结核分枝杆菌MPT83的免疫原性及其奶牛结核病血清学检测方法的建立

Immunogenicity evaluation of Mycobacterium tuberculosis MPT83 protein and establishment of serological diagnostic method for bovine tuberculosis detection

【目的】利用大肠杆菌系统表达并纯化结核分枝杆菌MPT83蛋白,通过小鼠模型评价其免疫原性,建立血清学间接ELISA方法用于牛结核病临床检测,评价其应用潜能。【方法】构建p ET30a(+)-mpt83重组质粒,转化BL21(DE3)诱导表达并纯化,经细胞表面分子的流式细胞术(Flow Cytometry,FCM)分析、ELISPOT试验等分析其在小鼠中的免疫原性,建立间接ELISA方法,检测临床奶牛血样,评价其用于牛结核病血清学检测的潜能。【结果】SDS-PAGE显示目的蛋白成功表达,Western blot证实其对兔抗H37Rv多抗血清具有良好免疫反应性;FCM结果显示其下调树突状细胞表面CD80分子的表达,上调小鼠脾脏CD4+和CD8+T细胞表面CD69的表达,ELISPOT结果表明其诱导的特异性IL-4分泌细胞数显著高于IFN-γ分泌细胞数,表现为Th2型免疫应答;建立了ELISA方法,检测临床奶牛血样200份,与牛结核外周血γ-干扰素体外释放试验结果的阳性符合率和阴性符合率分别为48.6%和90%。【结论】在大肠杆菌系统中高效可溶性表达MPT83蛋白,其在小鼠模型中主要呈现Th2型免疫应答,并以该蛋白为抗原建立了牛结核病血清学检测的间接ELISA方法。

[Objective] The aim of this study was to express Mycobacterium tuberculosis MPT83 protein and to evaluate its immunogenicity in murine model as well as the serological diagnosis potential value for bovine tuberculosis. [Methods]The fragment of mpt83 gene was amplified and constructed into p ET30a（ ＋）-mpt83 recombinant plasmid. MPT83 fusion protein was purified with His affinity chromatography column from strain of BL21（ DE3）-p ET30a（ ＋）-mpt83 after induced by IPTG,and then used to evaluate its immunogenicity in mice and the potential application in ELISA assay for the detection of bovine tuberculosis. [Results] SDS-PAGE and Western blot results show that MPT83 fusion protein was expressed successfully and possessed a good immunological reactivity. Flow cytometry（ FCM） analysis displayed decreased expression of CD80 on dendritic cells and up-regulation of CD69 expression on both splenic CD4＋and CD8＋T cells. Meanwhile,more IL-4 specific secreting cell spots rather than those of IFN-γ were detected by ELISPOT assay in C57 BL /6 mice injected with the fusion protein. Total 200 serum samples were detected by indirect ELISA based on MPT83 as antigen and the results showed 48. 6% positive coincidence rate and 90% negative’s compared to results of peripheral blood specific IFN-γ release assay in bovine tuberculosis detection. [Conclusions] MPT83 fusion protein was expressed successfully with capability of eliciting Th2 immune response in mice and could be used for ELISA assay to detect bovine tuberculosis as a serological diagnosis antigen.