结核分枝杆菌Ag85B/GST融合蛋白表达载体构建及在大肠杆菌中的表达

目的 构建结核分枝杆菌Ag85B／GSF融合蛋白表达质粒，并在大肠杆菌(E．coli)中诱导表达。方法 以质粒pUC18／Ag85B为模板，用PCR法扩增结核杆菌Ag85B基因，扩增的Ag85B上游含有EcoR Ⅰ酶切位点，下游含有SalⅠ酶切位点；将Ag85B基因定向插入质粒pGEX-4T-1中，构建原核表达质粒pGEX-Ag85B并转化E．coli B121，筛选阳性重组子，限制性内切酶切鉴定和DNA序列测定，异丙基硫代半乳糖苷(IPTG)诱导表达，SDS-PAGE和Westem blot鉴定结果。结果 成功构建原核表达质粒pGEX-Ag85B并表达出Mr约56000大小的Ag85B／GSY融合蛋白，表达量占总菌体的30％。结论 为进一步进行纯化Ag85B蛋白和研究其在膀胱肿瘤治疗中的作用奠定了基础。

Recombination of Mycobacterium tuberculosis Ag85B/GST fusion protein and its expressions in E.coli

Objective To construct Mycobacterium tuberculosis Ag85B/GST fusion protein expression vector and induce its expression in E.coli. Methods Ag85B encoding gene was cloned from the plasmid pUC18/Ag85B by PCR and inserted into expression plasmid pGEX-4T-1. The positive recombinants were identified by restriction endonuclease digestion and DNA sequencing, and expressed in E.coli induced by isopropyl-beta D-thiogalactopyranoside (IPTG). Results The desired Ag85B/GST fusion protein (about M r 56 000) was expressed and confirmed by SDS-PAGE and Western blot. Conclusion The results establish a groundwork for the further research of purifying Ag85B protein and evaluating its values in the immunotherapy of bladder tumor.