CN100354004C - Tubercle bacillus chimeric gene vaccine and preparation process thereof - Google Patents
Tubercle bacillus chimeric gene vaccine and preparation process thereof Download PDFInfo
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- CN100354004C CN100354004C CNB2004100843761A CN200410084376A CN100354004C CN 100354004 C CN100354004 C CN 100354004C CN B2004100843761 A CNB2004100843761 A CN B2004100843761A CN 200410084376 A CN200410084376 A CN 200410084376A CN 100354004 C CN100354004 C CN 100354004C
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A61P31/06—Antibacterial agents for tuberculosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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Abstract
The present invention provides a mosaic type tubercle bacillus gene vaccine which comprises encoded tubercle bacillus structural protein 85a genes disclosed in sequence 1 and encoded tubercle bacillus ESAT6 genes disclosed in sequence 2, wherein the ESAT6 genes are mosaicked in a sequence of the 85a genes connected with the inner part of a eukaryotic expression vector pVAX1. The present invention also provides a preparation method of the mosaic type tubercle bacillus gene vaccine (plasmid), which comprises the PCR amplification of the Ag85a genes, the construction of a gene vaccine HG85(plasmid), the PCR amplification of the ESAT6 genes, and the insertion of the ESAT6 genes in the plasmid HG85. The immunogenicity of the mosaic type tubercle bacillus gene vaccine of the present invention is enhanced, and the immune effect of the mosaic type tubercle bacillus gene vaccine is superior to that of a monogenic vaccine.
Description
Technical field
The present invention relates to the new generation vaccine technology of biological medicine category, be specifically related to adopt the tubercule bacillus mosaic type gene vaccine of the representative vaccine revolution for the third time of the chimeric technology development of gene.
Background technology
Because bacillus calmette-guerin vaccine does not have immanoprotection action to adult's pulmonary tuberculosis; add increasing day by day of anti-multiple antibiotics tulase strains separation rate; and the increasing of the concurrent mycobacterium tuberculosis infection case of acquired immune deficiency syndrome (AIDS), tuberculosis has become and has threatened one of human life and healthy the most dangerous infectious disease.Seek a kind ofly, become the focus and the task of top priority that world today research and development can prevent into human tuberculosis's novel vaccine than the better new generation vaccine of bacillus calmette-guerin vaccine immune protective effect.Only have 10 years historical dna vaccination (being also referred to as gene vaccine) technology, because its numerous advantage, caused vaccine revolution for the third time, be the infectious disease that to prevent more for a long time or preventive effect is not good, acquired immune deficiency syndrome (AIDS) for example, influenza, the prevention of malaria and tuberculosis etc. have been brought the dawn of wishing.
" naked engineering plasmid " that dna vaccination adopts escherichia coli to produce carries out immunity inoculation.Plasmid is also referred to as carrier or plasmid vector or vector plasmid, and being that Bacillus coli cells is internal energy is independent of the annular super coiled DNA that chromosome duplicates and expresses.Dna vaccination is through genetic engineering modified with escherichia coli plasmid, the proteinic gene of preferred antigens of coding pathogen, preferred immunostimulant cytokine have been inserted therein, as the gene or the CpG adjuvant motif of the interleukin 12 of encoding (IL-12), colony-stimulating factor (GM-CSF), and be suitable for gene recombinaton (engineering) plasmid that human cell's element such as promoter and suitable antibiotic resistance genes etc. make up.After the engineering plasmid DNA of purification is injected into positions such as human muscle and enters cell, this plasmid can be at relevant cell, antigen processed cell (Antigen Present Cell particularly, APC) express coded antigen in, induce body generation body fluid and cellullar immunologic response to react the infection of resisting pathogen.The plasmid DNA at the meticulous preparation of inhomogeneity pathogen of development at present, can adopt carrier for expression of eukaryon JW4303, or pcDNA3.1, or plasmid such as pVAX1 series, but the kalamycin resistance escherichia coli plasmid pVAX1 that can be used for human body that mainly adopts U.S. FDA to recommend transforms and forms.Eukaryotic expression plasmid is transformed in the escherichia coli (being called engineering bacteria this moment), under optimized conditions, the culturing engineering bacterium makes bacterial multiplication, plasmid replication in the bacterium becomes a plurality of copies. and antibacterial is collected in centrifugation in the time of suitably, and the cracking antibacterial discharges plasmid under suitable condition; Collect the back and remove impurity, plasmid purification promptly can be used as dna vaccination or gene vaccine and is used for inoculation.
Think the immune effect not ideal enough (Gregorialdis of monogenic tuberculosis dna vaccination in large animal experiment at present, " Genetic vaccines:strategies for optimization ", Pharmaceutical Research, 15:661-670,1998); The dual-gene vaccine that the method that two antigen encoding genes are merged in employing makes up, though be better than the single-gene vaccine, can reach with two kinds of antigenic purposes of difference of a kind of vector expression, but there is not collaborative potentiation between the two, also be the effective (Stevenson of the vaccine-induced immunne response of fusion gene unlike the single-gene vaccine, " DNA fusion gene vaccines against cancer:from laboratory to the clinic ", Immunology Research, 199:156-180,2004).Discovering in recent years, on cellular level with two kinds of virus chimerics together, sometimes it is little to produce a kind of side effect, and the stronger mosaic viral vaccine (Mathenge of immunogenicity, " Fusion PCR generated Japaneseencephalitis virus/dengue 4 virus chimera exhibits lack of neuroinvasivensess; attenuated neurovirulence; and a dual-flavi immune response in mice ", Journal of General Virology, 85:2503-2513,2004).Based on similar principle, the scientist who has will derive from same pathogenic microorganism on molecular level gene is entrenched togather and produces the mosaic gene vaccine, obtained effect (Domingo preferably, " Immunological propertiesof a DNA plasmid encoding a chimeric protein of herpes simplex virus type2 glycoprotein B and glycoprotein D ", Vaccine, 21 (25-26): 3565-3574,2003).The somebody attempts utilizing the mosaic gene technology gene that a kind of immunogenicity is more weak to be embedded in another pathogenic microorganism gene, improves the immunogenicity of poor antigen, and this is a heterologous mosaic gene technology.Its successful example has the chimeric encoding gene of HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) V3 in the encoding gene of hbs antigen (HbsAg).The antigenic V3 epi-position of HIV (human immunodeficiency virus) (Human Immunodeficiency Virus) coat protein is very important protective antigen decision a small bundle of straw, etc. for silkworms to spin cocoons on, but because its molecular weight is less, is difficult to induce stronger immunne response.Yet, the gene of coding HIVV3 is entrenched in the gene of coding hbs antigen (HbsAg), this mosaic gene of result can induce body fluid and the cellullar immunologic response (Bryder of the anti-HIV V3 of specificity, " Imoroved immunogenicity ofHIV-1 epitopes in HbsAg chimeric DNA vaccine plasmids by structuralmutations of HbsAg ", DNAand Cell Biology, 18 (3): 219-225,1999).
In the research work of tuberculosis new generation vaccine, someone is in the same place with the more intense tubercule bacillus protective antigen Ag85B gene fusion of coding immunogenicity at the poor tubercule bacillus protective antigen ESAT6 gene of immunogenicity of once will encoding, its expressed proteins antigen has bigger molecular weight, helps improving micromolecule ESAT6 immunogenicity of antigens; Yet, the gene of coding ESAT6 is connected the aminoterminal of coding Ag85B gene and the effect of c-terminus has very big difference, the latter is not as the former (Shi Changhong, " amalgamation and expression of mycobacterium tuberculosis secretory protein Ag85B-ESAT6 and purification ", China's tuberculosis and breathing magazine, 27 (2): 89-92,2004).And because proteinic epitope generally all concentrates on its aminoterminal and c-terminus, whether the fusion connected mode may influence expressed antigenic immunological characteristic is still waiting research.Yet chimeric method might be entrenched in a little gene middle part of big gene, does not influence the immunocompetence of big gene expression product when strengthening weak genetic immunization originality.But up to now, in the research of tubercle bacillus gene vaccine construction, do not see the report that adopts the mosaic gene technology and succeed.
The present inventor is surprisingly found out that; adopt will the encode ESAT6 gene of tubercule bacillus protective antigen of minimum of the chimeric technology of gene to be fitted in the antigenic gene of Ag85a of the tool immune protective effect of encoding; the novel tubercle bacillus chimeric gene vaccine that produces demonstrates the immune effect that is better than the single-gene vaccine in zoopery; the height that it induces the anti-ESAT6 antibody of generation and anti-Ag85a antibody all to produce than single-gene vaccine or two gene fusion bacterin, thus might apply in the actual vaccine.
Summary of the invention
The mosaic type tubercle bacillus gene vaccine that the object of the present invention is to provide a kind of immunogenicity to improve.
The present invention also aims to provide the preparation method of this mosaic type tubercle bacillus gene vaccine.
Mosaic type tubercle bacillus gene vaccine of the present invention comprises 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.
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 TCTACGCGGG 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
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 mosaic type tubercle bacillus gene vaccine of the present invention 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 a pair of primer amplification ESAT6 gene that has identical restriction endonuclease recognition sequence with the Ag85a 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.
Detailed Description Of The Invention
The present inventor searches for the epitope of tubercule bacillus structural protein Ag85a gene by the Epitope Informatics of the Intenet-based appliedbioinformatics company of computer software service company, find its epitope mainly concentrate on the aminoterminal of Ag85a and c-terminus (D ' Souza, " Mapping of murine Thl helper T-cell epitopesof mycolyl transferases Ag85A; Ag85B; and Ag85C from Mycobacteriumtuberculosis ", Infection and Immunity, 7 (1): 483-493,2003).Do not containing the Ag85a maternal gene centre portion of epitope, the sequence that can find following restricted enzyme to discern, they are respectively the Kpn I recognition sequences of 245-250 position; The Pst I recognition sequence of 325-330; And the Acc I recognition sequence of 430-435 position.
At the above-mentioned restriction endonuclease sites of tubercule bacillus 85a gene (sequence 1), promptly be positioned at 245-250 (
GGTACC) position restricted enzyme Kpn I recognition sequence, 325-330 (
CTGCAG) position restricted enzyme Pst I recognition sequence and/or 430-435 (
GTCTAC) the restriction enzyme A cc I recognition sequence of position, inserting other less gene order, the gene ESAT6 (sequence 2) as coding tubercule bacillus protective antigen can be built into mosaic type tubercle bacillus gene vaccine.
The present inventor designed have restricted enzyme Nhe I (
GCTAGC) and BamH I (
GGATCC) the primer a and the primer b of recognition site, with round pcr from tubercule bacillus chromosomal DNA amplification Ag85a maternal gene, and digest Ag85a gene and carrier for expression of eukaryon respectively with restricted enzyme Nhe I and BamH I, connect both digestion products then with ligase, be built into the plasmid that contains maternal gene Ag85a.
The present inventor has also designed the primer that carries Kpn I restriction endonuclease recognition sequence, PstI restriction endonuclease recognition sequence or Acc I restriction endonuclease recognition sequence at 5 ' end respectively, is respectively applied for the pcr amplification of ESAT6 mini-gene.
Digest carrier for expression of eukaryon that contains the Ag85a maternal gene and the ESAT6 mini-gene pcr amplification product that carries the primer acquisition of Kpn I restriction endonuclease recognition sequence at 5 ' end respectively with Kpn I restricted enzyme, digestion product with both couples together with ligase then, select the correct person of closure, be built into tubercule bacillus mosaic type gene vaccine HG856-1 plasmid.
Digest carrier for expression of eukaryon that contains the Ag85a maternal gene and the ESAT6 mini-gene pcr amplification product that carries the primer acquisition of Acc I restriction endonuclease recognition sequence at 5 ' end respectively with Acc I restricted enzyme, digestion product with both couples together with ligase then, select the correct person of closure, be built into tubercule bacillus mosaic type gene vaccine HG856-2 plasmid.
Digest carrier for expression of eukaryon that contains the Ag85a maternal gene and the ESAT6 mini-gene pcr amplification product that carries the primer acquisition of Pst I restriction endonuclease recognition sequence with 5 ' end respectively with Pst I restricted enzyme, digestion product with both couples together with ligase then, select the correct person of closure, be built into tubercule bacillus mosaic type gene vaccine HG856-3 plasmid.
Therefore, control of the present invention chimeric gene vaccine lungy is that a smaller tubercule bacillus ESAT6 gene is inserted into chimeric forming in the suitable site of a bigger tubercule bacillus Ag85a maternal gene.Wherein, be good to insert the gene constructed mosaic type tubercle bacillus gene vaccine HG856-1 that becomes of ESAT6 and the immunogenicity of HG856-2 plasmid in the Acc I recognition sequence site of Kpn I recognition sequence site, the 245-250 position of Ag85a maternal gene and 430-435 position.
The present invention adopts will the encode ESAT6 gene of minimum tubercule bacillus protective antigen of the chimeric technology of gene to be fitted in the antigenic gene of Ag85a of the tool immune protective effect of encoding; the novel tubercle bacillus chimeric gene vaccine that produces demonstrates the immune effect that is better than the single-gene vaccine in zoopery, the height that it induces the anti-ESAT6 of generation and anti-Ag85a antibody all to produce than single-gene vaccine or two gene fusion bacterin.
Description of drawings
Fig. 1 shows the difference of fusion gene and mosaic gene.
Fig. 2 is the design of graphics that contains the carrier for expression of eukaryon HG85-pVAX1 of tubercule bacillus Ag85a maternal gene.
Fig. 3 is the design of graphics of tubercule bacillus mosaic type gene vaccine HG856 of the present invention.
Fig. 4 shows that mosaic type tubercle bacillus gene vaccine HG856 is at external expressed tubercule bacillus Ag85a and ESAT6 chimeric protein.
Fig. 5 shows immunity printing and dyeing (Westernblot) experimental result of expressed Ag85a and ESAT6 chimeric protein.
Fig. 6 shows the variation of tubercule bacillus 85a specific antibody level in the mice serum behind the booster immunization.
Fig. 7 shows the variation of tubercule bacillus ESAT6 specific antibody level in the mice serum behind the booster immunization.
The specific embodiment
Below the present invention is further elaborated with embodiment.These embodiment only are used to illustrate the present invention, and scope of the present invention are not constituted any restriction.The main genetic engineering molecular biology cloning process that adopts routine among the embodiment, these methods are well known to those of ordinary skill in the art, for example: Lu Shengdong edits " modern molecular biology experimental technique ", (second edition, publishing house of China Concord Medical Science University, in December, 1999, Beijing); With J. Sa nurse Brooker, D.W. Russell work, Huang Peitang etc. translate: the relevant chapters and sections in " molecular cloning experiment guide " (Science Press published, Beijing the third edition, in August, 2002).Those of ordinary skills are according to following examples, the slightly modified and conversion be not difficult as the case may be and successfully implement the present invention, and these are revised and conversion all drops in the scope of the application's claim.
Among the embodiment primer of the PCR that is useful on by Shanghai give birth to that worker's biotechnology company limited is synthetic, purification and mass spectrography identify correct; Various restricted enzyme, other modification enzymes and the reagent such as reaction buffer that match are available from Shanghai TaKaRa company; Chemical reagent is all available from Shanghai chemical reagents corporation.
The acquisition of embodiment 1 Ag85a gene
Design primer a (sequence 3):
5’GACT
GCTAGCCACCATGGTTTCCCGGCCGGGCTTGCCGG-3’
5 ' end of primer a and Ag85a gene coded sequence is consistent, and with the 2nd to the 22nd the nucleotide sequence complementation (seeing sequence 1) of coding structure protein A g85a gene.Its 5 ' end contain a Nhe I (
GCTAGC) restriction endonuclease recognition sequence.
Design primer b (sequence 4):
5’GACT
GGATCCTTACTAGGCGCCCTGGGGCGCGG-3’
3 ' end of primer b and Ag85a gene coded sequence is consistent, and with the 869th to the 885th nucleotide sequence complementation of Ag85a gene.Its 5 ' end contain a BamH I (
GGATCC) restriction endonuclease recognition sequence.
Adopting primer a and primer b, is template with tubercule bacillus reference culture H37RV chromosomal DNA, adopts high-fidelity pfu archaeal dna polymerase to carry out the PCR reaction, amplification tubercule bacillus Ag85a gene.Wherein template amount, primer amount, enzyme dosage, used buffer are all undertaken (seeing J. Sa nurse Brooker, D.W. Russell work by the conventional method of genetic engineering molecule clone technology, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, the 597-632 page or leaf, in August, 2002, Science Press publishes, Beijing).The concentration of four kinds of dNTP is respectively 20 μ M, Mg
2+Concentration is 1.5mM; The temperature of degeneration, annealing, extension is respectively 94 ℃, 55 ℃, 72 ℃, and the time all is 1 minute, carries out 30 circulations altogether.Obtain the Ag85a gene DNA sequence.
In embodiment 1, introduced respectively among primer a and the primer b Nhe I (
GCTAGC) and BamH I (
GGATCC) restriction endonuclease sites, get tubercule bacillus Ag85a genetic fragment that round pcr obtains and carrier for expression of eukaryon pVAX1 plasmid (available from Invitrogen, Carlsbad, CA, USA) each 1 microgram, the Nhe I and the BamH I restricted enzyme that add 10 units respectively, be mixed in the reaction buffer of 50 microlitres (selecting buffer for use) by the description in each enzyme reagent kit, 37 ℃ digested 1 hour, DNA recovery method in conventional agarose gel electrophoresis separation and glue (press J. Sa nurse Brooker, D.W. Russell work, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, the 387-399 page or leaf, 404-407 page or leaf, in August, 2002, Science Press publishes, Beijing) obtain the Ag85a genetic fragment and the pVAX1 plasmid fragment of sticky end each other respectively.Then with T4 ligase (Shanghai TaKaRa biotechnology company product test kit, undertaken by operating instruction) above-mentioned two kinds of fragments are connected spend the night, add in escherichia coli DH1 or the DH5 α competent cell suspension, inoculation contains in 1% agar plate of kanamycin 37 ℃ cultivated 16-18 hour, obtained containing the conversion engineering bacteria of HG85 cloned plasmids.In containing the culture medium of kanamycin, cultivate this project bacterium, centrifugal collection, the alkaline lysis method of extracting plasmid, carry out enzyme action with two kinds of different enzymes of Nhe I and BamH I, the enzyme action product is made agar electrophoresis, verify correct positive colony, select the correct plasmid that contains tubercle bacillus gene Ag85a that can be used for follow-up chimeric operation.
Conventional agarose gel DNA electrophoresis: the sample loading buffer (Loading Buffer) that in the endonuclease reaction mixture of 50 μ l, adds 5 μ l, all add in the hole of agarose plate behind the mixing, adopt the TAE electrophoretic buffer, voltage is 80-90V/cm during electrophoresis, 30-45 minute.(see J. Sa nurse Brooker, D.W. Russell work, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, the 387-399 page or leaf, in August, 2002, Science Press publishes, Beijing).
The recovery of dna fragmentation in the agarose gel: the dna fragmentation fast purifying/recovery test kit that uses test kit to produce as Beijing ancient cooking vessel state biotech development center.Operate according to the test kit description, downcut the glue that contains required dna fragmentation behind the 1% low melting point gel electrophoresis, pack in the EP pipe, about 100 microlitres of every pipe add 400 microlitre buffer (200mM Tris-HCl, 1mM EDTA, pH8.0), 65 ℃ of heating dissolving in 5 minutes glue adds the saturated phenol extracting of equal-volume, centrifugal 5 minutes of 1500rpm; Get and reset and add the extracting of equal-volume chloroform, centrifugal 5 minutes of 1500rpm; Get supernatant and add 10 microlitre 3M NaCl (pH5).With behind 2 times of volume dehydrated alcohol mixings-20 ℃ placed centrifugal 5 minutes of 1500rpm 30 minutes; Sedimentary DNA washes once with 70% ethanol, eliminates liquid, uses the proper volume water dissolution, reclaims to obtain the Ag85aDNA fragment.Get 2 μ l recovery fragment and make agarose gel electrophoresis, the amount that efficient that estimation is reclaimed and follow-up coupled reaction need add.
The DNA coupled reaction: the test kit that adopts Promega to produce, the by specification operation: with the T4 ligase of 1 microlitre, 1 microlitre, 10 * buffer, the Ag85a dna fragmentation of 7 μ l, the pVAX1 plasmid fragment of 1 μ l, 14 ℃ of connections are spent the night behind the mixing.(referring to J. Sa nurse Brooker, D.W. Russell work, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, the 85-86 page or leaf, in August, 2002, Science Press publishes, Beijing).
The pVAX1 plasmid that contains the Ag85a gene (gene recombinaton HG85 cloned plasmids) that connects is joined in the competent cell of escherichia coli DG1 or DH5 α, and 37 ℃ of reactions transformed this cell in 1 hour.Take out 200 μ l inoculation and contain on the agar culture medium flat board of kanamycin, the antibacterial shop is even, cultivated 16-18 hour for 37 ℃.
Contain anti-kanamycin gene in the pVAX1 vector plasmid, therefore the escherichia coli that successfully transformed by gene recombinaton HG85 cloning vector plasmids can be grown to serve as bacterium colony on this culture medium.Select the monoclonal colony inoculation and contain in the culture fluid of kanamycin in 3ml, 37 ℃, the 200rpm wave and culture is about 14 hours.The bacterium liquid 12000rpm. that takes out 1ml collected thalline in centrifugal 1 minute, add 100 μ l solution I (50mM glucoses, 25mM Tris-HCl, 10mM EDTA, pH8.0) resuspended antibacterial, add 200 μ l solution II (2%SDS, 0.4molNaOH) the cracking antibacterial is 5 minutes, adds 150 μ l solution III (3M potassium acetate pH5.5) neutralizations; Centrifugal 10 minutes of 13000rpm; Draw the 3M sodium acetate pH5.3 that supernatant adds its 1/10 volume), the dehydrated alcohol of 2 times of volumes, room temperature reaction centrifugal 10 minutes of 13000rpm after 10 minutes; After the abandoning supernatant, ethanol is removed in the room temperature volatilization, and the DNA that obtains is done enzyme action and agarose gel electrophoresis evaluation with Nhe I and BamH I, obtains to contain the vector plasmid HG85-pVAX1 of tubercle bacillus gene HG85.
The structure of embodiment 3 mosaic type tubercle bacillus gene HG856-1 vector plasmids
Design primer c (sequence 5):
5’-GACT
GGTACCTAATGGCAGAGCAGCAGTGG-3’
5 ' end of primer c and ESAT6 gene coded sequence is consistent, and with the 1st to the 18th nucleotide sequence complementation of ESAT6 gene, contain the restriction endonuclease recognition sequence of a Kpn I at its 5 ' end.
Design primer d (sequence 6):
5’GACT
GGTACCTTGCGAACATCCCAGTGAC-3’
3 ' end of primer d and ESAT6 gene coded sequence is consistent, and with the 268th to the 285th nucleotide sequence complementation of ESAT6 gene, also contain the restriction endonuclease recognition sequence of a Kpn I at its 5 ' end.
With primer c and primer d, be template with tubercule bacillus reference culture H37RV chromosomal DNA, adopt high-fidelity pfu archaeal dna polymerase to carry out the PCR reaction, amplification tubercule bacillus ESAT6 gene.Wherein template amount, primer amount, enzyme dosage, used buffer are all undertaken (referring to J. Sa nurse Brooker by the conventional method of genetic engineering molecule clone technology, D.W. the Russell is outstanding, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, the 85-86 page or leaf, in August, 2002, Science Press publishes, Beijing).The concentration of four kinds of dNTP is respectively 20 μ M, Mg
2+Concentration is 1.5mM; The temperature of degeneration, annealing, extension is respectively 94 ℃, 55 ℃, 72 ℃, and the time all is 1 minute.Obtain the ESAT6 gene DNA sequence.
Obtain its fragment with the HG85-pVAX1 plasmid that contains tubercle bacillus gene Ag85a that obtains among the Kpn I digestion with restriction enzyme embodiment 2, dna sequencing fragment with the above tubercule bacillus ESAT6 gene that has Kpn I restriction endonuclease recognition sequence that obtains, according to two fragments being connected with the T4 ligase, be about to the ESAT6 gene DNA sequence and be inserted in 249 Kpn I enzyme recognition sites of tubercule bacillus Ag85a gene order with embodiment 2 basic similar methods (dna fragmentation reclaims and the DNA coupled reaction in agarose gel electrophoresis, the glue).The clone of reorganization is transformed in the competence escherichia coli as stated above, and inoculation kanamycin agar plate is cultivated; After selecting 3-5 positive bacterium colony and increasing bacterium in a small amount, the extracting plasmid DNA is as template; Sequence according to close ESAT6 gene 5 '-end junction in the Ag85a gene, design the go forward side by side performing PCR amplification of a pair of primer, check order in Ag85a gene and ESAT6 gene junction to amplified production, select the correct person of direction of insertion, be built into the gene vaccine HG856-1 plasmid that contains tubercule bacillus Ag85a and ESAT6 gene tabling.
The structure of embodiment 4 mosaic type tubercle bacillus gene HG856-2 vector plasmids
Design primer e (sequence 7):
5’-GACT
GTCTACTAATGGCAGAGCAGCAGTGG-3’
Primer e and ESAT6 gene coded sequence 5 ' consistent, and with the 1st to the 18th nucleotide sequence complementation of ESAT6 gene.The restriction endonuclease recognition sequence that contains an Acc I at its 5 ' end.
Design primer f (sequence 8):
5’-GACT
GTCTACTTGCGAACATCCCAGTGAC-3’
Primer f and ESAT6 gene coded sequence 3 ' consistent, and with the 268th to the 285th nucleotide sequence complementation of ESAT6 gene.The restriction endonuclease recognition sequence that also contains an Acc I at its 5 ' end.
With primer e and primer f, with tubercule bacillus reference culture H37RV chromosomal DNA is template, adopt high-fidelity pfu archaeal dna polymerase to carry out the PCR reaction, wherein template amount, primer amount, enzyme dosage, used buffer all by the conventional method of genetic engineering molecule clone technology carry out (referring to J. Sa nurse Brooker, D.W. Russell work, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, 85-86 page or leaf, in August, 2002, Science Press publishes, Beijing).The concentration of four kinds of dNTP is respectively 20 μ M, Mg
2+Concentration is 1.5mM, and the temperature of degeneration, annealing, extension is respectively 94 ℃, 55 ℃, 72 ℃, and the time all is 1 minute.Obtain the ESAT6 gene
Obtain its fragment with the HG85-pVAX1 plasmid that contains tubercle bacillus gene Ag85a that obtains among the Acc I digestion with restriction enzyme embodiment 2, dna sequencing fragment with the above tubercule bacillus ESAT6 gene that has Acc I inscribe restriction endonuclease recognition sequence that obtains, according to embodiment 2 basic similar methods (agarose gel electrophoresiies, dna fragmentation reclaims and the DNA coupled reaction in the glue) two fragments are connected with the T4 ligase, being about to the ESAT6 gene DNA sequence is inserted in 432 Acc I enzyme recognition sites of tubercule bacillus Ag85a gene order, the clone of reorganization is transformed in the competence escherichia coli as stated above, and inoculation kanamycin agar plate is cultivated; After selecting 3-5 positive bacterium colony and increasing bacterium in a small amount, the extracting plasmid DNA is as template; Sequence according to close ESAT6 gene 5 '-end junction in the Ag85a gene, design the go forward side by side performing PCR amplification of a pair of primer, check order in Ag85a gene and ESAT6 gene junction to amplified production, select the correct person of direction of insertion, be built into the gene vaccine HG856-2 plasmid that contains tubercule bacillus Ag85a and ESAT6 gene tabling.
The structure of embodiment 5 mosaic type tubercle bacillus gene HG856-3 vector plasmids
Design primer g (sequence 9):
5’-GACT
CTGCAGTAATGGCAGAGCAGCAGTGG-3’
5 ' end of primer g and ESAT6 gene coded sequence is consistent, and with the 1st to the 18th nucleotide sequence complementation of ESAT6 gene.The restriction endonuclease recognition sequence that contains a Pst I at its 5 ' end.
Design primer h (sequence 10):
5’-GACT
CTGCAGTTGCGAACATCCCAGTGAC-3’
3 ' end of primer h and ESAT6 gene coded sequence is consistent, and with the 268th to the 285th nucleotide sequence complementation of ESAT6 gene.The restriction endonuclease recognition sequence that also contains a Pst I at its 5 ' end.
Adopt technology and the method similar with embodiment 3, the sequence of design Pst I restriction enzyme site in primer, the ESAT6 gene PCR product Pst I digestion with restriction enzyme that will contain this restriction enzyme site, and then be inserted in the Pst I enzyme recognition site of the Ag85a gene order 325-330 position nucleotide in the HG85-pVAX1 plasmid, the clone of reorganization is transformed in the competence escherichia coli as stated above, and inoculation kanamycin agar plate is cultivated; After selecting 3-5 positive bacterium colony and increasing bacterium in a small amount, the extracting plasmid DNA is as template; Terminal sequence according to close ESAT6 gene 5 '-end junction in the Ag85a gene, design the go forward side by side performing PCR amplification of a pair of primer, check order in Ag85a gene and ESAT6 gene junction to amplified production, select the correct person of direction of insertion, be built into the 3rd the tubercule bacillus mosaic type gene vaccine HG856-3 plasmid that contains tubercule bacillus Ag85a and ESAT6 gene tabling.
The outer-gene of experimental example 1 mosaic type tubercle bacillus gene vaccine HG856 plasmid is expressed
(WI USA) verifies that mosaic type tubercle bacillus gene HG856 is in external expression for Promega, Madison to adopt TNT in vitro transcription and translation system test kit.According to the experimental procedure of describing in the U.S. Promega company description, in each 12.5 microlitre response systems, contain 0.25 microgram mosaic type tubercle bacillus gene HG856 plasmid DNA and 9 microlitre T
NT T7 fast reaction mother solution, with every milliliter contain 400 μ Ci[S
35] after the methionine of labelling mixes, hatched 90 minutes for 30 ℃.This mosaic gene expressed protein (is had [S
35] radioactivity) make conventional 10%SDS-PAGE electrophoresis, judge with the swimming position of autoradiography method observing protein whether the mosaic type tubercle bacillus gene expresses, and correctness of expression.
The results are shown in Figure 4.Swimming lane 1 to 5 is 5 expressed protein of mosaic type tubercle bacillus gene clone to be screened.According to the comparison of standard protein molecular weight 29kDa and 44kDa band, the protein molecular weight of clonal expression is about 42kDa in the 4th swimming lane.The molecular weight of tubercule bacillus Ag85a proteantigen is about 32kDa; And the molecular weight of tubercule bacillus ESAT6 proteantigen is about 10kDa, and the two addition is about 42kDa.Therefore the 4th swimming lane is the expressed product of mosaic type tubercle bacillus gene HG856.
Experimental example 2 immunity printing and dyeing (Western blot) experiments
Earlier 0.25 μ g tubercule bacillus proteantigen Ag85a and protein molecular weight standard are carried out the poly-third ethylene agarose gel vertical electrophoresis of SDS-, with 1.5 hours method of 100V electrotransfer protein transduction is moved on on the cellulose nitrate film then.With 5% defatted milk powder sealing 1 hour, then with 1: 1, the specificity mouse resisting anteserum of 000 dilution was hatched 1.5 hours at 37 ℃, with 0.1%PBS-Tween-20 washing 3 times; Adding was by PBS-Tween-20 buffer 1: 10, and the alkali phosphoric acid labelling goat anti-mouse igg antibody of 000 dilution (Cat#A3688), hatched 1.5 hours for 37 ℃, washs 3 times again by Sigma; The adding substrate (BCIP/NBT: AP Buffer=4: 1, BCIP/NBT is available from Sigma company) colour developing, stop with the PBS that contains 1mM EDTA.
The results are shown in Figure 5.Wherein,
The 1st row: protein standard molecular weight labelling;
The 2nd row: 85a single-gene vaccine;
The 3rd row: HG856-2 mosaic gene (being inserted in 432 AccI enzyme action points of 85a gene);
The 4th row: HG856-1 mosaic gene (being inserted in 249 KpnI enzyme action points of 85a gene).
The result shows that tubercule bacillus mosaic type gene vaccine and monogenic 85a nucleic acid vaccine can both produce the immunne response of specific anti-mycobacterium tuberculosis 85a proteantigen.
Inductive serum behind the experimental example 3 tubercule bacillus mosaic type gene HG856 plasmids inoculation mice
Specific antibody immune response (ELISA method mensuration)
Material and method:
1. laboratory animal
After 8 week ages female BALB/C mice random packet, raise at SPF (Specific Pathogen Free) the level Animal House that The 2nd Army Medical College animal center in Shanghai provides.
2. the immunity inoculation of laboratory animal
Adopt after three nucleic acid vaccine immunities, do last booster immunization with protein.
With 50 of female BALB/C mice, be divided into 10 groups at random, 5 every group: 1-2 group (ESAT6 single-gene vaccine), 3-4 group (85a single-gene vaccine), 5-6 group (the dual-gene vaccine of ESAT6+IL-12 coexpression), 7-8 group (ESAT6 is inserted in the chimeric gene vaccine in 85a432 site), 9-10 organize (ESAT6 is inserted in the chimeric gene vaccine in 85a249 site).The odd number group is the intramuscular injection group, injects 100 μ g/ time respectively at tibialis anterior, and the even numbers group is an electrotransfection group after the intramuscular injection, injects 10 μ g/ time respectively at tibialis anterior, every two all immunity once, and totally 3 times.After the last immunity the 10th day, the tail vein adopted and isolating serum is surveyed it with ELISA and tired.According to test result, determine that genetic immunization has produced specific immune response after, promptly for the third time behind the dna gene vaccine 8-12 days corresponding tubercule bacillus proteantigens of reuse carry out the abdominal cavity inoculation with booster immunization, 50 μ g/ are only.Will be after the Animal Anesthesia after one week put to death the results whole blood, the serum after centrifugal is in-20 ℃ of preservations.
3. electrotransfection method in the body
Clamp the injection site with electroded clip immediately after the intramuscular injection, carry out electrotransfection (voltage: 100 V in the body with WJ-2002 live body gene introducing apparatus (NingBo XinZhi Biology Science Co., Ltd); Pulse number: positive and negative each 6 times; Ripple is wide: 60 milliseconds; At interval: 10 milliseconds).
4. adopt conventional ELISA indirect method to carry out anti-85aIgG antibody and anti-ESAT-6 detection of antibodies
Reorganization Ag85a albumen or ESAT-6 albumen (respectively being 1.25 μ g/mL) with purification wrap by 96 hole ELISA Plate (50 μ l/ hole), and 4 ℃ of placements are spent the night, with 0.1%PBS-polysorbas20 washing 3 times; Every hole was sealed 1 hour with 0.5% bovine serum albumin of 100 μ l, with 0.1%PBS-polysorbas20 washing 3 times; Mice serum was with 1: 100 beginning doubling dilution to 1: 102400, add respectively in (50 μ l/ hole) in each hole, and hatched 2 hours for 37 ℃, with 0.1%PBS-polysorbas20 washing 3 times; Adding was by PBS-polysorbas20 buffer 1: 10, and the alkali phosphoric acid labelling goat anti-mouse igg antibody of 000 dilution (Cat#A3688), hatched 1.5 hours for 37 ℃, washs 3 times again by Sigma; Add the substrate colour developing, use 3M NaOH cessation reaction after 30 minutes, and make OD with microplate reader
105nmDetect.The results are shown in Table 1, table 2 and Fig. 6, Fig. 7.
Table 1: tubercule bacillus 85a specific antibody level detects (ELISA) in the mice serum of after the mycobacterial genomic vaccination and respective egg white matter vaccine reinforcement back
| The tubercle bacillus gene vaccine | The geometric mean titer (GMT) of specificity 85a antibody | |||
| Intramuscular injection group (100 μ g DNA) | Electrotransfection group after the intramuscular injection (10 μ g DNA) | |||
| Before protein is strengthened | After protein is strengthened | Before protein is strengthened | After protein is strengthened | |
| 85a single- |
55 | 360 | 65 | 420 |
| HG856-1 (85a+ESAT-6 mosaic gene) is inserted in 249 KpnI enzyme action points of |
38 | 187 | 64 | 482 |
| HG856-2 (85a+ESAT-6 mosaic gene) is inserted in 432 AccI enzyme action points of |
24 | 177 | 52 | 464 |
| Annotate: the GMT of immune matched group is not 9.7.With 50 μ g tubercule bacillus 85a proteantigen lumbar injections as booster immunization.This experiment is made substrate with alkali phosphatase, in the OD405nm reading. | ||||
Among Fig. 6, ■ is an intramuscular injection electricity transduction group (10 μ g), and is intramuscular injection group (100 μ g).
Before Ia is the vaccination of 85a single-gene, after Ib is the vaccination of 85a single-gene;
Before IIa is HG856-2 vaccination, after IIb is HG856-2 vaccination;
Before IIIa is HG856-1 vaccination, after IIIb is HG856-1 vaccination.
From table 1 and Fig. 6 as seen, adopt the electrotransfection method, the consumption of 85a gene vaccine inoculation has only 1/10 of simple intramuscular injection, and income effect is but suitable with it, even omits; This moment, the effect of 85a single-gene or chimeric gene vaccine did not have remarkable difference.And after strengthening with protein vaccine, the chimeric gene vaccine effect is not as the single-gene vaccine during simple intramuscular injection; But the chimeric gene vaccine effect of electrotransfection is better than the single-gene vaccine, and all is better than simple intramuscular injection.Point out this chimeric gene vaccine to make the electrotransfection basic vaccination, the reuse protein vaccine strengthens having the applications well prospect.
Table 2: the ESAT6 specific antibody level of tubercule bacillus in the mice serum of after the mycobacterial genomic vaccination and respective egg white matter vaccine reinforcement back
| The TB gene vaccine | The geometric mean titer (GMT) of specificity ESAT-6 antibody | |||||
| Intramuscular injection group (100 μ g) | Electrotransfection group after the intramuscular injection (10 μ g) | |||||
| ESAT-6 strengthens | ESAT-6+85a strengthens | |||||
| Before the reinforcement | After the reinforcement | Before the reinforcement | After the reinforcement | Before the reinforcement | After the reinforcement | |
| HG3 (ESAT-6 single-gene) | 19 ± 3 | 22 ± 6 | 12 ± 2 | 18 ± 3 | - | - |
| HG43 (85a+ESAT-6 mosaic gene) is inserted in 444 AccI enzyme action points of |
20 ± 11 | 50 ± 36 | 28 ± 17 | 241 ± 146 | 27 ± 18 | 178 ± 131 |
| HG44 (85a+ESAT-6 mosaic gene) is inserted in 252 KpnI enzyme action points of 85a gene | 17 ± 5 | 34 ± 9 | 22 ± 12 | 114 ± 167 | 26 ± 10 | 102 ± 51 |
| Annotate: the GMT of normal group is 7.0.This experiment is made substrate with alkali phosphatase, in the OD405nm reading. | ||||||
Fig. 7 shows the mycobacterial genomic vaccine immunity and strengthens the anti-ESAT-6 detection of antibodies of back mice serum with respective egg white matter vaccine, is expressed as average geometric and tires.
ESAT-6 antigen is poor antigen, than a little less than the 85a antigen many, difficultly behind the inoculation animal induce stronger immunne response, the back immunogenicity is strengthened among the 85a but it is entrenched in.
From table 2 and Fig. 7 as seen, adopt the electrotransfection method, the consumption of ESAT-6 single-gene vaccination has only 1/10 of simple intramuscular injection, and income effect is but suitable with it; The effect of ESAT-6 single-gene or chimeric gene vaccine was more or less the same before protein vaccine was strengthened.
And with after the protein vaccine reinforcement, simple muscle of ESAT-6 single-gene vaccine or electrotransfection injection effect are all not good; Mosaic gene HG43 vaccine effect is slightly better than ESAT-6 single-gene vaccine and mosaic gene HG44 vaccine during simple intramuscular injection; But the two kinds of mosaic gene HE43 and the HE44 vaccine effect of electrotransfection all obviously are better than the single-gene vaccine, and are better than simple intramuscular injection greatly.Point out this chimeric gene vaccine to make the electrotransfection basic vaccination, the reuse protein vaccine strengthens having the applications well prospect.For improving the Th1 cellullar immunologic response, booster immunization preferably adopts recombinant bacillus Calmette-Guerin vaccine, or contains the recombinant vaccinia of tubercule bacillus related gene.Mosaic type mycobacterial genomic vaccine as herein described is fit to do fundamental immunity.
The result of this experimental example shows: novel mosaic type tubercle bacillus gene vaccine can be induced anti-mycobacterium tuberculosis 85a and ESAT6 simultaneously in the mice body specific antibody, and effect is better than 85a single-gene vaccine and ESAT6 single-gene vaccine, is fit to do basic immunity.
Sequence table
＜110〉Li Zhongming
＜120〉tubercle bacillus 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〉tubercule bacillus 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〉tubercule bacillus 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
<210>4
<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
<211>29
＜212〉polynucleotide
＜213〉synthetic
<400>6
gactggtacc ttgcgaacat cccagtgac 29
<210>7
<211>30
<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
＜212〉polynucleotide
＜213〉synthetic
<400>10
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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| PCT/CN2005/001914 WO2006053485A1 (en) | 2004-11-19 | 2005-11-14 | A chimeric mycobacterium tuberculosis gene vaccine and the preparation method thereof |
| ZA200704459A ZA200704459B (en) | 2004-11-19 | 2007-05-30 | A chimeric mycobacterium tuberculosis gene vaccine and the preparation method thereof |
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| CN101376891B (en) * | 2007-08-27 | 2012-05-23 | 上海生物制品研究所有限责任公司 | Preparation of novel tuberculosis vaccine and use thereof |
| CN101376025B (en) * | 2007-08-29 | 2012-05-23 | 中国人民解放军总医院第二附属医院 | Bacillus tubercle gene vaccine for treating medicine-tolerant pulmonary tuberculosis |
| WO2010010577A1 (en) * | 2008-07-25 | 2010-01-28 | Department Of Biotechnology | Constructing a dna chimera for vaccine development against leishmaniasis and tuberculosis |
| CN101912605A (en) * | 2010-04-23 | 2010-12-15 | 上海市公共卫生临床中心 | Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof |
| CN102268446B (en) * | 2010-06-03 | 2013-01-02 | 上海海规生物科技有限公司 | Tubercle bacillus Ag85ab mosaic gene vaccine and preparation method and application thereof |
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| WO2002004018A2 (en) * | 2000-07-10 | 2002-01-17 | Colorado State University Research Foundation | Mid-life vaccine and methods for boosting anti-mycobacterial immunity |
| CN1539504A (en) * | 2003-05-09 | 2004-10-27 | 浙江省医学科学院 | Bacterin of tabling gene of E type hepatitis virus |
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| AU3593200A (en) * | 1999-02-09 | 2000-08-29 | Powderject Vaccines, Inc. | (mycobacterium tuberculosis), immunization |
| CN1467221A (en) * | 2002-07-14 | 2004-01-14 | 虹 王 | Preparation and application of tubercle bacillus CFP-10, ESAT-6 and CD40L fusion bacterin |
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| WO2002004018A2 (en) * | 2000-07-10 | 2002-01-17 | Colorado State University Research Foundation | Mid-life vaccine and methods for boosting anti-mycobacterial immunity |
| CN1539504A (en) * | 2003-05-09 | 2004-10-27 | 浙江省医学科学院 | Bacterin of tabling gene of E type hepatitis virus |
Non-Patent Citations (1)
| Title |
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| 结核杆菌Ag85A及ESAT-6双价抗原融合表达载体的构建及其在大肠杆菌中的高效表达 谢勇恩等人. 中国人兽共患病杂志，第18卷第2期 2002 * |
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