结核分枝杆菌NrdF1、PE_PGRS35、Rv1985c和Rv1986的原核表达及检测牛结核病抗体之应用

目的 在大肠杆菌中表达结核分枝杆菌RD2区域的NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白，并评价诊断牛结核病中的应用潜能。方法 以结核分枝杆菌H37Rv基因组DNA为模板，PCR 扩增nrdF1，pe_pgrs35，rv1985c 和 rv1986基因，并将其克隆到表达载体中，重组表达质粒转化E.coli BL21(DE3)，IPTG诱导目的蛋白表达，镍柱亲和纯化重组蛋白，SDS-PAGE和Western blotting试验鉴定目的蛋白的表达及其反应原性。以纯化的重组蛋白作为包被抗原，通过ELISA方法测定牛血清中特异性抗体，以之评价这些蛋白用于牛结核病抗体检测的价值。结果 成功表达了NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白，相对分子质量为42 ku、63 ku、46 ku和41 ku，对表达产物进行镍柱亲和纯化，获得了纯度较高的融合蛋白。重组蛋白均能与抗HIS单抗发生特异反应，表现出良好的反应原性。通过间接ELISA方法检测牛血清中特异性抗体，阳性检出率分别为7.35%、22.06%、16.18% 和16.18%。结论 结核分枝杆菌原核表达的NrdF1、PE_PGRS35、Rv1985c和Rv1986蛋白具有用作牛结核病血清学诊断试剂的潜能。

Prokaryotic expression of NrdF1, PE_PGRS35, Rv1985c and Rv1986 of Mycobacterium tuberculosis and the potential application for the detection of antibodies in bovine tuberculosis

Proteins encoded by regions of difference （RD） of Mycobacterium tuberculosis （MTB） constitute a potential source of specific antigens for vaccine development and immunodiagnosis .In the present study ,four genes named nrdF1 , pe_pgrs35 ,rv1985c ,and rv1986 from RD2 of MTB were cloned and overexpressed in E .coli with the induction of IPTG .Western blotting assay showed that these recombinant fusion proteins could well react with anti-His tag monoclonal antibody ,which in-dicated their good immunoreactivity .The serodiagnosis potential applications of these four proteins in bovine tuberculosis were further evaluated .An indirect ELISA assay was established by using fusion proteins as coating antigens for detection of their specific antibodies in bovine sera .The positive rates were 7 .35% in NrdF1 ,22 .06% in PE_PGRS35 ,16 .18% in Rv1985c and 16 .18% in Rv1986 respectively .All the results suggest that these fusion proteins have the potential application in serodiagnosis of bovine tuberculosis .