结核分枝杆菌Rv1886c的原核表达及其免疫生物学特性

Prokaryotic expression and immunological characteristics of Mycobacterium tuberculosis Rv1886c

【目的】Rv1886c基因编码的Ag85B是结核分枝杆菌(Mycobacterium tuberculosis,M.tb)感染早期的分泌蛋白,本研究对其所诱导的免疫应答特性进行了探索。【方法】对Ag85B进行原核表达和鉴定,并通过夹心ELISA、间接ELISA及ELISPOT方法测定其诱导的细胞免疫和体液免疫应答水平。【结果】SDS-PAGE及Western blot鉴定结果表明,以包涵体形式表达的Ag85B蛋白,经变性、复性后能与结核病人的抗血清及免疫重组李斯特菌LM-Ag85B的小鼠抗血清发生特异性反应,表明His-Ag85B融合蛋白具有较好的免疫活性。将纯化的Ag85B蛋白皮下免疫C57BL/6小鼠,夹心ELISA的测定结果表明,Ag85B蛋白免疫组诱导小鼠产生的特异性IFN-γ水平显著高于IL-4的水平(P<0.001),呈现Th1型细胞免疫应答趋势;以结核菌素PPD作为包被抗原,通过间接ELISA测定的血清抗体效价达到1∶6400,表明Ag85B也能诱导有效的体液免疫应答。此外,以尾静脉途径初次免疫小鼠42天时,ELISPOT测定结果显示,结核分枝杆菌H37Rv诱导小鼠产生Ag85B240-259特异性的IFN-γ水平极显著高于卡介苗(BCG)免疫组(P<0.001)。【结论】Ag85B蛋白能激发小鼠产生较强的Th1型细胞免疫应答和较好的体液免疫应答;BCG单次免疫后诱导小鼠产生的Ag85B特异的细胞免疫应答水平较低。本研究为揭示结核分枝杆菌的致病机理、新型疫苗的研制和早期诊断试剂的开发奠定了基础。

［Objective］Ag85B （Rv1886c） is secreted during the early stages of infection by Mycobacterium tuberculosis．The purpose of this study was probed into the immune response against Ag85B in vivo．［Methods］Ag85B was prokaryotic expressed and identified，its immunological characteristics were evaluated with indirect-ELSIA，Sandwich-ELISA and enzyme-linked immunospot assay （ELISPOT）．［Results］ Ag85B was mainly expressed in form of inclusion body confirmed by SDS-PAGE． Western blot analysis shows that the fusion protein had good specific reaction with serum of tuberculosis patient and serum of mice immunized with LM-Ag85B．C57BL/6 mice were subcutaneously immunized with Ag85B，the production of IFN-γ and IL-4 in the spleen cells was determined by Sandwich ELISA，the level of IFN-γ was significantly higher than that of IL-4（P＜0.001） in the Ag85B immunization group，it indicated the protein induced Th1-tendency immune responses．Furthermore，purified protein derivative （PPD） used as coating antigen，antibody titer against Ag85B in murine serum reached 1：6400，it was demonstrated that Ag85B could also induce humoral immune responses． Additionally，C57BL /6 mice were intravenously immunized with M．Tb H37Rv and bacillus Calmette-Guérin （BCG） respectively for 42 days，M．Tb H37Rv group intended to induce Ag85B specific Th1 type immune response，and its ability of eliciting cellular immunity was significantly stronger than BCG group（P＜0.001）．［Conclusion］Ag85B can affectively induce strongly Th1-tendency immune response and humoral response．Whereas，BCG prime vaccination only can elicit low levels of Ag85B240－259 specific immune response．The study laid foundation for probing the pathogenic mechanism，the development of novel vaccine and the establishment of clinical diagnostic method．