大肠杆菌毒素stx2eB-LTB-ST融合基因的构建与表达

Construction and expression of stx2eB-LTB-ST fusion gene

利用PCR技术,从病原性大肠杆菌中扩增Stx2e及LT毒素B亚基的基因并扩增ST基因,用复式PCR技术获得了这三段基因的两种融合结构,一种是三段基因直接相连,另一种是在这三段基因之间加入适当的Linker序列。在这两种融合基因两端加入适当的酶切位点并分别与pMD18-T载体相连,酶切后以正确的阅读框插入pET28a载体的多克隆位点中,转入受体菌BL21(DE3)进行表达,得到两种约28 Ku的蛋白,表达的融合蛋白均以包涵体的方式存在,约占全菌蛋白表达量的30%,将包涵体初步提纯,为下游工作奠定了基础。

In order to produce a candidate vaccine against edema disease and diarrhea of swine, the stx2eB, LTB and ST genes of avain E. coil strain were linked directly or indirectly with linkers by overlapping PCR. The PCR products were cloned into pMDI 8-T and sequenced. Two fusion genes were subcloned into pET28a to produce plasmids pET28a：：stx2eB-LTB-ST and pET28a：：stx2eB-LTB-ST （L）. The recombinant plasmids were subsequently transformed into host strain BL21 （DE3） for protein expression. SDS-PAGE and thin layer scanning showed that the expressed fusion proteins existed mainly in the form of inclusion body and accounted for about 30 % total bacteria protein.