人Endostatin基因的克隆及其在Hela细胞中的表达

Cloning of Human Endostatin and Its Expression in Hela Cells

利用RT-PCR克隆人Endostatin基因,分别构建到双顺反子表达载体pIRES2-EGFP和融合表达载体pEGFP-C1中.两个重组表达载体利用脂质体介导转染真核细胞Hela,48 h后可在荧光显微镜下观察到被转染细胞发出绿色荧光.传代10次后,绿色荧光仍持续表达,说明是稳定转染.以转染细胞的总DNA为模板PCR检测Endostatin基因已整合到细胞染色体上.利用兔抗人Endostatin抗体对已转染细胞进行免疫组化检测,显微镜下观察,转染的细胞显棕红色,表明Endostatin在转染细胞内稳定表达.本试验通过报告基因检测,DNA检测,免疫细胞化学检测三个水平证明Endostatin已整合到细胞的染色体上,为基因治疗以及联合治疗打下基础.

Human Endostatin cloned by RT-PCR was inserted into bicistronic expression vector pIRES2-EGFP and fusion expression vector pEGFP-Cl,which were transfected into Hela cells with lipofectamine TM regent. After 48 hours,under the fluorescence microscope,the green fluorescence was spreading in the transfected Hela cell. After culturing ten generations, the green fluorescence expressed persistently, which illustrated that the transfection was steady. On the level of total DNA,it can show that Endostatin integrated into total DNA by PCR of transfected Hela cell total DNA. Furthermore,after immunocytochemistry in the fixed tranfected Hela cell with rabbit anti- Endostatin, under the microscope, the transfected Hela cell became brown-red and the negative control was unstained,which meant that Endostatin has expressed in the transfected Hela cell. This experiment advantages gene therapy and therapeutic alliance.