增强型绿色荧光蛋白在DH5α大肠杆菌中的表达

Expression of Enhanced Green Fluorescent Protein in DH5α E.coli

采用PCR技术从pEGFPpA-CAN克隆载体中扩增出增强型绿色荧光蛋白基因(EGFP),插入到pGEM-Teasy载体中,构建pGT-EGFP重组质粒,其中的SP6启动子可用来表达EGFP蛋白。携带pGT-EGFP重组载体的DH5α大肠杆菌较普通DH5α大肠杆菌菌液略带淡绿色,在荧光显微镜下能够清晰观察该大肠杆菌的形态,带有绿色荧光。同时超声裂解携带pGT-EGFP重组载体的DH5α大肠杆菌,提取菌体总蛋白,SDS-PAGE显示EGFP蛋白是以可溶性蛋白形式存在的,分子质量大约为30kD,与其理论值大小一致。

To construct a recombinant plasmid（pGT-EGFP）,the fragment of EGFP（enhanced green fluorescent protein） gene was amplified by PCR from the cloning vector pEGFPpA-CAN,and then inserted into the pGEM-T easy vector.The expression of EGFP was regulated by the SP6 promoter in pGEM-T easy.Compared with the culture of DH5α E.coli,the culture of DH5α E.coli with pGT-EGFP was a little greener in color.Through fluorescence microscope,the morphological features of DH5α E.coli with green fluorescence could be clearly observed.Moreover,the extraction of total protein from the lysate of DH5α E.coli with pGT-EGFP derived from ultrasonic treatment was done.The SDS-PAGE analysis showed that EGFP was a soluble protein,with a molecular weight of approximately 30 kD,which was identical to the theoretical size.