双歧杆菌表达肠致病性大肠埃希菌EspA蛋白的体外研究

目的构建肠致病性大肠埃希菌(Enteropathogenic E.coli,EPEC)EspA蛋白双歧杆菌表达重组菌株,并验证蛋白表达。方法采用常规PCR扩增EspA片段,体外构建重组质粒pBAD-EspA,转化大肠杆菌BL21感受态细胞培养,挑取单克隆提取质粒,双酶切和PCR对重组质粒鉴定。pBAD-EspA电转化长双歧杆菌感受态细胞,挑取阳性克隆并PCR鉴定,经木聚糖诱导表达,蛋白质印迹试验(Western blot)分析验证EspA蛋白在双歧杆菌中的表达。结果经酶切后琼脂糖凝胶电泳及测序结果证明pBAD-EspA重组质粒构建成功。pBAD-EspA重组质粒转化双歧杆菌后经阿拉伯糖诱导,在诱导表达不同时间点菌液沉淀均有良好的蛋白表达。结论体外成功构建表达EPEC EspA蛋白的长双歧杆菌,为制备ESPA口服疫苗提供科学依据。

Research on the in vitro expression of Enteropathogenic E. coli EspA protein in Bifidobacteria

Objective To construct the expression vector of Enteropathogenic E.coli（EPEC）EspA gene and identify the induced expression of EspA protein in Bifidobacteria. Methods The EspA gene fragment was amplified and the recombinant plasmid pBAD-EspA was constructed in vitro, then were transformed it the BL21 competent bacteria. The positive bacteria cells was picked and verified by PCR. The expression of EspA protein in Bifidobacteria was induced by arabinose in vitro and detected by western blotting. Results The recombinant plasmid pBAD-EspA was successfully constructed and demonstrated by agarose gel electrophoresis and DNA sequencing. The expression of EspA protein in Bifidobacteria induced by arabinose was demonstrated by western blotting in vitro. Conclusion The recombinant Bifidobacteria expressing EspA protein was constructed in vitro.