绿色荧光蛋白表达载体的改造和表达  

Construction and expression of expression vector pEGP-N1-MSC

作　　者：潘求真[1] 徐曙光[1] 李佳[1] 张宝路[1] 田亮[1] 李海[1] 韩红兵[1] 连正兴[1] 杨宁[1] 

机构地区：[1]中国农业大学动物科技学院

出　　处：《黑龙江畜牧兽医》2007年第1期13-15,共3页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金资助项目(30371036)

摘　　要：为获得增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的真核表达载体pEG-FP-N1-MSC,并观察其在猪成纤维细胞中的表达情况,用NheⅠ和AgeⅠ消除pEGFP-N1质粒上的多克隆位点(MSC),然后补平连接,获得增强型绿色荧光蛋白编码基因的真核表达载体pEGFP-N1-MSC质粒,将pEGFP-N1-MSC质粒转化DH5α感受态细胞,于Kanr+LB平板上筛选阳性克隆。重组子经NheⅠ和AgeⅠ双酶切鉴定,将该载体转染猪成纤维细胞,24 h后观察EGFP表达情况,48 h后添加G418进行筛选。结果表明:改造的pEGFP-N1-MSC真核表达载体,成功转染猪成纤维细胞,在倒置荧光显微镜下呈现绿色光,获得可产生绿色荧光的pEGFP-N1-MSC载体,为构建猪双正选择同源重组载体奠定了基础。To get the eukaryotic expression vector pEGFP - N1 - MSC and to detect transient expression of enhanced green fluorescent protein （ EGFP ） in pigs fibroblast cells. The pEGFP - N1 was digested by Nhe Ⅰ/Age Ⅰ to delect MSC, and then it was transformed in to DH5α, a recombinant pEGFP - N1 - MSC was gotten and confirmed by Nhe Ⅰ/Age Ⅰ digestion. It＇s transient expression was observed in 24 h after it was transfected into pig＇s fibroblast cells, and it was selected by G418 after 48 h. The pEGFP - N1 - MSC was successfully reconstructed and GFP showing green fluorescence could be observed in pigs fibroblast cells under fluorescent microscopy. EGFP was successfully expressed in pigs fibroblast cells. It was build a good basic for construction other double positive selection marker vector in mammalian cells.

关 键 词：绿色荧光蛋白 表达载体 猪 改造 

分 类 号：S828.89[农业科学—畜牧学]