携带融合基因穿梭质粒的构建及在人肝癌细胞中的表达

构建携带胸苷激酶(tk)与增强型绿色荧光蛋白(EGFP)融合基因穿梭质粒,并观察其在肝癌细胞系HepG2细胞中的表达。应用基因重组技术,构建EGFP与tk的融合表达穿梭质粒载体,经限制酶切鉴定和测序分析,脂质体转染将其导入HepG2中,48h后用荧光显微镜观察荧光的表达,RT-PCR法检测融合蛋白tk和EGFP的mRNA表达,四甲基偶氮唑蓝(MTT)实验检测转染融合蛋白载体后不同浓度的更昔洛韦(GCV)对HepG2细胞的细胞毒作用。酶切鉴定和测序分析证实重组质粒中插入融合目的基因片断及载体DNA大小、方向和插入位点正确,在转染此穿梭质粒的HepG2细胞中检测到绿色荧光蛋白的表达,RT-PCR检测到融合蛋白tk和EGFP的mRNA表达;GCV对转染了携带tk与EGFP融合基因穿梭质粒载体有明显的细胞毒作用。成功构建携带tk与EGFP融合基因穿梭质粒载体,转染人肝癌细胞株HepG2中tk和EGFP的独立表达没有受到影响,该载体可利用绿色荧光蛋白作为报告基因监测tk的表达并可用于肝癌的自杀基因治疗。

Construction of the shuttle plasmid vector for fusion protein gene and expression in Hepatocellular Carcinoma cells

To construct the shuttle plasmid vector for tk and EGFP fusion protein gene and detect its expression in transfected liver cancer cell lines HepG2 cells.Recombinant expression vector was constructed by techniques of gene recombination and screening,and identified by restriction digestion and sequencing analysis.Then the recombinant shuttle plasmid was transfected into HepG2 cell by techniques of lipidosome transfection and its expression was detected by fluoroscopy and RTPCR.Cell killing after GCV application was determined by MTT.Identification of pDC316-tkEGFP-CMV by enzyme digestion and sequencing analysis showed that the length,inserted location and direction of the target gene which was inserted into the recombinant was correct and the expression of EGFP in transfected cell was observed.The MTT assess showed that the obvious cytotoxicity of GCV to HepG2 cells which was transfected the shuttle plasmid vector carrying the tk and EGFP fusion protein gene.The shuttle plasmid vector carrying the tk EGFP fusion protein gene has been constructed successfully and the individually expressing in transfected HepG2 cells of tk and EGFP was not effected.The shuttle plasmid vector can be used as report gene for monitoring the expressing of tk and as therapy gene for the suicide gene therapy of HCC.