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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。
Objective To construct prokaryotic expression vector of Shiga toxin 1B subunit (Stx1B) and obtain high purity Stx1B recombinant protein. Methods Stx1B was amplified by PCR and cloned into prokaryotic expression vector pGEX4T-2 by gene recombination technique. The recombinant plasmid was transformed into E. coli BL21 and induced by IPTG. The recombinant protein was purified by Glutathione Sepharose 4B affinity chromatography and analyzed by SDS- PAGE electrophoresis analysis and Western blot detection. Results The recombinant plasmid was induced to express a recombinant protein with a molecular weight of 34 kD. Western blot showed that the purity of the recombinant protein was about 90% after purified by affinity chromatography. Conclusion The recombinant plasmid Stx1B was successfully constructed and expressed and purified highly purified recombinant protein, which laid the foundation for the further study of biological properties.