大肠杆菌不耐热肠毒素B亚单位基因的原核表达

Prokaryotic Expression of the Escherichia coli Heat-labile Enterotoxin B Subunit Gene

大肠杆菌(Escherichia coli)不耐热肠毒素B亚单位(LTB)不仅是重要的黏膜免疫原,而且作为佐剂在黏膜免疫中起着重要的作用。试验采用PCR方法扩增了肠毒素性大肠杆菌的LTB基因,并将其克隆到pGEX-KG原核表达载体上,构建了重组质粒pKG-LTB,经酶切和测序鉴定后,转化到大肠杆菌BL21(DE3)进行IPTG诱导表达,经SDS-PAGE和Western-blot分析表明,克隆的LTB基因与谷胱甘肽转移酶(GST)基因获得了高效融合表达,表达的融合蛋白GST-LTB分子量约为35kDa,并具有免疫反应活性。这为进一步研究LTB蛋白黏膜免疫原性和黏膜佐剂的作用奠定了基础。

The Escherichia coli heat-labile enterotoxin B subunit （LTB） was one of the most powerful mucosal immunogens and mucosal adjuvants. In this study, the LTB gene was cloned from enterotoxigenlc E. coil and was fused with GST to construct prokaryotie expression plasmid pKG-LTB. After induction by IPTG, a high expression of fusion protein was ob- tained. SDS-PAGE analysis and Western blot showed that the fusion protein was 35kDa and the protein was specific to anti- CTB polyclonal antibody. The research laid a foundation for the further investigation of mucosal immunogenicity and mueosal adjuvant effect of protein LTB.