结核分枝杆菌ESAT-6蛋白的表达与纯化

Expression and purification of ESAT-6 protein of Mycobacterium tuberculosis

目的构建结核分枝杆菌esat-6基因原核表达载体,使其在大肠杆菌中表达融合重组蛋白,并纯化。方法用PCR方法从结核分枝杆菌H37Rv基因组中扩增出esat-6基因片段,克隆至pMD18-T载体,PCR筛选阳性克隆并测序;用限制性内切酶消化后,目的片段亚克隆至表达载体pGEX-4T-2,构建pGEX-esat-6重组质粒,将其转化入大肠杆菌JM109;PCR和双酶切鉴定转化菌落;将阳性菌株经IPTG诱导,SDS-PAGE和免疫印迹分析靶蛋白的表达;用谷胱甘肽-琼脂糖亲合层析法纯化融合蛋白。结果PCR扩增出esat-6 288bp的基因,克隆到pMD18-T载体中,经测序与GenBank中序列一致;随后亚克隆到表达载体pGEX-4T-2构建重组表达质粒,在JM109中表达了ESAT-6融合蛋白,表达的蛋白能被GST免疫血清识别;通过亲和层析纯化获得的蛋白能被结核病人血清识别。结论成功构建esat-6重组表达质粒,该质粒在JM109中表达ESAT-6融合蛋白,并获得较纯的蛋白。

To construct a recombinant prokaryotic expression vector carrying esat-6 gene and to purify the fusion protein of ESAT-6 expressed in E. Coli JM109, The gene riagment of esat-6 was amplified by PCR from Mycobacterium tuberculosis H37Rv genomic DNA and cloned to pMD18-T vector. The positive clone was screened by PCR and seguenced, and was digested with BamH I and Sal I, then subcloned to pGEX-4T-2 digested with the same restrictive endonucleareases. The recombinant plasmid pGEX-esat6 was transformed into E. coli JM109. After induced with IPTG, the expressed recombinant protein was confirmed by SDS-PAGE and Western-blotting. The GST-ESAT-6 was purified from lysates with Glutathionse SepharoseTM 4B column. The experimentat re- stilts showed that the esat-6 fragment was amplified by PCR and was subcloned to pGEX-4T-2 successfullly. Recombinant fusion protein was expressed with the induction of IPTG and could be recognized by goat polyclonal antibodies to Schistosma japonicum GST and by sera of patients with tuberculosis. It was concluded that the recombinant expression plasmid pGEX-esat-6 was successfully constructed and the purified fusion protein was obtained from the induced E. coli JM109 lysates.