致病性大肠杆菌绿色荧光蛋白标记

Enteropathogenic Escherichia coli(EPEC) marked with enhanced green fluorescent protein(EGFP)

目的:用增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标记致病性大肠杆菌(enteropathogenic escherichia coli,EPEC),以方便研究细菌的黏附。方法:以真核表达载体p EGFP-C1为模板设计引物,采用PCR方法扩增EGFP基因并测序后,克隆入原核表达质粒载体p Trc99a,转染EPEC,用荧光显微镜观察IPTG诱导后EPEC对Hep-2细胞的黏附及荧光表达情况。结果:成功构建原核表达质粒p Trc99a-EGFP和菌株EPEC/EGFP,黏附到Hep-2细胞表面的细菌克隆能够表达绿色荧光蛋白。结论:对EPEC进行了EGFP标记,为计数细菌的黏附提供方便。

Objective： To mark Enteropathogenic Escherichia coli（ EPEC） with Enhanced Green Fluorescent Protein（ EGFP）in order to study the adherence of EPEC conveniently. Methods： EGFP gene was amplified with PCR from the plasmid p EGFP-C1 template and subcloned into p Trc99 a,the recombinant plasmid p Trc99a-EGFP was transformed into EPEC,the adherence of EPEC with p Trc99a-EGFP to Hep-2 cells after the induction of IPTG was observed under fluorescence microscopy. Results： Plasmid p Trc99a-EGFP was constructed successfully,the adherence to Hep-2 cells of EPEC was observed under fluorescence microscopy after inducing with IPTG. Conclusion： EPEC is marked with fluorescence,which lays foundation for the study of the adherence of EPEC.