类志贺毒素Ⅱ型变异体A亚单位基因缺失株的构建及表达

Construction and expression of A subunit gene of Shiga-like toxin typeⅡ variant

采用PCR方法分别对类志贺氏菌毒素A亚单位基因第 2 98～ 80 5序列、818～ 12 0 1序列进行了扩增 ,使其第 80 6～ 817序列的碱基 (编码 16 7～ 170位氨基酸 )缺失 ,并将两片段连接后克隆至质粒载体pGEX 6P 1中 ,获得了含有重组质粒 pGEX Ade的重组大肠埃希氏菌 ;经SDS PAGE以及使用特异性抗SLT ⅡeA单克隆抗体的Western blotting ,证明该重组大肠埃希氏菌在IPTG诱导条件下可表达SLT ⅡeA。

The 298-805 bp and the 818-1 201 bp fragments of A subunit gene of Shiga-like toxin (typeⅡ) variant (SLT-Ⅱe A gene) were amplified by PCR, and the 806-817 bp fragment of the latter fragment was deleted by PCR. The PCR products were cloned into prokaryotic expression vector pGEX-6P-1 and the recombinant plasmid pGEX-A_(de) was constructed. The recombinant plasmid was transformed into E.coli BL21. SDS-PAGE and Western-blotting tests confirmed that the fusion protein named GST-ⅡeA had been expressed in E.coli BL21 from the SLT-ⅡeA gene.