融合自杀基因FCU1真核表达载体的构建与表达

Construction and Expression of a Fusion Suicide Gene FCU1 Vector Plasmid

目的 构建含有融合自杀基因FCU1的真核细胞表达载体 pEGFP FCU1,并转染肝癌细胞SMMC772 1,初步探讨FCU1/ 5 氟胞嘧啶 (5 FC)系统对体外培养细胞的生长抑制作用。方法 用SmaⅠ ,XhoⅠ两种限制性内切酶分别切割质粒 pEGFP C1和 pCIneoFCU1,所得载体片断和目的基因片断连接后转化 ,阳性重组子转染肝癌细胞SMMC772 1,以绿色荧光蛋白基因为报告基因 ,用G4 18筛选出阳性细胞克隆后 ,进行体外药物敏感实验。结果 酶切鉴定重组子阳性克隆率约为 6 0 % ,转染成功的细胞在荧光显微镜下可见绿色荧光 ;转染的细胞在 5 FC作用下其生长抑制率明显高于未转染细胞的生长抑制率 ,且旁观者效应显著。结论 自杀基因FCU1真核表达载体构建正确 ,转染细胞成功并获稳定表达 ,FCU1/ 5 FC系统对肝癌细胞SMCC772 1具有实验性的基因治疗作用。

Objective To construct a eukaryotic expression vector plasmid containing the novel fusion suicide gene FCU1 and transfect into hepatoma cell SMMC7721, so as to investigate the antitumor activity of FCU1 gene and prodrug 5-fluorocytosine (5-FC). Methods FCU1 was cloned into the eukaryotic expression vector pEGFP-C1. The recombinant was confirmed by restriction enzyme digestion. The positive recombinant was introduced into SMMC7721 cells by LipoGen, and the expression of EGFP-FCU1 was analyzed by fluorescence. Positive cells were got through G418 screening, and then their sensitivity to 5-FC was tested. Results Agrose electrophoresis showed that the positive recombinants were about 80 %. The cells successfully transfected with pEGFP-FCU1 could display green fluorescence under a fluorescence microscope. In the experiment in vitro, 10 % of the transduced cells could give rise to 60 % of the total cells to death. Conclusion The recombinant pEGFP-FCU1 was successfully constructed and expressesed in the hepatoma cells. FCU1/5-FC system possesses significant antitumor activity on hepatoma cell SMMC7721.