Stx1B基因克隆、表达及重组蛋白的纯化

目的构建志贺样毒素1B亚单位(Stx1B)的原核表达载体,获取高纯度的Stx1B重组蛋白。方法用PCR法扩增Stx1B,通过基因重组技术克隆入原核表达载体pGEX4T-2,将该重组质粒转化E.coli BL21,IPTG诱导表达,Glutathione Sepharose 4B亲和层析柱纯化重组蛋白,进行SDS-PAGE电泳分析及Westernblot检测。结果重组质粒经诱导后表达分子量为34kD的重组蛋白,Western blot检测显示特异性条带,亲和层析柱纯化后得到重组蛋白的纯度在90%左右。结论成功构建了Stx1B重组质粒,表达并纯化出高纯度重组蛋白,为进一步的生物性质研究奠定了基础。

Cloning and expression of the plasmid vector for Stx1B subunit gene and purification of its recombinant protein

In order to construct the prokaryotic expression plasmid of stxlB gene, to obtain the recombinant protein with high purity, the stx1B gene was amplified by PCR and was further cloned into the vector pGEX4T-2. The reconstructed plasmid was transformed into E. coli BL21. Induced by IPTG, the recombinant protein was analysised by SDS-PAGE, Western blotting and purified by an affinity column. The mass molecular relative （Mr） of the expressed products was found to be 34kD. And the expression of Stx1B was confirmed by Western blot assay. The purity of the fusion protein was about 90% after purification by affinity chromatography. In this study, we have successfully constructed the prokaryotic expression system for stx1B gene and the recombinant protein StxlB was purified.