【作者】 张海；

【导师】 徐志凯；

【作者基本信息】 第四军医大学 ， 病原生物学， 2005， 博士

【摘要】 结核病(Tuberculosis,TB)是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)所致以呼吸系统感染为主的慢性传染病,据WHO估计,目前全世界约有1/3人口感染MTB,每年有1000万新发患者和300万患者死亡。贫穷、人口增加、流动人口增多、耐药MTB增多及艾滋病流行等使TB问题雪上加霜。我国是全球22个TB高负担国家之一,现有MTB感染人数已达4亿,传染性TB患者达到200万。卡介苗(BCG)虽被广泛用于预防TB,但其效果仍不够满意。研究认为BCG可预防并减轻儿童的严重TB,但对成人TB的预防作用从0到80%不等。导致此现象的原因在于BCG菌株的变异使保护性抗原丢失、人群间遗传或营养的差异及环境等因素的影响。鉴于BCG自身的不足,以及当前TB流行的严重性,研究新的疫苗用于TB的预防已成为当前国内外研究热点。 MTB早期培养滤液蛋白(CFP)中的ESAT6和CFP10是重要的保护性抗原。本研究先后构建了融合表达ESAT6和CFP10蛋白的亚单位疫苗、基因疫苗以及重组耻垢分枝杆菌(mycobacterium smegmatis,M.S)疫苗,并比较了各自诱发的细胞免疫应答水平和对感染小鼠的保护力,更多还原

【Abstract】 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更多还原