CN101912605A

CN101912605A - Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof

Info

Publication number: CN101912605A
Application number: CN 201010156067
Authority: CN
Inventors: 范小勇; 马辉; 赵国屏; 李忠明
Original assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Current assignee: SHANGHAI PUBLIC HEALTH CLINICAL CENTER
Priority date: 2010-04-23
Filing date: 2010-04-23
Publication date: 2010-12-15

Abstract

The invention provides a reinforced mosaic gene recombinant bacillus calmette-guerin, which comprises a recombinant shuttle expression vector guided into bacillus calmette-guerin (BCG) by an electro-transformation method and cloned with a bacillus tubercle mosaic gene Ag85A-ESAT6. The invention also provides a preparation method for the reinforced mosaic gene recombinant bacillus calmette-guerin and application thereof to preparation of recombinant vaccines for preventing tuberculosis. A mycobacterium differential expression system or other shuttle expression plasmid is used as the vector so as to realize high-level expression of the bacillus tubercle mosaic gene in the bacillus calmette-guerin. The reinforced mosaic gene recombinant bacillus calmette-guerin can produce reinforced TH1-type immune response aiming at Ag85A and ESAT-6 antigenic specificities respectively, and is expected to become a novel tuberculosis vaccine for replacing the bacillus calmette-guerin.

Description

Reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof

Technical field

The invention belongs to microorganism and bioengineering field.Be specifically related to the mosaic gene recombiant vaccine, more specifically relate to mosaic gene recombinant bacillus calmette-guerin and preparation method thereof.

Background technology

Tuberculosis is a kind of main public's infectious disease that the whole world is paid close attention to.As far back as 1993, The World Health Organization (WHO) increased day by day with regard to the tuberculosis case because of the whole world and announces that the tuberculosis epidemic situation enters the state of emergency.At present, the whole world has 1/3 population (18.6 hundred million) to carry tubercule bacillus (Mycobacterium tuberculosis) approximately, and 8,000,000 newly-increased cases are arranged every year approximately, and 2,000,000 people die from tuberculosis; The nearly active tuberculosis patient 6,000,000 of China has 250,000 people to die from tuberculosis every year.According to the epidemics in 2005 that Ministry of Public Health is announced, in 27 kinds of legal first, Category B notifiable diseases, phthisical morbidity number has surpassed hepatitis B, and phthisical death toll has also surpassed rabies, and morbidity number and dead number average occupy first.And also (Multidrug-resistant, MDR) appearance of bacterial strain and acquired immune deficiency syndrome (AIDS) makes that originally just more and more serious tuberculosis epidemic situation becomes more complicated because of multiple drug resistance in control lungy.

At present, prevention tuberculosis unique effective vaccine be bacillus calmette-guerin vaccine (Bacillus Calmette-Guerin, BCG), a kind of attenuation cattle type mycobacterium (Mycobacterium bovis) of work.Yet regrettably, because BCG invention does not also have freeze drying technology at that time, the strain that is used for producing BCG goes down to posterity by cultured method in early days and preserves, this method is in use to the sixties in 20th century always, and the result has caused between the bacillus calmette-guerin vaccine strain that countries in the world use diversity greatly.According to statistics, carry out excessive little tens of times bacillus calmette-guerin vaccine clinical trial in countries in the world, yet result of the test shows, bacillus calmette-guerin vaccine is 0-80% to phthisical immune protective efficiency, diversity is (Brewer, T.F.and Colditz, G.A.Clin Infect Dis.1995 greatly; 20:126-35.Colditz, G.A., Brewer, T.F., et al.JAMA.1994; 271:698-702.).Therefore, a kind of tuberculosis novel vaccine of rendeing a service above BCG of protecting of research is imperative.

Although it is barely satisfactory that BCG renders a service the protection lungy of being grown up, because its good immunostimulating effect and the safety that confirmed already recent decades make it become possibility as the good bacterial expression vector that prevents tuberculosis and other infectious disease.Existing experiment report, the protectiveness candidate antigens gene of separate sources can be cloned in the BCG bacterium and be expressed, thereby makes up recombinant BCG vaccine (Stover, C.K., de la Cruz, V.F., et al.Nature.1991 relatively efficiently; 351:456-60.Stover, C.K., Bansal, G.P., et al.J Exp Med.1993; 178:197-209.Grode, L., Seiler, P., et al.J Clin Invest.2005; 115:2472-9.).Someone is used to come from origin of replication (Ranes, M.G., Rauzier, J., et al.JBacteriol, 1990 of Mfortuitum plasmid pAL5000; 172:2793-7.), made up multiple different escherichia coli-mycobacterium shuttle vector, and be used for that mycobacterium is carried out genetic manipulation and make it to have expressed heterologous gene (Stover, C.K., dela Cruz, V.F., et al.Nature.1991; 351:456-60.Timm, J., Lim, E.M.et al.JBacteriol.1994; 176:6749-53.Garbe, T.R., Barathi, J., et al.Microbiology, 1994; 140:133-8.).

In our previous work; once the ESAT-6 gene of small-molecular weight was inserted Kpn I, the Pst I of main protection antigen gene A g85A of tubercule bacillus and one or two site in the Acc I site; the Ag85A-ESAT6 mosaic gene that obtains is cloned among the carrier for expression of eukaryon pVAX1, has made up the mosaic gene nucleic acid vaccine.This invention has been awarded Chinese invention patent (ZL200410084376.1, Li Zhongming, tubercle bacillus chimeric gene vaccine and preparation method thereof), and submitted PCT application (PCT/2005/CN2005/001914 to, LI, Zhongming.A ChimericMycobacterium tuberculosis Gene Vaccine and the Preparation MethodThereof).Animal experiment shows, has all induced ideal immunne response (Li Z, Zhang H, Fan X, et al.Vaccine 2006 based on the dna vaccination of this kind mosaic gene in mice and Rhesus Macacus; 24:4565-8.Li Z, Song D, et al.DNA Cell Biol 2006; 25:25-30.).The ESAT-6 antigen molecular is little, though it is poor to have the very weak protection of immune response that the independent immune large animal of good immunogenicity produces in toy.Yet, we are in the Rhesus Macacus experiment, adopt this kind mosaic gene dna vaccination to carry out initial immunity, reuse reorganization ESAT-6 albumen booster immunization, surprisedly observed the anti-ESAT-6 production of antibodies of high titre, illustrate that this kind mosaic gene dna vaccination can strengthen the immunogenicity of ESAT-6, this is extremely important to resisting tuberculosis infection.Theoretically, in the Ag85A gene, insert the ESAT-6 gene and not only increased epitope, strengthened immunogenicity, and the embedding of form makes the configuration of Ag85A not be damaged in the frame, kept its specificity epitope to greatest extent, and Ag85A can be used as the carrier protein of ESAT-6 for it provides the active cell immunne response required t cell epitope, makes ESAT-6 also can induce good immune response in large animal.

A large amount of clinical research datas and animal experiment study result show, strengthen the IFN-γ secretion of Th1 type immune response and Th1 cell, body is set up protective immunity extremely important (Kaufmann SH, the Andersen P.Chem Immunol 1998 of opposing mycobacterium tuberculosis infection; 70:21-59.Andersen P.Trends Immunol 2001; 22:160-8).The immunoreation of enhanced Th1 type is defined as the secretion of high-level IFN-γ and a large amount of generations of IgG2a antibody subclass.IFN-γ is a kind of main anti-M.tb. protectiveness cytokine for mice or other animals, the Th1 cell of IFN-γ knock out mice is because can not secretion of gamma-IFN, mice tends to take place lethal tuberculosis infection (Cooper AM in M.tb. attack experiment, Dalton DK, et al.J Exp Med 1993; 178:2243-7.Flynn JL, Chan J, et al.J Exp Med 1993; 178:2249-54.).The main secretion of gamma-IFN of Th1 cell, and IFN-γ the raise differentiation to suppress the Th2 cell and IL-4, IL-9 expression of gene; Otherwise, the differentiation that IL-4 and IL-10 raise and then can reduce the Th1 cell.And the Th cellular products can also be regulated the conversion of IgG antibody subclass, and the antibody IgM that IL-4 mediation B cell produces is converted to IgG1 and IgE, and IFN-γ then mediates its conversion to IgG2a (Snapper CM, Paul WE.Science 1987; 236:944-7).Therefore, different antibody subclass and the production of cytokines special Th acknowledgement type that can react the generation of inducing in the immune animal; And enhanced Th1 type immune response or induce the high-level IFN-γ of generation all can cause the raising of body anti-mycobacterium tuberculosis infection ability.There are some researches show, in BCG, cross expression immunodominance antigen and also can induce the enhanced Th1 type immune response of generation (Dhar N, Rao V, Tyagi AK.Immunol Lett 2003; 88:175-84.Rao V, DharN, N, agiAK.Scand J Immunol 2003; 58:449-61.).

We are on the basis of granted patent ZL200410084376.1, utilize the constructed mycobacteria differential expression carrier (CN200810033549.5) of our laboratory to pass through a large amount of experiments and concentrating on studies again to art technology, successfully make up and screened novel tuberculosis prevention mosaic gene recombinant bacillus calmette-guerin, and in the mice body, induce and produced immunoreation of enhanced Th1 type and high-level IFN-γ, thereby finished the present invention.

Therefore, first purpose of the present invention provides a kind of reinforced mosaic gene recombinant bacillus calmette-guerin.

Second purpose of the present invention provides the preparation method of this reinforced mosaic gene recombinant bacillus calmette-guerin.

The 3rd purpose of the present invention provides and contains the application of this reinforced mosaic gene recombinant bacillus calmette-guerin on preparation prevention tuberculosis recombiant vaccine.

Summary of the invention

First aspect of the present invention provides a kind of reinforced mosaic gene recombinant bacillus calmette-guerin, has comprised by the clone among the electric method for transformation importing BCG shuttle expression carrier of tubercle bacillus chimeric gene Ag85A-ESAT6.

In the mosaic gene recombinant bacillus calmette-guerin of the present invention, described mosaic gene Ag85A-ESAT-6 comprises the coding mycobacterium tuberculosis ESAT-6 gene shown in coding tubercule bacillus structural protein Ag85A gene shown in the sequence 1 and the sequence 2, and wherein said ESAT-6 gene is entrenched on one or two site in the sequence that sequence that the 245-250 position restricted enzyme Kpn I of Ag85A gene discerned, sequence that 325-330 position restriction endonuclease Pst I is discerned and 430-435 position restriction endonuclease Acc I discerned.

Mosaic gene recombinant bacillus calmette-guerin of the present invention is preserved in Chinese typical culture collection center (CCTCC on January 25th, 2010, address: China. Wuhan. Wuhan University), preserving number is CCTCC M 2010025, and the called after cow mycobacteria belongs to bacillus calmette-guerin vaccine Denmark strain GW-A2/rBCG856 (Mycobacterium bovisBCG-Danish strain GW-A2/rBCG856).

In the preferred embodiment of the present invention, mosaic gene Ag85A-ESAT6 is the mosaic gene Ag856A2 that contains two ESAT-6 copy mini-genes.More preferably, two ESAT-6 copy mini-genes are entrenched on the Acc I site of Ag85A gene.

In the preferred embodiment of the present invention, described shuttle expression carrier is the mycobacteria differential expression system.

In the preferred embodiment of the present invention, described shuttle expression carrier is pMFA41.

Second aspect of the present invention provides the preparation method of reinforced mosaic gene recombinant bacillus calmette-guerin, comprises mosaic gene Ag85A-ESAT6 is cloned in the shuttle expression carrier, imports among the BCG by electric method for transformation again.

A preferred embodiment of the present invention is as target gene with mosaic gene Ag856A2 (the mosaic gene Ag85A-ESAT-6 that contains two ESAT-6 copy mini-genes), be cloned among the mycobacteria efficient expression vector pMFA41, it is imported among the BCG recombinant strain of BCG vaccine of screening and evaluation high level expression tubercule bacillus chimeric antigen by electric method for transformation.

Specifically, this preferred embodiment may further comprise the steps:

1) pcr amplification of tubercle bacillus chimeric gene Ag856A2: designing a pair of primer, is template with nucleic acid vaccine HG856A plasmid DNA, by pcr amplification to obtain mosaic gene Ag856A2.

2) structure of recombinant expression plasmid: mosaic gene Ag856A2 fragment and shuttle expression carrier pMFA41 that pcr amplification is obtained digest through BamHI-EcoR I double digestion, agarose gel electrophoresis separates back recovery, purification purpose fragment, and genes of interest is connected back transformed into escherichia coli DH5 α with carrier.

3) structure of mosaic gene recombinant bacillus calmette-guerin and screening: the clone is had in the method importing BCG competent cell of recombiant plasmid by the electricity conversion of mosaic gene, and coat on the Middlebrook 7H11 flat board that contains kanamycin and OADC, cultivate 3～4 weeks screening resistance recon for 37 ℃, and further verify the expression of chimeric antigen in BCG by the Western-blot method.

4) preparation of mosaic gene recombinant bacillus calmette-guerin: will identify that the rBCG bacterial strain that correctly contains recombinant expression plasmid is growing to logarithmic (log) phase in the Middlebrook 7H9 culture medium fully, centrifugal collection thalline, and be resuspended in the PBS-T80 buffer that contains 15% glycerol, preserve standby in-80 ℃ of refrigerators.

It is carrier that the present invention adopts mycobacteria differential expression system or other shuttle expression plasmids, can realize the high level expression of tubercle bacillus chimeric gene in bacillus calmette-guerin vaccine.

The 3rd aspect of the present invention provides the application of reinforced mosaic gene recombinant bacillus calmette-guerin in preparation prevention tuberculosis recombiant vaccine.

The present invention is with mosaic gene recombinant bacillus calmette-guerin immunity BALB/c mouse, and (i.p.) inoculated once in the abdominal cavity, and inoculates booster immunization once in the 10th all vaccine i.p. with same dosage.Measure the anti-Ag85A of different immune temporal inductions generations, the level and the subclass thereof of anti-ESAT-6 specific antibody by the ELISA method, to analyze its humoral immunoresponse(HI) effect; And after immune 12 weeks, break carotid artery and put to death mice, aseptic separating spleen, the preparation splenocyte is measured the IFN-γ level that Ag85A, ESAT-6 antigenic stimulus produce respectively by the ELISPOT method, to analyze its cellullar immunologic response effect.The result shows that than BCG (wtBCG) bacterial strain of wild type, mosaic gene recombinant bacillus calmette-guerin of the present invention can produce the enhancing Th1 type immunne response of antigen-specific, is embodied in the secretion of high-level IFN-γ and a large amount of generations of IgG2a antibody subclass.Vast amount of clinical data and zooperal result of study show that the protective immunity that the generation of immunoreation of enhanced Th1 type and IFN-γ is set up the opposing mycobacterium tuberculosis infection to body is extremely important.IFN-γ is a kind of anti-M.tb. protectiveness cytokine of necessity for mice or other animals, and IFN-γ knock out mice is attacked the tuberculosis infection that tends to take place lethal in the experiment because can not secretion of gamma-IFN at M.tb.Enhanced Th1 type immunoreation or induce the high-level IFN-γ of generation all can cause the raising of body anti-mycobacterium tuberculosis infection ability.

Description of drawings

Fig. 1 is the pcr amplification of embodiment 1 tubercle bacillus chimeric gene Ag856A2 and the structure collection of illustrative plates of recombinant expression plasmid.

Wherein, Lane 1:DNA marker DL-250plus; Lane 2: the double digestion collection of illustrative plates of the BamHI-EcoR I of recombinant expression plasmid; Lane 3: the pcr amplified fragment of mosaic gene Ag856A2; Lane 4:DNA marker DL-2000.

Fig. 2 is embodiment 2 mosaic gene recombinant expression carrier plasmid maps and protein fusion expression sequence thereof.

Fig. 2 A is a mosaic gene recombinant expression plasmid collection of illustrative plates, and wherein Kna represents the kalamycin resistance encoding gene, and OriM represents the mycobacteria replicon; OriE represents the escherichia coli replicon; PfurAma represents-the two fur A gene promoters that suddenly change of 35 districts/initiation codon that ag856a2 represents mosaic gene;

Fig. 2 B is the expressed sequence collection of illustrative plates of mosaic gene recombinant expression plasmid, and wherein DNA sequence is presented at the top, contains the Ag856A2 mosaic gene of pfurAma promoter and amalgamation and expression, and ribosome binding site (RBS) overstriking of promoter upstream and underscore show; Mosaic gene is cloned between the BamHI/EcoRI site of pMFA41 expression vector, six amino acid coding overstrikings of fur A gene head show, BamHI site italic underscore shows that the chimeric antigen amalgamation and expression is after fur A gene, and termination codon is represented with *.

Fig. 3 is that the Western-blot of embodiment 3 mosaic gene recombinant bacillus calmette-guerins analyzes collection of illustrative plates.

Wherein, Lane 1: dye albumen Marker in advance; Lane 2:wtBCG whole cell; Lane 3: mosaic gene recombinant bacillus calmette-guerin rBCG856 whole cell; The rAg856A2 albumen of purification among the Lane 4:E.coli.

Fig. 4 is that ELISA detects the antibody horizontal that mosaic gene recombinant bacillus calmette-guerin is induced generation.

Abdominal cavity inoculation BALB/c mouse (n=5), feminine gender and blank group are inoculated wtBCG or PBS respectively, and the 10th week is with the vaccine booster immunization of same dosage once; In specified time blood sampling, diluted its Anti-Ag85A that induces generation (A group), Anti-ESAT-6 (B group) or Anti-BCG whole cell albumen (C group) are detected in the back with ELISA antibody horizontal respectively in 1: 100,1: 100 or 1: 400, and analyze the significance of difference (*, P＜0.05 of wtBCG group and rBCG856A2 with ANOVA; *, P＜0.01).

Fig. 5 is that ELISA detects antibody subclass and the level that mosaic gene recombinant bacillus calmette-guerin is induced generation.

Abdominal cavity inoculation BALB/c mouse (n=5), feminine gender and blank group are inoculated wtBCG or PBS respectively, and the 10th week is with the vaccine booster immunization of same dosage once; Gather the serum of booster immunization front and back, it is anti-as two with the sheep anti-mouse igg 1 and the IgG2a of HRP labelling to dilute the back respectively in 1: 100,1: 100 or 1: 400, detect the Anti-Ag85A (A that it induces generation, the B group), Anti-ESAT-6 (C, D group) or the antibody subclass level of Anti-BCG whole cell albumen (E, F group), analyze the ratio of its IgG2a/IgG1 simultaneously, and analyze the significance of difference (*, P＜0.05 of wtBCG group and rBCG856A2 with ANOVA; *, P＜0.01).

Fig. 6 is the CD4 of the antigenic specificity secretion of gamma-IFN of ELISPOT quantitative assay generation that mosaic gene recombinant bacillus calmette-guerin is induced ⁺The T lymphocyte quantity.

After the immunity 12 weeks (3 weeks of booster immunization), put to death mice, aseptic separation splenocyte, add 2 μ g/mL rAg85A (A) respectively, 6 μ g/mL rESAT-6 (B), or10 μ g/mL BCG extract (C) stimulates lymphocyte, and with the T lymphocyte quantity of the secretion of gamma-IFN of mice ELISPOT test kit (eBioscience Inc.) quantitative assay antigen-specific.Cell number is presented in the following square brackets of each sky, and analyzes the significance of difference (*, P＜0.05 of wtBCG group and rBCG856A2 with ANOVA; *, P＜0.01).

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 skilled in the art.Those skilled in the art 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.

Pcr amplification and the clone of embodiment 1. tubercle bacillus chimeric gene Ag856A2

Design a pair of primer, its sequence is as follows:

85A-M1：5′-ATA? GGA?TCC?TTT?TCC?CGG?CCG?GGC?TTG?C-3′，

85A-M2：5′-TAA? GAA?TTC?CTA?GGC?GCC?CTG?GGG?CGC-3′。

Wherein 5 ' end primer (sequence 3 in the sequence table) is introduced BamH I restriction enzyme site in Ag85A structural gene upstream; 3 ' end primer (sequence 4 in the sequence table) is introduced the EcoRI restriction enzyme site in Ag85A gene downstream, the synthetic of primer finished by Shanghai Ying Jun biotech company.With nucleic acid vaccine HG856A plasmid DNA is template, the PCR response procedures is set is: 95 ℃ * 5min, (94 ℃ * 30s, 60 ℃ * 30s, 72 ℃ * 90s) * and 30Cycles, last 72 ℃ * 5min extends, pcr amplification obtains mosaic gene Ag85A (Fig. 1, swimming lane 3), DNA sequence is seen Fig. 2 B, and is committed to GenBank (Accession No.EU702753).

The structure of embodiment 2. mosaic gene recombinant expression carriers

Mosaic gene Ag856A2 fragment and shuttle expression carrier pMFA41 that pcr amplification is obtained digest through BamHI-EcoR I double digestion, agarose gel electrophoresis separates back recovery, purification purpose fragment, and genes of interest mixed with mol ratio with carrier at 3: 1, transform DH5 α competent cell in 22 ℃ after connecting 1h.Recombinant clone is seeded to overnight incubation in the LB fluid medium, the extracting recombiant plasmid, BamHI-EcoR I double digestion checking mosaic gene fragment has successfully been inserted (Fig. 1, swimming lane 2) among the mycobacteria expression vector pMFA41, and the success of mosaic gene construction of recombinant expression plasmid is described.Recombinant expression carrier pMFA856 plasmid map is seen Fig. 2 A, and the aminoacid sequence of chimeric protein amalgamation and expression is seen Fig. 2 B.

The structure and the screening of embodiment 3. mosaic gene recombinant bacillus calmette-guerins

1) preparation of BCG electricity transformed competence colibacillus cell: buy freeze dried strain of BCG vaccine from Shanghai Vaccine and Serum Institute, add the line activation on Middlebrook 7H11+10%OADC flat board of the resuspended back of sterilized water, cultivate 3-4 week for 37 ℃, until single bacterium colony occurring.Single colony inoculation of picking to 150mL Middlebrook 7H9+10%OADC fluid medium, in 37 ℃ of slight shaken cultivation to mid-log phase (OD ₆₀₀Be about about 0.8) after, triangular flask is placed 30min on ice, make culture medium be cooled to 0 ℃, in 4 ℃ 5, the centrifugal 5min of 000g reclaims thalline.With the resuspended bacterial sediment of 10% oil solution of 15mL ice pre-cooling, in 4 ℃ 5, the centrifugal 5min of 000g, washed cell 2 times.In 4 ℃ 5, the centrifugal 5min of 000g reclaims cell, and resuspended with initial incubation thing 1/20 volume pre-cooling 10% glycerite at last, and 200 μ L/ pipes are sub-packed in aseptic 1.5mL microcentrifugal tube and standby in-80 ℃ of preservations.

2) electricity transforms: getting two 200 μ L electricity changes competent cell, adds the recombinant expressed grain 5 μ L that the clone has mosaic gene in pipe, and forefinger flicks with the mixing content, places 10min on ice.Then it is transferred to the 2mm electric shock cup of ice pre-cooling, electric conversion instrument (Gene Pluser II is set, Bio-rad) parameter is 2.5kV, 25 μ F, 1,000 Ω, add 900 μ L Middlebrook7H9 culture medium after the discharge immediately, behind the mixing content is transferred to sterile test tube, places that incubation 20h makes bacteria resuscitation on 37 ℃ of shaking tables, and the antibiotics resistance gene of expression plasmid coding.The competent cell that has transformed is coated on the Middlebrook 7H11 culture medium of adding kanamycin.Place room temperature to liquid to be absorbed flat board, be inverted plate, and plate is placed sealed bag in case the culture medium water evaporates in 37 ℃ of cultivations, bacterium colony can occur behind the 3-4w.

The evaluation of embodiment 4. mosaic gene recombinant bacillus calmette-guerins

The picking recombinant clone is inoculated in the Middlebrook 7H9+10%OADC complete medium that contains 20 μ g/mL kanamycin, and (50r/min) cultivates the thoughtful OD of about 2-3 on 37 ℃ of slow shaking tables ₆₀₀Be about about 1.0.Centrifugal collection thalline and be resuspended in 1 * TBS buffer after the ultrasonic disruption cell, collect the lysate supernatant, measure its protein concentration with the Bradford method after, Western-blot identifies the expression of its chimeric antigen.

Respectively get equal protein (about 15 μ g) and run the 12%SDS-polyacrylamide gel electrophoresis, the half-dried transfer printing of 20V * 30min, pvdf membrane sealed in 10% defatted milk powder after 1 hour hatched 1 hour with mice chimeric protein Ag856A2 antiserum (1: 1000), (Santa Cruz) in hatch 45 minute at 1: 6000 in the sheep anti-mouse igg of HRP labelling again, react post-exposure in 1 minute in the X-ray sheet with chemical luminous substrate ECL at last.

The preparation and the mouse immune of embodiment 5. mosaic gene recombinant bacillus calmette-guerins

Mosaic gene recombinant bacillus calmette-guerin bacterial strain (rBCG856) and wtBCG are grown to logarithmic (log) phase in Middlebrook 7H9+10%OADC culture medium, 4, the centrifugal collection thalline of 000 * g, and be resuspended in the PBS-T80 buffer (1 * PBS that contains 15% glycerol, pH7.4,0.05%Tween80) ,-80 preserve standby in ℃ refrigerator.

The female BALB/c mouse in ages in SPF level 6～8 week is bought in the Shanghai City Experimental Animal Center, and 5/group, totally 3 groups, abdominal cavity inoculation (i.p.) 10 respectively ⁶CFU/0.4mL wtBCG or rBCG856, blank winding kind 0.4mL PBS-T80.Every 3 all tail vein bloods once, and in the 10th all vaccine i.p. inoculate booster immunization once with same dosage.Booster immunization kills under the mice aseptic condition after 3 weeks and wins spleen, isolated lymphocytes.

Embodiment 6.ELISA measures serum antibody level and subclass thereof

Other mice serum equal-volume mixing of each immune group that the different immunity time is collected dilutes the back application of sample in the removable ELISA Plate in 96 holes of antigen coated mistake respectively, measures the antibody horizontal that is produced in its serum by indirect ELISA method; Simultaneously, be two anti-with the goat anti-mouse igg 1 of HRP labelling and IgG2a respectively, analyze its antibody subclass separately.Concrete experimental procedure is as follows:

1) antigen coated: as to wrap by the removable ELISA Plate (MICROLON600 in 96 holes with 50mM carbonate buffer solution (pH9.6), Greiner Bio-one), antigen concentration is respectively 0.25 μ g/mL rAg85A, 1 μ g/mL rESAT-6,10 μ g/mL BCG extract, bag is 100uL by volume, and 37 ℃ of bags are spent the night by 2h or 4 ℃ of bags.

2) abandon antigen coated liquid, (0.05%Tween20) washing is 2～3 times for 1 * PBS, pH7.4 to add 200 μ L, 1 * PBS-T20 buffer.

3) sealing: add 100 μ L confining liquids (1%BSA, 1 * PBS-T20), 37 ℃ of sealing 1h.

4) abandon confining liquid, add 200 μ L, 1 * PBS-T20 buffer washing 1～2 time.

5) application of sample: blood serum sample to be diluted in sample diluting liquid (0.5%BSA at 1: 100,1 * PBS-T20), respectively getting 100 μ L adds in the ELISA Plate of rAg85A and the antigen coated mistake of rESAT-6, add BCG extract bag at 1: 400 by in the ELISA Plate of crossing, each sample is all answered the hole application of sample, 37 ℃ of reaction 1h.

6) abandon sample liquid, add 200 μ L, 1 * PBS-T20 buffer washing 5～6 times.

7) integrated enzyme reaction: with 1: 12,500 were diluted in sample diluting liquid with the goat anti-mouse igg (Santa Cruz Biotechnology) of HRP labelling, and every hole adds 100 μ L, 37 ℃ of reaction 1h.

8) abandon enzyme connection liquid, add 200 μ L, 1 * PBS-T20 buffer washing 6～7 times.

9) chromogenic reaction: equal-volume mixing TMB A, B component (KPL Inc.), every hole adds substrate 100 μ L, 37 ℃ of reaction 20min.

10) cessation reaction: every hole adds stop buffer 2M H ₂S0 ₄50 μ L.

11) reading: ELISA Plate is inserted in the microplate reader (Thermo Labsystem) reading under wavelength 450nm.

Preparation of embodiment 7. mouse spleen lymphocytes and IFN-γ ELISPOT measure

After 12 weeks of immunity, break carotid artery and put to death mice, aseptic separating spleen, and every group of mouse spleen concentrated on 200 order nylon net bags mill together, splenocyte of each group of preparation is with the T lymphocyte quantity of the secretion of gamma-IFN of mice ELISPOT test kit (eBioscience Inc.) quantitative assay antigen-specific.Concrete experimental procedure is as follows:

1) activation pvdf membrane: (every hole adds 15 μ L, 35% ethanol for MAIPS4510, Millipore) lid, and room temperature leaves standstill a moment with the pvdf membrane that soaks into and activation is wherein built-in carefully to open 96 hole ELISPOT plates in superclean bench.

2) carefully get rid of ethanol, and on sterile gauze, pat dry.

3) add 200 μ L coating buffers, flush away may remaining ethanol.Repeated washing 2 times.

4) carefully get rid of antigen coated liquid, and on sterile gauze, pat dry.

5) antigen coated: by specification dilution capture antibody (clone AN-18, eBioscience Inc.) is in antigen coated liquid, and final concentration is 4 μ g/mL, and every hole adds 100 μ L, and 4 ℃ of bags are spent the night.

6) carefully get rid of coating buffer, and on sterile gauze, pat dry; Add coating buffer 200 μ L/ holes, wash 3 times.

7) sealing: add 1640 complete culture solutions, the 200 μ L/ holes that contain 10%NBS, room temperature left standstill 1 hour, with the antigen site of sealing non-specific binding.

8) carefully get rid of deblocking liquid, and on sterile gauze, pat dry.

9) adjusting cell concentration is 4 * 10 ⁶/ mL, every hole adds 250 μ L, and promptly 1 * 10 ⁶/ hole, and adding antigenic stimulus thing to its final concentration is respectively rAg85A 2 μ g/mL, rESAT-66 μ g/mL, BCGextract 10 μ g/mL; 37 ℃ of 5%C0 ₂Middle cultured cell 24h.

* above step all needs the sterile working, and following steps are non-sterile operation.

10) abandon cell culture fluid, add 200 μ L/ hole 1 * PBS-T20 buffer, wash 3 times.

11) by specification dilute biotin labeled detection antibody (clone R4-6A2, eBioscienceInc.) in 1 * sample diluting liquid, final concentration is 2 μ g/mL, every hole adds 100 μ L, incubated at room 2h.

12) abandon antibody-solutions, add 200 μ L/ hole 1 * PBS-T20 buffer, wash 1min on the decolorization swinging table, repeated washing 5 times.

13) Streptavidin of by specification dilution HRP labelling is in 1 * sample diluting liquid, and final concentration is 0.4 μ g/mL, and every hole adds 100 μ L, incubated at room 45min.

14) abandon HRP-SA solution, add 200 μ L/ hole 1 * PBS-T20 buffer, wash 4 times; Add 200 μ L/ hole 1 * PBS, wash 3 times.

15) the AEC substrate solution (LabVision Corporation) of the adding 100 fresh configurations in μ L/ hole, incubated at room 10～30min, the Real Time Observation speckle forms situation.

16) abandon substrate solution, add 200 μ L/ hole deionized waters, wash 3 times.

17) air-dry ELISPOT plate in super-clean bench, and with the ELSIPOT calculating instrument (BioReader-4000 Biosys) counts speckle automatically and forms quantity (SFU).

SEQUENCE?LISTING

＜110〉Shanghai City public health clinical center

＜120〉reinforced mosaic gene recombinant bacillus calmette-guerin and preparation method thereof

<130>CN101104

<160>6

<170>PatentIn?version?3.3

<210>1

<211>888

<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

gactggtacc?agcccgcctg?cggcaaggcc?ggttgccaga?cttacaagtg?ggagaccttc 300

ctgaccagcg?agctgccggg?gtggctgcag?gccaacaggc?acgtcaagcc?caccggaagc 360

gccgtcgtcg?gtctttcgat?ggctgcttct?tcggcgctga?cgctggcgat?ctatcacccc 420

cagcagttcg?tctacgcggg?agcgatgtcg?ggcctgttgg?acccctccca?ggcgatgggt 480

cccaccctga?tcggcctggc?gatgggtgac?gctggcggct?acaaggcctc?cgacatgtgg 540

ggcccgaagg?aggacccggc?gtggcagcgc?aacgacccgc?tgttgaacgt?cgggaagctg 600

atcgccaaca?acacccgcgt?ctgggtgtac?tgcggcaacg?gcaagccgtc?ggatctgggt 660

ggcaacaacc?tgccggccaa?gttcctcgag?ggcttcgtgc?ggaccagcaa?catcaagttc 720

caagacgcct?acaacgccgg?tggcggccac?aacggcgtgt?tcgacttccc?ggacagcggt 780

acgcacagct?gggagtactg?gggcgcgcag?ctcaacgcta?tgaagcccga?cctgcaacgg 840

gcactgggtg?ccacgcccaa?caccgggccc?gcgccccagg?gcgcctag 888

<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>28

<212>DNA

＜213〉synthetic

<400>3

ataggatcct?tttcccggcc?gggcttgc 28

<210>4

<211>27

<212>DNA

＜213〉synthetic

<400>4

taagaattcc?taggcgccct?ggggcgc 27

<210>5

<211>1470

<212>DNA

＜213〉tubercule bacillus chromosomal DNA

<400>5

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

gactggtacc?agcccgcctg?cggcaaggcc?ggttgccaga?cttacaagtg?ggagaccttc 300

ctgaccagcg?agctgccggg?gtggctgcag?gccaacaggc?acgtcaagcc?caccggaagc 360

gccgtcgtcg?gtctttcgat?ggctgcttct?tcggcgctga?cgctggcgat?ctatcacccc 420

cagcagttcg?tctacatggc?agagcagcag?tggaatttcg?cgggtatcga?ggccgcggca 480

agcgcaatcc?agggaaatgt?cacgtccatt?cattccctcc?ttgacgaggg?gaagcagtcc 540

ctgaccaagc?tcgcagcggc?ctggggcggt?agcggttcgg?aggcgtacca?gggtgtccag 600

caaaaatggg?acgccacggc?taccgagctg?aacaacgcgc?tgcagaacct?ggcgcggacg 660

atcagcgaag?ccggtcaggc?aatggcttcg?accgaaggca?acgtcactgg?gatgttcgca 720

gtctacatgg?cagagcagca?gtggaatttc?gcgggtatcg?aggccgcggc?aagcgcaatc 780

cagggaaatg?tcacgtccat?tcattccctc?cttgacgagg?ggaagcagtc?cctgaccaag 840

ctcgcagcgg?cctggggcgg?tagcggttcg?gaggcgtacc?agggtgtcca?gcaaaaatgg 900

gacgccacgg?ctaccgagct?gaacaacgcg?ctgcagaacc?tggcgcggac?gatcagcgaa 960

gccggtcagg?caatggcttc?gaccgaaggc?aacgtcactg?ggatgttcgc?agtctacgcg 1020

ggagcgatgt?cgggcctgtt?ggacccctcc?caggcgatgg?gtcccaccct?gatcggcctg 1080

gcgatgggtg?acgctggcgg?ctacaaggcc?tccgacatgt?ggggcccgaa?ggaggacccg 1140

gcgtggcagc?gcaacgaccc?gctgttgaac?gtcgggaagc?tgatcgccaa?caacacccgc 1200

gtctgggtgt?actgcggcaa?cggcaagccg?tcggatctgg?gtggcaacaa?cctgccggcc 1260

aagttcctcg?agggcttcgt?gcggaccagc?aacatcaagt?tccaagacgc?ctacaacgcc 1320

ggtggcggcc?acaacggcgt?gttcgacttc?ccggacagcg?gtacgcacag?ctgggagtac 1380

tggggcgcgc?agctcaacgc?tatgaagccc?gacctgcaac?gggcactggg?tgccacgccc 1440

aacaccgggc?ccgcgcccca?gggcgcctag 1470

<210>6

<211>489

<212>PRT

＜213〉tubercule bacillus chimeric antigen protein sequence

<400>6

Phe?Ser?Arg?Pro?Gly?Leu?Pro?Val?Glu?Tyr?Leu?Gln?Val?Pro?Ser?Pro

1 5 10 15

Ser?Met?Gly?Arg?Asp?Ile?Lys?Val?Gln?Phe?Gln?Ser?Gly?Gly?Ala?Asn

20 25 30

Ser?Pro?Ala?Leu?Tyr?Leu?Leu?Asp?Gly?Leu?Arg?Ala?Gln?Asp?Asp?Phe

35 40 45

Ser?Gly?Trp?Asp?Ile?Asn?Thr?Pro?Ala?Phe?Glu?Trp?Tyr?Asp?Gln?Ser

50 55 60

Gly?Leu?Ser?Val?Val?Met?Pro?Val?Gly?Gly?Gln?Ser?Ser?Phe?Tyr?Ser

65 70 75 80

Asp?Trp?Tyr?Gln?Pro?Ala?Cys?Gly?Lys?Ala?Gly?Cys?Gln?Thr?Tyr?Lys

85 90 95

Trp?Glu?Thr?Phe?Leu?Thr?Ser?Glu?Leu?Pro?Gly?Trp?Leu?Gln?Ala?Asn

100 105 110

Arg?His?Val?Lys?Pro?Thr?Gly?Ser?Ala?Val?Val?Gly?Leu?Ser?Met?Ala

115 120 125

Ala?Ser?Ser?Ala?Leu?Thr?Leu?Ala?Ile?Tyr?His?Pro?Gln?Gln?Phe?Val

130 135 140

Tyr?Met?Ala?Glu?Gln?Gln?Trp?Asn?Phe?Ala?Gly?Ile?Glu?Ala?Ala?Ala

145 150 155 160

Ser?Ala?Ile?Gln?Gly?Asn?Val?Thr?Ser?Ile?His?Ser?Leu?Leu?Asp?Glu

165 170 175

Gly?Lys?Gln?Ser?Leu?Thr?Lys?Leu?Ala?Ala?Ala?Trp?Gly?Gly?Ser?Gly

180 185 190

Ser?Glu?Ala?Tyr?Gln?Gly?Val?Gln?Gln?Lys?Trp?Asp?Ala?Thr?Ala?Thr

195 200 205

Glu?Leu?Asn?Asn?Ala?Leu?Gln?Asn?Leu?Ala?Arg?Thr?Ile?Ser?Glu?Ala

210 215 220

Gly?Gln?Ala?Met?Ala?Ser?Thr?Glu?Gly?Asn?Val?Thr?Gly?Met?Phe?Ala

225 230 235 240

Val?Tyr?Met?Ala?Glu?Gln?Gln?Trp?Asn?Phe?Ala?Gly?Ile?Glu?Ala?Ala

245 250 255

Ala?Ser?Ala?Ile?Gln?Gly?Asn?Val?Thr?Ser?Ile?His?Ser?Leu?Leu?Asp

260 265 270

Glu?Gly?Lys?Gln?Ser?Leu?Thr?Lys?Leu?Ala?Ala?Ala?Trp?Gly?Gly?Ser

275 280 285

Gly?Ser?Glu?Ala?Tyr?Gln?Gly?Val?Gln?Gln?Lys?Trp?Asp?Ala?Thr?Ala

290 295 300

Thr?Glu?Leu?Asn?Asn?Ala?Leu?Gln?Asn?Leu?Ala?Arg?Thr?Ile?Ser?Glu

305 310 315 320

Ala?Gly?Gln?Ala?Met?Ala?Ser?Thr?Glu?Gly?Asn?Val?Thr?Gly?Met?Phe

325 330 335

Ala?Val?Tyr?Ala?Gly?Ala?Met?Ser?Gly?Leu?Leu?Asp?Pro?Ser?Gln?Ala

340 345 350

Met?Gly?Pro?Thr?Leu?Ile?Gly?Leu?Ala?Met?Gly?Asp?Ala?Gly?Gly?Tyr

355 360 365

Lys?Ala?Ser?Asp?Met?Trp?Gly?Pro?Lys?Glu?Asp?Pro?Ala?Trp?Gln?Arg

370 375 380

Asn?Asp?Pro?Leu?Leu?Asn?Val?Gly?Lys?Leu?Ile?Ala?Asn?Asn?Thr?Arg

385 390 395 400

Val?Trp?Val?Tyr?Cys?Gly?Asn?Gly?Lys?Pro?Ser?Asp?Leu?Gly?Gly?Asn

405 410 415

Asn?Leu?Pro?Ala?Lys?Phe?Leu?Glu?Gly?Phe?Val?Arg?Thr?Ser?Asn?Ile

420 425 430

Lys?Phe?Gln?Asp?Ala?Tyr?Asn?Ala?Gly?Gly?Gly?His?Asn?Gly?Val?Phe

435 440 445

Asp?Phe?Pro?Asp?Ser?Gly?Thr?His?Ser?Trp?Glu?Tyr?Trp?Gly?Ala?Gln

450 455 460

Leu?Asn?Ala?Met?Lys?Pro?Asp?Leu?Gln?Arg?Ala?Leu?Gly?Ala?Thr?Pro

465 470 475 480

Asn?Thr?Gly?Pro?Ala?Pro?Gln?Gly?Ala

485

Claims

1. a reinforced mosaic gene recombinant bacillus calmette-guerin has comprised by the clone among the electric method for transformation importing BCG reorganization shuttle expression carrier of tubercle bacillus chimeric gene Ag85A-ESAT6.

2. mosaic gene recombinant bacillus calmette-guerin as claimed in claim 1, wherein said mosaic gene Ag85A-ESAT6 comprises the coding mycobacterium tuberculosis ESAT-6 gene shown in coding tubercule bacillus structural protein Ag85A gene shown in the sequence 1 and the sequence 2, and wherein said ESAT-6 gene is entrenched on one or two site in the sequence that sequence that the 245-250 position restricted enzyme Kpn I of Ag85A gene discerned, sequence that 325-330 position restriction endonuclease Pst I is discerned and 430-435 position restriction endonuclease Acc I discerned.

3. mosaic gene recombinant bacillus calmette-guerin as claimed in claim 1, described mosaic gene recombinant bacillus calmette-guerin are the rBCG856 that is preserved in CCTCC, preserving number CCTCC M 2010025.

4. mosaic gene recombinant bacillus calmette-guerin as claimed in claim 1, wherein said mosaic gene Ag85A-ESAT6 is Ag856A2.

5. mosaic gene recombinant bacillus calmette-guerin as claimed in claim 1, wherein said expression vector are the mycobacteria shuttle expression carrier.

6. mosaic gene recombinant bacillus calmette-guerin as claimed in claim 5, wherein said mycobacteria shuttle expression carrier is pMFA41.

7. the preparation method of the described mosaic gene recombinant bacillus calmette-guerin of claim 1 is characterized in that: mosaic gene Ag85A-ESAT6 is cloned in the shuttle expression carrier, and the shuttle expression plasmid of will recombinating again imports among the BCG by electric method for transformation.

8. preparation method as claimed in claim 7, the preparation process when wherein said mosaic gene Ag85A-ESAT6 is Ag856A2 is: adopting primer b shown in primer a shown in the sequence 3 and the sequence 4, is that template is carried out pcr amplification with nucleic acid vaccine HG856A plasmid DNA.

9. preparation method as claimed in claim 7, the step that wherein said mosaic gene is cloned in the shuttle expression carrier is that pcr amplification gained mosaic gene is connected behind enzyme action with shuttle expression carrier, transformed into escherichia coli competent cell again, cell culture, and extracting recombiant plasmid.

10. as the described preparation method of one of claim 7-9, wherein said shuttle expression carrier is pMFA41.

11. the application of the described reinforced mosaic gene recombinant bacillus calmette-guerin of claim 1 in preparation prevention tuberculosis recombiant vaccine.