摘要
目的:构建甲胎蛋白(AFP)启动子介导的单纯疱疹病毒胸苷激酶(HSV-TK)重组真核表达载体,并检测其在AFP阳性肝癌细胞株的特异性表达。方法:PCR扩增AFP基因启动子、HSV-TK基因,将上述片段插入EGFP-N1载体的多克隆位点,构建pEGFP-AFP-TK重组表达载体,通过包裹PEG-PEI/Fe3O4纳米磁流体,经其介导,转染AFP阳性肝癌细胞株HepG2和AFP阴性肝癌细胞株SMMC7721,利用增强型绿色荧光蛋白及Western blotting法检测目的基因在2种细胞系中的表达。结果与结论:肝癌特异性真核表达载体pEGFP-AFP-TK构建成功。利用增强型绿色荧光蛋白及Western blotting法可以检测到嵌合AFP启动子的pEGFP-AFP-TK重组真核表达载体能够特异性在AFP阳性的肝癌细胞株表达,而在AFP阴性的肝癌细胞株中基本不表达。
Aim：To construct a recombinant eukaryotic expression vector containing α-fetoprotein （AFP） promotermediated HSV-TK suicide gene, and analyze its specific expression on AFP-positive hepatoma cells. Methods：The promoter of AFP gene,HSV-TK gene were amplified by PCR. The gene segments mentioned above were cloned into the multiple cloning sites of the eukaryotic expression vector pEGFP-N1 to construct pEGFP-AFP-TK. Then it was transfected into AFP- positive HepG2 and AFP-negative SMMC7721 hepatoma cells by PEG-PEI/Fe3O4 magnetic nanoparticles. Finally,the expression of the target gene in AFP-positive HepG2 and AFP-negative SMMC7721 hepatoma cells was detected by EGFP and Western blotting. Results and Conclusions：Hepatocellular carcinoma-specific eukaryotic expression vector carrying AFP promoter and HSV-TK suicide gene, pEGFP-AFP-TK, is successfully constructed. Using EGFP and Western blotting method, AFP promoter driven HSV-TK gene system could be specificly expressed in AFP-positive hepatoma HepG2 cells.
出处
《郑州大学学报（医学版）》
CAS
北大核心
2008年第3期473-477,共5页
Journal of Zhengzhou University(Medical Sciences)
基金
国家自然科学基金资助项目30672047
中国博士后科学基金资助项目20060390884
湖南省博士后科学基金资助项目2006FJ4240
