中国现代医学杂志2003，Vol.13Issue(22)：6-11,21,7.
含HSV-TK基因的真核表达载体的构建及其在HepG2细胞中的表达
Construction of eukaryotic expression plasmid carrying HSV-TK gene and its expression in HepG2 cells
张阳德 1孙颖2
作者信息
- 1. 中南大学卫生部肝胆肠外科研究中心,长沙,410008
- 2. 中国卫生部纳米生物技术重点实验室
- 折叠
摘要
Abstract
Objective:To construct a eukaryotic expression plasmid carrying the HSV-TK gene driven by AFP enhancer and CMV promoter for the purpose of targeted gene therapy for hepatocellular carcinoma (HCC).Methods:The minimal essential DNA fragment of AFP gene enhancer was amplified through PCR from genome DNA of HepG2 cells and cloned into the BglII site of plasmid pcDNA3.1-LUC to construct the recombinant plasmid pAFP-LUC. Then the full length cDNA of HSV-TK was cloned into EcoRI site of the recombinant plasmid pAFP-LUC instead of the Luciferase gene to construct pAFP-TK. The recombinant plasmid pAFP-LUC was transferred into AFP-producing hepatoma cell line (HepG2) and non-AFP-producing nonhepatoma cell line (HeLa) by means of lipofectamine. The expression of Luciferase was tested by Luciferase Assay. Results: The length and sequence of AFP enhancer amplified by PCR were confirmed by agarose gel electrophoresis and DNA sequencing.The length, position and orientation of inserted AFP enhancer in pAFP-LUC were all confirmed correct by the methods of restriction digestion and PCR. And it was confirmed by electrophoresis after restriction digestion that the full length HSV-TK was directedly and successfully cloned into the eukaryotic vector . The transcription of Luciferase gene was under the control of AFP enhancer. The expression of Luciferase gene was detected in HepG2 and HeLa cells. The expression of Luciferase is more potent in HepG2 than in HeLa (P＜0.05).Conclusions:Construction of a eukaryotic expression plasmid carrying HSV-TK gene driven by AFP enhancer and CMV promotor and its specific expression in HepG2 cells provide a sound basis for targeted gene therapy for HCC.关键词
肝细胞癌(HCC)/单纯疱疹病毒胸苷激酶(HSV-TK)/基因治疗/丙氧鸟苷(GCV)Key words
Hepatocellular Carcinoma (HCC)/Herpes Simplex Virus Thymidine Kinase (HSV-TK)/Gene therapy/Ganciclovir (GCV)分类
医药卫生引用本文复制引用
张阳德,孙颖..含HSV-TK基因的真核表达载体的构建及其在HepG2细胞中的表达[J].中国现代医学杂志,2003,13(22):6-11,21,7.
