结核分枝杆菌毒素蛋白higB的原核表达及分析

Prokaryotic Expression and Analysis of Toxin higB of Mycobacterium Tuberculosis

目的克隆编码结核分枝杆菌毒素基因higB并在大肠杆菌中表达。方法利用PCR法从结核分枝杆菌H37Rv基因组扩增higB基因;构建重组表达质粒pET-32a(+)-higB;筛选出阳性重组质粒后转化大肠杆菌,诱导目的蛋白表达;利用SDS-PAGE与Western-blot鉴定目的蛋白的表达。结果成功扩增出了higB基因,克隆于载体pET-32a(+)质粒中,PCR筛选和酶切鉴定获得阳性克隆,测序证实正确;转化大肠杆菌BL21后higB蛋白没有发生表达。结论已成功构建结核分枝杆菌毒素基因higB的原核表达栽体,但大肠杆菌BL21不能表达出higB蛋白。

Objective To clone higB gene from genome of Mycobacterium tuberculosis and observe the ex-pression of higB protein in E .coli BL21.Methods HigB gene was obtained by PCR from the genome of Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pET-32a（＋） to construct recombinant plasmid pET-32a（＋）-higB.The positive recombinant plasmid was transformed into E .coli BL21 for expression of higB protein under IPTG induction .Protein higB was identifed by SDS-PAGE and Western blot .Results HigB gene was successfully obtained and positive recombinant plasmid pET-32a（＋）-higB were identified by PCR ,restriction enzyme digestion anal-ysis and DNA sequencing .After induction with IPTG ,higB protein was not successfully expressed in E .coli BL21 .Con-clusion The recombinant expression plasmid pET-32a（＋）-higB was constructed successfully ,but higB protein was not expressed in E .coli BL21 .