WO1991004036A1 - TREPONEMA HYODYSENTERIAE ANTIGENS HAVING A MOLECULAR WEIGHT OF 39kDa AND DNA ENCODING THEREFOR - Google Patents

TREPONEMA HYODYSENTERIAE ANTIGENS HAVING A MOLECULAR WEIGHT OF 39kDa AND DNA ENCODING THEREFOR Download PDF

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Publication number
WO1991004036A1
WO1991004036A1 PCT/US1990/005129 US9005129W WO9104036A1 WO 1991004036 A1 WO1991004036 A1 WO 1991004036A1 US 9005129 W US9005129 W US 9005129W WO 9104036 A1 WO9104036 A1 WO 9104036A1
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Prior art keywords
protein
hyo
gene
dna
antigen
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PCT/US1990/005129
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French (fr)
Inventor
Jeffrey Gabe
Elizabeth Dragon
Michael Mccaman
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Ml Technology Ventures, L.P.
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Publication of WO1991004036A1 publication Critical patent/WO1991004036A1/en

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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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from 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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Abstract

A family of T. hyodysenteriae 39 kDa antigens are produced by recombinant techniques. Seven different genes and antigens have been identified. Such antigens may be used in a vaccine.

Description

TREPONEMA HYODYSENTERIAE ANTIGENS
HAVING A MOLECULAR WEIGHT OF 39kDa AND DNA ENCODING THEREFOR
This invention relates to Treponema
hyodysenterlae and more particularly to Treponema hyodysenterlae (T. hyo.) antigens, genes encoding for such antigens, cells genetically engineered with DNA encoding for such antigens and uses for such
antigens. Still more particularly, 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
mucohemorrhagic diarrhea, dehydration and weight loss.
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.
In accordance with one aspect of the present invention, there is provided 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.
Applicant has found that 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.
A comparison of the predicted amino acid
sequences from the mature peptides encoded by the 39kDa family genes is found in Appendix 1. The peptide sequence corresponding to the signal peptides of these proteins (Appendices 2A and 2B) has been removed for the purposes of this comparison.
Examination of this comparison reveals 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. (Kyte & Doolittle, Journal of Molecular Biology, 157,, 105 (1982))
Thus, in accordance with one aspect of the present invention, there is provided eight different antigens (or fragments or analogs thereof), 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.
In accordance with another aspect of the present invention, there is provided at least eight different genes, each of which encodes for a different T.hyo antigen having a molecular weight of about 39 kDa.
In accordance with yet another aspect of the present invention, there is provided 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.
In accordance with yet a further aspect of the present invention, there is provided 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
discontinuous polyacrylamide gel electrophoresis using the SDS buffer system described by Laemmli,
Nature, 227:680-85 (London, 1970) with an acrylamide concentration of 10-17% and a bis-acrylamide to acrylamide ratio of 1:29.
Thus, Applicant has found that there are at least eight different 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. Thus, for example, 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
generates antibodies which recognize an epitope of a 39 kDa antigen.
Similarly, 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 .
antigen as hereinabove described.
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. Thus, for example, the 39 kDa proteins of serotype B204 have minor
differences from those of serotype B234; however, such antigens, as well as the genes encoding such antigens are essentially identical to each other.
As a result, the term "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.
It is also to be understood that the DNA
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
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. For example, the DNA sequence may encode for the entire antigen; however, the expressed protein is a fragment of the antigen.
The term "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.
The term "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.
The term "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. Such vectors include chromosomal, nonchromosonal and synthetic DNA sequences; e.g., derivatives of SV40; bacterial plasmids; phage DNA's; yeast plasmids;
vectors derived from combinations of plasmids and phage DNAs, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, 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
synthesis. 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.
In addition, 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. As representative examples of appropriate hosts, there may be
mentioned: bacterial cells, such as E. coli.
Salmonella typhimurium; fungal cells, such as yeast; animal cells such as CHO or Bowes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein. As hereinabove indicated, 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.
antigen(s). For example, the desired DNA sequence may be fused in the same reading frame to a DNA sequence which aids in expression or improves
purification or permits expression of the appropriate protein.
When seeking to develop a vaccine neutralizing or protective antibodies could be targeted towards discontinuous, conformation-dependent epitopes of the native antigen. One must therefore consider whether the protein obtained from the recombinant expression system might have a three dimensional structure
(conformation) which differs substantially from that of the original protein molecule in its natural environment. Thus, dependent on the immunogenic properties of the isolated proteins, one might need to renature it to restore the appropriate molecular conformation. Numerous methods for renaturation of proteins can be found in the scientific literature and include; (1) denaturation (unfolding) of
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.
In accordance with another aspect of the present invention, one or more of the proteins produced from a genetically engineered host (genetically engineered with DNA encoding for a 39'KDa T. hyo antigen) 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
Infectious Disease Organization, Saskatoon, Canada) although not an adjuvant may be a suitable
immunostimulant when chemically conjugated to a 39 kDa T. hyo. antigen. As hereinabove indicated, 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
encompasses such fragments, derivatives and fusion products.
One or more of such 39kDa T. hyo. antigens may be employed in the vaccine. In a preferred
embodiment, 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. In general, each dose of the vaccine contains at least 5 micrograms and preferably at least 20 micrograms of such recombinant T. hyo. antigen(s). In most cases, the vaccine does not include such recombinant T . hyo. antigen in an amount greater than 20 milligrams.
The term "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.
If multiple doses are given, in general, they would not exceed 3 doses over a six week period.
The vehicle which is employed in conjunction with the recombinant T. hyo. antigen(s) may be any one of a wide variety of vehicles. As representative examples of suitable carriers, there may be
mentioned: mineral oil, alum, synthetic polymers, etc. vehicles for vaccines are well known in the art and the selection of a suitable vehicle is deemed to be within the scope of those skilled in the art from the teachings herein. The selection of a suitable vehicle is also dependent upon the manner in which the vaccine is to be administered. 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;
providing a tablet form, etc.
Other means for administering the vaccine should be apparent to those skilled in the art from the teachings herein; accordingly, the scope of the invention is not limited to a particular delivery form.
It is also to be understood that the vaccine may include active components or adjuvants in addition to the recombinant T. hyo. antigen or fragments thereof hereinabove described. In accordance with a further aspect of the present invention, there is provided 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.
More particularly, there is provided an
immunoassay for 39 kDa T . hyo. antibody in which a 39 kDa T. hyo. antigen is employed as a binder, in the assay, for specifically binding 39 kDa T. hyo.
antibody.
The assay technique which is employed is
preferably 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.
Thus, for example, 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.
In such a sandwich assay technique, the labeled antibody to 39 kDa T. hyo. antibody may be a
monoclonal antibody or a polyclonal antibody; e.g. 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
procedures known in the art; for example innoculating an appropriate animal with 39 kDa T. hyo. antibody. 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
teachings herein.
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. For example, 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.
ϊn a typical procedure, 39 kDa T. hyo . antigen, which is supported on a solid support is initially contacted with a sample containing or suspected of containing 39 kDa T. hyo. antibody to bind
specifically any of such antibody present in the sample to such antigen on 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.
Although 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.
In accordance with another aspect of the present invention, there is provided an assay or reagent kit for determining 39 kDa T . hyo. antibody which
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.
Although the present invention has been
particularly described with reference to use of the 39 kDa antigen(s) for imparting protection against T. hyo., 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.
Description of Appendices and Drawings
Appendix 1 is a comparison of Gene Products of the 39kDa gene Family without peptide signal
sequences from serotype B204 . 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; and
Figure 5 is a schematic of the construction of the pTrep PCR expression vehicle.
The present invention will be further described with respect to the following examples; however, the scope of the invention is not to be limited thereby. In the Examples, unless otherwise noted,
purifications, digestions and ligations are
accomplished as described in "Molecular Cloning, a laboratory manual" by Maniatis et al. Cold Spring Harbor Laboratory (1982). Pn the following examples, unless otherwise indicted, transformations are accomplished by the procedure of Cohen et al. PNAS 69
2110 (1973).
EXAMPLE 1 - Purification and Recovery of
Native Antigen
Treponema hyodysenteriae strain B204 was grown in. broth culture prepared as follows. Brain/Heart
Infusion (Difco) at 37 gms/liter distilled water was autoclaved allowed to cool, then sterile additions were made of a glucose solution (to a final
concentration of 5 gm/liter) and fetal calf serum (to final concentration of 5% vol/vol). 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
maintained at 6.8, and the culture was continuously perfused with the oxygen free gas (flow rate 50 mls/min/liter of culture).
Cells were removed from the fermentation when they had achieved a density of 5 × 108/ml or greater (measured by microscopic count). Cells were
concentrated by centrifugation then washed and recentrifuged twice in a buffer of 10mM potassium acetate pH 4.75, 150mM potassium chloride. The cells were then resuspended in 10mM potassium acetate pH 4.75 until an optical density of 25-30 (at 600nm) was achieved (as measured on solution dilutions) which is typically about 1/20 the original culture volume.
Extraction method: Tween-20 (a non-ionic detergent) was then added to the cell suspension to achieve a final
concentration of 0.2%. After gentle agitation for 10 minutes the cells were centrifuged (10,000 xg for 10 min). This supernatant fraction was discarded and the cells were resuspended in an equivalent volume of acetate buffer and then extracted by the addition of Tween-20 to a final concentration of 2.0%. After centrifugation the 2% Tween supernatant (detergent solubilized antigen pool) was saved and the cell pellet was resuspended and re-extracted with Tween-20 in a sequential manner for up to 5 additional cycles with the Tween-20 concentration increasing over the cycles from about 2% up to about 10%. The detergent solubilized supernatant fractions were pooled. This extraction procedure selectively (but not
quantitatively) solubilizes surface proteins of T. hyo without lysing or rupturing the bacteria.
To concentrate the antigen preparation,
supernatent fractions were subjected to
ultracentrifugation (100,000 × g) for 1.5 hours, and the recovered pellet material (HSP) was resuspended in 25mM Tris buffer pH 6.8 and dispersed by
sonication.
Antigen Purification
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
collected. 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.
It is possible to isolate a soluble form of the 39kDa antigen (39s) in addition to the sedimentable form (39p) that is isolated as the major protein component of the urea insoluble pellet (UP2)
described above. In order to produce the 39s antigen, 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. The
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
sedimentable membrane associated proteins which were also released by sonication. The supernatant (39*), which contains the 39s antigen as its predominant protein component , was then sterile filtered through a .2uM filter and stored at either 4C or frozen. If stored at 4C some proteolytic degredation of the 39s antigen occurs. Additional 39* can be isolated by repeating the above sonication and centrifugation steps on the 26,000 xg cell pellet. Approximately 4mg of 39* can be obtained per liter of original culture volume; a yield roughly equivalent to the yeild of UP2.
The electrophoretic mobility in SDS
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. Conversely, 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. (Marchalonis, et al. Biochemistry Journal; 24,
921 (1971)). Antisera from swine that have recovered from experimental infections of swine dysentery also recognize either the 39s or 39p form of the 39kDa antigen.
Example 2 - Protein Sequence of 39s and 39 antigens
The fermentation and protein purification are accomplished as in Example 1.
The insoluble material obtained by
centrifugation of the second urea extraction (UP2) 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. This
solution was subjected to gel filtration
chromatography over a 30 cm column of Sepharose 6B (from BioRad, Richmond, CA). The 39 kDa peak was identified by gel electrophoresis of column eluant fractions, the appropriate fractions were pooled and the protein concentrated by precipitation with acetone and collected by centrifugation. The pellet was dissolved in 1.1% SDS and then extracted with chloroform/methanol to remove residual SDS.
The 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
identity of the first 41 amino acids of the protein thus determined are shown below: 1 10
Met-Tyr-Gly-Asp-Arg-Asp- Ser-Trp- IIe-Asp-Phe-Leu-Thr-His -Gly-
20
Asn-Gln-Phe-Arg-Ala-Arg-Met-Asp-Gln-Leu-Gly-Phe-Val-Leu-Gly-
41
Asn-Asp-Thr- IIe-Lys -Gly-Thr-Phe- ? - ? -Arg-
Amino terminal peptide sequence of 39s was
obtained directly from a preparation of 39* which was concentrated by precipitation with acetone and
sequenced. Additional internal peptide sequence of
the 39s antigen was obtianed by digestion with
endoproteinase LysC (-1/100 w/w) in 50mM Tris-HCl
pH8.5, 0.1% SDS (37°C, 16 hrs.). Proteolytic
cleavage products were purified using reverse phase
HPLC and sequenced on a Vydac C4 column (250 mm × 4.6 mm, 5μM) developed with a linear gradient of 0%-100B%
(0% = 0.1% Trifluoracetic acid, 100B% = 67%
acetonetrile, 33% isopropanol, 0.1% trifluoroacetic
acid). Peptide sequence for the 39p antigen
extending that found above was determined in a
similar fashion. Some additional protein sequence
from 39* was also obtained from purified cleavage
products after digestion with endoproteinase
V8(-1/100 w/w) in 50mM NH4HCO3, pH 7.8, 0.1% SDS.
Internal sequences were also determined for the
39p antigen as follows:
An amino acid sequence was determined for an
HPLC purified peptide fragment derived from
proteolyitc digestion of the 39 kDa protein (of the
UP2 cell fraction)using endoproteinase Lys-C. 300μg of the 39 kDa protein was first precipitated with
acetone and then resuspended in a solution of 4 M
urea, 25 mM Tris pH 8.5 and digested with 2.5μg LysC (37°C, 16 hrs.). One peptide was obtained as a peak
of material eluting off of a C-4 reverse phase column
developed with a gradient of acetonitrile,
isopropanol (2:1) in 0.1% trifluroacetic acid.
The purified fragment had the following internal
sequence: val gln his sef leu ala trp gly ala
tyr ala glu leu tyr val arg pro val gln asp leu
glu glu tyr phe glu met asp ile asn. . .
The Amino acid sequence was also determined for
the protease resistant component of the 39 kDa
component of the UP2 fraction after its digestion
with chymotrypsin. A sonicated suspension of UP2
protein at 2mg/ml was incubated at 37 degrees C for
16 hrs. with 20 ug/ml chymotrypsin in a buffer of 25
mM Tris, pH 6.8, containing 0.1% Zwittergent 3-12
detergent. A protease resistant 27 kDa product was
isolated by electroelution after preparative gel
electrophoresis and precipitated and extracted with
chloroform/methanol prior to sequencing. The
component had the following sequence:
asp xxx xxx thr lysasp tyr met gly ile ser thr asp ile gln leu arg tyr tyr thr xxx ile asp ala phe asn ala ile arg leu tyr phe lys tyr gly gln xxx xxx phe
A summary and comparison of the amino acid
sequence data obtained from 39p and 39s is found in
Table 1 following the Examples. Although the
proteins have not been sequenced in their entirety,
none of the data identifies any difference in the
amino acid sequence of the 39p and 39s antigens.
Therefore, one can conclude that the amino acid
sequences of the 39p and 39s antigens are very
similar and may be identical. Example 3 - General Methods
Unless otherwise indicated, the General Methods described below were used in the following examples.
A. Construction of a Genomic Library of T.hyo DNA
48 μg of genomic DNA of T.hyo strain B204 was partially digested with Alu I. EcoRl linkers were kinased with P32 ATP according to manufacturers instructions (Pharmacia LKB Biotechnology,
Piscataway, New Jersey) and ligated to the Alu I partially digest T.hyo DNA at a linker concentration of 133 μg/ml using BMB ligase at a concentration of 50 units/ml. Following overnight ligation the ligase was heat inactivated and the reaction was digested with EcoRI.
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
(Madison, Wisconsin) and used according to
manufacturers specifications. 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.
B. Identification of a Recombinant Phage
In performing mixed oligonucleotide screening for the 39kDa gene, the procedure used was that of W.D. Benton & R.W. Davis Science 196, 180 (1977). Duplicate filters were hybridized with each
oligonucleotide probe. Approximately 10E6 cpm (1-2 ng probe) of probe was used for filter, overnight at 37C. The hybridization solution consisted of:
5X Denharts
0.1 um rATP
250 μg/ml E. coli tRNA
6X NET (lXNET=150mM NaCl, 15 mM Tris-HCl pH 7.5, 1 mM EDTA)
0.5% NP40
1 mM sodium Pyrophosphate
Prior to hybridization the filters were washed for 2 hours at 37C in hybridization solution.
Following hybridization the filters were washed twice at RT (20' /wash) and twice (20' /wash) at 37C in 6X NET, 0.1% SDS and once (20 '/wash) at 37C in 6X NET. The filters were then dried and exposed to X-ray film. Positive plaques were selected,
rescreened and plaque purified. Phage DNA was isolated using the technique of C. Helms, et al. (DNA 4 39, 1985).
C. PCR Protocol
1. 10ng genomic DNA (B204 or B234) was mixed with 10ul 10x reaction buffer (Perkin Elmer Cetus), 16ul 1.25mM dNTP (each), 25ul primer#l (4uM), 25 ul primer #2 (4uM) and brought to a final volume of 100ul with Q-H2O.
2. The mixture was denatured by heating at 94°C for 1.5 min. and annealed at 50°C for 2.5 min. 0.5ul of Taq polymerase (15U/μl) (Perkin Elmer Cetus) was added and polymerization was allowed to proceed at 55°C for 10 min. 3. One more round of denaturation, annealing and polymerization was performed with the time and temperature conditions specified in step #2.
4. Twenty three rounds of denaturation, annealing and polymerization were performed as in step #2 except that the polymerization temperature was increased to 65°C.
5. After the final round of amplification the mixture was extracted with phenol: chloroform (1:1) and chloroform and precipitated with ethanol.
6. After extraction and precipitation the sample was digested with appropriate enzymes (BamH1 and Hind3) and ligated with the desired vector (pUC8 or pUC9).
D. Dot Blot Screening Protocol
1. Grow an overnight culture of bacterial colony to be screened.
2. Spin down 50ul of the overnight culture and remove supernatant.
3. Resuspend the cell pellet in 200ul of 25mM Tris-Cl pH 8.0, 10mM EDTA + hen egg white lysozyme (1mg/ml).
4. Incubate for 5 min. at room temperature.
5. Sonicate briefly (3 seconds).
6. Add 20ul 3N NaOH and incubate 1 hour at 70C.
7. Let cool to room temperature, add 220ul 2M NH4OAc. Mix.
8. Apply to nitrocellulose filter with aid of vacuum.
9. Let filter air dry. Bake at 90C for 2hrs .
10. Probe filter with desired probe.
E. Preparation of Nick Translated Probe
1. Denature 50rig of the DNA fragment by boiling for 5 min. in 9ul TE. Chill on ice. 2. Add 5ul gamma dATP (6000 Ci/mMol,
10mCi/ml), 2ul degenerate hexamer in 10x reaction buffer (BMB), 3ul dNTP (25uM dG-,dT-, & dCTP final concentration), 2ul Klenow (2U/ul, BMB).
3. Incubate at 37C for 30 min. Stop with 30ul 10mM EDTA.
4. Separate labeled fragement from
unincorporated label with G-50 spin column (BMB).
F. Screening Procedure with Nick Translated Probes
The screening procedure was as follows:
1. Prewash baked nitrocellulose filters in 20mM Tris-Cl pH8.0, ImM EDTA, 0.1% SDS for 2 hrs. at 37°C.
2. Prehybridize filters for 2 hrs. at 42°C in 50% deionized formamide, 5X Denhardt's, 5X SSPE, 0.1% SDS, salmon sperm DNA (100ug/ml).
3. Denature nick translated probe by boiling for 5 minutes and chilling on ice.
4. Hybridize overnight at 42°C in
prehybridization solution and denatured probe.
5. Wash filters 2 times at room temperature in 2X SSC and 0.1% SDS.
6. Wash 1 time in 0.1X SSC at 42°C.
7. Dry filters and expose to x-ray film at -70°C with enhancing screen.
G. Cement Preparation
1. Resuspend cells from an overnight culture in 1/25th original volume in 25mM Tris-Cl pH 8.0, 10mM EDTA + 1mg/ml lysozyme.
2. Incubate 30-60 min. Sonicate to disrupt DNA and reduce viscosity. 3. Add 1/10th volume -20% Triton X-100.
Agitate on lab quake for 2 hours. Sonicate if necessary to reduce viscosity.
4. Centrifuge at 10,000×g for 10 min. to pellet cement.
5. Resuspend cement in 1/25th original volume in 20mM Tris-Cl pH 8.0, 5mM EDTA + 5% Triton X-100. Sonicate. Agitate on lab quake overnight.
6. Centrifuge at 10,000×g for 10 min. to pellet cement. Wash cement with 20mM Tris-Cl pH 8.0, 5mM EDTA. Centrifuge.
7. Resuspend in 1/50th original volume in 20mM Tris-Cl pH8.0, 5mM EDTA.
Example 4 - Identification of the Gene Encoding the Initial Member of the
39 kDa Antigen family
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.
Met-Tyr- Gly-Asp-Arg-Asp probe name = COD 555
ATG-TAT-GGT-GAT-AGT-GA
C C C C
A A degeneracy = 128 fold
G G (mix of 128 combinations)
10
Trp-IIe-Asp-Phe-Leu-Thr probe name = COD 553
TGG-ATT-GAT-TTT-TTT-AC
C C C C A A
G degeneracy = 96 fold
18
His- Gly- Asn- Gln- Phe- Arg probe name = COD 556 CAT-GGT-AAT-CAA-TTT-AG
C C C G CC
A degeneracy = 128 fold
G
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
hybridization to probes 553 and 555. The DNA was examined after digestion with EcoR1 and found to contain a 1.6 kb insert.
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
transcription-translation system containing
35 S-Methionine. SDS-gel electrophoresis of the protein products of this system showed 39 kDa protein species not seen with the parental plasmid lacking the T. hyo DNA insert. This suggests that the cloned DNA contains the complete coding sequence for the T. hyo 39 kDa antigen and that E. coli is capable of recognizing the treponemal promoter and ribosome binding site and directing the synthesis of this foreign protein.
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)). At various times during this digestion, 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.
PURIFICATION OF THE RECOMBINANT FORM OF THE
39 KDA ANTIGEN
E. coli strain CY-15,000 containing plasmid pTrep301 was grown in 250 mis of Luria broth
containing ampicillin at 200 μg/ml. The culture was grown for 18 hours at 37° C. The cells were
harvested by centrifugation then resuspended in 1/20 their original volume in a buffer of 25 mM Tris, 10 mM EDTA at pH 8.0 and containing lysozyme at 1 mg/ml. After a 30 minute incubation at room temperature the cells had lysed and were then further disrupted by sonication. The non-ionic detergent, Triton X-100 was added to a final concentration of 2%, the cell lysate was mixed for 1 hour at room temperature and then centrifuged at 10,000 ×g. The insoluble pellet fraction after these steps was saved. 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
recognized in a single sera. As hereinafter
indicated, 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.
As discussed in Example 4, infra, the T. hyo genome contains at least 7 genes encoding related antigens with molecular weights of about 39 kDa.
Although the product of only one of these genes is isolated from cells grown in vitro, it is possible that the other members of the gene family are expressed in vivo and are of immunological relevance to protecting against infection in the field. The observation that each of these proteins is preceded by a signal sequence indicates that they will all be exported from the cyooplasm of the cell when
expressed. When cells grown in vitro are surface labeled in the presence of I 125 and lactoperoxidase the 39kDA protein in the KGP fraction is the
predominant protein identified. Thus, cells
expressing other members of the 39kDa gene family would likely have a much different surface
architecture than cells expressing the Copy5 gene in vitro. An immune response mounted against one form of the 39kDa gene family could be only marginally effective against cells expressing one of the other forms. Example 5
Identification of the genes
encoding additional members of the
39 kDa antigen family
Internal amino acid sequence from 39p was divergent enough from the predicted amino acid sequence of pTrep301 to allow the selection and synthesis of a degenerate oligonucleotide (Cod664, Table 2) that could be used to distinguish between sequences encoding the gene product of pTrep 106 and those encoding the 3 kDa antigen.
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.
In addition a library constructed by the
ligation of a partial Sau3a digest of B204 genomic DNA and BamHI digested lambda EMBL3 was also screened with these probes. This screening identified a number of phages which hybridized to either Cod664, the nick translated probe 301 Sph-Bcl or both probes.
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.
Based on the DNA sequence from the above
subclones and the internal peptide sequences, it was determined that there were at least six related genes encoding similar 39 kDa proteins. Of these six genes, analysis of protein and nucleotide sequence data indicate that gene #5 most likely encodes the 39kDa antigen found in the UP-2 and 39* fractions.
None of the subclones or combinations of
subclones contained a full length copy of the #5 gene. Therefore, additional probes were prepared to isolate the remaining portion of the gene encoding the 39kDa antigen of the UP-2 and 39* fractions as well as other genes of the 39kDa family. These probes were based on additional internal sequences of the native antigen as well as DNA sequences of phages of Table 3.
A unique oligonucleotide probe specific for Gene #5 (Cod968) (Table 2) was synthesized and used to screen the GT11 library. Two degenerate
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.
Unique oligonucleotides were also synthesized and used to screen the GT11 library for full length genes corresponding to Gene #6 (Cod 934), Gene #7 (Cod 1010 and Cod 1011) and Gene #8 (Cod 1328).
A summary of all of the phages identified through this additional screening, their
hybridization patterns and subclones is contained in Table 4.
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.
The gene sequence and predicted amino acid sequence for each of the full length genes 1-8 is shown in Appendices 2 and 2A. In addition, 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. DNA sequence of Genes #1, 2, 3 (only 3') and 4 (only 5') indicated that these genes, although containing unique internal DNA sequences, contained identical 5' and 3' DNA
sequences. Thus, two linkered oligonucleotides corresponding to the 5' sequence (Cod 987), and the reverse complement of the 3' sequence (Cod 988), were synthesized to be used as primers for DNA synthesis. They were then mixed with a template of genomic DNA from either serotype B204 or B234. The
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
corresponding antigens. 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). In addition the clones were screened with a unique synthetic oligonucleotide which is common to all known forms of the 39kDa gene family (Cod 957). Some clones hybridized only to this nondiscriminant probe and upon DNA sequence analysis were found to correspond to Gene #7 even though there is a slight mismatch of DNA sequence between Cod 987 and the 5' and 3' ends of the gene. A summary of the subclones obtained along with their hydridization patterns is found in Table 4 following the Examples.
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
encoding a protein whose amino terminal amino acid sequence is identical to that of the 39s/39p antigen. 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
obtained to date. The 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.
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.
A schematic of the B204 expression clones derived from the PCR reaction (pTrep 345, 541, 604, 60.5, 620, 613) is found in Figure 5. The predicted amino acid sequences encoded by these clones are found in Appendix 5.
Expression of recombinant forms of the 39 kDa protein from genomic DNA subclones corresponding to Genes #2 and 6.
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
Appendix 8.
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 EcoR1 fragment from this clone, pTre p701, was then cloned into the EcoR1 site of pWHA142 to place the #6 sequence in the proper reading frame for expression from the Lac promoter. A schematic of the construction of pTrep702 is presented in Figure 3. The predicted protein sequence of the recombinant product encoded by pTrep702 is presented in Appendix 6.
An expression clone encoding the full length Copy #6 antigen, pTrep704, was constructed from pTrep702 by replacing its 430 bp Nsil-Ndel fragment with an 847 bp Nsil-Ndel fragment from pTrep508. 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
Figure 3. The predicted protein sequence of the recombinant product encoded by pTrep704 is presented in Appendix 7.
The recombinant products expressed by pTrep505, pTrep702, and pTrep704 as well as the PCR derived constructs are recovered as insoluble cements in
E.coli strain CY15000 after lysis of cells with lysozyme and extraction with Triton X-100 in the presence of EDTA.
These are immunoreactive with sera from animals experimentally infected with T.hyo (B204) and with sera from animals vaccinated with UP2 or the
electroeluted 39 kDa protein from UP2. The following 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.
Table 1 - Amino Acid Comparison of 39s and 39p Antigens
Sequence Sour
MYGDRDSWIDFLTHGNQFRARMDQLGFVLGN?TIKGTF??R 39p Nterm
MYGDRDSWIDFLTHGNQFRARMDQLGFVL?NGTIKGTFGF??Q?I 39* Nterm
P?S??TK?YMGISTDIQLRYYTGIDAFNAIRLYFKYGQAGFK 39P2,4
FPYS?STKDYMGISTDIQLRYYTGIDAFNAIRLYFKYGQAGFK 39*2
TANGASEYFAQSLGFEARFYFLNTPVGNVTINPFIKVVNTA 39p2
TANGASEYFAQSLGFEARFYFLNTPVGNVTINPFIKVVNTAL 39*2
AQAVLGITANSDVVSLYVEPSLGYQATYLGK 39p2
AQAVLGITANSDVVSLYVEPSL 39*
HISENPYLNIDSK 39p1
HISENPYLNIDSK 39*2
VQHSLAWGAYAELYVRPVQDLE?YFEMDIN 39p1
RNGVPVNFATSTGIT?YLPALGG?Q 39p2
MDINNSDSKRNGVPVNFATSTGITWYLPALGGAQ 39*3
Notes - -All sequence derived from fragments generated by digestion with LysC in the presence of SDS unless otherwise indicated.
1LysC digest in absence of SDS
2 Fraction gave muplitple sequences which were resolved on basi of intensity and DNA predicted amino acid sequence
3GluC digest in presence of SDS
4 Sequence begins with residue #2 due to machine failure and loss of residue #1
Single amino acid code used above is as follows:
A=ala H=his P=pro W=trp
C=cys I=Ile Q=gln Y=tyr
D=asp K= lys R= arg
E=glu L= leu S=ser
F=ρhe M=met T=thr
G=gly N=asn V=val Table 2
COD Sequence Source of Sequence Specific 664 ACG-AAG-GAT-TAT-ATG-GG 39p internal for Copy #
A A C C peptide sequence 5, 6
T
C
844 TTAATCCGCATGATA pTrep 106 1 908 GTTTCATCACAAGCAAA pTrep 333 3, 4 931 ATGAATATGACGGATAA pTrep 330 2 932 AAAGTTGATAAACAAGG pTrep 333 4 934 TATCATCCTTCTAATCCT pTrep 331 6 957 CCGAAAGTACCTTTAAT pTrep 106 ALI 968 TATAATCCTTATGATCCT pTrep 317 5 987 GAATTCCGGATCCATGTATGGpTrep 106 1-4
AGATCAGGACGATTGGATT
988 GTCGACAAGCTTATAATTAAApTrep 106 1-4
ATTCTGGCAAATACCAAGT
1010 ATATTGACTGATAGTAT pTrep 506 4 1111 AAATAATTTTGATATG pTrep 506 2 1020 GAT-AGA-AAA-AGG-AAT-GG 39* internal peptide 5
TCT A C sequence
C
1019 AAA-AGG-AAT-GGA-GTG-CC 39* internal peptide 6
A C T A sequence
T
C
1054 GAATTCCGGATCCATGTATGGCGACAG pTrep 326 5, 6
AGATTCTTGGATC
1055 GTCGACAAGCTTATAATTATTGAGCAC pTrep 510 5
CGCCTAAAGCAGG
1092 AGTATGTTTGAACCAATA pTrep 333 3 1151 TCATATGTATCGTGTATA pTrep 333 4 1095 GGAGTACCTAAACTTCAA pTrep 337 8 1248 CGACAGAGATTCTTGGA pTrep 613 5, 6 Tabla 2 (continued)
COD Sequence Source of Sequence Specific for Copy # 1328 GAATTCAATTACGGATT pTrep 537 8
1359 GTCGACCTGCAGTTATTApTrep 520 8
TTGTAAAGCAGGTAAATACCA
1438 GAATTCCGGATCCATGTATGGpTrep 537 8
TGCAGACAACACATGGCTT
Table #3
Table #3- -Identlfication of recombinant phage
and subclones
Identified by Screening with
Phage* pTrep 301 Sph-Bcl Cod664
9B^ 317 +
54.3 323 +
53-3B 326 + +
52-1A 327 + +
55-1 329 +
56-3 330 +
51-3 333 +
51-1B 331 + +
53-1B 337 + +
* All recombinant phage are from the GT11 library unless otherwise noted ^ From EMBL3 library
Table #4
Table #4- -ldenlificatlon of recombinant phage
and subclones
Identified by Screening with
Phage* pTrep 301Sph-Bcl 900 034 000 1010 1010 1020 1002 1005 1151 1240 1328 957
21 506 + +
17 509 + +
130 517 + +
544 525 + +
163 510 + +
2 631 +
519 520 +
109 515
441 349 +
101A 537 + +
Table 5
Expression Clones from PCR
Clone Serotype Cod957 Cod844 Cod908 Cod931 Cod932 Cod968 Cod1151 pTrep345 B204 + + pTrep605 B204 + +
pTrep604 B204 + + +
pTrep608 B234 + +
pTrep609 B234 + +
pTrep610 B234 + +
pTrep613 B204 + +
pTrep620 B204 +
pTrep651 B234 +
pTrep541 B204 +
Table 6
Clone Copy# Source Comments pTre p301 1 genomic pTrep505 2 genomic lacks amino
terminus pTre p604 3 per
pTrep345 4 per
pTrep613 5 per
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
Example 6
Use of 39* Antigen in a Vaccine
In two vaccination studies, purified 39* which includes 39s antigen and which is prepared according to the procedure of Example 1 (note pages 11 and 12) was tested in comparison with no challenge,
vaccination with adjuvant, or vaccination with the commercially available Hyguard (Haver Labs) product.
In the first study, six pigs per test group were used. The pigs averaged 22.6 lb and were
approximately 5-6 weeks of age. Five groups of pigs were given two doses, the first on day 0 and a booster on day 36. The injections were given I.M., in the neck with 1 mg/dose of native antigen.
Animals were challenged on day 50 by stomach
intubation using a pure culture of T. hyo. (B204) at 5.5 × 102 cfu per pig. The study was terminated on day 92.
Vaccines were given with Emulsigen adjuvant. Emulsigen was used as an adjuvant control, mixed with Dulbeco's PBS buffer. The Hyguard, a bacterin, was adminstered according to manufacturer's directions.
Animals were monitored daily for clinical signs of swine dysentery. Microbiological evaluation of routine weekly rectal swabs was conducted for T. hyo. and Salmonella. Animals showing signs of bloody diarrhea were swabbed and evaluated on that day.
Weekly postchallenge pigs were weighed and their feed intake determined. The experimental results are shown below.
Figure imgf000047_0001
Exatnple 7
Use of Native Antigen
A second study was conducted to confirm the results of Example 5 where the 39* vaccinates
performed exceptionally well when challenged
intra-gastrically with a measured dose of T.
hyodysenteriae. As shown below, 1 of 9 animals vaccinated with a 1 mg dose of 39* and challenged by stomach intubation with 4.4 × 102 CFU of T. hyo
(B204) developed clinical signs of swine disentery in comparison to 3 of 6 adjuvant vaccinates and 0 of 6
HyGuard vaccinates.
# of Breaks/
Vaccinate Group # Animals Day of Onset
No Challenge 0/6
Adjuvant 3/6 11, 11, 12
HyGuard 0/5
39*, 1mg 1/6 29
39* , 1mg- -new lot 0/3
Example 8
Production of Antibody
A cement preparation (Part G of Example 4) in an amount of 25-200 μg 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.
Numerous modifications and variations of the present invention are possible in light of the above teachnings; therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
Figure imgf000050_0001
FIGURE # --DNA Sequence of B204 Genes Encoding 39 kDa Antigens 1-4
1 GAATTCTATCAGAATATTTTTTTATAATATAAATATTTTTTTTATCTTCTATATCTATA
60 TATTTGCTGAGAT4ATCTTTTAATACATAATAANGCATTTTTTTNCATTNATTTTCANTT
120 TGTTAAATCATATATAATATTGTTAATATCATTAATTAAATTATCAATATTTAAATTATT
180 ACTATCTTGTTTTTTTATTTTTTATCATAGAT9AAGCACCAGCAGCTCCTATAGCCAATG
240 GTATACCTATCACAGGCAATAAGGTAGCTCCTAATACAGCACTAGTTGCTGCTGCCGTTG
300 CACCTATAGCACCAGCTGCTACTGCAGTACCGCCTAATAAACTAGAATATAAAGATGGTA
360 TTTGTTTTATTGATTCATTATTTTTAGATAATTCTTGCATCAAATATGTTAAGTTTTTTA
420 AAATAGGTATGAATTTTGATGAATATATTTCTTCTTCTATATTTTTTGTGCTATTAAAAC
480 AAGAAAATAAAATTTTTCCTATTGTGCTTTTTCCAGTGTTATTCTCTCCGGCTATTACAG
540 TAATACCATCTATTGTTATATTAGCTTCTTTGATTTTAGAAAAATTTTTTATATTCAATT
600 CCATTATATTTTATCCTTTAATAATTCTGTTATTATTATAACATAAAAAATATTTAATAA
660 AAATATATTAATTTTMATTAAATCTAATTCTTGAGCTATTTTATATTTTTAGTATAATAA
* Presumed translational start MetLysLysPhePheLeuIleMetThrVal 720 AAAATATAAACTTCAATTTAGGTATGTATAATGAAAAAGTTTTTTCTAATTATGACAGTA of Copy 1
LeuLeuSerMetSerTyzCysSecIlePheGlyMetTyzGlyAspGlnAspAspTzpIle
780 TTATTAAGTATGTCATATTGTTCAATTTTTGGTATGTATGGAGATCAGGACGATTGGATT
AspPheLeuThrAapGlyAanGlnPheArgAlaArgMetAspGlnLeuGlyPheValLeu
GlyAsnSerThrIleLysGlyThrPheGlyPheArgThrGlnSerSerSerThrGlnLeu 900 GGTAATAGTACTATTAAAGGTACTTTCGGTTTTAGAACTCAAAGTTCATCAACTCAATTA
GlyTyrIleLeuLeuAsnAsnAsnLeuGlyThrTyrLeuGlyAlaThrIleSerGlyGly 960 GGATATATTTTGTTGAATAATAATCTTGGTACTTATTTGGGAGCAACTATTTCTGGCGGT
IleGlyTyrThrSerGluAlspheSerIleGlyIleGlyTyrAsnTyrThrSerHisSer 1020 ATAGGATATACTTCTGAGGCTTTTAGTATAGGCATAGGCTATAATTATACCAGCCATTCC
LeuPheProThrSerAspAsnPheGlySerHisThrProValLeuMetIleAsnAlaLeu 1080 TTATTTCCTACTAGCGATAACTTTGGTTCTCATACTCCASTACTTATGATTAATGCTTTA
AsnAspAsnLeuArgIleValIleProValGlnIleLeuValHisAanGluSerIleAsp 1140 AATGATAATTTGAGGATAGTTATTCCTGTGCAAATATTAGTACATAATGAAAGTATTGAT
GlnLeuGlyTyrTyrArgAspAsnTyrLeuGlyIleSerThrAspThrGlnIleArgTyr 1200 CAACTTGGTTACTATAGAGATAATTATTTAGGTATAAGTACTGATACGCAAATAAGATAT
TyrThzGlyIleAspAlaPheAsnGluZleAzgLeuTyrValLysTyrGlyGlnLeuGly
1260 TATACAGGCATAGATGCTTTTAATGAAATAAGATTATATGTAAAATATGGGCAATTAGGA TyrLysIleAsnProHisAspThrIleAsnTyrThrGlnGluValLeuAlaArgSerPhe 1320 TATAAAATTAATCCGCATGATACTATAAATTATACACAAGAAGTTTTAGCAAGATCATTT
GlyPheGluThrArgPheTyrPheLeuAsnThrAlaValGlyAanValThrIleAanPro 1380 GGTTTTGAAACAAOATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCT
PheIleLysValAlaTyrAsnThrAlaLeuHisGlyTyrSerThrMetValArgAlaLeu 1440 TTTATTAAAGTAGCATATAATACAGCTTTGCATGGATATAGTACCATGGTAAGAGCATTG
AspGlyMetTyrGluGluIleGluGlyTyrTyrProAspSerProAlaGlnSerTyrGlu 1500 GATGGTATGTATGAASAAATAGAAGGTTATTATCCAGATAGTCCTGCTCAATCATATGAA
AspIleAsnValLysTrpAspLysAsnProTyrAspValThrValGlnAlaValLeuGly 1560 GATATTAATGTTAAATGGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTGGGA
ValThrAlaAsnSerAapIleValSerLeuTyrValGluProSerLeuGlyTyrArgAla 1620 GTAACTGCTAATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCT
LysTyrLeuGlyLysLeuThrTyrGluAspProAspGlyLysValAsnPheAspPheLys 1680 AAATATTTAGGAAAATTAACATATGAAGATCCAGATGGAAAAGTTAATTTTGATTTTAAA
ValAsnHisTyrLeuSerTrpGlyAlaTyrAlaGluLeuTyrIleThrProValLysAsp 1740 GTTAATCATTATTTATCTTGGGGTGCTTATGCAGAGCTTTATATAACACCGGTAAAAGAT LeuGluTrpTyrPheGluMetAspValAsnAsnSerAspSerAspSerThrGlyIlePro 1800 TTAGAATGGTATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCT
ValSerPheAlaSerThrThrGlyIleThrTrpTyrLeuProGluPheOC
1860 GTTAGTTTTGCTTCTACTACAGGAATAACTTGGTATTTGCCAGAATTTTAATTATAAAGC
1920 AAATTTTATATGATAAAATAAAAAATGTGGGGTATTTATTATTAAAAAATAAATACCCCA
1980 CATTTTATTAAATAATTTTTTCAGTAATTTTACATTTATATATTTTTTAGTATAATAAAA
* Presumed translational start of MetLysLysIlePheLeuIleMetThrValLeu 2040 ATATAAACTTAAATTTAGGTATATACAATGAAAAAAATTTTTCTAATTATGACAGTATTA
Copy 2
LeuSerMetSerTyrCysSerIlePheGlyMetTyrGlyAspGlnAspAspTrpIleAsp 2100 TTAAGTATGTCATATTGTTCAATATTTGGTATGTATGGAGATCAGGACGATTGGATTGAT PheLeuThrAspGlyAsnGlnPheArgAlaArgMetAspGlnLeuGlyPheValLeuGly
2160 TTTCTTCAGACGGCAATCAGTTTAGAGCTAGAATGGATCAATTAGGATTTGTTTTAGGT
AsnSerThrIleLysGlyThrPheGlyPheArgSerGlnSerLeuSerThrGlnLeuGly 2220 AATAGCACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCAATTAGGA
TyrIleLeuAlaIleTyrLysAspTyrThrTyrLeuGlyAlaThrIleSerGlyGlylle
2280 TATATTTTGGCTATATATAAAGATTATACTTATTTAGGAGCAACTATTTCCGGCGGTATA
GlyTyrThrSerGluAlaPheSerIleGlyLeuGlyTyrAsnTyrThrThrProLeuPro 2340 GGATATACTTCTGAGGCTTTTAGTATAGGTTTAGGTTATAATTATACTACACCGCTTCCT
IleSerTyrAsnPheGlySerHisThrProValLeuMetIleAsnAlaLeuAsnAapAsn 2400 ATTASTTATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAAT LeuArgIleValIleProValGlnIleLeuValHisAspGlyAanMetAsnMetThrAsp 2460 TTGAGGATAGTTATTCCTGTACAAATATTAGTACATGATGGTAATATGAATATGACGGAT
AsnIleAsnTyrLeuTyrAsnPheLeuGlyIleSerThrAspThrGlnIleArgTyrTyr 2520 WTATTAATTATTTATATAATTTTTTAGGTATAAGTACTGATACTCAAATAAGATATTAT
ThrGlyIleAspAlaPheAsnGluIleArgLeuTyrValLysTyrGlyGlnLeuGlyTyr 2580 ACAGGCATAGACGCTTTTAATGAAATAAGATTATATGTAAAATACGGACAATTAGGATAT
LysGlyGlySerTyrThrAspLysSerTyrAspGluGluPhePheAlaArgSerPheGly 2640 AAAGGCGGTTCATATACGGATAAAAGTTATGATGAAGAATTTTTTGCAAGATCATTTGGT
PheGluThrArgPheTyrPheLeuAsnThrAlaValGlyAsnValThrIleAsnProPhe
2700 TTTGAAACAAGATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCTTTT
IleLysValAlaTyrAanThrAlaLeuHisGlyPheSerThrMetValArgSerLeuAsp 2760 ATTAAAGTAGCATATAATACAGCTTTGCATGGATTTAGTACTATGGTAAGATCATTAGAT
SerValIleGluGluZleGluGlyTyrSerSerAspArgThrAlaLysAlaAlaGlyAsn 2820 AGTGTCATTGAAGAAATAGAAGGTTATAGTTCAGATCGTACCGCTAAAGCAGCAGGAAAT
ZleAsnAlaLysTrpAspLysAsnProTyrAspValThrValGlnAlaValLeuGlyVal 2880 ATTAATGCTAAATGGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTGGGAGTA
ThrAlaAsnSerAapIleValSerLeuTyrValGluProSerLeuGlyTyrArgAlaLys 2940 ACTGCTAATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCTAAA
TyrLeuGlyLysLeuThrTyrGluAspProAapGlyLysValAanPheAspPheLysVal 3000 TATTTAGGAAAATTAACATATGAAGATCCAGATGGAAAAGTTAATTTTGATTTTAAAGTT
AsnHisTyrLeuSerTrpCyaAlaTyrAlaGluLeuTyrIleThrProValLysAspLeu 3060 AATCATTATTTATCTTGGTGTGCTTATGCAGAGCTTTATATAACACCTGTAAAAGATTTS
GluTrpTyrPheGluMetAspValAsnAsnSerAspSerAspSerThrGlyIleProVal 3120 GAATGGTATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCTGTT
SerPheAlaSerThrThrGlyIleThrTrpTyrlLeuProGluPheOC
3180 AGTTTTGCTTCTACTACAGGAATAACTTGGTATTΓGCCAGAATTTTAATTATAAAGCAAA
3240 TTTTATATGACAAAATAAAAAATGGGGCATTTATTATTAAAAAATAAATACCCCACATTT
3300 TATTAAAIAACTTCTTAAATAATTTTACA4TRTATATTTTATTAGTATAATAAAATATAA
* Presumed translational start of Copy 3 MetLysLysSerPheLeuIleMetThrValLeuLeuSer 3360 AGTTAAATTTAGGTGTGTACAATGAAAAAAAGTTTTCTAATTATGACAGTATTATTAAGT
MetSerTyrCysSerIlePheGlyMetTyrGlyAspGlnAspAspTrpIleAspPheLeu 3420 ATGTCATATTGTTCAATATTTGGTATGTATGGAGATCAGGACGATTGGATTGATTTTCTT
ThrAspGlyAanGlnPheArgAlaArgMetAspGlnPheGlyPheValLeuGlyAsnSer 3480 ACAGACGGTAATCAGTTTAGAGCTAGAATGGATCAATTTGGATTCGTTTTAGGTAATAGC
ThrIleLysGlyThrPheGlyPheArgSerGlnSerLeuSerThrGlnLeuGlyTyrIle 3540 ACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCAATTAGGATATATT
LeuAlaIleTyrLysAspTyrThrTyrLeuGlyAlaThrIleSerGlyGlyIleGlyTyr 3600 TTGGCTATATATAAAGATTATACTTATTTAGGAGCAACTATTTCCGGCGGTATAGGATAT ThrSerGluAlaPheSerlleGlyLeuGlyTyrAsnTyrThrThrProLeuProIleSer 3660 ACTTCTGAGGCTTTTAGTATAGGTTTAGGTTATAATTATACTACACCGCTTCCTATTAGT
AspAsnPbeGlySerHisThrProValLeuMetIleAsnAlaLeuAsnAspAsnLeuArg 3720 GATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAATTTGAGG
ZleValIleProValGlnZleLeuValTyrAsnGlyAsnValGlnLysValAspLysGln 3780 ATAGTTATTCCTGTACAAATATTAGTATATAATGGTAATGTTCAAAAAGTTGATAAACAA
GlyAsnIleSerTyrSerHisAspTyrLeuGlyIleSerThrAspThrGlnIleArgTyr 3840 GGTAATATCTCTTATTCACATGATTATTTAGGTATAAGTACTGATACGCAAATAAGATAT
TyrThrGlyIleAspAlaPheAsnGluIleArgLeuTyrValLysTyrGlyGlnLeuGly 3900 TATACAGGTATAGATGCTTTTAATGAAATAAGATTATATGTAAAATATGGGCAATTAGGA
TyrLysAsnAlaProTyrValGlyLysAsnTyrGluGluGluPhePheSerArgSerPhe 3960 TATAAAAATGCTCCGTATGTTGGTAAGAATTATGAAGAAGAATTTTTTTCAAGGTCATTT
GlyPbeGluThrArgPheTyrPheLeuAanThrAlaValGlyAsnValThrZleAsnPro 4020 GGTTTTGAAACAAGATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCT
PhelleLysValAlaTyrAsnThrAlaLeuHisGlyPheSerThrMetIleArgAlaLeu 4080 TTTATTAAAGTAGCATATAATACAGCTTTGCATGGATTTAGTACCATGATAAGAGCATTA
AspSerMetPheGluProIleGluGlyTyrSerSerAspArgProValSerSerGlnAla 4140 GATAGTATGTTTGAACCAATAGAAGGTTATAGTCAGATCGTCCTGTTTCATCACAAGCA
AsnIleAsnAlaLysTrpAspLysAsnProTyrAspValThrValGlnAlaValLeuGly 4200 AATATTAATGCTAAATGGGATAAGAATCCTTATGATGTAACAGTGCAGGCAGTATTGGGA
ValThrAlaAanSerAapIleValSerLeuTyrValGluProSerLeuGlyTyrAzgAla 4260 GTAACCGCTAATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCT
LysTyrLeuGlyLysLeuThrTyrGluAspProAspGlyLysValAsnLeuAspPheLys 4320 AAATATTTAGGAAAATTAACATATGAAGATCCAGATGGAAAAGTTAATTTGGATTTTAAA
ValAsnHisTyrLeuSerTrpGlyAlaTyrAlaGluLeuTyrIleThrProValLysAsp 4380 GTTAATCATTATTTATCTTGGGGGGCTTATGCAGAGCTTTATATAACACCGGTAAAAGAT LeuGluTrpTyrPgeGluMetAspValAsnAanSerAspSerAapSerThrGlyIlePro 4440 TTGGAATGGTATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGAATACCT
ValSerPheAlaSerThrThrGlyIleThrTrpTyrLeuProGluPheOC
4500 GTTAGTTTTOCTTCTACTACAGGAATAACTTGGTATTTGCCAGAATTTTAATTATAAAGC
4560 AAATTTTATATGACAAAATAAAAAATGNGGGNTATTTATTATTAAAAAATAAATACCCCG
4620 TTTTTTATTAAATAACTTCTTAAATAATTTTACATTTTTATATTTTATTAGTATAATAAA
* Presumed translational start of MetLysLysPheLeuIlsMetThrValLeuLeu 4680 ATATAAAGTTAAATTTAGGTGTGTACAATGAAAAAGTTTCTAATTATGACAGTATTATTA
Copy 4
SerMetSerTyrCysSerIlePheGlyMetTyrGlyAspGlnAspAspTrpIleAspPhe 4740 AGTATGTCATATTGTTCAATATTTGGTATGTATGGAGATCAGGACGATTGGATTGATTTT LeuThrAspGlyAsnGlnPheArgAlaArgMetAspGlnPheGlyPheValLeuGlyAsn 4600 CTTACAGACGGTAATCAGTTTAGAGCTAGAATGGATCAATTTGGATTCGTTTTAGGTAAT
AsnThrlleLysGlyThrPheGlyPheArgSerGlnSerLeuSerThrHisLeuGlyTyr 4860 AACACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCACTTAGGCTAT
IleLeuLeuAsnAsnAsnPheGlyThrTyrPheGlyThrThrIleSerCysGlyIleGly 4920 ATTTTGTTAAATAATAATTTTGGTACTTATTTTGGAACAACTATATCATGCGGTATAGGA
TyrThrSerGluAlaPheSerIleGlylleGlyTyrAsnTyrThrThrProLeuProIle 4980 TATACTTCTGAGGCTTTTAGTATAGGTATAGGTTATAATTATACTACACCGCTTCCTATT
SerAspAsnPheGlySerHisThrProValLeuMetIleAsnAlaLeuAanAspAsnLeu 5040 AGTGATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAATTTG
ArgIleValIleProValGlnIleLeuValTyrAanGlyAsnIleβlnLyβValAspLys 5100 AGGATAGTTATTCCTGTACAAATATTAGTATATAATGGTAATATTCAAAAAGTTGATAAA
GlnGlyAsnIleHisAspThrTyrλspTyrLeuGlyIleSerThrAspThrGlnIleArg 5160 CAAGGTAATATACACGATACATATGATTATTTAGGTATAAGTACTGATACGCAAATAAGA
TyrTyrThrGlyZleAspAlaPheAsnGluZleArgLeuTyrIleLysTyrGlyGlnLeu 5220 TATTATACAGGTATAGATGCTTTTAATGAAATAAGATTATATATAAAATATGGACAATTA
GlyTyrLysAsnAlaProTyrValGlyLysAsnTyrGluGluGluLeuPheSerArgSer 5280 GGATATAAAAATGCTCCGTATGTTGGTAAAAATTATGAAGAAGAACTTTTTTCAAGGTCA
PheGlyPheGluThrArgPheTyrPheLeuAanThrThrValGlyAsnValThrIleAan 5340 TTTGGTTTTGAAACAAGATTCTATTTTTTGAATACTACTGTTGGAAATGTAACTATTAAT
ProPheIleLysValAlaTyrAsnThrAlaLeuHisGlyValGlyThrMetIleArgAla 5400 CCTTTTATTAAAGTAGCATATAATACAGCTTTGCATGGAGTTGGTACTATGATAAGAGCA LeuAapThrMetLeuGlnProIleGluAspTyrTyrProAspArgProValSerSerGln 5460 TTAGATACTATGCTTCAACCAATAGAAGATTATTATCCAGATCGTCCTGTTTCATCACAA
ValAapIleAspTyrLysLeuAspLysAsnProTyrAspValThrValGlnAlaValLeu 5520 GTAGATATTGATTATAAATTGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTG
GlyValThrAlaAsnSerAapIleValSerLeuTyrValGluProSerLeuGlyTyrLys 5580 GGAβTAACCGCTAATAGTGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAAG
AlaLysTyrLeuGlyLysMetGlnAspGluLysValAsnLeuAspPheLysValAsnHis 5640 GCTAAATATTTAGGAAAAATGCAAGATGAAAAAGTTAATTTGGATTTTAAAGTTAATCAT
TyrLeuSwrTrpGlyAlaTyrAlaGluLeuTyrIleThrProValLysAspLeuGluTrp 5700 TATTTATCTTGGGGTGCTTATGCAGAGCTTTATATAACACCTGTAAAAGATTTAGAATGG
TyrPheGluMetAspValAsnAsnSerAspSerAspSerThrGlyIleProValSerPhe 5760 TATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCTGTTAGTTTT
AlaSerThrThrGlyIleThrTrpTyrLeuProGluPheOC
5820 GCTTCTACTACAGGGATAACTTGGTATTTGCCAGAATTTTAATTATAAA6AAAATTTTAT
5880 ATGACAAAATAAAAAATAAGAGGTATTTATTTTTTTAATAATAAATACCTCTCTTATTTT
5940 ATTTATA8CTTTTTAATTTAATCTCTTCTTCTTTGTGCTAACATTAAAAOTCTTAAAAGT 6000 GTAAGTATAGCAGTAACAGCAGCAGCAACATAAGTCAAAGCAGCAGCAGATAAAACTTTT
6060 TTAGCTCCGTCAAGTTCTTCACTGTCTAAAAATCCGCCTTTATCCAATATTTTTATAGCT
Appendix 2 B
Figure # - -DNA Sequence of B204 Genes Encoding 39 kDa Antigen 5-8
*5' end of genomic DNA
1 CTGTATCTGTTTATTATCAGTCAGCATTTCATTCTTATTATTATGGCGATAAAAATATAA
61 AAATGGAAACTATAGTCGGAAACTTAATAGGAAGCGT4GCCAAAGAAAATAAAGATGATA
121 TACCAAAATTAAAGAACTATTTTAATAATTCTGTAAAAATAAAAGCTGAGAAATTTGGTA
181 AAAAATGGAAAGACTATTATGAAAGCAGAAATCTTATAAATAATTTTAATTTATAATATA
241 TTACCGATAAAAACAGCATTAATGTTTTTTATAATACTGTATTTCACAAAAATATAGCAT
301 TATGTATTAAATAAACTTTAATATATCTTTATATTTTATACTTTTGACTGTAATATTAAA
361 TTATCATTTATATAACTATACCTAAATAGATATATTACTATAATTGTATCACAAATATGA
421 TATAAACCATCATATTTAACATAAGAAAAAATATATTATTATTAAΛATATAAAAATTTTT
481 GTATTTTTTATAGAATTTTAA9AAAAATTTTTATATAATAATATTCATATATATTAGGA4
* Presumed translational start of Copy 8
MetLys PheLeuLeuThrValLeuAlaIleLeuThrIleAlaSerGly 541 AAAATAAAAATGAAAAA6TTTTTATTAACAGTGCTGGCTATTTTAACAATAGCTAGCGGA SerValPheGlyMetTyrGlyAlaAapAsnThrTrpLeuPhePheLeuIleHisGlyAsn 601 TCAGTGTTTGGTATGTATGGTGCAGACAACACATGGCTTTTCTTCCTCATACATGGCAAC
GlnPheArgAlaArgMetAsnGlnLeuGlyPheThrLeuGlyAsnGlyXleIleLysGly 661 CAATTCAGAGCTAGAATGAACCAATTAGGTTTCACTCTAGGTAACGGCATTATTAAAGGT
ThrPheGlyPheLysAlaAsnThrLeuIleAanGlySerIleLeuAsnThrGlyAsnLys 721 ACTTTCGGTTTCAAAGCTAATACσCTTATTAACGGAAGCATCTTAAATACAGGCAATAAA
GluAsnGlnλsnProLeuGluAlaThrXleSerAlaGlyXleGlyTyrThrGlyAspGly 781 GAAAACCAAAATCCATTAGAAGCTACTATTTCTGCTGGTATAGGTTACACAGGTGATGGT PheGlyValGlyValGlyTyrAsnTyrThrTyrThrAlaAlaAsnThrIleGlnThrLys 841 TTTGGTGTTGGTGTTGGTTATAACTATACTTATACTGCTGCAAATACTATTCAAACCAAA
AlaAlaLysGlyIleAsnThrHisThrPiroValIleThrPheAsnAlaValAsnAsnAsn 901 GCTGCTAAAGGAATAAATACTCATACACCTGTTATTACATTCAATGCTGTTAATAACAAT
LeuArgValAlaIleProValSerIleAlaValGluLysAspIleGlyLysLeuGlyAsn 961 TTAAGAGTAGCTATACCTGTAAGTATAGCTGTAGAAAAAGATATAGGTAAATTAGGTAAT
MetAspArgLysAspTyrLeuGlyLeuSerIleProAlaGlnIleArgTyrTyrThrGly 1021 ATGGATAGAAAAGATTATTTAGGTTTAAGCATACCTGCTCAAATAAGATATTATACAGGA
IleAspAlaPheAsnTyrIleArgPheGluPheAanTyrGlyLeuAsnLysTyrAsnGly 1081 ATAGATGCTTTCAACTATATAAGATTTGAATTCAATTACGGATTAAATAAATATAATGGT
ValGluAsnAsnThrThrThrGluTyrGlnAlaGlnThrIleSerPheGlnLeuArgLeu 1141 GTTGAAAACAATACAACTACAGAATATCAAGCACAGACTATAAGCTTCCAATTAAGACTT HisPheLeuAsnThrValLeuGlyAsrfAsnValThrValAsnProPheLeuArgValAsp 1201 CATTTCTTAAATACAGTTCTTGGAAATAATGTAACTGTAAACCCATTCTTAAGAGTTGAT
PheAlaSerThrValGlyAlaLysGlyLysGlyAsnValValPheProAlaAlaThrAla 1261 TTTGCTTCTACTGTAGGTGCTAAAGGAAAAGGAAATGTAGTATTCCCAGCAGCTACAGCT
PheAspGlyArgLeuThrAlaTrp AlaAsnAlaTrpAlaAspAspProHisSerIle 1321 TTTGATGGAAGACTTACAGCTTGGOCTGCAAATGCTTGGGCAGATGATCCACACAGCATT
TyrAspArgGluLeuTyrAspLeuLysIleIleProSerValSerLeuSerValAsnThr 1381 TATGACAGAGAACTTTATGACTTGAAAATCATACCTAGCGTATCTTTAAGTGTTAATACA
AspTyrValAsnLeuIlePheGluProGlyLeuGlyTyrArgValGlnAspAspGlyVal 1441 GACTATGTTAATTTAATATTTGAACCTGGTTTAGGATACAGAGTACAAGATGATGGTGTA
LysGlySerLysLeuThrHisThrLeuTyrTrpGlnAlaTyrGlyGluIleTyrIleArg 1501 AAAGGAAGCAAACTTACTCATACATTATACTGGCAGGCTTACGGAGAAATATATATCAGA
ProValGlnAspLeuGluTrpTyrPheGluMetAspValAsnAsnGlyValProLysLeu 1561 CCTGTTCAGGATCTTGAATGGTATTTCGAAATGGATGTTAATAACGGAGTACCTAAACTT
GlnGlyAsnProIleAlaSerGlyAsnSerMetProValValPheGlyAlaAsriThrGly 1621 CAAGGAAATCCTATTGCTTCAGGAAATTCAATGCCTGTTGTATTCGGAGCTAATACTGGT
IleThrTrpTyrLeuProAlaLeuGln
1681 ATAACTTGGTATTTACCTGCTTTACAATAATAAATAACAGTGAATAATCAGAAATAACTG
1741 ATTTTTAATACAAGGGGAGCTTCATTAAAAAGCTCCTCTTTTTTATGAGAAAAACAAATA
1801 TAAAGCTTNNGTTCATAATTATCAATTTAATCAAAACATACTAAGCAAATTCAAAATTTC
1861 ATAAAAAATAATTATTCAATAAAAATTCATAATTTTTTATTAAAAAAATATGYAAATATG
1921 TATTTGAATGACTATTTTTTTATATAATTCCTTTTATATGTATTGACATTTTATAACTTT
1981 TTCCTATAATGAAAGTAGAGGATCTTTAGGTGTCTATTGAGTTTTTAAGCGATCTCTCAA
* Presumed translational MetLysLysValLeuLeu. 2041 TAGACACTGTCAAAAGATTATAAAAAAATTAGGAGAAAAACAATGAAAAAAGTTTTATTG start of Copy 6
ThrAlaMetAlaLeuLeuThrIleAlaSerAlaSerAlaPheGlyMetTyrGlyAspArg 2101 ACAGCTATGGCATTATTGACTATAGCTAGTGCATCTGCTTTCGGTATGTATGGCGACAGA
AspSerTrpIleAspPheLeuThrHisGlyAsnGlnPheArgAlaArgMetAspGlnLeu 2161 GATTCTTGGATCGACTTCCTTACTCATGGTAATCAGTTCAGAGCTAGAATGGATCAATTA
GlyPheValLeuGlyAsnGlyThrIleLysGlyThrPheGlyPheArgSerGlnAlaIle 2221 GGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACTTTCGGTTTTAGATCTCAAGCTATT
GlyThrAlaLeuGlyAsnIleIleSerGlyAsnThrGlyAsnValAspLeuGlnThrThr 2281 GGAACAGCATTAGGTAATATCATTTCAGGTAATACTGGAAATGTAGATTTACAAACTACT IleSerAlaGlyIleGlyTyrThrSerGluProPheGlyIleGlyValGlyTyrAsnTyr 2341 ATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCGGTATTGGCGTAGGTTATAACTAC
ThrTyrValAsnProArgLeuGlyValHisThrProValLeuMetIleAsnAlaLeuAsn 2401 ACTTATGTAAATCCTAGATTAGGCGTTCATACTCCTGTACTTATGATCAATGCTTTAAAC
AsnAsnLeuArgIleAlaValProValGlnIleAlaValSerHisAspProPheAsnAsp 2461 AACAACTTAAGAATAGCAGTTCCTGTTCAAATAGCTGTAAGTCATGATCCTTTCAATGAT
SerAlaLysPheProTyrSerSerSerThrLysAspTyrMetGlyIleSerThrAspIle 2521 TCTGCTAAATTCCCTTATTCATCATCTACAAAAGATTATATGGGTATAAGCACTGATATA
GlnLeuArgTyrTyrThrGlyIleAspAlaPheAsnAlaIleArgLeuTyrPheLysTyr 2581 CAATTAAGATACTATACTGGTATAGATGCTTTCAATGCTATAAGATTATACTTCAAATAC
GlyGlnAlaGlyPheLysThrAlaAsnGlyAlaGlyAlaSerGluTyrPheAlaGlnSer 2641 GGACAAGCTGGATTTAAAACAGCTAACGGAGCTGGAGCTAGTGAGTATTTTGCTCAGTCA
LeuGlyPheGluAlaArgPheTyrPheLeuAsnThrProValGlyAsnValThrIleAsn 2701 TTAGGTTTTGAAGCTAGATTCTATTTCTTGAATACTCCTGTTGGAAACGTAACTATCAAT
ProPheIleLysValValTyrAsnThrAlaLeuLysGlyValSerArgThrValArgAla 2761 CCTTTCATCAAAGTTGTTTATAACACAGCTTTAAAAGGTGTAAGCAGAACTGTAAGAGCT
GlyGluAlaValGlnAsnThrValSerGlyTyrHisProSerAsnProAsnTyrLysLeu 2821 GGAGAAGCTGTACAAAATACTGTTTCTGGTTATCATCCTTCTAATCCTAATTATAAATTA
AspAlaPheAlaGlyArgTyrIleGlyLysAspPheLysTrpAspSerAsnProTyrAsp 2881 GATGCATTTGCTGGTAGATACATTGGTAAAGATTTCAAATGGGATTCAAATCCTTATGAT
ValLysAlaGlnAlaValLeuGlyIleThrAlaAsnSerAspValValSerLeuTyrVal 2941 GTAAAAGCTCAGGCTGTATTAGGTATCACTGCTAACAGCGATGTAGTATCTCTTTATGTT
GluProSerLeuGlyTyrGlnAlaThrTyrLeuGlyLysAsnIleSerGluAsnProTyr 3001 GAGCCTTCTTTAGGTTATCAAGCTACATATTTAGGAAAAAACATATCTGAAAATCCATAT
LeuAsnIleAspSerLysValGlnHisSerLeuAlaTrpGlyAlaTyrAlaGluLeuTyr 3061 TTAAATATAGATTCTAAAGTACAACATAGCTTAGCTTGGGGTGCTTATGCAGAACTTTAT
ValArgProValGlnAspLeuGluTrpTyrPheGluMetAspValAsnAsnGlyGlyThr 3121 GTAAGACCTGTTCAAGATCTTGAATGGTACTTCGAGATGGATGTTAATAATGGCGGTACA
ArgGlnGluSerGlyIleProValTyrPheLysSerThrThrGlyIleThrTrpTyrLeu 3181 AGACAAGAATCTGGTATCCCTGTATACTTTAAATCTACTACAGGTATAACTTGGTATTTA
ProAlaPheAsn
3241 CCTGCTTTCAATTAATTAGAAGTTAATTAATAGAAATTAATGAGGCTGGCCTTTAATAGG
3301 TTGGCCTCTTTTTTATTAATTTTCATATTGCAAAATAGTTTATTCATTATTATATAAGTT 3361 TACATTTTATTGTTTTGCAATATAATATTTTTATCTATAATCTACCTATATAAATTATAT * Presumed t rans lational
MetLysLys IleMetLeuAla 3421 AATAGAAGAAAATATTTTATGTTTAGGAGAACAGATAAAATGAAAAAAATTATGCTGGCA start of Copy 7
AlaIleAlaIleLeuThrIlePheSerAlaSerAlaLeuGlyMetTyrGlyAspGlnAsp 3481 GCTATTGCTATATTAACTATATTTAGTGCATGTGCTTTAGGAATGTATGGAGATCAAGAT
AspTrpIleAspPheLeuThrAspGlyAsnGlnLeuArgAlaArgMetAspGlnLeuGly 3541 GACTGGATTGATTTTCTTACAGATGGAAATCAATTAAGAGCCAGAATGGATCAATTAGGA
PheValLeuGlyAsnAsnThrIleLysGlyThrPheGlyLeuArgThrGlnAspAlaVal 3601 TTTGTACTTGGAAACAATACTATTAAAGGTACTTTCGGACTTAGAACTCAAGATGCCGTA
ThrSerLeuGlySerIleIleSerGlyLysThrAspAsnLeuGlyLeuAspAlaThrVal 3661 ACATCATTGGGAAGTATAATTTCAGGTAAAACAGATAATTTAGGATTAGATGCTACTGTT
SerMetGlyIleGlyTyrThrSerAspIlePheGlyIleGlyLeuGlyTyrAsnPheThr 3721 TCTATGGGAATAGGATACACTTCTGATATTTTCGGCATTGGCTTAGGATATAATTTTACA
TyrTyrAsnSerThrLeuGlyValHisThrProValLeuMetValAsnAlaLeuAsnAsn 3781 TATTATAACAGCACTTTAGGCGTTCATACTCCTGTACTTATGGTCAATGCTTTAAATAAT
AsnLeuArgIleAlaIleProIleGlnIleAlaAlaSerLysAspProPheGlyLysTyr 3841 AATTTAAGAATAGCAATACCTATACAAATAGCTGCATCAAAAGATCCTTTCGGAAAATAT
ThrIleSerGlnTyrLysAspTyrLeuGlyIleSerThrAspIleGlnIleArgTyrTyr 3901 ACTATCAGTCAATATAAAGACTATTTAGGAATAAGCACAGATATACAAATAAGATACTAT
ThrGluIleAspValPheAsnGlnValArgLeuTyrIleLysTyrGlyGlnSerGlyTyr 3961 ACAGAAATAGATGTATTCAATCAAGTAAGATTATACATCAAATATGGACAGTCAGGTTAT
LysAsnValLysAsnAsnPheAspMetPheAlaGlnSerPheGlyPheGluThrArgLeu 4021 AAAAATGTTAAAAATAATTTTGATATGTTTGCTCAATCATTTGGTTTTGAAACTAGACTA
TyrPheLeuAsnArgThrIleGlyAsnValAsnIleAsnProPheIleLysValSerTyr 4081 TATTTCTTAAACCGCACAATTGGAAATGTAAATATTAATCCTTTTATTAAAGTTTCATAT
AsnThrAlaLeuAlaSerSerAspValMetValArgAlaGlyGluSerLeuValAsnThr 4141 AATACAGCTTTAGCCAGCAGTGATGTAATGGTTAGAGCAGGAGAATCTCTTGTAAATACT
ThrTyrSerLysLysGluAsnLysTrpGluLysAsnProTyrAsnValThrAlaAlaAla 4201 ACTTATAGTAAAAAAGAAAATAAATGGGAAAAAAATCCTTATAATGTAACTGCTGCTGCT
ValLeuGlyLeuThrAlaAsnSerAspMetLeuSerLeuCysValGluProSerLeuGly 4261 GTATTAGGATTAACTGCTAACAGTGATATGCTATCTCTTTGTGTTGAACCTTCTTTAGGA
TyrAsnAlaValTyrLysGlyLysTyrLysThrAspSerLysTyrTyrLysValGlnHis 4321 TACAATGCCGTTTATAAAGGAAAATATAAAACTGATAGTAAATATTATAAAGTACAGCAT
AsnLeuTyrTrpGlyAlaTyrAlaGluLeuTyrIleThrProValGlnAspIleGluTrp 4381 AATTTATATTGGGGAGCTTATGCAGAACTTTATATTACTCCCGTTCAAGATATTGAATGG TyrPheGluMetAspIleAsnAsnGlyAsnSerArgGlnThrSerSerIleProIleTyr 4441 TATTTTGAAATGGACATTAATAATGGTAATTCAAGACAGACTTCTTCTATACCTATATAC
PheGluSerThrThrGlyIleThrTrpTyrLeuProGluLeu
4501 TTTGAATCTACTACAGGGATAACTTGGTATTTGCCTGAATTATAATAAAAATATTATTTT
4561 GTATAATTTAAACATTTATTGAATAAAAAAAATAAAAAATTAATAATTCTATAAAAAATT
4621 TAAATTAATACTTTACATATTTAAATAAATAGCTGTTTTTATTAAATCTAAGTATTATTT
4681 TAAAAAATAATAATTTTATTTATATTAGTGTTGACTTTTTTATGTAAATTCATTAGAATA
4741 AAAGTGTTGGTAAGATATAGACATTAAAAAAAGATTGCTTATAATGTCTATAACCATATT
4801 AGGAACAATAAAGATCCTCTAAATCCTTATTATTCCAATTGACACATTAGTAAAAACAGC
4861 ATAATTTTTCCATTTAAATATCTGTTTTTACGGAGAATAAAGATCCTCTAAATCTTATTA
4921 TTCTCACTGATTTTTTATAATCCGTTAGAATTATATAGATGTTCTAACTCATCTGGAAAT
4981 AACAAAGATCCTCTGTCTTTGTTGTTTCCTAAAATTATTTTGGAGTTACTACTTACAATG
*Presumed translational st MetLysLysValLeuLeuThrAlaMet 5041 AGTATTAACTATATAAAATTTTAGGAGAAAAATAATGAAAAAAGTTTTATTGACAGCTAT of Copy 5 * Amino terminus of Copy 5
AlaLeuLeuThrIleAlaSerAlaSerAlaPheGlyMetTyrGlyAspArgAspSerTrp 5101 GGCATTATTGACTATAGCTAGTGCATCTGCTTTCGGTATGTATGGCGACAGAGATTCTTG
IleAspPheLeuThrHisGlyAsnGlnPheArgAlaArgMetAspGlnLeuGlyPheVal 5161 GATCGACTTCCTTACTCATGGTAATCAGTTCAGAGCTAGAATGGATCAATTAGGTTTCGT
LeuGlyAsnGlyThrIleLysGlyThrPheGlyPheArgSerGlnAlaIleGlyThrAIa 5221 TTTAGGTAACGGTACTATTAAAGGTACTTTCGGTTTTAGATCTCAAGCTATTGGAACAGC
LeuGlyAsnIleIleSerGlyAsnThrGlyAsnValAspLeuGlnThrThrIleSerAla 5281 ATTAGGTAATATCATTTCAGGTAATACTGGAAATGTAGATTTACAAACTACTATTTCTGC
GlyIleGlyTyrThrSerGluProPheGlyIleGlyValGlyTyrAsnTyrThrTyrVal 5341 TGGTATAGGTTATACTTCTGAGCCTTTCGGTATTGGCGTAGGTTATAACTACACTTATGT
AsnProArgLeuGlyValHisThrProValLeuMetIleAsnAlaLeuAsnAsnAsnLeu 5401 AAATCCTAGATTAGGCGTTCATACTCCTGTACTTATGATCAATGCTTTAAACAACAACTT
ArgIleAlaValProValGlnIleAlaValSerHisAspProPheAsnAspSerAlaLys 5461 AAGAATAGCAGTTCCTGTTCAAATAGCTGTAAGTCATGATCCTTTCAATGATTCTGCTAA
PheProTyrSerSerSerThrLysAspTyrMetGlyIleSerThrAspIleGlnLeuArg 5521 ATTCCCTTATTCATCATCTACAAAAGATTATATGGGTATAAGCACTGATATACAATTAAG
TyrTyrThrGlyIleAspAlaPheAsnAlaIleArgLeuTyrPheLysTyrGlyGlnAla 5581 ATACTATACTGGTATAGATGCTTTCAATGCTATAAGATTATACTTCAAATACOGACAAGC GlyPheLysThrAlaAsnGlyAlaSerGluTyrPheAlaGlnSerLeuGlyPheGluAla 5641 TGGATTTAAAACAGCTAACGGAGCTAGTGAGTATTTTGCTCAGTCATTAGGTTTTGAAGC
ArgPheTyrPheLeuAsnThrProValGlyAsnValThrIleAsnProPheIleLysVal 5701 TAGATTCTATTTCTTGAATACTCCTGTTGGAAACGTAACTATCAATCCTTTCATCAAAGT
ValTyrAsnThrAlaLeuLysGlyValSerArgThrValArgAlaGlyGluAlaValGln 5761 TGTTTATAACACAGCTTTAAAAGGTGTAAGCAGAACTGTAAGAGCTGGAGAAGCTGTACA
AsnThrValSerGlyTyrAsnProTyrAspProAsnTyrLysLeuAspAlaPheAlaGly 5821 AAATACTGTTTCTGGTTATAATCCTTATGATCCTAATTATAAATTAGATGCATTTGCTGG
ArgTyrIleGlyLysAspPheLysTrpAspSerAsnProTyrAspValLysAlaGlnAla 5881 TAGATACATTGGTAAAGATTTCAAATGGGATTCAAATCCTTATGATGTAAAAGCTCAGGC
ValLeuGlyIleThrAlaAsnSerAspValValSerLeuTyrValGluProSerLeuGly 5941 TGTATTAGGTATCACTGCTAACAGCGATGTAGTATCTCTTTATGTTGAGCCTTCTTTAGG
TyrGlnAlaThrTyrLeuGlyLysHisIleSerGluAsnProTyrLeuAsnIleAspSer 6001 TTATCAAGCTACATATTTAGGAAAACACATATCTGAAAATCCATATTTAAATATAGATTC
LysValGlnHisSerLeuAlaTrpGlyAlaTyrAlaGluLeuTyrValArgProValGln 6061 TAAAGTACAACATAGCTTAGCTTGGGGTGCTTATGCAGAACTTTATGTAAGACCTGTTCA
AspLeuGluTrpTyrPheGluMetAspIleAsnAsnSerAspSerLysArgAsnGlyVal 6121 AGATCTTGAATGGTACTTCGAGATGGACATCAATAACTCTGATTCAAAAAGAAATGGTGT
ProValAsnPheAlaThrSerThrGlyIleThrTrpTyrLeuProAlaLeuGlyGlyAIa 6181 TCCTGTTAACTTCGCAACTTCTACAGGTATAACTTGGTACTTACCTGCTTTAGGCGGTGC
Gln
6241 TCAATAATTAATTTCTGTTAATTAAAGAATTTACAGAGGCTGGCTTTAATAAAAAAGTCA
6301 GTCTCTTTTTTTATCGCATATTTTCATATAATTAAACAAAAATATTTACATTATATAATT
Figure imgf000063_0001
Figure imgf000064_0001
Figure imgf000065_0001
Appendix 5
- -Predicted amino acid sequence from PCR derived T . hγo (B204) clones pTrep605- -Copy2
10 20 30 40 50 60
MTMITNRGSM YGDQDDWIDF LTDGNQFRAR MDQLGFVLGN STIKGTFGFR SQSLSTQLGY
70 80 90 100 110 120
ILAIYKDYTY LGATISGGIG YTSEAFSIGL GYNYTTPLPI SYNFGSHTPV LMINALNDNL
130 140 150 160 170 180
RIVIPVQILV HDGNMNMTDN INYLYNFLGI STDTQIRYYT GIDAFNEIRL YVKYGQLGYK
190 200 210 220 230 240
GGSYTDKSYD EEFFARSFGF ETRFYFLNTA VGNVTINPFI KVAYNTALHG FSTMVRSLDS
250 260 270 280 290 300
VIEEIEGYSS DRTAKAAGNI NAKWDKNPYD VTVQAVLGVT ANSDIVSLYV EPSLGYRAKY
310 320 330 340 350 360 LGKLTYEDPD GKVNFDFKVN HYLSWCAYAE LYITPVKDLE WYFEMDVNNS DSDSTGIPVS
370
FASTTGITWY LPEF
pTrep604 - -Copy3
10 20 30 40 50 60 MTMITNRGSM YGDQDDWIDF LTDGNQFRAR MDQFGFVLGN STIKGTFGFR SQSLSTQLGY
70 80 90 100 110 120 ILAIYKDYTY LGATISGGIG YTSEAFSIGL GYNYTTPLPI SDNFGSHTPV LMINALNDNL
130 140 150 160 170 180 RIVIPVQILV YNGNVQKVDK QGNISYSHDY LGΣSTDTQIR YYTGIDAFNE IRLYVKYGQL
190 200 210 220 230 240 GYKNAPYVGK NYEEEFFSRS FGFETRFYFL NTAVGNVTIN PFIKVAYNTA LHGFSTMIRA
250 260 270 280 290 300 LDSMFEPIEG YSSDRPVSSQ ANINAKWDKN PYDVTVQAVL GVTANSDIVS LYVEPSLGYR
310 320 330 340 350 360 AKYLGKLTYE DPDGKVNLDF KVNHYLSWGA YAELYITPVK DLEWYFEMDV NNSDSDSTGI
370 377
PVSFASTTGI TWYLPEF pTrep345 - - Copy4
10 20 30 40 50 60 MTMITNRGSM YGDQDDWIDF LTDGNQFRAR MDQFGFVLGN NTIKGTFGFR SQSLSTHLGY
70 80 90 100 110 120 ILLNNNFGTY FGTTISCGIG YTSEAFSIGI GYNYTTPLPI SDNFGSHTPV LMINALNDNL
130 140 150 160 170 180 RIVIPVQILV YNGNIQKVDK QGNIHDTYDY LGISTDTQIR YYTGIDAFNE IRLYIKYGQL
190 200 210 220 230 240 GYKNAPYVGK NYEEELFSRS FGFETRFYFL NTTVGNVTIN PFIKVAYNTA LHGVGTMIRA
250 260 270 280 290 300 LDTMLQPIED YYPDRPVSSQ VDIDYKLDKN PYDVTVQAVL GVTANSDIVS LYVEPSLGYK
310 320 330 340 350 360 AKYLGKMQDE KVNLDFKVNH YLSWGAYAEL YITPVKDLEW YFEMDVNNSD SDSTGIPVSF
370
ASTTGITWYL PEF
pTrep613- -Copy5
10 20 30 40 50 60 MTMITNRGSM YGDRDSWIDF LTHGNQFRAR MDQLGFVLGN GTIKGTFGFR SQAIGTALGN
70 80 90 100 110 120 IISGNTGNVD LQTTISAGIG YTSEPFGIGV GYNYTYVNPR LGVHTPVLMI NALNNNLRIA
130 140 150 160 170 180 VPVQIAVSHD PFNDSAKFPY SSSTKDYMGI STDIQLRYYT GIDAFNAIRL YFKYGQAGFK
190 200 210 220 230 240 TANGASEYFA QSLGFEARFY FLNTPVGNVT INPFIKVVYN TALKGVSRTV RAGEAVQNTV
250 260 270 280 290 300 SGYNPYDPNY KLDAFAGRYI GKDFKWDSNP YDVKAQAVLG ITANSDVVSL YVEPSLGYQA
310 320 330 340 350 360 TYLGKHISEN PYLNIDSKVQ HSLAWGAYAE LYVRPVQDLE WYFEMDINNS DSKRNGVPVN
370 378
FATSTGITWY LPALGGAQ pTrep620- -Copy7
10 20 30 40 50 60 MTMITNRGSM YGDQDDWIDF LTDGNQLRAR MDQLGFVLGN NTIKGTFGLR TQDAVTSLGS
70 80 90 100 110 120
IISGKTDNLG LDATVSMGIG YTSDIFGIGL GYNFTYYNST LGVHTPVLMV NALNNNLRIA
130 140 150 160 170 180 IPIQIAASKD PFGKYTISQY KDYLGISTDI QIRYYTEIDV FNQVRLYIKY GQSGYKNVKN
190 200 210 220 230 240 NFDMFAQSFG FETRLYFLNR TIGNVNINPF HCVSYNTALA SSDVMVRAGE SLVNTTYSKK
250 260 270 280 290 300 ENKWEKNPYN VTAAAVLGLT ANSDMLSLCV EPSLGYNAVY KGKYKTDSKY YKVQHNLYWG
310 320 330 340 349
AYAELYITPV QDIEWYFEMD INNGNSRQTS SIPIYFESTT GITWYLPEF
Figure imgf000068_0001
Appendix #6- - Predicted amino acid sequence of pTre p702
recombinant product
<- - - - - -Protein derive from
MetThrMetlleThrProSerLeuHisAlaCysArgSerThrLeuGluAspProArgVal 1 ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGT pUC polylinlcer- - - - - - > | | <- - - Copy 6 ORF- - - >
ProSerSerAsnTrpGluPheProMetAlaLeuLeuThrlleAlaSerAlaSerAlaPhe 61 CCGAGCTCGAATTGGGAATTCCCTATGGCATTATTGACTATAGCTAGTGCATCTGCTTT
GlyMetTyrGlyAspArgAspSerTrpIleAspPheLeuThrHisGlyAsnGlnPheAre 121 GGTATGTACGGCGACAGAGATTCTTGGATCGACTTCCTTACTCATGGTAATCAGTTCAG
AlaArgMetAspGlnLeuGlyPheValLeuGlyAsnGlyThrIleLysGlyThrPheGly 181 GCTAGAATGGATCAATTAGGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACTTTCGG
PheArgSerGlnAlalleGlyThrAlaLeuGlyAsnIlelleSerGlyAsnThrGlyAsp 241 TTTAGATCTCAAGCTATTGGAACAGCATTAGGTAATATCATTTCAGGTAATACTGGAAA
ValAspLeuGlnThrThrlleSerAlaGlylleGlyTyrThrSerGluProPheGlyIle 301 GTAGATTTACAAACTACTATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCGGTAT
GlyValGlyTyrAsnTyrThrTyrValAsnProArgLeuGlyValHisThrProValLeu 361 GGCGTAGGTTATAACTACACTTATGTAAATCCTAGATTAGGCGTTCATACTCCTGTACT
MetlleAsnAlaLeuAsnAsnAsnLeuArglleAlaValProValGlnlleAlaValSer 421 ATGATCAATGCTTTAAACAACAACTTAAGAATAGCAGTTCCTGTTCAAATAGCTGTAAG
HisAspProPheAsnAspSerAlaLysPheProTyrSerSerSerThrLysAspTyrMet 481 CATGATCCTTTCAATGATTCTGCTAAATTCCCTTATTCATCATCTACAAAAGATTATAT
GlylleSerThrAspIleGlnLeuArgTyrTyrThrGlylleAspAlaPheAsnAlaIle 541 GGTATAAGCACTGATATACAATTAAGATACTATACTGGTATAGATGCTTTCAATGCTAT
ArgLeuTyrPheLysTyrGlyGlnAlaGlyPheLysThrAlaAsnGlyAlaGlyAlaSer 601 AGATTATACTTCAAATACGGACAAGCTGGATTTAAAACAGCTAACGGAGCTGGAGCTAG
GluTyrPheAlaGlnSerLeuGlyPheGluAlaArgPheTyrPheLeuAsnThrProVal 661 GAGTATTTTGCTCAGTCATTAGGTTTTGAAGCTAGATTCTATTTCTTGAATACTCCTGT
GlyAsnValThrlleAsnProPhelleLysValValTyrAsnThrAlaLeuLysGlyVal 721 GGAAACGTAACTATCAATCCTTTCATCAAAGTTGTTTATAACACAGCTTTAAAAGGTGT
SerArgThrValArgAlaGlyGluAlaValGlnAsnThrValSerGlyTyrHisPro Ser 781 AGCΛGAACTGTAAGAGCTGGAGAAGCTGTACAAAATACTGTTTCTGGTTATCATCCTTC
AsnProAanTyrLysLeuAspAlaPheAlaGlyArgTyrlleGlyLysAspPheLysTre 841 AATCCTAATTATAAATTAGATGCATTTGCTGGTAGATACATTGGTAAAGATTTCAAATG
AspSerAsnProTyrAspValLysAlaGlnAlaValLeuGlylleThrAlaAsnSerAsp 901 GATTCAAATCCTTATGATGTAAAAGCTCAGGCTGTATTAGGTATCACTGCTAACAGCGA
ValValSerLeuTyrValGluProSerLeuGlyTyrGlnAlaThrTyrLeuGlyLysAsp 961 GTAGTATCTCTTTATGTTGAGCCTTCTTTAGGTTATCAAGCTACATATTTAGGAAAAAA End of Copy 6 ORF - - - > | | <- - - beta
IleSerGluAsnProTyrLeuAsnIleAspSerLysValGlnHisArgGluPheGlyAsn 1021 ATATCTGAAAATCCATATTTAAATATAGATTCTAAAGTACAACATAGGGAATTCGGCAAT galactosidase - - - >
SerLeuAlaValValLeuGlnArgArgAspTrpGluAsnProGlyValThrGlnLeuAsn 1081 TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAAT
ArgLeuAlaAlaHisProProPheAlaSerTrpArgAsnSerGluGluAlaArgThrAsp 1141 CGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGAT
ArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArgLeuMetArgTyrPheLeu 1201 CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTC
LeuThrHisLeuCysGlylleSerHisArgIleTrpCysThrLeuSerThrlleCysSer 1261 CTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGCTCT
AspAlaAlaAM
1321 GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGG
1381 GCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG
Appendix #7 - - Predicted protein sequence of pTrep704 recombinant product
<- - - - - - - - - -Protein derived from
MetThrMetlleThrProSerLeuHisAlaCysArgSerThrLeuGluAspProArgVal 1 ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA pUC polylinker- - - - - - > | | <- - - - - - Copy 6 ORF
ProSerSerAsnTrpGluPheProMetAlaLeuLeuThrlleAlaSerAlaSerAlaPhe 61 CCGAGCTCGAATTGGGAATTCCCTATGGCATTATTGACTATAGCTAGTGCATCTGCTTTC
GlyMetTyrGlyAspArgAspSerTrpIleAspPheLeuThrHisGlyAsnGlnPheArg 121 GGTATGTACGGCGACAGAGATTCTTGGATCGACTTCCTTACTCATGGTAATCAGTTCAGA
AlaArgMetAspGlnLeuGlyPheValLeuGlyAsnGlyThrlleLysGlyThrPheGly 181 GCTAGAATGGATCAATTAGGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACTTTCGGT
PheArgSerGlnAlalleGlyThrAlaLeuGlyAsnllelleSerGlyAsnThrGlyAsn 241 TTTAGATCTCAAGCTATTGGAACAGCATTAGGTAATATCATTTCAGGTAATACTGGAAAT
ValAspLeuGlnThrThrlleSerAlaGlylleGlyTyrThrSerGluProPheGlyIle 301 GTAGATTTACAAACTACTATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCGGTATT
GlyValGlyTyrAsnTyrThrTyrValAsnProArgLeuGlyValHisThrProValLeu 361 GGCGTAGGTTATAACTACACTTATGTAAATCCTAGATTAGGCGTTCATACTCCTGTACTT
MetIleAsnAlaLeuAsnAsnAsnLeuArglleAlaValProValGlnlleAlavalSer 421 ATGATCAATGCTTTAAACAACAACTTAAGAATAGCAGTTCCTGTTCAAATAGCTGTAAGT
HisAspProPheAsnAspSerAlaLysPheProTyrSerSerSerThrLysAspTyrMet 481 CATGATCCTTTCAATGATTCTGCTAAATTCCCTTATTCATCATCTACAAAAGATTATATG
GlylleSerThrAspIleGlnLeuArgTyrTyrThrGlylleAspAlaPheAsnAlaIle 541 GGTATAAGCACTGATATACAATTAAGATACTATAdTGGTATAGATGCTTTCAATGCTATA
ArgLeuTyrPheLysTyrGlyGlnAlaGlyPheLysThrAlaAsnGlyAlaGlyAlaSer 601 AGATTATACTTCAAATACGGACAAGCTGGATTTAAAACAGCTAACGGAGCTGGAGCTAGT
GluTyrPheAlaGlnSerLeuGlyPheGluλlaArgPheTyrPheLeuAsnThrProVal 661 C3AGTATTTTGCTCAGTCATTAGGTTTTGAAGCTAGATTCTATTTCTTGAATACTCCTGTT
GlyAanValThrlleAsnProPhelleLysValValTyrAsnThrAlaLeuLysGlyVal 721 GGAAACGTAACTATCAATCCTTTCATCAAAGTTGTTTATAACACAGCTTTAAAAGGTGTA
SβrArgThrValArgAlaGlyGluAlaValGlnAsnThrValSerGlyTyrHisProSer 781 AGCAGAACTGTAAGAGCTGGAGAAGCTGTACAAAATACTGTTTCTGGTTATCATCCTTCT
AsnProAsnTyrLysLevAspAl»aPheAlaGlyArgTyrIleGlyLysAspPheLysTrp 841 AATCCTAATTATAAATTAGATGCATTTGCTGGTAGATACATTGGTAAAGATTTCAAATGG
AspSerAsnProTyrAspValLysAlaGlnAlaValLeuGlylleThrAlaAsnSerAsp 901 GATTCAAATCCTTATGATGTAAAAGCTCAGGCTGTATTAGGTATCACTGCTAACAGCGAT
ValValSerLeuTyrValGluProSerLeuGlyTyrGlnAlaThrTyrLeuGlyLysAsn 961 GTAGTATCTCTTTATGTTGAGCCTTCTTTAGGTTATCAAGCTACATAtTTAGGAAAAAAC IleSerGluAsnProTyrLeuAsnlleAspSerLysValGlnHisSerLeuAlaTrpGly 1021 ATATCTGAAAATCCATATTTAAATATAGATTCTAAAGTACAACATAGCTTAGCTTGGGGT
AlaTyrAlaGluLeuTyrValArgProValGlnAspLeuGluTrpTyrPheGluMetAsp 1081 GCTTATGCAGAACTTTATGTAAGACCTGTTCAAGATCTTGAATGGTACTTCGAGATGGAT
ValAsnAsnGlyGlyThrArgGlnGluSerGlyIleProValTyrPheLysSerThrThr 1141 GTTAATAATGGCGGTACAAGACAAGAATCTGGTATCCCTGTATACTTTAAATCTACTACA
GlyIleThrTrpTyrLeuProAlaPheAsnOC
1201 GGTATAACTTGGTATTTACCTGCTTTCAATTAATTAGAAGTTAATTAATAGAAATTAATG
Appendix #8- - Predicted protein sequence of recombinant product of pTrep505
<- - - Protein derived from
MetThrMetlleThrProSerLeuHlsAlaCysArgSerThrLeuGluAspProArgVal 1 ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTA pUC polylinlcer- - - - - - > | | <- - - Copy 2 ORF - - - >
ProSerSerAsnTrpGluPheProArgMetAspGlnLeuGlyPheValLeuGlyAsnSer 61 CCGAGCTCGAATTGGGAATTCCCTAGAATGGATCAATTAGGATTTGTTTTAGGTAATAGC
ThrlleLysGlyThrPheGlyPheArgSerGlnSerLeuSerThrGlnLeuGlyTyrIle 121 ACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCAATTAGGATATATT
LeuAlalleTyrLysAspTyrThrTyrLeuGlyAlaThrlleSerGlyGlylleGlyTyr 181 TTGGCTATATATAAAGATTATACTTATTTAGGAGCAACTATTTCCGGCGGTATAGGATAT
ThrSerGluAlaPheSerlleGlyLeuGlyTyrAsnTyrThrThrProLeuProIleSer 241 ACTTCTGAGGCTTTTAGTATAGGTTTAGGTTATAATTATACTACACCGCTTCCTATTAGT
TyrAsnPheGlySerHisThrProValLeuMetlleAsnAlaLeuAsnAspAsnLeuArg 301 TATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAATTTGAGG
IleValIleProValGlnlleLeuValHisAspGlyAsnMetAsnMetThrAspAsnIle 361 ATAGTTATTCCTGTACAAATATTAGTACATGATGGTAATATGAATATGACGGATAATATT
AsnTyrLeuTyrAsnPheLeuGlylleSerThrAspThrGlnlleArgTyrTyrThrGly 421 AATTATTTATATAATTTTTTAGGTATAAGTACTGATACTCAAATAAGATATTATACAGGC
IleAspAlaPheAsnGluIleArgLeuTyrValLysTyrGlyGlnLeuGlyTyrLysGly 481 ATAGACGCTTTTAATGAAATAAGATTATATGTAAAATACGGACAATTAGGATATAAAGGC
GlySerTyrThrAspLysSerTyrAspGluGluPhePheAlaArgSerPheGlyPheGlu 541 GGTTCATATACGGATAAAAGTTATGATGAAGAATTTTTTGCAAGATCATTTGGTTTTGAA
ThrArgPheTyrPheLeuAsnThrAlaValGlyAsnValThrlleAsnProPhelleLys 601 ACAAGATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCTTTTATTAAA
ValAlaTyrAsnThrAlaLeuHisGlyPheSerThrMetValArgSerLeuAspSerVal 661 GTAGCATATAATACAGCTTTGCATGGATTTAGTACTATGGTAAGATCATTAGATAGTGTC
IleGluGluIleGluGlyTyrSerSerAspArgThrAlaLysAlaAlaGlyAsnIleAsn 721 ATTGAAGAAATAGAAGGTTATAGTTCAGATCGTACCGCTAAAGCAGCAGGAAATATTAAT
AlaLysTrpAapLysAsnProTyrAspValThrValGlnAlaValLeuGlyValThrAla 781 GCTAAATGGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTGGGAGTAACTGCT
AsnSerAspIleValSerLeuTyrValGluProSerLeuGlyTyrArgAlaLysTyrLeu 841 AATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCTAAATATTTA
GlyLysLeuThrTyrGluAspProAspGlyLysValAsnPheAspPheLysValAsnHis 901 GGAAAATTAACATATGAAGATCCAGATGGAAAAGTTAATTTTGATTTTAAAGTTAATCAT
TyrLeuSerTrpCysAlaTyrAlaGluLeuTyrlleThrProValLysAspLeuGluTrp 961 TATTTATCTTGGTGTGCTTATGCAGAGCTTTATATAACACCTGTAAAAGATTTAGAATGG
TyrPheGluMetAspValAsnAsnSerAspSerAspSerThrGlylleProValSerPhe
1021 TATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCTGTTAGTTTT AlaSerThrThrGlylleThrTrpTyrLeuProGluPheOC OC
1081 GCTTCTACTACAGGAATAACTTGGTATTTGCCAGAATTTTAATTATAAAGCAAATTTTAT
1141 ATGACAAAATAAAAAATGGGGCATTTATTATTAAAAAATAAATACCCCACATTTTATTAA
1201 ATAACTTCTTAAATAATTTTACA4TTTATATTTTATTAGTATAATAAAATATAAAGTTAA
1261 ATTTAGGTGTGTACAATGAAAAAAAGTTTTCTAATTATGACAGTATTATTAAGTATGTCCA
End of 1321 TATTGTTCAATATTTGGTATGTATGGACATCAGGACGATTGGATTGATTTTCTTACAGTC
T . hyo . insert- - - - - > |
1381 GGTAATCAGTTTAGAGGGAATTC

Claims

What is Claimed is:
1. A protein capable of eliciting at least one antibody capable of recognizinΛg at least one epitope of at least one T. hyo. antigen having a molecular weight of about 39 kDa.
2. The protein of Claim 1 encoded by gene 1.
3. The protein of Claim 2 encoded by the full length gene 1.
4. The protein of Claim 1 encoded by gene 2.
5. The protein of Claim 1 encoded by gene 3.
6. The protein of Claim 1 encoded by gene 4.
7. The protein of Claim 1 encoded by gene 5.
8. The protein of Claim 1 encoded by gene 6.
9. The protein of Claim 1 encoded by gene 7.
10. The protein of Claim 1 encoded by gene 8.
11. The protein of Claim 1 encoded by the full length gene 2.
12. The protein of Claim 1 encoded by the full length gene 3.
13. The protein of Claim 1 encoded by the full length gene 4.
14. The protein of Claim 1 encoded by the full length gene 5.
15. The protein of Claim 1 encoded by the full length gene 6.
16. The protein of Claim 1 encoded by the full length gene 7.
17. The protein of Claim 1 encoded by the full length gene 8.
18. DNA encoding at least one protein capable of eliciting at least one antibody recognizing at least one epitope of at least one T. hyo. antigen having a molecular weight of about 39 kDa.
19. The DNA of Claim 16 wherein said DNA is selected from the group consisting of genes 1, 2, 3, 4, 5, 6, 7 and 8 encoding a T. hyo. 39 kDa protein and mixtures thereof.
20. The DNA sequence of Claim 18 wherein the gene is a full length gene.
21. A host genetically engineered with DNA of
Claim 18.
22. A host genetically engineered with DNA of
Claim 19.
23. A host genetically engineered with DNA of
Claim 20.
24. An expression vehicle including DNA of
Claim 18.
25. An expression vehicle including the DNA of
Claim 19.
26. An expression vehicle including the DNA of
Claim 20.
27. Protein expressed by the host of Claim 21.
28. Protein expressed by the host of Claim 22.
29. Protein expressed by the host of Claim 23.
30. A process for protecting an animal against
T. hyo., comprising:
administering to an animal to be protected an effective amount of at least one protein of Claim 1.
31. A process for protecting an animal against T. hyo., comprising:
administering to an animal to be protected an effective amount of at least one protein of Claim 27.
32. A vaccine for protecting an animal against T. hyo. comprising:
at least one protein of Claim 1 in conjunction with a pharmaceutically acceptable carrier.
33. A vaccine for protecting an animal against T. hyo. comprising:
at least one protein of Claim 27 in conjunction with a pharmaceutically acceptable carrier.
34. Antibody which recognizes T. hvo antigen having a molecular weight of about 39 kDa.
PCT/US1990/005129 1989-09-13 1990-09-11 TREPONEMA HYODYSENTERIAE ANTIGENS HAVING A MOLECULAR WEIGHT OF 39kDa AND DNA ENCODING THEREFOR WO1991004036A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US40653589A 1989-09-13 1989-09-13
US406,535 1989-09-13

Publications (1)

Publication Number Publication Date
WO1991004036A1 true WO1991004036A1 (en) 1991-04-04

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PCT/US1990/005129 WO1991004036A1 (en) 1989-09-13 1990-09-11 TREPONEMA HYODYSENTERIAE ANTIGENS HAVING A MOLECULAR WEIGHT OF 39kDa AND DNA ENCODING THEREFOR

Country Status (4)

Country Link
EP (1) EP0491859A4 (en)
JP (1) JPH05502370A (en)
CA (1) CA2025230A1 (en)
WO (1) WO1991004036A1 (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
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
US6124098A (en) * 1992-04-30 2000-09-26 Institut Pasteur Rapid detection of antibiotic resistance in mycobacterium tuberculosis
US7012171B2 (en) 1989-12-21 2006-03-14 Advanced Technologies Cambridge Limited Modification of plant metabolism
US7074416B2 (en) * 1992-09-16 2006-07-11 University Of Tennessee Research Foundation Antigen of hybrid M protein and carrier for group A streptococcal vaccine
WO2008017636A2 (en) * 2006-08-09 2008-02-14 Spirogene Pty Ltd Genes and proteins of brachyspira hyodysenteriae and uses thereof
WO2008112970A2 (en) 2007-03-14 2008-09-18 Pioneer Hi-Bred International, Inc. Dominant gene suppression transgenes and methods of using same
EP2141239A1 (en) 2003-12-16 2010-01-06 Pioneer Hi-Bred International, Inc. Dominant gene suppression transgenes and methods of using same
CN103789327A (en) * 2007-08-03 2014-05-14 贝林格尔·英格海姆维特梅迪卡有限公司 Gene and protein of brachyspira hyodysenteriae and application of gene and protein
US9695221B2 (en) 2012-12-21 2017-07-04 Boehringer Ingelheim Vetmedica Gmbh Recombinant outer membrane proteins from Brachyspira hyodysenteriae and uses thereof

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JP2014087340A (en) * 2013-10-24 2014-05-15 Boehringer Ingelheim Vetmedica Gmbh Novel gene and protein of brachyspira hyodysenteriae and use thereof

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JPH0361488A (en) * 1988-06-29 1991-03-18 Ml Technol Ventures Lp Protective vaccine against comprising composition consisting of expression vehicle, host transformed by said composition, protein produced by said host and protein

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Infection and Immunity, Volume 26, No. 3, issued December 1979. D.H. BAUM, et al., "Partial Purification of a Specific Antigen of Treponema hyodysenteriae." Pages 1211-1213. See entire document. *
Infection and Immunity, Volume 54, No. 3, issued December 1986. L.A. JOENS, et al., "Molecular Characterization of Proteins from Porcine Spirochetes." Pages 893-896. See especially figure 2, page 894 and figure 4, page 895. *
Infection and Immunity, Volume 56, No. 12, issued December 1988. M.J. WANNEMUEHLER, et al., "Characterization of the Major Outer Membrane Antigens of Treponema hyodysenteriae." Pages 3032-3039. See especially figure 4, page 3035 and figure 5, page 3036. *
Infection and Immunity, Volume 56, No. 5, issued May 1988, S.N. CHATFIELD et al., "Identification of the Major Antigens of Treponema hyodysenteriae and Comparison with Those of Treponema innocens." Pages 1070-1075. See especially figure 4, page 1073. *
Infection and Immunity, Volume 57, No. 12, issued December 1989, D.A. BOYDEN, et al. "Cloning and Characterization of Treponema hydysenteriae Antigens and Protection in a CF-1 mouse Model by Immunization with a Cloned Endoflagellar Antigen." Pages 3808-3815. See especially figure 3 on page 3813. *
See also references of EP0491859A4 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5236708A (en) * 1989-11-03 1993-08-17 Iowa State University Research Foundation, Inc. Reduced-protein subunit vaccine for swine dysentery
US7012171B2 (en) 1989-12-21 2006-03-14 Advanced Technologies Cambridge Limited Modification of plant metabolism
US6124098A (en) * 1992-04-30 2000-09-26 Institut Pasteur Rapid detection of antibiotic resistance in mycobacterium tuberculosis
US7074416B2 (en) * 1992-09-16 2006-07-11 University Of Tennessee Research Foundation Antigen of hybrid M protein and carrier for group A streptococcal vaccine
US6068843A (en) * 1994-06-01 2000-05-30 Board Of Regents University Of Nebraska Nucleotide sequences and methods for detection of Serpulina hyodysenteriae
US5869630A (en) * 1994-06-01 1999-02-09 Board Of Regents, University Of Nebraska Lincoln Nucleotide sequences for detection of serpulina hyodysenteriae
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
EP2141239A1 (en) 2003-12-16 2010-01-06 Pioneer Hi-Bred International, Inc. Dominant gene suppression transgenes and methods of using same
WO2008017636A2 (en) * 2006-08-09 2008-02-14 Spirogene Pty Ltd Genes and proteins of brachyspira hyodysenteriae and uses thereof
WO2008112970A2 (en) 2007-03-14 2008-09-18 Pioneer Hi-Bred International, Inc. Dominant gene suppression transgenes and methods of using same
WO2008017636A3 (en) * 2007-08-03 2008-04-17 Novartis Ag Genes and proteins of brachyspira hyodysenteriae and uses thereof
CN103789327A (en) * 2007-08-03 2014-05-14 贝林格尔·英格海姆维特梅迪卡有限公司 Gene and protein of brachyspira hyodysenteriae and application of gene and protein
AU2007283667B2 (en) * 2007-08-03 2014-09-04 Boehringer Ingelheim Vetmedica Gmbh Genes and proteins of Brachyspira hyodysenteriae and uses thereof
US8992938B2 (en) 2007-08-03 2015-03-31 Matthew Bellgard Genes and proteins of Brachyspira hyodysenteriae and uses thereof
US9695221B2 (en) 2012-12-21 2017-07-04 Boehringer Ingelheim Vetmedica Gmbh Recombinant outer membrane proteins from Brachyspira hyodysenteriae and uses thereof

Also Published As

Publication number Publication date
EP0491859A4 (en) 1992-10-07
JPH05502370A (en) 1993-04-28
CA2025230A1 (en) 1991-03-14
EP0491859A1 (en) 1992-07-01

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