esat6基因表达载体的构建及其在戈登链球菌中的表达

为了构建结核分枝杆菌(MTb)esat6基因表达载体并在戈登链球菌GP251中进行分泌表达, 以结核杆菌H37Rv基因组DNA为模板扩增esat6基因, 将esat6基因TA克隆到pMD18-T, 构建pMD18-esat6重组载体。酶切消化pMD18-esat6, 将esat6基因亚克隆到质粒PSMB104, 生成PSMB104-esat6重组载体, 用于转化感受态戈登链球菌表达菌株GP251。用Tricine-SDS-PAGE和Western印迹检测esat6蛋白的表达, 并用ELISA技术检测该蛋白

Construction of the Recombinant Plasmid with esat6 Gene and Its Expression in Streptococcus gordonii

To construct expressing vector carrying esat6 gene and express this protein in Streptococcus gordonii GP251. esat6 gene was amplified by PCR with specific primer from genome of Mycobacterium tuberculosis (MTB)H37Rv. Inserted esat6 into the pMD18-T vector by T/A clone to get recombinant vector pMD18-esat6. Then digested pMD18-esat6 with restriction enzyme, esat6 was cloned to vector PSMB104 and expressed in Streptococcus gordonii GP251. The expression of esat6 protein was detected by Tricine-SDS-PAGE and Western-blot, ELISA technique was also used to detect its secretory volume. Restriction endonuclease, PCR, Tric ine-SDS-PAGE and Western-blot confirmed that esat6 gene was cloned into expressing vector successfully, and a 10 kD protein secreted in Streptococcus gordonii GP251, this protein has a good immunogenicity. The expression vector of esat6 gene was constructed, and esat6 protein expressed in Streptococcus gordonii1 successfully, it will be benefit for future study.