Construction Tuberculosis Vaccine Fused Expression ESAT6-CFP10 And Its Immunological Properties

Tuberculosis(TB) is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis(MTB). It is estimated that one-third of the world population are infected with MTB, causing over 10 million new TB cases and 3 million deaths annually. In China, the number of TB patients was the second in the world, approximately 400 million people infected, over 5 million got sick. BCG, the only available vaccine against TB, has been extensively evaluated and demonstrated a variable protective efficacy ranging from 0 to 80% in different field trials. Furthermore, due to following issues, such as the problem of TB multidrug-resistant (MDR) strains, co-infection with HIV, and increasing mobility of population, the word-wide situation of TB was deteriorating, which has created an urgent need for new vaccines to prevent TB .ESAT6 and CFP10 are both important protective antigen in the early culture filtrate protein(CFP) of MTB, and they were used in the diagnosis and vaccine widely of TB. In this study we had compared the levels ofcell-mediated immune responses and protective efficacy by recombinant vaccines fused expression ESAT6 and CFP10 inducing by the subunit vaccine, gene vaccine and recombinant mycobacterium smegmatis vaccine, and in order to search for a new effective TB vaccine .1. Expression and purification of ESAT6-CFP10 fusion proteinIn this study, cfp10 gene were amplified by polymerase chain reaction(PCR) from genome of MTB H37Rv strain, and inserted into cloning vector pGEM-7zf(+) for sequencing purpose with esat6. The genetic sequence of CFP10 were identical with that of Genbank reported, then digested by restriction endonuclease and cloned into expression vector pProEX HTb. The recombinant pPRO-e6c10 were transformed into E.coli DH5 a , induced with IPTG, expressed fusion protein of ESAT6 and CFP10 with relative molecular mass (Mr) of 28 kD were confirmed by western blot analysis with mouse-specific monoclonal antibody against 6 X His. Fused expression proteins were purified by Ni-NTA purification system. BALB/c mice were inoculated subcutaneously three times at 2 week interval by the purified recombinant ESAT6-CFP10 fusion protein, and the antibody titer of the immunized mice is 1:6400 by the ELISA method.2. Establishment of the stable P815 cell line expressed ESAT6-CFP10 fusionIn order to assess the level of the cell-mediated immune responseinduced by ESAT6-CFP10 fusion protein, we establish the stable expression cell line which can express fusion protein in P815 cell. esat6 and cfp10 gene were cloned into the eukaryotic expression vector pcDNA3.1(+), this recombinant plasimd was transfected into P815 cells(H-2d) by citation lipids whose genetic was identified with BALB/c. We got 11 strains positive cell clones by G418 selection. The specific mRNA of the fused protein was detected by RT-PCR, and the fused protein was expressed in the P815 cellplasim by indirect immunofluorescence technique (IFT).3. Study of immune characterization of the recombinant vaccineTo construct the recombinant mycobacterium smegmatis, esat6 and cfplO were cloned into shuttle plasmid pDE22 by electroporation by hygromycin resistance screening and PCR, recombinant mycobacterium smegmatis positive strains were identified. The fusion protein ESAT6-CFP10 could be secreted into supernatants of recombinant mycobacterium smegmatis by SDS-PAGE and Western-blot analysis.In order to assess the immune characterization of the recombinant vaccine, ESAT6-CFP10 fusion protein subunit vaccine, gene vaccine and recombinant vaccine were inoculated the BALB/c mice. The SI and the level of IFN- y and IL-2 stimulated by antigen-specific were detected by MTT method and indirect ELISA. Furthermore, the CTL specific lysis effect was measured by LDH method. The SI of the subunit vaccine group is 1.9, the gene vaccine group is 2.4, and the recombinant M.S is 2.8, the SI of these recombinant is lower than BCG(3.4) . IFN- Y concentration in cultured supernatant of spleen lymphocytes from mice immunized with subunit vaccine was 1721±19pg/mL, and the recombinant M.S is 2230+llpg/mL, the level of IFN- y of two recombinant vaccine is lower than BCG immunized group(2531±16pg/mL), but the gene vaccine was 2446 + 13pg/mL, which was the same as BCG immunized group. IL-2 concentration in cultured supernatant of spleen lymphocytes from mice immunized with recombinant vaccine were 211±llpg/mL, 196±16pg/mL and 221 ± 17pg/mL, respectively, significant greater than that of control group, but lower than that of BCG immunized group(295 ± 17pg/mL). The specific lysis...