牛型结核分枝杆菌ESAT-6原核表达载体的制备和蛋白纯化

The Construction of Prokaryotic Expression Vector of ESAT-6 from Mycobacterium Bovis and Protein Purification

本研究拟构建牛型结核分枝杆菌ESAT-6的原核表达载体,并纯化得到目的蛋白。将牛型结核分枝杆菌ESAT-6的PCR扩增产物连接到p NC-His E载体,构建ESAT6-p NC-His E重组质粒。将重组质粒转化至枯草芽孢杆菌,经PCR和限制性内切酶酶切鉴定为阳性;将该阳性枯草芽孢杆菌单菌落接种于培养基中,不需诱导即可分泌表达,离心取上清,并对上清进行纯化、检测,得到目的蛋白。本研究提供一种可快速、简便地获得大量枯草芽孢杆菌分泌性表达ESAT-6蛋白的方法。

The research aims to construct a prokaryotic expression vector of ESAT-6 from mycobacterium bovis and get the purified target protein. The PCR products of ESAT-6 were cloned into vector pNC-HisE, and the recombinant plasmid of ESAT6-pNC-HisE was con- structed. The recombinant plasmid was transformed into bacillus subtilis, the PCR result and restriction enzyme identification show that the recombinant plasmid of ESAT6-pNC-HisE is positive. The transformed cells were grown in proper medium and can perform the see- retory expression without induction. The target protein can be taken from supernatant by centfifugation, purification and detection. This research can provide a quick and simple mothed which can perform the high secretory expression of ESAT-6 by bacillus subtilis expression system.