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

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 ( . 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 leas " t 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. ( yte & 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_j_ 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 epitoρe(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 ρrotein(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 Appendi 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). 'In the following examples, unless otherwise indicted, transformations are accomplished by the procedure of Cohen et al. PNAS 6_9 2110 (1973). EXAMPLE I - 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 Tj_ 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 g they had achieved a density of 5 x 10 /ml or greater (measured by microscopic count). Cells were concentrated by centrifugation then washed and recentrifuged twice in a buffer of lOmM potassium acetate pH 4.75, 150mM potassium chloride. The cells were then resuspended in lOmM 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 x 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 xg 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 39ρ 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 lOmM 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 xg 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-reac€ive. 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 ρH6.8 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 x 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 NH 4 HC0 3 , 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 gin his se leu ala trp gly ala tyr ala glu leu tyr val arg pro val gin 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 lys- asp tyr met gly ile ser thr asp ile gin leu arg tyr tyr thr ile asp ala phe asn ala ile arg leu tyr phe lys tyr gly gin 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 EcoRl. 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. lOng genomic DNA (B204 or B234) was mixed with lOul lOx 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 lOOul with Q-H 2 0. 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 (BamHl 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, lOmM EDTA + hen egg white lysozyme (lmg/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 NH 4 0Ac. 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, lOmCi/ml), 2ul degenerate hexamer in lOx 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 lOmM 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 (lOOug/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 l/25th original volume in 25mM Tris-Cl pH 8.0, lOmM EDTA + lmg/ml lysozyme. 2. Incubate 30-60 min. Sonicate to disrupt DNA and reduce viscosity. 3. Add 1/lOth volume -20% Triton X-100. Agitate on lab quake for 2 hours. Sonicate if necessary to reduce viscosity. 4. Centrifuge at 10,000xg for 10 min. to pellet cement. 5. Resuspend cement in l/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 10,000xg for 10 min. to pellet cement. Wash cement with 20mM Tris-Cl pH 8.0, 5mM EDTA. Centrifuge. 7. Resuspend in l/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- Gin- 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 EcoRl linkered fragments derived from a partial Alul digest of genomic T.hyo DNA (strain B204 was screened with probes. One phage, 3-5C1 was identified by hybridization to probes 553 and 555. The DNA was examined after digestion with EcoRl and found to contain a 1.6 kb insert. The Eco Rl flanked, 1.6 kb segment of DNA from phage 3-5C1 was isolated by electroelution from an acrylamide gel and then ligated to plasmid pUC 19 which had been linearized by digestion with EcoRl. 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 ____ 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 BamHl, 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 Si, and this DNA was then religated and used to transform E. coli. Nucleotide sequencing (Sanger, et al., PNAS 24:5463 (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 Hindlll 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 ρTrep301 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 xg. 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 aei-d 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 jLn vitro., it is possible that the other members of the gene family are expressed n 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 cytoplasm 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 screeni was probed with Cod664 as well as a nick translated probe made f a 411 base pair Sphl-Bcll fragment encoding the amino terminal portion of the 39kDa protein from pTrep301. SUBSTITUTESHEET In addition a library ' constructed by the ligation of a partial Sau3a digest of B204 genomic DNA and BamHl 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 antx 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 f&und 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. Codl055 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 BamHl and Hind3 and cloned into either pUC8 or 9. Candidate clones were screened for hybridization to Cod968, Codl019, 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 BamHl and Sall 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 pUCl9 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 EcoRl fragment subcloned from the lambda GT11 library. This EcoRl 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 pUCl9 with a GAA reading frame across the EcoRl 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 pTreρ327 which contain overlapping regions of the #6 gene and share a common Bell site. pTrep501, which contains regions coding for the 5' portion of the #6 gene, wans- digested with Bell and Aatll and ligated with Bcll-Aatll fragment from pTrep 327 which contained the 3' sequences of the #6 gene. The EcoRl fragment from this clone, pTreρ701, was then cloned into the EcoRl 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 ρTreρ505, 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 MYGDRDSWIDFLTHGNQFRARMDQLGFVL?NGTIKGTFGF??Q?I 39* N P?S??TK?YMGISTDIQLRYYTGIDAFNAIRLYFKYGQAGFK 39P 2 ' FPYS?STKDYMGISTDIQLRYYTGIDAFNAIRLYFKYGQAGFK 39* 2 TANGASEYFAQSLGFEARFYFLNTPVGNVTINPFIKWNTA 39p 2 TANGASEYFAQSLGFEARFYFLNTPVGNVTINPFIKWNTAL 39* 2 AQAVLGITANSDWSLYVEPSLGYQATYLGK 39p 2 AQAVLGITANSDWSLYVEPSL 39* HISENPYLNIDSK 39P 1 HISENPYLNIDSK 39* 2 VQHSLAWGAYAELYVRPVQDLE?YFEMDIN 39P 1 RNGVPVNFATSTGIT?YLPALGG?Q 39p 2 MDINNSDSKRNGVPVNFATSTGITWYLPALGGAQ 39* 3 Notes--All sequence derived from fragments generated by digestion with LysC in the presence of SDS unless otherwis indicated. «LysC digest in absence of SDS Fraction gave muplitple sequences which were resolved on of intensity and DNA predicted amino acid sequence ?GluC digest in presence of SDS Sequence begins with residue #2 due to machine failure an loss of residue #1 Single amino acid code used above is as follows W=trp Y=tyr Tab e 2 COD Sequence Source of Sequence Specifj 664 ACG-AAG-GAT-TAT-ATG-GG 39p internal for Copy A A C C peptide sequence 5, T C 844 TTAATCCGCATGATA pTrep 106 908 GTTTCATCACAAGCAAA pTrep 333 931 ATGAATATGACGGATAA pTrep 330 932 AAAGTTGATAAACAAGG pTrep 333 934 TATCATCCTTCTAATCCT pTrep 331 r. 957 CCGAAAGTACCTTTAAT pTrep 106 ALl 968 TATAATCCTTATGATCCT pTrep 317 987 GAATTCCGGATCCATGTATGG- pTrep 106 1-Λ AGATCAGGACGATTGGATT 988 GTCGACAAGCTTATAATTAAA- pTrep 106 ATTCTGGCAAATACCAAGT 1010 ATATTGACTGATAGTAT pTrep 506 1111 AAATAATTTTGATATG pTrep 506 1020 GAT-AGA-AAA-AGG-AAT-GG 39* internal peptide TCT A C sequence C 1019 AAA-AGG-AAT-GGA-GTG-CC 39* internal peptide A C T A sequence T C 1054 GAATTCCGGATCCATGTATGGCGACAG pTrep 326 AGATTCTTGGATC 1055 GTCGACAAGCTTATAATTATTGAGCAC pTrep 510 CGCCTAAAGCAGG 1092 AGTATGTTTGAACCAATA pTrep 333 1151 TCATATGTATCGTGTATA pTrep 333 1095 GGAGTACCTAAACTTCAA pTrep 337 1248 CGACAGAGATTCTTGGA pTrep 613 5, Tabla 2 (continued) COD Sequence Source of Sequence Specif for Copy 1328 GAATTCAATTACGGATT pTrep 537 1359 GTCGACCTGCAGTTATTA- pTrep 520 TTGTAAAGCAGGTAAATA- • CCA 1438 GAATTCCGGATCCATGTATGG- pTrep 537 TGCAGACAACACATGGCTT Table #3 Table #3--ldβntlfication of recombinant phage and aubelonee Identified by Screening with Phage* pTrep 30 l Sph-Bcl CodS64 + + + + + + + + + + + + * All recombinant ph»age are from the QT11 library unless otherwise noted Λ From EMBL3 library IdAβ #4 Table #4∞ldenlificatlon of recombinant phage and subclones Identified by Screening with Phage* pTrep 30lSph-Bcl 900 034 000 1010 1010 1020 1002 1005 1151 1240 »*** Λζl + \o\i\ 5JT Table 5 Expression Clones from PCR Clone Serotype Cod957 Cod844 C»od908 Cod931 Cod932 Cod9β8 Co llδl Tabie 6 Example 6 Use of 39* AntiRen 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 2 5.5 x 10 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. Ov-stall during the 1-day pαatcfaallanfe paaiod Key: ADG; average daily weight gain in pounds Mean _ Recovery of T. hyo.; percentage of βaaples froe which T. hyo. parasite was recovered froa rectal swabls of challenged aniaalβ. 4 10708 1971SRSMS 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 2 stomach intubation with 4.4 x 10 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 Grou Animals Day of Onset 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 # --DNA Sequence of B204 Genes taeod±ne; 39 kDa Antigen* 1-4 1 GAATTCTATCAGAATATTTTtTTATAATATAAATATTTTTTTIATCTTCTATATCTATA 60 TATTTGCTGAGAT4ATCTTTTAATACAT^TAANGCATTTTTTTNCATTNATTTTCANTT 120 TGTTAAATCATATATAATATTGTTAATATCATTAATTAAATTATCAATATTTAAATTATT 180 ACTATCTTGTTTTTTTATTTTTTATCATAGAT9AΛGCΛCCAGCAGCTCCTATAGCCΛATG 2 0 GTATACCTATCACAGGCAATAAGGTAGCTCCTAATACAGCACTAGTTGCTGCTGCCGTTG 300 CACCTATAGCACCAGCTGCTACTGCAGTACCGCCTAATAAACTAGAATATAAAGATGGTA 360 TTTGTTTTATTGATTCATTATTTTTA β ATAArrCTTGCATCAAATATGTTAAGTTTTTTA 420 AAATAGGTATGAATTTTGATGAATATATTTCTTCTTCTATATTTTTTGTGCTATTAAAAC 480 AAGAAAATAAAATTTTTCCTATTGTGCTTTTTCCAGTGTTATTCTCTCCGGCTATTACAG 540 TAATACCATCTATTGTTATATTAGCTTCTTT6ATTTTAGAAAAATTTTTTATATTCAATT 600 CCATTATATTTTATCCTTTAATAATTCTGTTATTATTATAACATAAAAAATATTTAATAA 660 AAATATATTAATTTTMATTAAATCTAATTCTTGAGCTATrrTATATTTTTAGTATAATAA * Presumed translational start MβtLyβ ysPhePhβ βuIl»»MβtThrVal 720 AAAATATAAACTTCAATTTAGGTATGTATAATGAAAAAGTTTTTTCTAATTATGACAGTA of Copy 1 Leul^euSerMetSerTyzCysSecΣlePheβlyMetTyzβlyAβpGlnAspAspTzpZle 780 TTATTAAGTATGTCATATTGTTCAATTTTTGGTATGTATGGAGATCAGGACGATTGGATT AspP βlΛuT rAapGlyAanGlnPheArgAlaArςMβtAspGlnLβuGlyPhβVal eu GlyAβnSβrT rllβ yβGlyThrPhβGlyPhβArgThrGlnSβrSβrSβrThrGlnLeu 900 GGTAATAGTACTATTAAAβGTACTTTCGGTTTTAαAACTCAAAGTTCATCAACTCAATTA GlyTyrlle euLeuAsnAsnAsn euGlyT rTyrteuGlyJUaThrlleSerGlyGly 960 GGATATATTTTβTTOAAIAATAATCTTGGTACTTATTTGGGAGCAACTATTTCTGGCGGT ΣlβαiyTyrϊhr«er01uJU.a»hβSβrΣlβαiyΣlββlyTyrΛanTyrThrSβrHisSβr 1020 ATAGβATATACTTCTGAGβCTTTTAGTATAGGCATAGGCTATAATTATACCAGCCATTCC LβuPhβProThrSβrAspAsnP ^lySerHisThrProValLβuMβtllβAsnAlaLeu 1080 TTATTTCCTACTAβCGATAACTTTGGTTCTCATACTCCASTACTTATGλTTAATGCTTTλ AanAapAan βuJirgΣlβValΣlβProValGlnllβ βuValHisAanGluSβrllβAsp 1140 AATaATAATTTGAGGATAGTTATTCCTGTGCAAATATTAfiTACATAATGAAAGTATTGAT Glnl Λ uGlyTyrTyrArgAspAβnTyrlβuβlyllβSβrthrΛspT rβlnΣlβArςrryr 1200 CAACTTGGTTACTATAGAGATAATTATTTAGGTATAAGTACTGATACGCAAATAAGATAT TyrThzGlyΣleAspAlaPheAsnβluZleAz LeuTyrVal ysTyrGlyGlnLeuGly 1260 TATACAGGCATAGATGCTTTTAATGAAATAAGATTATATGTAAAATATGGGCAATTAGGA -2'- - TyrLysIl β A β nProHisAspThrll β AsnTyrThrGlnGluValLβuAlaArgSβrP β 1320 TATAAAATTAATCCGCATGATACTATAAATTATACACAAGAAGTTTTAGCAAGATCATTT GlyPbeOluTbrArςPhβTyrPhβLeuAsnThrAlaValGlyAanValThrlleAanPro 1380 GGT TTG.AAACAAOATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCT PheΣle ysValAlaTyrAsnThrAlaLeuMlsGlyTyrSerThrHetValAr AlaLeu 440 TTTATTAAAGTAGCATATAATACAGCTTTGCATGGATATAGTACCATGGTAAGAGCATTG AspGlyMetTyrGluGluΣleGluGlyTyrTyrProAspSerProAlaGlnSerTyrGlu 1500 GATGGTATGTATGAASAAATAGAAGGTTATTATCCAGATAGTCCTGCTCAATCATATGAA AspΣleAsnValX.ysTrpAspLysAsnProTyrAspValThrValGlnAlaValLeuGly 1560 GATATTAATGTTAAATGGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTGGGA ValThrAlaAsnS β rAapll β ValS β rl β uTyrValGluProSβrliβuGlyTyrArgΛla 1620 GTAACTGCTAATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCT ysTyrI«uGlyLysLeuThrTyrGluAspProAspGly ysValAsnPheAspPheI.ys 1680 AAATATTTAGGAAAATTAAC^TATGAAGATCCAGATGGAAAAGTTAATTTTGATTTTAAA ValAsnHisTyr βuSβrtrpGlyAlaTyrAlaGlu euTyrllβT rProVal yaAsp 1740 GTTAATCATTATTTATCTTGGGGTGCTTATGCAGAGCTTTATATAACACCGGTAAAAGAT LβuGluTrpTyrP βGluMβtAspValAsnAsnSβrAspSβrAspSβrThrGlyllβPro 1800 TTAGAATGGTATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCT ValSβrPhβAlaSβrThrThrGlyllβT rTrpTyrLeuProGluPhβOC 1860 GTTAGTTTTGCTTCTACTA ^GGAATAACrTGGTATTTGCCAGAATTTTAATTATAAAGC 1920 AAATTrrATATGATAAAATAAAAAATGTGGGGTATTTATTATTAAAAAATAAATACCCCA 1980 CATTTTATTAAATAATTTTTTCAGTAATTTTACATTTATATATTTTTTAGTATAATAAAA * Presumed translational start of Met ys ysΣlePheLeuΣleMetThrValLeu 2040 ATATAAACTTAAATTTAGGTATATACAATGAAAAAAATTTTTCTAATTATGACAGTATTA Copy 2 LeuSerMet8erTyrCys«erΣlePheQlyMetTyrGlyAspGlnAspAspTrpIl»aAsp 2100 TTAA<WATβTα«ATTβrrCAATATTTGGTATGTATGGAGAT W»»CGATTGGATTGAT Phβ ettThrAspβlyAsnOlnPheArgAlaArςMβtAspGlnLβuGlyP eValLeuGly 2160 rn rn Nwr*^hr\r *nTrr *<^^**^ M AsnSβrThrΣlβ yββlyThrPheGlyPhβAr SβrGlnSβr euSerThrGln euGly 2220 AATAOCAegATTAAAqOTACiα'l^∞IITTAGATCTCAGAGTTTATCAACTCAATTAGGA Tyrllβ euAlallβTyr ysAspTyrThrTyrLβuGlyAlaThrllβSβrGlyGlyll* 2280 TATATTTTGGCTATATATAAAGATTATACTΓATTTAGGAGCAACTATTTCCGGCGGTATA GlyTyrThrSβrGluAlaPhβSβrIlβGlyLeuGlyTyrAsnTyrThrT rProlΛuPro 2340 GGATATACTTCTGAGGCTTTTAGTATAGGTTTAGGTTATAATTATACTACACCGCTTCCT IleSerTyrAsnPheβlySerHisThrProVal eu.HetΣleAsnAlaLeuAsnλapAsn 2400 ATTASTTATAACTTTqGTgCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAAT -.» » - I*uAr9∑leValXleProValGlnΣleZ uValHisAspGlyAanMetAsnMetThrλsp 2460 TTGAGGATAOTTArrCCTGTACAAATATTAGTACATGATGGTAATATGAATATGACGGAT AsnΣl β ΛsnTyrL β uTyrΛsnP β β uGlyllβS β rT rAspThrGlnllβΛrgTyrTyr 2520 WTATTAATTATTTATATAATTTTTTAGGTATAAGTACTGATACTCAAATAAGATATTAT ThrGlyΣleAspAlaPheλsnGluΣleAr9 euTyrValI,ysTyrGlyGlnLeuGlyTyr 2580 ACAGGCATAGACGCTTrTAATGAAATAAGATTATATGTAAAATACGGACAATTAGGATAT LysGlyGlyS β rTyrThrAspLysS β rTyrAspGluGluP βP βAlaArgSβrP βGly 2640 AAAGGCGGTTCATATACGGATAAAAGTTATGATGAAGAATTTTTTGCAAGATCATTTGGT Ph β GluThrArgPh β TyrPhel β uAsnThrAlaValGlyAsnValThrΣlβΛsnProP β 2 00 TTTGAAACAAGATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCTTTT Zl β lysValAlaTyrAanThrΛlaLβuHisGlyPheSβrThrMβtValArgSβrLβuAsp 2760 ATTAAAGTAGCATATAATACAGCTTTGCATGGATTTAGTACTATGGTAAGATCATTAGAT SerValZleGluGluZleGluGlyTyrSerSerAspλrgThrAla ysAlaAlaGlyAsn 2820 AGTGTCATTGAAGAAATAGAAGGTTATAGTTCAGATCGTACCGCTAAAGCAGCAGGAAAT ZleAsnAla ysTrpAsp ysAsnProTyrAspValThrValGlnAlaVal euGlyVal 2880 ATTAATGCTAAATGGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTGGGAGTA T rAlaAsnSβrAapIlβValSβrLeuTyrValGluProSβrLeuGlyTyrArgAla ys 2940 ACTGCTAATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCTAAA TyrlβuGlylyβ βuThrTyrGluAspProAapGlyLyβValAanPheΛspPhβI.y3Val 3000 TATTTAGGAAAATTAACATATGAAGATCCAGATGGAAAAGTTAATTTTGATTTTAAAGTT AsnHisTyr βuSβrTrpCyaAlaTyrAlaGlu euTyrllβThrProVal yβ pLeu 3060 AATCATT TTTATCTTGβTGTGCTTATβCAGλfiCrrTATATAACACCTGTAAλλβATTTλ GluTrpTyrPhβGluHetAβpValAsnAβnSerAspSβrAβpSβrThrGlyllβProVal 3120 GAATGGTATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCTGTT SerPheAlaSβrThrThrGlyllβThrTrpTyrl-βuProGluPheOC 3180 AGTTTTGCTTCTACTACAGGAATAACTTGGTATTΓGCCAGAATTTTAATTATAAAGCAAA 3240 TRRTATATGACAAAATAAAAAATOGGGCΛTTTATTATTAAAAAATAAATACCCCACATTT 3300 TATTAAAIAACTTCTTAAATAATTTTACA4TRTATATTTTATTAGTATAATAAAATATAA • Presumed translational start of Copy 3 »4βtlyβI.yβSerPhβl Λ uΣl»eHβtThrValLβulβuSβr 3360 AGTTAAAITTJUWTOTOTACAATβAAAAAAAGTTrrCTAATTATGACAGTATTATTAAGT M β tS β rTyrCy β S β rll β PheGlyHβtTyrGlyAspGlnAspAspTEpIleAapPhβ eu 3420 ATGTCATATTGTTCAATATTTGGTATGTATGGAGATOU Ϊ GACβATTGGATTGATTTTCTT ThrAspβlyAanGlnP eAr9AlaAr9MβtAspGlnP eGlyP βValLβuGlyAsnSβr 3480 ACAGACGGTAATCA<rrτrAGAaCTAGAATGGATCAATTTGGAT'fCGTTTTAGGTAATAGC ThrΣlβ yββlyThrPhββlyPhβΛrgSβrβlnSβrl Λ uSβrThrβlnLβuβlyTyrllβ 3540 ACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCAATTAGGATATATT L β uAlalleTyrlys^pTyrt rTyr uGlyAlaThrllβSβrβlyβlyllβGlyTyr 3600 TTGGCTATATATAAAGATTATΛCTTATTTAGGAGCΛACTATTTCCGGCGβTATAββATAT -~- e. ~ ThrS β rGluAlaP β S β rll β GlyLβuGlyTyrAsnTyrT rT rPro βuProIlβSer 3660 ACTTCTS1W!GCTTTTAGIATAGGTTTAXWTATAATTATACTACACCGCTTCCTATTAGT AspAsn beGlySerHisTtarProVal «uMetΣleAsnAla euAsnAspλsnl>euAr9 3720 GATAACTTTGGTTCT -TACTCCTGTACTTATGATTAATGCTTTAAATGATAATTTGAGG ZleValΣleProValGlnZle euValTyrAsnGlyAsnValGlnLysValλsp ysGln 3780 A AGTTATTCCTGTACAAATATTAGTATATAATGGTAATGTTCAAAAAGTTGATAAACAA Gly»AsnIl β S β rTyrS β rHisAspTyrL β uGlyllβSβrThrAspThrGlnΣleArgTyr 3840 GGTAATATCTCTTATTCACATGATTATTTAGGTATAAGTACTGATACGCAAATAAGATAT TyrThr β lyZl β AspAlaPh β AsnGluIl β JUrg β uTyrValLysTyrGlyGlnLβuGly 3900 TΛTACAGGTATΛGATGCTTTTAΛTGAAΛTAΛGATTA ATGTAAΛΛTΛTGGGCΛATTAGGA TyrLysAsnAlaProTyrValGlyLysAsnTyrGluGluGluPhePheSerArgSerP e 3960 TATAAAAATGCTCCGTATGTTGGTAAGAATTATGAAGAAGAATTTTTTTCAAGGTCATTT GlyPbeGluThrArgPheTyrPhelΛuAanThrAlaValGlyAsnValThrZleAsnPro 4020 GGTTTTGAAACAAGATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCT P β ll β LysValAlaTyrAsnThrAlalΛuHisGlyP&βSβrThrMβtΣlβArgAlaLeu 4080 TTTATTAAAGTAGCATATAATACAGCTTTGCATGGATrrAGTACCATGATAAGAGCATTA AspSβrMβtPhββluProΣlβGluβlyTyrSβrSβrAspArgProValSβrSβrGlnAla 4140 GAT βTATGTTTGAACCAATAGAAGGTTATλOTTCAGATCβTCCTGTTTCλTCACAAGCA AsnΣlβAβnAlaLyβTrpAspLysAsnProTyrAspValThrValGlnAlaVal βuGly 4200 AATATTAATGCTAAATGGGATAAGAATCCTTATGATGTAACAGTG UWCAGTATTGGGA ValThrAlaAanSβrAapΣlβValSβrt*uTyrValβluProSβr.euβlyTyrAzgAla 4260 GTAACCGCTAATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCT LysTyrLΛuGly y«LβuThrTyrGluAspProAspGlyLya\ζ*lAan βuAspPhβLya 4320 AAATATTTAGGAAAATTAA ATATGAAβATOyiGATGβAAAAGTTAATTTGGATTTTAAA ValAsnHisTyrLeuSβrTrpGlyAlaTyrAlaGluLβuTyrIlβThrProValI.yβAsp 4380 GTTAATCATTATTTATCTrOGGσGGCTTATG UΪAGCTTTATATAACACCGGTAAAAGAT LβuβluTrpTyrPbeβluMetAβpValAsnAanSβrAspSβrAβpSerThrGlyllβPro 4440 TTGβAATO Ϊ TATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGAATACCT ValSer»ta*JUΛ*er*hrThzβlyΣlβThrtrpTyr βuProGluPheOC 4500 GTTAβTTTTOCTrCTACTACAβGAATAACTTGGTATTTGCCAGAATTTTAATTATAAAGC 4560 AAATTTTATATQACAAAATAAAAAATGIIGGqWTATTTATTATTAAAAAATAAATACCCCG 4620 TTTTTTATTAAATAACrTCTTAaUlTAATTTTACATTTTTATATTTTATTAGTATAATAAA * Preβuaed translational start of MetLys ysPhe euΣl-sMβtThrVal euLeu 4680 ATATAAAGTTAAATTTAGGTGTGTACAATGAAAAAOTTΓCTAATTATGACAGTATTATTA Copy 4 SerMetSerTyrCysSerΣleP eGlyMetTyrGlyλspGlnAspAspTrpΣl-sAspPhe 4740 AGTATGTCATATTGTTCAATATTTGGTATGTATGGAGATαUϊGACGATTGGATTGATTTT - . ;»- L β uThrAspGlyAβnGlnPheArgAlaArgMβtAspGlnPheGlyP βValLeuGlyAθn 4600 CTTACAGACGGTAATCAGTTTAGAGCTAGAATGGATCAATTTGGATTCGTTTTAGGTAAT A β nThrll β LysGlyThrP βGlyPhβArgSβrGlnSβrLeuSβrT rHis βuGlyTyr 4860.MCACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCACTTAGGCTAT ll β lΛulΛuAsnAsnA β nPh ββ lyThrTyrPheGlyThrT rllβSerCysGlyllβGly 4920 ATTTTGTTAAATAATAATTTTGGTACTTATTTTGGAACAACTATATCATGCGGTATAGGA TyrThrS β rGluAlaP β S β rΣl β Glyll β GlyTyrΛsnTyrThrThrProLβuProIle 4980 TATACTTCTGAGGCTTTTAGTATAGGTATAGGTTATAATTATACTACACCGCTTCCTATT S β rAspAsnPh β GlyS β rHisThrProVallΛuM β tlleAsnAlalβuAanAspAsnLβu 5040 .AGTGATAACTTTGGTTCTCATACTCCTGTΛCTTAT β ATTAATGCTTTAAATGATAATTTG Argll β ValΣl β ProValGlnZl β lΛuValTyrAanGlyAsnllββlnLyβValAspLys 5100 AGGATAGTTATTCCTGTACAAATATTAGTATATAATGGTAATATTCAAAAAGTTGATAAA GlnGlyAsnΣleHisAspThrTyrλspTyrLeuGlyΣleSerThrAspT rGlnlleArg 5160 CAAGGTAATATACACGATACATATGATTATTTAGGTATAAGTACTGATACGCAAATAAGA TyrTyrThrGlyZleAspAlaPheAsnGluZleArgLeuTyrΣle ysTyrGlyGlnLeu 5220 TATTATACAGGTATAGATGCTTTTAATGAAATAAGATTATATATAAAATATGGACAATTA GlyTyrLysAsnAlaProTyrValGlyLysAsnTyrβluGluβluLeuP eSerArgSer 5280 GGATATAAAAATGCTCCGTATGTTGGTAAAAATTATGAAGAAGAACTTTTTTCAAGGTCA PheGlyPheGluT rArgP βTyrPhβ βuAanThrThrValGlyAβnValThrllβAan 5340 TTTGGTTTTGAAACAAGATTCTATTTTTTGAATACTACTGTTGGAAATGTAACTATTAAT ProPhβllβ yβValAlaTyrAsnThrAlaLβuHisGlyValGlyT rMβtllβArgAla 5400 CCTTTTATTAAAGTAGCATATAATACAGCTTTGCATGGAGTTGGTACTATGATAAGAGCA βuAapThrMβtLβuGlnProIleGluAspTyrTyrProAspArgProValSβrSβrGlπ 5460 TTAGATACTATG^TCAACCAATAGAAGATTATTATCCAGATCGTCCTGTTTCATCACAA ValAapIlβAspTyr ys βuAsp yβAβnProTyrAspValThrValGlnAlaVal eu 5520 GTAGATATTGATTATAAATTGGATAAβAATCCTTATGATGTAACTGTGCAGGCAGTATTG GlyValThrAlaAsnSerAapllβValSβr βuTyrValGluProSβr βuGlyTyrLys 5580 GGAβTAACCGCTAATAβTβATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAAG AlaliysTyrLeuGly yβMβtGlnAspGlu ysValAsnLeuAspPhβLyjValAjnHls 5640 GCTAAATAgTTAaQAAAAATGCAAaATGAAAAAGTTAATTTGGATTTTAAAGTTAATCAT Tyr βu«βrTrpαiyAlaTyrAlaβluLβuTyrIlβThrProVal ysAsp euGluTrρ 5700 TATTTATCTTGβGGTθ fTATG «»GCTTTATATAACACCTGTAAAAGATTTAGAATGG TyrPheGluMetAspValAsnAsnSerAspSerAspSerT rβlyΣleProValSerP e 5760 TATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCTGTTAGTTTT AlaSerThrThrβlyΣlβThrTrpTyrLβuProGluPhβOC 5820 G<rTCTACTACAGGαATAλCrTβGTATTTGαyUSAATTTTAATTATAAA6AAAATTTrAT 5880 ATGACAAAATAAAAAATAAGAGGTATTTATTTTTTTAATAATAAATACCTCTCTTATTTT 5940 ATTTATA8CTTTTTAATTTAATCTCTTCTTCTTTGTGCTAACATTAAAAOTCTTAAAAGT 6000 CT**βTATA«aθTAACAGCAGCAαaUtfΛTAAGTCAAAGCAGCW 60β0 CTMCΪ <»»C** β TTCTTCACT β TCTAAAAATCCK / eføe ix Z 3 Figure # —DNA Se-quence of B204'Gfenes Encoding 39 kDa Antigen *5' end of genomic DNA 1 CTGTATCTGTTTATTATCAGTCAGCATTTCATTCTTATTATTATGGCGATAAAAAT 61 AAATGGAAACTATAGTCGGAAACTTAATAGGAAGCGT4GCCAAAGAAAATAAAGAT 121 TACCAAAATTAAAGAACTATTTTAATAATTCTGTAAAAATAAAAGCTGAGAAATTT 181 AAAAATGGAAAGACTATTATGAAAGCAGAAATCTTATAAATAATTTTAATTTATAA 2 1 TTACCGATAAAAACAGCATTAATGTTTTTTATAATACTGTATTTCACAAAAATATA 301 TATGTATTAAATAAACTTTAATATATCTTTATATTTTATACTTTTGACTGTAATAT 361 TTATCATTTATATAACTATACCTAAATAGATATATTACTATAATTGTATCACAAAT 421 TATAAACCATCATATTTAACATAAGAAAAAATATATTATTATTAAΛATATAAAAAT 81 GTATTTTTTATAGAATTTTAA9AAAAATTTTTATATAATAATATTCATATATATTA * Presumed translational start of Copy 8 Met ys PhβLeuLeuT rValLβuAlalleLeuThrlleAlaSe 5 1 AAAATAAAAATGAAAAA6TTTTTATTAACAGTGCTGGCTATTTTAACAATAGCTAGC SβrValPheGlyMβtTyrGlyAlaAapAsnThrTrpLβuPhePhβLeuΣleHisGly 601 TCAGTGTTTGGTATGTATGGTGCAGACAACACATGGCTTTTCTTCCTCATACATGGC GlnP eArgAlaArgMetAsnGln euGlyPheThrLeuGlyAsnGlyXlelleLys 661 CAATTCAGAGCTAGAATGAACCAATTAGGTTTCACTCTAGGTAACGGCATTATTAAA ThrPhβGlyP βLysAlaAsnT rLβuIlβAanGlySβrlleLeuAsnThrGlyAsn 21 ACTTTCGGTTTCAAAGCTAATACσCTTATTAACGGAAGCATCTTAAATACAGGCAA GluAsnGlnλsnPro euGluAlaT rXleSerAlaGlyXleGlyTyrT rGlyAsp 781 GAAAACCAAAATCCATTAGAAGCTACTATTTCTGCTGGTATAGGTTACACAGGTGAT P βGlyValGlyValGlyTyrAsnTyrT rTyrThrAlaAlaAsnThrllβGlnT r 841 TTTGGTGTTGGTGTTGGTTATAACTATACTTATACTGCTGCAAATACTATTCAAACC AlaλlaLysGlyllβAsnThrHisThrPiroValllβThrPheAsnAlaValAsnAsn 901 GCTGCTAAAGGAATAAATACTCATACACCTGTTATTACATTCAATGCTGTTAATAAC L β uArgValAlallβProValSerllβAlaValGluLysAspIlβGlyLysLeuGly 961 TTAAGAGTAGCTATACCTGTAAGTATAGCTGTAGAAAAAGATATAGGTAAATTAGG MβtAspArgLysAspTyrLeuGly βuSβrllβProAlaGlnlleArgTyrTyrT r 1021 ATGGATAGAAAAGATTATTTAGGTTTAAGCATACCTGCTCAAATAAGATATTATACA IleAspAlaP βAsnTyrllβArgPheGluPhβAanTyrGlyLeuAsnLysTyrAsn 1081 ATAGATGCTTTCAACTATATAAGATTTGAATTCAATTACGGATTAAATAAATATAAT ValGluAsnAsnThrThrThrGluTyrGlnAlaGlnThrllβSβrP βGlnLβuArg 11 1 GTTGAAAACAATACAACTACAGAATATCAAGCACAGACTATAAGCTTCCAATTAAGA HisPhe euAsnThrValLeuGlyAsrfAsnValThrValAsnProPhe euArgValAsp 1201 CATTTCTTAAATACAGTTCTTGGAAATAATGTAACTGTAAACCCATTCTTAAGAGTTGAT PheAlaSerThrValGlyAla ysGlyLysGlyAsnValValPheProAlaAlaT rAla 1261 TTTGCTTCTACTGTAGGTGCTAAAGGAAAAGGAAATGTAGTATTCCCAGCAGCTACAGCT PheAspGlyArglieuThrAlaTrp AlaAsnAlaTrpAlaAspAspProHisSerlle 1321 TTTGATGGAAGACTTACAGCTTGGOCTGCAAATGCTTGGGCAGATGATCCACACAGCATT TyrAspArgGluLeuTyrAspLeuLysIlβlleProSerValSer euSerValAsnThr 1381 TATGACAGAGAACTTTATGACTTGAAAATCATACCTAGCGTATCTTTAAGTGTTAATACA AspTyrValAsnLeuIlePheGluProGlyLeuGlyTyrArgValGlnAspAspGlyVal 1441 GACTATGTTAATTTAATATTTGAACCTGGTTTAGGATACAGAGTACAAGATGATGGTGTA LysGlySerLys euThrHisT rLeuTyrTrpGlnAlaTyrGlyGluIleTyrlleArg 1501 AAAGGAAGCAAACTTACTCATACATTATACTGGCAGGCTTACGGAGAAATATATATCAGA ProValGlnAspLeuGluTrpTyrP eGluMetAspValAsnAsnGlyValPro ys eu 1561 CCTGTTCAGGATCTTGAATGGTATTTCGAAATGGATGTTAATAACGGAGTACCTAAACTT GlnGlyAsnProIleAlaSerGlyAsnSerMetProValValPheGlyAlaAsriThrGly 1621 CAAGGAAATCCTATTGCTTCAGGAAATTCAATGCCTGTTGTATTCGGAGCTAATACTGGT IleT rTrpTyrLeuProAlaLeuGln 1681 ATAACTTGGTATTTACCTGCTTTACAATAATAAATAACAGTGAATAATCAGAAATAACTG 1741 ATTTTTAATACAAGGGGAGCTTCATTAAAAAGCTCCTCTTTTTTATGAGAAAAACAAATA 1801 TAAAGCTTNNGTTCATAATTATCAATTTAATCAAAACATACTAAGCAAATTCAAAATTTC 1861 ATAAAAAATAATTATTCAATAAAAATTCATAATTTTTTATTAAAAAAATATGYAAATATG 1921 TATTTGAATGACTATTTTTT ATATAATTCCTTTTATATGTATTGACATTTTATAACTTT 1981 TTCCTATAATGAAAGTAGAGGATCTTTAGGTGTCTATTGAGTTTTTAAGCGATCTCTCAA * Presumed translationa MetLys ysVal euLeu. 20 1 TAGACACTGTCAAAAGATTATAAAAAAATTAGGAGAAAAACAATGAAAAAAGTTTTATTG start of Copy 6 ThrAlaMetAlaLeuLeuThrlleAlaSerAlaSerAlaP eGlyMetTyrGlyAspArg 2101 ACAGCTATGGCATTATTGACTATAGCTAGTGCATCTGCTTTCGGTATGTATGGCGACAGA AspSerTrpIleAspPheLeuT rHisGlyAsnGlnPheArgAlaArgMetAspGlnLeu 2161 GATTCTTGGATCGACTTCCTTACTCATGGTAATCAGTTCAGAGCTAGAATGGATCAATTA GlyP eValLeuGlyAsnGlyT rlleLysGlyT rPheGlyPheArgSerGlnAlalle 2221 GGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACTTTCGGTTTTAGATCTCAAGCTATT GlyT rAlaLeuGlyAsnllelleSerGlyAsnT rGlyAsnValAsp euGlnThrThr 2281 GGAACAGCATTAGGTAATATCATTTCAGGTAATACTGGAAATGTAGATTTACAAACT CT - IleSerAlaGlylleGlyTyrT rSerGluProPheGlylleGlyValGlyTyrAsnTyr 2341 ATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCGGTATTGGCGTAGGTTATAACTAC ThrTyrValAsnProArgLeuGlyValHisThrProValLeuMetlleAsnAlaLeuAsn 2401 ACTTATGTAAATCCTAGATTAGGCGTTCATACTCCTGTACTTATGATCAATGCTTTAAAC AsnAsn euArglleAlaValProValGlnlleAlaValSerHisAspProPheAsnAsp 2461 AACAACTTAAGAATAGCAG.TTCCTGTTCAAATAGCTGTAAGTCATGATCCTTTCAATGAT SerAla ysPheProTyrSerSerSerThrLysAspTyrMetGlylleSerThrAspIle 2521 TCTGCTAAATTCCCTTATTCATCATCTACAAAAGATTATATGGGT TAAGCACTGAT TA Gln euArgTyrTyrT rGlylleAspAlaP eAsnAlalleArgLeuTyrPhe ysTyr 2581 CAATTAAGATACTATACTGGTATAGATGCTTTCAATGCTATAAGATTATACTTCAAATAC GlyGlnAlaGlyPheLysThrAlaAsnGlyAlaGlyAlaSerGluTyrPheAlaGlnSer 2641 GGACAAGCTGGATTTAAAACAGCTAACGGAGCTGGAGCTAGTGAGTATTTTGCTCAGTCA LeuGlyPheGluAlaArgPheTyrPheLeuAsnThrProValGlyAsnValThrlleAsn 2701 TTAGGTTTTGAAGCTAGATTCTATTTCTTGAATACTCCTGTTGGAAACGTAACTATCAAT ProPhelle ysValValTyrAsnThrAlaLeuLysGlyValSerArgThrValArgAla 2761 CCTTTCATCAAAGTTGTTTATAACACAGCTTTAAAAGGTGTAAGCAGAACTGTAAGAGCT GlyGluAlaValGlnAsnThrValSerGlyTyrHisProSerAsnProAsnTyrLysLeu 2821 GGAGAAGCTGTACAAAA ACTGTTTCTGGTTATCATCCTTCTAATCCTAATTATAAATTA AspAlaPheAlaGlyArgTyrlleGlyLysAspP eLysTrpAspSerAsnProTyrAsp 2881 GATGCATTTGCTGGTAGATACATTGGTAAAGATTTCAAATGGGATTCAAATCCTTATGAT ValLysAlaGlnAlaValLeuGlylleT rAlaAsnSerAspValValSerLeuTyrVal 2941 GTAAAAGCTCAGGCTGTATTAGGTATCACTGCTAACAGCGATGTAGTATCTCTTTATGTT GluProSerLeuGlyTyrGlnAlaThrTyr euGlyLysAsnlleSerGluAsnProTyr 3001 GAGCCTTCTTTAGGTTATCAAGCTACATATTTAGGAAAAAACATATCTGAAAATCCATAT LeuAsnlleAspSerLysValGlnHisSer euAlaTrpGlyAlaTyrAlaGluLeuTyr 3061 TTAAATATAGATTCTAAAGTACAACATAGCTTAGCTTGGGGTGCTTATGCAGAACTTTAT ValArgProValGlnAsp euGluTrpTyrPheGluMetAspValAsnAsnGlyGlyT r 3121 GTAAGACCTGTTCAAGATCTTGAATGGTACTTCGAGATGGATGTTAATAATGGCGGTACA ArgGlnGluSerGlylleProValTyrP eLysSerThrThrGlylleT rTrpTyr eu 3181 AGACAAGAATCTGGTATCCCTGTATACTTTAAATCTACTACAGGTATAACTTGGTATTTA ProAlaPheAsn 3241 CCTGCTTTCAATTAATTAGAAGTTAATTAATAGAAATTAATGAGGCTGGCCTTTAATAGG 3301 TTGGCCTCTTTTTTATTAATTTTCATATTGCAAAATAGTTTATTCAT T ATATAAGTT 3361 TACA T TTTATTGTTTTGCAATATAATATTTTTATCTATAATCTACCTATATAAATTATAT * Presumed t rans lationa Met ysLys IleMetLeuAla 3421 AATAGAAGAAAATATTTTATGTTTAGGAGAACAGATAAAATGAAAAAAATTATGCTGGCA start of Copy 7 AlalleAlalle euThrllePheSerAlaSerAlaLeuGlyMetTyrGlyAspGlnAsp 3481 GCTATTGCTATATTAACTATATTTAGTGCATGTGCTTTAGGAATGTATGGAGATCAAGAT AspTrpIleAspPheLeuThrAspGlyAsnGlnLeuArgAlaArgMetAspGln euGly 3541 GACTGGATTGATTTTCTTACAGATGGAAATCAATTAAGAGCCAGAATGGATCAATTAGGA P eValLeuGlyAsnAsnThrlleLysGlyThrPheGlyLeuArgThrGlnAspAlaVal 3601 TTTGTACTTGGAAACAATACTATTAAAGGTACTTTCGGACTTAGAACTCAAGATGCCGTA ThrSerLeuGlySerllelleSerGlyLysThrAspAsnLeuGlyLeuAspAlaT rVal 3661 ACATCATTGGGAAGTATAATTTCAGGTAAAACAGATAATTTAGGATTAGATGCTACTGTT SerMetGlylleGlyTyrThrSerAspIlePheGlylleGly euGlyTyrAsnPheT r 3721 TCTATGGGAATAGGATACACTTCTGATATTTTCGGCATTGGCTTAGGATATAATTTTACA TyrTyrAsnSerThr euGlyValHisThrProValLeuMetValAsnAlaLeuAsnAsn 3781 TATTATAACAGCACTTTAGGCGTTCATACTCCTGTACTTATGGTCAATGCTTTAAATAAT Asn euArglleAlalleProIleGlnlleAlaAlaSerLysAspProPheGlyLysTyr 3841 AATTTAAGAATAGCAATACCTATACAAATAGCTGCATCAAAAGATCCTTTCGGAAAATAT ThrlleSerGlnTyr ysAspTyrLeuGlylleSerT rAspIleGlnlleArgTyrTyr 3901 ACTATCAGTCAATATAAAGACTATTTAGGAATAAGCACAGATATACAAATAAGATACTAT ThrGluIleAspValPheAsnGlnValArgLeuTyrlle ysTyrGlyGlnSerGlyTyr 3961 ACAGAAATAGATGTATTCAATCAAGTAAGATTATACATCAAATATGGACAGTCAGGTTAT LysAsnValLysAsnAsnPheAspMetPheAlaGlnSerPheGlyPheGluThrArg eu 4021 AAAAATGTTAAAAATAATTTTGATATGTTTGCTCAATCATTTGGTTTTGAAACTAGACTA TyrPhe euAsnArgThrlleGlyAsnValAsnlleAsnProPhelleLysValSerTyr 4081 TATTTCTTAAACCGCACAATTGGAAATGTAAATATTAATCCTTTTATTAAAGTTTCATAT AsnThrAla euAlaSerSerAspValMetValArgAlaGlyGluSerLeuValAsnThr 4141 AATACAGCTTTAGCCAGCAGTGATGTAATGGTTAGAGCAGGAGAATCTCTTGTAAATACT ThrTyrSerLysLysGluAsnLysTrpGluLysAsnProTyrAsnValThrAlaAlaAla 4201 ACTTATAGTAAAAAAGAAAATAAATGGGAAAAAAATCCTTATAATGTAACTGCTGCTGCT Val euGly euThrAlaAsnSerAspMet euSer euCysValGluProSerLeuGly 4261 GTATTAGGATTAACTGCTAACAGTGATATGCTATCTCTTTGTGTTGAACCTTCTTTAGGA TyrAsnAlaValTyr ysGlyLysTyrLysT rAspSer ysTyrTyr ysValGlnHis 4321 TACAATGCCGTTTATAAAGGAAAATATAAAACTGATAGTAAATATTATAAAGTACAGCAT Asn euTyrTrpGlyAlaTyrAlaGluLeuTyrlleT rProValGlnAspIleGluTrp 4381 . AATTTATATTGGGGAGCTTATGCAGAACTTTATATTACTCCCGTTCAAGATATTGAATGG TyrPheGluMetAspIleAsnAsnGlyAsnSerArgGlnThrSerSerlleProIleTyr 4441 TATTTTGAAATGGACATTAATAATGGTAATTCAAGACAGACTTCTTCTATACCTA A AC PheGluSerThrThrGlylleT rTrpTyr euProGluLeu 4501 TTTGAATCTACTACAGGGATAACTTGGTATTTGCCTGAATTATAATAAAAATATTATTTT 4561 GTATAATTTAAACATTTATTGAATAAAAAAAAT AAAAATTAATAATTCTATAAAAAATT 4621 TAAATTAATACTTTACATATTTAAATAAATAGCTGTTTTTATTAAATCTAAGTATTATTT 4681 TAAAAAATAATAATTTTATTTATATTAGTGTTGACTTTTTTATGTAAATTCATTAGAATA 4741 AAAGTGTTGGTAAGATATAGACATTAAAAAAAGATTGCTTATAATGTCTATAACCA ATT 4801 AGGAACAATAAAGATCCTCTAAATCCTTATTATTCCAATTGACACATTAGTAAAAACAGC 4861 ATAATTTTTCCATTTAAATATCTGTTTTTACGGAGAATAAAGATCCTCTAAATCTTATTA 4921 TTCTCACTGATTTTTTATAATCCGTTAGAATTATATAGATGTTCTAACTCATCTGGAAAT 4981 AACAAAGATCCTCTGTCTTTGTTGTTTCCTAAAATTATTTTGGAGTTACTACTTACAATG ♦ Presumed translational st MetLysLysValLeuLeuThrAlaMet 5041 AGTATTAACTATATAAAATTTTAGGAGAAAAATAATGAAAAAAGTTTTATTGACAGCTAT of Copy 5 * Amino terminus of Cop Ala euLeuT rlleAlaSerAlaSerAlaPheGlyMetTyrGlyAspArgAspSerTrp 5101 GGCATTATTGACTATAGCTAGTGCATCTGCTTTCGGTATGTATGGCGACAGAGATTCTTG IleAspPheLeuThrHisGlyAsnGlnPheArgAlaArgMetAspGlnLeuGlyPhe al 5161 GATCGACTTCCTTACTCATGGTAATCAGTTCAGAGCTAGAATGGATCAATTAGGTTTCGT LeuGlyAsnGlyThrlle ysGlyThrPheGlyPheArgSerGlnAlalleGlyThrAIa 5221 TTTAGGTAACGGTACTATTAAAGGTACTTTCGGTTTTAGATCTCAAGCTATTGGAACAGC euGlyAsnllelleSerGlyAsnThrGlyAsnValAspLeuGlnThrThrlleSerAla 5281 ATTAGGTAATATCATTTCAGGT ATACTGGAAATGTAGATTTAC AACTACTATTTCTGC GlylleGlyTyrThrSerGluProPheGlylleGlyValGlyTyrAsnTyrThrTyrVal 5341 TGGTATAGGTTATACTTCTGAGCCTTTCGGTATTGGCGTAGGTTATAACTACACTTATGT AsnProArgLeuGlyValHisThrProVal euMetlleAsnAlaLeuAsnAsnAsnLeu 5401 AAATCCTAGATTAGGCGTTCATACTCCTGTACTTATGATCAATGCTTTAAACAACAACTT ArglleAlaValProValGlnlleAlaValSerHisAspProP eAsnAspSerAlaLys 5 61 AAGAATAGCAGTTCCTGTTCAAATAGCTGTAAGTCATGATCCTTTCAATGATTCTGCT A PheProTyrSerSerSerThr ysAspTyrMetGlylleSerThrAspIleGln euArg 5521 ATTCCCTTATTCATCATCTACAAAAGATTATATGGGTATAAGCACTGATATACAATTAAG TyrTyrThrGlylleAspAlaPheAsnAlalleArgLeuTy . rPheLysTyrGlyGlnAla 5581 AT CTATACTGGTATAG ' ATGCTTTCAATGCTATAAGATTATACTTCAAATACOGACAAGC GlyPhe ysThrAlaAsnGlyAlaStfrGluTyrPheAlaGlnSerLeuGlyPheGluAla 5641 TGGATTTAAAACAGCTAACGGAGCTAGTGAGTATTTTGCTCAGTCATTAGGTTTTGAAGC ArgPheTyrPhe euAsnThrProValGlyAsnValThrlleAsnProPhelleLysVal 5701 TAGATTCTATTTCTTGAATACTCCTGTTGGAAACGTAACTATCAATCCTTTCATCAAAGT ValTyrAsnThrAlaLeu ysGlyValSerArgThrValArgAlaGlyGluAlaValGln 5761 TGTTTATAACACAGCTTTAAAAGGTGTAAGCAGAACTGTAAGAGCTGGAGAAGCTGTACA AsnThrValSerGlyTyrAs-nProTyrAspProAsnTyr ys euAspAlaPheAlaGly 5821 AAATACTGTTTCTGGTTATAATCCTTATGATCCTAATTATAAATTAGATGCATTTGCTGG ArgTyrlleGlyLysAspPheLysTrpAspSerAsnProTyrAspVal ysAlaGlnAla 5881 TAGATACATTGGTAAAGATTTCAAATGGGATTCAAATCCTTATGATGTAAAAGCTCAGGC ValLeuGlylleThrAlaAsnSerAspValValSerLeuTyrValGluProSerLeuGly 5941 TGTATTAGGTATCACTGCTAACAGCGATGTAGTATCTCTTTATGTTGAGCCTTCTTTAGG * TyrGlnAlaT rTyrLeuGlyLysHisIleSerGluAsnProTyrLeuAsnlleAspSer 6001 TTATCAAGCTACATATTTAGGAAAACACATATCTGAAAATCCATATTTAAATATAGATTC ysValGlnHisSerLeuAlaTrpGlyAlaTyrAlaGluLeuTyrValArgProValGln 6061 TAAAGTACAACATAGCTTAGCTTGGGGTGCTTATGCAGAACTTTATGTAAGACCTGTTCA AspLeuGluTrpTyrPheGluMetAspIleAsnAsnSerAspSerLysArgAsnGlyVal 6121 AGATCTTGAATGGTACTTCGAGATGGACATCAATAACTCTGATTCAAAAAGAAATGGTGT ProValAsnPheAlaThrSerT rGlylleThrTrpTyrLeuProAlaLeuGlyGlyAIa 6181 TCCTGTTAACTTCGCAACTTCTACAGGTATAACTTGGTACTTACCTGCTTTAGGCGGTGC Gin 6241 TCAATAATTAATTTCTGTTAATTAAAGAATTTACAGAGGCTGGCTTTAATAAAAAAGTCA 6301 GTCTCTTTTTTTATCGCATATTTTCATATAATTAAACAAAAATATTTACATTATATAATT -61 - Nuc1«βttd ••quβnc* of T. hyo «.»*• mmmrt of øTr«e l »» ( l inl mr- I—T. hyo DNA — » . GAATTCC CTATTTTAT A TTTTT A GT A T AAT AA AA AATATAAACTTCAATTTAGJjVATGTAT CTT AA GG SATAAAATATAA A AATCAT A TTATTTTTTATATTTGAA β TTAAATCCATΔCATΛ 1 ECORl (Si«n*l p«ptιd« ••quβnc* — - l — -b#« ιn m*t rm protsnn — > Gl yflβtTyrβl yAspGt n Aap A«ø Trp I I »sA«pPH«L»uThrA«pβl yAβnβl nPh # Ar GGTATβTATββ*>βATCAββACGATTββATTGATTTTCTTACAαACββCAATCA6TTTAG CCATACATACCTCTAOTCCTβCTAACCTAACTAAAAOAATβTCTβCCβTTAGTCAAATC Al *Ar« MøtAsøβl nL»«uβ1 y*h#V*1 KiOt yA«nS#rThrI 1 *Cy«G1 yThrPh # Gl 6CTA β AATββATCAATTAββATTTSTTTTAββT»AATAβTACTATTAAAβSTACTTTCGG CGATCTTACCTAOTTAATCCTAAACAAAATC.CATTATCATOATAATTTCCATGAAAGCC Ph β Arf Thrβl nS» #rS#rS#rThr01 nL«» uG1 y Tyr XI p-L-auLtuAsnA-snAsnLøuθl TTT » A.aAACTC.AAAβTTCATC.AACTCAATTA.ββATATATTTTβTTβ^TAATAATCTTGG AAATCTTGAβTTTCAAβTAOTTβAβTTAATCCTATATAAAACAACTTATTATTAGAACC ThrTyrL-mAiβl yAt a Thr I I •Sβrβl yβl yll t>Q\ yTy ThrSβrβl uAl *Ph#S# I 1 ACTTATTTGOOAGCAACTATTTCTQOCMTATAOOATATACTTCTQAOOCTTTTAGTAT TGMTAAACCCTCGTTGATAAAβ»ACCGCCATATCCTATATβAAβACTCCGAAAATCATA CCGTATCCGATATTAATATββTCGβTAAββAATAAAββATGATCGCTATTGAAACCAAG Hi«Thr*roVal #urt#t X 1 »A*nA1 »*i.»uAsn A*p Aβn ø Arf 11 «V«1 11 e^roVa CATACTCCMTACTTATMTTAATβCTTTAAATβATAATTTβAββATAOTTATTCCTGT GTATGAGGTCATGAATACTAATTAC3AAATTTACTATTAAACTCCTATCAATAAGGACA Gl n I I «C«uV«1 HiβAβn€l uf« II •Aβpβl nL»*uOT yTy TyrArf, A»pA»nTyrLβ CAAATATTAβTACATAAT»lAAAαTATTβATCAACTTGαTTACTATAβ^i βATAATTATTT GTTTATAATCATiTATT^TTTCAT»AACT^TTβAACCAATβATATCTCTATTAATAAA Gl y 11 #S«rThrA»pThr81 nil #ArfTyrTyrThrβl yll «A«pA *PbβA*nG1 ull GGTATAAΘTACT3ATACβCAAATAAβATATTATACAβGCATAβATGCTTTTAATGAAAT CCATATTCATβACTATβCβTTTATTCTAT^TATβTCCOTATCT-ACβAAAATTACTTTA Ar«L»suTyrV«1 LyβTyrβl yβl n -βuβl yTyr yβll βAβnFroHiβAβ Thr I I β A β AGATTATATGTAAAATATβGβCAATTAββATATAΛAATTAATCCGCATGATACTATAAA TCTAATATACATTTTATAC-CCβTTAATreTATATTTT^TTAββCβTACTATGATATTT TyrThrβ! nβl uVal LβυAI _Ar*_ SβrPhrtl yPHββl u Thr»Arβj« .»TyrWι#L β uA* TATACACAA0»AA0TTTTAGCAAOATCATTTQ0TTTTQ«ΛACΛΛ3ATTCTATTTTTTGAA ATATβTQTTCTTCAAAATCSTTCTAβTAAACCAAAACTTTSTTCTAAβATAAAAAACTT -e>_- ThrAI aval βl yAanVal Thr ∑l eAsnPrePhell eLyaval AΪ aTyrAanThrAi a 723 ACT8CTttTTg » g AT8T » AACTATCAATCCTTTTATTAAAGTA0CATATAATACAGCTT TGACOACtACCTTTACATTGATAGTTAGGAAAATAATTTCATCGTATATTATGTCGAA Hiββl y y Se ThrWβtVal Λr _A ) ateύάepGl yfletTyrGl uGI ul I »G1 uGI T 783 CATGGATATAβTACCATββrAAGA β CArTGGATGGTATGTATGAAβAAATAGAAGGrT GTACCTATATCAT ββ TACCATTCTCGTAACCTACCATACATACTTCTTTATCTTCCAA TyrAroAepS β rAroAl aβl nS β rTyrβl uAep 11 «A«nV«l Ly«TroA«βLy«A«nP 843 TATCCAGATAGTCCTβCTCAATCATATGAAGATATTAATGTTAAATGGGATAAGAATC ATAββTCTATCAββACβAGTTAGTATACTTCTATAATTACAATTTACCCTATTCTTAG TyrA«pv«l Thr Val 01 nAl aVal Lauβl yV«t ThrAI aAanSerAeo 11 βVa) Ser L 903 TAT β Ar β TAACrβT β CA ββ CAOTATTGGOAOTAACr β CTAATA∞βATATAGTATCAC ATACTACATTGrf»CAC β TCCβTCATAACCCTCATT6ACβATTATCβCTATATCATAGT TyrVal βl yTyrAr*Al *Ly«TyrLβuβl yLyeLβuThrTyrGI u 9*3 TATβTTG-AβCCTTCTTTAββTTATAGββCTAAATATTTAββAAAATTAACATATGAA ATACAACTC30AΛ0AAATCCAATATCCCGATTTATAAATCCTTTTAATTGTATACTT • - r β A β pβl yLysVal A«nWneA»pPh » et.yaVal AβnHtaTyrLβuββrTrpβl yAI a 1023 CCAβATββAA.AAβTTAATTTTβATTTTAAAβTTAATCATTATTTATCTTβββGTΘCT GβTCTMCTTTTC-AATTAAAACTAAAATTrCAATTAOTAATAAATAG-AACCCCACβA Al aβl uLeuTyrll aThrProVal yaAβpLeuβl uTrpTyrr ti SI uftetAapVal 1083 βCAβAβCTTTATATAACACCOβTAAAAOATTTAOAATββTArTTTβAAATββATGTT CGTCTCGAAATATATTGTββCCATTTTCTAAATCTTACCATAAAACTTTACCTACAA A«nS#rA»pf#rAβpt#rThrβ1 yll e^ oVal I . βrPhβAI aSβrThrThrβl yll β 1143 AATAOT0ATTC» AOATrCT»ACA>9βTATACCT0rTAβTTTTαCTTCTACTACAββAATA TTATCACTAAβTCTAAβATβTCCATATGGACAATCAAAACGAAGATGATGTCCTTAT 1243 ββTATTTATTATTAAAAAAT»AAAT.ACCMACATTTTATTAAATAATTTTTTCAβTAA CCATAAATAATAATTTTTTATTTATββββTβT»AAAAT^TTTATTAAAAAAβTCATT (be* in tana encodinβ a rel ate protein 1323 TACATTTATATATTTTTTA» iTATAATAAAAATATAAACTTAAATTTAββTATATAC ATSTAΛATATAT»ΛAAAAATC» ATATTATTTTTATATTTβAATTTAAATCCATATATG Lya yall aFhaLaoΣl • » «•*. ThrVal LβuLβu§βrflβtf. βrTyrCyβββr 11 eFh 1383 AΛAAAAATTTTTCTAi»TTAT»9AC^TATTATTAAaTAT8TCATATTQTTCAATATT TTTTTTTAAAΛAβATT TACTβTCATAATAATTCATACAβTATAACAAβTTATAA ( Met Ty β 1 yAepθl nAepAa TrpII aAapPhet.au II eAepβl yAenβl n^heAr 1443 ATβTATββAβATC » AββACβATTββATTβATTTTCTTAT.AβAC3βTAATC^TTTAβAβ TACATACCTCT^TCCTMT » AΛCCT.Ai CTAAAAβAATATCTβCCATTAβTC.AAATCTC 1502 .6?- Affen ' x 4r Partial DNA -sequence of expression plasmid øTrβø3 . (plasmid PUC9--: • I — _ . coli lad ——— ι -r. .o MβtThrMetllβThrProSβrLβufilAI Sβr v— v* AATTTCACACA ββ AAACA β CTATGACCAT β ATTACGCCAAβCTTGCATGCCTCATATTGT TTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCGGTTCGAACGTACGGAGTATΔΔCΛ 1 39 HΪNOlil , sisnal ———>I <—mature T. hyo 39 kDa protein— SerItePheβlyHβ TyrβlyAepGlnAepAepTrpIleAepPheLeuThrAsoGl yA«n TCAATTTTTββTATGTATββMATCAββACβATTββATTβATTTTCTTACAGACGGCAAT AβTTAAAAACCATACATACCTCTA β rCCT β CTAACCTAACTAAAAβAATβTCTGCCGTTA GlnPha-ArfAl ArβriβtAβ GInLeuθlyPheValLeuGIyAβnSβrThrIJeLysGl v.. CAGTTTAGAGCTAGAATGGATCAATTAββATTTGTTTTAβGTAATAβTACTΔTTAAΔGGT.. GTCAAATCTCβATCTTACCTAβTTAATCCTAAACAAAATCCATTATCATβATAATTTCCA.. The first 25 amino aeidβ of thiβ recomOinant fuaion protein wer dete mined on protein iaolated from C. coli transformed with oi pTrepSOl . The protein was isolated aa a Triton X-100 insoluble aaareeata and **_ further purified by praparativa SDS 9βl aiact phoreβiβ. -• - tpfexdiX —Predicted amino acid sequence from PCR derived T. hγ (B204) clones pTrep605—Copy2 10 20 30 40 50 6 MTMITNRGSM YGDQDD IDF TDGNQFRAR MDQLGFVLGN STIKGTFGFR SQSLSTQLG 70 80 90 100 110 12 I AIYKDYTY LGATISGGIG YTSEAFSIGL GYNYTTPLPI SYNFGSHTPV LMINALNDN 130 140 " 150 160 170 18 RIVIPVQILV HDGNMNMTDN INYLYNFLGI STDTQIRYYT GIDAFNEIRL YVKYGQLGY 190 200 210 220 230 24 GGSYTDKSYD EEFFARSFGF ETRFYFLNTA VGNVTINPFI KVAYNTALHG FSTMVRSLD 250 260 270 280 290 30 VIEEIEGYSS DRTAKAAGNI NAKWDKNPYD VTVQAVLGVT ANSDIVSLYV EPSLGYRAK 310 320 330 340 350 36 LGKLTYEDPD GKVNFDFKVN HYLSWCAYAE LYITPVKDLE WYFEMDVNNS DSDSTGIPV 370 FASTTGITWY LPEF pTrep60 —Copy3 10 20 30 40 50 6 MTMITNRGSM YGDQDDWIDF LTDGNQFRAR MDQFGFVLGN STIKGTFGFR SQSLSTQLG 70 80 90 100 110 12 ILAIYKDYTY LGATISGGIG YTSEAFSIGL GYNYTTPLPI SDNFGSHTPV LMINALNDN 130 140 150 160 170 18 RIVIPVQILV YNGNVQKVDK QGNISYSHDY LGΣSTDTQIR YYTGIDAFNE IRLYVKYGQ 190 200 210 220 • 230 24 GYKNAPYVGK NYEEEFFSRS FGFETRFYFL NTAVGNVTIN PFIKVAYNTA LHGFSTMIR 250 260 270 280 290 30 LDSMFEPIEG YSSDRPVSSQ ANINAKWDKN PYDVTVQAVL GVTANSDIVS LYVEPSLGY 310 320 330 340 350 36 AKYLGKLTYE DPDGKVNLDF KVNHYLSWGA YAELYITPVK DLEWYFEMDV NNSDSDSTG 370 377 PVSFASTTGI TWYLPEF pTrep345—Copy4 10 20 30 ' • 40 50 6 MTMITNRGSM YGDQDDWIDF LTDGNQFRAR MDQFGFVLGN NTIKGTFGFR SQSLSTHL 70 80 90 100 110 12 ILLNNNFGTY FGTTISCGIG YTSEAFSIGI GYNYTTPLPI SDNFGSHTPV LMINALND 130 140 ' 150 160 170 18 RIVIPVQILV YNGNIQKVDK QGNIHDTYDY LGISTDTQIR YYTGIDAFNE IRLYIKYGQ 190 200 210 220 230 24 GYKNAPYVGK NYEEELFSRS FGFETRFYFL NTTVGNVTIN PFIKVAYNTA LHGVGTMIR 250 260 270 280 290 30 LDTMLQPIED YYPDRPVSSQ VDIDYKLDKN PYDVTVQAVL GVTANSDIVS LYVEPSLGY 310 320 330 340 350 36 AKYLGKMQDE KVNLDFKVNH YLSWGAYAEL YITPVKDLEW YFEMDVNNSD SDSTGIPVS 370 ASTTGITWYL PEF ρTrep613—Copy5 10 20 30 40 50 6 MTMITNRGSM YGDRDSWIDF LTHGNQFRAR MDQLGFVLGN GTIKGTFGFR SQAIGTALG 70 80 90 100 110 12 IISGNTGNVD LQTTISAGIG YTSEPFGIGV GYNYTYVNPR LGVHTPVLMI NALNNNLRI 130 140 150 160 170 18 VPVQIAVSHD PFNDSAKFPY SSSTKDYMGI STDIQLRYYT GIDAFNAIRL YFKYGQAGF 190 200 210 220 230 24 TANGASEYFA QSLGFEARFY FLNTPVGNVT INPFIKWYN TALKGVSRTV RAGEAVQNT 250 260 270 280 290 30 SGYNPYDPNY KLDAFAGRYI GKDFKWDSNP YDVKAQAVLG ITANSDWSL YVEPSLGYQ 310 320 330 340 350 36 TYLGKHΪSEN PYLNIDSKVQ HSLAWGAYAE LYVRPVQDLE WYFEMDINNS DSKRNGVPV 370 378 FATSTGITWY LPALGGAQ pTrep620—Copy7 10 " 20 30 40 50 MTMITNRGSM YGDQDDWIDF LTDGNQLRA MDQLGFVLGN NTIKGTFGLR TQDAVTS 0 80 90 100 110 IISGKTDNLG LDATVSMGIG YTSDIFGIGL GYNFTYYNST LGVHTPVLMV NALNNNL 130 140 150 160 170 IPIQIAASKD PFGKYTISQY KDYLGISTDI QIRYYTEIDV FNQVRLYIKY GQSGYKN 190 200 210 • 220 230 NFDMFAQSFG FETRLYFLNR TIGNVNINPF HCVSYNTALA SSDVMVRAGE SLVNTTY 250 260 270 280 290 ENKWEKNPYN VTAAAVLGLT ANSDMLSLCV EPSLGYNAVY KGKYKTDSKY YKVQHNL 310 320 330 340 349 AYAELYITPV QDIEWYFEMD INNGNSRQTS SIPIYFESTT GITWYLPEF :: ■ ;•:■ -y> *o w so ^"•eSs": BfiSSTa L IH5\J?=$W NELEFTL6NS IIKSTFsfK MTLIN6SI N -;, 30 -k_ 103 110 123 "iNt L-QN 1 ! »~Ξ I3Y "Ξ~TΪ3V; -NTTY ^T I3TWWK5H --»TPVHT Δ _v ; ;»v. :ι 22) 220 2*0 r iN ftWT T T« _ :Ξ= 2L s -j : s T ι5NNV*VNP c -MF«TVδ HKBCS VV** ",;• IS 290 200 -j?~L v UL'1?5VS. SWTu .1 :iv :: :: ■ : • 3*0 25 ) 2-so :-."2v'.si « _ τ -T_**i¥»~H : v : ϊS v£ -£ *f=2S N5 v W .L2BNP.fc s3NSH W?ϊ -NT;. K' Appendix #6—Predicted amino acid sequence of ρTreρ702 recombinant product _ < Protein derive MetThrMetlleT rProSerLeuHisAlaCysArgSerThrLeuGluAspPro 1 ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCC pUC polylinlcer >| |< Copy 6 ORF > ProSerSerAsnTrpGluP eProMetAlaLeuLeuThrlleAlaSerAlaSer 61 CCGAGCTCGAATTGGGAATTCCCTATGGCATTATTGACTATAGCTAGTGCATCT GlyMetTyrGlyAspArgAspSerTrpIleAspPheLeuThrHisGlyAsnGln 121 GGTATGTACGGCGACAGAGATTCTTGGATCGACTTCCTTACTCATGGTAATCAG AlaArgMetAspGlnLeuGlyP eValLeuGlyAsnGlyThrlleLysGlyThr 181 GCTAGAATGGATCAATTAGGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACT P eArgSerGlnAlalleGlyT rAlaLeuGlyAsnllelleSerGlyAsnThr 241 TTTAGATCTCAAGCTATTGGAACAGCATTAGGTAATATCATTTCAGGTAATACT ValAspLeuGlnT rThrlleSerAlaGlylleGlyTyrThrSerGluProPhe 301 GTAGATTTACAAACTACTATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTC GlyValGlyTyrAsnTyrT rTyrValAsnProArgLeuGlyValHisThrPro 361 GGCGTAGGTTATAACTACACTTATGTAAATCCTAGATTAGGCGTTCATACTCCT MetlleAsnAlaLeuAsnAsnAsnLeuArglleAlaValProValGlnlleAla 421 ATGATCAATGCTTTAAACAACAACTTAAGAATAGCAGTTCCTGTTCAAATAGCT HisAspProPheAsnAspSerAlaLysPheProTyrSerSerSerT rLysAsp 481 CATGATCCTTTCAATGATTCTGCTAAATTCCCTTATTCATCATCTACAAAAGAT GlylleSerThrAspIleGlnLeuArgTyrTyrThrGlylleAspAlaPheAsn 541 GGTATAAGCACTGATATACAATTAAGATACTATACTGGTATAGATGCTTTCAAT ArgLeuTyrPheLysTyrGlyGlnAlaGlyP ' eLysThrAlaAsnGlyAlaGly 601 AGATTATACTTCAAATACGGACAAGCTGGATTTAAAACAGCTAACGGAGCTGGA GluTyrP eAlaGlnSerLeuGlyP eGluAlaArgP eTyrPheLeuAsnThr 661 GAGTATTTTGCTCAGTCATTAGGTTTTGAAGCTAGATTCTATTTCTTGAATACT GlyAsnValThrllβAsnProPhelleLysValValTyrAsnThrAlaLeuLys 721 GGAAACGTAACTATCAATCCTTTCATCAAAGTTGTTTATAACACAGCTTTAAAA SerArgThrValΛrgAlaGlyGluAlaValGlnAsnThrValSerGlyTyrHis 781 AGCΛGAACTGTA.JU3.AGCTGGAGAAGCTGTACAAAATACTGTTTCTGGTTATCAT AsnProAanTyrLysLeuAspAlaPheAlaGlyArgTyrlleGlyLysAspPhe 8 1 AATCCTAATTATAAATTAGATGCATTTGCTGGTAGATACATTGGTAAAGATTTC AspSerAsnProTyrAspValLysAlaGlnAlaValLeuGlylleThrAlaAsn 901 GATTCAAATCCTTATGATGTAAAAGCTCAGGCTGTATTAGGTATCACTGCTAAC ValValSerLeuTyrValGluProSerLeuGlyTyrGlnAlaThrTyrLeuGly 961 GTAGTATCTCTTTATGTTGAGCCTTCTTTAGGTTATCAAGCTACATATTTAGGA End of Copy 6 ORF >| |< beta IleSerGluAsnProTyrLeuAsnlleAspSerLysValGlnHisArgGluP eGly 1021 ATATCTGAAAATCCATATTTAAATATAGATTCTAAAGTACAACATAGGGAATTCGGC galactosidase > - - SerLeuAlaValValLeuGlnArgArgAspTrpGluAsnProGlyValT rGlnLeu 1081 TCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTT ArgLeuAlaAlaHisProProPheAlaSerTrpArgAsnSerGluGluAlaArgT r 1141 CGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACC ArgProSerGlnGlnLeuArgSerLeuAsnGlyGluTrpArgLeuMetArgTyrPhe 1201 CGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTT LeuThrHisLeuCysGlylleSerHisArglleTrpCysThrLeuSerThrlleCys 1261 CTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACTCTCAGTACAATCTGC AspAlaAlaAM 1321 GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGA 1381 GCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGC _^c Appendix #7—Predicted protein sequence of pTrep704 recombina product < Protein derived MetThrMetlleThrProSerLeuHisAlaCysArgSerThrLeuGluAspProA 1 ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCC pUC polylinker >| |< Copy 6 ORF ProSerSerAsnTrpGluPheProMetAlaLeuLeuThrlleAlaSerAlaSerA 61 CCGAGCTCGAATTGGGAATTCCCTATGGCATTATTGACTATAGCTAGTGCATCTG GlyMetTyrGlyAspArgAspSerTrpIleAspPheLeuThrHisGlyAsnGlnP 121 GGTATGTACGGCGACAGAGATTCTTGGATCGACTTCCTTACTCATGGTAATCAGT AlaArgMetAspGlnLeuGlyP eValLeuGlyAsnGlyT rlleLysGlyThrP 181 GCTAGAATGGATCAATTAGGTTTCGTTTTAGGTAACGGTACTATTAAAGGTACTT PheArgSerGlnAlalleGlyThrAlaLeuGlyAsnllelleSerGlyAsnThrG 241 TTTAGATCTCAAGCTATTGGAACAGCATTAGGTAATATCATTTCAGGTAATACTG ValAspLeuGlnThrT rlleSerAlaGlylleGlyTyrThrSerGluProPheG 301 GTAGATTTACAAACTACTATTTCTGCTGGTATAGGTTATACTTCTGAGCCTTTCG GlyValGlyTyrAsnTyrThrTyrValAsnProArgLeuGlyValHisThrProV 361 GGCGTAGGTTATAACTACACTTATGTAAATCCTAGATTAGGCGTTCATACTCCTG MetIleAsnAlaLeuAsnAsnAsnLeuArglleAlaValProValGlnlleAlav 21 ATGATCAATGCTTTAAACAACAACTTAAGAATAGCAGTTCCTGTTCAAATAGCTGT HisAspProPheAsnAspSerAlaLysPheProTyrSerSerSerThrLysAspTy 481 CATGATCCTTTCAATGATTCTGCTAAATTCCCTTATTCATCATCTACAAAAGATT GlylleSerThrAspIleGlnLeuArgTyrTyrThrGlylleAspAlaPheAsnAl 5 1 GGTATAAGCACTGATATACAATTAAGATACTATAdTGGTATAGATGCTTTCAATGC ArgLeuTyrPheLysTyrGlyGlnAlaGlyP eLysThrAlaAsnGlyAlaGlyAl 601 AGATTATACTTCAAATACGGACAAGCTGGATTTAAAACAGCTAACGGAGCTGGAGC GluTyrPheAlaGlnSerLeuGlyP eGluλlaArgPheTyrP eLeuAsnT rPr 661 C3AGTATTTTGCTCAGTCATTAGGTTTTGAAGCTAGATTCTATTTCTTGAATACTCC GlyAanValThrlleAsnProP elleLysValValTyrAsnThrAlaLeuLysGl 721 GGAAACGTAACTATCAATCCTTTCATCAAAGTTGTTTATAACACAGCTTTAAAAGG SβrArgT rValArgAlaGlyGluAlaValGlnAsnThrValSerGlyTyrHisPr 781 AGCAGAACTGTAAGAGCTGGAGAAGCTGTACAAAATACTGTTTCTGGTTATCATCC AsnProAsnTyrLysLevAspAl»aPheAlaGlyArgTyrIleGlyLysAspP eLy 841 AATCCTAATTATAAATTAGATGCATTTGCTGGTAGATACATTGGTAAAGATTTCAA AspSerAsnProTyrAspValLysAlaGlnAlaValLeuGlylleT rAlaAsnSe 901 GATTCAAATCCTTATGATGTAAAAGCTCAGGCTGTATTAGGTATCACTGCTAACAG ValValSerLeuTyrValGluProSerLeuGlyTyrGlnAlaT rTyrLeuGlyLy 961 GTAGTATCTCTTTATGTTGAGCCTTCTTTAGGTTATCAAGCTACATAtTTAGGAAA IleSerGluAsnProTyrLeuAsnlleAspSerLysValGlnHiaSerLeuAlaTrpG 1021 ATATCTGAAAATCCATATTTAAATATAGATTCTAAAGTACAACATAGCTTAGCTTGGG AlaTyrAlaGluLeuTyrValArgProValGlnAspLeuGluTrpTyrPheGluMetA 1081 GCTTATGCAGAACTTTATGTAAGACCT5TTCAAGATCTTGAATGGTACTTCGAGATGG ValAsnAsnGlyGlyThrArgGlnGluSerGlylleProValTyrP eLysSerT rT 1141 GTTAATAATGGCGGTACAAGACAAGAATCTGGTATCCCTGTATACTTTAAATCTACTA GlylleT rTrpTyrLeuProAlaPheAsnOC 1201 GGTATAACTTGGTATTTACCTGCTTTCAATTAATTAGAAGTTAATTAATAGAAATTAA Appendix #8—Predicted protein sequence of recombinant product of pTrep505 < Protein derived MetThrMetlleThrProSerLeuHlsAlaCysArgSerThrLeuGluAspProAr 1 ATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCG pUC polylinlcer >I I< Copy 2 ORF > ProSerSerAsnTrpGluPheProArgMetAspGlnLeuGlyPheValLeuGlyAs 61 CCGAGCTCGAATTGGGAATTCCCTAGAATGGATCAATTAGGATTTGTTTTAGGTAA ThrlleLysGlyThrPheGlyP eArgSerGlnSerLeuSerT rGlnLeuGlyTy 121 ACCATTAAAGGTACTTTCGGTTTTAGATCTCAGAGTTTATCAACTCAATTAGGATA LeuAlalleTyrLysAspTyrThrTyrLeuGlyAlaT rlleSerGlyGlylleGl 181 TTGGCTATATATAAAGATTATACTTATTTAGGAGCAACTATTTCCGGCGGTATAGG T rSerGluAlaP eSerlleGlyLeuGlyTyrAsnTyrT rT rProLeuProIl 2 1 ACTTCTGAGGCTTTTAGTATAGGTTTAGGTTATAATTATACTACACCGCTTCCTAT TyrAsnPheGlySerHisT rProValLeuMetlleAsnAlaLeuAsnAspAsnLe 301 TATAACTTTGGTTCTCATACTCCTGTACTTATGATTAATGCTTTAAATGATAATTT IleValIleProValGlnlleLeuValHisAspGlyAsnMetAsnMetT rAspAs 361 ATAGTTATTCCTGTACAAATATTAGTACATGATGGTAATATGAATATGACGGATAA AsnTyrLeuTyrAsnP eLeuGlylleSerThrAspThrGlnlleArgTyrTyrTh 421 AATTATTTATATAATTTTTTAGGTATAAGTACTGATACTOUVATAAGATATTATAC IleAspAlaPheAsnGluIleArgLeuTyrValLysTyrGlyGlnLeuGlyTyrLys 481 ATAGACGCTTTTAATGAAATAAGATTATATGTAAAATACGGACAATTAGGATATAA GlySerTyrThrAspLysSerTyrAspGluGluPheP eAlaArgSerPheGlyP e 541 GGTTCATATACGGATAAAAGTTATGATGAAGAATTTTTTGCAAGATCATTTGGTTTT T rArgPheTyrPheLeuAsnT rAlaValGlyAsnValThrlleAsnProPhelle 601 ACAAGATTCTATTTTTTGAATACTGCTGTTGGAAATGTAACTATCAATCCTTTTATT ValAlaTyrAsnThrAlaLeuHisGlyPheSerThr.MetValλrgSerLeuAspSer 661 GTAGCATATAATACAGCTTTGCATGGATTTAGTACTATGGTAAGATCATTAGATAGT IleGluGluIleGluGlyTyrSerSerAspArgThrAlaLysAlaAlaGlyAsnlle 721 AT GAAGAAATAGAAGGTTATAGTTCAGATCGTACCGCTAAAGCAGCAGGAAATATT AlaLysTrpAapLysAsnProTyrAspValThrValGlnAlaValLeuGlyValThr 781 GCTAAATGGGATAAGAATCCTTATGATGTAACTGTGCAGGCAGTATTGGGAGTAACT AsnSerAspIleValSerLeuTyrValGluProSerLeuGlyTyrArgAlaLysTyr 841 AATAGCGATATAGTATCACTTTATGTTGAGCCTTCTTTAGGTTATAGGGCTAAATAT GlyLysLeuThrTyrGluAspProAspGlyLysValAsnP eAspP eLysValAsn 901 GGAAAATTAACATATGAAGATCCAGATGGAAAAGTTAATTTTGATTTTAAAGTTAAT TyrLeuSerTrpCysAlaTyrAlaGluLeuTyrlleThrProValLysAspLeuGlu 961 TATTTATCTTGGTGTGCTTATGCAGAGCTTTATATAACACCTGTAAAAGATTTAGAA TyrP eGluMetAsp V alAsnAsnSerAspSerAspSerThrGlylleProValSer 1021 TATTTTGAAATGGATGTTAATAATAGTGATTCAGATTCTACAGGTATACCTGTTAGT AlaSerThrThrGlylleThrTrpTyrLeuProGluPheOC OC 1081 GCTTCTACTACAGGAATAACTTGGTATTTGCCAGAATTTTAATTATAAAGCAAATTTT 1141 ATGACAAAATAAAAAATGGGGCATTTATTATTAAAAAATAAATACCCCACATTTTATT 1201 ATAACTTCTTAAATAATTTTACA4TTTATATTTTATTAGTATAATAAAATATAAAGTT 1261 ATTTAGGTGTGTACAATGAAAAAAAGTTTTCTAATTATGACAGTATTATTAAGTATGTC End 1321 TATTGTTCAATATTTGGTATGTATGGACATCAGGACGATTGGATTGATTTTCTTACAG T. hyo. inser —-—>l 1381 GGTAATCAGTT AGAGGGAATTC

Claims: What is Claimed is: 1. A protein capable of eliciting at least one antibody capable of recognizing 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 A host genetically engineered with DNA of A host genetically engineered with DNA of An expression vehicle including DNA of An expression vehicle including the DNA of An expression vehicle including the DNA of Protein expressed by the host of Claim 21. Protein expressed by the host of Claim 22. Protein expressed by the host of Claim 23. A process for protecting an animal against 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. hτo. comprising: at least one protein of Claim 1 in conjunction with a pharmaceutically acceptable carrier. 33. A vaccine for protecting an animal against . 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.

Inventors: DRAGON ELIZABETH, GABE JEFFREY, MCCAMAN MICHAEL, DRAGON, ELIZABETH, GABE, JEFFREY, MCCAMAN, MICHAEL
Applicants: ML TECHNOLOGY VENTURES, ML TECHNOLOGY VENTURES, L.P.
Application year: 1990
Main classification: A61K37
IPC codes: A61K39, C07K14, C07K16, C12N15, C12P21, C12R1