JPH05504067A - Shuttle plasmid for E. coli and mycobacteria - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Shuttle plasmids for E. coli and mycobacteria The present invention with respect to mycobacteria and Niscillichia coli (Eschr. DNA shuttle vector that can replicate in ichia coli) cells The present invention also relates to bacterial hosts containing such NA vectors, It further relates to vaccines containing such bacteria.

Mycobacterium bovis BCG (hereinafter referred to as BCG) is used as a vaccine against tuberculosis (TB). It has been used for many years. Vaccination programs begin with BCG as a long-term cell-mediated stimulates the immune system, and secondly, it has a remarkable safety record, making it suitable for humans. It was very effective for controlling TB. These characteristics are unique to recombinant BCG is a good candidate to form the basis of a live administration system for Make it.

Many issues are involved in DNA manipulation involving mycobacteria.

These are much rarer than bacteria such as E. coli. (one) plasmid vector, slow growth rate and low morphology of mycobacteria Includes transformation rate. Genetic manipulation day in and day out, given these problems can be replicated in standard bacterial workhorses, such as E. coli, and In mycobacteria at or near the final stage of genetic manipulation, further reproduction is possible. It is desirable to generate shuttle vectors that can In this case, the gene inserted into mycobacteria and involved in mycobacterial growth. rupture and transformation can be largely avoided. Gicquel-5anzey etc. . (Acta Lepologica 1989.7.5uppl(1): 208-211) have two starting points for reproduction (one for mycobacteria). and the other one is for E. coli), known as pAL8. A Mycobacterium E. coli plasmid shuttle vector has been described. multiple Mycobacterium hector E. , mycobacteria and E. coli cannot grow, and vice versa. , was necessary due to the evolutionary distance between it and Cori. Such a vector has two origins. The inclusion of points has the problem that they are of considerable size, which makes cloning efficiency, the size of the desired DNA fragment that can be inserted into such a plasmid. This reduces the number of unique restriction sites that can be inserted into the plasmid. enhance

The applicant has demonstrated the replication ability of the vector in mycobacterial and E. coli cells. To provide a DNA shuttle vector carrying a single origin of replication Thus, the problems associated with prior art shuttle vectors have been overcome. described after this In a particularly preferred aspect of the invention, the replication region is corynebacterium plasmid pNG2 or a fragment thereof corresponds to the replication area.

According to a first aspect of the invention, a single origin of replication (this origin of replication is a Mycobacterium (enables replication of the vector in E. coli and E. coli cells) and selectable matrices. A DNA shuttle vector carrying a reproduction region comprising a marker is provided. . The shuttle vector contains one or more vectors for insertion of the desired nucleotide sequence. It further comprises a nucleotide sequence containing a number of restriction sites.

Preferably, the reproduction region is as described above in mycobacteria and E, co+, I. Vector's? Corynebacterial plasmid pNG2 or its equivalent that enables production of Jj corresponds to the reproduction region of the fragment.

Surprisingly, the reproduction region of plasmid p~G2; Differently, conferring the ability to reproduce in various bacterial species.

[1114 shuttle vectors may further have one or more restriction nucleotide sequences. When inserted into the site, the promoter allows DNA transcription to proceed. , a promoter and one or more restriction endonuclease sites downstream thereof. It consists of DNA shuttle vectors contain multiple promoters and downstream restriction enzymes. Can include a donuclease site.

As used herein, the term "shuttle vector" refers to a double-stranded linear or double-stranded Includes plasmid DNA which can be a full circular strand.

Shuttle vectors can be used using many techniques well known in the art, such as combination, mobilization, transformation, transfection, transduction They can be introduced into bacterial cells by injection or electroporation.

As used herein, the term "selectable marker" refers to the nucleotide sequence Refers to any selectable feature provided by or encoding a column.

Suitable selectable markers include antibiotics or enzymes or immunologically detectable proteins. capable of producing a detectable reaction when supplied with a protein or suitable substrate. Examples of resistance to chemicals that can cause Icin, penicillin, hygronucin, kanamycin, and the like, Includes lactosidase, urease, alkaline phosphatase and the like. can be done.

The term "promoter" is widely used and is used to bind RNA polymerase and and the downstream (3' or "operably linked) nucleotide sequences of the promoter. Refers to any nucleotide sequence that directs transcription of a sequence. proper promo The promoter is a prokaryotic promoter, such as the P1 promoter of -, trp promoter, lac promoter, transposon Tn903 and Kanamycin resistance promoter of transposon Tn5, mycobacterial protein promoter, such as the promoter of the common mycobacterial 65Kd antigen; Icobacterial liposome I? NA promoter, M, bovis antigen, MPB7 0. MPB59 and MPB64 promoters, eukaryotic promoters and prokaryotic promoters hybrids between motors and eukaryotic promoters, e.g. the growth hormone promoter, and the like.

Restriction endonuclease sites provided on the vector may include one or more known restriction endonuclease sites. Endonucleases, such as EcoRl, BamHl, Pstl.

C1al, Kpnl, HindlI[, HincI and HincII, and the like. Restriction endonuclease sites are Closely grouped ilJ! I or ? containing the endonuclease site? J number can be supplied in the form of a polylinker.

The nucleotide sequence of interest can be obtained using methods well known in the art and e.g. Sambrook et al. (Mo Iecalar Cloning: A L aborat.

ry Manual, Co1d Spring Harbor Labora by the method described in Tory, 2nd Edition, 1989). DNA fragments with complementary “sticky ends” to enable their annealing. or by ligation of the ``flush'' ends (i without the terminal pair of nucleotides) The endonuclease provided on the vector by ligation of the nucleotide sequence with enzyme cleavage site. Nucleotides for insertion into vectors of the invention The sequence contains a promoter to direct transcription of downstream (3') sequences. be able to. Promoter is the natural promoter of the gene to be transcribed. or a different promoter. As mentioned above, the shuttle vector of the present invention vector encodes one or i number of restriction endonuclease cleavage sites. itself contains one or more promoters upstream (5°) from the sequence Can be done. In such 6[, the desired nucleotide lacking a promoter is The code sequence is inserted into the vector and transcription of the desired nucleotide sequence is directed to it. is driven by a promoter present on the vector.

The nucleotide sequences for insertion into the vectors of the invention are suitable for disease-causing organisms. Bacteria, viruses or parasites, such as Taenia obihe/L7 * urosis (h embryo Mycobacterium L brae (M) cobacterium le rae), mycobacterium baratu cobacteria + aratuberculosis , Bacterodgs nodosus is Bordetella (B.

rdetella). desired Nucleotide sequences that contain hormones, such as LH1? H1 growth hormone or an epitope or homolog thereof; or a nucleotide sequence recombinant with the bacterial host cell. or a nucleotide sequence capable of mutating a DNA sequence within a host cell. columns can be coded. The shuttle vector of the present invention can be used as an expression vector. If functional, the nucleotide sequence encoding the desired product is A signal or leader sequence that enables insertion into the cell membrane or secretion from the host cell. Can contain columns. The absence of a secretory leader prevents anti-inflammatory activity within the cytoplasm of the bacterial host cell. antigen or other protein products (where the antigen or other The protein product is recovered by well known methods).

According to a particular embodiment of the invention, when measured by agarose electrophoresis, about 3 ．． DNA with the size of IKb, glossy vector ρEP2, Koriho Hatachiri An approximately 1.85 Kb complex containing a single origin of replication derived from the replicon pNG2. production region, the antibiotic IFI award gene resistant to kanamycin, and the DNA sequence of interest. A nucleotide sequence containing numerous restriction endonuclease cleavage sites for insertion of sequences. columns are supplied. 4. Is the 5 Kbr plasmid pEP3 the same as pEP2? J [Produced sequences and effective hygroma in both E. coli and mycobacteria.] Contains a marker that coats icin resistance.

The shuttle vector of the present invention is described in Bergey's Manual of Det. erminatiVe Bacteriology, 8th Edition , PP, 599-861. Mainly, for example, Mycobacterium, E.

coli, Bacterium coli and Actinomycetes can be replicated in species s). The vector of the present invention is derived from Bacillus ) W does not seem to replicate. Any of the bacterial strains of the present invention to the art to ascertain whether or not vectors can be reproduced therein. can be easily tested according to methods well known in the art. Advantageously, the present invention The shuttle vector has been used in vaccination programs throughout the world, as described above. Adjuva is used intensively and has the potential to stimulate long-term cell-mediated immunity. bacterial strains, such as a weakened strain of Mycobacterium bovis BCG. can be reproduced to a high copy number.

The shuttle vector that incorporates the origin of replication of pNG2 is Particularly effective in objects.

According to another aspect of the invention, a shuttle vector as defined herein A total of 100 W hosts are provided, including a host. That shuttle vector is It can exist as a single copy or more preferably multiple copies thereof within the bacterial host.

Preferably, the bacterial host is a Mycobacterium, a Mycobacterium coli strain 2 or an E. coli strain, For example, C. pseudotuberculosis (C. pseudotuberculosis) sis), M. smegmatis (t" macis) and M. bovis BCG Yes, but not limited to this. The vector is isolated from the host cell as described above. As an excretory product from the cells, antigens or other proteins are released into the bacterial host for their expression. can be used to carry proteinaceous genes. Proteins are placed on plasmid vectors It can be expressed while present or after recombination or insertion into the chromosome. other On the other hand, the expressed product is inserted into or participates in the host cell membrane or cell wall. , or within the host cell itself.

A bacterial host expressing the desired antigen can be used to create a vaccine, e.g. M, bovis BCG. Based on immunization, the immune response , such as M. bovis BCG and disease-causing bacteria, viruses or parasites. The desired antigen can be accumulated.

In another aspect of the invention, a shuttle vehicle, as defined herein, The polypeptide is supplied when expressed by a bacterial host cell, including a bacterial vector.

The present invention provides for replication in Mycobacterium and E. coli. into the replication region of pNG2, unless modifications such as preclude the ability of such sequences to nucleotide sequences or deletions therefrom.

Techniques for insertion or deletion of nucleotide sequences are well known in the art. There is. The mutants can be used in Gram-negative and Gram-positive host cells, such as E. coli and Coli. can be easily tested for the ability to confer reproduction in Lineobacteria .

The invention also provides that, after insertion into a suitable vector, the mycobacterial and E, of ρN62 itself or a fragment thereof capable of replication in E. coli. Expand to duplicate area as well.

E. coli culture II containing plasmid pEP2 was prepared using Au5tralian Go Vernsent Analytical Laboratory (AGAL) , Pymble, New 5outh Waies, Buda in Au5tra1ia Deposited on February 23, 1990 under the PES Treaty and of N901007080. A deposit number was obtained.

The invention will be illustrated by the following non-limiting figures and examples.

drawing Figure 1 shows the structure of plasmid pEP2, plasmid pNG2 (14,5 ) was digested with EcoRI and the largest fragment was transformed into pUC4. 'Connected to the cartridge. E, obtained after electroporation into coli The extracted plasmid DNA was extracted and partially digested with Pstl, and then and electroporated again with E. coli. Obtained Ka One of the n' colonies contained plasmid pEP2. Restriction site: B (Bag old), E (EcoRI), H (HindI[I), Hc (HincI[) .

P (PstI), 5 (Sal r), Kan are resistant kana from pUC4K. Mention the mycinogenic gene.

Figure 2A shows the ring shape of Mycobacterium E. coli shuttleherata ρEP2. It's a map. The restriction map gives the appropriate cleavage site: B (BamHI), E (EcoRI), H (HindlI [), Hc (H4ncII), K ri, P (Pstl), 5 (Sal I). Calamycin resistance gene is pUC This is the \\\\ part from 4K. Figure 2B: Sequencing method. The arrow is M13 Hector shows the extent and direction of the sequences generated from restriction fragments cloned into . X indicates a sequence derivatized using an oligonucleotide primer.

FIG. 3 is the complete nucleotide sequence of the replication region of ρEP2. Appropriate restriction The position is marked for comparison with FIG. 1 (E (Eco RI)).

H(Hindl[[), Flc(Hinc[I), KHALI), IR and O R1 inverted and direct repeat sequences, respectively, l? Bs, putative liposome binding Synthesis site;---11←----1 putative rho-dependent transcription terminus terminus; 2-fold axis of symmetry involving T. 0RFA, major reading frame.

Figure 4 shows the gene probed with plasmid pEP2 extracted from E. coli. agarose gel (plate on the right) and Southern blotting (plate on the left) (A) Undigested pEP2 DNA from E. coli, 5 00ng, (B) Pst I digested, E. coli cartilage pEP2 DNA, 200 ng, (C) Complete PstI-digested M, bovis BCG Total NA extract, 2.5 μg, (D) Pstl digested M, bovis B CG DNA.

2.5μg, (E)M, undigested DNA extract of Bovis BCG pEP2, 2.5 μg.

(F) M, undigested DNA extract of Bovis BCG, 2.5 tl g, (G) Contains the λ DNA marker digested by HindI [[.

Example 1 11Ω Dan”: Unless otherwise noted, manipulation of engineered molecules and preparation of solutions follow standard known techniques. evening. Such techniques include Salbrook et al. ing, A Laboratory Manual, Co1d Sprin g Harbor Laboratory.

Co1d Spring Harbor (1989), PP, 1-500 It is described in

and plasmid: Mycobacterium bovis BCG mutant C5L, Com+*onwealth Serutm Labora turies (C5L) Aus trali It was obtained as a lyophilized human vaccine from A.

M, Smegmatis (M, see 5tatic) and M, Feli (Muga Rainbow V) , Fairfield Ho5pital, Melbourne, Au5 tralia and E. coli JM109 was obtained from Promega (Madiso n, Wisconsin, U.S.A.). Plasmid pNG 2 (Serwold-Davis et al., 1987. Proc, Natl. Acad, Sci.

USA 84:4964-4968) and pPB3, Dr. Ph1lip B ird (Monash University Faculty of Me dicine, Alfred Ho5pita1. from Melbourne) Obtained.

Plasmid pAL8 (Gicquel-5anzey et al., 1989 Ac ta Leprol, 7: 207-211), Or, Bridgett e Gicquel-5anzey (Pasteur In5tituto, and plasmid pUGc4K was obtained from Pharmacia Obtained from LKB (Uppsala, Sweden).

Yutaka: E. coli strain in Luria broth (LB: 10 g of tryptone, 5 g of Yeast extract, 10g NaCl/I! ). coryneform bacteria were grown in B medium (Oxoid), and Mycobacterium sp. Grow in bos or 7H11 medium (Oxoid, Au5tralia) .

Electroporation of: Mycobacterium species and Corynebacterium luculosis, Gene available from Bio-Rad Laboratories Inc. Using Putser, Lugos i (Tubercule 70:1 59-170 (1989)). transformation The body was treated with 100 μg/d of kanamycin or 8200 μg/rd of hygromine. 7H11 or on nutrient medium.

DNA and hybridization: DNA is isolated from yeast. Mycobacterium was extracted using a modification of the method used for and Jeffrey, Anal.

Biochen+, 1981.178: 82-87). mycobacteria Cells were harvested by centrifugation from 400-mL Dubos broth and incubated for 5 d. It was resuspended in TEI buffer and heat killed by treatment at 70°C for 1 hour. gala Add Speed (3-61111) and simmer the mixture for a minimum of 30 seconds. Stir thoroughly to disperse the bacteria. The bacterial suspension is then precooled in a bath of liquid nitrogen. Transfer to liquid nitrogen in a cooled quilter and finely grind using a pre-chilled pestle. Grinded into fine powder. Cell crushing was performed in a /NN Imzard hood. . The crushed frozen cells were lysed (6.6mM Tris, 30gM) and E DTA, 1.2%-/v sodium lauroyl sarcosinate') 15d It was added little by little (with a spatula).

Add 5 mg of protease and ink the mixture for 90 minutes at 37°C. The mixture was then extracted with an equal volume of phenol-chloroform. and the aqueous phase was precipitated with isopropanol for 10 min at 4”C. I met. Beret, 1. Dissolved in Od's TEi buffer and phenol-chloro Extracted with chloroform and then water-saturated ether, then extracted with ether. Tanol precipitated and resuspended in TE.

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

pNG2RI DNA was digested with various restriction enzymes, and the fragments were Hybond N (Aersham) Southern plot on nylon filter (Re+dC”,)e filter with random hexamer ply Hybridize overnight at 37°C with pEP2 labeled with 32P using Soybean, washed with increasing stringency (up to 65°C) if necessary, and An X-ray film (Fuji RX) was irradiated. Restriction digested whole genome D NA was isolated from M. bovis BCG and transformed with pEP2. Transfer the DNA to nylon and add pEP labeled as above. 2. Other transformed Mycobacterium species were transferred to D! IA Using Tondoblot hybridization, with ρEP2 probe analyzed. C. luculosis transformants were analyzed on the same B birch.

E for kanamycin or hygromycin resistance using whole cellular DNA; E. coli was isolated and transformed.

Saranijichi Shibunkaji: Nucleotide sequence analysis was performed using denatured T7 DNA polymerase (Pharsa dideoxo chain using cia LKB, Uppsula, Sweden) How to stop (Sanger et al., 1977, Proc, Natl, Acad , Sci, USA 74: 5463-5467). pE The complete nucleotide sequence of P2 can be converted into a generic or oligonucleotide primer. was used on both strands (Figure 1B). The latter is Gene Assembly er+DNA Synthesizer (Pharmacia LKB.

(described above). Examine DIIA and amino acid sequence data, and DNA5IS and PRO! l+Is soft fair package (Pharmac ia LKB).

Example 2 Structure and analysis of shuttle vector: The 1.3 Kb EcoR1 fragment from pUc4K carrying the kanamycin resistance gene The fragment was ligated to EcoRl-digested pNG2 DNA. That series The ligation mixture was electroborated into E. coli JM109 and the transformants were , selected on LB plates supplemented with 50 μg kanamycin/−. vinegar All transformants have a 9.5 Kb pNG2 EcoR1 fragment. Contains plasmids. Plasmid DNA from one of these clones (2u g) (pNG2R1) was partially digested with PstI and T4 DNA poly Planted using merase. NA fragment smaller than 10Kb was purified from a 1% agarose gel and R6 coli Jl'1109 was purified from Kanama resistant used for icinic transformation. isolated from transformants, obtained The restriction map of the plasmid (pEP2) was prepared using standard methods (e.g. Sambrook). ) and is shown in FIG. 1A.

Plasmid pEP2, as determined by agarose gel electrophoresis, 3. I It has a molecular weight of Kb. This plasmid carries one of the polylinkers in pUC4. and therefore the unique Pstl, SalI, BanHl and It has an EcoR1 site. Hybridization analysis performed on plasmid pEP2 and pNG2R [(pNG2 after digestion with EcoRI) are both 800 bp ndln fragment. pNG with pEP plasmid A Southern blotting of the digest of subclone 2 was carried out using the region of pNG2 shown in Figure 1. region is the region integrated into the ρEP2 plasmid, and therefore the region is It was shown that it contains the starting point of the production.

Hygromine resistance in Mycobacterium species and C. Hyg The romycin phosphotransferase gene (hph) was introduced into mycobacterial It was cloned into Shuttle Hector pEP.

Hygromycin phosphotransferase gene (hph) from pPB3 To clone pUc4K into pEP2, pUc4K DNA was linearized with XhoT. and then using the Flenow fragment of DNA polymerase turned into a plant. pPB3 DNA into 5 units of individual Alul.

Digested with Hae III and Rsa I for 30 seconds on ice. 2. OK A fragment of size b was gel-mounted using Geneclean, Bresa. (tech), and was isolated by plant-terminated linear pUc4K DNA. Connected in the evening.

The ligation mixture was electroporated into JM109 and the recombinant LB protein supplemented with 50 μg hygromycin B (Sigma)/r11. Selected on rate. 1.7 Kb Sal I-C1 from chimera to pUC4 The aI insert was inserted into Sal　r　-C1a within the kanamycin resistance gene of pEP2. ligated into the r site. JM109 was hygroconjugated with the ligation mixture as described above. The kanamycin-resistant plasmid (pEP3) was transformed into a mycin-resistant plasmid (pEP3). Guided the restriction map. Plasmid pEP3 has a unique 5a11 site; It has a size of about 4.5 Kb.

The nucleotide sequence of pEP2, which does not contain the kanamycin resistance gene, is shown in Figure 3. It will be done. Examination of the DNA sequence showed a single open reading frame (ORF). many Potential translation initiation codons can be identical within this region, but only one is preceded by a putative liposome binding site. The upstream sequence of this ORF ( ORFA) does not have an E. coli consensus promoter, but a putative A rho-dependent transcription terminator was found downstream of the stop codon (Figure 2). . This putative terminator is similar to other rho-dependent terminators. As described (Rosenberg et al., Nature 272.414- 428 [1978)), 90% pine in the TAATC AATAT consensus sequence Chishiku Ryder, In1tia ion of DNA Replic ation (1981), ^ academic Press.

New York), and is preceded by a region of two-fold symmetry axis (FIG. 2).

Therefore, 0RFA can encode a protein of 28 KDa, which is large. The size is consistent with that reported for other Rapmil proteins.

In addition to the predicted size of the 0RFA protein, the inventors have also Additional evidence suggesting that the derivatized amino acid sequence Ru. First, the codon bias for the putative 0RFA product (Phe, TT C; Asp, GAC; Arg, CG; Ile, ATC; Val.

GTC; Ala GCC; Thr, ACC) are other Corynebacterium is the same as the codon for protein. Second, coded by 0RFA The predicted proteins that will be included in the Importantly, the characteristics of Rep proteins (and other D1-binding proteins) It exhibits high basicity in nature (19% basic residues).

DataHase research has revealed the complete 1.85Kb nucleotide sequence and prediction of 0RFA. However, no significant homology was found. It wasn't done. Furthermore, a more detailed analysis of the pEPZRepel region and Corynebacterium Therium (pBLl, Martin et al., Biotechnol.

etc., GenelT: 315-321 r1988)). It showed no similarity to any of the related plasmids.

A plasmid replication region simply contains an origin of replication. In most cases, the origin is located in the code region, has direct and inverted repeat groups, and is enriched in A+T. (Kamio et al., J.

Bacteriol, 258:307-312 [1984), 5cotl etc., Microbiol, Rev.

48: 1-23 (1984), Rosenato, Mo1. Gen, Genet, 179: 527-537 [1980], Rauzie r etc., Gene71: 315-321 [1988)), 0RF The upstream region of A does not contain an ORF and contains many direct and inverted repeats D)1 A sequence exists (Figure 2). Furthermore, the area between nt86 and 171 is completely A total of 63.5% A compared to an average of 45% for the full Rep SJI range. +T (Figure 2). Therefore, this appears to be the pEP2 origin of replication.

The replicated region of pEP2 can encode a single 28KDa protein; and the monomer that allows plasmid replication in Gram-negative and Gram-positive bacteria. It has one starting point. Therefore, pEP2 is a unique shuttle vector and For example, in genetic analysis of Mycobacterium and live recombinant vaccines. As such, it should be a useful tool in the development of M, Bovis BCG.

Electroporation of mycobacteria with EP2 and EP3: pEP2 Electroporation of Mycobacterium species by DNA This resulted in transformation to namycin resistance (Table 1).

To confirm that the plasmid is present in mycobacteria, total cell preparation Products were made from kanamycin resistant transformants.

Figure 4 shows the total amount extracted from the BCG CSL strain transformed with pEP2. Figure 3 shows a combination of agarose gel and Southern blonot analysis of cellular DNA. moreover, The relative intensity of the gel bands indicates that the pEP plasmid is high in these bacteria. Copy number suggests replication. Furthermore, it was isolated from the industrially resistant transformant. When electroborating E. coli using the total genomic DNA obtained, approximately 1 ．． 0x10' kanamycin-resistant clones were obtained per 1 μg of ON. Ta. - These data taken together indicate that the PEP2 replicon is present in these bacteria. This suggests that high copy numbers promote stable plasmid replication.

Plasmid pEP3 contains a small amount of M. smegmatis and M. bovis BCG C3L. can be transformed to be resistant to at least 200 μg of hygromycin/−. (Table 1). M. smegmatis transformed with ρEP3 Total cellular DNA can transform E. coli to hygromycin resistance. Ta. This is because the Php gene encoding hygromycin resistance is mycobacterial. and E. coli, and thereby kill those bacteria. is shown to constitute a useful marker for.

Table 1 Electroporated Plasmids for Mycobacteria and Related Species Troporation efficiency: Kan' CFU/1 μg of plasmid DNA Bacterial species pEP2 pAL8 pEP3BCG var CSL 10” 10 '10'N/D = Not done, --- = Transformation to resistant phenotype It didn't exist.

Different numbers for individual electroporations indicate the results of separate experiments. vinegar.

Electroporation of seeds by EP2 and EP3: pEP replicon pEP2 and 3 plasmid DNA was used to determine the host range of Corynebacterium spp. Electroporated into T. tuberculosis. Transformation is , which occurred in C. pseudotusherculosis (Table 1), these results , indicating that the pEP2 plasmid has a broad host range.

Example 3 EP26: Protein from mycobacterial 65KDa heat shock protein (0.6Kb) The PCR-derived DNA fragment carrying the motor was converted into Pstl-R of pEP2. am old site, generating a plasmid referred to as pEP5.

This promoter is known to function in E. coli and mycobacteria. and is therefore useful in shuttle herater expression systems. .

Chloramphenicol-acetyltransferase (CAT) code The gene was cloned into the BamH1 site of pEP5. 65KDa promo CAT gene expression driven by PhaIIlacia (registered trademark) Mark, Pharmacia, Pitcataway, N, J,, IJ, S, A ,) CAT detection was detected using the detection sensor. According to this assay, 64KDa The promoter of E. coli and C. It has a strength comparable to that of the induced Iac promoter.

? 1PB70 is the main secreted protein of M. bovis and a component of PPD. child The gene and its signal sequence were integrated into the pEP2 expression system. MPB A truncated form of the 70 gene removes its promoter, but its liposomal Generated by PCR which leaves the binding site (RBS) intact. this is , in pEP2, Ps t4-BaIll)! 1 fragment (0.5Kb) was cloned. Next, the 65KDa promoter was transformed into Pstl-5cal was cloned upstream of the MP870 BS on the fragment.

After electroporation into E. coli and C. , expression was examined by Western plot and Elisa. This construct is , was not expressed in E. coli. but.

Therefore, it is expressed in C. pseudotusherculosis and the product was detected in both the sonicated material and the culture filtrate. This is MPB7011Bs is inactive in E. coli and in C. pseudoherculosis. It shows that it is functional.

Those skilled in the art will appreciate that the invention described herein is susceptible to modifications and variations other than those specifically described. I understand that I may receive qualifications. The present invention is within the scope of the claims. It is to be understood that such changes and modifications are included. The present invention also includes the book All steps, features, compositions and compounds mentioned or illustrated in the specification or collectively and encompassing all combinations of any of the aforementioned steps or features.

ABCDEF ABCDEFG abstract DNA fragments that can replicate in Mycobacteria, E. coli, and other bacterial hosts. Torvector is described. The shuttle vector is a complex vector in many bacterial species. It comprises a single origin of replication that confers the ability to create products. Also, the desired polypeptide , for example, molecules encoding antigens of disease-causing bacteria, viruses, and parasites. A torque vector is disclosed.

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Claims (21)

[Claims] 1. A single origin of replication, which originates from mycobacteria and E. coli cell odor to enable replication of the vector; and carry a replication region comprising a selectable marker. DNA shuttle vector with 2. a first nucleotide sequence operably linked to said origin of replication; , and one or more restriction enzymes for insertion of the desired second nucleotide sequence. The shuttle vector according to claim 1, comprising a donuclease cleavage site. 3. one or more operably linked to a nucleic acid sequence encoding a desired polypeptide. The shuttle vector according to claim 1 or 2, comprising a promoter. 4. said selectable marker confers resistance to antibiotics or enzymes, or An immunosorbent that can cause a detectable reaction when supplied with an appropriate substrate. Claim 1 which encodes an epidemiologically detectable protein or chemical compound Shuttle vector. 5. The selectable marker is ampicillin, streptomycin, penicillin. , hygromycin or kanamycin. Shuttle vector. 6. The selectable marker is β-galactosidase, urease or alkali. The shuttle vector according to claim 4, which is a gene encoding phosphatase. Tar. 7. The promoter is P1 promoter of bacteriophage λ, trp promoter, promoter, lac promoter, transposon Tn903 or transposon Kanamycin resistance promoter of Tn5, common mycobacterial 65Kd anti-promoter The original mycobacterial promoter, mycobacterial ribosomal RNA promoter Motor, M. Promoter of bovis antigens MPB70, MPB59 and MPB64 -, metallotionine promoter, growth hormone promoter or eukaryotic and prokaryotic A shuttle according to claim 3 selected from hybrids between promoters. vector. 8. The desired peptide is isolated from disease-causing bacteria, viruses or parasites. 4. The shuttle vector of claim 3 encoding one or more antigens. 9. The one or more antigens are Taenia bovis, rotavirus, Babesia bovis. , Mycobacterium bovis, Mycobacterium tuberculosis, Mycobacterium Cuterium leprae, Mycobacterium paratubeculosis, Bacteriodes The shuttle according to claim 8, which is selected from antigens of Nodosus or Bordetella. vector. 10. Claims wherein the desired polypeptide is a hormone or an epitope thereof. The shuttle vector according to paragraph 3. 11. The origin of replication is mycobacteria and E. In coli, the vector Corynebacterium plasmid pNG2 or a fragment thereof that allows the replication of The shuttle vector according to claim 1, comprising a replication region of . 12. Any of claims 1 to 11 that can be reproduced in Actinomycetes The shuttle vector according to item 1. 13. Any one of claims 1 to 11 that can be replicated in corynebacteria. Shuttle vector as described in section. 14. Shuttle vector selected from pEP2 and pEP3. 15. Bacteria comprising the shuttle vector according to any one of claims 1 to 14. host cell. 16. Mycobacteria, E. Cori, Corynebacteria and Actinomycetes 16. The host cell of claim 15 selected from: 17. M. Frey, M. Smegmatis, M. Bovis BCG, E. Cori JM109 ,C. The host according to claim 15, which is selected from Pseudotuberculosis. cell. 18. Origin of replication of Corynebacterium plasmid pNG2 or a fragment thereof a DNA sequence encoding a point, the plasmid or fragment thereof comprising: Mycobacteria and E. The replication of the vector containing the above DNA sequence in coli A DNA sequence characterized in that it enables. 19. Claim 17 comprising an origin of replication operatively coupled to said origin of replication. DNA sequence described in section. 20. The DNA sequence is a desired polypeptide operably linked to a promoter. 19. The DNA sequence according to claim 18, which encodes tido. 21. A polypeptide expressed by a host cell, said polypeptide comprising: The system according to any one of claims 4, 8 and 9, which is resident in the host cell. A polypeptide characterized in that it is encoded by a vector.