结核分枝杆菌重组Bb-ESAT-6疫苗的构建、鉴定和表达及其保护力

目的构建和鉴定结核分枝杆菌(MTB)重组双歧杆菌(Bifidobacteria bifidum,Bb)-ESAT-6疫苗,研究该疫苗对鼠结核杆菌感染的免疫保护效果。方法以结核分枝杆菌H37Rv标准株基因组DNA为模板,通过PCR扩增得到ESAT-6抗原编码基因;将该基因定向克隆到大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-ESAT-6;用电穿孔法将该质粒导入Bb及BL21(DE3),构建结核分枝杆菌重组Bb-ESAT-6疫苗。用IPTG诱导重组BL21(DE3)菌表达ES-AT-6/GST融合蛋白;SDS-PAGE及Western blot对表达产物进行鉴定。65只BALB/c雌性小鼠,随机分为5组:A组(SC)、B组(IM)、C组(IN)、D组(Bb)、E组(PBS)。分别于免疫后8周用5×105CFU MTB H37Ra减毒株悬浮于10μL PBS中进行鼻腔粘膜接种感染。攻击感染后6w剖杀各组小鼠8只,计数肝、肺组织荷菌量,常规ELISA检测血清IgG及其亚类和IgE,MTT法检测特异性脾淋巴细胞增殖。结果PCR成功扩增出288bp的ESAT-6基因;双酶切证实ESAT6基因成功插入pGEX-1λT中;PCR证实rBb-ESAT-6疫苗构建成功。免疫印迹分析发现重组质粒pGEX-ESAT-6的表达产物在相对分子量(35kDa)处有明显的蛋白表达条带,表达效率为20%,且能被活动性结核病人血清特异识别。鼻腔内接种组的肝、肺组织荷菌量明显低于其他免疫组。疫苗接种组IgG、IgG1、IgG2a和IgG2b明显升高,IgG3和IgE无明显变化;疫苗接种组的脾淋巴细胞明显增殖。结论成功构建了结核分枝杆菌重组Bb-ESAT-6疫苗,重组Bb-ESAT-6疫苗在抗结核分枝杆菌感染过程中具有一定免疫保护作用。

Dynamic observations on the production of cytokines in spenocytes of BALB/c mice after immunization with recombinant Bifidobacterium bifidum-ESAT-6-MPT 64 vaccine of Mycobacterium tuberculosis

The changes in the production of cytokines in spenocytes of BALB/c mice after immunization with recombinant Bifidobacterium bifidum(Bb)-ESAT-6-MPT64 vaccine of Mycobacterium tuberculosis were dynamically observed in the present study,in which BALB/c mice were immunized with recombinant Bb-ESAT-6-MPT64 vaccine intranasally and subcutaneously each 4 mice were killed before and 2,4,6,8 and 10 weeks after immunization respectively,and the lymphocytes suspensions from spleen were prepared and cultivated in vitro with MTB-Ag and Con-A to induce the production of IL-12,IL-10 and IFN-γ,while,MTB and LPS were used to induce the production of TNF-α.ELISA assay was used to detect the presence of various cytokines in the lymphocyte suspensions.The experimental results showed that the levels of IFN-γ,IL-12,TNF-α and IL-10 in group of mice inoculated with vaccine rose up 2-10 weeks after immunization,reaching their peaks during 6-8 weeks.On the same time point,the cytokine levels induced by Con-A or LPS were comparatively higher than that induced by MTB-Ag(P<0.05 or 0.01).It is concluded that the mixed type of immne responses is induced at early course of immunization after inoculation with recombinant Bifidobacterium bifidum-ESAT-6-MPT64 vaccine in mice.