CN101376025B - Bacillus tubercle gene vaccine for treating medicine-tolerant pulmonary tuberculosis - Google Patents

Abstract

The invention relates to a method for preparing a mycobecterium tuberculosis gene vaccine used for curing drug-resistant tuberculosis, and is characterized in that the mycobacterium tuberculosis gene vaccine against the drug-resistant tuberculosis clones a coding gene sequence of the mycobacterium tuberculosis Ag85A to the eukaryotic expression vector pVAX1, or clones coding gene sequences of the Ag85A and the ESAT6 together to the eukaryotic expression vector pVAX1 to form the ESAT6-Ag85A chimeric gene vaccine. Separated immunity or combined chemotherapy can obviously relieve pathology change in lungs of mice with drug-resistant tuberculosis and can obviously reduce bacteria numbers in lungs and spleens of little mice.

Description

Be used to prepare the tubercle bacillus gene vaccine of treating resistant tuberculosis

Technical field

The present invention relates to be used to prepare the tubercle bacillus gene vaccine of treating resistant tuberculosis.

Background technology

So far tuberculosis remains first " killer " of global infectious disease; Main cause is that bacillus calmette-guerin vaccine can not effectively prevent lung in adult tuberculosis; Tulase drug resistance problem is more and more serious, and social problems such as AIDS, immigrant and poverty make tuberculosis in some developed country's bottom outs.The World Health Organization's existing tuberculosis patient 2,000 ten thousand in the report whole world, annual newly-increased tuberculosis patient 800-1000 ten thousand, annual 200-300 ten thousand people die from tuberculosis.Tuberculosis epidemic situation of China and drug resistance situation are all quite serious, and the tuberculosis patient numerical digit occupies the second in the world, is only second to India.2000 the 4th time national tuberculosis epidemiological random sampling survey result shows that China has 5.5 hundred million people to infect tulase, existing pulmonary tuberculosis patient 4,510,000, and annual death toll is about 130,000, is positioned at first of the national various infectious disease.Resistant tuberculosis people is many, and the total resistant rate of mycobacterium tuberculosis is 27.8%, and wherein initial drug resistant rates is 18.6%, and the acquired drug-resistance rate is 46.5%, and anti-multiple medicines rate is 10.7%.Tuberculosis be a kind of infect, aspects such as immunity, prevention and treatment are full of contradictions and the chronic infectious disease of challenge; Rationally, the chemotherapy of rule generally can be killed the most tulases of intralesional in 1～2 month; But still have a small amount of bacterium residual; Especially the tulase that parasitizes in the macrophage is difficult for being killed, and needs to continue treatment 3～4 months, even the longer time.Because the toxic and side effects of anti-multiple medicines popular, chemotherapeutics lungy; And the body's immunity that reason such as the use of HIV's infection, immunosuppressant and senile tuberculosis causes is low; Intractable tuberculosis is increased; Antituberculosis therapy faces great challenge; Especially anti-multiple medicines tuberculosis (MDR-TB), extensively resistant tuberculosis (XDR-TB) faces the hard case that pasts medical help, and makes the control of tuberculosis epidemic situation of China not very good, and tuberculosis number, number of the infected, death toll all are positioned at first of the national various infectious disease.Therefore, the new anti-tuberculosis preparation of development is imperative.

Because the antituberculotics that development is new not only has high input, the cycle is long, and is more difficult than the development of new therapeutic vaccine, and new drug also possibly produce drug resistance very soon.And vaccine therapy is only injected several pins in some months, and more than the convenience of taking medicine every day, and side effect is less, and expense is low.Therefore, become one of focus of research through immune modulating treatment tuberculosis in recent years, the research and development of therapeutic vaccine become a crucial research direction.

The application of therapeutic vaccine is different with preventative vaccine, the former is used for tubercle bacillus affection person or tuberculosis patient, and the latter is used for normal population.Effectively preventative vaccine might not be used as therapeutic vaccine, and it is undesirable to be used for therapeutic effect like research proof bacillus calmette-guerin vaccine.The tuberculosis immunity mainly is cell-mediated immunoreation, and vaccine therapy can reach the purpose of treatment disease through the potentiality of regulating or optionally inducing the tuberculosis patient immune system to contain.

The immunomodulator that is used for the tuberculosis auxiliary treatment at present mainly contains two types: one type is nonspecific cytokine such as IFN-γ, IL-2 etc., and the half-life is short, needs injection repeatedly, and expense is high.Another kind of is specific immunomodulator: (1) mycobacterium vaccine (trade name microcaloire vaccine): produced by Anhui Long Kema Biology Pharmacy Co., Ltd; It is the acellular immunomodulator that the cow mycobacteria is processed behind the high-temperature inactivation purification; Its active component is main with cell wall; Also having with protein is main cytokine induction material, and has stronger immunocompetent DNA polymer.II, III phase clinical research result show that the microcaloire vaccine can strengthen lunger's cellular immune function, can accelerate with the chemotherapy coupling that sputum conversion, focus absorb and the cavity dwindles the speed of closing, and shorten SCC course of treatment.Its untoward reaction is few and slighter, and is safe in utilization, and relapse rate is low.This vaccine is in clinical practice, as the auxiliary treatment of phthisical immunization therapy and SCC at present.(2) Mycobacterium phlei preparation (trade name mycobacterium phlei F.U.36 injection utilin ' s): by the development of the healthy pharmaceutcal corporation, Ltd of Chengdu Venus; It is the immunomodulator that the Mycobacterium phlei of deactivation is processed; Can significantly improve lunger's cellular immune function, promote the absorption of pulmonary tuberculosis focus to take a turn for the better.(3) BCG vaccine polysaccharide nucleic acid injection (trade name Si Qikang): produce by Changsha Jiu Zhitang; Be to adopt hot phenol method to remove the bacterium protein that the bacillus calmette-guerin vaccine thalline can be induced delayed hypersensitivity; The reuse ethanol precipitation extracts the stronger thalline lipopolysaccharide composition of immunocompetence, processes sterile saline solution then, can strengthen lunger's cellular immunization and humoral immune function; T lymphocyte and natural killer cell function in the remarkable enhancing body; Improve the level that induces of IL-2, IL-2 receptor expression and IFN-γ, promote the absorption of cloudy commentaries on classics of antibacterial and TB focus to take a turn for the better, improved the curative effect of combined chemotherapy.These three kinds of vaccines all are killed vaccines, can only induce humoral immunization and Th1 type cellullar immunologic response, can not reply by inducing specific cytotoxic T lymphocyte (CTL).

Do not see the research report of using nucleic acid vaccine treatment resistant tuberculosis at present both at home and abroad as yet.How to use nucleic acid vaccine treatment resistant tuberculosis and just become the technical barrier that this technical field urgent need will solve.

Summary of the invention

The purpose of this invention is to provide a kind of tubercle bacillus gene vaccine of treating resistant tuberculosis that is used to prepare.

Above-mentioned purpose of the present invention reaches through following technical scheme:

Be used to prepare the tubercle bacillus gene vaccine of treating resistant tuberculosis, it is characterized in that: the tubercle bacillus gene vaccine of described resistant tuberculosis is for to be cloned into mycobacterium tuberculosis Ag85A coding gene sequence on the carrier for expression of eukaryon pVAX1; Or Ag85A and ESAT6 coding gene sequence be cloned on the carrier for expression of eukaryon pVAX1 together, forms the ESAT6-Ag85A chimeric gene vaccine.

The applying step of the tubercle bacillus gene vaccine of above-mentioned preparation treatment resistant tuberculosis of the present invention is following:

One, the preparation of Ag85A nucleic acid vaccine:

(1) gene order:

The gene order of the described tubercule bacillus Ag85A nucleic acid vaccine that is used to treat resistant tuberculosis is following:

1 T TTTCCCGGC?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 ACTGGTACC AGCCCGCCTG?CGGCAAGGCC?GGTTGCCAGA?CTTACAAGTG?GGAGACCTTC

301 CTGACCAGCG?AGCTGCCGGG?GTGGCTGCAG?GCCAACAGGC?ACGTCAAGCC?CACCGGAAGC

361 GCCGTCGTCG?GTCTTTCGAT?GGCTGCTTCT?TCGGCGCTGA?CGCTGGCGAT?CTATCACCCC

421 CAGCAGTTCG?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?CACCGGGC CC?GCGCCCCAGG?GCGCCTAG

The gene order of the Ag85A of the described tubercule bacillus ESAT6-Ag85A chimeric gene vaccine that is used for treating resistant tuberculosis is the same, and the gene order of ESAT6 is following:

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?AGGCAAC GTC?ACTGGGATGT?TCGCATAG

(2) through technique for gene engineering clone Ag85A gene:

1, design of primers and synthetic

Forward primer 15 '-GACT GCTAGCCACCATGGTTTCCCGGCCGGGCTTGCCGG-3 '

Downstream primer 25 '-GACT GGATCCTTACTAGGCGCCCTGGGGCGCGG-3 '

Primer 1 is positioned at Ag85A gene order 2-22 bit base, its 5 ' end contain a Nhe I ( GCTAGC) restriction enzyme site; Primer 2 is positioned at Ag85A gene order 869-885 bit base, its 5 ' end contain a BamH I ( GGATCC) restriction enzyme site.

2, pcr amplification Ag85A gene

With primer 1,2, under the effect of high-fidelity pfu archaeal dna polymerase, be template with mycobacterium tuberculosis H37Rv genomic DNA, amplification Ag85A gene.The PCR response procedures: 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 1min circulate 30 times; Last 72 ℃ are extended 7min.Identify the amplification of DNA fragments of 923bp in 1% agarose gel electrophoresis;

3, segmental enzyme action of Ag85A gene amplification and recovery:

Get tubercule bacillus Ag85A gene amplification fragment and carrier for expression of eukaryon pVAX1 plasmid (available from Invitrogen, Carlsbad, CA, USA) each 1 microgram; Add the restricted enzyme NheI and the BamH I of 10 units respectively, be mixed in the reaction buffer of 50 microlitres, in 37 ℃ of digestion 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, 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 respectively.

4, the Ag85A genetic fragment is connected with pVAX1 plasmid fragment:

Ag85A genetic fragment behind the purification is connected with cloning vehicle pVAX1 plasmid fragment, and target gene fragment was mixed with the carrier segment in 3: 1 in molar ratio, and the 10ul reaction system is following:

2 * connection buffer, 5 μ l

PVAX1 plasmid fragment 1 μ l

Ag85A genetic fragment 0.6 μ l

T ₄Dna ligase 1 μ l

Sterilized water is mended to 10 μ l

5. the preparation of escherichia coli DH1 or DH5 α competent cell:

The single colony inoculation of picking bacillus coli DH 5 alpha (or DH1) is put in 37 ℃ of shaken cultivation casees in the 200rpm overnight incubation in 5ml LB culture fluid; Change by 1/100 inferior morning and plant in 100ml LB culture fluid, put in 37 ℃ of shaken cultivation casees and continue to cultivate 2-3h, treat bacteria concentration OD in 200rpm ₆₀₀Be 0.6～0.8 o'clock, put into ice-cold big centrifuge tube, 4 ℃, 4000rpm, centrifugal 10min are abandoned supernatant; The resuspended bacterium deposition of the 0.1mol/LCaCl2 of 20ml ice pre-cooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant; With the resuspended bacterium deposition of 4ml 0.1mol/L CaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerol of 1ml inferior morning, the piping and druming mix homogeneously, per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is subsequent use to put-70 ℃ of preservations;

6. connect the conversion of product:

Segmental each the 5 μ l of product that are connected add in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively with Ag85A genetic fragment and pVAX1 plasmid; Put into 42 ℃ of water bath heat shock 90s, take out ice bath 2min rapidly; Add LB culture fluid 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains kanamycin.Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h;

7. the extraction of plasmid:

According to blue white macula screening, the picking white colony is 8 at random, is inoculated in 5ml respectively and contains in the LB culture medium of kanamycin, and 37 ℃ of shaken cultivation are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount;

(1) plasmid extracts the preparation of reagent in a small amount

Solution I: 50mmol/L glucose

25mmol/L?Tris·CL(pH?8.0)

10mmol/L?EDTA(pH?8.0)

At 10lbf/in ²(6.895 * 10 ⁴Pa) steam sterilization 15 minutes under the high pressure; Be stored in 4 ℃ subsequent use;

Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation

Solution III: 5mol/L potassium acetate 60ml

Glacial acetic acid 11.5ml

Deionized water 28.5ml

Be stored in 4 ℃, subsequent use;

(2) extract plasmid in a small amount

Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol that adds 2 times of volumes fully mixes, and places 2h deposit D NA in-20 ℃; In 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant; With 1ml 70% washing with alcohol deposition once, room temperature intensive drying; Deposition is dissolved in the 20 μ l TE buffer, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use;

8. evaluation recombiant plasmid:

(1) pcr amplification is identified: with the bacterium colony DNA of selecting is template, carries out pcr amplification with primer 1,2 and identifies.Amplified production is electrophoresis in 1% agarose gel, positive recombiant plasmid called after pVAX1-Ag85A;

(2) enzyme action is identified: get recombiant plasmid 5 μ l, use restricted enzyme Nde I, BamH I double digestion 2h respectively; Electrophoresis in 1% agarose gel.With dna molecular amount standard and amplified production is contrast, produces two fragments behind the enzyme action, and one consistent with carrier segment size, and one consistent with this gene amplification segment;

(3) sequencing: directly select a clone and send order-checking, sequencing result is consistent with the genome sequence of report.Thereby obtained the Ag85A nucleic acid vaccine.

(3) through technique for gene engineering clone ESAT6-Ag85A mosaic gene:

1, design of primers and synthetic

Forward primer 35 '-GACT GGTACCTAATGGCAGAGCAGCAGTGG-3 '

Downstream primer 45 '-GACT GGTACCTTGCGAACATCCCAGTGAC-3 '

Primer 3 is positioned at ESAT6 gene order 1-18 bit base, its 5 ' end contain a Kpn I ( GGTACC) restriction enzyme site; Primer 4 is positioned at ESAT6 gene order 268-285 bit base, and its 5 ' end also contains a Kpn I restriction enzyme site.

2, pcr amplification ESAT6 gene

With primer 3,4, under the effect of high-fidelity pfu archaeal dna polymerase, be template with mycobacterium tuberculosis H37Rv genomic DNA, amplification ESAT6 gene.The PCR response procedures: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min circulate 30 times; Last 72 ℃ are extended 7min.Identify the amplification of DNA fragments of 308bp in 1% agarose gel electrophoresis;

3, Ag85A and segmental enzyme action of ESAT 6 gene amplifications and recovery:

Get recombiant plasmid pVAX1-Ag85A and tubercule bacillus ESAT 6 gene amplification fragments, add restricted enzyme Kpn I respectively, be mixed in the reaction buffer of 50 microlitres; In 37 ℃ of digestion 1 hour, DNA recovery method in conventional agarose gel electrophoresis separation and glue (was pressed J. Sa nurse Brooker, D.W. Russell work; Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, 387-399 page or leaf; The 404-407 page or leaf; In August, 2002, Science Press publishes, Beijing) obtain the pVAX1-Ag85A genetic fragment and ESAT 6 genetic fragments of sticky end respectively.

4, the pVAX1-Ag85A genetic fragment is connected with ESAT 6 genetic fragments:

PVAX1-Ag85A genetic fragment behind the purification is connected with ESAT 6 genetic fragments, the ESAT6 gene order is inserted on 249 Kpn I restriction enzyme sites of Ag85A gene order, the 10ul reaction system is following:

2 * connection buffer, 5 μ l

ESAT 6 genetic fragments 1 μ l

PVAX1-Ag85A genetic fragment 1 μ l

T ₄Dna ligase 1 μ l

Sterilized water is mended to 10 μ l

5. the preparation of bacillus coli DH 5 alpha competent cell:

Picking bacillus coli DH 5 alpha list colony inoculation is put in 37 ℃ of shaken cultivation casees in the 200rpm overnight incubation in 5ml LB culture fluid; Change by 1/100 inferior morning and plant in 100ml LB culture fluid, put in 37 ℃ of shaken cultivation casees and continue to cultivate 2-3h, treat bacteria concentration OD in 200rpm ₆₀₀Be 0.6～0.8 o'clock, put into ice-cold big centrifuge tube, 4 ℃, 4000rpm, centrifugal 10min are abandoned supernatant; The resuspended bacterium deposition of the 0.1mol/L CaCl2 of 20ml ice pre-cooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant; With the resuspended bacterium deposition of 4ml 0.1mol/LCaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerol of 1ml inferior morning, the piping and druming mix homogeneously, per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is subsequent use to put-70 ℃ of preservations;

6. connect the conversion of product:

Each the 5 μ l of product that are connected of pVAX1-Ag85A genetic fragment and ESAT 6 genetic fragments are added in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively; Put into 42 ℃ of water bath heat shock 90s, take out ice bath 2min rapidly; Add LB culture fluid 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains kanamycin.Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h;

7. the extraction of plasmid:

According to blue white macula screening, the picking white colony is 6 at random, is inoculated in 5ml respectively and contains in the LB culture medium of kanamycin, and 37 ℃ of shaken cultivation are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount;

(1) plasmid extracts the preparation of reagent in a small amount

Solution I: 50mmol/L glucose

25mmol/L?Tris·CL(pH?8.0)

10mmol/L?EDTA(pH?8.0)

At 10lbf/in ²(6.895 * 10 ⁴Pa) steam sterilization 15 minutes under the high pressure; Be stored in 4 ℃ subsequent use;

Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation

Solution III: 5mol/L potassium acetate 60ml

Glacial acetic acid 11.5ml

Deionized water 28.5ml

Be stored in 4 ℃, subsequent use;

(2) extract plasmid in a small amount

Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol that adds 2 times of volumes fully mixes, and places 2h deposit D NA in-20 ℃; In 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant; With 1ml 70% washing with alcohol deposition once, room temperature intensive drying; Deposition is dissolved in the 20 μ l TE buffer, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use;

8. evaluation recombiant plasmid:

(1) enzyme action is identified: get recombiant plasmid and use restricted enzyme Nde I, BamH I double digestion 2h respectively; Electrophoresis in 1% agarose gel.With dna molecular amount standard is contrast, produces two fragments behind the enzyme action, and one is the carrier segment, and one is reorganization ESAT 6-Ag85A genetic fragment;

(2) sequencing: directly select four clones and send order-checking, select the correct recombiant plasmid of closure as the ESAT6-Ag85A chimeric gene vaccine.

Three, the preparation of nucleic acid vaccine and storage:

The purified recombinant plasmid that obtains is dissolved in the normal saline, and concentration is 1mg/ml; Or be prepared as the lyophilized formulations storage, with before being dissolved in the normal saline.

Four, the application of nucleic acid vaccine:

Every mice bilateral tibialis anterior is slowly injected mycobacterium tuberculosis nucleic vaccine Ag85A gene vaccine or ESAT6-Ag85A chimeric gene vaccine 50 μ g/50 μ l, adds up to every each intramuscular injection 100 μ g/100 μ l of mice, injection in per 15 days 1 time, totally 5 times.The degree of depth of vaccine intramuscular injection is 2mm.

Beneficial effect of the present invention:

The present invention is cloned into mycobacterium tuberculosis Ag85A coding gene sequence or Ag85A and ESAT6 coding gene sequence on the carrier for expression of eukaryon pVAX1 through technique for gene engineering together, makes up Ag85A gene vaccine or ESAT6-Ag85A chimeric gene vaccine as the resistant tuberculosis therapeutic vaccine.

First Application Ag85A gene vaccine of the present invention or ESAT6-Ag85A chimeric gene vaccine are treated anti-multiple medicines tuberculosis effectively; Tuberculosis model lung pathology is changed to be alleviated; The main organs bacterial population obviously reduces; The curative effect of Ag85A gene vaccine group obviously is better than normal saline group, pVAX1 vehicle treatment group, rifampicin (RFP) treatment group, ESAT6-Ag85A chimeric gene vaccine group and microcaloire vaccine group; For resistant tuberculosis has been opened up a new treatment approach, to the propagation and popular the having great importance of effective control resistant tuberculosis.

Dna vaccination of the present invention generates endogenous antigen in cell; Can not only induce humoral immunization and Th1 type cellullar immunologic response, can also reply by inducing specific CTL, can regulate resistant tuberculosis people's immunologic function specifically; Activate the potentiality of containing in its immune system; Raising is replied the specific immune of tulase, eliminates immunologic tolerance, thus the tuberculosis that infects in the treatment cell.

Gene vaccine is compared with immunomodulator commonly used at present, has the following advantages: (1) gene vaccine is produced easy, quick, has avoided the complicated processes of traditional vaccine preparation.(2) gene vaccine can be expressed in vivo steadily in the long term, and the persistent period of induction of immunity significantly is longer than routine immunization.(3) gene vaccine is similar with natural infection in the process of host cell inner expression proteantigen; Press usual channel processing, handle, modify; And be with native conformation and pass host immune system, the immunity of humoral immunization and Th1 type can be induced simultaneously, and the CTL immunne response can be induced; But killed vaccine such as microcaloire vaccine can only be induced humoral immunization and Th1 type cell response, can not induce CTL to reply usually.(4) gene vaccine is insensitive to heat, can lyophilizing preserve, and in saline solution, can recover original activity, preservation, convenient transportation, and need not refrigerated condition can be transported to all over the world, but thereby extensive use.(5) a few cada gene vaccines of injection in some months are more convenient and inexpensive than the cytokine of administration every day; (6) side effect is less, uses more safe and reliable.

Through the accompanying drawing and the specific embodiment the present invention is further specified below, but and do not mean that restriction protection domain of the present invention.

Description of drawings

Fig. 1 is the photo that normal saline group mouse lung general pathology changes.

Fig. 2 is the photo that pVAX1 vehicle treatment group mouse lung general pathology changes.

Fig. 3 is the photo that rifampicin treatment group mouse lung general pathology changes.

Fig. 4 is the photo that microcaloire bacterination group mouse lung general pathology changes.

Fig. 5 is the photo that RFP+Ag85A nucleic acid vaccine treatment group mouse lung general pathology changes.

Fig. 6 is the photo that Ag85A nucleic acid vaccine treatment group mouse lung general pathology changes.

Fig. 7 is the photo that RFP+Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group mouse lung general pathology changes.

Fig. 8 is the photo that Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group mouse lung general pathology changes.

Fig. 9 is the photo of each treatment group mouse lung histo pathological change.

The specific embodiment

Embodiment 1

Be used to prepare the tubercle bacillus gene vaccine of treating resistant tuberculosis, one of which is that mycobacterium tuberculosis Ag85A coding gene sequence is cloned on the carrier for expression of eukaryon pVAX1.

(1) gene order:

The gene order of the described tubercule bacillus Ag85A nucleic acid vaccine that is used to treat resistant tuberculosis is following:

1 T TTTCCCGGC?CGGGCTTGCC?GGTGGAGTAC?CTGCAGGTGC?CGTCGCCGTC?GATGGGCCGT

61 GACATCAAGG?TCCAATTCCA?AAGTGGTGGT?GCCAACTCGC?CCGCCCTGTA?CCTGCTCGAC

121 GGCCTGCGCG?CGCAGGACGA?CTTCAGCGGC?TGGGACATCA?ACACCCCGGC?GTTCGAGTGG

182 TACGACCAGT?CGGGCCTGTC?GGTGGTCATG?CCGGTGGGTG?GCCAGTCAAG?CTTCTACTCC

242 ACTGGTACC AGCCCGCCTG?CGGCAAGGCC?GGTTGCCAGA?CTTACAAGTG?GGAGACCTTC

301 CTGACCAGCG?AGCTGCCGGG?GTGGCTGCAG?GCCAACAGGC?ACGTCAAGCC?CACCGGAAGC

361 GCCGTCGTCG?GTCTTTCGAT?GGCTGCTTCT?TCGGCGCTGA?CGCTGGCGAT?CTATCACCCC

421 CAGCAGTTCG?TCTACGCGGG?AGCGATGTCG?GGCCTGTTGG?ACCCCTCCCA?GGCGATGGGT

481 CCCACCCTGA?TCGGCCTGGC?GATGGGTGAC?GCTGGCGGCT?ACAAGGCCTC?CGACATGTGG

541 GGCCCGAAGG?AGGACCCGGC?GTGGCAGCGC?AACGACCCGC?TGTTGAACGT?CGGGAAGCTG

602 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?CACCGGGC CC?GCGCCCCAGG?GCGCCTAG

(2) through technique for gene engineering clone Ag85A gene:

1, design of primers and synthetic

Forward primer 15 '-GACT GCTAGCCACCATGGTTTCCCGGCCGGGCTTGCCGG-3 '

Downstream primer 25 '-GACT GGATCCTTACTAGGCGCCCTGGGGCGCGG-3 '

Primer 1 is positioned at Ag85A gene order 2-22 bit base, its 5 ' end contain a Nhe I ( GCTAGC) restriction enzyme site; Primer 2 is positioned at Ag85A gene order 869-885 bit base, its 5 ' end contain a BamH I ( GGATCC) restriction enzyme site.

2, pcr amplification Ag85A gene

With primer 1,2, under the effect of high-fidelity pfu archaeal dna polymerase, be template with mycobacterium tuberculosis H37Rv genomic DNA, amplification Ag85A gene.The PCR response procedures: 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 1min circulate 30 times; Last 72 ℃ are extended 7min.Identify the amplification of DNA fragments of 923bp in 1% agarose gel electrophoresis;

3, segmental enzyme action of Ag85A gene amplification and recovery:

Get tubercule bacillus Ag85A gene amplification fragment and carrier for expression of eukaryon pVAX1 plasmid (available from Invitrogen, Carlsbad, CA, USA) each 1 microgram; Add the restricted enzyme NheI and the BamH I of 10 units respectively, be mixed in the reaction buffer of 50 microlitres, in 37 ℃ of digestion 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, 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 respectively.

4, the Ag85A genetic fragment is connected with pVAX1 plasmid fragment:

Ag85A genetic fragment behind the purification is connected with cloning vehicle pVAX1 plasmid fragment, and target gene fragment was mixed with the carrier segment in 3: 1 in molar ratio, and the 10ul reaction system is following:

2 * connection buffer, 5 μ l

PVAX1 plasmid fragment 1 μ l

Ag85A genetic fragment 0.6 μ l

T ₄Dna ligase 1 μ l

Sterilized water is mended to 10 μ l

5. the preparation of escherichia coli DH1 or DH5 α competent cell:

The single colony inoculation of picking bacillus coli DH 5 alpha (or DH1) is put in 37 ℃ of shaken cultivation casees in the 200rpm overnight incubation in 5ml LB culture fluid; Change by 1/100 inferior morning and plant in 100ml LB culture fluid, put in 37 ℃ of shaken cultivation casees and continue to cultivate 2-3h, treat bacteria concentration OD in 200rpm ₆₀₀Be 0.6～0.8 o'clock, put into ice-cold big centrifuge tube, 4 ℃, 4000rpm, centrifugal 10min are abandoned supernatant; The resuspended bacterium deposition of the 0.1mol/LCaCl2 of 20ml ice pre-cooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant; With the resuspended bacterium deposition of 4ml 0.1mol/L CaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerol of 1ml inferior morning, the piping and druming mix homogeneously, per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is subsequent use to put-70 ℃ of preservations;

6. connect the conversion of product:

Segmental each the 5 μ l of product that are connected add in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively with Ag85A genetic fragment and pVAX1 plasmid; Put into 42 ℃ of water bath heat shock 90s, take out ice bath 2min rapidly; Add LB culture fluid 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains kanamycin.Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h;

7. the extraction of plasmid:

According to blue white macula screening, the picking white colony is 8 at random, is inoculated in 5ml respectively and contains in the LB culture medium of kanamycin, and 37 ℃ of shaken cultivation are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount;

(1) plasmid extracts the preparation of reagent in a small amount

Solution I: 50mmol/L glucose

25mmol/L?Tris·CL(pH?8.0)

10mmol/L?EDTA(pH?8.0)

At 10lbf/in ²(6.895 * 10 ⁴Pa) steam sterilization 15 minutes under the high pressure; Be stored in 4 ℃ subsequent use;

Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation

Solution III: 5mol/L potassium acetate 60ml

Glacial acetic acid 11.5ml

Deionized water 28.5ml

Be stored in 4 ℃, subsequent use;

(2) extract plasmid in a small amount

Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol that adds 2 times of volumes fully mixes, and in 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant in-20 ℃ of placement 2h deposit D NA; With 1ml 70% washing with alcohol deposition once, room temperature intensive drying; Deposition is dissolved in the 20 μ l TE buffer, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use;

8. evaluation recombiant plasmid:

(2) pcr amplification is identified: with the bacterium colony DNA of selecting is template, carries out pcr amplification with primer 1,2 and identifies.Amplified production is electrophoresis in 1% agarose gel, positive recombiant plasmid called after pVAX1-Ag85A;

(2) enzyme action is identified: get recombiant plasmid 5 μ l, use restricted enzyme Nde I, BamH I double digestion 2h respectively; Electrophoresis in 1% agarose gel.With dna molecular amount standard and amplified production is contrast, produces two fragments behind the enzyme action, and one consistent with carrier segment size, and one consistent with this gene amplification segment;

(3) sequencing: directly select a clone and send order-checking, sequencing result is consistent with the genome sequence of report.Thereby obtained the Ag85A nucleic acid vaccine.

9. the preparation of nucleic acid vaccine and storage:

The purified recombinant plasmid that obtains is dissolved in the normal saline, and concentration is 1mg/ml; Or be prepared as the lyophilized formulations storage, with before being dissolved in the normal saline.

Embodiment 2

Be used to prepare the tubercle bacillus gene vaccine of treating resistant tuberculosis, it is cloned on the carrier for expression of eukaryon pVAX1 for mycobacterium tuberculosis Ag85A and ESAT6 coding gene sequence, forms the ESAT6-Ag85A chimeric gene vaccine.

(1) gene order:

The gene order of the described tubercule bacillus Ag85A nucleic acid vaccine that is used to treat resistant tuberculosis is following:

1 T TTTCCCGGC?CGGGCTTGCC?GGTGGAGTAC?CTGCAGGTGC?CGTCGCCGTC?GATGGGCCGT

61 GACATCAAGG?TCCAATTCCA?AAGTGGTGGT?GCCAACTCGC?CCGCCCTGTA?CCTGCTCGAC

121 GGCCTGCGCG?CGCAGGACGA?CTTCAGCGGC?TGGGACATCA?ACACCCCGGC?GTTCGAGTGG

183 TACGACCAGT?CGGGCCTGTC?GGTGGTCATG?CCGGTGGGTG?GCCAGTCAAG?CTTCTACTCC

243 ACTGGTACC AGCCCGCCTG?CGGCAAGGCC?GGTTGCCAGA?CTTACAAGTG?GGAGACCTTC

301 CTGACCAGCG?AGCTGCCGGG?GTGGCTGCAG?GCCAACAGGC?ACGTCAAGCC?CACCGGAAGC

361 GCCGTCGTCG?GTCTTTCGAT?GGCTGCTTCT?TCGGCGCTGA?CGCTGGCGAT?CTATCACCCC

421 CAGCAGTTCG?TCTACGCGGG?AGCGATGTCG?GGCCTGTTGG?ACCCCTCCCA?GGCGATGGGT

481 CCCACCCTGA?TCGGCCTGGC?GATGGGTGAC?GCTGGCGGCT?ACAAGGCCTC?CGACATGTGG

541 GGCCCGAAGG?AGGACCCGGC?GTGGCAGCGC?AACGACCCGC?TGTTGAACGT?CGGGAAGCTG

603 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?CACCGGGC CC?GCGCCCCAGG?GCGCCTAG

The gene order of the Ag85A of the described tubercule bacillus ESAT6-Ag85A chimeric gene vaccine that is used for treating resistant tuberculosis is the same, and the gene order of ESAT6 is following:

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?AGGCAAC GTC?ACTGGGATGT?TCGCATAG

(2) through technique for gene engineering clone ESAT6-Ag85A mosaic gene:

1, design of primers and synthetic

Forward primer 35 '-GACT GGTACCTAATGGCAGAGCAGCAGTGG-3 '

Downstream primer 45 '-GACT GGTACCTTGCGAACATCCCAGTGAC-3 '

Primer 3 is positioned at ESAT6 gene order 1-18 bit base, its 5 ' end contain a Kpn I ( GGTACC) restriction enzyme site; Primer 4 is positioned at ESAT6 gene order 268-285 bit base, and its 5 ' end also contains a Kpn I restriction enzyme site.

2, pcr amplification ESAT6 gene

With primer 3,4, under the effect of high-fidelity pfu archaeal dna polymerase, be template with mycobacterium tuberculosis H37Rv genomic DNA, amplification ESAT6 gene.The PCR response procedures: 94 ℃ of 1min, 55 ℃ of 1min, 72 ℃ of 1min circulate 30 times; Last 72 ℃ are extended 7min.Identify the amplification of DNA fragments of 308bp in 1% agarose gel electrophoresis;

3, Ag85A and segmental enzyme action of ESAT 6 gene amplifications and recovery:

Get recombiant plasmid pVAX1-Ag85A and tubercule bacillus ESAT 6 gene amplification fragments, add restricted enzyme Kpn I respectively, be mixed in the reaction buffer of 50 microlitres; In 37 ℃ of digestion 1 hour, DNA recovery method in conventional agarose gel electrophoresis separation and glue (was pressed J. Sa nurse Brooker, D.W. Russell work; Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, 387-399 page or leaf; The 404-407 page or leaf; In August, 2002, Science Press publishes, Beijing) obtain the pVAX1-Ag85A genetic fragment and ESAT 6 genetic fragments of sticky end respectively.

4, the pVAX1-Ag85A genetic fragment is connected with ESAT 6 genetic fragments:

PVAX1-Ag85A genetic fragment behind the purification is connected with ESAT 6 genetic fragments, the ESAT6 gene order is inserted on 249 Kpn I restriction enzyme sites of Ag85A gene order, the 10ul reaction system is following:

2 * connection buffer, 5 μ l

ESAT 6 genetic fragments 1 μ l

PVAX1-Ag85A genetic fragment 1 μ l

T ₄Dna ligase 1 μ l

Sterilized water is mended to 10 μ l

5. the preparation of bacillus coli DH 5 alpha competent cell:

Picking bacillus coli DH 5 alpha list colony inoculation is put in 37 ℃ of shaken cultivation casees in the 200rpm overnight incubation in 5ml LB culture fluid; Change by 1/100 inferior morning and plant in 100ml LB culture fluid, put in 37 ℃ of shaken cultivation casees and continue to cultivate 2-3h, treat bacteria concentration OD in 200rpm ₆₀₀Be 0.6 ~ 0.8 o'clock, put into ice-cold big centrifuge tube, 4 ℃, 4000rpm, centrifugal 10min are abandoned supernatant; The resuspended bacterium deposition of the 0.1mol/L CaCl2 of 20ml ice pre-cooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant; With the resuspended bacterium deposition of 4ml 0.1mol/LCaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerol of 1ml inferior morning, the piping and druming mix homogeneously, per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is subsequent use to put-70 ℃ of preservations;

6. connect the conversion of product:

Each the 5 μ l of product that are connected of pVAX1-Ag85A genetic fragment and ESAT 6 genetic fragments are added in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively; Put into 42 ℃ of water bath heat shock 90s, take out ice bath 2min rapidly; Add LB culture fluid 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains kanamycin.Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h;

7. the extraction of plasmid:

According to blue white macula screening, the picking white colony is 6 at random, is inoculated in 5ml respectively and contains in the LB culture medium of kanamycin, and 37 ℃ of shaken cultivation are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount;

(1) plasmid extracts the preparation of reagent in a small amount

Solution I: 50mmol/L glucose

25mmol/L?Tri?s·CL(pH?8.0)

10mmol/L?EDTA(pH?8.0)

At 10lbf/in ²(6.895 * 10 ⁴Pa) steam sterilization 15 minutes under the high pressure; Be stored in 4 ℃ subsequent use;

Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation

Solution III: 5mol/L potassium acetate 60ml

Glacial acetic acid 11.5ml

Deionized water 28.5ml

Be stored in 4 ℃, subsequent use;

(2) extract plasmid in a small amount

Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol that adds 2 times of volumes fully mixes, and places 2h deposit D NA in-20 ℃; In 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant; With 1ml 70% washing with alcohol deposition once, room temperature intensive drying; Deposition is dissolved in the 20 μ l TE buffer, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use;

8. evaluation recombiant plasmid:

(1) enzyme action is identified: get recombiant plasmid and use restricted enzyme Nde I, BamH I double digestion 2h respectively; Electrophoresis in 1% agarose gel.With dna molecular amount standard is contrast, produces two fragments behind the enzyme action, and one is the carrier segment, and one is reorganization ESAT 6-Ag85A genetic fragment;

(2) sequencing: directly select four clones and send order-checking, select the correct recombiant plasmid of closure as the ESAT6-Ag85A chimeric gene vaccine.

9. the preparation of nucleic acid vaccine and storage:

The purified recombinant plasmid that obtains is dissolved in the normal saline, and concentration is 1mg/ml; Or be prepared as the lyophilized formulations storage, with before being dissolved in the normal saline.

Application implementation example 1

1, the selection of infection strain and bacterium amount:

Carry out mycobacteria cultivation, strain identification and drug sensitive test by " diagnosis of tuberculosis bacteriological analysis rules ".Anti-INH standard: 10 μ g/ml and 1 μ g/ml are respectively high and low degree drug resistance; Anti-RFP standard: 250 μ g/ml and 50 μ g/ml are respectively high and low degree drug resistance.1 routine tuberculosis patient sputum specimen is cultivated positive through traditional mycobacteria, carrying out traditional strain identification is mycobacterium tuberculosis, and it is numbered mycobacterium tuberculosis clinical separation strain HB361.Use traditional drug sensitive test method and identify that mycobacterium tuberculosis clinical separation strain HB361 is the anti-multiple medicines strain (seeing table 1) of high anti-rifampicin, low anti-isoniazid.

Table 1 drug sensitivity test result

Well-grown mycobacterium tuberculosis clinical separation strain HB361 grinds to form bacteria suspension with the glass grinding device on the improvement Luo Shi ovum gallinaceum culture medium, behind the bacteria suspension with normal saline preparation 3mg/ml, carries out 10 times of serial dilutions again, with 10 ^-1, 10 ^-2, 10 ^-3, 10 ^-4Bacterium liquid is respectively got 0.1ml and is inoculated in 2 improvement Luo Shi ovum gallinaceum plate culture medium, cultivates for 4 weeks in 37 ℃.Offensive HB361 bacteria suspension contains 5.5 * 10 for every milliliter ⁵CFU, every mouse tail vein injection 0.4ml, every mice actual attack bacterium amount is 2.2 * 10 ⁵CFU (seeing table 2).

Table 2 mycobacterium tuberculosis clinical separation strain HB361 bacteria suspension colony counting result

The bacteria suspension extension rate	Clump count	Colony counting (CFU/ml)
			10 -1	+++，+++
10 -2	+++，+++
			10 -3	50，60
10 -4	5，6	5.5×10 5

2, animal is selected:

The body weight of buying the quality certification from Military Medical Science Institute is 105 of the female Balb/c mices in 6-8 age in week of 17-19g, is no more than 2g with batch test body weight difference, puts Animal Lab. and feeds 1 day.

3, route of infection:

Every mouse tail vein injection 0.4ml bacteria suspension infecting mouse.

4, experiment is divided into groups:

After mice weighed, be divided into 8 groups at random equably, 10 every group.Infect the 3rd day begin treatment in back.

(1) normal saline group: every slow injecting normal saline 50 μ l of mice bilateral tibialis anterior, each intramuscular injection normal saline 100 μ l, injection in per 15 days 1 time, totally 5 times.

(2) pVAX1 vehicle treatment group: every mice bilateral tibialis anterior is slowly injected pVAX1 vector plasmid DNA 50 μ g/50 μ l, every the each intramuscular injection pVAX1 of mice vector plasmid DNA 100 μ g/100 μ l, injection in per 15 days 1 time, totally 5 times.The degree of depth of vaccine intramuscular injection is 2mm.

(3) rifampicin (RFP) treatment group: specification: 100/bottle, the 0.15g/ grain, lot number: 0611071, producer: Shenyang Hongqi Pharmaceutical Co., Ltd..By per kilogram of body weight 20mg every day (0.02mg/g) administration, mice is calculated every mice 0.4mg by 20 grams.Get 1 of rifampicin capsules, pour out medicated powder, add water 187.5ml dissolving, rifampicin concentration is 0.8mg/ml.Infect the back and played every mice hello rifampicin every day on the 3rd day once, each 0.5ml.

(4) microcaloire bacterium mattress treatment group: adult 22.5ug/ml/60kg, i.e. 0.000375ug/g, every of mice press 20g and calculates the microcaloire of injecting 0.0075ug.Method: get 22.5ug/ and prop up 1 of microcaloire, add the 10ml sterilized water for injection, mixing; Concentration is 2.25ug/ml, gets the good microcaloire of 1ml dilution again, adds the 9ml sterile water for injection; Concentration is 0.225ug/ml, gets the microcaloire of 1ml 0.225ug/ml again, adds the 2ml sterile water for injection; Concentration is 0.075ug/ml, each intramuscular injection microcaloire 0.0075ug

(5) RFP+Ag85A nucleic acid vaccine treatment group: the treatment of rifampicin is with (3).The infection back was played every mice bilateral tibialis anterior on the 3rd day and is slowly injected Ag85A nucleic acid vaccine 50 μ g/50 μ l, every the each intramuscular injection Ag85A of mice nucleic acid vaccine 100 μ g/100 μ l, injection in per 15 days 1 time, totally 5 times.The degree of depth of vaccine intramuscular injection is 2mm.

(6) Ag85A nucleic acid vaccine treatment group: the infection back was played every mice bilateral tibialis anterior on the 3rd day and is slowly injected Ag85A nucleic acid vaccine 50 μ g/50 μ l, every the each intramuscular injection Ag85A of mice nucleic acid vaccine 100 μ g/100 μ l, injection in per 15 days 1 time, totally 5 times.The degree of depth of vaccine intramuscular injection is 2mm.

(7) RFP+Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group: the treatment of rifampicin is with (3).The infection back was played every mice bilateral tibialis anterior on the 3rd day and is slowly injected Ag85A/ESAT-6 mosaic gene nucleic acid vaccine 50 μ g/50 μ l; Every the each intramuscular injection Ag85A/ESAT-6 of mice mosaic gene nucleic acid vaccine 100 μ g/100 μ l; Injection in per 15 days 1 time, totally 5 times.The degree of depth of vaccine intramuscular injection is 2mm.

(8) Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group: the infection back was played every mice bilateral tibialis anterior on the 3rd day and is slowly injected Ag85A/ESAT-6 mosaic gene nucleic acid vaccine 50 μ g/50 μ l; Every the each intramuscular injection Ag85A/ESAT-6 of mice mosaic gene nucleic acid vaccine 100 μ g/100 μ l; Injection in per 15 days 1 time, totally 5 times.The degree of depth of vaccine intramuscular injection is 2mm.

5, observation index:

(1) mice body weight change: weigh before the treatment 1 time, weigh weekly after the treatment 1 time;

(2) record dead mouse number, calculating mortality rate;

(3) lung, liver, splenomegaly body pathological observation;

(4) lung, liver, spleen ponderal index: mouse lung, liver, spleen weight are respectively divided by the mice body weight;

(5) count plate of lung and spleen:

After mice is weighed,, observe lung, liver and splenomegaly body pathology, get left lung, right lung, liver, last spleen and following spleen and weigh with drawing the neck marrow-breaking to put to death mice.Get left lung and last spleen and respectively add 3ml and 2ml normal saline respectively, grind to form tissue suspension with tissue grinder, lung is got the 1ml suspension and is added 1ml 6%NaOH digestion 30min and shake up, and becomes 10 with the normal saline serial dilution ^-1, 10 ^-2, 10 ^-3, 10 ^-4Bacteria suspension is with 10 ^-2, 10 ^-3, 10 ^-4Bacteria suspension 0.1ml is inoculated on the modified Russell medium, and 2 culture medium of each dilution factor inoculation are smeared evenly, cultivate for 4 weeks in 37 ℃; Spleen need not digest, and becomes 10 with the normal saline serial dilution ^-1, 10 ^-2, 10 ^-3, 10 ^-4Bacteria suspension is with bacteria suspension 10 ^-2, 10 ^-3, 10 ^-4Bacteria suspension 0.1ml is inoculated on the modified Russell medium that contains amphotericin B, and 2 culture medium of each dilution factor inoculation are smeared evenly, cultivate for 4 weeks in 37 ℃.

6, result

Gene vaccine is used to treat the effect observation of the anti-multiple medicines tuberculosis of mice model:

1. mice body weight change:

Mice is after mycobacterium tuberculosis is attacked, and each is organized the mice body weight and slowly increases no significant difference.

2. respectively organize the dead mouse situation:

Rifampicin treatment group before killing Mus dead 1, all the other each groups are not all seen dead mouse.

3. stop to kill in 3 weeks of treatment back rat meats observe examine lung, liver, splenomegaly body pathological change (table 3, Fig. 1):

As shown in Figure 1, be the photo that normal saline group mouse lung general pathology changes.(digital camera of using the panasonic of Panasonic 7,200,000 pixels is taken, optical zoom 3.6,15 centimetres of distances by Fig. 1; Fig. 2-Fig. 8 together.) can find out that in the normal saline group, 3 mouse lungs are light moderate swelling, all the other are the lung atrophy; 2 full lungs of mice are not seen the nodositas pathological changes, 3 visible 10-20 nodositas focuses of mouse lung, 5 visible more than 40 nodositas focuses of mouse lung; 2 mouse lungs are not seen the caseous necrosis kitchen range, 8 take a favourable turn only, in, the caseous necrosis of severe, average every mouse lung caseous necrosis area reaches 43%.Liver does not see obviously unusual.Spleen is light, moderate swelling.Hilar lymph node is light, moderate swelling.A large amount of ascites appear in 1 mice.

As shown in Figure 2, be the photo that pVAX1 vehicle treatment group mouse lung general pathology changes.Can be known by Fig. 2: 1 mouse lung is light moderate swelling, and all the other are the lung atrophy; All visible many nodositas focuses of 10-40 of mouse lung; 8 mouse lungs are not seen the caseous necrosis kitchen range, 2 take a favourable turn only, the caseous necrosis of moderate, and average every mouse lung caseous necrosis area reaches 37.5%.Liver does not see obviously unusual.Spleen is light, moderate swelling.Hilar lymph node is light, moderate swelling.

As shown in Figure 3, be the photo that rifampicin treatment group mouse lung general pathology changes.Can be found out that by Fig. 3 in the rifampicin treatment group, 2 mouse lungs are light moderate swelling, all the other are the lung atrophy; All visible many nodositas focuses of 10-40 of mouse lung; 2 mouse lungs are not shown in cheese appearance necrosis region, 7 take a favourable turn only, the caseous necrosis of moderate, and average every mouse lung caseous necrosis area reaches 21%.Liver does not see obviously unusual.Spleen is light, in, the severe enlargement.Hilar lymph node is light, moderate swelling.

As shown in Figure 4, be the photo that microcaloire bacterination group mouse lung general pathology changes.Can be found out that by Fig. 4 in the microcaloire bacterination group, 2 mouse lungs are silght enlargement, all the other are the lung atrophy; 7 visible more than 10 nodositas focuses of mouse lung, 3 visible more than 40 nodositas focuses of mouse lung; 4 mouse lungs are not seen the caseous necrosis kitchen range, 5 caseous necrosises of degree of taking a favourable turn only, and the caseous necrosis of 1 visible severe, average every mouse lung caseous necrosis area reaches 31%.Liver does not see obviously unusual.Spleen is light, moderate swelling.Hilar lymph node light (30%), moderate (70%) enlargement.

As shown in Figure 5, be the photo that RFP+Ag85A nucleic acid vaccine treatment group mouse lung general pathology changes.Can be found out that by Fig. 5 rifampicin adds in the Ag85A nucleic acid vaccine treatment group, 2 mouse lungs are light moderate swelling, and all the other are the lung atrophy; 2 visible more than 10 nodositas focuses of mouse lung, 8 visible more than 40 nodositas focuses of mouse lung; 7 mouse lungs are not seen the caseous necrosis kitchen range, 3 take a favourable turn only, the caseous necrosis of moderate, and average every mouse lung caseous necrosis area reaches 23%.Liver does not see obviously unusual.Spleen is light, moderate swelling.Hilar lymph node is light, moderate swelling.

As shown in Figure 6, be the photo that Ag85A nucleic acid vaccine treatment group mouse lung general pathology changes.Can be known that by Fig. 6 in the Ag85A nucleic acid vaccine treatment group, 2 mouse lungs are silght enlargement, all the other are the lung atrophy; 2 visible more than 10 nodositas focuses of mouse lung, 8 visible more than 40 nodositas focuses of mouse lung; 8 mouse lungs are not seen the caseous necrosis kitchen range, 2 take a favourable turn only, the caseous necrosis of moderate, and average every mouse lung caseous necrosis area reaches 20%.Liver does not see obviously unusual.Spleen is light, moderate swelling.Hilar lymph node is light, moderate swelling.

As shown in Figure 7, be the photo that RFP+Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group mouse lung general pathology changes.Can know that by Fig. 7 rifampicin adds in the Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group, the equal atrophy of mouse lung; All visible many nodositas focuses of 10-40 of mouse lung; 7 mouse lungs are not seen the caseous necrosis kitchen range, 3 caseous necrosises of degree of taking a favourable turn only, and average every mouse lung caseous necrosis area reaches 15%.Liver does not see obviously unusual.Spleen is light, in, the severe enlargement.Hilar lymph node is light, moderate swelling.

As shown in Figure 8, be the photo that Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group mouse lung general pathology changes.Can be known that by Fig. 8 in the Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group, 3 mouse lungs are light moderate swelling, all the other are the lung atrophy; 4 visible more than 10 nodositas focuses of mouse lung, 6 visible more than 40 nodositas focuses of mouse lung; 7 mouse lungs are not seen the caseous necrosis kitchen range, 3 caseous necrosises of degree of taking a favourable turn only, and average every mouse lung caseous necrosis area reaches 16.7%.Liver does not see obviously unusual.Spleen is light, in, the severe enlargement.The hilar lymph node moderate swelling.

3. organ weights index:

Each organizes the difference with insignificance of organ weights index and matched group, nonsensical (seeing table 3).

4. lung tissue pathological examination:

Be embedded in the paraffin after lung tissue is fixed with 10% paraformaldehyde and cut into slices.Tissue slice is with hematoxylin and eosin reagent dyeing, and observation analysis result under optical microscope (4 times of amplifications) sees Fig. 9.

5. can find out by Fig. 9 that normal saline group, pVAX1 vehicle treatment group lung tissue pathological changes are extensive, not see or rare proliferative tuberosity; Rifampicin treatment group, rifampicin add Ag85A nucleic acid vaccine treatment group, Ag85A nucleic acid vaccine treatment group, rifampicin, and to add Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group, Ag85A/ESAT-6 mosaic gene nucleic acid vaccine treatment group, microcaloire treatment group lung tissue pathological changes lighter; extent of disease limitation; visible more tubercle mainly is proliferative and changes.

6. the count plate of lung and spleen:

Ag85A gene vaccine group, RFP+Ag85A gene vaccine group, ESAT6-Ag85A chimeric gene vaccine group, RFP+ESAT6-Ag85A chimeric gene vaccine group and the colony counting of microcaloire vaccine group lung spleen all reduce 2-5 doubly than normal saline group, pVAX1 vehicle treatment group, rifampicin (RFP) treatment group; Wherein the curative effect of Ag85A gene vaccine group and RFP+Ag85A gene vaccine group obviously is better than ESAT6-Ag85A chimeric gene vaccine group, RFP+ESAT6-Ag85A chimeric gene vaccine group and microcaloire vaccine group (seeing table 3).

Experiment conclusion: gene vaccine has therapeutical effect to the mice resistant tuberculosis, and the therapeutical effect of Ag85A gene vaccine is the most remarkable.

Sequence table

Claims (1)

1. be used to treat the tubercle bacillus gene vaccine of resistant tuberculosis; It is characterized in that: the described tubercle bacillus gene vaccine that is used to treat resistant tuberculosis is for to be cloned into carrier for expression of eukaryon pVAX1 with mycobacterium tuberculosis Ag85A coding gene sequence, the Ag85A gene vaccine of formation; Or Ag85A and ESAT6 coding gene sequence are cloned on the carrier for expression of eukaryon pVAX1 ESAT6-Ag85A chimeric gene vaccine of formation together;

The gene order of described mycobacterium tuberculosis Ag85A is following:

1 T TTTCCCGGC?CGGGCTTGCC?GGTGGAGTAC?CTGCAGGTGC?CGTCGCCGTC?GATGGGCCGT

61 GACATCAAGG?TCCAATTCCA?AAGTGGTGGT?GCCAACTCGC?CCGCCCTGTA?CCTGCTCGAC

121?GGCCTGCGCG?CGCAGGACGA?CTTCAGCGGC?TGGGACATCA?ACACCCCGGC?GTTCGAGTGG

184?TACGACCAGT?CGGGCCTGTC?GGTGGTCATG?CCGGTGGGTG?GCCAGTCAAG?CTTCTACTCC

244?ACTGGTACC AGCCCGCCTG?CGGCAAGGCC?GGTTGCCAGA?CTTACAAGTG?GGAGACCTTC

301?CTGACCAGCG?AGCTGCCGGG?GTGGCTGCAG?GCCAACAGGC?ACGTCAAGCC?CACCGGAAGC

361?GCCGTCGTCG?GTCTTTCGAT?GGCTGCTTCT?TCGGCGCTGA?CGCTGGCGAT?CTATCACCCC

421?CAGCAGTTCG?TCTACGCGGG?AGCGATGTCG?GGCCTGTTGG?ACCCCTCCCA?GGCGATGGGT

481?CCCACCCTGA?TCGGCCTGGC?GATGGGTGAC?GCTGGCGGCT?ACAAGGCCTC?CGACATGTGG

541?GGCCCGAAGG?AGGACCCGGC?GTGGCAGCGC?AACGACCCGC?TGTTGAACGT?CGGGAAGCTG

604?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?CACCGGGC CC?GCGCCCCAGG?GCGCCTAG

The gene order of Ag85A in the described tubercule bacillus ESAT6-Ag85A chimeric gene vaccine is the same, and the gene order of ESAT6 is following:

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?AGGCAAC GTC?ACTGGGATGT?TCGCATAG。

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