CA2076753A1 - Shuttle plasmid for escherichia coli and mycobacteria - Google Patents
Shuttle plasmid for escherichia coli and mycobacteriaInfo
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- CA2076753A1 CA2076753A1 CA 2076753 CA2076753A CA2076753A1 CA 2076753 A1 CA2076753 A1 CA 2076753A1 CA 2076753 CA2076753 CA 2076753 CA 2076753 A CA2076753 A CA 2076753A CA 2076753 A1 CA2076753 A1 CA 2076753A1
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- 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
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Abstract
DNA shuttle vectors which are capable of replication in Mycobacteria, E. coli and other bacterial hosts are described. The shuttle vectors comprise a single origin of replication which confers the ability of replication in a number of bacterial species.
Also disclosed are shuttle vectors encoding desired polypeptides, such as antigens of disease causing bacteria, viruses and parasites.
Also disclosed are shuttle vectors encoding desired polypeptides, such as antigens of disease causing bacteria, viruses and parasites.
Description
20767~
WO91/13157 PCT/AU91/00064 :
' 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 ~-20 hosts containing such DNA vectors, and further relates to `~
vaccines containing such bacter:ia. ~ :
Mvcobacterium bovis BCG (hereinafter BCG) has been ~:
used for many years as a ~accine against tuberculosis tTB). Th~ vaccination programme has been extremely 25 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 ~ .
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WO91/13157 PCT/AU91/00064 :
' 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 ~-20 hosts containing such DNA vectors, and further relates to `~
vaccines containing such bacter:ia. ~ :
Mvcobacterium bovis BCG (hereinafter BCG) has been ~:
used for many years as a ~accine against tuberculosis tTB). Th~ vaccination programme has been extremely 25 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 ~ .
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further capable of replication in Mycobacterium at or near tAe 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 (l): 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 betwsen 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 s~tes which may be introduced into the plasmids.
The presen~ applicant has overcome problPms 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 25 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 accordan~e witA 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 addltionally comprise a nucleotide sequence containing one or more restriction site~ for the insertion of a ., . . :: ~ . : -- - ~
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207~7~
WO9~/13ls7 PCT/~U91/00064 desired nucleotide sequence.
Preferably, the repllcation reglon corresponds to the replication region of the corynebacteria plasmid pNG2 or a fragment thereof which permits repllcation of said vector in mycobacteria and E. coli.
Surprislngly, the replication region of plasmid pNG2 confers the ability of replication in various bacterial species, apart from Corynebacteria.
The DNA shut~le 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 promotsrs and downstream restriction endonuclease sites.
The term "shuttle vector" as used herein includes plasmid DNA which may be double-stra~ded 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 marke!r" 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 provid~d with a suitable substrate. Examples include resistance to ampicillin, streptomycin, penicillin, hygromycin, kanamycin, and the like, ~-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 .
.
, WO91/131~7 2 0 7 6 7 ~ ~ PCT/AU91/000~
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promoters such as the Pl promoter of bacterlophage lambda, trp promoter, lac promoter, kanamycin resistance promoters of transposon Tn903 and transposon Tn5, mycobacterial promoters such as that of the promoter of the com~on mycobacterial 65Kd antigen, rtbosomal RNA
promoter of mycobacteria, promoters of M.bovis antigens MPB70, MPB59 and MPB64 and the like, hybrids betwPen 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, Kpnl, HindIII, 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 ~equences of interest may be inserted into the endonuclease cleava~e 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 Sambroo~ et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd Edition, 1989). A nucleotide sequence fDr 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 ::.
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, . , WO91/13157 2 0 7 6 7 ~ ~ PCT/AU91/00064 sequence belng 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 in~o membranes of a suitable host cell or secretion from a host cell. Absence of a secretory leader will cause th,e 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 ~here is provided a DNA shuttle vector pEP2, said vector having a size of about 3.l kh, as detsrmined by agarose ~el electrophoresis, a replication region of about l.85 kb comprising single origin of replication derived from the Corynebacterium replicon pNG2, an antibiotic r sistance gene to kanamycin, and a nucleotide sequence containing a number of restriction endonuclease cleavage sites for the insertion o 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 .
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WO91/13157 ~ 7 6 7 .S C) PCT/AU91/00064 --replication in species of both gram nega~ive and gram positive hosts, such as Mvcobacterium, E. ccli, Corynebacterlum, and Ac-tinomvcetes, as defined in Bergey's Manual of Determinative Bacteriology, 8th Edition, pp. 599-861. ~he 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 no~ 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 shu~tle vector as herein defined. The shuttle vector may be present as a single copy, or ~ore pre~erably as multiple copies thereof within the bacterial host. Preferably, but in no way limiting the invention, the bacterial host is a Mvcobacteria, Corynebacteria or E. coli strain, such as C.
~seudotuberculosis, M. sme~matis, and M. bovis BCG. ~he 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 reside~t within the host cell :
'~ . ' `'. ',' , '' ~ `' ' ',' ' , ' ' ' , ''' :'' . ' ' ' ' :: ' ' , ' ' ' ' ' ~ ' ' W0~1/13157 20767r~ Pcl/~u9l/ono64 itself. Bacterlal hosts expressing deslred an~igens 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 expre~sed by a bact~rial host cell containing a shuttle vector as herein defined.
This invention contemplates deletions or insertions j~A
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 Eo coli. Techniques for lr.sertion or deletion of nucleotide sequences are well known in the art. Mutants could be readily tested for the ability to ; confar r~plication in gram negative and gram positive host cells, such as E. coli and corynebacteria.
This invention also extends to replication region of 20 pNG2 itself or fragments thereof, which, on inser~ion ~-intc suitable vector are capable of permitting replication of said vector mycobacteria and E. coli.
A culture of Eschrichia coli containing plasmid p~P2 was deposited under the terms and conditions of the Budapest Treaty at the Australian Government Analytical Laboratory (AGAL), Pymble, New 'outh Wales, Australia on 23rd February, l990 and accorded Accession No.
N90/007030.
~his invention will now be illustrated with 30 reference to the following non-limiting Figures and ~-Examples.
FIGURES
Figure 1 shows the construction of plasmid pEP2.
Plasmid pN~2 (14.5) was digested with EcoRI and the largest fragment ligated to the Kanr cartridge of pUC4K.
Following electroporation into E.coli the resulting plasmid DNA was extracted and partially digested with :. : , :, - , ., . : :, , :: :- .
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; Pstl, and then religated and again electroporated wlth E.coli. One of the resulting Kan' colonies contained plasmid pEP2. Restriction siles: BtBamHl)~ E( EcoRI), ~(HindIII), Hc(HlncII), P(PstI), 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 (HindIII), Hc (HincII), K (X~nI), p (PstI), S (SalI).
Kanamycin reslstance gene \\\\\ from pUC4K. -B Sequencing strategy. Arrows indicate the extent and direction of sequence generated from restriction fragments cloned in Ml3 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 l (E (EcoRI), H
(HindIII), Hc (HincII), K (K~nI). IR and DR, inverted and direct repeat sequences respectively: i~BS, 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 , 25 plasmid pEP2 extracted from E.coli. Tracks contain (A) Undigested pEP2 DNA from E.coli 500ng, (B) Pst l diyested pEP2 DNA from E.coli 200n~, ~C) PstI digested whole DNA
extract of M. bovis BCG pEP2, 2.5ug, (D) PstI digested M.
bovis BCG DNA, 2.5 ug (E) Undigested DNA extract of M.
bovis ~CG pEP2, 2.5ug, (F) Undigested DNA extract of M.
- bovis BCG, 2.5ug, (G) HindIII digested lambda DNA
markers.
' EXAMPLE 1 Recombinant DNA Procedures:
~ 35 Unless otherwise specified herein, manipulation of ; recombinant molecules and the preparation of solutions , are by standard known techniques. Such techniques are .', ~
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g described in Sambrook et al. Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor [1989], pp. 1-500.
Bacterial S~rains and Plasmids:
S Mycobacterium bovis BCG variant CSL was obtained as lyophylised human vaccine from the Commonwealth Serum Laboratories (CSL) Australia. M. sme~matis and M. Dheli 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 B4: 4964-4968) and pPB3 was obtained from Dr. Philip Bird (Monash University Faculty of Medicine, Alfred ~ospi~al, Melbourne). Plasmid pAL8 (Gicquel-Sanzey et al. 1989 15 Acta Leprol. 7: 207-211) was obtained from Dr. Brigette Gicquel-Sanzey (Pasteur Institute, Paris) and plasmid pUGC4~ 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:
Mvcobacter~um speci~s and CorYnebacterium ~seudo-tuberculosis were electroporated according to Lugosi et al. (~uber~ule 70. 159-170 tl98g]) using a Gene Pulser -~
commercially available from Bio-Rad Laboratories Inc.
Transformants were selected on 7Hll or nutrient media containing 100 ~g kanamycin per ml or 200~g 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. ~iochem. 1981, 178: 82-87).
Mycobacterial cells were harvested from 400 mls of Dubos " . -: : : .: , : . - .: .: . ~ : :
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WO91/131~7 2 0 7 S 7 ~ ~ PCT/AU91/000~
broth by centrifugatlon, resuspended in 5 ml of TE buffer and heat-killed by treatment at 70C for l 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 liquld nitrogen in a mortar pre-cooled in a bath of liquid nitrogen and crushed into a fine powder uslng a pre-cooled 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 bufer (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 4C for l0 minutes. The pellet was dissolved in l.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. Dseudotuberculosis c as previously described (Hodgson et al., l990).
pNG2RI DNA was digested with various restriction enzymes and the fragments Southern blotted (Reid et al.) ~;
to Hybond N (Amersham) nylon filters. ~ilters were 25 hybridised overnight at 37C with pEP2 labelled with 32P
using random hexamer primers, washed at increasing stringency as necessary (up to 65C) 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 do~
`~ blot hybridisation. C~ pseudotuberculosis transformants were analysed in the same fa~hion.
Total cell DNA was used to isolate and transform E.coli to kanamycin or hygromycin resistance.
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Nucleotide sequence analysis was determined by the dideoxy chain termination method (Sanyer et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) using modified T7 DNA polymerase (Pharmaoia LKB, Uppsula, Sweden). The complete nucleotide sequence of pEP2 was derived on both strands using universal or oligonucleotide primers (Figure lB). The laLter 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).
Shuttle Vector Construction a~d Analysi~ Thereof:
The l.3 kb EcoRl fragment from pUC4K carrying the ~anamycin resistance gene was ligated to pNG2 DNA
digested wi~h EcoRl. The ligation mix was electroporated into E.coli JMlO9 and transformant selected on L~ plates supplemented with 50,ug kanamycin per ml. All transformants contained plasmids with the 9.5 kb pNG2 EcoRl fragment. Plasmid DNA (2 ~g) from one of these clones (pNG2~I) was partially digested with PstI and blun~ed using T4DNA polymerase. DNA fra~ments smaller than lO kb were purified from a l~ agarose gel and used to transform E. coli JMl09 to kanamycin resistance. A
restriction map of a resulting plasmid tpEP2) isolated from a transformant was derived using standard procedures (Sambrook et al., Supra) and is shown in Figure lA.
Plasmid pEP2 has a molecular weight of 3.l kb as determined by agarose gel electrophoresis. This plasmid retains one of the pUC4K polylinkers and hence has unique PstI, SalI, BamHl and EcoRI sites. Hybridization analysis shows that plasmids pEP2 and pNG2RI (pNG2 after digestion with EcoRI) both contain an 800 bp HindIII
fragment. Southern blotting of digests of sub-clones of pNG2 with the pEP2 plasmid showed that the region of PNG2 marked in Figure l was that incorporated in the pEP2 - . -. , . :, , , . :.
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WO91/131~7 2 Q 7 6 7 A~ ~ PCT/AU91/00064 -plasmid, and thus the area containing the origin of replication.
To determine whether hygromycin resistance could be used as a selectable marksr in Mycobacteria species and ~
5 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 l0 XhoI and then blunted using the Klenow fragment of DNA - -polymerase I. pPB3 DNA was digested for 30s on ice using 5 units each of AluI, HaeIII and RsaI. Fragments ~.Okb in size were gel purified (Geneclean, Bresatech) and ligated overnight with the linear, blunted pUC4K DNA. The ligation mix was electroporated into JMlO9 and recombinants were selected on LB plates supplemented wi~h 150ug hygromycin B (Sigma) per ml. A l.7kb SalI-ClaI~ ~
insert from a pUC4K chimera was ligated into the SalI- ~ .
ClaI site within the kanamycin resistance gene of pEP2.
20 JMl09 was transformed to hygromycin resistance with ~he ~.
ligation mix as described above and a res~riction map of :~
a hygromycin resistance plasmid (pEP3) was derived. The plasmid pEP3 has a unique SalI site and $s 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 D~A 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 9O~ match to the TAATCAATAT
consensus sequence tRyder et al., Initiation of D~A
Replication [1981], Academic Press, New York) and as has `'', ~:
WO91/13157 2 0 7 6 7 ~ ~ PCT/AU~1/OOn64 bePn descrlbed 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 tharefore 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; Ile, ATC; Val~ GTC; Ala, Gcc; Thr, ACC) is the same as that for other Corvnebacterium proteins. Secondly, the predicted ~; 15 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 inco~patibility.
Database searches were performed usin~ 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 Corvnebacterium (pBL1, Martin et al., Biotechnol. 5:
137-146 [1987]) and MYcobacterium (pAL5000, Rauzier et al., Gene II: 315-321 [19883).
;! .
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 praceded 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 C1980], 35 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).
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WO91/13l~7 PC~/AU91/000~
2~767~3 14 -In addition, the region between nt 86 and 171 ls 63.5 A~T compared with an average o 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. p~P2 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 Mvcobacterium 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 resistan~
transformants. Figure 4 shows the results of agarose gel and Southern blot analysis of ~otal 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 relat:Lve intensity of ~he gel bands suggests that the pEP plasmids replicate to high copy number in these bacteria. In additlon, 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 l). Total cellular DNA
isolated from pEP3 transformed M. s0eamatis was capable of transforming E. coli to hygromycin resistance. This shows that the php gene encoding hygromycin resistance is ::
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W091/13157 2 0 7 6 7 ~ ~ PCT/AU91/000~
effective in both mycobacteria and E, coll and thereby constitutes a useful marker for these bacteria.
~ABI.~ 1 EL~CTROPORATIO~ 0~ MYCOBACT~RIA AND RE~AT~D SPECIES
_____________________________ _____ _ --Plasmid electroporation efficiency:~anr CFU
per ~icrogram of plasm~d DNA
pEP2 pAL8 pEP3 Bacterl~l Specie~
Mvcobact@rium Dhlei --- 103 Mycobacterium smeqmatis lo2 lol lo2 ~Y5353~3~3~9~ bovis 1o2 1o2 N/D
lO2 103 BCG v~r CSL lo2 104 Cor~nebacterium ovis 103 ___ lO2 t pseudot.uberculosis ) 104 ________________________ N~D = Not done, ---= no transformation ~o resistant phenotype :~
Different f~gures for each electroporatlon represent the rosult of ~epar~te expcrimente.
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WO9l/13t~7 ~ 7 6 7~ ~ PCT/AU91/00064 lectroporation of Other Bacterial Species with pEP2 and ~EP3 ~ . _ To determine the host range of the pEP replicon, pEP2 and 3 plasmid DNA was electroporated into Corn~bacterium ~eudotuberculosis. Transformation occurred in C. pseudo tuberculosis tTable 1). These results indicate that the pEP2 plasmids have a wide host ,~
range.
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 Pstl-BamHl 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-~ran~ferase (CAT) was cloned in~o the BamHl site of pEP5.
Expression of the CAT gene driven by the 65kDa promoter was detected using the Pharmacia (Registered trademarX, i Pharmacia, Pitcataway, N.J., U.';.A.) C~T detection kit. ~;
~ccording to this assay the 64kl)a 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 PStl~
BamHl fragmen~ (O.SKb). The 65kDa promoter was then cloned upstream of the MPB70 BS on a Pstl-Scal fragment.
After electroporation into E. coli and C.
Dseudotuberculosis, expression was tested for by western blots and Elisas. This construct did not express in E. ~;~
~.
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.
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WO91/131~7 PCT/AU91/00064 coli. It did however express ln C. pseudotube.rculosis 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
pseudotuberculosls.
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 ~he 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.
,:
,
Multiple origins of replication have been necessary due to the evolutionary distance betwsen 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 s~tes which may be introduced into the plasmids.
The presen~ applicant has overcome problPms 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 25 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 accordan~e witA 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 addltionally comprise a nucleotide sequence containing one or more restriction site~ for the insertion of a ., . . :: ~ . : -- - ~
.
207~7~
WO9~/13ls7 PCT/~U91/00064 desired nucleotide sequence.
Preferably, the repllcation reglon corresponds to the replication region of the corynebacteria plasmid pNG2 or a fragment thereof which permits repllcation of said vector in mycobacteria and E. coli.
Surprislngly, the replication region of plasmid pNG2 confers the ability of replication in various bacterial species, apart from Corynebacteria.
The DNA shut~le 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 promotsrs and downstream restriction endonuclease sites.
The term "shuttle vector" as used herein includes plasmid DNA which may be double-stra~ded 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 marke!r" 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 provid~d with a suitable substrate. Examples include resistance to ampicillin, streptomycin, penicillin, hygromycin, kanamycin, and the like, ~-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 .
.
, WO91/131~7 2 0 7 6 7 ~ ~ PCT/AU91/000~
_ a, .
promoters such as the Pl promoter of bacterlophage lambda, trp promoter, lac promoter, kanamycin resistance promoters of transposon Tn903 and transposon Tn5, mycobacterial promoters such as that of the promoter of the com~on mycobacterial 65Kd antigen, rtbosomal RNA
promoter of mycobacteria, promoters of M.bovis antigens MPB70, MPB59 and MPB64 and the like, hybrids betwPen 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, Kpnl, HindIII, 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 ~equences of interest may be inserted into the endonuclease cleava~e 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 Sambroo~ et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd Edition, 1989). A nucleotide sequence fDr 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 ::.
.: .,. , : , - . -.: :. .: ,., .. , ., :-.. , ... - . :.
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, . , WO91/13157 2 0 7 6 7 ~ ~ PCT/AU91/00064 sequence belng 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 in~o membranes of a suitable host cell or secretion from a host cell. Absence of a secretory leader will cause th,e 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 ~here is provided a DNA shuttle vector pEP2, said vector having a size of about 3.l kh, as detsrmined by agarose ~el electrophoresis, a replication region of about l.85 kb comprising single origin of replication derived from the Corynebacterium replicon pNG2, an antibiotic r sistance gene to kanamycin, and a nucleotide sequence containing a number of restriction endonuclease cleavage sites for the insertion o 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 .
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.
WO91/13157 ~ 7 6 7 .S C) PCT/AU91/00064 --replication in species of both gram nega~ive and gram positive hosts, such as Mvcobacterium, E. ccli, Corynebacterlum, and Ac-tinomvcetes, as defined in Bergey's Manual of Determinative Bacteriology, 8th Edition, pp. 599-861. ~he 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 no~ 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 shu~tle vector as herein defined. The shuttle vector may be present as a single copy, or ~ore pre~erably as multiple copies thereof within the bacterial host. Preferably, but in no way limiting the invention, the bacterial host is a Mvcobacteria, Corynebacteria or E. coli strain, such as C.
~seudotuberculosis, M. sme~matis, and M. bovis BCG. ~he 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 reside~t within the host cell :
'~ . ' `'. ',' , '' ~ `' ' ',' ' , ' ' ' , ''' :'' . ' ' ' ' :: ' ' , ' ' ' ' ' ~ ' ' W0~1/13157 20767r~ Pcl/~u9l/ono64 itself. Bacterlal hosts expressing deslred an~igens 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 expre~sed by a bact~rial host cell containing a shuttle vector as herein defined.
This invention contemplates deletions or insertions j~A
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 Eo coli. Techniques for lr.sertion or deletion of nucleotide sequences are well known in the art. Mutants could be readily tested for the ability to ; confar r~plication in gram negative and gram positive host cells, such as E. coli and corynebacteria.
This invention also extends to replication region of 20 pNG2 itself or fragments thereof, which, on inser~ion ~-intc suitable vector are capable of permitting replication of said vector mycobacteria and E. coli.
A culture of Eschrichia coli containing plasmid p~P2 was deposited under the terms and conditions of the Budapest Treaty at the Australian Government Analytical Laboratory (AGAL), Pymble, New 'outh Wales, Australia on 23rd February, l990 and accorded Accession No.
N90/007030.
~his invention will now be illustrated with 30 reference to the following non-limiting Figures and ~-Examples.
FIGURES
Figure 1 shows the construction of plasmid pEP2.
Plasmid pN~2 (14.5) was digested with EcoRI and the largest fragment ligated to the Kanr cartridge of pUC4K.
Following electroporation into E.coli the resulting plasmid DNA was extracted and partially digested with :. : , :, - , ., . : :, , :: :- .
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; Pstl, and then religated and again electroporated wlth E.coli. One of the resulting Kan' colonies contained plasmid pEP2. Restriction siles: BtBamHl)~ E( EcoRI), ~(HindIII), Hc(HlncII), P(PstI), 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 (HindIII), Hc (HincII), K (X~nI), p (PstI), S (SalI).
Kanamycin reslstance gene \\\\\ from pUC4K. -B Sequencing strategy. Arrows indicate the extent and direction of sequence generated from restriction fragments cloned in Ml3 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 l (E (EcoRI), H
(HindIII), Hc (HincII), K (K~nI). IR and DR, inverted and direct repeat sequences respectively: i~BS, 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 , 25 plasmid pEP2 extracted from E.coli. Tracks contain (A) Undigested pEP2 DNA from E.coli 500ng, (B) Pst l diyested pEP2 DNA from E.coli 200n~, ~C) PstI digested whole DNA
extract of M. bovis BCG pEP2, 2.5ug, (D) PstI digested M.
bovis BCG DNA, 2.5 ug (E) Undigested DNA extract of M.
bovis ~CG pEP2, 2.5ug, (F) Undigested DNA extract of M.
- bovis BCG, 2.5ug, (G) HindIII digested lambda DNA
markers.
' EXAMPLE 1 Recombinant DNA Procedures:
~ 35 Unless otherwise specified herein, manipulation of ; recombinant molecules and the preparation of solutions , are by standard known techniques. Such techniques are .', ~
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~767~3 WO9l/l31~7 PCT/AU91/000~
g described in Sambrook et al. Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor [1989], pp. 1-500.
Bacterial S~rains and Plasmids:
S Mycobacterium bovis BCG variant CSL was obtained as lyophylised human vaccine from the Commonwealth Serum Laboratories (CSL) Australia. M. sme~matis and M. Dheli 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 B4: 4964-4968) and pPB3 was obtained from Dr. Philip Bird (Monash University Faculty of Medicine, Alfred ~ospi~al, Melbourne). Plasmid pAL8 (Gicquel-Sanzey et al. 1989 15 Acta Leprol. 7: 207-211) was obtained from Dr. Brigette Gicquel-Sanzey (Pasteur Institute, Paris) and plasmid pUGC4~ 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:
Mvcobacter~um speci~s and CorYnebacterium ~seudo-tuberculosis were electroporated according to Lugosi et al. (~uber~ule 70. 159-170 tl98g]) using a Gene Pulser -~
commercially available from Bio-Rad Laboratories Inc.
Transformants were selected on 7Hll or nutrient media containing 100 ~g kanamycin per ml or 200~g 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. ~iochem. 1981, 178: 82-87).
Mycobacterial cells were harvested from 400 mls of Dubos " . -: : : .: , : . - .: .: . ~ : :
: ' '''' ' ' ' : .. ' ' ' ,, . :
. .
WO91/131~7 2 0 7 S 7 ~ ~ PCT/AU91/000~
broth by centrifugatlon, resuspended in 5 ml of TE buffer and heat-killed by treatment at 70C for l 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 liquld nitrogen in a mortar pre-cooled in a bath of liquid nitrogen and crushed into a fine powder uslng a pre-cooled 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 bufer (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 4C for l0 minutes. The pellet was dissolved in l.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. Dseudotuberculosis c as previously described (Hodgson et al., l990).
pNG2RI DNA was digested with various restriction enzymes and the fragments Southern blotted (Reid et al.) ~;
to Hybond N (Amersham) nylon filters. ~ilters were 25 hybridised overnight at 37C with pEP2 labelled with 32P
using random hexamer primers, washed at increasing stringency as necessary (up to 65C) 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 do~
`~ blot hybridisation. C~ pseudotuberculosis transformants were analysed in the same fa~hion.
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 (Sanyer et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) using modified T7 DNA polymerase (Pharmaoia LKB, Uppsula, Sweden). The complete nucleotide sequence of pEP2 was derived on both strands using universal or oligonucleotide primers (Figure lB). The laLter 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).
Shuttle Vector Construction a~d Analysi~ Thereof:
The l.3 kb EcoRl fragment from pUC4K carrying the ~anamycin resistance gene was ligated to pNG2 DNA
digested wi~h EcoRl. The ligation mix was electroporated into E.coli JMlO9 and transformant selected on L~ plates supplemented with 50,ug kanamycin per ml. All transformants contained plasmids with the 9.5 kb pNG2 EcoRl fragment. Plasmid DNA (2 ~g) from one of these clones (pNG2~I) was partially digested with PstI and blun~ed using T4DNA polymerase. DNA fra~ments smaller than lO kb were purified from a l~ agarose gel and used to transform E. coli JMl09 to kanamycin resistance. A
restriction map of a resulting plasmid tpEP2) isolated from a transformant was derived using standard procedures (Sambrook et al., Supra) and is shown in Figure lA.
Plasmid pEP2 has a molecular weight of 3.l kb as determined by agarose gel electrophoresis. This plasmid retains one of the pUC4K polylinkers and hence has unique PstI, SalI, BamHl and EcoRI sites. Hybridization analysis shows that plasmids pEP2 and pNG2RI (pNG2 after digestion with EcoRI) both contain an 800 bp HindIII
fragment. Southern blotting of digests of sub-clones of pNG2 with the pEP2 plasmid showed that the region of PNG2 marked in Figure l was that incorporated in the pEP2 - . -. , . :, , , . :.
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WO91/131~7 2 Q 7 6 7 A~ ~ PCT/AU91/00064 -plasmid, and thus the area containing the origin of replication.
To determine whether hygromycin resistance could be used as a selectable marksr in Mycobacteria species and ~
5 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 l0 XhoI and then blunted using the Klenow fragment of DNA - -polymerase I. pPB3 DNA was digested for 30s on ice using 5 units each of AluI, HaeIII and RsaI. Fragments ~.Okb in size were gel purified (Geneclean, Bresatech) and ligated overnight with the linear, blunted pUC4K DNA. The ligation mix was electroporated into JMlO9 and recombinants were selected on LB plates supplemented wi~h 150ug hygromycin B (Sigma) per ml. A l.7kb SalI-ClaI~ ~
insert from a pUC4K chimera was ligated into the SalI- ~ .
ClaI site within the kanamycin resistance gene of pEP2.
20 JMl09 was transformed to hygromycin resistance with ~he ~.
ligation mix as described above and a res~riction map of :~
a hygromycin resistance plasmid (pEP3) was derived. The plasmid pEP3 has a unique SalI site and $s 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 D~A 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 9O~ match to the TAATCAATAT
consensus sequence tRyder et al., Initiation of D~A
Replication [1981], Academic Press, New York) and as has `'', ~:
WO91/13157 2 0 7 6 7 ~ ~ PCT/AU~1/OOn64 bePn descrlbed 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 tharefore 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; Ile, ATC; Val~ GTC; Ala, Gcc; Thr, ACC) is the same as that for other Corvnebacterium proteins. Secondly, the predicted ~; 15 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 inco~patibility.
Database searches were performed usin~ 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 Corvnebacterium (pBL1, Martin et al., Biotechnol. 5:
137-146 [1987]) and MYcobacterium (pAL5000, Rauzier et al., Gene II: 315-321 [19883).
;! .
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 praceded 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 C1980], 35 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).
.,. ;~'~.
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WO91/13l~7 PC~/AU91/000~
2~767~3 14 -In addition, the region between nt 86 and 171 ls 63.5 A~T compared with an average o 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. p~P2 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 Mvcobacterium 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 resistan~
transformants. Figure 4 shows the results of agarose gel and Southern blot analysis of ~otal 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 relat:Lve intensity of ~he gel bands suggests that the pEP plasmids replicate to high copy number in these bacteria. In additlon, 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 l). Total cellular DNA
isolated from pEP3 transformed M. s0eamatis was capable of transforming E. coli to hygromycin resistance. This shows that the php gene encoding hygromycin resistance is ::
;.
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. .
W091/13157 2 0 7 6 7 ~ ~ PCT/AU91/000~
effective in both mycobacteria and E, coll and thereby constitutes a useful marker for these bacteria.
~ABI.~ 1 EL~CTROPORATIO~ 0~ MYCOBACT~RIA AND RE~AT~D SPECIES
_____________________________ _____ _ --Plasmid electroporation efficiency:~anr CFU
per ~icrogram of plasm~d DNA
pEP2 pAL8 pEP3 Bacterl~l Specie~
Mvcobact@rium Dhlei --- 103 Mycobacterium smeqmatis lo2 lol lo2 ~Y5353~3~3~9~ bovis 1o2 1o2 N/D
lO2 103 BCG v~r CSL lo2 104 Cor~nebacterium ovis 103 ___ lO2 t pseudot.uberculosis ) 104 ________________________ N~D = Not done, ---= no transformation ~o resistant phenotype :~
Different f~gures for each electroporatlon represent the rosult of ~epar~te expcrimente.
. .
.
- . .
: ; : . ; ~ ~ . ; ' .
' .
, :, . ~ . , ~ , , .:
WO9l/13t~7 ~ 7 6 7~ ~ PCT/AU91/00064 lectroporation of Other Bacterial Species with pEP2 and ~EP3 ~ . _ To determine the host range of the pEP replicon, pEP2 and 3 plasmid DNA was electroporated into Corn~bacterium ~eudotuberculosis. Transformation occurred in C. pseudo tuberculosis tTable 1). These results indicate that the pEP2 plasmids have a wide host ,~
range.
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 Pstl-BamHl 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-~ran~ferase (CAT) was cloned in~o the BamHl site of pEP5.
Expression of the CAT gene driven by the 65kDa promoter was detected using the Pharmacia (Registered trademarX, i Pharmacia, Pitcataway, N.J., U.';.A.) C~T detection kit. ~;
~ccording to this assay the 64kl)a 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 PStl~
BamHl fragmen~ (O.SKb). The 65kDa promoter was then cloned upstream of the MPB70 BS on a Pstl-Scal fragment.
After electroporation into E. coli and C.
Dseudotuberculosis, expression was tested for by western blots and Elisas. This construct did not express in E. ~;~
~.
', `, " . : : : . ~ .
.
~ u ~
WO91/131~7 PCT/AU91/00064 coli. It did however express ln C. pseudotube.rculosis 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
pseudotuberculosls.
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 ~he 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 (21)
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 .beta.-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 bovis, 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.
pseudotuberculosis.
18. A DNA sequence encoding the origin of replication of Corynebacterium 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 d aims 4 and 8 to 9, resident within said host cell.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AUPJ881590 | 1990-02-26 | ||
| AUPJ8815/90 | 1990-02-26 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2076753A1 true CA2076753A1 (en) | 1991-08-27 |
Family
ID=3774519
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2076753 Abandoned CA2076753A1 (en) | 1990-02-26 | 1991-02-25 | Shuttle plasmid for escherichia coli and mycobacteria |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0517751A4 (en) |
| JP (1) | JPH05504067A (en) |
| CA (1) | CA2076753A1 (en) |
| NZ (1) | NZ237217A (en) |
| WO (1) | WO1991013157A1 (en) |
Families Citing this family (34)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2720076B1 (en) | 1994-05-20 | 1996-08-02 | Inst Nat Sante Rech Med | Shuttle vectors for the introduction of DNA into mycobacteria and use of these bacteria as vaccines. |
| US7288254B2 (en) | 1995-10-30 | 2007-10-30 | The United States Of America As Represented By The Secretary, Department Of Health And Human Services, Nih | Use of immunotoxins to induce immune tolerance to pancreatic islet transplantation |
| US7696338B2 (en) | 1995-10-30 | 2010-04-13 | The United States Of America As Represented By The Department Of Health And Human Services | Immunotoxin fusion proteins and means for expression thereof |
| WO1998039363A2 (en) | 1997-03-05 | 1998-09-11 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Immunotoxins and methods of inducing immune tolerance |
| US7517527B2 (en) | 1995-10-30 | 2009-04-14 | The United States Of America As Represented By The Department Of Health And Human Services | Immunotoxin with in vivo T cell suppressant activity and methods of use |
| US7125553B1 (en) | 1996-04-15 | 2006-10-24 | The United States of America as represented by the Department of Health and Human Services c/o Centers for Disease Control and Prevention | Methods of inducing immune tolerance using immunotoxins |
| AU736501B2 (en) * | 1997-03-05 | 2001-07-26 | Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services, The | Novel vectors and expression methods for producing mutant proteins |
| US6686339B1 (en) | 1998-08-20 | 2004-02-03 | Aventis Pasteur Limited | Nucleic acid molecules encoding inclusion membrane protein C of Chlamydia |
| CA2340330A1 (en) | 1998-08-20 | 2000-03-02 | Aventis Pasteur Limited | Nucleic acid molecules encoding inclusion membrane protein c of chlamydia |
| US6693087B1 (en) | 1998-08-20 | 2004-02-17 | Aventis Pasteur Limited | Nucleic acid molecules encoding POMP91A protein of Chlamydia |
| JP2003512829A (en) | 1999-10-22 | 2003-04-08 | アヴェンティス パストゥール リミテッド | Modified GP100 and uses thereof |
| EP1792995A3 (en) | 2000-05-08 | 2007-06-13 | Sanofi Pasteur Limited | Chlamydia secretory locus orf and uses thereof |
| EP1741782B1 (en) | 2000-05-10 | 2011-06-22 | Sanofi Pasteur Limited | Immunogenic polypeptides encoded by MAGE minigenes and uses thereof |
| ATE357461T1 (en) | 2000-08-25 | 2007-04-15 | Aventis Pasteur | INNER-CORE OLIGOSACCHARIDE EPITOPES FROM LIPOPOLYSACCHARIDES OF HAEMOPHILUS INFLUENZA AS VACCINES IN THE PROPHYLACTIC TREATMENT OF HAEMOPHILUS INFLUENZAE INFECTIONS |
| US7060462B2 (en) | 2000-11-02 | 2006-06-13 | National University Of Singapore | AopB gene, protein,homologs, fragments and variants thereof, and their use for cell surface display |
| WO2003050268A2 (en) | 2001-12-12 | 2003-06-19 | Aventis Pasteur Limited | Enhancement of the immune response using cd36-binding domain |
| PT1496939E (en) | 2002-04-09 | 2007-11-22 | Sanofi Pasteur Ltd | Modified cea nucleic acid and expression vectors |
| US20090017061A1 (en) | 2005-01-18 | 2009-01-15 | Bernard Jan Appelmelk | Mycobacteria with Mannose Cap-Deficient Lipoarabinomannan |
| CN106008679A (en) | 2008-12-24 | 2016-10-12 | 荷兰王国卫生福利和运动部国家公共卫生和环境研究所 | Modified Steptococcus pneumonia pneumolysin (PLY) polypeptides |
| WO2012101509A2 (en) | 2011-01-28 | 2012-08-02 | Sanofi Pasteur Sa | Immunological compositions against hiv |
| RU2458130C1 (en) * | 2011-04-13 | 2012-08-10 | Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" (ФБУН ГНЦ ВБ "Вектор") | RECOMBINANT PLASMID DNA pTB323 CODING HYBRID POLYPEPTIDE CST-DELTAMPT64 WITH PROPERTIES OF SPECIES-SPECIFIC MYCOBACTERIAL ANTIGEN MPT64 (MPB64), RECOMBINANT ESCHERICHIA COLI BACTERIAL STRAIN - PRODUCER OF HYBRID POLYPEPTIDE CST-DELTAMPT64 AND RECOMBINANT POLYPEPTIDE CST-DELTAMPT64 |
| AU2012294454B2 (en) | 2011-08-08 | 2017-02-02 | Curelab Oncology, Inc. | Methods and compositions relating to p62 for the treatment and prophylaxis of cancer |
| US9303071B2 (en) | 2012-09-17 | 2016-04-05 | Novartis Tiergesundheit Ag | Salmonid alphavirus and uses thereof |
| US9809863B2 (en) | 2012-11-02 | 2017-11-07 | Elanco Tiergesundheit Ag | Flavivirus associated with Theiler's disease |
| WO2014140938A2 (en) | 2013-03-14 | 2014-09-18 | Centre Hospitalier Universitaire Vaudois | Immunological methods |
| SG11201605316VA (en) | 2013-12-29 | 2016-07-28 | Curelab Oncology Inc | Methods and compositions relating to p62/sqstm1 for the treatment and prevention of inflammation-associated diseases |
| PL3177644T3 (en) | 2014-08-05 | 2021-06-14 | MabQuest SA | Immunological reagents binding to pd-1 |
| US10730933B2 (en) | 2015-12-05 | 2020-08-04 | Centre Hospitalier Universitaire Vaudois | HIV binding agents |
| WO2017125815A2 (en) | 2016-01-22 | 2017-07-27 | MabQuest SA | Immunological reagents |
| WO2020012435A1 (en) | 2018-07-13 | 2020-01-16 | Lausanne University Hospital | Hiv binding agents |
| JP2022541900A (en) | 2019-07-15 | 2022-09-28 | ローザンヌ ユニヴァーシティ ホスピタル | HIV binding agent |
| TW202206098A (en) | 2020-08-11 | 2022-02-16 | 美商碩騰服務公司 | Anti-coronavirus vaccines |
| CN114315985A (en) | 2020-09-29 | 2022-04-12 | 硕腾服务有限责任公司 | Attenuated porcine epidemic diarrhea virus |
| CA3230802A1 (en) | 2021-08-31 | 2023-03-09 | Zoetis Services Llc | Attenuated alphavirus |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5504005A (en) * | 1987-03-02 | 1996-04-02 | Albert Einstein College Of Medicine Of Yeshiva University | Recombinant mycobacterial vaccine |
| EP0681026A1 (en) * | 1987-03-02 | 1995-11-08 | Whitehead Institute For Biomedical Research | Recombinant mycobacterial vaccine |
| US4952500A (en) * | 1988-02-01 | 1990-08-28 | University Of Georgia Research Foundation, Inc. | Cloning systems for Rhodococcus and related bacteria |
| EP0486495A1 (en) * | 1989-03-08 | 1992-05-27 | Commonwealth Scientific And Industrial Research Organisation | Expression system for actinomycetes and related organisms |
-
1991
- 1991-02-25 WO PCT/AU1991/000064 patent/WO1991013157A1/en not_active Application Discontinuation
- 1991-02-25 EP EP19910904701 patent/EP0517751A4/en not_active Ceased
- 1991-02-25 CA CA 2076753 patent/CA2076753A1/en not_active Abandoned
- 1991-02-25 JP JP3504156A patent/JPH05504067A/en active Pending
- 1991-02-26 NZ NZ23721791A patent/NZ237217A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| EP0517751A1 (en) | 1992-12-16 |
| JPH05504067A (en) | 1993-07-01 |
| EP0517751A4 (en) | 1993-03-31 |
| WO1991013157A1 (en) | 1991-09-05 |
| NZ237217A (en) | 1992-07-28 |
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