EP0527771A1 - Polypeptides utiles au niveau du diagnostic et du traitement contre les infections d es au mycoplasma chez les animaux - Google Patents

Polypeptides utiles au niveau du diagnostic et du traitement contre les infections d es au mycoplasma chez les animaux

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Publication number
EP0527771A1
EP0527771A1 EP91907676A EP91907676A EP0527771A1 EP 0527771 A1 EP0527771 A1 EP 0527771A1 EP 91907676 A EP91907676 A EP 91907676A EP 91907676 A EP91907676 A EP 91907676A EP 0527771 A1 EP0527771 A1 EP 0527771A1
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EP
European Patent Office
Prior art keywords
protein
dna
dna construct
mycoplasma
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP91907676A
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German (de)
English (en)
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EP0527771A4 (en
Inventor
Jerry M. Kuner
Christine Ko
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Amgen Boulder Inc
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Synergen Inc
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Publication of EP0527771A1 publication Critical patent/EP0527771A1/fr
Publication of EP0527771A4 publication Critical patent/EP0527771A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to a class of polypeptides useful in diagnostic assays to determine the presence of antibodies to Mycoplasma organisms in mammals, particularly in pigs or hogs.
  • the invention also relates to recombinant-DNA methods for the manufacture of these polypeptides and recombinant phage clones containing DNA sequences suitable for use in the recombinant methods.
  • the invention also relates to vaccination compositions and methods of vaccination to inhibit Mycoplasma infections in animals.
  • Enzootic pneumonia of pigs also known as virus pneumonia, infectious pneumonia, anterior lobe pneumonia, enzootic virus pneumonia and mycoplasmal pneumonia of swine, rarely causes death, but often results in severe morbidity and reduced performance in weight gain of swine.
  • the causative agent was Mycoplasma hvopneumoniae, also known as Mycoplasma suipneumoniae.
  • the disease is transmitted from pig to pig through the nasal passages by airborne organisms expelled from infected pigs.
  • the Mycoplasma establish themselves deep in the apical and cardiac lobes of the lungs where they cause visible plum colored or gray lesions and cause difficulty in breathing and reduced weight gain.
  • M. hvopneumoniae may be followed by secondary infection by other mycoplasma species (M ⁇ . hvorhinus and M ⁇ . floculare) as well as bacterial pathogens (Pasteurella and Bordetella species) .
  • the Mvcoplasmas are prokaryotic cells smaller and simpler in structure than bacteria, but more complex than viruses. Unlike viruses, they are capable of a free living existence, though they are often found in association with eukaryotic cells. They are bounded by a cell membrane but not by a cell wall. They have an extremely small genome, approximately 750,000 base pairs in length.
  • the present invention also relates to recombinant-DNA methods for manufacturing the polypeptides. These recombinant-DNA methods utilize DNA sequences contained in various recombinant phage clones which are described herein.
  • Another object of the present invention is to provide a vaccine composition and a method of vaccination effective for preventing certain Mycoplasma infections in animals.
  • Proteins A, B, C, D, and E have been disclosed.
  • the DNA corresponding to portions of these proteins is contained on various lambda phages which also are identified herein.
  • the entire gene for Protein C has been provided herein.
  • a recombinant-DNA method for the manufacture of polypeptides analogous to Mycoplasma surface proteins is disclosed. These proteins are capable of crs ⁇ txny ⁇ ii ⁇ f ⁇ -uno i ⁇ gnostic co ⁇ u ex Wusn expos ⁇ u to sera from swine infected with Mycoplasma hyopneumoniae and certain other mycoplasma organisms.
  • This method comprises: (a) Preparation of a DNA sequence coding for a polypeptide possessing antigenic properties analogous to those possessed by a polypeptide produced by Mycoplasma organisms;
  • FIGURE 1 depicts the entire DNA sequence and translated amino acid sequence of the Mycoplasma hvopneumoniae genomic DNA insert in the phage ⁇ gtll clone R69.
  • the DNA sequence (upper line) is divided into three base codons which align with the proper reading frame of the gene.
  • the amino acid sequence (lower line) is the translation of the DNA codon directly above, written in the single letter amino acid code as described herein.
  • FIGURE 2 depicts the Mycoplasma hvopneumoniae DNA sequence from the recombinant plasmid clone pUC18::28C2 which contains the entire gene for Protein C. A total of 4547 base pairs were sequenced. Approximately 1700 base pairs of DNA downstream were not sequenced. The Protein C gene starts at nucleotide 1801 and ends at nucleotide 3672 of the insert.
  • FIGURE 3 depicts the amino acid sequences of peptides derived from endoproteinase digested purified Protein C and the positions of these sequences in the amino acid sequence of the intact protein. The sequences are written in the single letter amino acid code as described herein.
  • FIGURE 4 depicts the entire DNA sequence and amino acid sequence of the Protein C gene.
  • the DNA sequence (upper line) was directly determined (see Example VIII-B) and the amino acid sequence (lower line) was determined by translation of the DNA sequence above.
  • the underlined segments of the amino acid sequence denote the parts of the sequence for which there is direct amino acid sequencing data (see Example VIII-C and D and Figure 3).
  • the three UGA codons (TGA in the DNA sequence) are enclosed in boxes.
  • the position of the ⁇ gtll clone R69 insert (starting at nucleotide 455 and ending at nucleotide 976) is delineated by marks above the DNA sequence.
  • the amino acid sequence is written in the single letter amino acid code as described herein.
  • FIGURE 5 depicts the modifications made to the 5' end of the Protein C gene for recombinant expression of E ⁇ . coli using the plasmid vector pT5T.
  • the initial 25 bases encode a BamHl restriction site for connection to the pT5T vector with the TC3 translational coupler ending with the translational stop TAA. Following the TC3 coupler is the restart methionine codon ATG, followed by the Protein C gene.
  • Both the original and modified DNA sequences are shown with the unchanged nucleotides depicted in lower case letters and the modified nucleotides depicted in capital letters in the modified sequence.
  • the translation of these sequences (unchanged between the original and modified sequences) is shown in bold below the DNA sequences.
  • the shaded boxes indicate restriction endonuclease recognition sites in the sequences, with the name of the restriction endonuclease noted above or below the box.
  • FIGURE 6 depicts the DNA sequence and the translated amino acid sequence of the insert in the expression plasmid pT5T: :M852 which produces the recombinant full length Protein C.
  • Translation starts at the ATG restart at position 1 which is preceded by the TC3 coupler (See Figure 5). Translation of the sequence proceeds to the natural stop codon TAA at nucleotide 1876.
  • the three UGA codons (TGA in the DNA sequence) in the Protein C gene have been changed to UGG (TGG) in this clone to permit expression in E . coli.
  • TGG's are denoted by boxes in the sequence.
  • the numbering system is offset by three nucleotides from Figure 4 because of the addition of the start codon ATG to the 3'-end.
  • FIGURE 7 depicts the DNA sequence and the translated amino acid sequence of the insert in the expression plasmid pT5T::M851 which produces the recombinant truncated Protein C.
  • Translation starts at the ATG restart at position 1 which is preceded by the TC3 coupler (See Figure 5).
  • Translation of the Protein C sequence proceeds to the HindiII restriction site at nucleotides 1294 to 1299 where the insert is joined to the pT5T vector which encodes three more amino acids (glycine, threonine and aspartic acid) before a translational stop codon.
  • FIGURE 8 is a photograph of a Coo assie Blue stained polyacrylamide gel showing the electrophoretically separated proteins in M , ⁇ hvopneumoniae extracts SI, 7S, and 7P (See Example X-A) .
  • FIGURE 9 depicts the restriction map of clone pUC18::28C2, the M. hvopneumoniae genomic clone containing the entire Protein C gene.
  • the arrows below the main figure depict the sequencing strategy, showing the direction and extent of the sequencing done.
  • Also depicted are the positions of the sequences of clone R69, the original ⁇ gtll clone isolated which encodes part of Protein C, and R68, another ⁇ gtll clone which starts before the coding region for Protein C and reads into the coding sequence.
  • the positions of the three UGA codons in Protein C coding sequence are also shown.
  • FIGURE 10 depicts certain features of the bacterial expression construct pT5T: :M852 which expresses the full length recombinant Protein C. Features are representative only and not drawn to exact scale. The shaded part indicates the M_i. hvopneumoniae segment containing the Protein C gene. Expression construct pT5T::M851, which expresses the truncated Protein C, is essentially the same except that it lacks the ⁇ 2 kb HindiII fragment.
  • the present invention relates to a class of polypeptides which are useful, inter alia, for in vitro diagnosis of mycoplasma infection in swine.
  • the present invention also relates to vaccine compositions and methods of vaccination including the above-mentioned class of polypeptides.
  • These substantially purified proteins are analogous to various Mycoplasma hvopneumoniae proteins which are capable of inducing an immune response when present in swine tissue. Because an immune response has been mounted in infected swine against analogous antigens, the sera of such infected swine will contain antibodies which will recognize one or more of the polypeptides of the present invention.
  • the instant polypeptides may serve, either in combination or individually, as the active ingredient in an in vitro diagnostic assay to determine the presence in swine sera of antibodies directed toward various Mycoplasma species.
  • the instant polypeptides either in combination or individually, may also be used in vaccine compositions to illicit an immune response in animals to prevent Mycoplasma infections in the vaccinated animals.
  • analogous when used in connection with a protein, antigen or polypeptide, is intended to mean a polypeptide which is capable of detecting antibodies raised in response to an infection with natural Mycoplasma proteins in swine. A polypeptide possessing analogous antigenic properties will thus exhibit some homology to the native Mycoplasma protein. It should be noted that “analogous" polypeptides, as the term is used herein, may raise an immune response which is stronger than, the same as, or weaker than the response raised by natural Mycoplasma proteins.
  • substantially homologous is meant a degree of homology to the protein of interest in excess of that displayed by any previously reported, purified, substantially homologous protein composition.
  • the degree of homology is in excess of 60%, and more preferably 75%, with particularly preferred proteins being in excess of 85% or 90% homologous with the native protein.
  • the degree of homology as described above is calculated as the percentage of amino acid residues found in the smaller of the two sequences that align with identical amino acid residues in the sequences being compared when four gaps in a length of 100 amino acids may be introduced to assist in that alignment as set forth by Dayhoff, M.O. in Atlas of Protein Seguences and Structure, Vol. 5, page 124 (1972), National Biochemical Research Foundation, Washington, D.C.
  • the protein of the present invention is either isolated from a natural source or is a synthetic polypeptide.
  • synthetic polypeptide is intended to mean an amino acid sequence which has not previously been isolated from nature in a substantially purified form.
  • synthetic encompasses, among others, polypeptides created by recombinant-DNA methods or synthesized in whole or in part in vitro.
  • synthetic polypeptides are contemplated in which 1 or 2 amino acids differ from those set forth in the preferred sequences set forth below.
  • pure form or “purified form,” when used to refer to the protein of interest disclosed herein, shall mean substantially free of other proteins which are not the protein of interest.
  • the protein of the present invention is at least 50% pure, more preferably 70% pure and even more preferably 80% or 90% pure.
  • Protein A a 105kd protein of M. hyopneumoniae
  • Protein B a 90kd protein of JJ . hvopneumoniae
  • Protein C an 85kd protein of M. hvopneumoniae
  • Protein D a 70kd protein of M. hvopneumoniae
  • Protein E a 43kd protein of M. hvopneumoniae. It should be noted that the molecular weights associated with the proteins disclosed herein are not to be interpreted as absolute values.
  • each of the proteins A through E is a protein present on the surface of the Mycoplasma organism.
  • a protease trypsin
  • each of these proteins contain one or more specific portions which may serve as an antigenic determinant capable of binding to at least one antibody present in sera of Mycoplasma infected swine.
  • These specific antigenic portions either singly or in various combinations, would be, therefore, capable of serving as the basis for an in. vitro diagnostic assay.
  • the present inventors have also shown that at least Protein C (an 85 kd protein) and a fragment thereof are useful in vaccine compositions to prevent Mycoplasma infection in animals. It is also believed that the other disclosed proteins and variants thereof can also ellicit an immune response, and may also protect animals from Mycoplasma infection. Such proteins or fragments thereof may be used individually or in a mixture in a vaccine composition.
  • DNA sequences encoding portions of these proteins are contained on the lambda gtll clones identified herein.
  • the DNA sequences coding for the entire proteins are contained in the same lambda gtll library from which the above clones were derived, and methods are described below that will allow the identification and isolation of such clones.
  • a portion of the gene encoding polypeptide A (105kd) is contained on the lambda gtll clone R60b which contains an insert of Mycoplasma DNA of 1.5 kilobases. This fragment can be excised using the restriction endonucleases Kpnl and Sacl which cut in the flanking vector sequences but not within the insert.
  • the corresponding expression plasmid R60b-a has been constructed by insertion of the Kpnl/Sacl insertion fragment of the lambda gtll clone into the plasmid vector pSEV6.
  • a portion of the gene encoding polypeptide B (90kd) is contained on the lambda gtll clone LMHC1-9 which contains an insert of Mycoplasma DNA of 0.45 kilobases. This fragment can be excised using the restriction endonucleases Kpnl and Sacl which cut in the flanking vector sequences but not within the insert.
  • the corresponding expression plasmid LMHCl-9a has been constructed by insertion of the Kpn/Sac insertion fragment of the lambda gtll clone into the plasmid vector pSEV6.
  • a portion of the gene encoding polypeptide C (85kd) is contained on the lambda gtll clone R69 which contains an insert of Mycoplasma DNA of 0.5 kilobases (specifically 522 bases) .
  • This fragment can be excised using the restriction endonucleases Kpnl and Sacl which cut in the flanking vector sequences but not within the insert.
  • the corresponding expression plasmid R69b (also designated pSEV6::R69) has been constructed by insertion of the Kpnl/Sacl insertion fragment of the lambda gtll clone into the plasmid vector pSEV6.
  • a portion of the gene encoding polypeptide D (70kd) is contained on the lambda gtll clone 86-4 which contains an insert of Mycoplasma DNA of 3.2 kilobases. This fragment can be excised using the restriction endonucleases Kpnl and Saul which cut in the flanking vector sequences but not within the insert.
  • the corresponding expression plasmid 86-4C has been constructed by insertion of the Kpnl/Saul insertion fragment of the lambda gtll clone into the plasmid vector pSEV6.
  • a portion of the gene encoding polypeptide E (43kd) is contained on the lambda gtll clone Pi which contains an insert of Mycoplasma DNA of 0.5 kilobases. This fragment can be excised using the restriction endonucleases Kpnl and Sacl which cut in the flanking vector sequences but not within the insert.
  • the corresponding expression plasmid Pic has been constructed by insertion of the Kpnl/Sacl insertion fragment of the lambda gtll clone into the plasmid vector pSEV6.
  • DNA encoding the proteins or the proposed antigenic determinants may be expressed in mammalian systems.
  • the DNA of interest is excised from the DNA contained on the lambda gtll phage clone and inserted, in a suitable form, into a microbial expression system.
  • the antigenic polypeptides are produced by a method comprising:
  • M ⁇ hvopneumoniae is a prokaryote
  • genomic DNA may be used directly without concern about introns.
  • other Mycoplasma species have been shown to utilize the normal stop codon UGA as a tryptophan codon in protein synthesis.
  • expression in other systems e.g., E ⁇ coli
  • the DNA prepared in accordance with the above methods is inserted into an expression vector suitable for use in the intended expression system.
  • Embodiments of the present invention are envisioned as employing other known or currently undiscovered vectors which would contain one or more of the DNA sequences encoding antigenic polypeptides described herein.
  • these vectors have some or all of the following characteristics: (1) possess a minimal number of hostorganism sequences; (2) be stable in the desired host; (3) be capable of being present in a high copy number in the desired host; (4) possess a regulatable promoter; and (5) have at least one DNA sequence coding a selectable trait present on a portion of the plasmid separate from that where DNA sequence encoding for ' the antigenic polypeptide will be inserted.
  • E. coli pUC8 Many selectable replicons pUC9 have been characterized.
  • pBR322 Maniatis, T. et al. (1982) pGW7 Molecular Cloning: A placid Laboratory Manual, Cold pDP8 Spring Harbor Laboratory.
  • PSEUDOMONAS RSF1010 Some vectors useful in . aeruginosa Rmsl49 broad host range of gram- P. putida pKT209 negative bacteria including
  • These "operational elements,” as discussed herein, include but are not limited to at least one promoter, at least one ribosome-binding sequence and at least one transcription terminator.
  • these "operational elements” also include at least one operator, at least one leader sequence for proteins to be exported from the intracellular space, at least one regulator and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector DNA.
  • an EL. coli vector system is preferred in one embodiment as a cloning vector.
  • several vector plasmids which autonomously replicate in a broad range of Gram negative bacteria are preferred for use as cloning vehicles in hosts of the genera Pseudomonas. These are described by Tait, R.C., Close, T.J., Lundquist, R.C., Hagiya, M., Rodriguez, R.L., and Kado, C.I. in Biotechnology, May, 1983, pp. 269-275; Panopoulos, N.J. in Genetic Engineering in the Plant Sciences, Praeger Publishers, New York, New York, pp. 163-185, (1981); and Sakaguchi, K. in Current Topic in Microbiology and Immunolo gy . .96.:31-45, (1982).
  • RSF1010 plasmid RSF1010 and derivatives thereof as described by Bagdasarian, M. , Bagdasarian, M.M. , Coleman, S., and Timmis, K.N. in Plasmids of Medical, Environmental and Commercial Importance. Timmis, K.N. and Puhler, A., eds., Elsevier, North Holland Biomedical Press, (1979).
  • the advantages of RSF1010 are that it is relatively small, high copy number plasmid which is readily transformed into and stably main ⁇ tained in both E_j. coli and Pseudomonas species. In this system, it is preferred to use the Tac expression system as described for Escherichia.
  • E_ j _ coli trp promoter is readily recognized by Pseudomonas RNA polymerase as set forth by Sakaguchi, K. in Current Topics in Microbiology and Immunology, j)j5:31-45 (1982) and Gray, G.L., McKeown, K.A., Jones, A.J.S., Seeburg, P.H., and Heyneker, H.L. in Bio/Technology, Feb. 1984, pp. 161-165. Transcriptional activity may be further maximized by requiring the exchange of the promoter with, e.g., an _____ coli or 2- ⁇ . aeruginosa trp promoter.
  • Pj. aeruginosa is transformed with vectors directing the synthesis of the antigenic polypeptides as either an intracellular product or as a product coupled to leader sequences that will effect its processing and export from the cell.
  • these leader sequences are preferably selected from the group consisting of beta-lactamase, OmpA protein, and that of carboxyeptidase G2 from Pseudomonas.
  • Translation may be coupled to translation initiation for any of the E . coli proteins as well as to initiation sites for any of the highly expressed proteins of the host to cause intracellular expression of the antigenic polypeptides.
  • the antigenic polypeptides of the present invention as either an intracellular or a secreted protein.
  • the present embodiments include both systems. Shuttle vectors that replicate in both Bacillus and E. coli are available for constructing and testing various genes as described by Dubnau, D., Gryczan, T., Contente, S., and Shivakumar, A.G. in Genetic Engi- neering. Vol. 2, Setlow and Hollander eds., Plenum Press, New York, New York, pp. 115-131, (1980). For the expression and secretion of anti ⁇ genic polypeptides from B_j.
  • the signal sequence of alpha-amylase is preferably coupled to the coding region for the antigenic polypeptide.
  • the portable DNA sequence will be translationally coupled to the riboso e binding site of the alpha-amylase leader sequence.
  • alpha-amylase promoter or a derivative thereof.
  • This derivative contains the RNA polymerase recognition sequence of the native alpha-amylase promoter but incorporates the lac operator region as well.
  • Similar hybrid promoters constructed from the penicillinase gene promoter and the lac operator have been shown to function in Bacillus hosts in a regulatable fashion as set forth by Yansura, D.G. and Henner in Genetics and Biotechno ⁇ logy of Bacilli, Ganesan, A.T. and Hoch, J.A., eds., Academic Press, pp. 249-263, (1984).
  • the lad gene of lacl q also would be included to effect regulation.
  • Clostridium One preferred construction for expression in Clostridium is in plasmid pJU12 described by Squires, C. H. et al in J. Bacteriol., 159:465-471 (1984), transformed into C. perfringens by the method of Heefner, D. L. et al. as described in J. Bacteriol. , 159:460-464 (1984). Tran ⁇ scription is directed by the promoter of the tetracycline resistance gene. Translation is coupled to the Shine- Dalgarno sequences of this same tet gene in a manner strictly analogous to the procedures outlined above for vectors suitable for use in other hosts.
  • yeast Maintenance of foreign DNA introduced into yeast can be effected in several ways. See, for example, Botstein, D., and Davis, R. W. , in The Molecular Biology of the Yeast Saccharomvces, Cold Spring Harbor Laboratory, Strathern, Jones and Broach, eds., pp. 607-636 (1982).
  • One preferred expression system for use with host organisms of the genus Saccharomvces harbors the antigenic polypeptide gene on the 2 micron plasmid.
  • the advantages of the 2 micron circle include relatively high copy number and stability when introduced into cir° strains.
  • These vectors preferably incorporate the replication origin and at least one antibiotic resistance marker from pBR322 to allow replication and selection in Ej_ coli.
  • the plasmid will preferably have 2 micron sequences and the yeast LEU2 gene to serve the same purposes in LEU2 mutants of yeast.
  • the regulatable promoter from the yeast GAL1 gene will preferably be adapted to direct transcription of the antigenic polypeptide gene. Translation of the DNA sequence in yeast will be coupled to the leader sequence that directs the secretion of yeast alpha-factor. This will cause formation of a fusion protein which will be processed in yeast and result in secretion of the desired antigenic polypeptide. Alternatively, a methionylantigenic polypeptide will be translated for inclusion within the cell.
  • various operational elements may be present in each of the preferred vectors of the present invention. It is contemplated any additional operational elements which may be required may be added to these vectors using methods known to those of ordinary skill in the art, particularly in light of the teachings herein.
  • each of these vectors in a way that allows them to be easily isolated, assembled, and interchanged. This facilitates assembly of numerous functional genes from combinations of these elements and the coding region of the antigenic polypeptide. Further, many of these elements will be applicable in more than one host.
  • At least one origin of replication recognized by the contemplated host microorganism, along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the DNA encoding for the antigenic polypeptide are contemplated as being included in these vectors. It is additionally contemplated that the vectors, in certain preferred embodiments, will contain DNA sequences capable of functioning as regulators ("operators"), and other DNA sequences capable of coding for regulator proteins. In preferred vectors of this series, the vectors additionally contain ribosome binding sites, transcription terminators and leader sequences.
  • regulators in one embodiment, will serve to prevent expression of the DNA sequence encoding for the antigenic polypeptide in the presence of certain environmental conditions and, in the presence of other environmental conditions, allow transcription and subsequent expression of the protein coded for by the DNA sequence.
  • regulatory segments be inserted into the vector such that expression of the DNA sequence will not occur in the absence of, for example, isopropylthio-beta-d-galactoside.
  • the transformed microorganisms containing the DNA of interest may be grown to a desired density prior to initiation of the expression of the antigenic polypeptides.
  • expression of the desired antigenic polypeptide is induced by addition of a substance to the microbial environment capable of causing expression of the DNA sequence after the desired density has been achieved.
  • Additional operational elements include, but are not limited to, ribosome-binding sites and other DNA sequences necessary for microbial expression of foreign proteins.
  • the operational elements as discussed herein can be routinely selected by those of ordinary skill in the art in light of prior literature and the teachings contained herein. General examples of these operational elements are set forth in B. Lewin, Genes, Wiley & Sons, New York (1983). Various examples of suitable operational elements may be found on the vectors discussed above and may be elucidated through review of the publications discussing the basic characteristics of the aforementioned vectors.
  • an additional DNA sequence is located immediately preceding the DNA sequence which codes for the antigenic polypeptide.
  • the additional DNA sequence is capable of functioning as a translational coupler, i.e., it is a DNA sequence that encodes an RNA which serves to position ribosomes immediately adjacent to the ribosome binding site of the antigenic polypeptide RNA with which it is contiguous.
  • the vectors are assembled by methods generally known to those of ordinary skill in the art. Assembly of such vectors is believed to be within the duties and tasks performed by those with ordinary skill in the art and, as such, is capable of being performed without undue experimentation. For example, similar DNA sequences have been ligated into appropriate cloning vectors, as set forth in Schonert et al.. Proceedings of the National Academy of Sciences U.S.A. , £1.:5403-5407 (1984).
  • the host organism would produce greater amounts per vector of the desired antigenic polypeptides.
  • the number of multiple copies of the DNA sequence which may be inserted into the vector is limited only by the ability of the resultant vector, due to its size, to be transferred into and replicated and transcribed in an appropriate host microorganism.
  • the cloning vector contain a selectable marker, such as a drug resistance marker or other marker which causes expression of a selectable trait by the host microorganism.
  • a selectable marker such as a drug resistance marker or other marker which causes expression of a selectable trait by the host microorganism.
  • a drug resistance or other selectable marker is intended in part to facilitate in the selection of transformants.
  • the presence of such a selectable marker on the cloning vector may be of use in keeping contaminating microorganisms from multiplying in the culture medium. In this embodiment, such a pure culture of the transformed host microorganisms would be obtained by culturing the microorganisms under conditions which require the induced phenotype for survival.
  • non-translated sequences include those which stabilize the mRNA or enhance its transcription and those that provide strong transcriptional termination signals which may stabilize the vector as they are identified by Gentz, R. , Langner, A., Chang, A.C.Y., Cohen, S.H., and Bujard, H. in Proc. Natl. Acad. Sci. USA. .78.:4936-4940 (1981).
  • the vector thus obtained is then transferred into the appropriate host microorganism. It is believed that any microorganism having the ability to take up exogenous DNA and express those genes and attendant operational elements may be chosen. It is preferred that the host microorganism be an anaerobe, facultative anaerobe or aerobe. Particular hosts which may be preferable for use in this method include yeasts and bacteria. Specific yeasts include those of the genus Saccharomvces, and especially Saccharomvces cerevisiae.
  • Specific bacteria include those of the genera Bacillus and Escherichia and Pseudomonas. Various other preferred hosts are set forth in Table I, supra. In other, alternatively preferred embodiments of the present invention. Bacillus subtilis, Escherichia coli or Pseudomonas aeruginosa are selected as the host microorganisms.
  • the vector is transferred into the host organism using methods generally known by those of ordinary skill in the art. Examples of such methods may be found in Advanced Bacterial Genetics by R. W. Davis et al. , Cold Spring Harbor Press, Cold Spring Harbor, New York, (1980). It is preferred, in certain embodiments, that the transformation occur at low temperatures, as temperature regulation is contemplated as a means of regulating gene expression through the use of operational elements as set forth above. In another embodiment, if osmolar regulators have been inserted into the vector, regulation of the salt concentrations during the transformation would be required to insure appropriate control of the synthetic genes.
  • the cloning vector first be transferred into Escherichia coli, where the vector would be allowed to replicate and from which the vector would be obtained and purified after amplification. The vector would then be transferred into the yeast for ultimate expression of the antigenic polypeptide.
  • the host microorganisms are cultured under conditions appropriate for the expression of the antigenic polypeptide. These conditions are generally specific for the host organism, and are readily determined by one of ordinary skill in the art, .n light of the published literature regarding the growth conditions for such organisms, for example Bergev's Manual of Determinative Bacteriology. 8th Ed., Williams & Wilkins Company, Baltimore, Maryland.
  • any conditions necessary for the regulation of the expression of the DNA sequence, dependent upon any operational elements inserted into or present in the vector, would be in effect at the transformation and culturing stages.
  • the cells are grown to a high density in the presence of appropriate regulatory conditions which inhibit the expression of the DNA sequence encoding for the antigenic polypeptide.
  • the environmental conditions are altered to those appropriate for expression of the DNA sequence. It is thus contemplated that the production of the antigenic polypeptide will occur in a time span subsequent to the growth of the host cells to near optimal density, and that the resultant antigenic polypeptide will be harvested at some time after the regulatory conditions necessary for its expression were induced.
  • the transcription terminators contemplated herein serve to stabilize the vector.
  • those sequences as described by Gentz et al. in Proc. Natl. Acad. Sci. , USA 78: 4936-4940 (1981), are contemplated for use in the present invention.
  • a clone containing the entire gene for Protein C (pUC18: :28C2) has been isolated and sequenced.
  • the insert from clone R69 was used as a DNA hybridization probe to isolate a plasmid clone called pUC18::28C2 was made by inserting M. hvopneumoniae DNA, partially digested with the restriction enzyme Sau3a, into the plasmid vector pUC18 cut with BamHl. (See Example VIII).
  • the entire gene and part of the surrounding DNA was sequenced (See Example VIII-B and Figure 2) .
  • the amino terminal sequence of the intact Protein C purified from Mj. hvopneumoniae cells, was determined to give a stating point (See Example VIII-C),
  • the amino acid sequence was determined for several peptide fragments of Protein C, which were generated by Endopeptidase digestion and purified by HPLC (See Example VIII-D). The sequences of these peptides are shown in Figure 3. These amino acid sequences were used to determine the proper reading frame of the DNA sequence.
  • the translated DNA sequence for the entire Protein C is shown in Figure 4, with the positions of the sequenced peptides underlined.
  • the amino terminal sequence of the intact Protein C was found to lie in the middle of an extensive open reading frame. It appears that the gene for Protein C actually codes for a larger protein (as much as 20 kd larger) which may be processed to the 85 kd size. Since mature Protein C (85kd) has been determined to be protective, the present inventors have not studied extensively the region which is thought to be processed away. The gene for Protein C (85kd) (excluding the processed portion) encodes a protein which is 624 amino
  • SUBSTITUTESHEET acids in length with a calculated molecular weight of 70.5 kd.
  • the discrepancy between the calculated and apparent molecular weights as determined by SDS-PAGE is not unusual.
  • the gene for Protein C contains three in-frame UGA codons (shown boxed in Figure 4) TGA in the DNA, which code tryptophan (See Example VIII-E) .
  • the use of UGA as a tryptophan codon is common among the mycoplasmas, but in other organisms (including E ⁇ ..coli.) UGA is used as a translation termination signal.
  • the UGA codons were changed to E ⁇ . coli UGG (tryptophan) codons (See the discussion below and Example IX).
  • the original clone for Protein C, R69, is contained completely within the coding sequence for Protein C, starting at nucleotide 455 and extending to nucleotide 976 in the sequence shown in Figure 4.
  • Expression clones were made that produce recombinant Protein C.
  • a recombinant plasmid (pT5T::M852) was constructed which expressed the entire Protein C (85kd) in E. coli.
  • Expression of the entire Protein C in an Ej_ coli expression vector required the replacement of the three inframe UGA codons with UGG codons, the normal tryptophan codon in E ⁇ . coli. Operationally, this was acheived by replacing the final "A” with a "G” in each of these codons. This was accomplished using a technique called "site directed in vitro mutagenesis" (Kunkel et al., Methods in Enzvmol.
  • SUBSTITUTESHEET incorporated and subsequent replication yields DNA molecules with both strands substituted. This technique was used to change all three of the UGA codons to UGG in the gene for Protein C (See Example IX-A) .
  • Example X-A2 Mycoplasma hvopneumoniae cells when subjected to low pH conditions.
  • 7S Proteins present in the SI extract which remain soluble when the pH is raised to 7.0.
  • Example X-A3 Can be denatured by adding SDS to 1.25% or urea to 6M.
  • PROTEIN C NATURAL 85 kd PROTEIN
  • FULL LENGTH RECOMBINANT PROTEIN C Described in Example IX and X-C.
  • TRUNCATED RECOMBINANT PROTEIN C Described in Section IX and X-D.
  • the j . hvopneumoniae genomic expression library was obtained in four consecutive steps. First, genomic DNA was obtained from M ⁇ . hvopneumoniae cells. Second, random fragments were generated by sonication, the ragged ends were blunted using phage T4 polymerase, internal EcoRI restriction sites were protected by EcoRI methylase, EcoRI linkers were added to the ends and excess linkers cleaved off. Third, the prepared fragments were ligated into the lambda gtll vector
  • M_j_ hvopneumoniae cells were thawed in ice and transferred to a polypropylene tube. The volume of cells was 0.8 ml. To this was added a ml of proteinase K solution: 0.075 M Tris pH8; 0.17 M EDTA pH8; 0.15% Triton X 100; and 400 ug/ml Proteinase K (BOEHRINGER MANNHEIM BIOCHEMICALS, Indianapolis, Indiana). The cells were incubated with the proteinase K solution at 65°C for 30 minutes. 2.0 ml of phenol was added to the mixture and the tube was gently rocked at 4°C for 20 min. The sample was then centrifuged in a BECKMAN JA20 rotor at 5000 rpm for 5 min at 4°C. The aqueous phase was removed with a large bore
  • the aqueous phase was then re-extracted with phenol followed by chloroform/isoamyl alcohol as described in the above paragraph.
  • the extracted aqueous phase was adjusted to 0.3 M sodium acetate by adding a 1/10 volume of a 3.0 M sodium acetate stock solution.
  • DNA was precipitated from this solution by adding 2.5 volumes of cold ethanol. Gentle mixing with the ethanol yielded a large, viscous mass of nucleic acid that was "spooled” out of solution using a small glass rod. This material was then rinsed twice with 70% ethanol and allowed to air-dry. The dry nucleic acids were resuspended in 0.5 ml of TE buffer by rocking the solution for 16 hr at 4°C.
  • the nucleic acids were treated with RNaseA.
  • Five-tenths milliliter of the sporozoite nucleic acid preparation was treated with 2.5 ug/ml of RNaseA (MILES LABORATORIES, Elkhart, Indiana) at 65°C for 30 min.
  • the aqueous phase was adjusted to 3.2 M ammonium acetate by adding a 0.75 volume of a 7.5 M stock solution.
  • the DNA was precipitated from this solution with the addition of 0.54 volumes of isopropanol. Mixing of the isopropanol yielded a large viscous pellet that was spooled from the solution and rinsed twice with 80% ethanol. After air drying, the DNA was resuspended in 0.2 ml of TE buffer.
  • Integrity of the Mj. hvopneumoniae DNA was analyzed by electrophoresis through an agarose gel and subsequent staining of the gel with ethidi ⁇ m bromide. These results indicated that the DNA was of high molecular weight and relatively free of RNA. The concentration of the DNA was determined by optical absorbance at 260 nm. This particular preparation yielded 90 ⁇ g of purified DNA.
  • M. hvopneumoniae DNA was sonicated with a small tipped probe of a Branson Sonifier cell disrupter 200 set at the lowest power setting. 80 ⁇ g of DNA in 200 ⁇ l total volume was sonicated in three 3-second bursts. This generated fragments ranging in size from 0.3 to 23 Kb in size with the highest proportion in the 1.3 to 4.4 Kb range.
  • T4-DNA polymerase (NEW ENGLAND BIOLABS; Beverly, Mass.).
  • the reaction was carried out in 33 mM Tris Acetate pH7.8; 66 mM Potassium Acetate, 10 mM Magnesium Acetate, 0.1 mg/ml Bovine Serum Albumin, 0.5 mM dithiothreitol, and 0.1 mM of each of the deoxynucleotide triphosphates, dATP, dGTP, dCTP, dTTP.
  • 20 units of T4 polymerase was reacted for 1 hour at 37°. The reaction was stopped by heating the sample for 10 min at 68°C. Excess salts were removed by passing the reaction mix over a BIOGEL P30 (BIO RAD) column equilibrated in TE.
  • Short oligonucleotide EcoRI linkers (NEW ENGLAND BIOLABS) were added to the blunt ended fragments in a ligation reaction consisting of 66 mM Tris pH7.6, 5 mM MgCl 2 , 5mM dithiothreitol, 10 mM ATP, with the linker at 5-10 uM.
  • T4 DNA ligase (P.L. BIOCHEMICALS) was added and the reaction proceeded at 14°C for 16 hours. The ligation reaction was terminated by heating the mixture at 70° for 10 min.
  • Phosphatased and EcoRI-cleaved lambda gtll DNA was mixed with the prepared fragments of M ⁇ hvopneumoniae DNA. These DNAs were ligated with T4 DNA ligase (P.L BIOCHEMICALS) overnight at 14°C. A small aliquot of the ligation reaction mixture was analyzed by gel electrophoresis to monitor the ligation reaction. The mixture was heated at 70°C for 5 min and then mixed with lambda in. vitro packaging extracts (VECTOR CLONING SYSTEMS, San Diego, CA) . The packaging reaction was allowed to proceed for 60 minutes at room temperature and then a drop of chloroform was added to prevent bacterial growth. Titration of this mix yielded a library with a complexity of 1.5 X 10 recombinants.
  • phage were diluted with lambda dil and adsorbed to 12.
  • coli strain Y1088 as described by Young, R. and Davis, R. in Science, 222:778-782 (1983). Amplification of the library on this strain ensures that the beta- galactosidase gene is not expressed; therefore, any phage containing coding sequences that might be deleterious to the host E_ s . coli cell are not expressed and not lost from the library. Amplifications of the library yield a stock that g was 6 X 10 phage per ml.
  • the lambda gtll:M. hvopneumoniae expression library was plated at densities of 5,000 through 20,000 phage per
  • the filters were then incubated individually or in pairs for 2 hr with 10-20 ml of primary antibody (e.g., immune swine serum, hyperimmune rabbit antimycoplasma serum, mouse monoclonal antimycoplasma antibodies) typically diluted 1:200 (1:500 for monoclonals) with TBS to which had been added 2% bovine serum albumin (BSA).
  • primary antibody e.g., immune swine serum, hyperimmune rabbit antimycoplasma serum, mouse monoclonal antimycoplasma antibodies
  • TBS bovine serum albumin
  • the filters were washed 3 x 10 min with TBS containing 0.1% NP-40 (SIGMA), then incubated singly or in pairs for 60 min with 10-20 ml solution of a second antibody (e.g., peroxidase-conjugated goat anti-rabbit IgG, CAPPEL LABORATORIES) diluted 1:500 in TBS + 2% bovine serum albumin.
  • a second antibody e.g., peroxidase-conjugated goat anti-rabbit IgG, CAPPEL LABORATORIES
  • Filters were batch-washed 3 x 10 min in TBS and stained in a solution comprising 200 ml TBS, 2.5 ml of a 3% hydrogen peroxide solution and 40 ml of a 3 mg/ml solution of 4- chloro-1-napthol in methanol. Staining was quenched by removing the filters to water. Positive staining plaques were subjected to several rounds of rescreening with antibody as described above
  • proteins When proteins are immobilized on nitrocellulose membranes such as Western blot transfers of proteins separated on polyacrylamide gels or replicas of phage plaques taken from agar plates, it is possible to bind antibodies which specifically recognize the immobilized proteins. Antibodies which do not specifically bind to the immobilized proteins remain in solution and can be easily washed away, leaving behind only those specifically bound.
  • the bound antibodies can be eluted by rinsing the filters in a low pH buffer (5 mM glycine, pH 2.3, 0.5 M NaCl, 0.5% Tween 20, 0.01% BSA) , which dissociates the antibody- antigen complex. If the eluted antibodies are immediately neutralized, i.e., using a 50 mM Tris HCl, final concentration, they retain full activity and can be used for a variety of analytical purposes.
  • a low pH buffer 5 mM glycine, pH 2.3, 0.5 M NaCl, 0.5% Tween 20, 0.01% BSA
  • Antibodies eluted from plaque replicas of a purified recombinant clone were used to determine which Mycoplasma protein corresponded to that clone. By eluting antibodies bound to plaque replicas of the recombinant clones and using those antibodies to probe Western blots of Mycoplasma proteins, it was possible to determine which protein is encoded in the recombinant clone.
  • Plasmid pLG2 was obtained from Dr. L. Guarente (MIT) (Guarente, L., in Cell, 20: 543-553 (1980).
  • This vector is a pBR322 derivative which, like lambda gtll, has lac operator and promoter sequences in addition to a wild-type beta- galactosidase gene containing a single EcoRI site near the 3' end of the gene.
  • pLG2 contains the lac repressor gene.
  • Moving _j. hvopneumoniae DNA inserts from lambda gtll into the EcoRI site of this vector yields an identical fusion protein to that initially identified in the phage.
  • Plasmid pLG2 was modified to remove an extra EcoRI site prior to its use for expression. Plasmid pLG2 was partially digested with EcoRI restriction endonuclease to linearize the plasmid. The plasmid DNA was then displayed on a preparative agarose gel and the linear-sized DNA band was eluted from the gel. The eluted DNA was precipitated by making the solution 0.3 M with sodium acetate and adding 2.5 volumes of ethanol. The DNA was pelleted by centrifugation and the pellet was resuspended in TE buffer. The Klenow fragment of E coli DNA Polymerase I was mixed with the DNA in the presence of dATP and dTTP to fill in the EcoRI cohesive ends.
  • T4 DNA ligase (P.L. BIOCHEMICALS) was added and the mixture was incubated at 4°C for 16 hr. The ligated DNA was then used to transform JS ⁇ coli AMA1004. Casadaban, M. , et al., in Methods in Enzvmology. 100:293 (1983).
  • Transformants were selected on ampicillin plates in the presence of a chromagenic substrate for beta- galactosidase activity (X-GAL) .
  • Transformants with beta- galactosidase activity were screened by cleaving the DNA with EcoRI.
  • a plasmid, pSEV4 that had only a single EcoRI site near the carboxy-terminus of the beta-galactosidase gene was identified from the transformants and characterized.
  • Plasmid pSEV4 has a unique EcoRI site near the carboxy- terminus of the beta-galactosidase gene.
  • Plasmid pSEV4 contains the wild-type lac operator, promoter and repressor in addition to the beta-galactosidase gene.
  • a successor to pSEV4 was constructed to allow polarized "cassette" subcloning of DNA inserts from lambda gtll directly into a plasmid expression vector. Because EcoRI inserts could be subcloned in either orientation of pSEV4, each pSEV4 subclone must be screened for its antigenic reaction. Polarized subcloning using pSEV6 obviates the need for this extra analysis.
  • Extensive mapping of pSEV4 located five restriction endonuclease sites in the lac operon 5' to the beta- galactosidase gene's unique EcoRI site. Three of these sites are unique and two were made unique by deletion of the superfluous DNA between the lad gene and the pBR322-derived amp gene. Only one useful restriction site was found 3' to the EcoRI site, the Ncol site, therefore, additional restriction enzyme sites were inserted in this region using a chemically synthesized polylinker. The construction of pSEV6 was done in two steps. First, pSEV4 was shortened by approximately 5,700 bp to eliminate superfluous DNA.
  • Plasmid pSEV4 was cleaved with SphI restriction endonuclease, and the enzyme was inactivated by heating at 70°C for 10 min. The DNA was then partially digested with Aat.II and the resulting digest was displayed by electrophoresis on a preparative agarose gel. The 7,620 bp fragment was excised from the gel and electroeluted.
  • the electroeluted DNA was precipitated from a 0.3 M sodium acetate solution by adding 2.5 volumes of ethanol and incubating at -70°C for 30 min.
  • the DNA was concentrated by pelleting in a Brinkman microcentrifuge for 15 minutes and the pellet was resuspended in TE buffer.
  • T4 DNA polymerase (NEW ENGLAND BIOLABS) was added to blunt-end the cohesive ends generated by the Aatll digest. After heat inactivation of the T4 DNA polymerase, T4 DNA ligase (P.L. BIOCHEMICALS) was added to ligate the blunt ends of the DNA fragment.
  • the ligated DNA was used to transform competent AMA1004 E_;_ ⁇ olj cells. Lac+ transformants were screened for the 7,620 bp plasmid.
  • One plasmid, pSEV5 was identified and characterized as having the appropriate structure.
  • DNA from pSEV5 was purified by standard methods, and subsequently cleaved with the restriction endonuclease Ncol, which cleaved at a unique site 3' of the beta-galactosidase gene.
  • An oligonucleotide adapter molecule would regenerate the Ncol site and which also contained Bgl.II and Kpn I sites was chemically synthesized. The sequence of this adapter molecule is as follows:
  • beta-galactosidase :M. hvopneumoniae antigen fusion proteins have been purified either by use of a substrate analog affinity column for beta-galactosidase or by classical methods of protein purification.
  • the cells were resuspended in 20 mL of breaking buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM MgCl 2 , 5% glycerol, 1 mM phenymethyl sulfonyl fluoride (PMSF)) at 4°C and centrifuged again at 5000 x G for 15 min.
  • the cells were again suspended in 20-40 mL of breaking buffer.
  • the cells could be frozen at this point and stored at -20°C if desired.
  • the unfrozen or thawed cells were broken with two passes through a French pressure cell (AMINCO) at 20,000 psi. Cell debris was removed by centrifugation at 30,000 x G for 30 min.
  • breaking buffer 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM MgCl 2 , 5% glycerol, 1 mM phenymethyl sulfon
  • the precipitate solution was stirred for one hour and the precipitate was removed by centrifugation at 30,000 x G for 15 min.
  • the pellet was redissolved in 10 to 15 ml of starting buffer (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol (DTT) and 0.1% Triton X-100), and then dialyzed overnight against 500 mL of starting buffer.
  • starting buffer 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol (DTT) and 0.1% Triton X-100
  • Affinity resin p- aminophenyl-beta-D-Thiogalactopyranoside-agarose, obtained from SIGMA
  • SIGMA SIGMA
  • the column was washed with 10 column volumes of starting buffer of 50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10 mM MgCl 2 , and 1.0 mM dithiothreitol (DTT) and 0.1 Triton -X100 before use.
  • the column can be regenerated after use by washing extensively with elution buffer 0.1 sodium borate, pH 10.0, 250 mM NaCl, 1 mM DTT or by washing with 6M guanidine hydrochloride in 50 mM Tris-HCl, pH 7.5. After washing, the column is reequilibrated with 10 column volumes of starting buffer. For affinity chromatography, dialyzed material was applied to the pre-equilibrated affinity column at a flow rate of about 0.2 ml/min.
  • the column was washed with 15 ml of starting buffer at the same flow rate, then with 30 ml of starting buffer at about 0.5 ml/min followed with 180 ml starting buffer at about 1 ml/min. Finally, the column was washed with 120 ml of starting buffer without TRITON at the same flow rate.
  • the absorbed protein was eluted with 0.1 sodium borate, pH 10.0, 250 mM NaCl, 1 mM DTT using 120 ml at a flow rate of about 1 ml/min.
  • the peak-protein containing fractions were pooled and could be concentrated if desired to about 10 ml using an AMICON ultrafiltration device (Model 8050) containing an YM-30 membrane.
  • pSEV4 :CH2-13
  • pSEV4 :CH2-13
  • the dialyzed material is subjected to ultracentrifugation at 100,000 x G for 30 min.
  • the pellet containing the bulk of the fusion protein was redissolved in a small volume of dialysis buffer 50 mM Tris-HCl, ph 7.5, 250 mM NaCl, 10 mM MgC12 and 1.0 mM dithiothreitol (DTT) and 0.1 M TRITON -X 100.
  • This method yields material that is not as pure as that generated by the affinity column when judged by SDS-polyacrylamide electrophoresis.
  • the purified materials obtained by these methods were analyzed for protein by the BIO-RAD (Richmond, CA) protein method as recommended by the manufacturer. It was also subjected to analysis by SDS-polyacrylamide electrophoresis. These gels are visualized either by protein staining or by Western blot analysis. Protein staining was typically done with either the silver stain method as described by Wray, W.P. et al. in Anal. Biochem.. 118:197-203 (1981) or the BIO- RAD protein stain. Western blot analysis is carried out as described by Remart, J. et al. in PNAS (USAU 16.:3116 (1979).
  • the Western blot analysis involves electrophoretic transfer of the resolved protein bands to nitrocellulose, blocking the nitrocellulose paper with BSA, probing with a specific antibody (either anti-betagalactosidase or anti ⁇ mycoplasma sera). After washing, the blots are probed with the appropriate peroxidase conjugated second antibody, followed by color development using the peroxidase catalyzed reaction.
  • the recombinant M ⁇ . hvopneumoniae clones that were picked as reactive with various antimycoplasma sera contain only a portion of the entire coding region for that particular polypeptide, because of the requirement that the insert sequence be in frame with the beta-galactosidase gene in lambda gtll and the limited number of clones screened. In some cases it may be important to have cloned the entire coding sequence of a given antigenic M. hvopneumoniae polypeptide in order to maximize the immune response or modulate the response.
  • a method is outlined below that will allow the isolation of clones containing such full length fragments.
  • the lambda gtll expression library described above may also be viewed as a simple genomic library if one does not require that the inserted segments of M ⁇ hvopneumoniae be expressed as fusion proteins. Some of these clones should contain the entire coding region for a particular protein even though it is not in frame with the beta galactosidase expression system.
  • clones may be detected using DNA hybridization probes derived from the clones already picked by antibody methods and known to correspond to particular mycoplasma proteins.
  • the insert fragments from these clones is "nick translated" using E ⁇ . coli DNA polymerase to incorporate 32p- labeled deoxynucleotides into the DNA.
  • This labeled DNA is then used as a radioactive probe to select an homologous M. hvopneumoniae sequence from the recombinant lambda gtll library.
  • Phages selected from the recombinant library by this method are plaque purified, DNA is prepared, and the M. hvopneumoniae specific insert is mapped with various restriction endonucleases. The resulting map is compared with a similar map derived from the initial clone to confirm the identity of the new genomic phage.
  • Proteins which are exposed on the surface of a mycoplasma cell have been shown to be susceptible to digestion by a protease such as trypsin when whole, intact cells are lightly treated with that enzyme (Klinkert, M., Herrmann, R., and Schaller, H. , Infection and Immunity, 49; 329-335 (1985)).
  • This technique in combination with the elution of antibodies from clones, allows the rapid determination of whether a particular clone corresponds to a trypsin sensitive surface protein.
  • Total proteins from trypsinized and non-trypsinized mycoplasma cells are placed in adjacent lanes and separated by SDS polyacrylamide slab gel electrophoresis.
  • the displayed proteins are then electroblotted onto nitrocellulose membrane by the western blot procedure.
  • This blot is then probed using antibodies from a polyclonal serum which have been affinity purified from a specific clone using the antibody elution technique described above in Example II-B.
  • the specific mycoplasma protein on the Western blot corresponding to the clone will be revealed by staining of the bound antibody in the lane with proteins from non- trypsinized cells, showing up as a specific stained band. If that protein is trypsin sensitive, the corresponding position in the lane with proteins from trypsinized cells will be blank, whereas a non-trypsin sensitive band will stain as in the untreated cells.
  • the present inventors have shown trypsin sensitivity for Proteins A, B. C, D and E, indicating that they are surface proteins.
  • the recombinant DNA lambda gtll clone R69 encodes part of protein C.
  • the M ⁇ . hvopneumoniae DNA insert in this clone was sequenced.
  • the insert in this clone was excised with EcoRI and subcloned into the single stranded sequencing vector M13mpl8 (See Example VIII-B for references to this vector and sequencing procedures) .
  • Subclones with the insert in each orientation were isolated and the sequence determined in both directions.
  • the 5'—>3' orientation of the R69 insert and the proper reading frame were determined by sequencing directly from the double stranded lambda gtll-R69 clone (Chen and Seeburg, DNA, 4.:165(1985) ) using lambda gtll forward and reverse primers (NEW ENGLAND BIOLABS).
  • the DNA insert in this clone is 522 nucleotides long and encodes 173 amino acids.
  • the DNA sequence and translated amino acid sequence which comprises part of protein C are depicted in Figure 1. This DNA sequence is contained entirely within the coding sequence of Protein C, starting at nucleotide 455 and extending to nucleotide 976 in the sequence of the whole gene shown in Figure 4.
  • the pUC18 Mi. hvopneumoniae genomic library was constructed to provide a recombinant library with larger insert fragments than the lambda gtll library (Example I), so that the entire Protein C gene would be contained on a single clone.
  • Genomic Mycoplasma hvopneumoniae DNA was partially digested with the restriction endonuclease Sau3a and size fractionated on a 35 ml 10->40% Sucrose gradient in 1M NaCl, 0.02M Tris pH 8. 0.005 M EDTA.
  • the digested DNA sample was layered on top of the gradient and centrifuged at 100,000 x g for 24 hours.
  • One ml fractions were collected from the bottom of the gradient and samples from selected fractions were electrophoresed on a 0.5% agarose gel in TAE buffer (O.04 M Tris Acetate, 0.001M EDTA) alongside DNA size standards and stained with Ethidium Bromide to determine the size range of DNA fragments in each fraction.
  • Fractions enriched for DNA fragments in the 8-12 kilo basepair range were pooled, concentrated and dialyzed against TE8 buffer (0.01M Tris, 0.001M EDTA pH 8).
  • the vector, pUC18 was prepared by complete digestion with the restriction endonuclease BamHl, followed by dephosphorylation with the enzyme Calf Intestinal Phosphatase (0.01 Units of BOEHRINGER-MANNHEIM Calf Intestinal Phosphatase per pmol of vector, 2 X 30 min at 37°C, in CIP buffer ImM ZnCl 2 , ImM MgCl 2 , lOmM Tris Cl pH 8.3). This treatment was followed by phenol extraction, ethanol precipitation and resuspension in TE8 buffer.
  • Each array was then transferred to several nitrocellulose discs on Luria agar Ampicillin plates using a 48 tine prong apparatus and grown overnight at 37°C.
  • the nitrocellulose discs with the arrays of colonies growing on them were processed for probing by DNA hybridization. Colonies were lysed and DNA denatured by floating the discs on puddles of 0.5 M NaOH, 1.5 M NaCl for
  • the discs were blotted between sheets of Whatman 3MM paper, then baked at 80°C for 2 hours. Discs were then stored at room temperature until probed.
  • a 32 P labelled hybridization probe was made from the M_j_ hvopneumoniae DNA insert in the R69 clone.
  • the source of this insert was the clone pSEV6::R69, which was constructed by "cassette" subcloning of the insert in ⁇ gtll- R69.into pSEV6, as described in Example II-B.
  • the 0.6 kb insert fragment was generated by digesting the pSEV6::R69 plasmid DNA with the restriction endonuclease EcoRI, releasing the insert, followed by separation by agarose gel electrophoresis (0.7% agarose, TAE buffer, with gel and buffer containing 0.5 ⁇ g/ml Ethidium Bromide, 30V, 3h) .
  • the insert band was visualized under long wave UV light, a slit was made with a scalpel blade just below the desired band and a small piece of Whatman NA50 paper was inserted into the slit below the band.
  • the insert fragment migrated onto and bound to the NA50 paper, which was removed and washed in NET buffer (20mM Tris pH 8, O.lmM EDTA) .
  • the fragment was eluted from the paper in NET buffer containing l.OM NaCl, extracted with butanol, precipitated in ethanol, and finally resuspended in TE buffer.
  • the purified fragment was labelled by nick translation with 32 P- ⁇ dC using a BIORAD nick translation kit, following the instructions for that kit.
  • the labelled fragment was separated from the unincorporated nucleotides by gel filtration using BIORAD P30 resin in TE buffer.
  • the hybridization mix contained 50% formamide, 5X SSPE, 1% SDS, 0.1% Sodium Pyrophosphate, 0.15 mg/ml tRNA, 0.0125 mg/ml sonicated salmon sperm DNA. To this was added 400,000 dpm/ml of the labeled probe, after it was denatured by incubation in a boiling water bath for 5 minutes, then chilled. All 36 of the gridded arrays were probed with this mix. Hybridization was carried out in a shaking incubator at 42°C for 16 hours.
  • the filter discs were then washed 4 X 15 min in 0.1X SSPE, 1% SDS at 65°C, blotted dry and autoradiographed on KODAK XAR-5 X-ray film at -70°C using a DUPONT CRONEX LIGHTNING PLUS INTENSIFYING SCREEN. The positions with positive dark signals were marked on the film and the corresponding clones picked from the master plates, streaked out for single colonies, and three such colonies for each positive clone were retested by the same filter hybridization process described above.
  • Hindlll/PstI etc. were subcloned into the M13mpl8 or M13mpl9 phage vectors (Messing et al.. Gene. .26.:101-106, 1983) and sequenced using the enzyme sequenase, specifically engineered for chain- termination DNA sequencing by the procedure of Tabor and Richardson (Tabor et al.. PNAS 84:4767-4771, (1987)). DNA sequenase sequencing kits were purchased from United States Biochemical Corp. and the reactions were performed according to the manufacturer's instructions. Reactions were analyzed on 6% acrylamide buffer gradient gels (Biggin et al. , PNAS, 8O:3963-3965, (1983)).
  • DNA sequences were analyzed using the Pustell and Kafatos (Pustell et al. , Nucleic Acid Res. , .12.:643-655, (1984)) algorithms obtained as software provided by INTERNATIONAL BIOTECHNOLOGIES INC. (New Haven, Conn.). DNA sequence for the coding region of the gene for Protein C was obtained for both strands of the DNA. Primers used for the DNA sequencing reactions were either the Ml3 universal primer or synthetic oligonucleotides designed from sequences obtained using the APPLIED BIOSYSTEM DNA Sequencer.
  • the gel purified Protein C in NH 4 HC0 3 buffer (See Example X-B below) was applied directly to a glass fiber filter and dried under a stream of argon gas to remove the NH4HCO3.
  • the sequencer with this sample was precycled and then sequenced.
  • the amino-terminal sequence obtained was QQQEANSTNSSP, which matched the translated DNA sequence beginning at nucleotide 1801 of the pUC18::28C2 insert, defining the 5' end of the gene.
  • Protein C was obtained using a combination of ion exchange column purification followed by preparative gel purification by SDS-PAGE.
  • the Protein C was partially purified using a Pharmacia Mono Q ion exchange column using the CHAPS method described in Example X-B2.
  • the column fractions enriched for the Protein C were pooled.
  • Preparative gel purification was performed essentially as described in Example X-B4, except that the sample was reduced with 2.5% 2-mercaptoethanol before loading onto the gel.
  • the protein used for endoprotease digestions was eluted from the gel slices in 0.5 X running buffer (0.0125 M Tris, 0.096 M glycine, 0.05% SDS), as described in Example X-B4.
  • the gel slices were soaked in 0.05 M NH4HCO3, 0.1% SDS, then placed in ISCO sample concentrator cups and eluted in the same buffer at 50 V for 5 hours. The concentrated sample was then diluted 50 fold in 0.01 M NH4HCO3, 0.05% SDS and then recon ⁇ entrated in ISCO sample concentrator cups in the same buffer (50 V, 5 hours). 3. GENERATION AND PURIFICATION OF PEPTIDE FRAGMENTS
  • the protein was digested with 2% (w/w) endoproteinase Lys-C (BOEHRINGER-MANNHEIM) in 0.1 M Tris, pH 8.6, at 37°C for 15 h.
  • the protein was also digested with 2% (w/w) Chymotrypsin (BOEHRINGER-MANNHEIM) in 0.25 M Ammonium Bicarbonate buffer, pH 7.8, for 4 h at 37°C.
  • the peptides generated were purified by reverse phase HPLC on a 5 ⁇ m Vydac C4 (4.5 x 250 mm) column (The Separations Group) in 0.1% TFA in water with a 0—>50% acetonitrile gradient (0.5% per minute). Isolated peptides were applied to a glass fiber filter and sequenced.
  • the Protein C peptide YLKQNEWD contains a tryptophan
  • TGA UAA in the RNA
  • hvopneumoniae as has been shown in other mycoplasma species.
  • Oligonucleotide-directed site-specific in. vitro mutagenesis was carried out to change the UGA to UGG codons. Mutagenesis was carried out using the MUTA-GENE IN VITRO MUTAGENESIS KIT from BIORAD based on a method described by Kunkel (Kunkel et al. , Methods Enzvmol. , 154:367-382 (1987)).
  • the host cell used for genetic selection for mutagenesis is the E_i_ coli strain CJ236 (Genotype:dut, ung, thi, rel Al, pCJ105[cap r ]) which provides a very strong selection against the non-mutagenized strand of a double-stranded DNA due to the dut (dUTPase) and ung (uracil N-glycosylase) mutations.
  • Template DNA, substituted with uracil and used for mutagenesis is selectively destroyed when transformed into host cells that contain wild-type ung loci, such as JM103 in this case, thus allowing preferential replication of the newly synthesized mutated DNA.
  • Mutagenesis of the three UGA codons was carried out in two parts.
  • the first part involved the simultaneous changing of the A to G in the first two UGA codons (nucleotide positions 231 and 1068) contained in the DNA fragment from the beginning of the gene for Protein C to the Hindlll recognition site at nucleotide #1294.
  • the second part involved the changing of nucleotide A to G in the third UGA codon at nucleotide position 1413.
  • clone pSEV6: :R68SKH was used.
  • pSEV6: :R68SKH was obtained by joining the ⁇ 2.1 kb Hpal fragment from the genomic clone pUC18::28C2 (see Figure 9) to the Hpal site in pSEV6::R68.
  • the expression clone pSEV6::R68 is a ⁇ - galactosidase fusion clone which encodes part of Protein C. It was derived from the ⁇ gtll clone, R68, by "cassette" subcloning into the plasmid pSEV6 (See Example III-B) .
  • the insert in R68 begins before the 5' end of the Protein C coding sequence (as defined by the amino-terminus of the protein), but is in frame with the protein coding sequence, making a fusion protein which is partially Protein C and partially from the region upstream of the Protein C coding sequence.
  • the correctly mutagenized SstI/ HindiII fragment was subcloned back into the original vector, pSEV6:R68SKH, and the mutagenized clone was designated as pSEV6: :R68SKHM.
  • the mutagenesis was also confirmed by increase in size of the ⁇ -galactosidase fusion protein, expected for replacing the E ⁇ . coli termination codons (UGA) with tryptophan (UGG) codons.
  • HindiII fragment from clone 2Ha was ligated into HindiII cut and dephosphorylated pSEV6: :R68SKHM.
  • the resulting plasmid, designated pSEV6: :M852a-l, contains the entire gene for
  • the Mycoplasma codons were changed to E_ ⁇ coli-preferred codons, the first two Spel sites were eliminated, and an EcoRI and a Nhel restriction sites were engineered in, all without changing the original amino acid sequence of the Protein C.
  • the four oligonucleotides used were:
  • Oligonucleotides NA and NB are complementary to each other and oligonucleotides NC and ND are complementary to each other.
  • the four oligonucleotides were kinased, NA and NB were annealed to each other and NC and ND were annealed to each other.
  • pSEV6::M851 was digested with Spel and the digested DNA was ligated to annealed oligos NC and ND. This DNA was then ligated to annealed NA and NB oligos to complete the reconstruction of the 5'-end of the gene.
  • the ligated DNA mix was digested with BamHl and HindiII to obtain the DNA fragment containing the 5'-end of the gene to the HindiII recognition sequence at nucleotide position 1294. This DNA fragment was used for expression of the truncated form of
  • Protein C described below. The reconstructed region of 5'- end is shown in Figure 5.
  • the T7 promoter based expression vector pT5T is essentially the same as pJU1003 (Squires, et al. , J. Biol. Chem.. 263:16297-16302 (1988)), except that there is a short stretch of DNA between the unique Belli site 5' to the T7 promoter and the Clal site in the tetracycline resistance gene.
  • the sequence of this DNA is:
  • the vector pT5T was linearized with BamHl and HindiII restriction enzymes and gel-purified.
  • the ⁇ 2kb HindiII gel-purified fragment (excised from clone 2Ha, see Example IX-A), containing the third mutagenized UGA codon and the C-terminus of the gene, was ligated to pT5T: :M851 (digested with HindiII restriction enzyme and treated with alkaline phosphatase to dephosphorylate the vector to minimize self- ligation of the vector). Because the HindiII fragment could ligate to the digested vector in either orientation, transformants were restriction site mapped to ascertain the correct orientation of the HindiII fragment. This resulted in a construct designated pT5T::M852, which is illustrated in
  • PROTEIN C BOTH TRUNCATED PROTEIN C AND THE FULL-LENGTH PROTEIN C
  • pT5T: :M851-B2B The clone found to be expressing an IPTG-inducible protein migrated at a molecular weight of ⁇ 50kd (Truncated Protein C) by pT5T: :M851 is designated pT5T: :M851-B2B, and the clone found to be expressing an IPTG-inducible protein migrating at a molecular weight of ⁇ 85kd (full length Protein C) by pT5T::M852 is designated pT5T: :M852-1.
  • pT5T::M852-l produces a recombinant protein which co-migrates on SDS polyacrylamide gels with natural 85kd protein isolated from mycoplasma. Both the short 85kd and the full- length 85kd recombinant proteins are immuno-reactive with sera from pigs immunized with gel-purified natural 85kd protein on Western blots (
  • DNA sequencing of pT5T::M851 and pT5T: :M852 confirmed that the sequences of the recombinants were correct.
  • Amino terminal amino acid sequencing of the intact recombinant full length Protein C yielded the sequence MQQQEANSTNSSPT confirming the correct initial sequence with a methionine added to the amino terminus (See Figure 4).
  • a sample of the insoluble pellet following French Press disruption of the cells See Example X-C. was solubilized in sample buffer and electrophoresed on a polyacrylamide gel using the MZE system 3328.IV, described by Moos et al. (J. Biol. Chem.. 263:6005-6008 (1988)).
  • SI extract contains a variety of proteins depicted in the photograph of a SDS-polyacrylamide gel in Figure 8. The major protein components are 87 kd, 85 kd (Protein C), 65 kd, 50 kd, as well as several other minor proteins.
  • the SI extract was concentrated and desalted using an AMICON ultrafiltration cell
  • ULTRAFILTRATION membrane operated at 40 psi at 4°C.
  • Initial concentration of the extract to 1/100 of the starting volume took several hours and left a thick protein coating on the filtration membrane.
  • 1/2 the starting volume of 2 mM glycine pH 2.5 was added to the vessel and stirred, the protein coating released from the membrane in sheets which dissolved in the 2 mM glycine.
  • the second concentration step to 1/100 of the starting volume proceeded rapidly at 40 psi, usually taking ⁇ 1 hour.
  • This concentrate was removed from the vessel and the membrane washed 2X with 2-3 ml of 2 mM glycine pH 2.5 and the washes were pooled with the concentrate resulting in a desalted concentrate that was about 1/50 the original volume.
  • This concentrated SI extract, called Sic was indistinguishable from the SI extract in its protein composition, as judged by the protein bands on Coomassie blue stained SDS polyacrylamide gels.
  • the redissolved pellet contained the remaining 60% of the protein and was enriched for the 87 kd protein with half of the 50 kd protein and several minor proteins. Neither fraction was completely free of proteins contained in the other fraction. The proteins in each fraction are depicted in the Coomassie stained gel in Figure 8.
  • the natural Protein C was purified by ion exchange chromatography using two different methods: 1) the CHAPS method utilized a buffer containing the zwitterionic detergent CHAPS and the 7S extract as the starting material; and 2) the Urea method utilized a buffer containing 6M Urea and the Sic extract as the starting material. Purification using either of these agents was adequate, though the urea containing buffer gave somewhat better resolution in elution profiles. Without any detergent or denaturant, resolution of elution peaks was extremely poor.
  • the starting material for purification of the natural Protein C was the extract known as 7S— the pH 7 supernatant derived from the concentrated low pH extract — SI (see descriptions of extracts, Example X-A.
  • the 7S is enriched for the 85 kd protein (Protein C) as well as a 65kd protein and a 50 kd protein and also contains other minor contaminants.
  • Ion exchange chromatography was performed using a PHARMACIA Mono Q HR5/5 column ( 1 ml bed volume) connected to a PHARMACIA FPLC system.
  • the CHAPS buffer consisted of 0.05% CHAPS (3-[ (3-Cholamidopropyl)dimethylammonio]-1- propanesulfonate), 0.02 M BIS-TRIS (bis[2- Hydroxyethyl]iminotris-[hydroxymethyl]methane) , pH 6.0.
  • a typical sample consisted of 34 ml 7S extract, 3.05 ml H20, 0.76 ml 1M BIS-TRIS pH 6.0, 0.19 ml 10% CHAPS, for a total of 38 ml with 0.02 M BIS-TRIS pH 6, 0.05% CHAPS.
  • This sample was filtered through a GELMAN ACRODISC disposable filter assembly with a Q.2 ⁇ pore size. The sample was loaded onto the column at 1 ml/min, the column was washed with 5-10 column volumes of CHAPS buffer, then eluted with a linear gradient of NaCl in the same buffer. The gradient ran from 0 to 0.25 m NaCl over a volume of 40 ml.
  • the 85 kd protein eluted at 0.08 -0.10 M NaCl with some trailing of the peak into the higher [NaCl] fractions.
  • Samples from selected fractions were analyzed by SDS-PAGE and the gels were silver stained to reveal those fractions containing the 85 kd protein.
  • the fractions which were enriched for the 85 kd protein were pooled. These pools contained >60% 85 kd protein and the rest a variety of contaminating proteins, as estimated visually by Coomassie stained gels. Protein concentrations were determined using a BIORAD Protein Assay kit.
  • the urea buffer consisted of 6M urea (BRL, Ultrapure, Enzyme grade) in 0.02 M BIS-TRIS pH 6.0.
  • the starting material was the concentrated low pH extract — the Sic.
  • a typical sample contained 4ml Sic, 3.8 ml H 2 0, in which was dissolved 3.85 g Urea. After the Urea was dissolved, 0.2 ml 1M BIS-TRIS pH 6.0 was added, yielding ⁇ 10 ml of solution containing 6M Urea, 0.02 M BIS-TRIS pH 6.0. This sample was filtered through a GELMAN ACRODISC disposable filter assembly with a 0.2 ⁇ m pore size.
  • the sample was loaded onto the Pharmacia Mono Q HR 5/5 column at 1 ml/min, the column was washed with 5-10 column volumes of Urea buffer, then eluted with a linear gradient of NaCl in the same buffer. The gradient ran from 0 to 0.25 M NaCl over a volume of 40 ml.
  • the 85kd protein eluted at 0.04 -0.08 M NaCl in fairly sharp peaks without much trailing into higher fractions.
  • Samples from selected fractions were analyzed by SDS-PAGE and the gels were silver stained to reveal those fractions containing the 85 kd protein.
  • the fractions which were enriched for the 85 kd protein were pooled. These pools contained 50% 85 kd protein with a major contaminant at ⁇ 50 kd and a variety of minor contaminating proteins, as estimated visually by Coomassie stained gels. Protein concentrations were determined using a BIORAD Protein Assay Kit.
  • the Running buffer contained 0.025 M Tris base, 0.192 M glycine, 0.1% SDS, pH 8.3.
  • the sample buffer contained 10% w/v glycerol, 3% SDS, 0.0625 M Tris-HCl pH 6.8. No reducing agent or dye was used for these samples.
  • the sample (pooled column fractions enriched for the 85kd protein) was mixed with an equal volume of sample buffer and heated in a boiling water bath for 10 min, then cooled to room temperature. Five ml of this mix was loaded into the gel apparatus and electrophoresed at 50 mA until the ion front reached the bottom of the gel ( ⁇ 2.5 hours).
  • the back of the nitrocellulose sheet was marked with a black VWR lab marker to indicate the position of the edges of the gel, so that the gel could be aligned with the blot after staining.
  • the gel was then aligned with the marks on the nitrocellulose and the area over the darkest and thickest staining band was excised from the gel using a razor blade.
  • the slice was crushed by forcing it through a disposable plastic syringe with no needle attached into a tube containing 0.5X Running Buffer (see above). The crushed gel slices were stored in this buffer at 4°C.
  • Elution of the protein from the gel slices was accomplished using ISCO sample concentrator cups.
  • the crushed gel slices were placed in the cups in 0.5X Running Buffer and electroeluted at 1 Watt per cup for 3-4 hours.
  • the concentrated sample was removed using a pipetman and pooled with samples from other cups.
  • the concentration of protein in these samples was estimated by running different amounts of the sample along side different amounts of a known standard on SDS-PAGE and comparing the intensity of Coomassie Blue staining of the protein bands.
  • the concentrated samples were pooled, diluted to a concentration of 1.0 mg/ml with 0.5 X Running Buffer, and stored at 4°C until used for vaccine testing.
  • Strain pT5T::M852-l (See EXAMPLE IX-C) was grown in shaker flasks (350 ml in 21 baffled flasks, 250-350 rpm, 37°C).
  • Strain pT5T: :M851-B2B (See EXAMPLE IX-C) was grown in shaker flasks (350 ml in 21 baffled flasks, 250-350 rpm, 37°C).
  • the supernatant was mixed 1:1 with gel sample buffer and purified by preparative polyacrylamide gel electrophoresis as described for the natural Protein C purified from Mi. hvopneumoniae cells (See GEL PURIFICATION; Example X- B4) .
  • Healthy, 6-week-old conventional pigs were purchased and assigned to pens of 5-6 pigs each. Pigs in 3 pens (a total of 15-18 pigs per group) were vaccinated by intramuscular injection with 3.0 ml/pig of experimental vaccine or a placebo, in a water-in-oil emulsion adjuvant. Pigs received 4 vaccinations (50,100,200, and finally 400 micrograms total protein per injection) at 7 day intervals.
  • Mycoplasma hvopneumoniae infected donor pigs were placed in each pen to provide a natural source of infection for the vaccinates.
  • the donor pigs were artificially infected with Mi. hvponeumoniae by intranasal infusion of a homogenate made from infected lung tissue, which results in a serious infection.
  • This infection was started at the same time that the vaccinate pigs received their first injection, so that a serious infection was present at the time of mixing.
  • the vaccinates and the donors were kept separated until mixing. Ventilation in the building was reduced to facilitate transmission of the disease. Blood samples were collected from each pig, one prior to the first injection, and one after the last injection but before mixing with the donor pigs.
  • each pig was killed and the lungs were examined for severity of the disease.
  • the primary criterion was the percent of gross pneumonic lesions in the lungs as estimated by visual inspection.
  • Lung tissue was also collected for an Indirect Fluorescent Antibody (IFA) test to determine whether the gross lesions contained Mycoplasma hvopneumoniae infections (See Example X-G) . These samples were also cultured for aerobic bacteria other than mycoplasma. Animals which had visible gross lesions and tested positive in the IFA test were considered to be infected when determining the incidence of the disease in the different groups.
  • IFA Indirect Fluorescent Antibody
  • the average lesion score (mean % lesions) was also used as a measure of the effectiveness of a vaccine. For the purpose of averaging, those animals which were scored as having >0% but ⁇ 1% lesions were given a lesion score of 0.5%.
  • the pneumonic lesion data were statistically analyzed using the square root transformation and the means shown in the Table 2 were transformed to the original scale.
  • the pneumonic lesion data were statistically analyzed using the square root transformation and the means shown in the Table 2 were transformed to the original scale.
  • INCIDENCE A ratio of the number of pigs in a group which have gross pneumonic lesions and score positive on the IFA test to the total number of pigs in the group. This ratio indicates the degree to which the transmission of the disease is reduced by the vaccination.
  • MEAN % The average lesion score of all the pigs in a particular group. This number allows comparison of the severity of the disease between different groups.
  • IFA POSITIVE The IFA (Indirect Fluorescent
  • Antibody test detects the presence of M ⁇ hvopneumoniae cells in the lung tissue, particularly in the gross lesions. This indicates whether the lesions seen are in fact caused by jML. hvopneumoniae or some other agent (other mycoplasma species or bacteria). It is expressed as a ratio of the number of pigs in a group scoring positive in the IFA test to the total number of pigs in the group. Detailed description of this test in Example X-G. General conclusions based of the results obtained are listed below:
  • SI vaccinates showed significant reduction in incidence, mean percent lesion scores, and numbers of pigs which were IFA positive, when compared to nonvaccinated controls.
  • Results were combined between experiments 8802 (in which protein C was purified by ion exchange chromatography) and 8902 (in which Protein C was further purified by preparative polyacrylamide gel electrophoresis) . There was no statistically significant difference between the results of these two tests, so they were combined. Significant reductions in incidence, means % lesions, and IFA positive scores are exhibited compared to placebo vaccinated controls.
  • tissue samples were taken from lesions in infected lungs or from the tips of the lobes of the lungs in animals with no detectable gross lesions. This is the region of the lung where infection is most likely to occur. Frozen sections of these tissues were made, mounted and acetone fixed on glass slides. A rabbit anti-M. hvopneumonia serum was bound to the sections, washed, and followed by a Fluorescein-linked goat anti-rabbit IgG. Unbound antibodies were washed off and the sections were observed under a fluorescence microscope. Sections which displayed bright fluorescent rings around the alveoli were scored as positive. Control sera showed only background fluorescence.
  • Serum from pigs vaccinated with placebo showed a slight inhibition in metabolism compared to the normal medium controls.
  • serum from pigs vaccinated with natural Protein C, recombinant full length Protein C, or truncated Protein C all strongly inhibited metabolism of M. hvopneumoniae, as indicated by delay or lack of color change in these tubes.
  • Measurement of pH at the time when the normal media control had changed color (3 days for 100 x dilution of inoculum) showed that acid production was substantially reduced in the Protein C vaccinates.
  • the normal medium control was pH 6.7 while the average pH of 5 sera from each vaccinate group was 7.5, 7.6, and 7.5 for the natural, full length recombinant, and truncated Protein C's respectively.
  • the placebo vaccinated control sera averaged ph 7.0.
  • Normal uninoculated Friis medium is pH 7.5.
  • results of these blots indicated that sera from pigs vaccinated with natural Protein C strongly bound to: 1) an 85 kd band in western blot lanes containing separated proteins from E_j_ coli stain pT5T:M852-l, which expresses the full- length recombinant Protein C; and 2) a ⁇ 50 kd band in lanes containing proteins from E ⁇ . coli strain pT5T:M851-B2B, which expresses the truncated Protein C. Both of these bands correspond to the recombinant Protein C products in these strains. Conversely, sera from pigs vaccinated with either of the recombinant Protein C's bind strongly to an 85 kd band in lanes containing M JL hvopneumoniae extract 7S (see Example
  • Samples of lung were collected and evaluated microscopically for lesions of Mycoplasma pneumonia from three groups of 15 pigs (recombinant Protein C vaccinates, truncated Protein C vaccinates, and placebo vaccinates). A grossly non-involved sample of lung from each pig and one representative lesion were examined and graded microscopically except for one pig in which no lesion remained after sampling for IFA and culture.
  • Mycoplasma hvopneumoniae is the causative agent of enzootic pneumonia in swine. Other mycoplasmas cause similar respiratory diseases in other organisms. For example: M. pneumoniae in humans; ⁇ gallisepticum in chickens; M ⁇ bovis in cattle; etc. It is very possible that surface proteins homologous to polypeptide C exist in these other Mycoplasmas that may be effective as vaccines against these infective agents. The genes encoding these homologous proteins could be isolated by using the gene for polypeptide C as a probe for DNA hybridization at low stringency to a recombinant DNA library made from another mycoplasma species.
  • the recombinant library could be constructed like the pUC18 library described in Example VIII-A1, and the library screened with the R69 probe as described in Example VIII-A2 except under different stringency conditions.
  • DNA hybridization could be carried out in low stringency conditions at 32° in 30% formamide, 5X SSPE, 1% SDS, 0.1% sodium pyrophosphate, 0.15 mg/ml tRNA, 0.125 mg/ml sonicated salmon sperm DNA.
  • An initial wash could be carried out in the same buffer (minus probe, tRNA, and sonicated salmon sperm DNA) at 32°C (3 X 10 min washes) and the filters exposed to X-ray film for autoradiography to reveal clones which hybridize to the probe.
  • the filters could be washed under higher stringency conditions by raising the formamide concentration in 5% increments, and/or raising the wash temperature in 5°C increments, with each increment being followed by exposure to film to determine the number of "positive” hybridizing clones.
  • Inserts from the newly isolated positives would have to be subcloned into sequencing vectors and sequenced (See Example VIII-B for procedures and general strategy) .
  • the amino terminus of the coding sequence and the reading frame could be determined by comparison with the sequence for the Protein C gene. Once the positions of any UGA codons was determined, changes in these codons and other modifications to facilitate expression could be done in the same general manner as described for the Protein C gene (See Example IX-A and 1X-B). Expression, purification, and testing of these recombinant proteins would depend on the properties of the proteins (likely to be similar to polypeptide C, described in Example IX-C4, X-B, X-C, and X-D) and the animals being tested.
  • Protein C itself from M ⁇ hvopneumoniae might be protective against other species.
  • the recombinant Protein C could be tested directly in vaccine trials with other animals subsequently challenged with the corresponding Mycoplasma species.
  • fragments of Protein C might be useful either as vaccines or for diagnostic purposes. Such fragments might be produced in a variety of ways: 1) cloning of a section of the Protein C gene into a recombinant expression vector and producing the fragment as a recombinant protein much as the truncated version of Protein C was produced.
  • a peptide fragment to be used as a vaccine would be required to elicit an immune response directed against the natural Protein C (when administered as a vaccine to swine, See Example X-E).
  • Such a response could be detected by Western blot analysis, showing that serum from pigs vaccinated with the fragment(s) recognize an 85 kd protein band on Western blots containing Protein C.
  • a cellular immune response directed against Protein C could be detected using a T-lymphocyte proliferation assay in a method analogous to that described by Schwartz et. al., J. of Immunol.. U5.:1330 (1975).
  • a fragment to be used as a diagnostic should be recognized by sera from pigs infected with M ⁇ hyopneumonia in an analytical procedure such as the ELISA test See Example X- H.

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Abstract

L'invention concerne une classe de polypeptides utiles pour le diagnostic in vitro d'une infection dûe au Mycoplasma chez les animaux. Ces polypeptides sont également capables d'induire une réponse immunitaire chez les porcs qui n'ont pas été précédemment exposés au Mycoplasma. Les polypeptides sont constitués d'une protéine antigènique de 50 kd et de plusieurs formes tronquées de cette protéine, ainsi que de fusions de B- galactosidase. En outre, plusieurs fragments de polypeptides sont prévus pour venir s'ajouter aux séquences de 5' ADN pour les clones génomiques. L'expression s'effectue dans E. Coli. Des procédés de recombinaison d'ADN pour la production de ces polypeptides et certains vecteurs phages utiles dans ces procédés sont également décrits. L'invention concerne également des procédés pour vacciner les animaux avec une composition de vaccin qui renferme des polypeptides.
EP19910907676 1990-04-02 1991-04-01 Polypeptides useful in diagnosis of and treatment against mycoplasma infections in animals Withdrawn EP0527771A4 (en)

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WO1988000977A1 (fr) * 1986-07-25 1988-02-11 Synergen, Inc. Polypeptides utiles dans le diagnostic d'infections du mycoplasma chez les porcs, et procedes d'adn recombinant pour leur fabrication
EP0283840A2 (fr) * 1987-03-26 1988-09-28 Ml Technology Ventures, L.P. Antigène de mycoplasma hyopneumoniae et ses utilisations

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US4530901A (en) * 1980-01-08 1985-07-23 Biogen N.V. Recombinant DNA molecules and their use in producing human interferon-like polypeptides
US4894332A (en) * 1985-03-27 1990-01-16 Biogen, Inc. DNA sequences coding for Mycoplasma hypopneumoniae surface antigens, methods of use to make the corresponding proteins

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WO1988000977A1 (fr) * 1986-07-25 1988-02-11 Synergen, Inc. Polypeptides utiles dans le diagnostic d'infections du mycoplasma chez les porcs, et procedes d'adn recombinant pour leur fabrication
EP0283840A2 (fr) * 1987-03-26 1988-09-28 Ml Technology Ventures, L.P. Antigène de mycoplasma hyopneumoniae et ses utilisations

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