-~897 5 - ~4 TE~EPOMEMA HYODYSEMTERIAE ~NTIGENS
HAVING A MOL~CULAR WEIGHT OF 39 kDa AND DNA ~NCODING THEREFOR
Thls lnventlon relates to Treponema hyodysenterlae and more partlcularly to Treponema hyodysenteriae lT. hyo.~ antigen~, genes enco~lng for sUch antigenS, cells genetically englneered wlth D~A encoding for such antlgens and uses for such antigens.
Still more partlcularly, this lnventlon relates to Treponema hyodysenteriae antlgens havlng a rnolecular welght of 39 kDa and to the production thereof by recomblnant technique~.
Swlne dysentery is a severe, infectlous dlsease found ln all ma~or pig-rearing countrles. The symptoms o~ swlne dysentery are severe mucohemorrhaglc dlarrhea, dehydratlon and weight loss.
The present lnventlon ls dlrected to certaln antlgens whlch are useful ln determlnlng and/or treatlng Treponema hyodysenteriae and to recomblnant or genetlc engineerlng technlques for produclng such antlgens.
In accordance with one aspect of the present lnventlon, there ls provlded a proteln comprlslng at least one epltope of at least one T.hYo. antlgen havlng a molecular welght of about 39 kDa.
Applicant has found that T.hYo. includes DNA whlch encodes for a plurallty of proteins each having a molecular welght of about 39 kDa. Stlll more partlcularly, Appllcant has found that there are at least elght dlfferent genes, each of which encodes for a T.hyo. protein havlng a molecular weight of about 39 kDa. The proteln products encoded by such genes have been found to have conserved reglons whlch are interspersed wlth varlable A~
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2 fi~975-84 reglons. It has been ~oun~ that the varlable re~ions are generally located in the more hydrophllic portions of the proteln whereas the conserved reglons are located ln the more hydrophoblc portlons of the proteln.
- ~ comparison of the predicted amino acid sequences frorn the mature peptides encoded by the 39 kDa famlly genes is found ln Appendix 1. The peptide sequence correspondlng to the slgnal peptl~es of these protein (Appendlces 2A and 2B) has been removed for the purposes of thls comparison. Examlnation of thls comparlson reveals each gene encodes a protein product of slmilar molecular welght and that there are reglons of conserved proteln sequence punctuated by regions of variable sequence. The conserved reglons are generally ln the more hydrophobic portions of the protelns whlle the varlable reglons tend to be ln the more hydrophlllc portlons. (Kyte & Doollttle, Journal of Molecular Blology, 157, 105 (1982)) Thus, ln accordance with one aspect of the present inventlon, there ls provlded elght different antigens (or fragments or analogs thereof), whlch are T.hyo. antigens whlch have a molecular welght of about ~,,.,"~
` .1 , . -39 kDa. Such seven genes are hereinafter sometimes referred to as genes 1-8 or copies 1-8.
In accordance wlth another aspect of the present inventlon, there ls provlded at least elght dlfferent genes, each - of which encodes for ~ different T hvo. antigen having a molecular weight of about 39 kDa.
In accordance with yet another aspect of the present inventlon, there ls provided an expression or clonlng vehicle which lncludes a DNA sequence which encodes for a T.hyo. antlgen (or fragment or analog thereof!, which has a molecular weight of about 39 kDa.
In partlcular, there is provided a DNA sequence independent of the T.hyo. organism, said DNA se~uence encodlng a T.hYo. antlgen havlng a molecular welght of about 39 kDa, or a derivative or fragrnent of sald antigen, whereln sald derivatlve or fragment of said antigen elicits at least one antlbody whlch recognlzes sald T.hYo. antlgen.
In accordance wlth 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 welght of about 39 kDa.
The molecular weight for characterizing the 39 kDa T.hYo. antigen or protein is obtalned by discontinuous polyacrylamide gel electrophoresis using the SDS buffer system described by Laemmli, Nature, 227:680-8S (London, 1970! wlth an acrylamide concentration of 10-17% and a bis-acrylamide to acrylamide ratio of 1:29.
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3a 6897 5 - 84 Thus, Applicant has found that there are at least elght dlfferent T.h~o. an~lgens, each of which has a molecular welght of about 39 kDa, which are encoded by elght dlfferent genes.
The D~A sequence may encode for a proteln whlch ls the entire 39 kDa antlgen, or a Eragment or derlvatlve oE the antlgen, or a fuslon product of the antlgen or fragment and another proteln, provlded that the proteln whlch is produced from such DNA
sequence ellclts antlbodles after lmmunizatlon whlch C' ~3 recognize an epitope of a as kDa T. hyo. antigen.
Thus, for example, the DNA sequence may encode for a protein which i~ ~r contain~ within it a fragment of a 39 kDa antigen provided that ~uch fragment generates anti~odies which recognize an epitope of a 39 kDa anti~en.
Similarly, the VNA sequence may encode for a protein which i~ 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 r~cognize an epitope(s) of a 39 kDa ~.hyo.
antigen as hereinabove described.
The DNA sequenoe may encode a protein which is a fu~ion product o~ (i) a protein which produce3 antlbodies which recognize an epitope(~) of a noted 39 kDa T.hyo. antigen and (ii~ another protein (for example chymo~in).
The 39 ~Da antigens may vary somewhat between specific strains of T. hyo. Thus, for example, the 39 kDa proteins of Aerotype B204 have minor differ~nces ~rom those of serotype B234; however, ~uch an~igens, as well a~ the genes encodin~ such antigens are e~sen~ially identical to each other.
AB a result, the tesm "DNA sequence which encodeY for a protein which produc~s antibodies which recognize an epitopets) of a noted 39 kDa T. hYo.
anti~en" encompasses DNA ~equences which ~ncode for and/or express in appropriate transformed cells, proteins whlch may be the appropriate antigen, antlgen 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 tha DNA
sequence present in the vector when introduced into a cell may express only a portion of the protein which ,; . ~ ~ , , ; -, -5- f~ t~ u g~
i9 encoded by such DNA s~q~ence, and ~uch DNA
sequance is within the noted terminology, provided that the protein portion expressed elicits antibodie~
which recognize an epitope(s) of one or more o~ the noted 39 kDa T; hyo. anti~en~. For example, the DNA
sequence may encode for the eneire antigen; however, the expressed protein is a fragment of the antigen.
The term "gene (1, 2, 3, 4, 5, 6, 7 or ~) encoding a T. hyo. 39 kDa protein" mean~ the ~ntire or full length gene sequence or an an~log, fragment or derivative thereof whlch encodes a protein which is capable of eliciting at least one antibody which recognize9 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. h~o. 39 kDA protein encoded by the entire or full length gene or an analogue, fragment or derivative o~ ~uch protein which i8 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 varicty of vectors or pla~mids. Such vector~ include chromosomal, nonchromosonal and synthetic DNA sequences; e.g., derivatives o SY40;
bacterial plasmid~; pha~e DNA'~; 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 saquence m~y be lnserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an ;. . :
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appropriate restriction endanuclease site(~) by proced~res known in the art. Such procedure9 and others are deemed to be within the scope of those skilled in the art.
The DNA sequence ~n the expression vector i~
operati~ely linked to an appropriate expression control sequence(s) (promoter) to direct mRNA
synthesis. As representative example~ of ~uch promoters, there may be mentloned: 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 prokaryotlc or eukaryotic cells or their viruse9. The expression vector al~o contsins a riboso~e binding 8ite for translation initiation and a transcription terminator. Th~
vector msy also include appropriate ~equence3 ~or amplifying expr~ssion.
In addition, the expression ~ectors preferably contain a gene to provide a phenotypic trait for selection of transformed ho3t cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or ~uch as tetracycline or ampicillin resi~tance in E. coli.
The vector containing the appropriate ~NA
~equence a3 hereinabove described, as well as an appropriate promoter or control sequence, may be employed to tran~form an appropriate host to permit the host to éxpress the protein. As repre~entative example~ of appropriate hosts, there may be mentioned: bacterial cell~, such a~ E. coli, Salmonella tYPhimurium; fungal cell8, such as yeast;
animal cells such a~ CH0 or ~owes melanoma; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those ~killed in the art from the teachings herein.
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As hereinabove indicated, the expression vehicle including the appropriate DNA sequence inserted at the selected site may include a DNA or gene ~equesloe which i3 not part of the gene coding for the protein which is capabIe of producing antibodies which recognize an epitope(s) of the noted T. hYo.
antigen(s). For example, the de~ired DNA sequence may be fused in the same reading frame to a DNA
sequence which aids in expreqsion or improve~
p~rif~ cation or permits expres~ion of the appropriate protein.
When seeking to develop a vaccine neutrali~ing or protective antibodies could be targeted towards discontinuous, conformation-dependent epitopes of the native antigen. One mu~t therefore consider whather the protein obtained from the recombinant expre~sion 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 neet 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, chaotrope~, organic solvent~ and ionic detergents followed by a renaturation step achieved by dilution, dialysi , or pH ad~ustment to remove the denaturant, and ~2) reconstitution of proteins into a lipid bila~er 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 protein~ produced from a zenetically engineered host (genetically engineered R 6 89 7 5 - ~34 wlth DN~ enco~lng for a 39 kDa ~y_. antlgen) may he employed irl con~unctlon With a pharmaceutically acceptable carrier or may be ~irectly con~ugated to a carrler or lmmunostlmulant to provlde protectlon agalnst swine dysentery, and ln partlcular swlne dysentery lnduced by T.hyo.. The Rotavlrus VP6 carrler system developed by VIDO (Veterinary Infectious Disease Organlzatlon, Saskatoon, Canada) although not an ad~uvant may be a suitable lmmunostlmulant when chemically con~ugated to a 39 kDa T.hyo.
antlgen. As he~elnabove lndicated, such proteln~s) ls capable of ellclting antibodles whlch recognize an epitope(s) of one or more of the hereinabove noted 39 kDa T.hYo. antl~ens. Such expressed proteln Wlll be sometimes herelnafter referred to as a "recornblnant T.hyo. antlgen," however, as hereinabove lndicated, such protein may not correspond to a T.hYo. antlgen ln that lt may also be a fragment, derlvatlve or fuslon product. The term "recomblnant T.hYo. antlgen" also encompasses such fragments, derlvatives and fuslon products.
The present inventlon also provldes a vacclne for protectlng an anlmal against T.hYo. comprlsing at least one proteln capable of ellcitlng at least one antlbody capable of recognlzlng at least one epltope of at least one T.hYo. antigen having a molecular weight of about 39 kDa, ln adml~ture wlth a pharmaceutlcally acceptable carrler.
One or more of such 39 kDa T.hyo. antlgens may be employed in the vaccine. In a preferred embodiment, all of the 39 kDa T.hyo. anti~ens are employed in formulatlng a vacclne (l.e., the seven antlgens or fragments or derlvatlves thereof encoded by the seven dlfferent T~hYo~ genes).
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: . , ~a 68975-84 The recombinant T.h~o. antlgen(s) ls employed ln the vaccine ln an a~ount ePfective to provlde protectlon agalnst swlne dysentery. In general, each dose of the vacclne contains at least 5 micrograms and preferably at least 20 micrograms of such recombinant T.hyo. antigen(s). In most cases, the ,.
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- ~ . , , _9_ vaccin~ does not include s~i~h recombinant ~
anti~en in an amount greater than 20 milligra~s.
The term "pro~ection" or "protecting" when ~ed with re3pect to the vaccine fo~ ~wine dysentery described herein means that the vaccine prevents swine dysentery andtor reduces`the severity oE swine dysentery.
If multiple doses are given, in general, they would not exceed 3 doses o~er a six week period.
The vehicle which is employed in con~unction with the recombinant T. h~o. antigen( 8 ) may be any one of a wide variety of vehicle3. As repre~entative example~ of suitabl~ 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 suit~ble vehicle is deemed to be within the scope of those skilled in the art from the teachings herein. The selection of a ~uitable vehicle is also dependent upon the manner in which the vaccine is to be administered. The vaccine may be in the form of an in~ectable dose and may be admini~tered intra-mu~cularly, intraYenously, or by sub-cutaneous admini~tration. It is also possible to administer the vaccine intranasally or orally by mixing the active eomponents 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 teaching3 herein; accordingly, the scope of the invention is not limited to a particular delivery form.
It i3 also to be understood that the vaccine may include act:Lve component3 or ad~uvant~ in addition to the recombinant T. hYo. antigen or fragment~ thereof hereinabove described.
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In accordance with a ~urther aspect of the present invent:Lon, there i9 provided an a~ay for detection or determination of antibody to 39 kDa T.
hyo. antigan which employs a 39 kDa T. hyo. protein antigen, of the type hereina~ove de~cribed, as a ~pecific binder in the assay.
More particularly, there i~ provided an immunoassay for 39 kDa T. hyo. antibody in which a 39 kDa T. hYo. antigen i9 employed a~ a binder, in the assay, for specifically binding 39 kDa T. h~o.
antibody.
The a~qay technique which i9 employed ls preferably a ~andwich type of assay wherein the 39 kDa T. hyo. antigen i9 ~upported on a solid support, as a binder, to bind 39 kDa T. h~o. specific antibody pre~ent in a ~ampla, with the bound antibody then being determined by ui~e of an appropriatQ trac~r.
The tracer i~ comprised of a ligand labeled with a detectable label. The li~and i~ one which i9 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 ~uch a ~andwich 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 i9 ~peci~ic or 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.
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The detectable label may be any o a wide ~ariety of detectable label9, including enzyme~, radioactive labels, ohromogens (including both fluore~cent a~d/or absorbl~g dye~) and the l~ke. The ~election of ~ detectable label i~ deemed to be within ~cope cf tho~e ~killed in the art from teachlng~ herein.
The solid support for the antigen may be any one of a wide variety of solld ~upports and the 9election of a suitable support is deemed to be within the scope of those ~killed in the art from the teachings herein. For example, the solld support may bs a microtiter plate; a tube, a particle9 etc.; however, the scope of the invention is not limited to any representative support. Th~ antigen may be ~upported on the ~pport by techniques known in the art; e.g., by coating; covalent coupline, etc. The ~lection of a suitsble technique i~ deemd to be within the ~cope of tho~e skilled in the art from the teaching~
herein.
The sandwich as~ay may be accomplished by variou~ technique~; e.g., "forward"; rever~e"; or "simultaneous"; however, the forward technique is preEerred.
In a typical procedure, 39 kDa ~ y~ antigen, which i~ ~upported on a ~olid support i~ initially contacted with a sample containing or suspected of containing 39 kDa T hYo. antibody to bind ~pecifically any of such antibody present in the sample to such antigen on the support.
After washing of the solid ~upport, the support i9 contacted with a tracer which binds to 39 kDa T~
hyo. antibody. If ~uch antibody were present in the sample~ the tracer becomes bound to ~uch antibody bound to ~uch antigen on the solid support, and the .
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presence of tracer on the ~olid support i~ indicative of the prasenca of 39 kDa T. IlYo~ antibody in the ~ample. The presence of tracer may be datermined by determining the presence of the detectable label by procedures known in the art.
Althou~h the preferred pr~cedure i~ a sandwich a3s~y, it is to be understood that the 39 kDa T. hY.
antigen(s) may be used in othar assay technique3, e.g., an agglutination assay wherein the antigen is used on a s~lid particle ~uch a~ a latex particle.
In accordance with another aspect of the pre~ent invention, there i3 provided an assay or reagent kit for determining 39 kDa T. hyo. antibody which includes 39 kDs T. hYo. antigen, as hereinabove described, and a tracer comprised of a ligand and a detectable label. The li~and of the tracer i9 bound by 39 kDa T. h~o. antibody. The r~agent~ 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 a~ a probe by use of techniques known in the art.
Although the pre~nt invention has been particularly deacribed with reference to use of the 39 kDa anti~en(3) for imparting protection against T.
hyo., one or more of such anti8en~ may be used to produce antibodies (monoclonal and/or polyclonal) by procedures known in the art and such antlbodies may be used in a vaccine to impart protection against T.
hyo.
Description of APPendices and Drawin~s Appendix 1 is a comparison of Gene Products of the 39kDa glene Family witho~t peptide signal sequences from serotype B2~4 .
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Appendi~ 2A is the DN~ sequence of gene~ 1-4 encoding anti8enes 1-4 of the ~9kDa Xene family from Yerotype B204.
Appendix 2B is the DNA sequence o~ genes 5-8 encoding antigens 5-8 oE the 39 kDa gene family from serotype ~204.
Appendix 3 is the nucleotide sequence of T.hyo gene insert of pTrep 106.
Appendix 4 is a partial DNA ~equence of plasmid pTrep 301.
Appendix 5 i9 predicted amino acid seq~ence~
from PCR derived T.hyo. ~B204) clones.
Appendix 6 i9 the predicted protein ~eqeunce encoded by pTrep 702.
Appendix 7 i~ the predicted protein ~equence encoded by pTrep 704.
Appendix 8 is the predicted amino acid sequence for pTrep 505.
Figure 1 i~ a map of the gene family and ~ub-clones obtained from screening for 39 kDa gene;
Figure 2 is a pla~mid map of pTrep 505;
Figure 3 is a schematic of the construction o pTrep 70~;
Figure 4 i~ a schematic of the construction oP
pTrep 704; and Figure 5 is a schematic of the constr~ction of the pTrep PCR expre3sion vehicle.
The pre~ent invention will be ~urther described with respect to the following examples; however, the scope of t~e invention is not to be limited thereby.
In the Examples, unle~s otherwise noted, purification~, digestion~ and ligation~ are accomplished as de~cribed in "Molecular Cloning, a labora~ory manual" by Maniatis et al. Cold Spring .
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, r Harbor Laboratory (1982). ~n the following example~, unles~ otherwise indicted, transformations are accomplished by the procedure of Cohen et al. PNAS 69 2110 (1973).
EXAMPLE I - Purification and Recover~ of - NatiYe Anti~en Treponema hYodys~nteriae strain B204 was grown in broth culture prepared as follows. Brain/Heart Infusion (Difco) at 37 gms/liter distilled water wa~
autoclaved allowed to cool, then 9terile 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 wa~
then prereduced (made anaeroblc) by 24 hours of perfu~ion with a ~tream of gas composed of 90~
nitrogen, 10% carbon dioxi~e. The complete media was then inoculated with a 1-10% volume of aotively growing T. hYo culture, the temperature was maintained at 37C-39C, the culture pH wa9 maintained at 6.8, and the culture wa~ continuously perfused with the oxygen free gas (flow rate 50 ml~/min/liter of culture).
Cells were removed from the fermentation when they had achieved a density of 5 x lU8/ml or greater (measured by mlcro~copic count). Cell~ were concentrated by centrifugation then washed and recentrifuged twice in a buffer of lOmM potassium acetate pH 4.75, 150mM potaxsium chloride. The cells were then resu~pended in lOmM potassium acetate pH
4.75 until an optical density of 2S-30 (at 600nm) was achieved (is mea~ured on solution dilutions) which is typically abo~t 1/20 the original culture ~olume.
Extraction method:
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, Tween-20 (a non-ionicrdetergent) w~ then added to the cell suspension to achieve a final concentration of 0.2%. After ge~tle agitation for lO
min~tes the cells were centrifuged (lO,000 xg for 10 min). This supernatant fraction wa~ 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 concent~ation of 2.0%. After centrifugation the 2% Tween ~upernatant (deter~ent solubilized antigen pool) was saved and the cell pellet wa~ resuspended and re-extracted with Tween-20 in a sequential manner for up to 5 additional cycleY
with the Tween-20 concentr~tion incr~asing 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 ~urface protein~ o T, hYo without lysing or rupturing the bacteria.
To concentrate the antigen preparation, supernatent fractions were sub~ected to ultracentrifuRati~n ~100,000 x ~) for 1.5 hours, and the recovered pellet material (HSP) wa9 resuspended in 25mM Tri~ buffer pH 6.8 and di~persed by sonication.
Anti~en Purification The re~uspended HSP was then mixed with 15 volumes of Tris-HCl pH 6.8, 6M urea which had been filtered through a 0.45uM Eilter. This wa~ ~tirred at roo~ temperature for several hours. This wa9 centrifuged at 100,000 xg and the supernatant (USl) set a~ide. The pellet fraction from this step (VPl) wa~ resu9pendet and extracted with urea a second time. This material wa~ centrifuged a~ before and the supernatant (US2) and pellet (UP2) were collected.
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The predominant proteIn constitutent of UP2 i9 39 KDa protein sometime~ referred to as the 39p antigen. The 39p antigen which was further purified by molecular ~iev~ column chromatography in the pre~ence of SDS or electroelution from acrylamide gels.
It i9 po3sible to i901ate a soluble form of the 39kDa antigen (39q) in addition to the sedimentable form (39p) that i~ i~olated as the maJor protein component of the urea in~oluble pell~t (VP2) described above. In order to produce the 39~
antigen, T.hyo cell~ (B204) were extractd w1th Tween 20 as de~cribed above. After the final Tween 20 extraction the residual cell pellet wa9 re~u~pended in approximately 2ml of lOmM pota~sium acetate, pH
4.75 per gram wet weight and ~onicated. The sonlcated cell pellet was 3eparated from the 39~
antigen, by centrifugation at 26,000 xg for 15' at 4C. The ~upernatant was then centrifuged at 100,000 xg for 2 hours at 20C to pellet any of the sedimentable membrane a~oriated protein~ which were also relea~ed by sonication. The supernatant (39*), which contain~ the 39s antigen as it~ predominant protein component, was then sterile filtered through a .2uM filter and ~tored at eithrr 4C or frozen. If ~torad at 4C ~ome proteolytic degredation of the 39s antigen occurs. Additional 39* can be isolated by repeating the above sonication and centri$ugation 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 mobillty in SDS
polyacrylamide gels of the 399 protein and the 39p protein is identical. The two proteins are al30 .: :
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immunologically cros~-reactive. Anitsera raised against UP2, or gel purified 39p, will recogniæe 399 on Western blots. Conversely, anti~era 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 I125. (Marchalo~i~, et al. Biochemi~try Journal; 24, 921 (1971)). Antisera from swine that have recovered from experimental infect~ons of swine dysentery also recognize either the 39s or 39p form of the 39kDa antigen.
ExamPle 2 - Protein Sequence of 399 and 39 anti~ens The Eermentation and protein purification are accompli~hed a~ in Example 1.
The insoluble material obtained by centrifugation of the Yecond urea extrsction (UP2) contains a single ma~or protein component which is 39p antigen. This inQoluble protein wa~ solubilized by boiling in 25 mM Tri9-HCl pH6.8 containing 3%SDS, 1 mM EDTA, and 70 mM 2-mercaptoethanol. This solution was sub~ected to gel filtration chromatography over a 30 cm column of Sepharose 6B
(from BioRad, Richmond, CA). The 39 kDa pe~k wa~
identified by gel electrophore~is of column eluant fraction~, the appropriate fraction~ were p~oled 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 removP re~idual SDS.
The amino acid sequence of the amino-terminu~ of the 39 kDa protein prepared sbove was determined using sequential Edman degradation in an nutomated Applied Biosy~tem3 gas phase sequenator. The identity of the fir~t 41 amino acid9 of the protein thu~ determLned are shown below:
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Met-Tyr-Gly-Asp-Arg-Asp-ser-Trp-IIe-Asp-Phe-Leu-Thr-His-Gly-Asn-Gln-Phe-Arg-Ala-Arg-Met-Asp-Gln-Leu-Gly-Phe-Val-Leu-Gly-~ 4~
Asn-A~p-Thr-I}e-Lys-Gly-Thr-Phe-?-?-Arg-Amino terminal peptlde ~equence of 399 was obt~ined directly from a preparation of 39* which was concentrated by precipîtation with ac~tone and sequenced. Additional internal peptide sequence of the 399 antigen was obtianed by dige9tion with endoproteinase Ly~C ~-1/100 w/w) in 50mM Tri~-HCl pH8.S, O.lZ SDS (~7C, 16 hrs.). Proteolytic cleavage product~ were purified using rever~e pha~e HPLC and sequenced on a Vydac C4 column (250 mm x 4. 6 mm, 5~M) developed with a linesr gradient of 0~-lOOB%
(0% = 0.1% Trifluoracatic acid, 100~% = 67%
acetonetrile, 33% i~opropanol, 0.1% trifluoroacetic acid). Peptide sequence Eor the 39p antigen e~tending that found abova was determined in a similar fashion. Some sddit1Onal protein sequence from 39* wa9 8180 obtained from purified cleavage product~ after dige~tion with endoproteinase V8(~1/100 w/w) in SOmM NH4HC03, pH 7.8, 0.170 SDS.
Internal sequences were also determined for the 39p antigen as foll ow9:
An amino acid seq~ence was determined for an HPLC purified peptide fragment derived from proteolyit~ dige~tion of the 39 kDa protein (of the UP2 cell fraction)u~ing endoproteinase Lys-C. 300~g of the 39 kDa protein was flrst precipitated with ace~one and then resu~pended in a solution of 4 M
urea, 25 mM Tris pH 8.S and dlgested with 2.S~g LysC
~rQIe m~rk ..
- . ~
.. ~ ~ . ~ . . -1 g ~ . ~
(37C, 16 hrs.). One peptl~e was obtained as a peak o~ material eluting off of a C-4 reverse pha~e column daveloped with a gradient of acetonitrile, isopropanol (Z:l) in 0.1% trlfluroacetic acid.
The purified ~ragment had the following internal - sequence: val gln his ser leu ala trp gly ala tyr ala glu leu tyr val arg pro val gln asp leu glu glu tyr phe glu met asp ile asn. . .
The Amino acid sequence ~WB9 also determined for the protease resistant component of the 39 kDa component of the UP2 fract~on after its dige~tion with chymotryp9in. A ~onicated ~u~pensiorl of UP2 protein at 2mg/ml was incubated at 37 degree9 C for 16 hrs. with 2~ ug/ml chymotrypsin in a buffer of 2 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 ~equence:
asp xxx xxx thr lys-asp tyr met gly ile ser thr asp ile gln leu arg tyr tyr thr xxx-ile asp ala phe asn ala ile arg leu tyr phe lys tyr gly gln xxx-xxx phe A summary and comparison of the amino acid sequence data obtained from 39p and 39s is found in Tabla 1 following the Example~. Although the protein~ have not been sequenced in their entirety, none of the tata identifie~ any difference in the amino acid 3aquence oE the 39p and 399 anti~ens.
Therefore, one can conclude that the amino acid sequences of the 39p and 39g antigens are very similar and may be identical.
.. . . .
~ .
- ., Example 3 - Ge~eral Methods Unles~ otherwi~e indicated, the General Method~
described below were used in tha following example~.
A. Con~truction of a Genomlc LibrarY of T.hYo DNA
- 48 ug of genomic DNA of T;hYo strain B204 was partially digested with Alu I. EcoRI linkers were kina~ed with P3Z ATP according to manufacturer~
instructions (Pharmacia LKB Biotechnology, Pi~cataway, New Jer~ey) and ligated to the Alu I
partially digest T.hYo DNA at a linker conc~ntr~tion of 133 ~g/ml using BMB liga~e at a concentra~ion of 50 units/ml. Followine overnight ligation the liga~e was hea$ insctivated and the reaction wa~ digested with EcoRI.
The DNA was fractionated on an~S-200 (Pharmacia) column using 0.3 NaCl, 0.05 m Tri9-HCl pH 8.0, 1 mM
ED~A, 0.06% sodium azide as a column buffer, in order to remove free linkers and free ATP. The recovered T.h~o DNA was then ligated to dephosph~rylated lambda gtll EcoRI arm~ obtained from PRomega~Biotec~
(Madison, Wisconsin) and used according ~o manufacturer~ specifications. The lieation was then packaged into lambda bactariophage p~rtlcle3 using the in vitro packaging kit, ~Gigpack,"obtained from Stratagene (San Diego, CA). The phage wa~ then titered on a stationary phage culture of E. coli strain Y1090r-(Promega Biotech~. The number of white plaque~ indicated that the original phage stock contained 1.4 X lOE7 pfu/ml ~n a total of 0.5ml.
B. Identification of à Recombinant Yha~e In performin~ mixed oligonucleotide screening for the 39kDa gene, the procedure u~ed was that of W.D. Benton & R.W. Davi9 Science 196, 180 (1977).
~)rad~nar~;
--Duplicate filterq were hyb~idized with each oligon~cleotide probe. Approximately 10E6 CPm (1-2 ng probe) of probe was uged for filter, overnight at 37C. The hybridization solut:lon consisted of:
5X Denharts - 0.1 um rATP
250 ~g/ml E. coli tRNA
6X NET (lXNET=150mM NaCl, 15 mM Tris-HCl pH 7.S, 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 Eilters were washed twice at RT (20'/wash) and twice (20'/wa~h) 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 ~-ray film. Positive plaque~ were selected, rescreened and plaque purified. Phage DNA was isolated using the technlque of C. Helms, et al. ~DNA
4 39, 1985).
C. PCR Protocol 1. lOng genomic DNA ~B204 or ~234) was mixed with lOul lOx reaction buffer (Perkin Elmer Cetus), 16ul 1.2~mM dNTP teach), 25~1 primer#l (4uM), 2S ul primer #2 (4uM) and brought to a final volume of lOOul w~th ~-H20.
2. The mixture was denatured by heating at 94C for 1.5 min. and`annealed at 50~C for 2.5 min.
0.5ul of Taq polymerase (15U/~l) (Perkin Elmer Cetus) was added and polym rization was allowet to proceed at 55C for 10 min.
, , ' ,, ,' ' ' '. -: ~:
. . , -22- f;~`r)~
3. One moxe round o~ denaturation, annealing and polymerization was performed with the time and temperature conditions specif:Led in step #2.
4. Twenty three rounds of denaturationl annealing and polymerization were performed as in - step ~Z except that the pQlymerization temperature was increased to 65C.
5. After the final round of amplification the mixture was extracted with phenol:chloroform (1:1) and chloroform and precipitated with ethanol.
6. After extractio~ and precipitation the sample was digested with appropriate enzymes (samHl and Hind3) and ligated with the desired vector (pUC8 or pUC9).
D. Dot 810t Screeni~Protocol 1. Grow an overnight culture of bacterial colon~ to be screened.
2. Spin down 50ul of the overnight culture and remove supernatant.
3. Re~uspend the cell pellet in ZOOul of 25mM
Tris-Cl pH 8.0, lOmM EDTA l hen egg white lysozyme (lmg/ml).
4. Incubste 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
NH40Ac. Mi~.
8. Apply to nitrocellulose filter with aid of vacuum.
9. ~et filter air dry. 8ake at 90C for 2hrs.
10. Probe filter with desired probe.
E. PreParation of Nick Translated Probe 1. Denature 50ng of the DNA fragment by boiling for 5 min. in 9ul TE. Chill on ice.
' .
-23- ~ f ,~ J
2. Add 5ul gamma dATP (6000 Ci/mMol, lOmCi/ml), 2ul de8enerate hex~mer in 10~ reaction buffer (E~MB), 3ul dNTP (Z5uM dG- ,dT-, & dCTP final concentration~, 2ul Klenow (2U/ul, BMB).
3. Incu~ate at 37C for 30 min. Stop with 30ul lOmM EDTA.
4. Separate labeled fragement ~rom unincorporated label with G-50 spin column (BMB).
F. Screenin~ Procedure with Nick Tran~lated Probe~
The screening procedure was as followY:
1. Prewash baked nitrocellulose filter~ in ZOmM Tris-Cl pH8.0, lmM EDTA, ~.1% SDS for 2 hrs. at 37C.
2. Prehybridize filters for 2 hrs. at 42C in 50% deioniz~d formamide, 5X Denhardt's, 5X SSPE, 0.1%
SDS, salmon sperm DNA tlOOug/ml).
3. Denature nic~ translated probe by boiling for 5 minutes and chilling on ice.
4. Hybridize overnight at 42C in prehybridization ~olution and denatured probe.
S. Wash filters 2 times ~t room temperature in 2X SSC and 0.1% SDS.
6. Wash 1 time in O.lX SSC at 42C.
7. Dry filters and expose to x-ray film at -70C with enhancitlg ~creen.
G. Cement PreParation 1. ReYuspend cells from an overnight culture ~n 1/25th original volume in 25mM Tris-Cl pH 8.0, lOmM EDTA ~ lmg/ml ly~ozyme.
2. Incubate 30-60 min. Sonicate to disrupt DNA and reduce vlscosit~.
. .
~ ~ , , . :
-24- ~ J~
3. Add l/lOth v~lum~.20% Triton X-100.
Agitnte on lab quake for 2 hours. Sonicate if necessary to reduce viscosity.
4. Centrifuge at l~,O~Oxg for 10 min. to pellet cement.
5. Resuspend cement in ~/2Sth original vo~ume in 20mM Tri9-Cl pH 8.0, 5mM EDTA -I 5% Trlton X-100.
Sonicate. Agitate on lab quake overnight.
6. Centrifuge at lO,OOOxg for 10 min. to pellet cement. Wash cement with 20mM Tris-Cl pH 8.0, 5mM EDTA. Centrifuge.
7. Resuspend in 1/50th orig.inal volume in 20mM
Tris-Cl pHB.0, 5mM EDTA.
Example 4 - Identification of the Gene Encodin~ the Initial Member of the 39 kDa Anti~en familY
A set of DNA probe~ were synthesiæPd u9ing the amino terminal amino acid sequence data shown in Example 2. Each o~ them were compri~ed oP a pool of degenerate sequences which encompass all the possible combinations of nucleotides which could encode the amino acld sequence of the target region as indicated below. Each probe i~ 17 nucleotide~ in length.
:-:
. . .
,~ C,i ~
,~. .
Met-Tyr- Gly-Asp-Arg-Asp lprobe nnme = COD 555 ATa-TAT-aaT-GAT-AaT-GA
C C C C
A A degeneracy = 128 fold ~ a (mix of 128 comb~atlon~) Trp-IIe-Asp-Phe-Leu-Thr probe name - COD 553 Tt~G-ATT-GAT-TTT-TTT-AC
C C
A A
G degeneracy = 96 fold His- Gly- Asn- Gln- Phe- Arg probe name = COD 556 CAT-GGT-AAT-CAA-TTT-AG
C C C G CC
A degeneracy = 128 fold G
A lambda GTll library con~aining EcoRI linkered fragments derived from a partial AluI digest of genomic T.hyo DNA tstrain B204 was screened with probes. One phage, 3-5Cl wa~ ldentified by hybridization to probe~ 553 and 555. The DNA was examined after digestion with EcoRl and found to contain a l.6 kb in~ert.
The Eco Rl f1anked, 1. 6 kb 9egment of DN~ from pha~e 3-5Cl was isolated by electroelution from an acrylsmide gel and then ligated to pla~mid pUC l9 which had been linearized by digestion with EcoRl.
These DNAs were the ligated together, transEormPd into E. coli, and a clone containing recombinant pla~mid pTrep 106 (Appendix 3) wa3 identified by analysi~ or re~trictlon dige~ts of plasmid DNA.
Plasmld pTrep 1~6 was used to direct protein synthesis in an in ~itro coupled tran~cription-translation ~ystem containing 35S-Methionine. SDS-gel electrophoresis of the ' , c~ r~
_ -2~-protein product~ of this 9~em showed 39 kDa protein species not seen with the parental pla~mid lacking the T. hYo DNA insert. Thi~ suggests that the cloned DNA contain~ the complete coding sequence for the T.
hyo 39 kDa anti-gen and that E. coli i9 cap~ble of recognizing the treponemal pro~oter and riboso~e binding ~ite and directin~ the ~ynthesis of thi~
forei~n protein.
E. coli. ~trains transformed with plasmid pTrep 106 did not produce ~ignificant amounts of the desired 39 kDa T. hyo. ~ntlgen. Therefore, plasmid construction allowing high level expre~sion of the recombinant antigen was made as follows. The Eco RI
flanked, 1.6 kb fragment of pTrep 106 wa5 ligated to plasmid pUC 18 linearized by digastion with Eco RI.
The resulting plasmid, pTrep 112, wa9 then cut with PstI and samHI, then treated with exonuclease III to remove (in a unidirectional manner~ the non-coding DNA sequence up~tream of t~e predicted ATG start codon of the 39 kDa T. hy~ antigen (Henikoff, Gene 28 p. 351-59 (1984)). At variou3 times during thi~
digestion, DNA aliquots were removed, the exo III
inactivated by phenol extraction, the remaining DNA
rendered blunt ended by digestion with nuclease Sl, and this DNA wa9 then religated and used to transform E. coli. Nucleotide sequencing (Sanger, et al., PNAS
74:~463 (1977)) of plasmid DNA from ~ne such new clone, pTrep 112-1, indicated that a contiguous sequence of 372 codons encodine the mature T. h~o. 39 kDa protein and 7 amino acids from the signal sequence were fu~ed downstream of the Hind III site of the parental pUC 18 plasmid. The fu~ion was in a reading frame to encode a ~uslon protein whose expression would be regulated by the lac promoter after the orientation of the cloned fragment wa9 . . .
.
:
27- ~ ~s~
in~erted (see Appendix 4) b~ cloning into pUC 9 ~rom the HindIlI to Eco RI site. E. coli. transformed with the resulting plasmid, pTrep 301, produced an insoluble 39 kDs antigen which reacts with sera from swine (both those recovered from swine dynsentery as well as animals immuni~ed with the 39 kDa protein purified from T. hYo.) in both an immunoblot and plate ELISA assay.
PURIFICATION OF THE RECOMBINANT FORM OF THE
E. coli strain CY-15,000 containing plasmid pTrep30~ was grown in 250 mls of Luria broth contsining ampicillin at 200 ~g/ml. The culture wa9 grown for lB hours at 37 C. The cells were harvested by centrifugation then resuspended in l/20 their original volume in a buffer of 25 mM Tris, 10 mM EDTA at pH 8.0 and containing ly~o~yme at 1 mg/ml.
After a 30 minute incubation at room temperature the cells had lysed snd 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 lnsoluble pellet fraction after these steps wa9 saved. The ma~or protein component of this fraction had a Mr of about 40 kDa as 3udged by Commassie blue staining of sample~ after SDS-gel electrophoresis. This same proteln component was reoognized in Western blot analysi3 by swine and mouse antisera raised against the authentic 39 kDa T. hyo protein obtained from the UP2 fraction. Thi~ recombinant protein was al~o recognized in immunoblots probed with sera from pigs that had recovered from experimentally induced swine dysentery.
.
The predicted amino aoid sequence of the 3~kDa recombinant antigen obtained in this Example clo~ely resembles but is not identical to the amiho acid sequence of the 39 kDa antigen of the UP2 fraction of T. hyo~; however, they have common epitopes recognized in a single sera. ~s hereinafter indicated, the 39 kDa recombinant produced in this Example corresponds to a protein encoded by gene 1, one o~ the multiple genes encoding different T. hYo antigens, each having a molecular weight of about 39kDa.
As discussed in Example 4, infra, the ~ X~
genome contains at least 7 genes encoding relat~d antigens with molecular weights of about 39 kDa.
Although the product of only one of these genes i9 isolated from cells grown ~n vitro, it i9 possible that the other members of the gene family are expressed ln vivo and are of immunological rele~ance to protecting again~t infection in the field. The observation that each of these proteins is preceded by a signal sequence indicates that they will all be expor~ed from the cytopla~m of the cell when expressed. When cells grown i vitro are surface labeled in the presence of ll25 and lactoperoxidase the 39kDA protein in the KGP fraction i~ the prednminant protein identified. Thus, cells expressing other members of the 39kDa gene family would likely have a much different surface architecture than cells expressing the CopyS gene in vitro. An immune response mounted against one form of the 39kDa gene family could be only marginally effective again~t cells expressing one of the other forms.
.
.
....
~ 3~
Example 5 Identification of ~he ~enes encodin~ additional members of the 39 kDa anti~en familY
Internal amino acid ~eque!nce from 39p was divergent enough from the predicted amino acid sequence of pTrep301 to allow the ~election and synthe~i~ of a clegenerate oligonucleotide (Cod664, Table 2) that could be used to distinguish between ~equenceR
encoding the gene prod~ct of pTrep 106 and those en~oding the 39 kDa antigen.
The lambda GTll/ B204 library used in the pTrep 106 screenil-r was probed with Cod664 as well a 3 a nick translated probe made fr~
a 411 base pair Sphl-Bcll fragment encoding the amino terminal portion of the 39kDa protein from pTrep301.
.
- -30~ r~t ~
In addition a libraryrcon9tructed by the ligation of a partial Sau3a digest of ~204 genomic DNA and ~amHI digested lambda 'EM~L3 was also screened with these probe~. This screeming 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 pVC8, 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 Example~.
Based on the DNA ~equence from the above subclones and the internal peptide sequences, it wa~
determined that there were at least six related'gene~
encoding similar 39 kDa proteins. Of these ~ix genes, analysis of protein and nucleotide sequence data indicate that gene #5 most likely encode8 the 39kDa antigen found in the UP-2 and 39* fraction9.
None oE the ~ubclones or combinations of ~ubclone~ contained a full length copy of the #5 gene. Therefore, additional probes were prepared to isolate the remaining portion of the gene encodin~
thP 39kDa antigen of the UP-2 and 39* fractions a~
well a~ other genes of the 39kDa family. These probe~ were based on additional lnternal sequence~ of the nati~e snti~en a~ well as DNA sequence~ of phage~
of Table 3.
A uniqus oligonucleotide probe specific ~or Gene #5 (Cod968~ (Table 2) was synthesized and used to screen the GTll library. Two degenerate oligonucleo'tides (Cod 1019 and Cod 1020) derived from peptide sequence of a carboxy terminal fragment from the 399 and 39p antigen~ were al80 used to screen the GTll library to obtain a phage~s) which contained more extended coding ~equences for the #5 gene. The :
31 ~ J~.~
degeneracy of Cod lOl9 an~ 1020 wa~ decrea~ed by assuming that codon u9age ~or some amino acid~ would be 3imilar to that found in other genes in the 39 kDa family.
Unique oligonucleotides were al~o ~ynthesi~ed and used to screen the GTll library for full length genes corresponding to Gene #6 tCod 934), Gene #7 (Cod lOlO and Cod lOll) and Gene #8 (Cod 1328).
A summary oE all of tha phages identified through this additional screening, their hybridization pattern~ and 5ubclone~ i9 contained in Table 4.
DNA sequence derived from overlapping sublcones indicates that the 39 k~a ~enas are found in two subfamilie~ of tandemly repeated 39kDa gene9. Family l contain~ 4) and Family 2 contains (5-8). Each gene encodes a protein with a pre~umptive ~ignal ~equence directing transport of the protein through the inner bacterial membrane.
The gene sequence and predicted amino acid ~equence for eacb o~ the full length genes 1-8 i9 shown in Appandices 2 and 2A. In addition, Figure l of the drawings iA a map o~ the gene family and sub-clone~ obtained from screening the two libraries.
Appendi~ l ~hows the relationship between the predicted ~mino a~id ~equences of proces3ed product~
of genes 1-7 encoding seven different full length 39 kDa T. hYo antigen~ as well a3 the carboxy terminal fragment of gene #8.
The Perkin-Elmer/Cetus polymerase chain reaction system wa~ u~ed as a supplement to ~creening phage libraries to identify clones containing full length copies of 39 kDa genes. DNA sequence of Genes #l, 2, 3 (only 3') and 4 (only 5') lndicated that these genes, although containing unique internal DNA
, . ~
:. ~
'.
.
sequences, contained ident'ical 5' and 3 ' DNA
sequencas. Thus, two linkered oligonucleotides corresponding to the 5' ~equence (Cod 987), and the reverse complement of the 3' sequence (Cod 9B8), were synthesized to be used as primers for DNA synthesi3.
They were then mixed with a tlemplate of genomic DNA
from either serotype B204 or ]~234. The oligonucleotide/DNA mixtures were pas~ed through 25 cycles of heat denaturation, annealling and Taq polymerase directed DNA synthesis to amplify genomic DNA ~equences between the two oligonucleotide primer~. The newly synthesized amplified sequences were dige~ted 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 followet by a full-length copy of the mature forms oE the corresponding anti~en~. Clones were initially screenad 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 ~ene ~3 (Cod 908), and ~ene #4 (Cod 932, 1151). In addition the clones were screened with a unique synthetic oligonucleotide which is common to all known form3 of the 39kDa gene family (Cod 957). Some clones hybridized only to this nondiscriminant probe and upon DNA ~equence analysi3 were found to correspond to Gene #7 even though there is a slight mismatch of DNA ~equence between Cod 987 and the S' and 3' end~ of the gene.
A summary of the subclones obtalned along with their , : ;~
~ ' ' ; ' ~
- ::
hydridization patterns is ~ound in Table 4 following the Examples.
The PCR system was also used to ~ynthesize and clone the #5 gene encoding th~s full length 39p/39s antigen. The oli~omers used Ln this procedure were Cod 1054 and Cod 1055. Cod lOS4 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 39~/39p antigen.
CodlO55 wa~ derived from the reverse complement of the DNA sequence encoding the carboxy terminal peptide of the 39s/39p antigen. This sequence distinguishes the #S gene from any other gene obtained to date. The oligonucleotides were then mixed with genomic DNA from either ~204 or B234 and passed through 25 cycle~ o~ heat denaturation, annealling and DNA synthesis in the presence of Taq polymerase in order to amplify intervenlng sequences.
The amplified mixture was digested with BamHl and Hind3 and cloned into either pUC8 or 9. Candidate clones were screened for hybrldization to Cod968, CodlOl9, Cod 1020 and Cod 957. One of tho~e clones, pTrep 613, includes the entire coding sequence for gene which is expressed under control of the beta-galactosida~e promoter of pUC.
The PCR ~ystem wa~ al~o u~ed 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 2S cycle~ of heat denaturation, annealing, and DNA synthesis in the presence of Taq polymerase in order to amplify intervening sequence3.
The amplified mi~ture wa~ digested with ~amHI and - ~ .
.
!~ . , _ !
-3q-SalI and cloned into eithe~ pUC8 or 9. Candidate clone~ were screened for hybridization to Cod 957.
One of these clone~, pTrep 541, includes the entire coding ~equence for the gene which i~ expressed under control of the`beta-galactosidase promoter of pUC.
A schematic o~ the B204 expression clone~
derived from the PCR reaction (pTrep 345, 541, 604, 605, 620, 613) is found in Figure 5. The pr~dicted smino acld sequences encoded by theqe clone~ are found in Appendix S.
E~pres~ion of recombinant forms of the 39 kDa Protein from Renomic DNA subclones corresPondin-~ to Genes ~2 and 6.
pTrep505, a pUCl9 based plasmid directs the expression of all but the first 19 amino acid9 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 GTll library. Thi~ EcoRl fragment wa~ subcloned into pWHA142 to place it in the proper orientation and readin~ frame for expression from the Lac promotèr.
pWHA142 i~ a derivativa of pUCl9 with a GAA reading frame across the EcoRl site. A plasmid map of pTrep505 i~ presented in Figure 2. The predictet protein ~equance from pTrep 505 i5 presented in Appendix 8.
pTrep 702, a pUC19 ba~ed plasmid direct~ the expres~ion of 13 amino acids from the signal sequenca of Gene ~6 plu~ the first 315 amino acid~ of the mature protein fu~ed to the Lac~ complementing peptide in pUC. It wa~ constructed in two steps from pTrep501 and pTrep327 which contain overlappin~
region3 o~ the #6 gene and ~hare a common Bcll site.
pTrep501, which cont~in~ region9 coding for the ~' .
.: : .
- -35- ~, t;~ J ~
portion of the #6 gene, wa~.dige~ted with Bcll and Aatll and ligated with Bcll-Aatll ragment from pTrep 327 which contained the 3' sequences of the ~6 gene.
The EcoRl fragment from this clone, pTrep701, wa9 then cloned into the EcoRl site of pWHA142 to place the #6 seq~ence in the proper reading frame for ~xpre~sion from the Lac promoter. A schematic of the construction of pTrep702 i9 presented in Figure 3.
The predicted protein sequence of the recombinnnt product encoded by pTrep702 i~ presented in Appendl~
6.
An expres~ion clone encoding the f~ll length Copy ~6 antigen, pTrep704, wa9 constructed from pTrep702 by replacing its 430 bp Nsil-Ndel fragment with an 847 bp N~il-Ndel fragment from pTrep508. The 3' cloning site of pTrep508 is downstream of the cloning si~e contained within pTrep702 and thu~
contains the DNA sequence~ encodin~ 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 Appendi~ 7.
The recombinant products expres~ed by pTrep505, pTrep702, and pTrep704 as well as the PCR derived constructs are recovered as insoluble cements in E.coli strain CY15000 after lysis o cells with ly~ozyme and e~trartion wlth Triton X-lOO~in the presence of EDTA.
These are immunoreactive with ~era from animals experimentally infected with T.hro (B204) and with sera from animals vacci~ated with UP2 or the electroeluted 39 kDa protein from UP2.
~ Tra~ a~
, -3B~
The following Table 5~tabulatas the 39 kDa expression clone~ for expressing seven different T.
hyo. antigens having a molecular weight of about 39 kDa, ~ith reference to the different gene~ or copie~.
;
' Table 1 - Amino Acid Compar~son of 39~ and 39p Antigen~
Sequence Source MYGDRDSWIDFLTHGNQFRARMDQLGFVLGN?TIKGTF'7?R 39p Nterm MYGDRDSWIDFLTHGNQFRARMDQLGFVL?NGTIKGTFGF??Q?I 39* Nterm P?S7?TK?YMGISTDIQLRYYTGIDAFN.AIRLYFKYGQAGFK 3gp2, 4 FPYS?STKDYMGISTDIQ~RYYTGIDAFN.AIRLYFKYGQAGFK 3 9 *2 TANGASEYFAQSLGFEARFYFLNTPVGNV~INPFIKWNTA 39p2 TANGASEYFAQSLGFEARFYFLNTPVGNVTINPFIKWNTAL 39*2 AQAVLGITANSDWSLYVEPSLGYQATYLGK 39p2 AQAVIIGITANSDWSLWEPSL 39*
HISENPYLNIDSK 39pl HISENPYLNIDSK 39*2 VQHSLAWGAYAELYVRPVQDLE?YFEMDIN 39pl RNGVPVNFATSTGIT?YLPAL(:G?Q 39p2 MDINNSDSKRNGVPVNFATSTGITWYLPALGGAQ 39*3 Notes--All sequence derived from fraE~ments ~enerated by dige~tion with LysC in the pre~ence o~ SDS unless otherwise ~ ndl cat ed .
2Ly~C dige~t in ab~ence oiE SDS
Fraction gave muplitple ~equences which were resolved on basis ~f intensity and DNA predicted amino acid ~equence 4GluC digest in presence of SDS
Sequence begin~ with residue #2 due to machine failure and lo~ of residue #l Single amino acid code uYed above i~ a~ follows:
A=ala H=his P=pro W=trp C=cy~ I=Ile Q=gln Y=tyr D=asp K=ly~ R-arg E;= g lu L= leu S - ~ e r F=phe M=met T=thr G=gly N=asn V=val ~, : .
,J.~ 3 Table 2 COD Sequence Souree o Sequence Specif i, 664 AcG-AAa-GAT-TAT-ATG-GG 39p internalfor Copy A A c c peptide ~equence 5, T -C
844 TTAATCCGCAI'GATA pTrep 106 908 GTTTCATCACAAGCAAA pTrep 333 3, ', 931 ATGAATATGACGGATAA pTrep 330 7 932. AAAGTTGATAAACMGG pTrep 333 /, 934 TATCATCCTTCTAATCCT pTrep 331 fi 957 CCGAAAGTACCTTTAAT pTrep 106 ALl.
968 TATAATCCTTATGATCCT pTrep 317 r~
987 GAATTCCGGATCCATGTATGG- pTrep 106 1-~
AGATCAGGACGATTGGATT
988 GTCGACAAGCTTATAATTAAA- pTrep 106 1-ATTCTGGCAAATACCAAGT
1010 ATATTGACTGATAGTAT pTrep 506 i 1111 AAATAATTTTGATATG pTrep 506 1020 GAT-AGA-AAA-AGG-AAT-GG 39* internal peptide r`
TCT A C sequence C
1019 MA-AGG-AAT-GGA-GTG-CC 39* internal peptide r`
A G T A sequence T
C
1054 GAATTCCGGATCCATGTATGGCGACAG- pTrep 326 5, AGATTCTTGGATC
1~55 GTCGACAAGCTTATAATTATTGAGCAC- pTrep 510 r`
CGCCTAAAGCAGG
1092 AGTATGTTTGMCCMTA pTrep 333 11~1 TCATATGTATCGTGTATA pTrep 333 ', 1095 GGAGTACCTAAACTTCAA pTrep 337 11 1248 CGACAGAGATTCTTGGA pTrep 613 S, fi -S s ?. ~3 Tsblet 2 (continued?
COD Sequence Source of Sequence Specif;-for Copy 11 1328 GAATTCAATTACGGA~T pTrep S37 ~3$9 ~TCGACCTGCAGT~ATTA- pTrep S2 TTGTARAGCAGGTAAATA-CCA
1438 GAATTCCGGATCCATGTATGG- pTrep 537 TGCAGACAACACATGGCTT
, ~ c~
.. .. . . .. . _ . .. . . . . ... . ... ~ .. . . . . .
Table ~3 . .
Tahls *3~1den~1tlc~1an oS roc~m~Ja1 ph~o~
~nd ~ubclan~
Id~ntllled by Screenln0 w11h Pha~e' pTrep 301 Sph~Bcl Cod664 9B^ 3 17 +
54-3 323 +
53-3B 326 ~ +
56-3 ~30 51-3 333 ` +
51-18 331 +
53- 1 9 337 + +
All recombinanl phag~ ar~ ~rom Iho GT11 library unbss o~h~ls2 nel~
^ From EM8L3 libraly , :, (~.J ~ J I~J ~.f 41 r O +
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n ~ a3 c~ c~
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E C
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o c .~
c o ~ + +
o ~ ~ en .
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a + +
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Table 5 Express~on Clones from PC~
CloneSerotype Cod957Cod8~4 ICod908 Cod~31 Cod932 Cod968 Codll51 pTrep345 B204 +
pTrep605 1~204 I +
pTrep604 ~3204 1 .
pTrep60~ ~3234 pTrep609 ~3234 pTrep610 B234 ~ ~
pTrep613 E~204 ~ +
pTrep620 B204 pTrep651 B234 +
pTrep541 B20~ +
" ` `' ` ' . ` '~.
:
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Tabl2 6 Clone CoPy# Source Comments pTre p3~1 - 1 genomlc pTrep505 2 genomic lacks amino terminus pTrep604 3 pcr pTrep345 4 pcr pTrep613 5 pcr pTrep702 6 genomic lack~ carboxy terminus pTrep704 6 genomic full 1ength pTrep620 7 pcr l amino acid substitution at C terminus relative to ~enomic ~equence pTrep605 2 pcr full length pTrep541 3 pcr full length .
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J ~ 3 E r i Use of 39* AntiRen in a Vaccine In two vaccination s~udies, purified 39* which includes 393 antigen and which is prepared according ta the proc~dule of Example 1 (note pages 11 and 12) was tested in compari30n with no challenge, vaccination with ad~uvant, or V8CC~ nation w~th the commercially a~ailable Hyguard (Haver Labs) product.
In the first ~t~dy, six pi~s per test group were used. The pig9 averaged 22.6 lb and were approximately S-6 weeki~ of age. Five group~ of pig9 were given two doses, the first on day 0 and a booster on day 36. The in~ections were ~iven I.M., in the neck with 1 mg/dose of native antigen.
Animals were challenged on day 50 by ~tomach intubation using a p~re rulture of T. hyo. tB204) at 5.5 x 10 cfu per pig. The st~dy was terminated on day 92.-Vaccines were given with Emulsi~en ad~uvant.Emulsigen was ui~ed as an ad~uvant control, mixed with Dulbeco's PBS buffer. The Hyguard, a bacterin, wa adminstered according to manufacturer'i~ directions.
Animals were monitored daily for clinical ~igns of swine dysentery. Microbiological evaluation of routine weekly rectal swabs wa9 conducted for T. hYo.
snd Salmo~ella. Animals showing 9ign9 of bloody diarrhea were swabbed and evaluated on that day.
Weekly postchallenge pig9 were weighed and their feed intake determlned. The experimental results ars shown below.
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E~ample 7 Use oE Native Anti~en A ~econd study was conducted to confirm the re~ults of Example 5 where the 39* vaccinates performed exceptionally well when challenged intra-gastrically with a measured dose of T
hYodysenteriae. As ~hown below, 1 of 9 animals vaccinated with a l mg dose o;E 39* and cha1lenged by stomach intubation with 4.4 x lO2 CFU of T. hx~
(B204) developed clinlcal si~ns of swine disentery in comparison to 3 of 6 ad~uvant vaccinate~ and 0 of 6 HyGuard vaccinstes.
# of Break~/
Vaccinate Group # AnimalsDay of Onset No Challenge 0/6 Ad~uvant 3/6ll, ll, 12 HyGuard 0/5 39*, lm~ l/6 29 39*, lmg--new lot 0/3 Example 8 Production o~ AntibodY
A cement preparation (Part G of Example 4) in an amount of 25-200 ~g in Emulsigen is intermu~cularly in~ected into pig~. Two weeks later, the pig i9 boosted with an identical dose. Two weeks sfter the boost, the pig i~ bled and the blood is allowed to clot. Immune serum i9 separated by centrifugstion at 4C.
Numerou3 modification~ and variation~ of the present invention are po~sible in light of the aSove teachnings; therefore, within the scope of the " .~ .
, ' 3 ~
-g7-appended claims, tha inventlon may be practiced otherwise than as particularly described.