FimH_(1-156)融合基因的构建及其原核蛋白表达与纯化

Construction,prokaryotic expression and purification of FimH_(1-156) fusion protein

目的构建含有与溶酶体膜蛋白2(LAMP-2)P41-49完全同源序列的尿路致病性大肠杆菌(UPEC)Ⅰ型菌毛FimH1-156为目的基因的原核载体,表达并纯化FimH1-156融合蛋白。方法采用PCR克隆pPKL241质粒获取FimH1-156基因,插入原核表达载体pET28a(+);将重组表达质粒转染感受态E.coli BL21(DE3),经IPTG诱导表达FimH1-156融合蛋白,超声裂解细菌,NiNTA层析法进行纯化,聚丙烯酰胺凝胶电泳(SDS-PAGE)分析、蛋白免疫印迹法(Western blot)对其进行鉴定。结果成功构建表达载体,经对融合蛋白表达条件的优化,在IPTG浓度为1mmol/L,诱导4h目的蛋白表达量最高,主要以包涵体形式存在;Western blot证实该FimH1-156融合蛋白可与抗6×His单克隆抗体发生结合反应。结论成功克隆FimH1-156融合蛋白表达基因并构建至原核表达载体,获得了包涵体纯化的FimH1-156融合蛋白,为下一步建立实验动物模型奠定了基础。

Objective To construct and express a prokaryotic expression vector carrying the gene of FimH 1-156 that comprises human lysosome membrane protein 2 P41-49 gene ,and to express and purify the fusion protein .Methods FimH1-156 gene was cloned from plasmid pPKL241 by PCR ,and inserted into vector pET-28a（＋ ） to obtain prokaryotic expression plasmid pET-28a-FimH . After transforming Escherichia coli BL21（DE3） with pET-28a-FimH ,fusion protein FimH1-156 was expressed under induction .The target fusion protein was purified ,and its antigenicity was detected through Western blot .Results The expressed recombinant pro-tein was purified ,the expression of protein was the highest when IPTG was 1 mmol/L and 4h after induction ,it was expressed as include body form ,and the expressed protein was identified to react with monoclonal antibodies 6×His by Western blotting .Conclu-sion We cloned FimH1-156 fusion protein expressed genes successfully ,constructed prokaryotic expression vector ,and won the in-clusion body purification of FimH1-156 fusion protein .