EP0486495A1 - Systeme d'expression pour des actinomycetes et des organismes apparentes - Google Patents

Systeme d'expression pour des actinomycetes et des organismes apparentes

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Publication number
EP0486495A1
EP0486495A1 EP90904182A EP90904182A EP0486495A1 EP 0486495 A1 EP0486495 A1 EP 0486495A1 EP 90904182 A EP90904182 A EP 90904182A EP 90904182 A EP90904182 A EP 90904182A EP 0486495 A1 EP0486495 A1 EP 0486495A1
Authority
EP
European Patent Office
Prior art keywords
dna
promoter
mycobacterium
recombinant dna
mpb70
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Withdrawn
Application number
EP90904182A
Other languages
German (de)
English (en)
Other versions
EP0486495A4 (fr
Inventor
Anthony John Radford
Paul Richard Wood
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Commonwealth Scientific and Industrial Research Organization CSIRO
Original Assignee
Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Commonwealth Scientific and Industrial Research Organization CSIRO filed Critical Commonwealth Scientific and Industrial Research Organization CSIRO
Priority claimed from AU52626/90A external-priority patent/AU627011B2/en
Publication of EP0486495A4 publication Critical patent/EP0486495A4/fr
Publication of EP0486495A1 publication Critical patent/EP0486495A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • 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
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • 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 generally to a recombinant DNA expression system applicable to actinomycetes and related organisms. More particularly, the present invention is directed to the MPB70 gene, its secretory signal and its promoter region, or combinations thereof, in expression, secretion and/or fusion vectors for actinomycetes and related organisms.
  • the actinomycetes and related organisms contain members capable of causing serious infection in mammals, including man.
  • Notable human pathogenic agents belonging to this group are Mycobacterium tuberculosis and Mycobacterium leprae, the causative agents of tuberculosis and leprosy, respectively.
  • the present invention now provides a recombinant DNA expression system for the actinomycetes and related organisms enabling the expression, for example, of recombinant antigens for use in vaccine development.
  • the present invention provides the MPB70 gene, its secretory signal and its promoter region attached to by gene fusion, or in front of, any foreign (i.e.
  • heterologous gene or nucleotide sequence inserted into any member of the actinomycetes or related organisms via bacteriophage or plasmid vector or by insertion into the chromosome or any combination thereof for the purpose of expressing the polypeptide or protein encoded by the foreign gene or nucleotide sequence.
  • one aspect of the present invention is directed to a DNA vector comprising one or more origins of replication, at least one selectable marker and the promoter region or derivatives thereof from the MPB70 gene, said promoter region capable of directing the transcription of nucleotide sequences operably linked downstream thereof.
  • Another aspect of the present invention is directed to a DNA fusion vector comprising one or more origins of replication, at least one selectable marker and the MBP70 gene and its promoter and optionally its secretory signal and/or their derivatives and one or more restriction endonuclease sites downstream of said MBP70 gene such that transcription and translation of a nucleotide sequence inserted into said sites results in a fusion polypeptide between MPB70 protein or part thereof and the transcribed and translated product of any nucleotide sequence downstream thereof.
  • Yet another aspect of the present invention relates to a DNA promoter probe vector comprising one or more origins of replication, at least one first selectable marker and the secretory signal or derivatives thereof from the MPB70 gene fused to or in front of a second selectable marker, said vector further comprising one or more restriction endonuclease sites upstream of said secretory signal such that the insertion of a promoter in the correct orientation in said sites permits the transcription of said second selectable marker.
  • Figure 1 is a restriction map of the MPB70 region of the Mycobacterium bovis chromosome, the MPB70 gene being indicated by the rectangular box.
  • EcoRl, BamHl, Pstl and Smal are restriction endonucleases; Kb, kilobase pairs.
  • Figure 2 is the nucleotide and corresponding amino acid sequence encoding the mature MPB70 protein and the nucleotide sequence of the MPB70 gene promoter.
  • the nucleotide sequence is given in single letter code according to standard practice in the 5'$3' direction.
  • A refers to deoxyadenosine-5'-phosphate
  • C refers to deoxycytidine-5'-phosphate
  • G refers to deoxyguanosine-5'-phosphate
  • T refers to deoxythymidine-5'-phosphate.
  • the amino acid sequence of the MPB70 protein is listed above the gene sequence, where ALA is Alanine, ARG is Arginine, ASN is Asparagine, ASP is Aspartic acid, CYS is Cysteine, GLN is Glutamine, GLU is Glutamic acid, GLY is Glycine, HIS is Histidine, ILE is Isoleucine, PHE is Phenylalanine, PRO is Proline, SER is Serine, THR is Threonine, TRP is Tryptophan, TYR is Tyrosine, and VAL is Valine.
  • Figure 3 is a photographic representation depicting primer extension of MPB70 mRNA.
  • the band arrowed represents the extension product after primed extension of the MPB70 mRNA using reverse transcriptase.
  • the sequence marked is that of the MPB70 gene upstream (5' ) region.
  • Figure 4 is a photographic representation showing the expression of the MPB70 gene in Mycobacterium smegmatis. Probing protein sonicate samples for M.smegmatis transformed with the MPB70 gene.
  • A coo assie stained polyacrylamide gel
  • B A western blot of the gel in (A) probed with the SB10 monoclonal antibody. Track (1) Bio- Rad low molecular weight markers; (2)
  • M.smegmatispEP2MPB70 Mycobacterium phlei; (4) M. phleipEP2; (5) E.coli; (6) M ⁇ bovis BCG strain Japan.
  • the present invention is predicated, at least in part, on the cloning and characterization of the MPB70 gene, including its secretory signal and its promoter region.
  • the MPB70 gene refers to the polynucleotide sequence encoding the MPB70 protein in Mycobacterium bovis BCG.
  • secretory signal means the nucleotide sequence encoding the signal peptide which facilitates the secretion of the MPB70 protein out of the cell. The signal peptide is cleaved off the MPB70 protein during the secretion process.
  • Promoter is used in its broadest sense and, in accordance with the present invention, refers to the nucleotide sequence located upstream of the MPB70 gene which binds RNA polymerase and directs same to the correct transcriptional start site whereupon the MPB70 gene is transcribed.
  • the MPB70 gene and its secretory signal sequence is said to be relative to the promoter meaning that said promoter directs the transcription of both the signal sequence and MPB70 gene in the correct reading frame. This is also referred to herein as being operably linked to said promoter.
  • the nucleotide sequence of the MPB70 gene, its secretory sequence and promoter are shown in Figure 2.
  • the present invention extends to any and all single or multiple nucleotide additions, deletions and/or substitutions to the MPB70 gene and its secretory signal and promoter.
  • the present invention also extends to natural recombinant and synthetic forms of the MPB70 gene and its secretory signal and promoter. All such variations to the MPB70 gene; its secretory signal and 5. its promoter are referred to herein as "derivatives”.
  • the present invention is directed to a DNA vehicle or vector carrying the promoter of the MPB70 gene located thereon and 0 further having one or more restriction endonuclease sites downstream of said promoter such that when a nucleotide sequence is inserted into one or more of these sites, the promoter will cause the nucleotide sequence to be transcribed.
  • a 5 DNA vehicle is defined to include plasmid DNA, lysogenic phage DNA and/or transposon DNA, in double or single stranded linear or double or single stranded circular form and may or may not be self transmissible or mobilizable and will contain one or more origins of 0 replication.
  • At least one origin of replication will allow replication in a prokaryotlc cell and preferably in an actinomycetes or related organism and more preferably in a species of mycobacterium such as Mycobacterium bovis BCG.
  • the DNA vehicle is a vector in the 5 sense that it carries the subject promoter and provides the genetic information necessary for said vehicle to replicate autonomously in a cell or to integrate into the cell's genome and may be introduced into a cell by any of a number of techniques such as conjugation, mobilization, 0 transformation, transfection, transductlon or electroporation amongst others.
  • a eukaryotic or prokaryotlc cell transformed with the vectors and DNA such vectors or constructs introduced by any means and either integrated into the cells genome or 5 existing e trachromosomally (e.g. as an autonomously replicating plasmid).
  • the DNA vehicle will also contain at least one selectable marker as discussed below.
  • the present invention includes use of the aforementioned DNA vehicle carrying the promoter of the MPB70 gene as an expression vector in the actinomycetes and related organisms.
  • actinomycetes and related organisms bacteria of the genera Mycobacteria. Nocardia, Streptomvces. Brevibacterium. Corvnebacterium and Arthrobacter amongst others as defined in Bergey's Manual of Determinative Bacteriology, 8th ed, pp 599-861. Accordingly, a nucleotide sequence encoding a particular polypeptide or protein when inserted downstream of the MPB70 promoter via one or more of the restriction endonuclease sites and then, by any number of means, introduced into an appropriate microorganism or other cell, will be transcribed and translated into the said polypeptide or protein. Thus the nucleotide sequence is said to be expressed by way of the expression vector.
  • Polypeptides or proteins synthesized in vivo may or may not, depending on the molecule and the host bacterium, be easily exported out of the cell. Accordingly, to facilitate secretion out of the cell of polypeptides or proteins synthesized by the expression vector, a further embodiment of the present invention is to include on the expression vector the secretory signal sequence of the MPB70 gene positioned relative (i.e. operably linked) to the promoter. Hence, expression of an inserted nucleotide sequence in vivo will result in a polypeptide or protein carrying the signal peptide of the MPB70 protein. The polypeptide or protein will now be readily exported out of the cell.
  • the expression vector will comprise a DNA vehicle carrying the MPB70 gene promoter and the MPB70 gene secretory signal sequence relative thereto such that the promoter will direct the synthesis of the signal peptide fused to the polypeptide or protein encoded by a nucleotide sequence inserted downstream of the promoter and signal sequence.
  • the signal peptide may be cleaved to form the polypeptide or protein during export from the cell.
  • the present invention provides a promoter probe vector.
  • a promoter probe vector is used to identify and clone promoters from actinomycetes and related species.
  • the promoter probe vector comprises a DNA vehicle carrying the MPB70 gene secretory signal sequence fused to, or in front of, a nucleotide sequence encoding a detectable marker.
  • the vehicle further comprises one or more restriction endonuclease sites located upstream of the secretory sequence such that when a DNA fragment containing a promoter from an actinomycete or related organisms is inserted via the aforementioned endonuclease sites, said promoter will cause the expression of the secretory sequence fused to the detectable marker.
  • Suitable detectable markers include resistance to antibiotics or chemicals resistance to phage infection, sensitivity to streptomycin or enzymes capable of causing a detectable reaction when provided with a suitable substrate. Examples include resistance to ampicillin, streptomycin, penicillin, tetracycline, kanamycin and the like and ⁇ -galactosidase, alkaline phosphatase or urease, amongst others.
  • the present invention provides fusion vectors for the production of polypeptides or proteins fused to the MPB70 protein.
  • the fusion of such polypeptides or proteins to the MPB70 protein may be important, for example, to protect them from host proteases or other hydrolyzing enzymes or to add antigenic determinants to the MPB70 fusion protein, or to enhance the antigenicity of any protein fused to the MPB70 protein.
  • the fusion vector may comprise a DNA vehicle carrying the MPB70 gene promoter and, in relative position thereto, the MPB70 gene and its secretory sequence.
  • the fusion vector further comprises one or more restriction endonuclease sites located at the 3' end of the MPB70 gene or downstream thereof but before its stop codon.
  • nucleotide sequence encoding a polypeptide or protein is inserted into the fusion vector via the aforementioned restriction endonuclease sites and upon expression, a polypeptide or protein fused to the MPB70 protein or derivative thereof, is produced.
  • Fusion vectors comprising the MPB70 gene can be constructed by those skilled in the art by a variety of means and detailed, for example, in Sambrook et al. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, 2nd Edition, 1989.
  • Linker sites comprising one or more restriction enzyme sites can be introduced into the gene at any point after the promoter region, by site directed mutagenesis, partial cleavage and linker mutagenesis, transposition of restriction sites or other techniques.
  • Such polylinkers can be inserted so that fusions with foriegn antigens can be made with the signal sequence of MPB70 or part thereof, or the signal sequence plus part or all of the MPB70 gene, thus influencing the secretion of the antigen-MPB70 fusion product and its immunogenicity as MPB70 is a major immunogenic protein of MN. bovis BCG.
  • linker sites can be inserted such that only the promoter region of MPB70 is used.
  • DNA vehicles can be from known and already characterized plas ids, bacteriophages or transposons of the actinomycetes and related organisms.
  • One skilled in the art could isolate any indigenous plasmid, lysogenic bacteriophage or transposon from any of these bacteria and use these as a source of the DNA vehicle or at least part thereof.
  • any one DNA vehicle is not limited to those which only replicate in actinomycetes and related organisms since it is within the scope of the present invention to include shuttle vectors.
  • a shuttle vector as used herein is meant a DNA vehicle capable, naturally or by design, of replication in both the actinomycetes and related species and in bacteria such as Escherichia coli. Bacillus sp. or Pseudomonas sp. and the like and/or eukaryotic cells such as mammalian, yeast or fungal cells.
  • the vectors contemplated herein will contain at least one origin of replication wherein where necessary replication can occur in a prokaryotic cell and further origins of replication for replication in different prokaryotic species and/or eukaryotic species.
  • the present invention is useful for any actinomycete or related organisms, it is particularly directed to any species of Mycobacteria. and preferably M. bovis BCG.
  • the present invention therefore extends to DNA constructs comprising the MPB70 gene, its secretory signal and/or its promoter region and/or derivatives thereof attached to by gene fusion or in front of a heterologous nucleotide sequence.
  • the DNA constructs also may comprise separately the MPB70 gene, its secretory signal or its promoter region and/or their derivatives whether with or without additional heterologous nucleotide sequences.
  • the present invention also extends to actinomycetes or related organisms carrying such DNA constructs and particularly species of mycobacterium and even more particularly M.bovis BCG. Furthermore, such DNA constructs may be inserted, fused or associated with plasmid, bacteriophage, chromosomal DNA or combinations thereof.
  • MPB70 genes for MPB70 could involve the cloning of the MPB70 promoter by either digesting the DNA comprising the promoter area with restriction enzymes and cloning the promoter by ligation to other DNA using well established techniques, or by the use of polymerase chain technology to prepare the gene for cloning, such techniques are outlined in Sambrook et al. supra.
  • mycobacterial promoters or promoters effective in actinomycetes and related species including mycobacteria could be cloned into the MPB70 gene or fusion vector systems containing segments of the MPB70 gene as described such as to initiate the transcription of the sequences of the MPB70 gene which may facilitate both translation, secretion and immunogenicity of the foreign antigen cloned in the MPB70 fusion vector.
  • DNA constructs of the present invention are described in terms of DNA, it would be readily apparent to one skilled in the art that the constructs could be maintained and in some cases used in corresponding RNA form without falling outside the scope of the present invention.
  • the present invention contemplates using these nucleotide sequences to delete or insert the MPB70 gene, or parts thereof, in M. bovis. M. bovis BCG or any other mycobacterial species for the purpose of creating a vaccine inducing a serological or cell-mediated immune response diagnostically distinguishable from a response induced by wild type infection with a pathogenic mycobacterium, including M. bovis. M. tuberculosis. M. leprae. M. paratuberculosis. M. avium. M. intracellulare and M. ulcerans species.
  • This Example describes the cloning of the MPB70 gene from M. bovis.
  • Mycobacterium bovis AN5 was from the Commonwealth serum Laboratories, Parkville, Australia, originally obtained form the Ministry of Agriculture Weybridge Laboratories, England. Escherichia coli strains Y1090, Y1089 (Young and Davis, Science 222: 778-782, 1983), NM538 and NM539 (Frischauf et al. , J. Mol. Biol. 170: 827-842, 1983) have been previously described. Lambda phage gtll and EMBL3 were from Promega Biotec (Wisconsin), and M13n.pl8 was obtained from Bresatec (Sth. Australia). The origin and sequence of lambda gtll clone C4a has been previously publishd (Radford et al. , Infect. Immun. 56: 921-925, 1988.)
  • a long fragment library of M. bovis AN5 DNA was cloned in the bacteriophage vector EMBL3.
  • AN5 DNA was partially digested with restriciton enzyme Sau3a and size selected on an agarose gel for fragments greater than 10Kb, which were then extracted from the gel using the Geneclean (BiolOl) process. Insert was then ligated with EMBL3 DNA that had been prepared with BamHl cohesive ends. Packaging and plating of the phage gave 1.5 x 10 -5 pfu and plating the phage on the recombinant restrictive host E. coli NM539 revealed that the library was greater than 50% recombinant phage.
  • the library was propagated on the restrictive NM539 host.
  • EXAMPLE 1 The 1.85 Kbp fragment to which the C4a clone hybridised (EXAMPLE 1) was subcloned into MI3mpl8 and the DNA sequenced according to Sanger et al. Supra.
  • the exact origin of the MPB70 promoter has been mapped by primer extension of an oligonucleotide annealed to RNA derived from M.bovis.
  • the primer used was specific to MPB70 mRNA. After annealing the primer to the mRNA addition of reverse transcriptase permitted the primer to be extended in the direction of the 5' end of the mRNA, making a complementary DNA copy of the MPB70 mRNA.
  • the sample was then denatured and run on a 6% (w/v) polyacrylamide sequencing gel simultaneously with the products of a primer extension sequencing reaction performed on a cloned gene of MPB70, using the same primer.
  • the mRNA copied cDNA copy terminates at the 5' end, or transcription initiation point, of the mRNA. The exact point can be ascertained by comparison with the parallel sequence.
  • Figure 3 shows the results of the primer extension experiment, and shows that the mRNA transcription start site is at nt 207 (see Figure 2). Based on the E_ ⁇ coli concensus model for the structure of bacterial promoters the MPB70 promoter would be located between nt 168 and 202.
  • the MPB70 gene was cloned into the pEP2 E. coli/Mycobacteria shuttle vector on an EcoRl fragment derived from the EMBL3 clone carrying the MPB70 gene.
  • This DNA was extracted from E.coli and electroplated into a strain of M ⁇ smegmatis, which was selected for the kanamycin resistance character carried on the pEP2 plasmid.
  • a kanamycin resistant colony was isolated and streaked to purity. This isolate was grown in broth, centrifuged, and the pellet sonicated to produce protein extracts. Aliquots of this sample and controls were run on SDS polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. These membranes were immunoblotted with the MPB70 specific monoclonal antibody SB10 (Wood et al., J. Gen. Micro. 134: 2599- 2604, 1988).
  • Figure 4 shows that the sample from the M ⁇ smegmatis containing the MPB70 gene expressed a protein of the same molecular weight as that of the native protein expressed from ⁇ bovis BCG strain Japan. Although the level of expression was less than that of the BCG Japan strain, no MPB70 was dis ⁇ erible in non-transformed M ⁇ smegmatis or the related fast growing mycobacteria, Mycobacterium phlei.
  • MPB70 gene will express in other strains of mycobacteria as well as M.bovis BCG.
  • MPB70 gene, its secretory signal sequence and its promoter can undergo single or multiple nucleotide base deletions, insertions or substitutions which may or may not affect their respective functions. All such modifications are considered to be within the scope of the present invention. It is also considered within the scope of the present invention to include cell-free in vitro expression systems using the nucleic acids contemplated herein.

Abstract

La présente invention se rapporte généralement à un système d'expression d'ADN recombinant applicable aux actinomycètes et aux organismes apparentés. Plus particulièrement, elle est dirigée vers le gène MPB70, son signal et sa région promotrice, ou des combinaisons de ceux-ci, dans des vecteurs d'expression, de secrétion et/ou de fusion pour des actinomycètes et des organismes apparentés.
EP90904182A 1989-03-08 1990-03-05 Systeme d'expression pour des actinomycetes et des organismes apparentes Withdrawn EP0486495A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AU3099/89 1989-03-08
AUPJ309989 1989-03-08
AU52626/90A AU627011B2 (en) 1989-03-08 1990-03-05 Expression system for actinomycetes and related organisms

Publications (2)

Publication Number Publication Date
EP0486495A4 EP0486495A4 (fr) 1991-12-13
EP0486495A1 true EP0486495A1 (fr) 1992-05-27

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WO (1) WO1990010701A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5591632A (en) * 1987-03-02 1997-01-07 Beth Israel Hospital Recombinant BCG
JPH05504067A (ja) * 1990-02-26 1993-07-01 コモンウェルス　サイエンティフィック　アンド　インダストリアル　リサーチ　オーガナイゼイション 大腸菌及びマイコバクテリアのためのシャトルプラスミド
US5637303A (en) * 1990-10-25 1997-06-10 Commonwealth Scientific And Industrial Research Organisation Use of a phospholipase D mutant of Corynebacterium pseudotuberculosis for vaccination
EP0499003A1 (fr) * 1991-02-14 1992-08-19 N.V. Innogenetics S.A. Polypeptides et peptides, en particulier polypeptides et peptides recombinants, acides nucléiques les encodantes, et leur utilisation pour le diagnostic de la tuberculose
US6566121B1 (en) 1991-06-13 2003-05-20 Albert Einstein College Of Medicine Of Yeshiva University Insertional mutations in mycobacteria
EP0521220A1 (fr) * 1991-06-14 1993-01-07 Institut Pasteur Actinomycétale immunogène recombinant
FR2720076B1 (fr) 1994-05-20 1996-08-02 Inst Nat Sante Rech Med Vecteurs navettes pour l'introduction d'ADN dans des mycobactéries et utilisation de ces bactéries comme vaccins.
GB9517494D0 (en) * 1995-08-25 1995-10-25 Mini Agriculture & Fisheries Recominent DNA expression system
EP2284257A1 (fr) 2005-01-18 2011-02-16 Vereniging voor Christelijk Hoger Onderwijs, Wetenschappelijk Onderzoek en Patiëntenzorg Mycobactéries dont le lipoarabinomanan ne contient pas le cap de manose

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006626A1 (fr) * 1987-03-02 1988-09-07 Whitehead Institute For Biomedical Research Vaccin mycobacterien recombinant
AU628329B2 (en) * 1988-03-31 1992-09-17 Commonwealth Scientific And Industrial Research Organisation Diagnosis of mycobacterium bovis infection

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
No relevant documents disclosed. *
See also references of WO9010701A1 *

Also Published As

Publication number Publication date
WO1990010701A1 (fr) 1990-09-20
EP0486495A4 (fr) 1991-12-13

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