摘要
目的 利用含有人甲胎蛋白启动子的腺相关病毒载体 ,构建能在人肝癌细胞株中特异表达目的基因的质粒。方法 通过设计含有特定酶切位点的引物 ,选择性地从人基因组中扩增出人甲胎蛋白启动子 (AFPpromoter)序列并克隆到含有绿色荧光蛋白 (GFP)报告基因的质粒pTR UF5上 ,构建成含报告基因的重组腺相关病毒质粒rAAV AFP GFP ;再通过平端连接的方法 ,构建成含p5 3基因的质粒rAAV AFP 5 3。通过用rAAV AFP GFP转染表达AFP的HepG2 细胞株和不表达AFP的 2 93细胞株 ,以检测AFP启动子的功能。rAAV AFP 5 3转染HLE肝癌细胞株后 ,用流式细胞分析检测对细胞周期的影响。结果 rAAV AFP GFP和经测序、酶切鉴定证实为含有人AFP启动子的质粒。细胞转染表明rAAV AFP GFP能特异地在AFP阳性细胞中高效表达目的基因 (质粒转染效率为 36 .5 % ) ;rAAV AFP 5 3使HLE的凋亡率达到 73 .88%。结论 成功构建了以腺相关病毒为载体、受人AFP启动子调控表达人野生型p5 3基因和报告基因的质粒。
Objective To construct plasmids that express target genes in hepatoma cell line using adeno associated virus (AAV)vectors containing human AFP promoter. Methods Primers containing specific enzyme cutting sites were designed to amplify the alpha fetoprotein promoter(AFP promoter) from human genome. The promoter was cloned into pTR UF5, a plasmid containing GFP reporter gene,resulting in the recombinant AAV plasmid containing the reporter gene (rAAV AFP GFP). Blunted ligation was used to construct the recombinant AAV vector plasmid containing human wild p53 gene (rAAV AFP 53). The plasmid rAAV AFP GFP was used to transfect the AFP expressing Hep G 2 and non AFP expressing 293 cell lines, respectively, to measure the function of the cloned AFP promoter. Flow cytometry was used to measure the effect of rAAV AFP 53 on hepatoma cell line HLE. Results rAAV AFP 53 and rAAV AFP GFP were verified by DNA sequencing and enzyme digestion to carry human AFP promoter. Cell transfection of rAAV AFP GFP showed selective expression in AFP positive hepatoma cell lines with a transfection rate of 36.5%; rAAV AFP 53 induced apoptosis rate was 73.88%. Conclusion Two adeno associated virus plasmids are successfully constructed that carry p53 gene and reporter gene, respectively, guided by AFP promoter. The former one showes a hepatoma specific apoptosis inducing effect.
出处
《中华医学杂志》
CSCD
北大核心
2000年第6期461-463,共3页
National Medical Journal of China
关键词
腺相关病毒
甲胎蛋白
启动子
基因治疗
肝癌
Adeno Associated viruses
Alpha fetoprotein
Promoter region (Genetics)
Gene therapy
