结核分枝杆菌ag85b-il.docVIP

结核分枝杆菌Ag85B/IL 【关键词】 Ag85B；IL 　　Prokaryotic expression, purification and identification of Mycobacterium tuberculosis Ag85B/IL2 fusion protein and detection of its biological activity 　　【Abstract】 AIM： To express Mycobacterium tuberculosis Ag85B/ IL2 fusion protein in E.coli, to purify and identify it, and to detect its biological activity. METHODS： By using genetic engineering techniques, we constructed a recombinant expression plasmid pGEXAg85B/IL2, and expressed Ag85B/IL2GST fusion protein in E.coli BL21 under the induction of IPTG. Moreover, we also studied the optimal expression conditions concerning IPTG concentration and induction time. After renatured by urea in a concentration gradient, and purified by GSTSepharose affinity chromatography and digested by thrombin, the Ag85B/IL2 fusion protein was further purified by anionexchange chromatography and RPHPLC and identified with Western blot. Its Nterminal amino acid sequence and IL2 bioactivity were measured as well. RESULTS： A novel fusion protein Ag85B/IL2GST was expressed in E.coli in a way of inclusion body,accounting for 30% of total lysate protein of bacteria. After purified by GSTSepharose affinity chromatography, anionexchange chromatography and RPHPLC, the desired Ag85B/IL2 fusion protein with purification degree of 98.32% was acquired and confirmed by Western blot. Its Nterminal amino acid sequence was identical to the anticipation, and its specific IL2 bioactivity was 2500 u/mg. CONCLUSION： Ag85B/IL2 fusion protein was successfully expressed in E.coli and purified. This results established a groundwork for the further researches of Ag85B/IL2 fusion protein in the immunotherapy of bladder tumor. 　　【Keywords】 Ag85B; interleukin2; prokaryotic expression; fusion protein; purification 　　【摘要】 目的：在大肠杆菌中表达结核分枝杆菌Ag85B/IL2融合蛋白，并对其进行纯化、鉴定和活性初步测定. 方法：将构建的pGEXAg85B/IL2重组菌BL21扩增后接种于LB培养基中，异丙基硫代半乳糖苷(IPTG)诱导重组融合蛋白的表达，筛选最适诱导剂浓度和最适诱导时间；梯度浓度尿素复性包涵体，经GSTSepharose亲和层析、凝血酶切、阴