结核分枝杆菌Ag85B-ESAT6融合蛋白在小鼠体内诱导的免疫应答及其保护力

Immunity responses and protective efficacy induced by Mycobacterium tuberculosis Ag85B-ESAT6 fusion protein in mice

目的 :研究Ag85B与ESAT6融合蛋白在小鼠体内诱导的体液和细胞免疫应答以及对MTB感染小鼠的保护力 .方法 :采用皮下包埋的方法 ,将预先转移到硝酸纤维素膜上的表达蛋白免疫小鼠 3次 ,每次间隔 2wk,用MTB培养上清滤液蛋白 (culturefiltrateproteins,CFP)作为抗原 ,ELASA测定免疫小鼠血清特异性抗体的滴度 .为了检测融合蛋白免疫小鼠引起的细胞免疫应答 ,最后一次免疫完成后 4wk ,取一部分免疫小鼠分离脾淋巴细胞 ,体外用抗原刺激 ,MTT法检测淋巴细胞刺激反应 ,ELISA检测悬液中IFN γ水平 .另一部分免疫的BALB/c小鼠经尾静脉感染MTB毒株H37Rv ,4wk后计数脾脏细菌负荷数 .结果 :Ag85B ESAT6蛋白免疫小鼠血清特异性抗体滴度为 1∶10 0 0 ,ESAT6 Ag85B蛋白免疫小鼠血清特异性抗体滴度为 1∶5 0 0 0 .融合蛋白Ag85B ESAT6和ESAT6 Ag85B免疫组淋巴细胞刺增殖指数分别为 2 .4 0± 0 .17和 2 .6 0± 0 .2 5 ,而生理盐水组只有 0 .90± 0 .2 1;相对应的IFN γ含量分别为 (3.5 1± 0 .30 ) μg/L和 (4 .0 5± 0 .4 1) μg/L ,显著高与生理盐水对照组 (0 .5 0± 0 .2 5 ) μg/L ,P <0 .0 5 ,但不及BCG免疫组 (5 .5 5±0 .31) μg/L .与生理盐水免疫组 (细菌负荷6 .31± 0 .13)相比较 ,融合蛋白免疫的BALB/c小鼠 。

AIM: To evaluate humoral and cell-mediated immune response by Ag85B and ESAT6 fusion proteins and to test their protective efficacy against Mycobacterium tuberculosis (MTB) challenge. METHODS: BALB/c mice were immunized three times at a 2-week interval subcutaneously on the mouse back respectively with fusion protein Ag85B-ESAT6 and ESAT6-Ag85B that were transferred to membrane beforehand. The spleen lymphocytes of the immunized mice stimulation index (SI) were measured by MTT method and the level of secreted IFN-γwhich upon antigen-specific stimulation was detected by ELISA. The fusion proteins vaccinated BALB/c mice were intravenously infected with 10 5 CFU MTB H37Rv. Four weeks later the numbers of CFU in spleens were determined. RESULTS: The titer of sera specific antibody in BALB/c mice immunized with fused expression protein Ag85B-ESAT6 was 1∶1000 and that of ESAT6-Ag85B was 1∶5000. The SI of fusion proteins immunized groups was significantly higher (which was 2.40±0.17 and 2.60±0.25 for Ag85B-ESAT6 and ESAT6-Ag85B immunized groups respectively) than that of saline immunized group (0.90±0.21). The IFN-γlevels in culture supernatant of spleen lymphocytes from the fusion proteins immunized mice were (3.51±0.30) μg/L and (4.05 ±0.41) μg/L for Ag85B-ESAT6 and ESAT6-Ag85B immunized groups respectively, significant different from those of saline immunized group (0.50±0.25) μg/L, P<0.05, which were lower than that of Bacillus CalmetteGuerin (BCG) immunized group (5.55±0.31) μg/L. Compared with the saline immunized mice(bacteria load was 6.31± 0.13) dramatic reductions of MTB replication were observed in the spleen (bacteria load was 5.04± 0.11 and 5.15± 0.29 respectively, P<0.05) of BALB/c mice immunized with fusion proteins following a subsequent challenge, but the protection efficacy of the mice immunized with Ag85B-ESAT6 or ESAT6-Ag85B was higher than that of BCG vaccination group. CONCLUSION: Ag85B and ESAT6 fusion protein can be used as novel components of the new TB vaccine.