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目的 构建AFP增强子CMV启动子调控下的HSV -TK真核表达质粒用于肝细胞癌的靶向基因治疗。方法 采用PCR方法从HepG2细胞基因组DNA中扩增AFP基因增强子最小的功能片断 ,插入pcDNA3.1-LUC质粒的BglII位点 ,从而构建重组表达质粒 pAFP -LUC。HSV -TKcDNA全长序列替换pAFP -LUC质粒中EcoRI位点的LuciferasecDNA全序列构建重组表达质粒 pAFP -TK。质粒 pAFP -LUC采用脂质体法转染AFP阳性的肝癌细胞系HepG2及AFP阴性的非肝癌细胞系HeLa ,用Luciferase分析试剂盒分析Luciferase的表达情况。结果 PCR扩增所得AFP增强子片段的长度和序列通过琼脂糖凝胶电泳、DNA测序得到证实。采用限制性内切酶酶切及PCR方法证实质粒pAFP -LUC中插入的AFP增强子大小、位置及方向均正确。酶切后凝胶电泳分析证实HSV -TK已成功地定向克隆入真核表达载体中。Luciferase报道基因的表达受AFP增强子的调控 ,在AFP阳性肝癌细胞HepG2中高效表达 ,而在AFP阴性的HeLa细胞中表达很低 ,这种差异具有显著性 ,P <0 .0 5。结论 AFP增强子CMV启动子调控的HSV -TK真核表达质粒的构建及其在HepG2细胞中的特异性表达为肝细胞癌的靶向基因治疗提供了实验基础。
Objective To construct HSV-TK eukaryotic expression plasmid under the control of AFP enhancer CMV promoter for targeted gene therapy of hepatocellular carcinoma. Methods The minimal functional fragment of AFP gene enhancer was amplified from HepG2 cells by PCR and inserted into BglII site of pcDNA3.1-LUC plasmid to construct recombinant expression plasmid pAFP-LUC. Full-length HSV-TK cDNA sequence replacement of pAFP -Luc plasmid EcoRI site Luciferase cDNA sequence was constructed recombinant expression plasmid pAFP-TK. Plasmid pAFP-LUC was transfected into AFP-positive HepG2 and AFP-negative HeLa cells by lipofectamine. Luciferase expression was analyzed by Luciferase assay kit. Results The length and sequence of AFP enhancer fragment obtained by PCR amplification were confirmed by DNA sequencing using agarose gel electrophoresis. Restriction endonuclease digestion and PCR confirmed that the size of the AFP enhancer inserted into the plasmid pAFP-LUC was correct. After digestion gel electrophoresis analysis confirmed HSV-TK has been successfully cloned into the eukaryotic expression vector. The expression of Luciferase reporter gene was highly regulated by AFP enhancer in AFP positive HepG2 cells and low in AFP negative HeLa cells. The difference was significant (P <0.05). Conclusion The construction of HSV-TK eukaryotic expression plasmid regulated by AFP enhancer CMV promoter and its specific expression in HepG2 cells provide the experimental basis for targeted gene therapy of hepatocellular carcinoma.