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【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 encoded by Rv1886c gene is an early secretory protein of Mycobacterium tuberculosis (M.tb) infection. In this study, the immune response characteristics induced by Mycobacterium tuberculosis (M.tb) were explored. 【Method】 Prokaryotic expression and identification of Ag85B were performed. The cellular and humoral immune responses induced by Ag85B were determined by sandwich ELISA, indirect ELISA and ELISPOT. [Results] The results of SDS-PAGE and Western blot showed that the Ag85B protein expressed in inclusion bodies could be denatured and refolded to react with the antiserum of the tuberculosis patients and the mouse antiserum of the immunologically recombinant Listeria monocytogenes LM-Ag85B Specific reaction, indicating that His-Ag85B fusion protein has good immunocompetence. The purified Ag85B protein was immunized subcutaneously in C57BL / 6 mice. The results of sandwich ELISA showed that the specific IFN-γ level induced by Ag85B protein immunization group was significantly higher than that of IL-4 (P <0.001) Th1-type cell immune response trend; tuberculin PPD as coating antigen by indirect ELISA serum antibody titer reached 1: 6400, indicating that Ag85B can induce a valid humoral immune response. In addition, ELISPOT assay showed that the level of IFN-γ specific to Ag85B240-259 induced by M. tuberculosis H37Rv was significantly higher than that of BCG immunized mice (P < 0.001). 【Conclusion】 Ag85B protein can stimulate mice to produce stronger Th1-type cellular immune response and better humoral immune response. The Ag85B-specific cellular immune response induced by BCG was lower after BCG immunization. This study laid the foundation for revealing the pathogenesis of Mycobacterium tuberculosis, the development of new vaccines and the development of early diagnostic reagents.