CN100354004C - Tubercle bacillus chimeric gene vaccine and preparation process thereof - Google Patents

Abstract

A chimeric tubercle bacillus gene vaccine is provided, which comprises the gene encoding tuberculosis structural protein 85a shown in sequence 1 and the gene encoding tuberculosis ESAT6 shown in sequence 2, wherein the ESAT6 gene is chimeric in the sequence of the 85a gene, The 85a gene is connected to the eukaryotic expression vector pVAX1. Also provided is a preparation method of the chimeric Mycobacterium tuberculosis gene vaccine (plasmid), including PCR amplification of the Ag85a gene; construction of the gene vaccine HG85 (plasmid); PCR amplification of the ESAT6 gene and insertion of the ESAT6 gene into the plasmid HG85. The immunogenicity of the chimeric Mycobacterium tuberculosis gene vaccine of the invention is enhanced, and the immune effect is better than that of the single gene vaccine.

Description

Mycobacterium tuberculosis chimeric gene vaccine and preparation method thereof

technical field

The invention relates to a novel vaccine technology in the field of biomedicine, in particular to a tubercle bacillus chimeric gene vaccine representing the third vaccine revolution developed by using gene chimeric technology.

Background technique

Because BCG vaccine has no immune protection effect on adult pulmonary tuberculosis, and the isolation rate of multi-antibiotic-resistant tuberculosis strains is increasing day by day, as well as the number of cases of AIDS complicated with tuberculosis infection, tuberculosis has become one of the most dangerous infectious diseases that threaten human life and health one. Finding a new type of vaccine with better immune protection effect than BCG has become a hot spot and urgent task in the research and development of new vaccines that can prevent tuberculosis in adults in today's world. The DNA vaccine (also known as gene vaccine) technology with a history of only 10 years has triggered the third vaccine revolution due to its many advantages. , the prevention of malaria and tuberculosis, among others, brings glimmers of hope.

DNA vaccines use "naked engineered plasmids" produced by Escherichia coli for immunization. Plasmids, also known as vectors or plasmid vectors or vector plasmids, are circular supercoiled DNA that can replicate and express independently of chromosomes in E. coli cells. DNA vaccine is the Escherichia coli plasmid through genetic engineering, in which the gene encoding the preferred antigen protein of the pathogen, the preferred immune enhancing cytokine, such as encoding interleukin 12 (IL-12), cell colony-stimulating factor (GM -CSF) genes or CpG adjuvant motifs, elements such as promoters suitable for human cells, and appropriate antibiotic resistance genes to construct gene recombination (engineering) plasmids. After the purified engineered plasmid DNA is injected into human muscle and other parts into the cells, the plasmid can express the encoded antigen in the relevant cells, especially the antigen processing cells (Antigen Present Cell, APC), and induce the body to produce body fluids and cells. The immune response responds to defend against infection by pathogens. Eukaryotic expression vector JW4303, or pcDNA3.1, or pVAX1 series of plasmids can be used to prepare carefully prepared plasmid DNA for different types of pathogens, but mainly use the kanamycin resistance recommended by the US FDA and can be used in humans Escherichia coli plasmid pVAX1 was transformed. Transform the eukaryotic expression vector plasmid into Escherichia coli (referred to as engineering bacteria at this time), and cultivate the engineering bacteria under optimized conditions to make the bacteria proliferate, and the plasmid in the bacteria will be copied into multiple copies. When appropriate, the bacteria will be collected by centrifugation , Under proper conditions, the bacteria are lysed to release the plasmid; after collection, impurities are removed, and the plasmid is purified, which can be used as a DNA vaccine or gene vaccine for vaccination.

At present, it is believed that the immune effect of the single-gene tuberculosis DNA vaccine in large animal experiments is not ideal (Gregorialdis, "Genetic vaccines: strategies for optimization", Pharmaceutical Research, 15: 661-670, 1998); Although the double-gene vaccine constructed by the method is superior to the single-gene vaccine, it can achieve the purpose of expressing two different antigens with one vector, but there is no synergistic synergistic effect between the two, that is, the immune response induced by the fusion gene vaccine No better than single-gene vaccines (Stevenson, "DNA fusion gene vaccines against cancer: from laboratory to the clinic", Immunology Research, 199:156-180, 2004). Studies in recent years have found that chimeric virus vaccines with less side effects and stronger immunogenicity can sometimes be produced by chimerizing two viruses at the cellular level (Mathenge, “Fusion PCR generated Japanese encephalitis virus/ dengue 4 virus chimera exhibits lack of neuroinvasivensess, attenuated neurovirus, and a dual-flavi immune response in mice", Journal of General Virology, 85:2503-2513, 2004). Based on similar principles, some scientists have chimerically chimeric genes from the same pathogenic microorganism at the molecular level to produce chimeric gene vaccines, which have achieved good results (Domingo, "Immunological properties of a DNA plasma encoding a chimeric protein of herpes simplex virus type2 glycoprotein B and glycoprotein D", Vaccine, 21(25-26): 3565-3574, 2003). There are also attempts to use chimeric gene technology to chimera a gene with relatively weak immunogenicity into another pathogenic microorganism gene to improve the immunogenicity of weak antigens, that is, heterologous chimeric gene technology. A successful example is the chimera coding gene of HIV V3 in the coding gene of hepatitis B surface antigen (HbsAg). The V3 epitope of the HIV coat protein antigen is a very important protective epitope, but it is difficult to induce a strong immune response due to its small molecular weight. However, the gene encoding HIV V3 is chimerized in the gene encoding hepatitis B surface antigen (HbsAg), and as a result, this chimeric gene can induce specific anti-HIV V3 humoral and cellular immune responses (Bryder, "Imoroved immunogenicity of HIV -1 epitopes in HbsAg chimeric DNA vaccine plasmads by structural mutations of HbsAg", DNA and Cell Biology, 18(3):219-225, 1999).

In the research of new tuberculosis vaccines, someone once fused the ESAT6 gene encoding the Mycobacterium tuberculosis protective antigen with relatively poor immunogenicity and the Ag85B gene encoding the Mycobacterium tuberculosis protective antigen with relatively strong immunogenicity, and the protein antigen expressed by it It has a larger molecular weight, which is conducive to improving the immunogenicity of the small molecule ESAT6 antigen; however, there is a big difference in the effect of linking the gene encoding ESAT6 to the amino-terminal and carboxyl-terminal of the gene encoding Ag85B, and the latter is not as good as the former (Shi Changhong , "Fusion expression and purification of Mycobacterium tuberculosis secretory protein Ag85B-ESAT6", Chinese Journal of Tuberculosis and Respiratory Medicine, 27(2):89-92, 2004). Moreover, since the antigenic epitopes of proteins are generally concentrated at their amino and carboxyl terminals, whether the fusion linking method may affect the immunological properties of the expressed antigen remains to be studied. However, the chimeric method may chimera a small gene in the middle of a large gene, which can enhance the immunogenicity of the weak gene without affecting the immunological activity of the expression product of the large gene. But so far, in the research on the construction of Mycobacterium tuberculosis gene vaccine, there is no report of using chimeric gene technology and achieving success.

The inventors surprisingly found that the ESAT6 gene encoding the smallest Mycobacterium tuberculosis protective antigen was chimerized into the gene encoding the Ag85a antigen with the most immune protection effect by using gene chimeric technology, resulting in a new type of Mycobacterium tuberculosis chimeric gene In animal experiments, the vaccine has shown that the immune effect is better than that of the single-gene vaccine, and the anti-ESAT6 antibody and anti-Ag85a antibody induced by it are higher than those produced by the single-gene vaccine or the two-gene fusion vaccine, so it may be used in actual vaccines.

Summary of the invention

The purpose of the present invention is to provide a chimeric Mycobacterium tuberculosis gene vaccine with improved immunogenicity.

The object of the present invention is also to provide a preparation method of the chimeric tubercle bacillus gene vaccine.

The chimeric Mycobacterium tuberculosis genetic vaccine of the present invention comprises the gene encoding Mycobacterium tuberculosis structural protein 85a shown in sequence 1 and the gene encoding Mycobacterium tuberculosis ESAT6 shown in sequence 2, wherein the ESAT6 gene is chimeric at No. 245-250 of the 85a gene One or two sites in the sequence recognized by restriction endonuclease Kpn I, the sequence recognized by endonuclease Pst I at position 325-330 and the sequence recognized by endonuclease Acc I at position 430-435 Above, the 85a gene is connected to the eukaryotic expression vector.

sequence 1

1 TTTTCCCGGC CGGGCTTGCC GGTGGAGTAC CTGCAGGTGC CGTCGCCGTC GATGGGCCGT

61 GACATCAAGG TCCAATTCCA AAGTGGTGGT GCCAACTCGC CCGCCCTGTA CCTGCTCGAC

121 GGCCTGCGCG CGCAGGACGA CTTCAGCGGC TGGGACATCA ACACCCCGGC GTTCGAGTGG

181 TACGACCAGT CGGGCCTGTC GGTGGTCATG CCGGTGGGTG GCCAGTCAAG CTTCTACTCC

241 ACT GGTACC AGCCCGCCTG CGGCAAGGCC GGTTGCCAGA CTTACAAGTG GGAGACCTTC

301 CTGACCAGCG AGCTGCCGGG GTGG CTGCAG GCCAACAGGC ACGTCAAGCC CACCGGAAGC

361 GCCGTCGTCG GTCTTTCGAT GGCTGCTTCT TCGGCGCTGA CGCTGGCGAT CTATCACCCC

421 CAGCAGTTC G TCTAC GCGGG AGCGATGTCG GGCCTGTTGG ACCCCTCCCA GGCGATGGGT

481 CCCACCCTGA TCGGCCTGGC GATGGGTGAC GCTGGCGGCT ACAAGGCCTC CGACATGTGG

541 GGCCCGAAGG AGGACCCGGC GTGGCAGCGC AACGACCCGC TGTTGAACGT CGGGAAGCTG

601 ATCGCCAACA ACACCCGCGT CTGGGTGTAC TGCGGCAACG GCAAGCCGTC GGATCTGGGT

661 GGCAACAACC TGCCGGCCAA GTTCCTCGAG GGCTTCGTGC GGACCAGCAA CATCAAGTTC

721 CAAGACGCCT ACAACGCCGG TGGCGGCCAC AACGGCGTGT TCGACTTCCC GGACAGCGGT

781 ACGCACAGCT GGGAGTACTG GGGCGCGCAG CTCAACGCTA TGAAGCCCGA CCTGCAACGG

841 GCACTGGGTG CCACGCCCAA CACCGGGCCC GCGCCCCAGG GCGCCTAG

sequence 2

1 ATGGCAGAGC AGCAGTGGAA TTTCGCGGGT ATCGAGGCCG CGGCAAGCGC AATCCAGGGT

61 AATGTCACCT CCATTCATTC CCTCCTTGAC GAGGGGAAGC AGTCCCTGAC CAAGCTCGCA

121 GCGGCCTGGG GCGGTAGCGG TTCGGAGGCG TACCAGGGTG TCCAGCAAAA ATGGGACGCC

181 ACGGCTACCG AGCTGAACAA CGCGCTGCAG AACCTGGCGC GGACGATCAG CGAAGCCGGT

241 CAGGCAATGG CTTCGACCGA AGGCAACGTC ACTGGGATGT TCGCATAG

The preparation method of chimeric type tuberculosis gene vaccine of the present invention comprises the following steps:

(1) using primer a shown in sequence 3 and primer b shown in sequence 4 to amplify the Ag85a gene sequence by polymerase chain reaction;

(2) digest the Ag85a gene and the eukaryotic expression vector with endonuclease Nhe I and BamH I respectively, and connect the digestion products of the two with ligase, and construct the plasmid containing gene Ag85a;

(3) using a pair of primers with the same endonuclease recognition sequence as the Ag85a gene to amplify the ESAT6 gene;

(4) Digest the plasmid containing the gene Ag85a and the ESAT6 gene with an endonuclease that can recognize the above-mentioned endonuclease recognition sequence;

(5) Ligating the digestion product of step (4) with ligase to obtain the chimeric gene vaccine HG856.

Detailed description of the invention

The inventor searches for the antigenic epitopes of the Mycobacterium tuberculosis structural protein Ag85a gene through Epitope Informatics of the computer software service company Intenet-based applied bioinformatics company, and finds that its antigenic epitopes are mainly concentrated in the amino and carboxyl terminals of Ag85a (D'Souza, "Mapping of murine Thl helper T-cell epitopes of mycolyl transferases Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis", Infection and Immunity, 7(1):483-493, 2003). In the middle segment of the Ag85a parental gene that does not contain antigenic epitopes, the sequences recognized by the following restriction endonucleases can be found, which are the Kpn I recognition sequence at position 245-250; the Pst I recognition sequence at position 325-330 and the Acc I recognition sequence at positions 430-435.

In the above-mentioned restriction endonuclease site of Mycobacterium tuberculosis 85a gene (sequence 1), namely the restriction endonuclease Kpn I recognition sequence at the 245-250 ( GGTACC ) position, the restriction at the 325-330 ( CTGCAG ) position Recognition sequence of sex endonuclease Pst I and/or restriction endonuclease Acc I recognition sequence of position 430-435 ( GTCTAC ), inserted into other smaller gene sequences, such as the gene ESAT6 (sequence 2), can be constructed into a chimeric Mycobacterium tuberculosis gene vaccine.

The present inventors designed primer a and primer b with recognition sites for restriction endonucleases Nhe I ( GCTAGC ) and BamH I ( GGATCC ), amplified Ag85a maternal gene from Mycobacterium tuberculosis DNA by PCR technique, and used restriction The endonucleases Nhe I and BamH I digested the Ag85a gene and the eukaryotic expression vector respectively, and then connected the digested products with ligase to construct a plasmid containing the parent gene Ag85a.

The present inventor has also designed primers carrying Kpn I restriction endonuclease recognition sequence, PstI restriction endonuclease recognition sequence or Acc I restriction endonuclease recognition sequence respectively at the 5' end, respectively for ESAT6 minigene PCR amplification.

Use Kpn I restriction endonuclease to digest the eukaryotic expression vector containing the Ag85a parent gene and the ESAT6 minigene PCR amplification product obtained by the primer carrying the Kpn I restriction endonuclease recognition sequence at the 5' end respectively, and then the two The digested products were connected with ligase, and those with the correct connection direction were selected to construct the HG856-1 plasmid of the chimeric gene vaccine of Mycobacterium tuberculosis.

The eukaryotic expression vector containing the Ag85a parent gene and the ESAT6 minigene PCR amplification product obtained by the primers carrying the Acc I restriction endonuclease recognition sequence at the 5' end were respectively digested with Acc I restriction endonuclease, and then the two The digested products were connected with ligase, and those with the correct connection direction were selected to construct the HG856-2 plasmid of the chimeric gene vaccine of Mycobacterium tuberculosis.

The eukaryotic expression vector containing the Ag85a parent gene and the ESAT6 minigene PCR amplification product obtained by using the primers carrying the Pst I restriction endonuclease recognition sequence at the 5' end were respectively digested with Pst I restriction endonuclease, and then the two The digested products were connected with ligase, and those with the correct connection direction were selected to construct the HG856-3 plasmid of the chimeric gene vaccine of Mycobacterium tuberculosis.

Therefore, the chimeric gene vaccine for preventing and treating tuberculosis of the present invention is formed by inserting a relatively small Mycobacterium tuberculosis ESAT6 gene into an appropriate site of a relatively large Mycobacterium tuberculosis Ag85a maternal gene. Among them, chimeric Mycobacterium tuberculosis genetic vaccines HG856-1 and HG856 were constructed by inserting the ESAT6 gene at the 245-250th Kpn I recognition sequence site and the 430-435th Acc I recognition sequence site of the Ag85a maternal gene. The immunogenicity of -2 plasmid is better.

The present invention uses gene chimeric technology to chimerize the ESAT6 gene encoding the smallest Mycobacterium tuberculosis protective antigen into the gene encoding the most immune-protective Ag85a antigen, and the resulting novel Mycobacterium tuberculosis chimeric gene vaccine has shown in animal experiments It is better than the immune effect of single-gene vaccine, and the anti-ESAT6 and anti-Ag85a antibodies induced by it are higher than those produced by single-gene vaccine or two-gene fusion vaccine.

Description of drawings

Figure 1 shows how fusion genes differ from chimeric genes.

Fig. 2 is a construction diagram of the eukaryotic expression vector HG85-pVAX1 containing the parental gene of Mycobacterium tuberculosis Ag85a.

Fig. 3 is a construction diagram of the Mycobacterium tuberculosis chimeric genetic vaccine HG856 of the present invention.

Figure 4 shows the chimeric protein of Mycobacterium tuberculosis Ag85a and ESAT6 expressed in vitro by the chimeric Mycobacterium tuberculosis gene vaccine HG856.

Figure 5 shows the results of Western blot experiments of the expressed Ag85a and ESAT6 chimeric proteins.

Figure 6 shows the changes in the level of Mycobacterium tuberculosis 85a-specific antibody in serum of mice after booster immunization.

Figure 7 shows the changes in the level of Mycobacterium tuberculosis ESAT6-specific antibody in mouse serum after booster immunization.

Detailed ways

The present invention is described further below with embodiment. These examples are only for illustrating the present invention and do not constitute any limitation to the scope of the present invention. Mainly adopt conventional genetic engineering molecular biology cloning method in the embodiment, these methods are well known to those of ordinary skill in the art, for example: " modern molecular biology experimental technology " edited by Lu Shengdong, (second edition, published by Peking Union Medical College Medical University Press, December 1999, Beijing); and J. Sambrook, D.W. Russell, translated by Huang Peitang, etc.: "Molecular Cloning Experiment Guide" (third edition, August 2002, published by Science Press, Beijing) relevant chapters in . According to the following examples, those skilled in the art can easily implement the present invention by slightly modifying and transforming according to specific conditions, and these modifications and transforms all fall within the scope of the claims of the present application.

All the primers used for PCR in the examples were synthesized, purified and identified by mass spectrometry by Shanghai Sangong Bioengineering Technology Co., Ltd.; reagents such as various restriction endonucleases, other modified enzymes and matching reaction buffers were purchased from Shanghai TaKaRa Company; chemical reagents were purchased from Shanghai Chemical Reagent Company.

Embodiment 1 The acquisition of Ag85a gene

Design primer a (SEQ ID NO: 3):

5' GACT GCTAGC CACCATGGTTTCCCGGCCGGGCTTGCCGG-3'

Primer a is consistent with the 5' end of the Ag85a gene coding sequence, and is complementary to the 2nd to 22nd nucleotide sequence of the Ag85a gene encoding the structural protein (see sequence 1). It contains a NheI ( GCTAGC ) recognition sequence at its 5' end.

Design primer b (SEQ ID NO: 4):

5'GACT GGATCC TTACTAGGCGCCCTGGGGCGCGG-3'

Primer b is consistent with the 3' end of the Ag85a gene coding sequence, and is complementary to the 869th to 885th nucleotide sequence of the Ag85a gene. It contains a restriction recognition sequence of BamH I ( GGATCC ) at its 5' end.

Primer a and primer b were used to amplify the Mycobacterium tuberculosis Ag85a gene by using high-fidelity pfu DNA polymerase to carry out PCR reaction with the chromosomal DNA of the standard strain H37RV of Mycobacterium tuberculosis as template. Wherein the template amount, primer amount, enzyme amount, and buffer used are all carried out according to the conventional method of genetic engineering molecular cloning technology (see J. Edition, pp. 597-632, August 2002, published by Science Press, Beijing). The concentrations of the four dNTPs were 20 μM, and the Mg ²⁺ concentration was 1.5 mM; the denaturation, annealing, and extension temperatures were 94°C, 55°C, and 72°C, respectively, and the time was 1 minute, and a total of 30 cycles were performed. The DNA sequence of Ag85a gene was obtained.

Example 2 Construction of the plasmid vector HG85-pVAX1 containing the Mycobacterium tuberculosis gene Ag85a

Nhe I ( GCTAGC ) and BamH I ( GGATCC ) restriction endonuclease sites have been introduced into primer a and primer b respectively in Example 1, and the Mycobacterium tuberculosis Ag85a gene fragment obtained by PCR technology and the eukaryotic expression vector pVAX1 1 microgram of each plasmid (purchased from Invitrogen, Carlsbad, CA, USA), 10 units of Nhe I and BamH I restriction endonucleases were added, and mixed in 50 microliters of reaction buffer (according to the instructions in each enzyme kit) Instructions select buffer), digested at 37°C for 1 hour, separated by conventional agarose gel electrophoresis and recovered DNA in the gel (according to J. Sambrook, DW Russell, translated by Huang Peitang, "Molecular Cloning Experiment Guide", The third edition, pages 387-399, pages 404-407, August 2002, published by Science Press, Beijing) respectively obtained Ag85a gene fragment and pVAX1 plasmid fragment with cohesive ends. Then use T4 ligase (the product kit of Shanghai TaKaRa Biotechnology Co., Ltd., according to the operation manual) to connect the above two fragments overnight, add Escherichia coli DH1 or DH5α competent cell suspension, and inoculate 1% kanamycin-containing Cultivate in the agar plate for 16-18 hours at 37° C. to obtain transformed engineering bacteria containing the HG85 clone plasmid. Cultivate the engineered bacteria in a medium containing kanamycin, collect by centrifugation, extract the plasmid by alkaline lysis, digest with two different enzymes Nhe I and BamH I, and perform agar electrophoresis on the digested product to verify the correct positive clone , select the correct plasmid containing the Mycobacterium tuberculosis gene Ag85a that can be used for subsequent chimeric operations.

Conventional agarose gel DNA electrophoresis: Add 5 μl of loading buffer (Loading Buffer) to 50 μl of enzyme digestion reaction mixture, mix well and add all to the wells of the agarose plate, use TAE electrophoresis buffer, and the voltage during electrophoresis is 80-90V/cm, 30-45 minutes. (See J. Sambrook, D.W. Russell, translated by Huang Peitang, etc. "Molecular Cloning Experiment Guide", third edition, pages 387-399, August 2002, published by Science Press, Beijing).

Recovery of DNA fragments in agarose gel: Use the kit for the rapid purification/recovery kit of DNA fragments produced by Beijing Dingguo Biotechnology Development Center. Operate according to the kit instructions, cut out the gel containing the required DNA fragment after 1% low-melting point gel electrophoresis, put it into an EP tube, about 100 microliters per tube, add 400 microliters of buffer (200mM Tris-HCl, 1mM EDTA, pH8.0), heated at 65°C for 5 minutes to dissolve the gel, added an equal volume of saturated phenol for extraction, centrifuged at 1500rpm for 5 minutes; took the supernatant and added an equal volume of chloroform for extraction, and centrifuged at 1500rpm for 5 minutes; took the supernatant and added 10 microliters 3M NaCl (pH5). After mixing with 2 times the volume of absolute ethanol, place it at -20°C for 30 minutes, centrifuge at 1500 rpm for 5 minutes; wash the precipitated DNA once with 70% ethanol, remove the liquid, dissolve it with an appropriate volume of water, and recover the Ag85a DNA fragment. Take 2 μl of recovered fragments for agarose gel electrophoresis to estimate the efficiency of recovery and the amount to be added in subsequent ligation reactions.

DNA ligation reaction: use the kit produced by Promega, operate according to the instructions: mix 1 μl of T4 ligase, 1 μl of 10× buffer, 7 μl of Ag85a DNA fragment, 1 μl of pVAX1 plasmid fragment, mix well and connect at 14°C overnight. (see J. Sambrook, D.W. Russell, Huang Peitang et al. translation "Molecular Cloning Experiment Guide", third edition, 85-86 pages, August 2002, published by Science Press, Beijing).

The ligated pVAX1 plasmid containing the Ag85a gene (recombinant HG85 cloning plasmid) was added to the competent cells of Escherichia coli DG1 or DH5α, and the cells were transformed at 37° C. for 1 hour. Take out 200 μl and inoculate it on the agar medium plate containing kanamycin, spread the bacteria evenly, and incubate at 37°C for 16-18 hours.

The pVAX1 vector plasmid contains the anti-kanamycin gene, so the Escherichia coli successfully transformed by the recombinant HG85 cloning vector plasmid can grow into colonies on this medium. Select a single clone colony and inoculate it in 3ml of culture solution containing kanamycin, and culture it at 37°C with shaking at 200rpm for about 14 hours. Take out 1ml of the bacterial solution and centrifuge at 12000rpm for 1 minute to collect the bacterial cells, add 100μl solution I (50mM glucose, 25mM Tris-HCl, 10mM EDTA, pH8.0) to resuspend the bacteria, add 200μl solution II (2% SDS, 0.4molNaOH) Lyse the bacteria for 5 minutes, add 150 μl solution III (3M potassium acetate pH5.5) to neutralize; centrifuge at 13000rpm for 10 minutes; absorb the supernatant and add 1/10 volume of 3M sodium acetate pH5.3), 2 times the volume of anhydrous Ethanol, react at room temperature for 10 minutes and then centrifuge at 13000rpm for 10 minutes; after discarding the supernatant, evaporate the ethanol at room temperature, use Nhe I and BamH I for enzyme digestion and agarose gel electrophoresis identification to obtain the gene containing Mycobacterium tuberculosis HG85 vector plasmid HG85-pVAX1.

Example 3 Construction of Chimeric Mycobacterium tuberculosis Gene HG856-1 Vector Plasmid

Design primer c (SEQ ID NO: 5):

5'-GACT GGTACC TAATGGCAGAGCAGCAGTGG-3'

Primer c is consistent with the 5' end of the ESAT6 gene coding sequence, and is complementary to the 1st to 18th nucleotide sequence of the ESAT6 gene, and contains a KpnI enzyme recognition sequence at its 5' end.

Design primer d (SEQ ID NO: 6):

5'GACT GGTACC TTGCGAACATCCCAGTGAC-3'

Primer d is consistent with the 3' end of the ESAT6 gene coding sequence, and is complementary to the 268th to 285th nucleotide sequence of the ESAT6 gene, and also contains a KpnI restriction recognition sequence at its 5' end.

Using primer c and primer d, the tuberculosis standard strain H37RV chromosomal DNA was used as a template, and the high-fidelity pfu DNA polymerase was used for PCR reaction to amplify tuberculosis ESAT6 gene. Wherein the template amount, primer amount, enzyme amount, and buffer used are all carried out according to the conventional method of genetic engineering molecular cloning technology (see J. Sambrook, DW Russell, Huang Peitang et al. Edition, pp. 85-86, August 2002, published by Science Press, Beijing). The concentrations of the four dNTPs were 20 μM, and the Mg ²⁺ concentrations were 1.5 mM; the denaturation, annealing, and extension temperatures were 94°C, 55°C, and 72°C, respectively, and the time was 1 minute. Get the DNA sequence of ESAT6 gene.

The HG85-pVAX1 plasmid containing the Mycobacterium tuberculosis gene Ag85a obtained in Example 2 is digested with Kpn I restriction endonuclease to obtain its fragment, and the DNA sequence fragment of the Mycobacterium tuberculosis ESAT6 gene with the Kpn I restriction endonuclease recognition sequence obtained above According to the method substantially similar to Example 2 (agarose gel electrophoresis, DNA fragment recovery and DNA ligation reaction in the gel), the two fragments are connected with T4 ligase, that is, the ESAT6 gene DNA sequence is inserted into the Mycobacterium tuberculosis Ag85a gene sequence 249 in the Kpn I enzyme recognition site. Transform the recombined clone into competent Escherichia coli according to the above method, and inoculate it on a Kanamycin agar plate for culture; select 3-5 positive colonies for small-scale enrichment, and extract the plasmid DNA as a template; Design a pair of primers for the sequence at the junction of the 5'-end of the gene and perform PCR amplification, sequence the junction of the Ag85a gene and the ESAT6 gene of the amplified product, select the one with the correct insertion direction, and construct a gene containing Mycobacterium tuberculosis Ag85a and ESAT6 A chimeric gene vaccine HG856-1 plasmid.

Example 4 Construction of Chimeric Mycobacterium tuberculosis Gene HG856-2 Vector Plasmid

Design primer e (SEQ ID NO: 7):

5'-GACT GTCTAC TAATGGCAGAGCAGCAGTGG-3'

Primer e is consistent with the 5' of the ESAT6 gene coding sequence, and is complementary to the 1st to 18th nucleotide sequence of the ESAT6 gene. It contains an Acc I restriction recognition sequence at its 5' end.

Design primer f (SEQ ID NO: 8):

5'-GACT GTCTAC TTGCGAACATCCCAGTGAC-3'

Primer f is consistent with the 3' of the ESAT6 gene coding sequence, and is complementary to the 268th to 285th nucleotide sequence of the ESAT6 gene. It also contains an Acc I restriction recognition sequence at its 5' end.

Using primer e and primer f, using the chromosomal DNA of the standard strain of Mycobacterium tuberculosis H37RV as a template, high-fidelity pfu DNA polymerase was used to carry out PCR reaction. Conventional methods (see J. Sambrook, DW Russell, "Molecular Cloning Experiment Guide" translated by Huang Peitang, third edition, pp. 85-86, August 2002, published by Science Press, Beijing). The concentrations of the four dNTPs were 20 μM, the Mg ²⁺ concentrations were 1.5 mM, the denaturation, annealing, and extension temperatures were 94° C., 55° C., and 72° C. for 1 minute. get ESAT6 gene

The HG85-pVAX1 plasmid containing the Mycobacterium tuberculosis gene Ag85a obtained in Example 2 is digested with Acc I restriction endonuclease to obtain its fragment, and the DNA of the Mycobacterium tuberculosis ESAT6 gene with the Acc I endonuclease recognition sequence obtained above Sequence fragment, according to the method substantially similar to embodiment 2 (agarose gel electrophoresis, DNA fragment recovery and DNA ligation reaction in the gel) two fragments are connected with T4 ligase, be about to insert ESAT6 gene DNA sequence into Mycobacterium tuberculosis Ag85a gene sequence In the 432-bit Acc I enzyme recognition site, the recombinant clone was transformed into competent Escherichia coli according to the above method, and inoculated on a Kanamycin agar plate for culture; after selecting 3-5 positive colonies for small-scale enrichment, extract Plasmid DNA was used as a template; according to the sequence of the Ag85a gene near the 5'-end junction of the ESAT6 gene, a pair of primers were designed and PCR amplified, and the amplified product was sequenced at the junction of the Ag85a gene and the ESAT6 gene, and the insertion direction was selected correctly. Or, construct the gene vaccine HG856-2 plasmid containing the chimeric gene of Mycobacterium tuberculosis Ag85a and ESAT6.

Example 5 Construction of Chimeric Mycobacterium tuberculosis Gene HG856-3 Vector Plasmid

Design primer g (SEQ ID NO: 9):

5'-GACT CTGCAG TAATGGCAGAGCAGCAGTGG-3'

Primer g is consistent with the 5' end of the ESAT6 gene coding sequence and is complementary to the 1st to 18th nucleotide sequence of the ESAT6 gene. It contains a Pst I enzyme recognition sequence at its 5' end.

Design primer h (SEQ ID NO: 10):

5'- GACTCTGCAGTTGCGAACATCCCAGTGAC -3'

Primer h is consistent with the 3' end of the ESAT6 gene coding sequence and is complementary to the 268th to 285th nucleotide sequence of the ESAT6 gene. It also contains a Pst I enzyme recognition sequence at its 5' end.

Using techniques and methods similar to those in Example 3, the sequence of the Pst I restriction site is designed in the primer, and the ESAT6 gene PCR product containing the restriction site is digested with the Pst I restriction endonuclease, and then inserted Into the PstI enzyme recognition site of the Ag85a gene sequence 325-330 nucleotides in the HG85-pVAX1 plasmid, the recombinant clone was transformed into competent Escherichia coli as described above, and inoculated on a Kanamycin agar plate for culture; After selecting 3-5 positive colonies for small-scale enrichment, extract the plasmid DNA as a template; design a pair of primers according to the end sequence of the Ag85a gene near the 5'-end junction of the ESAT6 gene and carry out PCR amplification. The junction of Ag85a gene and ESAT6 gene of the product was sequenced, and the one with the correct insertion direction was selected to construct the third tubercle bacillus chimeric gene vaccine HG856-3 plasmid containing the chimeric gene of Mycobacterium tuberculosis Ag85a and ESAT6 gene.

EXPERIMENTAL EXAMPLE 1 In Vitro Gene Expression of Chimeric Mycobacterium tuberculosis Gene Vaccine HG856 Plasmid

TNT in vitro transcription and translation system kit (Promega, Madison, WI, USA) was used to verify the expression of chimeric Mycobacterium tuberculosis gene HG856 in vitro. According to the experimental procedure described in the instructions of Promega Corporation of the United States, each 12.5 microliter reaction system contains 0.25 micrograms of chimeric Mycobacterium tuberculosis gene HG856 plasmid DNA and 9 microliters of _TNT T7 rapid reaction master solution, and each milliliter contains 400 μCi[ S ³⁵ ]-labeled methionine was mixed and incubated at 30°C for 90 minutes. Perform regular 10% SDS-PAGE electrophoresis on the protein expressed by the chimeric gene (with [S ³⁵ ] radioactivity), and use autoradiography to observe the swimming position of the protein to determine whether the chimeric Mycobacterium tuberculosis gene is expressed, and the expression correctness.

The results are shown in Figure 4. Swimming lanes 1 to 5 are the proteins expressed by 5 chimeric Mycobacterium tuberculosis gene clones to be screened. According to the comparison with the standard protein molecular weight bands of 29kDa and 44kDa, the molecular weight of the protein expressed by the clone in the fourth lane is about 42kDa. The molecular weight of Mycobacterium tuberculosis Ag85a protein antigen is about 32kDa; while the molecular weight of Mycobacterium tuberculosis ESAT6 protein antigen is about 10kDa, the sum of the two is about 42kDa. Therefore, the fourth lane is the product expressed by the chimeric Mycobacterium tuberculosis gene HG856.

Experimental example 2 immunoblotting (Western blot) experiment

0.25 μg Mycobacterium tuberculosis protein antigen Ag85a and protein molecular weight standard were subjected to vertical electrophoresis on SDS-polyethylene agarose gel, and then the protein was transferred to nitrocellulose membrane by 100V electrotransfer for 1.5 hours. Block with 5% skimmed milk powder for 1 hour, then incubate with specific mouse antiserum diluted 1:1,000 at 37°C for 1.5 hours, wash with 0.1% PBS-Tween-20 for 3 times; add PBS-Tween-20 buffer Alkaline phosphate-labeled goat anti-mouse IgG antibody (Sigma, Cat#A3688) diluted 1:10,000, incubated at 37°C for 1.5 hours, and washed 3 times; add substrate (BCIP/NBT:AP Buffer=4:1, BCIP /NBT (purchased from Sigma Company) for color development, was terminated with PBS containing 1 mM EDTA.

The results are shown in Figure 5. in,

Column 1: protein standard molecular weight marker;

Column 2: 85a monogenic vaccine;

Column 3: HG856-2 chimeric gene (inserted in the 432 AccI restriction site of 85a gene);

Column 4: HG856-1 chimeric gene (inserted at the KpnI restriction site at position 249 of the 85a gene).

The results showed that both the tuberculosis chimeric gene vaccine and the single-gene 85a nucleic acid vaccine could produce specific immune responses against the tuberculosis 85a protein antigen.

Experimental Example 3 Serum Induced After Mice Inoculated with Mycobacterium Tuberculosis Chimeric Gene HG856 Plasmid

Specific antibody immune response (as determined by ELISA method)

Materials and methods:

1. Experimental animals

Eight-week-old female BALB/C mice were randomly divided into groups and raised in the SPF (Specific Pathogen Free) animal room provided by the Animal Center of Shanghai Second Military Medical University.

2. Immunization of experimental animals

After three times of nucleic acid vaccine immunization, the final booster immunization was done with protein.

50 female BALB/C mice were randomly divided into 10 groups, 5 in each group: Group 1-2 (ESAT6 monogene vaccine), Group 3-4 (85a monogene vaccine), Group 5-6 (ESAT6+IL-12 co-expression double gene vaccine), group 7-8 (chimeric gene vaccine with ESAT6 inserted at 85a432 site), group 9-10 (chimeric gene vaccine with ESAT6 inserted at 85a249 site) . The single group was the intramuscular injection group, which was injected 100 μg/time in the tibialis anterior muscle, and the double group was the electrotransfection group after intramuscular injection, which was injected 10 μg/time in the tibialis anterior muscle, and immunized once every two weeks, a total of 3 times. On the 10th day after the last immunization, the titer of the serum collected and separated from the tail vein was measured by ELISA. According to the test results, after confirming that the gene immunization has produced a specific immune response, that is, 8-12 days after the third gene vaccine immunization, the corresponding Mycobacterium tuberculosis protein antigen is used for intraperitoneal inoculation to boost the immunization, 50 μg per mouse. One week later, the animals were anesthetized and sacrificed to harvest whole blood, and the centrifuged serum was stored at -20°C.

3. In vivo electrotransfection method

Immediately after the intramuscular injection, the injection site was clamped with a clip with electrodes, and the in vivo electrotransfection was performed with the WJ-2002 in vivo gene introduction instrument (Ningbo Xinzhi Biotechnology Co., Ltd.) (voltage: 100 V; pulse frequency: 6 positive and 6 negative) times; wave width: 60 milliseconds; interval: 10 milliseconds).

4. Detection of anti-85aIgG antibody and anti-ESAT-6 antibody by conventional ELISA indirect method

Coat 96-well ELISA plates (50 μl/well) with purified recombinant Ag85a protein or ESAT-6 protein (1.25 μg/mL each), place overnight at 4 ° C, and wash 3 times with 0.1% PBS-Tween 20; The wells were blocked with 100 μl of 0.5% bovine serum albumin for 1 hour, washed 3 times with 0.1% PBS-Tween 20; the mouse serum was diluted to 1:102400 from 1:100, and added to each well (50 μl /well), incubated at 37°C for 2 hours, washed 3 times with 0.1% PBS-Tween 20; added alkaline phosphate-labeled goat anti-mouse IgG antibody (Sigma, Cat. #A3688), incubate at 37°C for 1.5 hours, and wash 3 times; add the substrate to develop color, stop the reaction with 3M NaOH after 30 minutes, and use a microplate reader for OD _105nm detection. The results are shown in Table 1, Table 2 and Figures 6 and 7.

Table 1: Detection of Mycobacterium tuberculosis 85a-specific antibody levels in serum of mice after inoculation with tuberculosis gene vaccine and after corresponding protein vaccine booster (ELISA)

Mycobacterium tuberculosis genetic vaccine Geometric mean titer (GMT) of specific 85a antibody Intramuscular injection group (100μg DNA) Electrotransfection group after intramuscular injection (10μg DNA) Before protein boost After protein boost Before protein boost After protein boost 85a single gene vaccine 55 360 65 420 HG856-1 (85a+ESAT-6 chimeric gene) was inserted at the KpnI restriction site at position 249 of the 85a gene 38 187 64 482 HG856-2 (85a+ESAT-6 chimeric gene) was inserted at the 432 AccI restriction site of the 85a gene twenty four 177 52 464 Note: The GMT of the unimmunized control group was 9.7. 50μg Mycobacterium tuberculosis 85a protein antigen was injected intraperitoneally as booster immunization. In this experiment, alkaline phosphatase was used as a substrate and read at OD405nm.

In Fig. 6, ■ is the intramuscular injection electrotransduction group (10 μg), and □ is the intramuscular injection group (100 μg).

Ia is before 85a single gene vaccination, Ib is after 85a single gene vaccination;

IIa is before HG856-2 vaccination, IIb is after HG856-2 vaccination;

IIIa is before HG856-1 vaccination, and IIIb is after HG856-1 vaccination.

It can be seen from Table 1 and Figure 6 that the dosage of 85a gene vaccination is only 1/10 of that of simple intramuscular injection by electroporation, but the effect obtained is equivalent to it, or even slightly better; at this time, 85a single gene or chimeric gene vaccine effect was not significantly different. After boosting with protein vaccine, the effect of chimeric gene vaccine is not as good as that of single gene vaccine in simple intramuscular injection; however, the effect of electrotransfected chimeric gene vaccine is worse than that of single gene vaccine, and both are worse than simple intramuscular injection. It is suggested that the chimeric gene vaccine has a good application prospect as the basic vaccination of electrotransfection, and then boosted with protein vaccine.

Table 2: ESAT6-specific antibody levels of Mycobacterium tuberculosis in mouse serum after inoculation with the tuberculosis gene vaccine and after boosting with the corresponding protein vaccine

TB Gene Vaccine Geometric mean titer (GMT) of specific ESAT-6 antibody Intramuscular injection group (100μg) Electrotransfection group after intramuscular injection (10μg) Enhanced ESAT-6 Enhanced ESAT-6+85a before strengthening After strengthening before strengthening After strengthening before strengthening After strengthening HG3 (ESAT-6 single gene) 19±3 22±6 12±2 18±3 - - HG43 (85a+ESAT-6 chimeric gene) was inserted at the 444th AccI restriction site of the 85a gene 20±11 50±36 28±17 241±146 27±18 178±131 HG44 (85a+ESAT-6 chimeric gene) was inserted at the KpnI restriction site at position 252 of the 85a gene 17±5 34±9 22±12 114±167 26±10 102±51 Note: The GMT of the normal group is 7.0. In this experiment, alkaline phosphatase was used as a substrate and read at OD405nm.

Figure 7 shows the detection of anti-ESAT-6 antibody in mouse serum after immunization with tuberculosis gene vaccine and boosted with corresponding protein vaccine, expressed as mean geometric titer.

The ESAT-6 antigen is a weak antigen, much weaker than the 85a antigen, and it is difficult to induce a strong immune response after inoculating animals, but its immunogenicity is enhanced after being chimerized in 85a.

It can be seen from Table 2 and Figure 7 that the dosage of ESAT-6 single-gene vaccine was only 1/10 of that of simple intramuscular injection by electroporation, but the effect was equivalent; Synthetic vaccines are similarly effective.

However, after boosting with protein vaccine, the effect of ESAT-6 single-gene vaccine alone intramuscular or electrotransfection injection is not good; the effect of chimeric gene HG43 vaccine is slightly better than ESAT-6 single-gene vaccine and chimeric gene HG44 vaccine when simple intramuscular injection ; But the effects of the two chimeric gene HE43 and HE44 vaccines by electrotransfection were significantly better than those of single-gene vaccines, and much better than that of simple intramuscular injection. It is suggested that the chimeric gene vaccine has a good application prospect as the basic vaccination of electrotransfection, and then boosted with protein vaccine. In order to improve the Th1 cell immune response, it is best to use recombinant BCG or recombinant vaccinia containing genes related to Mycobacterium tuberculosis for booster immunization. The chimeric tuberculosis gene vaccine described herein is suitable for basic immunization.

The results of this experiment show that the new chimeric Mycobacterium tuberculosis gene vaccine can simultaneously induce specific antibodies against Mycobacterium tuberculosis 85a and ESAT6 in mice, and the effect is better than that of 85a single-gene vaccine and ESAT6 single-gene vaccine. Basic immunizations.

sequence listing

<110> Li Zhongming

<120> Mycobacterium tuberculosis chimeric gene vaccine and preparation method thereof

<130>021194

<140>

<141>2004-11-19

<160>10

<170>PatentIn version 3.2

<210>1

<211>887

<212>DNA

<213> Mycobacterium tuberculosis chromosomal DNA

<400>1

ttttcccggc cgggcttgcc ggtggagtac ctgcaggtgc cgtcgccgtc gatgggccgt 60

gacatcaagg tccaattcca aagtggtggt gccaactcgc ccgccctgta cctgctcgac 120

ggcctgcgcg cgcaggacga cttcagcggc tgggacatca acaccccggc gttcgagtgg 180

tacgaccagt cgggcctgtc ggtggtcatg ccggtgggtg gccagtcaag cttctactcc 240

actggtacca gcccgcctgc ggcaaggccg gttgccagac ttacaagtgg gagaccttcc 300

tgaccagcga gctgccgggg tggctgcagg ccaacaggca cgtcaagccc accggaagcg 360

ccgtcgtcgg tctttcgatg gctgcttctt cggcgctgac gctggcgatc tatcaccccc 420

agcagttcgt ctacgcggga gcgatgtcgg gcctgttgga cccctcccag gcgatgggtc 480

ccaccctgat cggcctggcg atgggtgacg ctggcggcta caaggcctcc gacatgtggg 540

gcccgaagga ggacccggcg tggcagcgca acgacccgct gttgaacgtc gggaagctga 600

tcgccaacaa cacccgcgtc tgggtgtact gcggcaacgg caagccgtcg gatctgggtg 660

gcaacaacct gccggccaag ttcctcgagg gcttcgtgcg gaccagcaac atcaagttcc 720

aagacgccta caacgccggt ggcggccaca acggcgtgtt cgacttcccg gacagcggta 780

cgcacagctg ggagtactgg ggcgcgcagc tcaacgctat gaagcccgac ctgcaacggg 840

cactgggtgc cacgcccaac accgggcccg cgccccaggg cgcctag 887

<210>2

<211>288

<212>DNA

<213> Mycobacterium tuberculosis chromosomal DNA

<400>2

atggcagagc agcagtggaa tttcgcgggt atcgaggccg cggcaagcgc aatccagggt 60

aatgtcacct ccattcattc cctccttgac gaggggaagc agtccctgac caagctcgca 120

gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcatag 288

<210>3

<211>39

<212> polynucleotide

<213> Synthetic

<400>3

gactgctagc caccatggtt tcccggccgg gcttgccgg 39

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<211>33

<212> polynucleotide

<213> Synthetic

<400>4

gactggatcc ttactaggcg ccctggggcg cgg 33

<210>5

<211>30

<212> polynucleotide

<213> Synthetic

<400>5

gactggtacc taatggcaga gcagcagtgg 30

<210>6

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<212> polynucleotide

<213> Synthetic

<400>6

gactggtacc ttgcgaacat cccagtgac 29

<210>7

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<212>DNA

<213> polynucleotide

<400>7

gactgtctac taatggcaga gcagcagtgg 30

<210>8

<211>29

<212> polynucleotide

<213> Synthetic

<400>8

gactgtctac ttgcgaacat cccagtgac 29

<210>9

<211>30

<212> polynucleotide

<213> Synthetic

<400>9

gactctgcag taatggcaga gcagcagtgg 30

<210>10

<211>29

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gactctgcag ttgcgaacat cccagtgac 29

Claims (9)

1. mosaic type tubercle bacillus gene vaccine, it is characterized in that: comprise the coding tubercule bacillus ESAT6 gene shown in coding tubercule bacillus structural protein 85a gene shown in the sequence 1 and the sequence 2, wherein said ESAT6 gene is entrenched on one or two site in the sequence that sequence that the 245-250 position restricted enzyme Kpn I of 85a gene discerned, sequence that 325-330 position restriction endonuclease Pst I is discerned and 430-435 position restriction endonuclease Acc I discerned, and described 85a gene is connected in the carrier for expression of eukaryon.

2. mosaic type tubercle bacillus gene vaccine as claimed in claim 1, wherein said Kpn I recognition sequence is GGTACCDescribed Pst I recognition sequence is CTGCAGDescribed Acc I recognition sequence is GTCTAC

3. mosaic type tubercle bacillus gene vaccine as claimed in claim 1, wherein said carrier for expression of eukaryon is JW4303, or pcDNA3.1, or pVAX1 series.

4. mosaic type tubercle bacillus gene vaccine as claimed in claim 1, wherein said carrier for expression of eukaryon is a pVAX1 series.

5. the preparation method of mosaic type tubercle bacillus gene vaccine according to claim 1 is characterized in that, may further comprise the steps:

(1) use primer b shown in primer a shown in the sequence 3 and the sequence 4 with PCR amplification Ag85a gene order;

(2) digest Ag85a gene and carrier for expression of eukaryon respectively with restriction endonuclease Nhe I and BamH I, and connect the two digestion product, be built into the plasmid that contains gene A g85a with ligase;

(3) with having the primer that is selected from Kpn I restriction endonuclease recognition sequence, Pst I restriction endonuclease recognition sequence and Acc I restriction endonuclease recognition sequence to amplification ESAT6 gene;

(4) digest plasmid and the ESAT6 gene that contains gene A g85a respectively with the restriction endonuclease that can discern above-mentioned restriction endonuclease recognition sequence;

(5) with the digestion product of ligase Connection Step (4), obtain chimeric gene vaccine HG856.

6. as the preparation method of mosaic type tubercle bacillus gene vaccine as described in the claim 5, wherein said restriction endonuclease recognition sequence is: the sequence of Kpn I identification GGTACC, Pst I identification sequence CTGCAGOr the sequence of Acc I identification GTCTAC

7. as the preparation method of mosaic type tubercle bacillus gene vaccine as described in the claim 5, wherein said primer is to being primer d shown in primer c shown in the sequence 5 and the sequence 6.

8. as the preparation method of mosaic type tubercle bacillus gene vaccine as described in the claim 5, wherein said primer is to being primer f shown in primer e shown in the sequence 7 and the sequence 8.

9. as the preparation method of mosaic type tubercle bacillus gene vaccine as described in the claim 5, wherein said primer is to being primer h shown in primer g shown in the sequence 9 and the sequence 10.

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