CN101875913B - Recombinant Mycobacterium smegmatis strain expressing Mycobacterium tuberculosis Ag85B and ESAT-6 fusion protein and application thereof - Google Patents

Abstract

The invention relates to a recombinant mycobacterium smegmatis strain capable of expressing mycobacterium tuberculosis Ag85B and ESAT-6 fusion protein and application thereof. The recombinant plasmid containing Ag85B and ESAT-6 fusion protein gene is electrically transformed into Mycobacterium smegmatis, abbreviated as AE-MS, and the preservation number is as follows: CCTCCM 2010097. The recombinant Mycobacterium smegmatis strain AE-MS obtained by screening is used for immunizing mice, and can induce a stronger immune response level than that of Mycobacterium smegmatis. The invention also relates to application of the constructed strain in preparation of a preparation or a medicament for preventing and treating tuberculosis. The recombinant mycobacterium expressing the Ag85B and ESAT-6 fusion gene integrates the advantages of a target antigen and a live carrier, and can stimulate stronger immune response of an organism after immunization and improve the immune protection of the organism, so the recombinant mycobacterium has good application prospect.

Description

Recombinant Mycobacterium smegmatis strain expressing Mycobacterium tuberculosis Ag85B and ESAT-6 fusion protein and application thereof

technical field

The invention belongs to the field of tuberculosis vaccines. The invention relates to a recombinant mycobacterium smegmatis strain expressing Ag85B and ESAT-6 fusion protein which can improve the immune effect of vaccines. The invention also relates to the application of the constructed bacterial strain in the preparation of preparations or medicines for the prevention and treatment of tuberculosis. the

Background technique

Research status of tuberculosis and genetic engineering vaccines

According to the WHO report, about 2 billion people in the world have been infected with Mycobacterium tuberculosis (MTB), among which those infected by drug-resistant strains may reach 50 million, with about 10 million new patients and 3 million deaths per year , the death rate of tuberculosis (TB) is second only to AIDS in all infectious diseases. WHO lists TB, together with AIDS and malaria, as the most important infectious killer of human beings. my country is one of the 22 countries with a high burden of TB in the world. The number of deaths due to TB is about 250,000 each year. It ranks first among the 27 legally notifiable infectious diseases in my country and is 2 times the total number of deaths from various other infectious diseases and parasitic diseases. times. Therefore, the Ministry of Health of my country has listed TB as one of the major diseases under national key control. the

In terms of TB prevention, Bacillus Calmette-Guérin vaccine (BCG) is currently the only effective vaccine for TB prevention. It is mainly used as a preventive vaccine before MTB infection, and it is aimed at the early infection of MTB. It cannot prevent and control latent infection. The relapse of state MTB, and its short protection period, weak protective immune response and other problems, so it is urgent to develop a new type of TB vaccine that can be used for infected people and prevent relapse. In terms of TB treatment, due to the long course of TB chemotherapy for more than 6 months, patients cannot adhere to medication and have poor compliance, and often cause drug resistance, especially the emergence of extensive drug-resistant tuberculosis and super-drug-resistant tuberculosis. important reason for the method. With the gradual deepening of the understanding of the nature of MTB, it has been confirmed that TB patients have a low Th1-type cellular immune response, so therapeutic vaccines that can improve the immune function of the body, especially the Th1-type response, have a good application prospect. the

The new TB vaccines currently under development mainly include subunit vaccines, gene vaccines, recombinant BCG vaccines, attenuated or enhanced whole-bacteria live vaccines, auxotrophic live vaccines, and vaccines combined with DC, etc., but there is no new type of TB vaccine yet. Vaccines can replace traditional BCG, and no vaccine is widely used for immunotherapy of TB. Recombinant live vaccines are currently the most studied TB vaccines, which consist of introducing genes encoding MTB protective antigens into live microbial vectors. In the study of live vaccines, how to obtain vaccine strains with ideal immune effects is the most concerned issue for researchers. The main measures are: first, select the carrier with immune adjuvant effect; second, select the appropriate target antigen. the

vector andexpression protocol for fast and efficient recombinant protein expression inMycobacterium smegmatis.Protein Expression and Purification，2008；57(1)：81-87.)等构建了快速高效的MS表达载体，克隆基因本身的启动子作用下 能稳定地表达重组分泌型蛋白，且其产量为BCG的5～10倍。胡佳杰(重组M.S-Sj26GST疫苗有关毒性实验研究。华南预防医学，2002；28(5)：44-45)等检测了重组BCG-hsp70和重组MS-sj26GST对小鼠的毒性，结果表明这两种重组分枝杆菌所诱导淋巴细胞增殖指数较对照组高，小鼠体重、各脏器重量、各血象指标、血清肌酐和ALT含量与对照组无差异。说明此疫苗能提高小鼠的免疫功能，且对小鼠无系统毒性作用。因此，MS是分枝杆菌活疫苗优良载体。  Introducing exogenous genes into mycobacteria to construct recombinant mycobacterial live vaccines is one of the important directions of new TB vaccine research. Most domestic and foreign studies use BCG as the carrier of mycobacterial live vaccines, but recombinant BCG vaccines still exist as follows: Disadvantages: slow growth, high nutritional requirements, more than 10 hours to cultivate one generation; thick cell wall, rich in lipids, hindering molecular transport including exogenous DNA; low transformation efficiency; special vectors are required for exogenous gene expression. Mycobacterium smegmatis (Mycobacterium smegmatis, MS) is a non-pathogenic mycobacterium with fast growth and high transformation efficiency and a strong cellular immune adjuvant. It has similar immunological properties to BCG, such as Zhu DY, et al. (RecombinantM.smegmatis vaccine targeted delivering IL-12/GLS into macrophages caninduce specific cellular immunity against M.tuberculosis in BALB/c mice.Vaccine, 2007; 25(4): 638-648.) et al constructed the expression IL-12 and GLS The rMS fusion gene, the recombinant live vaccine can stimulate the proliferation of T lymphocytes, initiate Th1 cell immune response, promote the secretion of various cytokines (IFN-γ, IL-2, IL-12, etc.), and improve the body's phagocytosis of pathogens and killing effect. Harth et al. (High extracellular levels of mycobacterium tuberculosis glutamine synthetase and superoxide dismutase in actively growing cultures aredue to high expression and extracellular stability rather than to a protein-specific export mechanism. Immunol Cell Biol. 2003: 2003, 87) The four secreted proteins of MTB were transferred into MS for expression, and various index tests showed that the biochemical immune characteristics of the recombinant protein and the natural protein were almost identical. Rachael (A new Gateway

vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis. Protein Expression and Purification, 2008; 57(1): 81-87.) etc. have constructed a fast and efficient MS expression vector, which can be stabilized under the action of the promoter of the cloned gene itself. Highly express recombinant secreted protein, and its yield is 5-10 times that of BCG. Hu Jiajie (Experimental research on the toxicity of recombinant MS-Sj26GST vaccine. South China Preventive Medicine, 2002; 28(5): 44-45) etc. detected the toxicity of recombinant BCG-hsp70 and recombinant MS-sj26GST to mice, and the results showed that the two The lymphocyte proliferation index induced by the recombinant mycobacteria was higher than that of the control group, and the mouse body weight, weight of various organs, various blood indicators, serum creatinine and ALT levels were not different from those of the control group. It shows that the vaccine can improve the immune function of mice, and has no systemic toxicity to mice. Therefore, MS is an excellent carrier for live mycobacterial vaccines.

Choosing an appropriate target antigen can significantly improve the protective efficiency of the vaccine. Secreted protein is a group of proteins secreted and released by MTB in the early stage of logarithmic growth. It is a very important specific antigen in the early stage of MTB infection, and it is also a type of protein that can induce protective immunity among the currently recognized MTB proteins. The Ag85 complex includes three components, Ag85A, Ag85B and Ag85C, and its content occupies the first place in mycobacterial secretory proteins. Among them, Ag85B is related to the synthesis of mycobacterial cell walls, and is involved in the pathogenic process after binding to human fibronectin. The antigenicity of the antigen is strongest in the secreted protein. Comparative genomics found that the existing BCG strains all lost the ESAT-6 gene, and the researchers found that the ESAT-6 subunit vaccine could stimulate a strong ESAT-6-specific T cell response protection, which was comparable to that of BCG; ESAT- 6 is also one of the main target antigens of immune memory effector cells, which can induce rapid proliferation and release high levels of INF-γ in the early stage of MTB re-infection, activate monocyte-macrophages, and control infection. Olsen et al. (Protection of Mice with a Tuberculosis Subunit VaccineBased on a Fusion Protein of Antigen 85B and ESAT-6. Infect Immun. 2001, 69(5): 2773-2778.) expressed Ag85B-ESAT-6 fusion in Escherichia coli protein, and immunized mice with purified ESAT-6, Ag85B, ESAT-6-Ag85B, Ag85B-ESAT-6, BCG and adjuvant MPL-DDA respectively. The results showed that the protective effect induced by the recombinant fusion protein expressed in E. coli was superior. on Ag85B, ESAT-6 protein and BCG alone. Furthermore, Ag85B-ESAT-6 fusion protein can induce long-term anti-MTB immune memory in mice. Therefore, the fusion protein of Ag85B and ESAT-6 has better immunogenicity. the

Contents of the invention

The purpose of the present invention is to obtain a recombinant Mycobacterium smegmatis strain expressing Ag85B and ESAT-6 fusion protein. The bacterial strain can be used in the development of new preventive and/or therapeutic vaccines (preparations) for tuberculosis. the

The present invention is achieved like this:

To select the dominant antigens Ag85B and ESAT-6 of Mycobacterium tuberculosis in the reproductive period, firstly, a 48bp (Gly3Ser)4 hydrophobicity was connected between the Ag85B and ESAT-6 protein genes by using restriction enzyme sites and PCR primer design. linker, clone the fusion gene into the mycobacterium-Escherichia coli shuttle expression vector to obtain a recombinant plasmid expressing the fusion protein of Ag85B and ESAT-6. Prepare Mycobacterium smegmatis competent cells, transform the recombinant plasmids into bacteria, screen positive clones with hygromycin resistance plate, identify their genotypes by PCR and restriction enzyme analysis, and analyze their phenotypes by Western-blot The positive strain was named as the recombinant Mycobacterium smegmatis strain expressing the fusion protein of Mycobacterium tuberculosis Ag85B and ESAT-6 (Ag85B-ESAT-6-Mycobacterium smegmatis), referred to as AE-MS. Bacterial strain AE-MS was sent to China Center for Type Culture Collection on April 22, 2010 for preservation, and its preservation number is: CCTCC M2010097. the

The application of the recombinant mycobacterium smegmatis strain in the preparation of vaccines and preparations for preventing and treating tuberculosis. the

The result of the recombinant Mycobacterium smegmatis strain stimulating the body's immune response: After the strain of the present invention was expanded and cultured, 10 ⁷ mice were subcutaneously immunized, and the level of humoral immune response was detected including antibody titer and antibody subtype determination; Response level, including peripheral blood CD4 ⁺ /CD8 ⁺ ratio, splenic lymphocyte killing effect, ELISPOT detection of IFN-γ producing cell frequency. The test results showed that the recombinant Mycobacterium smegmatis strain expressing the fusion protein of Mycobacterium tuberculosis Ag85B and ESAT-6 obtained through screening and immunized mice could induce higher levels of humoral and cellular immune responses than Mycobacterium smegmatis. The recombinant mycobacterium strain expressing the Ag85B and ESAT-6 fusion gene has concentrated the advantages of the target antigen and the live carrier, and can stimulate a stronger immune response of the body after immunization and improve the immune protection of the body, so it has a good application prospect.

Description of drawings:

Figure 1: PCR results of AE-MS genotype identification. the

Figure 2: AE-MS genotype identification results of enzyme digestion identification. the

Figure 3: Western-blot analysis results of AE-MS phenotype identification. the

Figure 4: Schematic diagram of detection data of CD4+/CD8+ ratio in peripheral blood of mice immunized with AE-MS. the

Figure 5: Schematic diagram of detection data of spleen lymphocyte killing effect in AE-MS immunized mice. the

Figure 6: Schematic diagram of the frequency data of IFN-γ-producing cells detected by ELISPOT in AE-MS immunized mice. the

Detailed ways

Construction of recombinant plasmid pDE-AEL:

According to the genome sequence of MTB H37Rv strain, 2 pairs of primers were designed. The primers for the ag85b sequence are:

P1: 5'-TAGGATCCATGACCGCGGGCGCGTTCTC-3', containing BamHI restriction site;

P2: 5'-GCGAAGCTTTCATGCGAACATCCCAGTGA-3', containing HindIII restriction site. the

The primers for the esat-6 sequence are:

P1: 5'-GCATCGATGGTGGCTCAGGTGGCTCCGGTGGAGGCGGAAGCGGCGGTGGA GGA TC AACAGAGCAGCAGTGGAATTT-3', containing ClaI restriction site and 48bp linker sequence;

P2: 5'-GCGAAGCTTTCATGCGAACATCCCAGTGA-3', containing HindIII restriction site. the

The PCR product of the target gene was cloned into the pGEM-T vector for sequencing. After the sequence was identified correctly, it was cloned into the pDE22 vector (mycobacterial secreted expression vector) according to their different restriction sites. The sequence determination showed that the fusion protein was successfully constructed. The Escherichia coli-mycobacterium shuttle expresses the recombinant plasmid pDE-AEL, and its complete gene sequence is shown in SEQ ID NO:1. the

Preparation of electroporation competent cells:

Preparation of MS Competence: Pick an inoculation loop of MS from the strains preserved in the modified Roche medium, inoculate it in 5ml 7H9 medium, cultivate overnight at 30°C with 100rpm shaking, and transfer to 200ml medium at a ratio of 1:100 , continue to cultivate until OD600nm = 0.8 ~ 1, ice bath 1.5h. Centrifuge at 4°C, 5 000rpm for 10min, collect bacteria, suspend the pellet in pre-cooled 10% glycerol solution, centrifuge at 4°C, 5 000rpm for 10min, wash three times in total, and reduce the volume of 10% glycerol solution by half each time. Finally, the bacterial pellet was resuspended in 10% glycerol solution at a ratio of 100:1, and stored at -70°C until use. the

Competent preparation of E.coli: Inoculate activated E.coli in 100ml LB culture medium, culture at 37°C until OD ₆₀₀ is about 0.6, collect bacteria by centrifugation, wash twice with 10mM HEPES solution, and use pre-cooled 10% glycerol solution Wash once, collect bacteria by centrifugation, resuspend in 10% glycerol solution, store in -70°C.

Recombinant plasmid electrotransformation MS competent cells:

Take 0.4ml of MS competent cells to melt in an ice bath, mix with 5μg of ethanol-precipitated recombinant plasmid, place on ice for 10min, transfer to a 0.2cm pre-cooled electroporation cup, and electroporate once, the parameters are: voltage 2.5kV, capacitance 25μF, resistance 1 000Ω, ice bath for 2min, transfer to 5ml 7H9 medium, culture at 30℃ for 2h, spread on 7H10 plate containing OADC and hygromycin 150μg/ml, set blank plasmid as control, incubate at 30℃ for 5～ 7d until visible colonies are formed. the

AE-MS genotype identification:

Pick a single colony on the electroporation MS plate and detect it by PCR method. The amplified target band can be seen at about 1200bp, as shown in Figure 1. Resuspend it in 20 μl of 10% glycerol solution, take 1 μl of bacterial solution and 40 μl of E.coli electroporation cells, mix well, put them in ice bath for 10 minutes, and transform MS-E.coli by direct electroporation, parameters: 0.2cm electroporation cuvette, Voltage 2.5kV, capacitance 45μF, ice-bath for 2min, transfer to 4ml LB medium, incubate at 37℃ for 1h, smear on a plate, and incubate for 12-16h until colonies form. Colonies were picked, cultured by conventional methods, plasmids were extracted, and identified by enzyme digestion, about 1 200 bp fragments could be excised, as shown in Figure 2. The positive clones identified by both PCR and enzyme digestion were named the recombinant Mycobacterium smegmatis strain expressing the fusion protein of Mycobacterium tuberculosis Ag85B and ESAT-6, referred to as AE-MS. the

AE-MS phenotype identification:

The AE-MS strains with positive genotype identification were picked and inoculated in 7H9 medium containing hygromycin, cultivated until the OD ₆₀₀ was greater than 1, and centrifuged at 6000rpm for 10min to harvest the bacteria. PEG 6 000 concentrated bacterial culture supernatant, set aside. Separately take bacteria and resuspend them in PBS, and ultrasonically break them in an ice bath with a power of 300W, each time for 10s, for a total of 3 minutes, to fully lyse the bacteria, and take samples for SDS-PAGE analysis. The culture supernatant of the recombinant bacteria was concentrated with sucrose and PEG 6 000, respectively, and then subjected to SDS-PAGE. Take the pre-treated PVDF membrane and stick it to PAGE gel, transfer electrophoresis under the condition of constant voltage 100V for 1h, after blocking with 50g/L skimmed milk, add anti-ESAT-6 monoclonal antibody and anti-Ag85B polyclonal antibody, overnight at 4℃; after washing with PBST , add HRP-goat anti-mouse IgG, and place at 37°C for 1h; after washing with PBS, add substrate solution to develop color. An obvious target band can be seen at about 40kDa, and it is expressed in both the supernatant and the precipitate of the cell lysate, as shown in Figure 3. The identification results showed that the recombinant Mycobacterium smegmatis strain that could secrete and express the fusion protein of Mycobacterium tuberculosis Ag85B and ESAT-6 was constructed successfully. Its deposit number is: CCTCC M2010097.

AE-MS immune response level detection:

Grouping and immunization of experimental animals: 60 C57BL/6 mice were randomly divided into 3 groups (20 in each group), and were subcutaneously inoculated with AE-MS strains, common MS and normal saline respectively. The doses of AE-MS and MS immunizations were equal It is 10 ⁷ CFU/only.

1. Antibody level of AE-MS immunized mice

The results of ELISA detection of specific antibodies in the sera of immunized mice in each group showed that antibodies could be detected two weeks after AE-MS immunization, and the antibody level gradually increased after booster immunization, reaching a maximum of 1:6 4 weeks after the initial immunization 400, exceeding the antibody levels of the BCG group and the MS immune group, and comparable to the antibody level of the recombinant Ag85B and ESAT-6 fusion protein immune group. The results of antibody subclass analysis showed that the ratio of IgG2a/IgG1 in the AE-MS immunization group was significantly increased to 2.1, which was significantly higher than that in the MS immunization group and normal saline control group (P<0.01). the

2. Cellular immunoassay of AE-MS immunized mice

(1) Peripheral blood CD4+/CD8+ cell ratio detection:

From the 1st week to the 6th week after immunization, 0.5 ml of blood was collected from the tail every week, anticoagulated with heparin, 3 mice/group. The erythrocytes were lysed with erythrocyte lysate for 6 minutes, centrifuged at 2 000 rpm for 5 minutes, washed 3 times with PBSD cell washing solution (3ml/time/tube). The cell pellet was resuspended in serum-free RPMI 1640 medium, and stained with FITC: CD4/RPE: CD8 double labeling in the dark for 40 min. Wash the cells 3 times as above, add 0.5ml/tube of cell fixative to be tested. The cell samples were detected by flow cytometry, and the results are shown in Table 1. As shown in Figure 4, the results of analyzing the ratio of CD4+/CD8+ cells in each group showed that the ratio of CD4+/CD8+ cells in each group tended to increase with the prolongation of immunization time. AE-MS experiment Group showed a significant increase trend (NS: normal saline immunization; AE: AE protein immunization; BCG: BCG immunity; MS: Mycobacterium smegmatis immunity). the

(2) Spleen lymphocyte killing effect:

Six weeks after immunization, immunize mice in each group, separate spleen lymphocytes, and adjust the concentration of isolated lymphocytes to 2×10 ⁷ /ml and 1×10 ⁷ /ml with RPMI 1640 complete culture medium containing 10% FCS, respectively. ml, 5×10 ⁶ /ml and 2.5×10 ⁶ /ml, add 100 μl/well into a 96-well plate, adjust the concentration of target cell P815-AE to 4×10 ⁵ /ml, add 50 μl/well, 37°C 5% CO ₂ After culturing for 5 hours, centrifuge at 1 000 rpm for 5 minutes, take 50 μl of supernatant from each well, add 50 μl/well of LDH analysis solution, and stop the reaction after 30 minutes of action. 490nm visible light detection OD value. The results are shown in Table 2, as shown in Figure 5, when the ratio of effector cells to target cells was 1:50, the killing effect of CTL in the immune group was the highest (P<0.01).

(3) Analyze the frequency of IFN-γ-producing cells: 6 weeks after immunization, immunize mice in each group, separate spleen lymphocytes, and adjust the concentration of isolated lymphocytes to 2.5 with RPMI 1640 complete culture medium containing 10% FCS. ×10 ⁶ /ml, stimulated with the corresponding antigen, and detected the frequency of IFN-γ producing cells by ELISPOT. As shown in Table 3 and Figure 6, the frequency of IFN-γ-producing cells in mice in the AE-MS immunization group was significantly higher than that in the control group (P<0.01), indicating that AE-MS immunization can induce an increase in the level of IFN-γ in mice.

sequence listing

<110> Fourth Military Medical University of Chinese People's Liberation Army

<120> Recombinant Mycobacterium smegmatis strain capable of expressing Mycobacterium tuberculosis Ag85B and ESAT-6 fusion protein and its application

<160>1

<210>1

<211>1221

<212>DNA

<213>Artificial

<220>

<223> contains the genes encoding Ag85B and ESAT-6 proteins. A 48bp Linker sequence is inserted between the two genes through primer design to facilitate the correct folding of each domain of the fusion protein.

<220>

<221> CDS

<222>(7)...(876)

<223>Ag85B protein

<220>

<221> CDS

<222>(931)...(1215)

<223> ESAT-6 protein

<400>1

ggatccatga ccgcgggcgc gttctcccgg ccggggctgc cggtcgagta cctgcaggtg 60

ccgtcgccgt cgatgggccg cgacatcaag gttcagttcc agagcggtgg gaacaactca 120

cctgcggttt atctgctcga cggccagcgc gcccaagacg actacaacgg ctgggattc 180

aacacccccgg cgttcgagtg gtactaccag tcgggactgt cgatagtcat gccggtcggc 240

gggcagtcca gcttctacag cgactggtac agcccggcct gcggtaaggc tggctgccag 300

acttacaagt gggaaacctt cctgaccagc gagctgccgc aatggttgtc cgccaaacagg 360

gccgtgaagc ccaccggcag cgctgcaatc ggcttgtcga tggccggctc gtcggcaatg 420

atcttggccg cctaccaccc ccagcagttc atctacgccg gctcgctgtc ggccctgctg 480

gacccctctc aggggatggg gcctagcctg atcggcctcg cgatgggtga cgccggcggt 540

tacaaggccg cagacatgtg gggtccctcg agtgacccgg catgggagcg caacgaccct 600

acgcagcaga tccccaagct ggtcgcaaac aacacccggc tatgggttta ttgcgggaac 660

ggcaccccga acgagttggg cggtgccaac atacccgccg agttcttgga gaacttcgtt 720

cgtagcagca acctgaagtt ccaggatgcg tacaacgccg cgggcgggca caacgccgtg 780

ttcaacttcc cgcccaacgg cacgcacagc tgggagtact ggggcgctca gctcaacgcc 840

atgaagggtg acctgcagag ttcgttaggc gccggcatcg atggtggctc aggtggctcc 900

ggtggaggcg gaagcggcgg tggaggatca acagagcagc agtggaattt cgcgggtatc 960

gaggccgcgg caagcgcaat ccagggaaat gtcacgtcca ttcattccct ccttgacgag 1020

gggaagcagt ccctgaccaa gctcgcagcg gcctggggcg gtagcggttc ggaggcgtac 1080

cagggtgtcc agcaaaaatg ggacgccacg gctaccgagc tgaacaacgc gctgcagaac 1140

ctggcgcgga cgatcagcga agccggtcag gcaatggctt cgaccgaagg caacgtcact 1200

gggatgttcg catagaagct t 1221

Claims (2)

1. the recombinant mycobacterium smegmatis strain (recombinant Mycobacterium smegmatis) that can express mycobacterium tuberculosis Ag 85 B and ESAT-6 fusion rotein abbreviates AE-MS as, and its deposit number is: CCTCC M2010097.

2. the recombinant mycobacterium smegmatis strain described in the claim 1 is used for preventing and treating the application of vaccine lungy and preparation in preparation.

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