EP0517751A1

EP0517751A1 - Shuttle plasmid for escherichia coli and mycobacteria

Info

Publication number: EP0517751A1
Application number: EP91904701A
Authority: EP
Inventors: Anthony John Radford; Adrian L. M. Hodgson
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1990-02-26
Filing date: 1991-02-25
Publication date: 1992-12-16
Also published as: EP0517751A4; JPH05504067A; NZ237217A; CA2076753A1; WO1991013157A1

Abstract

Vecteurs navettes d'ADN capables de réplication dans Mycobateria, E.coli et autres hôtes bactériens. Ils comprennent une origine unique de réplication qui les rend capables de se répliquer dans plusieurs espèces bactériennes. La présente invention décrit en outre des vecteurs navettes encodant les polypeptides voulus, par exemple antigènes de bactéries, virus, et parasites à l'orifice de maladies.DNA shuttle vectors capable of replication in Mycobateria, E.coli and other bacterial hosts. They include a unique origin of replication which makes them capable of replicating in several bacterial species. The present invention further describes shuttle vectors encoding the desired polypeptides, for example antigens of bacteria, viruses, and parasites at the orifice of diseases.

Description

SHUTTLE PLASMID FOR ESCHERICHIA COLI AND MYCOBACTERIA

This invention relates to DNA vectors, and is particularly concerned with DNA shuttle vectors which are capable of replication in mycobacteria and Eschrichia coli cells. The invention also relates to bacterial hosts containing such DNA vectors, and further relates to vaccines containing such bacteria.

Mycobacterium bovis BCG (hereinafter BCG) has been used for many years as a vaccine against tuberculosis (TB). The vaccination programme has been extremely effective in controlling human TB firstly because BCG stimulates long term cell-mediated immunity and secondly because it has had an outstanding safety record. These characteristics make BCG a good candidate to form the basis of a live delivery system for recombinant vaccines.

A number of problems are associated with DNA

manipulations involving mycobacteria. These include very few (one) plasmid vectors, poor growth rates of

mycobacteria and low transformation rates when compared to bacterium such as E. coli. Given these problems, it is desirable to produce a shuttle vector which is capable of replication in a standard bacterial work horse such as E. coli for day to day genetic manipulations, and is further capable of replication in Mycobacterium at or near the final stage of genetic manipulation. In this way, genes can be inserted into mycobacteria and time delays associated with Mycobacterium growth and

transformation can be largely avoided. Gicquel-Sanzey et al. (Acta Lepologica 1989, 7, Suppl (1): 208-211) have described a mycobacteria-E. coli plasmid shuttle vector known as pAL8 which comprises two origins of replication, the first for mycobacteria and the second for E. coli. Multiple origins of replication have been necessary due to the evolutionary distance between mycobacteria and E. coli such that a mycobacterial plasmid having a

mycobacterial origin is not capable of growth in E. coli and vice versa. Such vectors suffer from the problem that they are a considerable size due to the inclusion of two origins of replication, this decreasing cloning efficiency, the size of desired DNA fragments which may be inserted into such plasmids, and also increasing the number of unique restriction sites which may be

introduced into the plasmids.

The present applicant has overcome problems

associated with prior art shuttle vectors by providing a DNA shuttle vector which carries a single origin of replication which confers the ability of the vector to replicate in mycobacteria and E. coli cells. In a particularly preferred aspect of this invention as will be described hereinafter, the replication region

corresponds to that of the corynebacterial plasmid pNG2 or fragments thereof.

In accordance with a first aspect of this invention, there is provided a DNA shuttle vector carrying a

replication region comprising a single origin of

replication, said origin of replication allowing

replication of said vector in Mycobacteria and E. coli cells; and a selectable marker. The shuttle vector may additionally comprise a nucleotide sequence containing one or more restriction sites for the insertion of a desired nucleotide sequence.

Preferably, the replication region corresponds to the replication region of the corynebacteria plasmid pNG2 or a fragment thereof which permits replication of said vector in mycobacteria and E. coli.

Surprisingly, the replication region of plasmid pNG2 confers the ability of replication in various bacterial species, apart from Corynebacteria.

The DNA shuttle vector may further comprise a promoter with 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 allows DNA transcription to proceed. The DNA shuttle vector may contain multiple promoters and

downstream restriction endonuclease sites.

The term "shuttle vector" as used herein includes plasmid DNA which may be double-stranded linear or double-stranded circular. The shuttle vector may be introduced into a bacterial cell by any number of

techniques well known in the art, such as conjugation, mobilisation, transformation, transfection, transduction or electroporation.

The term "selectable marker" as used herein refers to any selectable characteristic provided by or encoded for, by a nucleotide sequence. Suitable detectable markers include resistance to antibiotics or enzymes or immunologically detectable proteins or chemicals capable of causing a detectable reaction when provided with a suitable substrate. Examples include resistance to ampicillin, streptomycin, penicillin, hygromycin,

kanamycin, and the like, β-galactosidase, urease,

alkaline phosphatase and the like.

The term "promoter" is used in its broadest sense and refers to any nucleotide sequence which binds to RNA polymerase and which directs the transcription of

nucleotide sequences downstream (3' "or operably linked") to the promoter. Suitable promoters include prokaryotic promoters such as the P1 promoter of bacteriophage lambda, trp promoter, lac promoter, kanamycin resistance promoters of transposon Tn903 and transposon Tn5,

mycobacterial promoters such as that of the promoter of the common mycobacterial 65Kd antigen, ribosomal RNA promoter of mycobacteria, promoters of M.bovis antigens MPB70, MPB59 and MPB64 and the like, hybrids between eukaryotic and prokaryotic promoters, and eukaryotic promoters such as the metallothionine promoter, growth hormone promoter, and the like.

Restriction endonuclease sites provided on the vector may correspond to the cleavage of one or more known restriction endonucleases, such as EcoRl, BamHl, Pstl, Clal, Kp,l, Hindlll, HincI and HinciI, and the like. Restriction endonuclease sites may be provided in the form of one or more polylinkers which contain a number of closely grouped restriction endonuclease sites.

Nucleotide sequences of interest may be inserted into the endonuclease cleavage sites provided on the vector by ligation of DNA fragments having complementary "sticky ends" to allow annealing thereof, or by ligation of nucleotide sequence having "flush" ends (ends having no unpaired nucleotides) by methods well known in the art, and described for example, in Sambrook et al.

(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd Edition, 1989). A nucleotide sequence for insertion into the vector of this invention may include a promoter to direct transcription of downstream ( 3') sequences. Promoters may be the natural promoter of the gene to be transcribed or may be a different promoter. As previously mentioned, the shuttle vector of this invention may itself contain one or more promoters upstream ( 5') from nucleotide sequences encoding one or more restriction endonuclease cleavage sites. In such an embodiment, a desired nucleotide sequence lacking a promoter may be inserted into the vector with transcription of the desired nucleotide sequence being driven by the promoter resident on the vector.

Nucleotide sequences for insertion into the vector of this invention may, for example, encode one or more antigens from disease causing bacteria, viruses or parasites, such as Taenia ovis, rotavirus, Babesia bovis, Mycobacterium bovis, Mycobacterium tuberculosis,

Mycobacterium leprae, Mybacterium paratuberculosis,

Bacteriodes nodosus or Bordetella. Desired nucleotide sequences may also encode hormones such as LHRH, growth hormone or epitopes or analogues thereof; or DNA

sequences capable of recombination with nucleotide sequences within a bacterial host cell; or nucleotide sequences capable of mutagenising DNA sequences within a host cell. Where the shuttle vector of this invention functions as an expression vector, nucleotide sequences encoding desired products may include a signal or leader sequence to allow insertion into membranes of a suitable host cell or secretion from a host cell. Absence of a secretory leader will cause the accumulation of antigen or other protein product within the cytoplasm of a bacterial host cell, where it may be recovered by well known methods.

In accordance with a specific embodiment of this invention there is provided a DNA shuttle vector pEP2, said vector having a size of about 3.1 kb, as determined by agarose gel electrophoresis, a replication region of about 1.85 kb comprising a single origin of replication derived from the Corynebacterium replicon pNG2, an

antibiotic resistance gene to kanamycin, and a nucleotide sequence containing a number of restriction endonuclease cleavage sites for the insertion of a DNA sequence of interest. The 4.5 kbr plasmid pEP3 contains the same sequences of replication as pEP2 plus a marker encoding hygromycin resistance effective in both E.coli and

mycobacteria.

Shuttle vectors of this invention are capable of replication in species of both gram negative and gram positive hosts, such as Mycobacterium, E. coli,

Corynebacterium, and Actinomycetes, as defined in

Bergey's Manual of Determinative Bacteriology, 8th

Edition, pp. 599-861. The vectors of this invention do not appear to replicate in Bacillus species. Any

bacterial strain may be readily tested according to methods well known in the art, to ascertain whether or not the shuttle vector of this invention is capable of replication therein. Advantageously, the shuttle vectors of this invention are capable of replication to high copy numbers in bacterial strains such as in the attenuated strain of Mycobacterium bovis BCG, which as previously stated has been extensively used in vaccination

programmes throughout the world, and is a potent

adjuvant, which stimulates long-term cell mediated immunity.

Shuttle vectors incorporating the origin of

replication of pNG2 are particularly efficient in

organisms of the genus Corynebacterium.

In accordance with a further aspect of this

invention, there is provided a bacterial host which contains a shuttle vector as herein defined. The shuttle vector may be present as a single copy, or more

preferably as multiple copies thereof within the

bacterial host. Preferably, but in no way limiting the invention, the bacterial host is a Mycobacteria,

Corynebacteria or E. coli strain, such as C.

pseudotuberculosis, M. smegmatis, and M. bovis BCG. The vector may be used to deliver antigen or other protein genes into the bacterial host for expression thereof, as an excreted product from the host cell as previously described. Proteins may be expressed while residing on the plasmid vector or after recombination or insertion into the chromosome. Alternatively, expressed products may be inserted into or associated with the host cell membrane or cell wall or resident within the host cell itself. Bacterial hosts expressing desired antigens may be provided as vaccines, for example, as M. bovis BCG, expressing a desired antigen. On immunisation, an immune response would be mounted to the host, such as M. bovis BCG as well as the desired antigen of the disease causing bacterium, virus or parasite.

In a further aspect of this invention there is provided a polypeptide when expressed by a bacterial host cell containing a shuttle vector as herein defined.

This invention contemplates deletions or insertions of nucleotide sequences to or from the replication region of pNG2 as long as such modifications do not prevent the ability of such sequences to confer replication in

Mycobacterium and E. coli. Techniques for insertion or deletion of nucleotide sequences are well known in the art. Mutants could be readily tested for the ability to confer replication in gram negative and gram positive host cells, such as E. coli and corynebacteria.

This invention also extends to replication region of pNG2 itself or fragments thereof, which, on insertion into suitable vector are capable of permitting

replication of said vector mycobacteria and E. coli.

A culture of Eschrichia coli containing plasmid pEP2 was deposited under the terms and conditions of the

Budapest Treaty at the Australian Government Analytical Laboratory (AGAL), Pymble, New South Wales, Australia on 23rd February, 1990 and accorded Accession No.

N90/007080.

This invention will now be illustrated with

reference to the following non-limiting Figures and

Examples.

FIGURES

Figure 1 shows the construction of plasmid pEP2.

Plasmid pNG2 (14.5) was digested with EcoRI and the largest fragment ligated to the Kan^r cartridge of pUC4K. Following electroporation into E.coli the resulting plasmid DNA was extracted and partially digested with Pst1, and then religated and again electroporated with E.coli. One of the resulting Kan^r colonies contained plasmid pEP2. Restriction siles: B(BamHl), E(EcoRI), H( HindΙII ) , Hc(HincII ) , P( PstΙ ) , S( SalI ) . Kan refers to kanamycin resistance gene from pUC4K.

Figure 2A is a circular map of the Mycobacterium-E. coli shuttle vector pEP2. The restriction map is given only for relevant cleavage sites: B (BamHI), E. (EcoRI), H (Hindlll), Hc (HincII), K (Kpnl), p (Pstl), S (SaIl). Kanamycin resistance gene \\\\\ from pUC4K.

B Sequencing strategy. Arrows indicate the extent and direction of sequence generated from restriction

fragments cloned in M13 vectors. X denotes sequence derived using oligonucleotide primers.

Figure 3 is a complete nucleotide sequence of the replication region of pEP2. Relevant restriction sites are marked for comparison with Figure 1 (E (EcoRI), H (Hindlll). Hc (HincII). K (Kpnl). IR and DR, inverted and direct repeat sequences respectively; RBS, putative ribosome binding site; -------> <--------- dyad symmetry associated with putative rho-dependent transcriptional terminator, T. ORFA, major open reading frame.

Figure 4 shows an agrose gel (right hand plate) and a Southern blot (left hand plate) of that gel probed with plasmid pEP2 extracted from E.coli. Tracks contain (A) Undigested pEP2 DNA from E.coli 500ng, (B) Pst 1 digested pEP2 DNA from E.coli 200ng, (C) Pstl digested whole DNA extract of M. bovis BCG pEP2, 2.5ug, (D) Pstl digested M. bovis BCG DNA, 2.5 ug (E) Undigested DNA extract of M. bovis BCG pEP2, 2.5ug, (F) Undigested DNA extract of M. bovis BCG, 2.5ug, (G) Hindlll digested lambda DNA

markers.

EXAMPLE 1

Recombinant DNA Procedures:

Unless otherwise specified herein, manipulation of recombinant molecules and the preparation of solutions are by standard known techniques. Such techniques are described in Sambrook et al. Molecular Cloning, A

Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor [1989], pp. 1-500.

Bacterial Strains and Plasmids:

Mycobacterium bovis BCG variant CSL was obtained as lyophylised human vaccine from the Commonwealth Serum Laboratories (CSL) Australia. M. smegmatis and M. pheli were obtained from the Fairfield Hospital, Melbourne, Australia and Escherichia JM109 was obtained from Promega (Madison, Wisconnsin, U.S.A.). Plasmids pNG2 (Serwold- Davis et al. 1987, Proc. Natl. Acad. Sci. USA 84: 4964- 4968) and pPB3 was obtained from Dr. Philip Bird (Monash University Faculty of Medicine, Alfred Hospital,

Melbourne). Plasmid pAL8 (Gicquel-Sanzey et al. 1989 Acta Lepirol. 7: 207-211) was obtained from Dr. Brigette Gicquel-Sanzey (Pasteur Institute, Paris) and plasmid pUGC4K was purchased from Pharmacia LKB (Uppsala,

Sweden).

Media:

E . coli strains were grown Luria broth, (LB: 10 grams tryptone, 5 grams yeast extract, 10 grams NaCl per litre). Coryneform bacteria were cultured in LB media (Oxoyd) and mycobacterium species were grown in Dubos or 7H11 media (Oxoid, Australia).

Electroporation of Bacteria:

Mycobacterium species and Corynebacterium pseudo- tuberculosis were electroporated according to Lugosi et al. (Tubercule 70: 159-170 [1989]) using a Gene Pulser commercially available from Bio-Rad Laboratories Inc.

Transformants were selected on 7H11 or nutrient media containing 100 μg kanamycin per ml or 200μg hygromyan B per ml.

DNA Isolation and Hybridization Analysis:

DNA was extracted from Mycobacterium using a

modification of a method used to isolate DNA from yeast (Mann and Jeffrey, Anal. Biochem. 1981, 178: 82-87).

Mycobacterial cells were harvested from 400 mis of Dubos broth by centrifugation, resuspended in 5 ml of TE buffer and heat-killed by treatment at 70ºC for 1 hour. Glass beads (3-6 mm) were added and the mix vortexed vigorously for a minimum of 30 seconds to disperse the bacteria.

Bacterial suspensions were then transferred to liquid nitrogen in a mortar pre-cooled in a bath of liquid nitrogen and crushed into a fine powder using a precooled pestle. The crushing of cells was performed in a biohazard hood. Frozen crushed cells were added in small portions (spatula loads) to 15 ml of lysis buffer (6.6 mM Tris, 30 mM EDTA, 1.2% w/v sodium lauroylsarkosinate). Five mg of protease K was added and the mixture incubated for 90 minutes at 37°C. The mix was then extracted with an equal volume of phenol-chloroform and the aqueous phase precipitated with isopropanol at 4°C for 10

minutes. The pellet was dissolved in 1.0 ml of TE buffer and extracted with phenol-chloroform and then water- saturated ether prior to ethanol precipitation and resuspension in TE.

Genomic DNA was isolated from C. pseudotuberculosis as previously described (Hodgson et al., 1990).

pNG2RI DNA was digested with various restriction enzymes and the fragments Southern blotted (Reid et al.) to Hybond N (Amersham) nylon filters. Filters were hybridised overnight at 37°C with pEP2 labelled with 32P using random hexamer primers, washed at increasing stringency as necessary (up to 65ºC) and exposed to X-ray film (Fuji RX). Restriction digested total genomic DNA isolated from M. bovis BCG and that transformed with pEP2 was Southern blotted to nylon and probed with labelled pEP2 as described above. Other transformed Mycobacterium species were analysed with the pEP2 probe using DNA dot blot hybridisation. C. pseudotuberculosis transformants were analysed in the same fashion.

Total cell DNA was used to isolate and transform E. coli to kanamycin or hygromycin resistance. DNA Sequence Analysis:

Nucleotide sequence analysis was determined by the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) using modified T7 DNA polymerase (Pharmacia LKB, Uppsula, Sweden). The complete nucleotide sequence of pEP2 was derived on both strands using universal or oligonucleotide primers

( Figure 1B). The latter was synthesized using a Gene Assembler plus DNA Synthesizer (Pharmacia LKB, Supra). DNA and amino acid sequence data were collated and an analysed using the DNASIS and PROSIS software packages (Pharmacia LKB).

EXAMPLE 2

Shuttle Vector Construction and Analysis Thereof:

The 1.3 kb EcoR1 fragment from pUC4K carrying the kanamycin resistance gene was ligated to pNG2 DNA

digested with EcoRI. The ligation mix was electroporated into E.coli JM109 and transformant selected on LB plates supplemented with 50μg kanamycin per ml. All

transformants contained plasmids with the 9.5 kb pNG2 EcoRI fragment. Plasmid DNA (2 μg) from one of these clones (pNG2RI) was partially digested with Pstl and blunted using T4DNA polymerase. DNA fragments smaller than 10 kb were purified from a 1% agarose gel and used to transform E. coli JM109 to kanamycin resistance. A restriction map of a resulting plasmid (pEP2) isolated from a transformant was derived using standard procedures (Sambrook et al., Supra) and is shown in Figure 1A.

Plasmid pEP2 has a molecular weight of 3.1 kb as determined by agarose gel electrophoresis. This plasmid retains one of the pUC4K polylinkers and hence has unique Pstl , SaIl , BamH1 and EcoRI sites. Hybridization

analysis shows that plasmids pEP2 and pNG2RI (pNG2 after digestion with EcoRI ) both contain an 800 bp Hindlll fragment. Southern blotting of digests of sub-clones of pNG2 with the pEP2 plasmid showed that the region of PNG2 marked in Figure 1 was that incorporated in the pEP2 plasmid, and thus the area containing the origin of replication.

To determine whether hygromycin resistance could be used as a selectable marker in Mycobacteria species and C. pseudotuberculosis a hygromycin phosphotransferase gene (hph) was cloned into the Mycobacterium shuttle vector pEP.

To clone the hygromycin phosphotransferase gene (hph) from pPB3 into pEP2, pUC4K DNA was linearised, with Xhol and then blunted using the Klenow fragment of DNA polymerase I. pPB3 DNA was digested for 30s on ice using 5 units each of Alul, Haelll and Rsal. Fragments 2.0kb in size were gel purified (Geneclean, Bresatech) and ligated overnight with the linear, blunted pUC4K DNA. The

ligation mix was electroporated into JM109 and

recombinants were selected on LB plates supplemented with 150ug hygromycin B (Sigma) per ml. A 1.7kb Sall-Clal insert from a pUC4K chimera was ligated into the Sall- Clal site within the kanamycin resistance gene of pEP2. JM109 was transformed to hygromycin resistance with the ligation mix as described above and a restriction map of a hygromycin resistance plasmid (pEP3) was derived. The plasmid pEP3 has a unique Sail site and is approximately 4.5 kb in size (Fig. 2).

The nucleotide sequence of pEP2 excluding the kanamycin resistant gene is presented in Figure 3.

Examination of the DNA sequence revealed a single open reading frame (ORF). A number of potential translational start codons can be identified within this region but only one is preceded by a putative ribosome binding site (Figure 2). Although the sequence upstream of this ORF (ORFA) does not possess an E. coli consensus promoter, a putative rho-dependent transcriptional terminator was found downstream of the stop codon (Figure 2). This putative terminator has a 90% match to the TAATCAATAT consensus sequence (Ryder et al., Initiation of DNA Replication [1981], Academic Press, New York) and as has been described for other rho-dependent terminators

(Rosenberg et al., Nature 272, 414-428 [1978]), and is preceded by a region of dyad symmetry ( Figure 2). ORFA is therefore capable of coding for a 28kDa protein, a size consistent with that reported for other Rep

proteins.

In addition to the predicted size of the ORFA protein, further evidence to suggest that we have

identified the legitimate translated region arises from an examination of the derived amino acid sequence.

Firstly, the codon bias for the putative ORFA product (Phe, TTC; Asp, GAC; Arg, CGC; lIe, ATC; Val, GTC; Ala, Gcc; Thr, ACC) is the same as that for other

Corynebacterium proteins. Secondly, the predicted protein encoded by ORFA is highly basic in nature (19% basic residues) which is a characteristic of Rep proteins

(and other DNA binding proteins) thought to be important in the role they play in replication and incompatibility.

Database searches were performed using both the complete 1.85kb nucleotide sequence and the predicted amino acid sequence of ORFA, however no significant homologies were found. In addition, more detailed analyses revealed no similarities between the pEP2 Rep region and either of the potentially related plasmids from Corynebacterium (pBL1, Martin et al., Biotechnol. 5: 137-146 [1987]) and Mycobacterium (pAL5000, Rauzier et al., Gene II: 315-321 [1988]).

Plasmid replication regions invariably have an origin of replication. Most commonly, origins are located in non-coding regions, possess clusters of direct and inverted repeats and may be preceded by A+T rich sequences (Kamio et al., J. Bacteriol. 258: 307-312

[1984], Scott et al., Microbiol. Rev. 48: 1-23 [1984], Rosen et al., Mol. Gen. Genet. 179: 527-537 [1980],

Rauzier et al., Gene 71: 315-321 [1988]). The region upstream of ORFA contains no ORFs and possesses a number of direct and inverted repeat DNA sequences ( Figure 2). In addition, the region between nt 86 and 171 is 63.5% A+T compared with an average of 45% for the entire Rep region (Figure 2). We therefore believe this to be the pEP2 origin of replication.

The replication region of pEP2 is capable of

encoding a single 28kDa protein and possesses a single origin allowing plasmid replication in both Gram-positive and negative bacteria. pEP2 is therefore a unique shuttle vector and should be a useful tool, for example, in the genetic analysis of Mycobacterium and in the development of M. bovis BCG as a live recombinant

vaccine.

Electroporation of Mycobacteria with pEP2 and pEP3:

Electroporation of Mycobacterium species with pEP2 DNA resulted in transformation to kanamycin resistance respectively (Table 1). To confirm that the plasmids were present in the mycobacteria, total cellular DNA

preparations were made from the kanamycin resistant transformants. Figure 4 shows the results of agarose gel and Southern blot analysis of total cell DNA extracted from BCG CSL strains that had been transformed with pEP2. The pEP2 plasmid is clearly present in the transformed strains. Furthermore, the relative intensity of the gel bands suggests that the pEP plasmids replicate to high copy number in these bacteria. In addition, when total genomic DNA isolated from the drug resistant

transformants was used to electroporate E. coli,

approximately 1.0 X 10⁵ kanamycin resistant clones were obtained per ug DNA. Taken together these data suggest that the pEP2 replicon promotes stable plasmid

replication to high copy number in these bacteria.

Plasmid pEP3 was capable of transforming M.

smegmatis and M. bovis BCG CSL to resistance to at least 200ug hygromycin per ml ( Table 1). Total cellular DNA isolated from pEP3 transformed M. smeσmatis was capable of transforming E. coli to hygromycin resistance. This shows that the php gene encoding hygromycin resistance is effective in both mycobacteria and E. coli and thereby

constitutes a useful marker for these bacteria.

N/D = Not done, ---= no transformation to resistant phenotype

Different figures for each electroporation represent the result of separate experiments.

Electroporation of Other Bacterial Species with

pEP2 and pEP3:

To determine the host range of the pEP replicon, pEP2 and 3 plasmid DNA was electroporated into

Cornybacterium pseudotuberculosis. Transformation occurred in C. pseudo tuberculosis (Table 1). These results indicate that the pEP2 plasmids have a wide host range.

EXAMPLE 3

Generation and Expression of pEP2 Constructs:

A PCR derived DNA fragment carrying the promoter from the Mycobacterial 65kDa heat shock protein (0.6Kb) was cloned into the Pst1-BamH1 sites of pEP2, generating a plasmid referred to as pEP5. This promoter is known to function in E. coli as well as Mycobacteria and is therefore useful in this shuttle vector expression system.

A gene encoding for chloramphenicol-acetyl- transferase (CAT) was cloned into the BamH1 site of pEP5. Expression of the CAT gene driven by the 65kDa promoter was detected using the Pharmacia (Registered trademark, Pharmacia, Pitcataway, N.J., U.S.A.) CAT detection kit. According to this assay the 64kDa promoter has a strength comparable with the induced lac promoter in both E. coli and C. pseudotuberculosis.

MPB70 is the major secreted protein of M. bovis and a component of PPD. This gene and its signal sequence was incorporated into the pEP2 expression system. A truncated form of the MPB70 gene was generated by PCR removing its promoter but leaving its ribosome binding site (RBS) intact. This was cloned into pEP2 as a Pst1- BamH1 fragment (0.5Kb). The 65kDa promoter was then cloned upstream of the MPB70 BS on a Pst1-Scal fragment.

After electroporation into E. coli and C.

pseudotuberculosis, expression was tested for by western blots and Elisas. This construct did not express in E. coli. It did however express in C. pseudotuberculosis with product detected in both the solicate and culture filtrate. Ths indicated that the MPB70 RBS was inactive in E. coli yet was recognised and functional in C.

pseudotuberculosis.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically

described. It is to be understood that the invention includes all such variations and modifications which fall within its spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification,

individually or collectively, and any and all

combinations of any two or more of said steps or

features.

Claims

CLAIMS :

1. A DNA shuttle vector carrying a replication region comprising a single origin of replication, said origin of replication allowing replication of said vector in Mycobateria and E.coli cells; and a selectable marker.

2. A shuttle vector according to claim 1

comprising a first nucleotide sequence operably linked to said origin of replication, and containing one or more restriction endonuclease cleavage sites for the insertion of a desired second nucleotide sequence.

3. A shuttle vector according to any one of claims 1 or 2 which comprises one or more promoters having operably linked thereto a nucleic acid sequence encoding a desired polypeptide.

4. A shuttle vector according to claim 1 wherein said selectable marker confers resistance to an

antibiotic or enzyme, or encodes an immunologically detectable protein or chemical compound capable of causing a detectable reaction when provided with a suitable substrate.

5. A shuttle vector according to claim 4, wherein said selectable marker confers resistance to ampicillin, streptomycin, penicillin, hygromycin or kanamycin.

6. A shuttle vector according to claim 4, wherein said selectable marker is a gene encoding β- galactosidase, urease or alkaline phosphotase.

7. A shuttle vector according to claim 3, wherein said promoter is selected from the P1 promoter of bacteriophage lambda, trp promoter, lac promoter, kanamycin resistance promoters of transposon Tn903 or transposon Tn5, mycobacteria promoter of the common mycobacterial 65Kd antigen, ribosomal RNA promoter of mycobacteria, promoter of M.bovis antigens MPB70, MPB59 and MPB64, metallothionine promoter, growth hormone promoter or hybrids between eukaryotic and prokaryotic promoters.

8. A shuttle vector according to claim 3, wherein said desired peptide encodes one or more antigens from disease causing bacteria, viruses or parasites.

9. A shuttle vector according to claim 8, wherein said one or more antigens are selected from antigens of Taenia ovis, rotavirus, Babesia bovis, Mycobacterium bovi,. Mycobacterium tuberculosis, Mycobacterium leprae, Mybacterium paratuberculosis, Bacteriodes nodosus or Bordetella.

10. A shuttle vector according to claim 3, wherein said desired polypeptide is a hormone or an epitope thereof.

11. A shuttle vector according to claim 1, wherein said origin of replication comprises the replication region of the Corynebacterium plasmid pNG2 or a fragment thereof which permits replication of said vector in mycobacteria and E.coli.

12. A shuttle vector according to any one of claims 1 to 11, capable of replication in Actinomycetes.

13. A shuttle vector according to any one of claims 1 to 11, capable of replication in Corynebacteria.

14. A shuttle vector selected from pEP2 and pEP3.

15. A bacterial host cell containing a shuttle vector according to any one of claims 1 to 14.

16. A host cell according to claim 15 selected from Mycobacteria, E. coli, Corynebacteria, and Actinomycetes.

17. A host cell according to claim 15 selected from M. pheli, M. smegmatis, M. bovis BCG, E. coli JM109, C. pseudotuberculosis.

18. A DNA sequence encoding the origin of

replication of Corynebacteriurn plasmid pNG2 or a fragment thereof which permits replication of a vector containing said DNA sequence in Mycobacteria and E. coli.

19. A DNA sequence according to claim 17 comprising an origin of replication operably linked to said origin of replication.

20. A DNA sequence according to claim 18 where the DNA sequence encodes a desired polypeptide is operably linked to said promoter.

21. A polypeptide expressed by a host cell wherein said polypeptide is encoded by a shuttle vector according to any one of claims 4 and 8 to 9, resident within said host cell.