一种原位示踪外源目的基因表达载体的构建

应用分子克隆技术 ,分别将增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)、内部核糖体进入位点 (internalribosomeentrysite,IRES)和编码H-ras基因C端 2 0个氨基酸的DNA(rasc2 0 )片段插入真核表达载体pcDNA3,构建真核重组表达载体并将其命名为pZX。通过脂质体介导将该载体转染人宫颈癌细胞系HeLa ,培养过夜后在荧光显微镜下观察绿色荧光蛋白在细胞内的分布 ,并与pEGFP-C3质粒DNA转染该细胞系进行比较。结果表明 ,转染pZX载体的实验组细胞膜发出绿色荧光 ,而对照组绿色荧光则均匀弥散于整个细胞中 ,工具性载体pZX已构建成功

Construction of pZX, a Vector for in situ Labeling Expression of Transgene

The DNA fragments encoding the enhanced green fluorescent protein (EGFP),the internal ribosome entry site (IRES) and the C-terminal 20 amino acids of H-ras (rasc20) were inserted into the eukaryotic expression vector pcDNA3 at its multiple cloning sites.A new construct which could simultaneously express the EGFP tagged at C-terminal with rasc20 and a gene of interest was generated.The new plasmid (pZX) was transfected into human HeLa cells with pEGFP-C3 as control. The subcellular localization of green fluorescence signals was detected after overnight incubation.Green fluorescence signals were detected on plasma membrane in cells transfected with pZX,while the signals were distributed homogeneously in cells transfected with control plasmid.The new pZX plasmid could be used to label the expression of transgene in situ.;