高效转染HepG2细胞的逆转录病毒载体系统的构建及应用  

Construction and application of retroviral vector system useful for high-efficiency transfection into HepG2 cells

作　　者：董海燕[1] 陈剑锋[1] 邵敬伟[1] 王涛[1] 

机构地区：[1]福州大学药物生物技术与工程研究所,福建福州350002

出　　处：《福州大学学报（自然科学版）》2007年第6期941-944,共4页Journal of Fuzhou University(Natural Science Edition)

基　　金：福建省教育厅科研资助项目(JB05020)

摘　　要：为构建含绿色荧光蛋白(EGFP)和人胰岛素原基因的逆转录病毒表达载体,并检测其在人肝癌细胞HepG2中的表达,将IRES-EGFP片段克隆到含调控元件的人胰岛素原基因逆转录病毒表达载体(pLXSN-GI-Ins)中,构建得到表达质粒pLXSN-GI-Ins-EGFP.经脂质体介导转染HepG2细胞后,各孔分别加入含有30.0 mmol/L葡萄糖的培养液继续培养24 h,在荧光显微镜下观察EGFP基因的表达,检测细胞上清液中的胰岛素值.数据显示,成功构建逆转录病毒表达质粒pLXSN-GI-Ins-EGFP.转染HepG2细胞后48 h,表达EGFP基因的细胞数目占总细胞数目的比值为(38.0±5.0)%.结果表明,构建了含EGFP和调控元件的人胰岛素原基因逆转录病毒表达载体,并且能够在HepG2细胞中成功表达.To construct the retroviral expression vector carrying green fluorescent protein gene（EGFP） and human insulin gene and study expression of the vector in HepG2 cells. The fragment encoding IRES - EGFP was cloned into the retroviral expression vector carrying human insulin gene with regula- tory element（ pLXSN - GI - Ins）. The expression plasmid of pLXSN - GI - Ins - EGFP were constructed. The plasmid was transfected into HepG2 ceils by using lipofectamine. To observe the expression of EGFP gene by fluorescence microscope cells cultured in medium with glucose at concentration of 30. 0 mmol/L. Our results suggest that the retroviral expression plasmid of pLXSN - GI - Ins - EGFP was successfully constructed and the expression of EGFP gene was （38.0 ± 5.0） %. Thus, the retro- viral expression vector carrying green fluorescent protein gene （EGFP） and human insulin gene with regulatory element was successfully constructed and expressed in HepG2 cells.

关 键 词：绿色荧光蛋白 胰岛素 逆转录病毒载体 HEPG2细胞 

分 类 号：Q78[生物学—分子生物学] R587.1[医药卫生—内分泌]