大肠杆菌志贺毒素stxB融合基因的构建及其高效表达

Construction and High-level Expression of EHEC stxB Fusion Gene

利用 PCR方法 ,从肠出血性大肠杆菌 O15 7∶ H7的基因组 DNA中 ,分别扩增出 Stx1B和 Stx2 B亚基的结构基因片段 ,然后再通过 PCR将这 2个片段连接起来 ,并定向克隆到表达质粒 p ET2 8a的 Nco 和 Eco R 位点之间 ,构建了重组表达质粒 p MB1。将重组质粒转化到宿主菌 BL2 1(DE3)中 ,对重组菌株 BL2 1(p MB1)用 IPTG进行诱导表达 ,并对最佳诱导时间进行了探索。 SDS- PAGE电泳检测结果表明 ,重组菌株表达出了 16 30 0的目的蛋白 Stx2 B- L-Stx1B;经薄层扫描分析表明 ,表达量约占菌体总蛋白的 6 8.4 % ,且目的蛋白为包涵体表达 ,表达量约占细菌沉淀的84 .6 %。研究还表明 ,未用 IPTG诱导的 BL2 1(p MB1)菌株也可有少量目的蛋白的基础表达。不同诱导时间的结果表明 ,在 3～ 7h的诱导时间内 。

In this study,the structure sequences of Shiga toxin 2B(stx2B) and Shiga toxin 1B(stx1B) were respectively amplified from EHEC O157∶H7 by PCR.stx2B-L-stx1B fusion gene was constructed by a 12 bp linker ligating these two fragments.After digested with restriction endonuclease NcoⅠ and EcoRⅠ,the fusion gene was orientally inserted into expression vector pET28a.The resultant recombinant plasmid,terming pMB1,was transformed into E.coli BL21(DE3).IPTG was used to induce the expression of the target protein.After the harvested bacteria were dispersed with sonication,the inclusion bodies were extracted with centrifugation.SDS-PAGE was used to analyze the expression of fusion protein.The recombinant Stx2B-L-Stx1B fusion protein was high level expressed in inclusion bodies of E.coli and covered above 68.4% of total bacterial proteins.The recombinant Stx2B-L-Stx1B fusion protein can be employed in immunization against EHEC O157∶H7.