牛分枝杆菌Mb0950c的免疫特性及其血清学检测方法的建立

Immunogenicity evaluation of Mycobacterium bovis Mb0950c protein and establishment of serological diagnostic method for bovine tuberculosis detection

【目的】利用原核表达系统对牛分枝杆菌Mb0950c蛋白进行表达和纯化,通过小鼠模型评价其免疫原性,建立血清学间接ELISA方法用于牛结核病的临床检测。【方法】构建pET32a-Mb0950c原核表达质粒,并转化至BL21(DE3)中诱导蛋白的表达,对蛋白进行纯化。使用流式细胞术(flow cytometry,FCM)、ELISA等对该蛋白在小鼠中的免疫原性进行分析。建立基于Mb0950c的间接ELISA方法,评价该方法的临床检测潜力。【结果】SDS-PAGE和Western blotting结果显示,成功获得了可溶性Mb0950c蛋白,且具有良好免疫反应性;FCM结果显示,Mb0950c蛋白上调了T细胞表面CD69分子的表达。细胞因子和抗体结果表明,该蛋白能够诱导特异性的IFN-γ和IL-4的分泌,同时能诱导机体分泌特异性的抗体,且以IgG1型为主。建立了ELISA检测方法应用于牛结核临床检测,结果显示,该方法与牛结核外周血IFN-γ外释放试验和皮试试验结果的阳性符合率、阴性符合率和总符合率分别为65.7%、97.9%和72.4%。【结论】在原核表达系统中可溶性表达Mb0950c蛋白,它在小鼠模型中诱导Th1和Th2型免疫应答,基于此蛋白建立了牛结核病血清学检测的间接ELISA方法。

[Objective] The purpose of this study is to express Mb0950 c protein by prokaryotic expression system, and to evaluate its immunogenicity by mouse model, further to establish a serological indirect enzyme-linked immunosorbent assay(ELISA) for clinical detection of bovine tuberculosis. [Methods] The prokaryotic expression plasmid of pET32a-Mb0950c was constructed and transformed into BL21(DE3) to induce protein expression. The immunogenicity of the protein in mice was analyzed by flow cytometry(FCM) and ELISA. And then an indirect ELISA method based on Mb0950c was established and used in clinical trial. [Results] SDS-PAGE and Western blotting showed that Mb0950 c protein was successfully obtained and had good immunogenicity. The analysis of FCM showed that Mb0950c protein upregulated the expression of CD69 on the surface of T cells. The results of ELISA showed that the protein could induce the secretion of IFN-g and IL-4, at the same time, it could induce the body to secrete specific antibodies, and it mainly belongs to IgG1. Total 192 serum samples were detected by indirect ELISA based on Mb0950 c and the results showed that the positive coincidence rate, negative coincidence rate and total coincidence rate were 65.7%, 97.9% and 72.4% respectively. [Conclusion] Mb0950 c protein is expressed in the prokaryotic expression system and it induces Th1-and Th2-type immune response in mouse models. Based on this protein, an indirect ELISA method for serological detection of bovine tuberculosis has been established.