Meng Chuang, Wan Ting, Xu Zhengzhong, Shan Fa, Fan Feng, Chen Xiang, Jiao Xin'an
Wei Sheng Wu Xue Bao. 2015 Feb 4;55(2):220-6.
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.
The fragment of mpt83 gene was amplified and constructed into pET30a(+)-mpt83 recombinant plasmid. MPT83 fusion protein was purified with His affinity chromatography column from strain of BL21(DE3)-pET30a(+)-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.
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 C57BL/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.
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.
本研究旨在表达结核分枝杆菌MPT83蛋白，评估其在小鼠模型中的免疫原性以及对牛结核病的血清学诊断潜在价值。
扩增mpt83基因片段并构建到pET30a(+)-mpt83重组质粒中。用IPTG诱导BL21(DE3)-pET30a(+)-mpt83菌株后，通过His亲和层析柱纯化MPT83融合蛋白，然后用于评估其在小鼠中的免疫原性以及在ELISA检测牛结核病中的潜在应用。
SDS-PAGE和Western blot结果表明MPT83融合蛋白成功表达并具有良好的免疫反应性。流式细胞术（FCM）分析显示树突状细胞上CD80表达降低，脾脏CD4+和CD8+ T细胞上CD69表达上调。同时，在注射融合蛋白的C57BL/6小鼠中，ELISPOT检测发现分泌IL-4的特异性细胞斑点多于分泌IFN-γ的细胞斑点。以MPT83为抗原通过间接ELISA检测了200份血清样本，结果显示在牛结核病检测中与外周血特异性IFN-γ释放试验结果相比，阳性符合率为48.6%，阴性符合率为90%。
MPT83融合蛋白成功表达，能够在小鼠中引发Th2免疫反应，可作为血清学诊断抗原用于ELISA检测牛结核病。
