增强型绿色荧光蛋白在DH5_大肠杆菌中的表达.pdfVIP

200 2010, Vol. 31, No. 21 食品科学 ※生物工程 增强型绿色荧光蛋白在DH5 α大肠杆菌中的表达 王晓辉 (中国农业大学食品科学与营养工程学院，北京 100083) 摘 要：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(E GFP) ，插入到pGEM-T easy 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-E GFP 重组载体的DH5 α 大肠杆菌较普通DH5 α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-E GFP 重组载体的DH5 α大肠杆菌，提取菌体总蛋白，SDS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。 关键词：增强型绿色荧光蛋白；p GEM-T easy ；DH 5 α大肠杆菌 Expression of Enhanced Green Fluorescent Protein in DH5 αE. coli WANG Xiao-hui (College of Food Science and Nutritional Engineering, China Agricultural University, Beij ing 100083, China) Abstract：To construct a recombinant plasmid (pGT-E GFP), the fragment of E GFP (enhanced green fluorescent protein) gene was amplified by PCR from the cloning vector pEGFPpA-CAN , and then in serted 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 DH5 αE. coli, the culture of DH5 αE. coli with pGT-E GFP was a little greener in color. Through fluorescence microscope, the morphological features of DH5 αE. coli with green fluorescence could be clearly ob served. Moreover, the extraction of total protein from the lysate of DH5 αE. coli with pGT-E GFP derived from ultrasonic treatment was done. The SDSanalysis showed that EGFP was a soluble protein, with a molecular weight of approximately 30 kD, which was identical to the theoretical size. Key words ：enhanced green fluorescent protein ；pGEM-T easy ；DH5 αE . coli 中图分类号：Q8 19 文献标识码：A 文章编号：1002-6630(20 10)2 1-0200-04 绿色荧光蛋白(green fluorescent protein ，GFP )是 达，扩大了G