative bacteria
    • C07K16/1207—Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to Treponema
  • Treponema hyodysenterlae T. hyo.
  • genes encoding for such antigens genes encoding for such antigens, cells genetically engineered with DNA encoding for such antigens and uses for such
  • this invention relates to Treponema hyodysenterlae antigens having a molecular weight of 39kDa and to the production thereof by recombinant techniques.
  • Swine dysentery is a severe, infectious disease found in all major pig-rearing countries. The symptoms of swine dysentery are severe
  • the present invention is directed to certain antigens which are useful in determining and/or treating Treponema hyodysenterlae and to recombinant or genetic engineering techniques for producing such antigens.
  • a protein which is capable of eliciting at least one antibody which recognizes an epitope of at least one T.hyo antigen having a molecular weight of about 39kDa.
  • T.hyo includes DNA which encodes for a plurality of proteins each having a molecular weight of about 39kDa. Still more particularly, Applicant has found that there are at least eight different genes, each of which encodes for a T.hyo protein having a molecular weight of about 39 kDa.
  • the protein products encoded by such genes have been found to have conserved regions which are interspersed with variable regions. It has been found that the variable regions are generally located in the more hydrophilic portions of the protein whereas the conserved regions are located in the more hydrophobic portions of the protein.
  • each gene encodes a protein product of simlar molecular weight and that there are regions of conserved protein sequence punctuated by regions of variable sequence.
  • the conserved regions are generally in the more hydrophobic portions of the proteins while the variable regions tend to be in the more hydrophilic portions.
  • antigens which are T.hyo antigens which have a molecular weight of about 39 kDa.
  • Such seven genes are hereinafter sometimes referred to as genes 1-8 or copies 1-8.
  • At least eight different genes each of which encodes for a different T.hyo antigen having a molecular weight of about 39 kDa.
  • an expression or cloning vehicle which includes a DNA sequence which encodes for a T.hyo antigen (or fragment or analog thereof), which has a molecular weight of about 39 kDa.
  • a host cell or organism which is genetically engineered with DNA which encodes for a T.hyo antigen (or fragment or derivative thereof), which has a molecular weight of about 39 kDa.
  • the molecular weight for characterizing the 39 kDa T. hyo. antigen or protein is obtained by
  • T.hyo antigens each of which has a molecular weight of about 39 kDa, which are encoded by eight different genes.
  • the DNA sequence may encode for a protein which is the entire 39 kDa antigen, or a fragment or derivative of the antigen, or a fusion product of the antigen or fragment and another protein, provided that the protein which is produced from such DNA sequence elicits antibodies after immunization which recognize an epitope of a 39 kDa T. hyo. antigen.
  • the DNA sequence may encode for a protein which is or contains within it a fragment of a 39 kDa antigen provided that such fragment
  • the DNA sequence may encode for a protein which is a derivative of the antigen e.g., a mutation of one or more amino acids in the peptide chain, as long as such derivative elicits antibodies which recognize an epitope(s) of a 39 kDa T .hyo .
  • the DNA sequence may encode a protein which is a fusion product of (i) a protein which produces antibodies which recognize an epitope(s) of a noted 39 kDa T .hyo. antigen and (ii) another protein (for example chymosin).
  • the 39 kDa antigens may vary somewhat between specific strains of T. hyo.
  • the 39 kDa proteins of serotype B204 have minor
  • DNA sequence which encodes for a protein which produces antibodies which recognize an epitope(s) of a noted 39 kDa T. hyo. antigen encompasses DNA sequences which encode for and/or express in appropriate transformed cells, proteins which may be the appropriate antigen, antigen fragment, antigen derivative or a fusion product of such antigen, antigen fragment or antigen derivative with another protein.
  • sequence present in the vector when introduced into a cell may express only a portion of the protein which is encoded by such DNA sequence, and such DNA
  • the DNA sequence is within the noted terminology, provided that the protein portion expressed elicits antibodies which recognize an epitope(s) of one or more of the noted 39 kDa T. hyo. antigens.
  • the DNA sequence may encode for the entire antigen; however, the expressed protein is a fragment of the antigen.
  • gene (1, 2, 3, 4, 5, 6, 7 or 8) encoding a T. hyo . 39 kDa protein means the entire or full length gene sequence or an analog, fragment or derivative thereof which encodes a protein which is capable of eliciting at least one antibody which recognizes at least one epitope of the full length T. hyo. 39 kDa antigen encoded by such full length gene.
  • protein encoded by gene 1, 2, 3, 4, 5, 6, 7 or 8 means a T . hyo . 39 kDa protein encoded by the entire or full length gene or an analogue, fragment or derivative of such protein which is capable of eliciting at least one antibody which recognizes at least one epitope of the full length T. hyo. 39 kDa antigen encoded by such full length gene.
  • 39 kDa T . hyo. antigen or protein means a T. hyo. antigen or protein having a molecular weight of about 39 kDa.
  • the appropriate DNA sequence may be included in any of a wide variety of vectors or plasmids.
  • vectors include chromosomal, nonchromosonal and synthetic DNA sequences; e.g., derivatives of SV40; bacterial plasmids; phage DNA's; yeast plasmids;
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA
  • LTR or SV40 promoter As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain a gene to provide a phenotypic trait for selection of transformed host cells such as
  • dihydrofolate reductase or neomycin resistance for eukaryotic cell culture or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate hosts there may be
  • bacterial cells such as E. coli.
  • the expression vehicle including the appropriate DNA sequence inserted at the selected site may include a DNA or gene sequence which is not part of the gene coding for the protein which is capable of producing antibodies which recognize an epito pe(s) of the noted T. hyo.
  • the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in expression or improves
  • improperly folded proteins using agents such as alkali, chaotropes, organic solvents and ionic detergents followed by a renaturation step achieved by dilution, dialysis, or pH adjustment to remove the denaturant, and (2) reconstitution of proteins into a lipid bilayer or liposome to re-create a membrane like environment for the immunogenic protein.
  • agents such as alkali, chaotropes, organic solvents and ionic detergents
  • one or more of the proteins produced from a genetically engineered host may be employed in conjunction with a pharmaceutically acceptable carrier or may be directly conjugated to a carrier or immunostimulant to provide protection against swine dysentery, and in particular swine dysentery induced by T. hyo..
  • the Rotavirus VP6 carrier system developed by VIDO Veterinary Medicine
  • immunostimulant when chemically conjugated to a 39 kDa T. hyo. antigen such protein(s) is capable of eliciting antibodies which recognize an epitope(s) of one or more of the hereinabove noted 39 kDa T. hyo. antigens.
  • Such expressed protein will be sometimes hereinafter referred to as a "recombinant T. hyo. antigen,” however, as hereinabove indicated, such protein may not correspond to a T. hyo . antigen in that it may also be a fragment, derivative or fusion product.
  • the term "recombinant T. hyo . antigen" also
  • One or more of such 39kDa T. hyo. antigens may be employed in the vaccine.
  • One or more of such 39kDa T. hyo. antigens may be employed in the vaccine.
  • a preferred embodiment a preferred use of the antigens described herein.
  • all of the 39kDa T. hyo. antigens are employed in formulating a vaccine (i.e., the seven antigens or fragments or derivatives thereof encoded by the seven different T. hyo. genes).
  • the recombinant T. hyo. antigen(s) is employed in the vaccine in an amount effective to provide protection against swine dysentery.
  • each dose of the vaccine contains at least 5 micrograms and preferably at least 20 micrograms of such recombinant T. hyo. antigen(s).
  • the vaccine does not include such recombinant T . hyo. antigen in an amount greater than 20 milligrams.
  • protection or “protecting” when used with respect to the vaccine for swine dysentery described herein means that the vaccine prevents swine dysentery and/or reduces the severity of swine dysentery.
  • the vehicle which is employed in conjunction with the recombinant T. hyo. antigen(s) may be any one of a wide variety of vehicles.
  • suitable carriers there may be
  • the vaccine may be in the form of an injectable dose and may be administered intra-muscularly, intravenously, or by sub-cutaneous administration. It is also possible to administer the vaccine intranasally or orally by mixing the active components with feed or water;
  • the vaccine may include active components or adjuvants in addition to the recombinant T. hyo. antigen or fragments thereof hereinabove described.
  • an assay for detection or determination of antibody to 39 kDa T. hyo. antigen which employs a 39 kDa T. hyo. protein antigen, of the type hereinabove described, as a specific binder in the assay.
  • a sandwich type of assay wherein the 39 kDa T. hyo. antigen is supported on a solid support, as a binder, to bind 39 kDa T. hyo. specific antibody present in a sample, with the bound antibody then being determined by use of an appropriate tracer.
  • the tracer is comprised of a ligand labeled with a detectable label.
  • the ligand is one which is immunologically bound by the 39 kDa T. hyo. antibody and such ligand may be labeled by techniques known in the art.
  • the 39 kDa T. hyo. antibody bound to the 39 kDa T. hyo. antigen on the solid support may be determined by the use of an antibody for 39 kDa T. hyo. antibody which is labeled with an appropriate detectable label.
  • the labeled antibody to 39 kDa T. hyo. antibody may be a
  • the polyclonal antibody may be anti-swine IgG or may be an antibody which is specific for 39 kDa T. hyo. antibody, which antibody may be produced by
  • the detectable label may be any of a wide variety of detectable labels, including enzymes, radioactive labels, chromogens (including both fluorescent and/or absorbing dyes) and the like. The selection of a detectable label is deemed to be within scope of those skilled in the art from
  • the solid support for the antigen may be any one of a wide variety of solid supports and the selection of a suitable support is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the solid support may be a microtiter plate; a tube, a particle, etc.; however, the scope of the invention is not limited to any representative support.
  • the antigen may be supported on the support by techniques known in the art; e.g., by coating; covalent coupling, etc. The selection of a suitable technique is deemd to be within the scope of those skilled in the art from the teachings herein.
  • the sandwich assay may be accomplished by various techniques; e.g., "forward”; reverse”; or “simultaneous”; however, the forward technique is preferred.
  • the support After washing of the solid support, the support is contacted with a tracer which binds to 39 kDa T. hyo. antibody. If such antibody were present in the sample, the tracer becomes bound to such antibody bound to such antigen on the solid support, and the presence of tracer on the solid support is indicative of the presence of 39 kDa T. hyo. antibody in the sample.
  • the presence of tracer may be determined by determining the presence of the detectable label by procedures known in the art.
  • the preferred procedure is a sandwich assay, it is to be understood that the 39 kDa T. hyo. antigen(s) may be used in other assay techniques, e.g., an agglutination assay wherein the antigen is used on a solid particle such as a latex particle.
  • an assay or reagent kit for determining 39 kDa T . hyo. antibody which
  • the 39 kDa T. hyo. antigen includes 39 kDa T. hyo. antigen, as hereinabove described, and a tracer comprised of a ligand and a detectable label.
  • the ligand of the tracer is bound by 39 kDa T. hyo. antibody.
  • the reagents may be included in a suitable kit or reagent package, and may further include other components, such as buffers etc.
  • the 39 kDa T. hyo. antigen is preferably supported on a solid support.
  • DNA fragments may be used as a probe by use of techniques known in the art.
  • one or more of such antigens may be used to produce antibodies (monoclonal and/or polyclonal) by procedures known in the art and such antibodies may be used in a vaccine to impart protection against T. hyo.
  • Appendix 1 is a comparison of Gene Products of the 39kDa gene Family without peptide signal
  • Appendix 2A is the DNA -sequence of genes 1-4 encoding antigenes 1-4 of the 39kDa gene family from serotype B204.
  • Appendix 2B is the DNA sequence of genes 5-8 encoding antigens 5-8 of the 39 kDa gene family from serotype B204.
  • Appendix 3 is the nucleotide sequence of T.hyo gene insert of pTrep 106.
  • Appendix 4 is a partial DNA sequence of plasmid pTrep 301.
  • Appendix 5 is predicted amino acid sequences from PCR derived T .hyo. (B204) clones.
  • Appendix 6 is the predicted protein seqeunce encoded by pTrep 702.
  • Appendix 7 is the predicted protein sequence encoded by pTrep 704.
  • Appendix 8 is the predicted amino acid sequence for pTrep 505.
  • Figure 1 is a map of the gene family and sub-clones obtained from screening for 39 kDa gene
  • Figure 2 is a plasmid map of pTrep 505
  • Figure 3 is a schematic of the construction of pTrep 702
  • Figure 4 is a schematic of the construction of pTrep 704.
  • Figure 5 is a schematic of the construction of the pTrep PCR expression vehicle.
  • Treponema hyodysenteriae strain B204 was grown in. broth culture prepared as follows. Brain/Heart
  • the media was then prereduced (made anaerobic) by 24 hours of perfusion with a stream of gas composed of 90% nitrogen, 10% carbon dioxide.
  • the complete media was then inoculated with a 1-10% volume of actively growing T. hyo culture, the temperature was maintained at 37°C-39°C, the culture pH was
  • Cells were removed from the fermentation when they had achieved a density of 5 ⁇ 10 8 /ml or greater (measured by microscopic count). Cells were
  • the resuspended HSP was then mixed with 15 volumes of Tris-HCl pH 6.8, 6M urea which had been filtered through a 0.45uM filter. This was stirred at room temperature for several hours. This was centrifuged at 100,000 ⁇ g and the supernatant (US1) set aside. The pellet fraction from this step (UP1) was resuspended and extracted with urea a second time. This material was centrifuged as before and the supernatant (US2) and pellet (UP2) were
  • the predominant protein constitutent of UP2 is 39 KDa protein sometimes referred to as the 39p antigen.
  • the 39 p antigen which was further purified by molecular sieve column chromatography in the presence of SDS or electroelution from acrylamide gels.
  • T.hyo cells B204 were extractd with Tween 20 as described above. After the final Tween 20 extraction the residual cell pellet was resuspended in approximately 2ml of 10mM potassium acetate, pH 4.75 per gram wet weight and sonicated.
  • sonicated cell pellet was separated from the 39s antigen, by centrifugation at 26,000 xg for 15' at 4C. The supernatant was then centrifuged at 100,000 ⁇ g for 2 hours at 20°C to pellet any of the
  • polyacrylamide gels of the 39s protein and the 39p protein is identical.
  • the two proteins are also immunologically cross-reactive.
  • Anitsera raised against UP2, or gel purified 39p will recognize 39s on Western blots.
  • antisera raised against 39* will recognize 39p on Western blots.
  • 39S and 39p also comigrate with the predominant protein on the surface of intact T.hyo cells labelled with I 125 .
  • Antisera from swine that have recovered from experimental infections of swine dysentery also recognize either the 39s or 39p form of the 39kDa antigen.
  • centrifugation of the second urea extraction contains a single major protein component which is 39p antigen.
  • This insoluble protein was solubilized by boiling in 25 mM Tris-HCl pH6.8 containing 3%SDS, 1 mM EDTA, and 70 mM 2-mercaptoethanol.
  • amino acid sequence of the amino-terminus of the 39 kDa protein prepared above was determined using sequential Edman degradation in an automated Applied Biosystems gas phase sequenator.
  • the purified fragment had the following internal
  • the DNA was fractionated on an S-200 (Pharmacia) column using 0.3 NaCl, 0.05 m Tris-HCl pH 8.0, 1 mM EDTA, 0.06% sodium azide as a column buffer, in order to remove free linkers and free ATP.
  • the recovered T.hyo DNA was then ligated to dephosphorylated lambda gtll EcoRl arms obtained from PRomega Biotec
  • the ligation was then packaged into. lambda bacteriophage particles using the in vitro packaging kit, "Gigpack, "obtained from Stratagene (San Diego, CA).
  • the phage was then titered on a stationary phage culture of E . coli strain Y1090r- (Promega Biotech) .
  • the number of white plaques indicated that the original phage stock contained 1.4 X 10E7 pfu/ml in a total of 0.5ml.
  • oligonucleotide probe Approximately 10E6 cpm (1-2 ng probe) of probe was used for filter, overnight at 37C.
  • the hybridization solution consisted of:
  • Phage DNA was isolated using the technique of C. Helms, et al. (DNA 4 39, 1985).
  • step #2 Twenty three rounds of denaturation, annealing and polymerization were performed as in step #2 except that the polymerization temperature was increased to 65°C.
  • the screening procedure was as follows:
  • a set of DNA probes were synthesized using the amino terminal amino acid sequence data shown in Example 2. Each of them were comprised of a pool of degenerate sequences which encompass all the possible combinations of nucleotides which could encode the amino acid sequence of the target region as indicated below. Each probe is 17 nucleotides in length.
  • Trp-IIe-Asp-Phe-Leu-Thr probe name COD 553
  • a lambda GT11 library containing EcoRI linkered fragments derived from a partial AluI digest of genomic T.hyo DNA (strain B204 was screened with probes. One phage, 3-5Cl was identified by
  • the Eco R1 flanked, 1.6 kb segment of DNA from phage 3-5Cl was isolated by electroelution from an acrylamide gel and then ligated to plasmid pUC 19 which had been linearized by digestion with EcoR1. These DNAs were the ligated together, transformed into E. coli, and a clone containing recombinant plasmid pTrep 106 (Appendix 3) was identified by analysis or restriction digests of plasmid DNA.
  • Plasmid pTrep 106 was used to direct protein synthesis in an in vitro coupled
  • E. coli. strains transformed with plasmid pTrep 106 did not produce significant amounts of the desired 39 kDa T. hyo. antigen. Therefore, plasmid construction allowing high level expression of the recombinant antigen was made as follows. The Eco RI flanked, 1.6 kb fragment of pTrep 106 was ligated to plasmid pUC 18 linearized by digestion with Eco RI. The resulting plasmid, pTrep 112, was then cut with Pstl and BamHI, then treated with exonuclease III to remove (in a unidirectional manner) the non-coding DNA sequence upstream of the predicted ATG start codon of the 39 kDa T.
  • hyo. antigen Henikoff, Gene 28 p. 351-59 (1984)).
  • DNA aliquots were removed, the exo III inactivated by phenol extraction, the remaining DNA rendered blunt ended by digestion with nuclease S1, and this DNA was then religated and used to transform E. coli.
  • Nucleotide sequencing (Sanger, et al., PNAS 74:5463 (1977)) of plasmid DNA from one such new clone, pTrep 112-1, indicated that a contiguous sequence of 372 codons encoding the mature T. hyo.
  • 39 kDa protein and 7 amino acids from the signal sequence were fused downstream of the Hind III site of the parental pUC 18 plasmid.
  • the fusion was in a reading frame to encode a fusion protein whose expression would be regulated by the lac promoter after the orientation of the cloned fragment was inverted (see Appendix 4) by. cloning into pUC 9 from the HindIII to Eco RI site.
  • E. coli. transformed with the resulting plasmid, pTrep 301 produced an insoluble 39 kDa antigen which reacts with sera from swine (both those recovered from swine dynsentery as well as animals immunized with the 39 kDa protein purified from T. hyo.) in both an immunoblot and plate ELISA assay.
  • E. coli strain CY-15,000 containing plasmid pTrep301 was grown in 250 mis of Luria broth
  • the culture was grown for 18 hours at 37° C.
  • the cells were
  • the major protein component of this fraction had a Mr of about 40 kDa as judged by Commassie blue staining of samples after SDS-gel electrophoresis.
  • This same protein component was recognized in Western blot analysis by swine and mouse antisera raised against the authentic 39 kDa T. hyo protein obtained from the UP2 fraction.
  • This recombinant protein was also recognized in immunoblots probed with sera from pigs that had recovered from experimentally induced swine dysentery.
  • the predicted amino acid sequence of the 39kDa recombinant antigen obtained in this Example closely resembles but is not identical to the amino acid sequence of the 39 kDa antigen of the UP2 fraction of T. hyo.; however, they have common epitopes
  • the 39 kDa recombinant produced in this Example corresponds to a protein encoded by gene 1, one of the multiple genes encoding different T. hyo antigens, each having a molecular weight of about 39kDa.
  • the T. hyo genome contains at least 7 genes encoding related antigens with molecular weights of about 39 kDa.
  • the lambda GT11/ B204 library used in the pTrep 106 screening was probed with Cod664 as well as a nick translated probe made from a 411 base pair Sphl-Bcll fragment encoding the amino terminal portion of the 39kDa protein from pTrep301.
  • Hybridizing phage were purified and subcloned into pUC8, 9, 18 or 19 for sequencing and additional manipulations for expression.
  • the recombinant lambda phage, their hybridization patterns, and subclones are elaborated in Table 3 following the Examples.
  • oligonucleotides (Cod 1019 and Cod 1020) derived from peptide sequence of a carboxy terminal fragment from the 39s and 39p antigens were also used to screen the GT11 library to obtain a phage(s) which contained more extended coding sequences for the #5 gene.
  • the degeneracy of Cod 1019 and 1020 was decreased by assuming that codon usage for some amino acids would be similar to that found in other genes in the 39 kDa family.
  • DNA sequence derived from overlapping sublcones indicates that the 39 kDa genes are found in two subfamilies of tandemly repeated 39kDa genes.
  • Family 1 contains (1-4) and Family 2 contains (5-8).
  • Each gene encodes a protein with a presumptive signal sequence directing transport of the protein through the inner bacterial membrane.
  • Figure 1 of the drawings is a map of the gene family and sub-clones obtained from screening the two libraries.
  • Appendix 1 shows the relationship between the predicted amino acid sequences of processed products of genes 1-7 encoding seven different full length 39 kDa T. hyo antigens as well as the carboxy terminal fragment of gene #8.
  • the Perkin-Elmer/Cetus polymerase chain reaction system was used as a supplement to screening phage libraries to identify clones containing full length copies of 39 kDa genes.
  • oligonucleotide/DNA mixtures were passed through 25 cycles of heat denaturation, annealling and Taq polymerase directed DNA synthesis to amplify genomic DNA sequences between the two oligonucleotide primers.
  • the newly synthesized amplified sequences were digested with Bam HI and Hind III, and cloned into pUC8 or pUC9. If cloned into pUC8 the fragments were oriented in the proper direction and in the proper reading frame for expression from the Lac promoter of a fusion protein comprised of 9 amino acids encoded by the pUC polylinker followed by a full-length copy of the mature forms of the
  • Clones were initially screened by the Dot Blot Screening Protocol with unique and discriminating synthetic oligonucleotides derived from clones containing the full-length sequence of Gene #1 (Cod 844), or Gene #2 (Cod 931), or the partial sequence of Gene #3 (Cod 908), and Gene #4 (Cod 932, 1151).
  • the clones were screened with a unique synthetic oligonucleotide which is common to all known forms of the 39kDa gene family (Cod 957).
  • the PCR system was also used to synthesize and clone the #5 gene encoding the full length 39p/39s antigen.
  • the oligomers used in this procedure were Cod 1054 and Cod 1055.
  • Cod 1054 was derived from the DNA sequence of the 5' end of gene #6, a gene
  • Cod1055 was derived from the reverse complement of the DNA sequence encoding the carboxy terminal peptide of the 39s/39p antigen. This sequence distinguishes the #5 gene from any other gene
  • oligonucleotides were then mixed with genomic DNA from either B204 or B234 and passed through 25 cycles of heat denaturation, annealling and DNA synthesis in the presence of Taq polymerase in order to amplify intervening sequences.
  • the amplified mixture was digested with BamH1 and Hind3 and cloned into either pUC8 or 9.
  • Candidate clones were screened for hybridization to Cod968, Cod1019, Cod 1020 and Cod 957.
  • One of those clones, pTrep 613 includes the entire coding sequence for gene which is expressed under control of the
  • beta-galactosidase promoter of pUC beta-galactosidase promoter of pUC.
  • the PCR system was also used to synthesize and clone the #8 gene encoding the full length Copy 8 Antigen.
  • the oligomers used in this procedure were Cod 1359 and Cod 1438, corresponding to the 3' and 5' ends of the gene, respectively.
  • the oligonucleotides were mixed with genomic DNA from either B204 or B234 and passed through 25 cycles of heat denaturation, annealing, and DNA synthesis in the presence of Taq polymerase in order to amplify intervening sequences.
  • the amplified mixture was digested with BamHI and SalI and cloned into either pUC8 or 9.
  • Candidate clones were screened for hybridization to Cod 957.
  • One of these clones, pTrep 541 includes the entire coding sequence for the gene which is expressed under control of the beta-galactosidase promoter of pUC.
  • pTrep505 a pUC19 based plasmid directs the expression of all but the first 19 amino acids of Gene #2 of the 39 kDa gene family from the Lac promoter. It was constructed from pTrep 323 which contained an EcoR1 fragment subcloned from the lambda GT11 library. This EcoR1 fragment was subcloned into pWHA142 to place it in the proper orientation and reading frame for expression from the Lac promoter. pWHA142 is a derivative of pUC19 with a GAA reading frame across the EcoR1 site.
  • a plasmid map of pTrep505 is presented in Figure 2. The predicted protein sequence from pTrep 505 is presented in
  • pTrep 702 a pUC19 based plasmid directs the expression of 13 amino acids from the signal sequence of Gene #6 plus the first 315 amino acids of the mature protein fused to the LacZ complementing peptide in pUC. It was constructed in two steps from pTrep501 and pTre p327 which contain overlapping regions of the #6 gene and share a common Bcll site.
  • pTrep501 which contains regions coding for the 5' portion of the #6 gene, was digested with Bcll and Aatll and ligated with Bcll-Aatll fragment from pTrep 327 which contained the 3' sequences of the #6 gene.
  • the 3' cloning site of pTrep508 is downstream of the cloning site contained within pTrep702 and thus contains the DNA sequences encoding the carboxy terminal portion and stop codon of the Copy #6 antigen which are lacking in pTrep702.
  • a schematic of the construction of pTrep704 is presented in
  • Table 5 tabulates the 39 kDa expression clones for expressing seven different T. hyo. antigens having a molecular weight of about 39 kDa, with reference to the different genes or copies.
  • pTrep702 6 genomic lacks carboxy terminus pTrep704 6 genomic full length pTre p620 7 per 1 amino acid substitution at C terminus relative to genomic sequence pTrep605 2 per full length pTrep541 8 per full length
  • Emulsigen adjuvant was used as an adjuvant control, mixed with Dulbeco's PBS buffer.
  • the Hyguard a bacterin, was adminstered according to manufacturer's directions.
  • a cement preparation (Part G of Example 4) in an amount of 25-200 ⁇ g in Emulsigen is intermuscularly injected into pigs. Two weeks later, the pig is boosted with an identical dose. Two weeks after the boost, the pig is bled and the blood is allowed to clot. Immune serum is separated by centrifugation at 4°C.
  • FIGURE # DNA Sequence of B204 Genes Encoding 39 kDa Antigens 1-4
  • IleGlyTyrThrSerGluAlspheSerIleGlyIleGlyTyrAsnTyrThrSerHisSer 1020 ATAGGATATACTTCTGAGGCTTTTAGTATAGGCATAGGCTATAATTATACCAGCCATTCC
  • GlnLeuGlyTyrTyrArgAspAsnTyrLeuGlyIleSerThrAspThrGlnIleArgTyr 1200 CAACTTGGTTACTATAGAGATAATTATTTAGGTATAAGTACTGATACGCAAATAAGATAT
  • IleSerTyrAsnPheGlySerHisThrProValLeuMetIleAsnAlaLeuAsnAapAsn 2400 ATTASTTATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAAT LeuArgIleValIleProValGlnIleLeuValHisAspGlyAanMetAsnMetThrAsp 2460 TTGAGGATAGTTATTCCTGTACAAATATTAGTACATGATGGTAATATGAATATGACGGAT
  • ThrAspGlyAanGlnPheArgAlaArgMetAspGlnPheGlyPheValLeuGlyAsnSer 3480 ACAGACGGTAATCAGTTTAGAGCTAGAATGGATCAATTTGGATTCGTTTTAGGTAATAGC
  • TyrLysAsnAlaProTyrValGlyLysAsnTyrGluGluGluPhePheSerArgSerPhe 3960 TATAAAAATGCTCCGTATGTTGGTAAGAATTATGAAGAAGAATTTTTTTCAAGGTCATTT
  • ProPheIleLysValAlaTyrAsnThrAlaLeuHisGlyValGlyThrMetIleArgAla 5400 CCTTTTATTAAAGTAGCATATAATACAGCTTTGCATGGAGTTGGTACTATGATAAGAGCA LeuAapThrMetLeuGlnProIleGluAspTyrTyrProAspArgProValSerSerGln 5460 TTAGATACTATGCTTCAACCAATAGAAGATTATTATCCAGATCGTCCTGTTTCATCACAA
  • GlnPheArgAlaArgMetAsnGlnLeuGlyPheThrLeuGlyAsnGlyXleIleLysGly 661 CAATTCAGAGCTAGAATGAACCAATTAGGTTTCACTCTAGGTAACGGCATTATTAAAGGT
  • LysGlySerLysLeuThrHisThrLeuTyrTrpGlnAlaTyrGlyGluIleTyrIleArg 1501 AAAGGAAGCAAACTTACTCATACATTATACTGGCAGGCTTACGGAGAAATATATATCAGA
  • GlnGlyAsnProIleAlaSerGlyAsnSerMetProValValPheGlyAlaAsriThrGly 1621 CAAGGAAATCCTATTGCTTCAGGAAATTCAATGCCTGTTGTATTCGGAGCTAATACTGGT
  • GlyPheValLeuGlyAsnGlyThrIleLysGlyThrPheGlyPheArgSerGlnAlaIle 2221 GGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACTTTCGGTTTTAGATCTCAAGCTATT
  • GlyMetTyrGlyAspArgAspSerTrpIleAspPheLeuThrHisGlyAsnGlnPheAre 121 GGTATGTACGGCGACAGAGATTCTTGGATCGACTTCCTTACTCATGGTAATCAGTTCAG
  • ValAspLeuGlnThrThrlleSerAlaGlylleGlyTyrThrSerGluProPheGlyIle 301 GTAGATTTACAAACTACTATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCGGTAT
  • GlylleSerThrAspIleGlnLeuArgTyrTyrThrGlylleAspAlaPheAsnAlaIle 541 GGTATAAGCACTGATATACAATTAAGATACTATACTGGTATAGATGCTTTCAATGCTAT
  • GlyMetTyrGlyAspArgAspSerTrpIleAspPheLeuThrHisGlyAsnGlnPheArg 121 GGTATGTACGGCGACAGAGATTCTTGGATCGACTTCCTTACTCATGGTAATCAGTTCAGA
  • ValAspLeuGlnThrThrlleSerAlaGlylleGlyTyrThrSerGluProPheGlyIle 301 GTAGATTTACAAACTACTATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCGGTATT
  • GlylleSerThrAspIleGlnLeuArgTyrTyrThrGlylleAspAlaPheAsnAlaIle 541 GGTATAAGCACTGATATACAATTAAGATACTATAdTGGTATAGATGCTTTCAATGCTATA
  • TyrAsnPheGlySerHisThrProValLeuMetlleAsnAlaLeuAsnAspAsnLeuArg 301 TATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAATTTGAGG
  • TyrLeuSerTrpCysAlaTyrAlaGluLeuTyrlleThrProValLysAspLeuGluTrp 961 TATTTATCTTGGTGTGCTTATGCAGAGCTTTATATAACACCTGTAAAAGATTTAGAATGG

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  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
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  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
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Abstract

On produit une famille d'antigènes de 39 kDa contre T. hyodysenteriae, à l'aide de techniques de recombinaison. On a identifié sept gènes et antigènes différents. On peut utiliser lesdits antigènes dans un vaccin.
EP19900914865 1989-09-13 1990-09-11 Treponema hyodysenteriae antigens having a molecular weight of 39kda and dna encoding therefor Withdrawn EP0491859A4 (en)

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US5236708A (en) * 1989-11-03 1993-08-17 Iowa State University Research Foundation, Inc. Reduced-protein subunit vaccine for swine dysentery
AU644619B2 (en) 1989-12-21 1993-12-16 Advanced Technologies (Cambridge) Limited Modification of plant metabolism
US5633131A (en) * 1992-04-30 1997-05-27 Institut Pasteur Rapid detection of isoniazid resistance in mycobacterium tuberculosis probes for selecting nucleic acid encoding isoniazid resistance, and methods and kits
IL107026A0 (en) * 1992-09-16 1993-12-28 Univ Tennessee Res Corp Antigen of hybrid m protein and carrier for group a streptococcal vaccine
US5698394A (en) * 1994-06-01 1997-12-16 Board Of Regents Of The University Of Nebraska Nucleotide sequences and methods for detection of Serpulina hyodysenteriae
RU2141339C1 (ru) * 1996-03-26 1999-11-20 Пожарская Виктория Олеговна Способ получения антигенов из культуральных бледных трепонем
US20070169227A1 (en) 2003-12-16 2007-07-19 Pioneer Hi-Bred International Inc. Dominant Gene Suppression Transgenes and Methods of Using Same
ES2339559T3 (es) 2003-12-16 2010-05-21 Pioneer Hi-Bred International, Inc. Transgenes de supresion de gen dominante y metodos de uso de los mismos.
KR20140120946A (ko) 2007-08-03 2014-10-14 베링거 인겔하임 베트메디카 게엠베하 브라키스피라 하이오디센테리애의 유전자와 단백질 및 이의 용도
CN103789327A (zh) * 2007-08-03 2014-05-14 贝林格尔·英格海姆维特梅迪卡有限公司 猪痢疾短螺旋体的基因和蛋白质及其用途
AU2013360773B2 (en) 2012-12-21 2018-04-05 Boehringer Ingelheim Vetmedica Gmbh Novel recombinant outer membrane proteins from Brachyspira hyodysenteriae and uses thereof
JP2014087340A (ja) * 2013-10-24 2014-05-15 Boehringer Ingelheim Vetmedica Gmbh ブラキスピラ・ヒオディセンテリアの新規な遺伝子とタンパク質及びその使用

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JPH0361488A (ja) * 1988-06-29 1991-03-18 Ml Technol Ventures Lp 発現用ビークルから成る組成物とそれにより形質転換された宿主とそれにより生産される蛋白質及び蛋白質から成るトレポネマ・ヒオジセンテリエーに対する保護用ワクチン

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WO1991004036A1 (fr) 1991-04-04
EP0491859A4 (en) 1992-10-07
JPH05502370A (ja) 1993-04-28
CA2025230A1 (fr) 1991-03-14

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