AU627011B2 - Expression system for actinomycetes and related organisms - Google Patents
Expression system for actinomycetes and related organismsInfo
- Publication number
- AU627011B2 AU627011B2 AU52626/90A AU5262690A AU627011B2 AU 627011 B2 AU627011 B2 AU 627011B2 AU 52626/90 A AU52626/90 A AU 52626/90A AU 5262690 A AU5262690 A AU 5262690A AU 627011 B2 AU627011 B2 AU 627011B2
- Authority
- AU
- Australia
- Prior art keywords
- dna
- promoter
- mycobacterium
- mpb70
- recombinant dna
- 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.)
- Ceased
Links
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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
Description
EXPRESSION SYSTEM FOR ACTINOMYCETES AND
RELATED ORGANISMS
he 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.
Recombinant DNA technology has been an important tool in developing vaccines against a number of mammalian and plant pathogens. 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. In particular, 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.
Accordingly, 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.
In the Figures:
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 and 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; (3) 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. In reference to the MPB70 gene, "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. As used in the present specification, 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".
Accordingly, in one embodiment, 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. As used in the present specification, "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. In other words, 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. Reference herein to 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.
In a further embodiment 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. By
"actinomycetes and related organisms" is meant 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. In this embodiment, 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.
In yet a further embodiment, the present invention provides a promoter probe vector. Such a vector is used to identify and clone promoters from actinomycetes and related species. In this case, 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 β-galactosidase, alkaline phosphatase or urease, amongst others.
In a further embodiment, 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. Accordingly, a 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. Alternatively linker sites can be inserted such that only the promoter region of MPB70 is used.
Techniques required to construct the aforementioned vectors are well known in the art. For example, these techniques can be found in Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, ppl-545 and Sambrook et al supra. The source of 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, however, 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. The choice of 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. By 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. In this case, 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. Additionally, although 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.
Other mechanisms for using the MPB70 gene is an expression system 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. Alternatively, other 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.
Although the 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.
In addition to using the MPB70 gene, its secretory signal and its promoter region, or combinations thereof, in the construction of a variety of vector molecules, 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 has
important consequences in diagnostic research and disease management in that individuals vaccinated, for example, with a derivative of M. bovis BCG carrying a deletion mutation in the MPB70 gene, will mount a serological and cell-mediated immune response diagnostically distinguishable from an individaul vaccinated with a strain expressing the MPB70 gene. Techniques for inserting genes or inducing insertional or deletional mutations are well known in the art.
EXAMPLE 1
This Example describes the cloning of the MPB70 gene from M. bovis.
Bacteria and Cloning vectors.
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.)
DNA techniques.
All enzymes and phage vectors were purchased from Promega Biotec except where noted otherwise. M. bovis DNA extraction was after the method of Shoemaker et al ., Am. Rev. Respr. Pis. 134: 210-213, 1986. Cloning and DNA techniques were as described by Maniatis et al., Supra. Sequence analysis was by the primer extension dideoxy termination method of Sanger et al., J. Mol.
Biol. 143: 161-178, 1980, after DNA was cloned in M13mpl8. Both reverse transcriptase and T7 DNA polymerase (Sequenase, USB) were used, and sequencing reactions were analyzed on 6%(w/v) polyacrylamide-8M urea gels.
Cloning MPB70 gene.
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.
This library was examined using the MPB70 partial clone C4a (Radford et al., Supra) as a hybridisation probe, and two reactive phage isolated. The restriction map of the MPB70 region of the M. bovis chromosome was then established using these two clones, and is shown in Figure (1). Southern blot analysis of restriction enzyme Pstl digested AN5 DNA had shown that the C4a clone hybridised with a 1.85 Kb Pstl fragment, which was found to be present in both reactive phage.
EXAMPLE 2
Nucleotide sequence of MPB70 gene.
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 sequence is written in the 5'-»3' direction and
is shown in Figure 2 together with the corresponding amino acid sequence.
EXAMPLE 3
Mapping the MPB70 Promoter
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.
EXAMPLE 4
Expression of the MPB70 gene in Mycobacterium smegmatis
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 disσerible in non-transformed M^ smegmatis or the related fast growing mycobacteria, Mycobacterium phlei.
This indicates that the MPB70 gene will express in other strains of mycobacteria as well as M.bovis BCG.
The skilled artisan will immediately recognise that the 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.
Claims (1)
- CLAIMS :1. 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 operatively linked downstream thereof.2. The recombinant DNA vector according to claim 1 wherein at least one origin of replication allows replication in a prokaryotic cell.3. The recombinant DNA vector according to claim 2 wherein the origin of replication allows replication in an actinomycete or related organism.4. The recombinant vector according to claim 3 wherein the actinomycete is a species of mycobacterium.5. The recombinant vector according to claim 4 wherein the mycobacterium is Mycobacterium bovis BCG.6. The recombinant DNA vector according to claim 1 wherein the selectable marker comprises resistance to an antibiotic, resistance to phage infection, sensitivity to streptomycin or an enzyme.7. The recombinant DNA vector according to any one of the preceding claims in single or double stranded form and linear or circular form.8. The recombinant DNA vector according to claim 7 further comprising one or more unique restriction endonuclease sites downstream of said promoter which permit a nucleotide sequence inserted into one or more of said sites to be transcribed by said promoter. 9. The recombinant DNA vector according to claim 8 wherein said nucleotide sequence is the MPB70 gene or derivatives thereof.iθ. The recombinant DNA vector according to any one of the preceding claims further comprising the nucleotide sequence encodingthe secretory signal or its derivatives for the MPB70 gene operably linked to the promoter.11. A DNA fusion vector comprising one or more origins of replication, at least one selectable marker and the MPB70 gene and its promoter and optionally its secretory signal and/or their derivatives and one or more restriction endonuclease sites downstream of said MPB70 gene such that transcription and translation of a nucleotide sequence inserted into said sites results in a fusion polypeptide between the MPB70 protein or its derivatives and the transcription and translation product of any nucleotide sequence downstream thereof.12. The recombinant DNA fusion vector according to claim11 wherein at least one origin of replication allows replication in a prokaryotic host.13. The recombinant DNA fusion vector according to claim12 wherein the origin of replication allows replication in an actinomycete or related organism.14. The recombinant DNA fusion vector according to claim13 wherein the actinomycete is a species of mycobacterium.15. The recombinant DNA fusion vector according to claim 14 wherein the mycobacterium is Mycobacterium bovis BCG.16. The recombinant DNA fusion vector according to claim 11 wherein the selectable marker is resistance to an antibiotic, resistance to phage infection, sensitivity to streptomycin or an enzyme.17. The recombinant DNA fusion vector according to any one of claims 11 to 16 in single or double stranded form and linear or circular form.18. 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.19. The recombinant DNA promoter probe vehicle according to claim 18 wherein at least one origin of replication allows replication in a prokaryotic cell.20. The recombinant DNA promoter probe vehicle according to claim 19 wherein the origin of replication allows replication in an actinomycete or related organism.21. The recombinant DNA promoter probe vehicle according to claim 20 wherein the actinomycete is a species of mycobacterium.22. The recombinant DNA promoter probe vehicle according to claim 21 wherein the species of mycobacterium is Mycobacterium bovis BCG.23. The recombinant DNA promoter probe vehicle according to claim 18 wherein the first and second selectable markers comprise resistance to an antibiotic, resistance to phage infection, sensitivity to streptomycin or an enzyme.24. The recombinant DNA promoter probe vehicle according to any one of claims 18 to 23 in a single or double stranded form and linear or circular form.25. A DNA construct comprising the MPB70 gene, its secretory signal and its promoter region or derivatives thereof attached to by gene fusion or in front of a heterologous nucleotide sequence.26. The DNA construct according to claim 25 inserted into a plasmid, bacteriophage, chromosomal DNA or any combinations thereof in an actinomycete or related organism.27. The DNA construct according to claim 26 carried in a species of mycobacterium.28. The DNA construct according to claim 27 carried in Mycobacterium bovis BCG.29. A DNA construct comprising the MPB70 gene promoter attached to by gene fusion or in front of a heterologous nucleotide sequence.30. The DNA construct according to claim 29 inserted into a plasmid, bacteriophage or chromosomal DNA or any combinations thereof.31. The DNA construct according to claim 30 carried by an actinomycete or related organism.32. The DNA construct according to claim 31 carried by a species of mycobacterium. 33. The DNA construct according to claim 32 carried by Mycobacterium bovis BCG.34. A eukaryotic or prokaryotic organism transformed with a vector or DNA construct according to any one of the preceding claims.35. A vaccine capable of inducing a serological or cell-mediated immune response diagnostically distinguishable from a response induced by wild-type infection with a pathogenic mycobacterium said vaccine comprising a species of mycobacterium carrying a deletion, insertion and/or modification of the MPB70 gene.36. The vaccine according to claim 35 wherein the Mycobacterium species is Mycobacterium bovis, M.bovis BCG, M.leprae. M.paratuberculosis, M.avium. M.intracellulare or .ulcerans.
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AU52626/90A AU627011B2 (en) | 1989-03-08 | 1990-03-05 | Expression system for actinomycetes and related organisms |
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AU643842B2 (en) * | 1990-10-25 | 1993-11-25 | Commonwealth Scientific And Industrial Research Organisation | Corynebacteria and related organisms as vaccine vectors |
JPH06501847A (en) * | 1990-10-25 | 1994-03-03 | コモンウェルス サイエンティフィク 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 | Corynebacterium and related organisms as vaccine vectors |
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