HBV启动子调控的GFP在肿瘤细胞中的表达  

Expressions of GFP in Tumor Cells under the Direction of HBV Promoters

作　　者：茆灿泉[1] 黄常志[2] 赵玫[2] 范云霞[2] 徐枫[2] 杜菲[2] 杨树德[1] 

机构地区：[1]卫生部北京医院临检中心,北京100730 [2]中国协和医科大学中国医学科学院肿瘤医院肿瘤研究所

出　　处：《中国肿瘤生物治疗杂志》2001年第1期55-58,共4页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金! (3 9670 696)

摘　　要：目的 :研究HBV启动子调控下的GFP报告基因在肿瘤细胞中的表达。方法 :采用DNA重组技术 ,将GFPs65T和HBV的EⅡmCMV启动子 (增强子 )亚克隆到哺乳动物表达载体pcDNA3 中 ,构建了pcDNA3 GFP ,pcDNA3 GFP EⅡ ,pcDNA3 GFP EⅡ w3种含GFP的重组质粒 ,在Lipofectin介导下分别转染Hela和Bel740 2肿瘤细胞 ,经G418抗性筛选 ,挑选阳性克隆并扩大培养用于荧光和Westernblotting检测 ,利用GELDoc2 0 0 0数字成像系统对GFP蛋白表达进行定量分析。结果 :酶切和电泳表明重组质粒构建正确 ;获得了稳定转染各质粒的阳性细胞克隆 ;经Westernblotting检测 ,亲本和空载细胞的GFP值在本底范围 ,GFP转染的各细胞 ,均可检测到 2 7kD的GFP目的蛋白 ;EⅡmCMV启动子调控的Hela GFP EⅡ ,Hela GFP EⅡ w表达的GFP量均低于Hela GFP ;pcDNA3 GFP EⅡ w在Bel740 2细胞中的表达量明显高于其在Hela细胞中的表达量。结论 :HBV启动子调控下的GFP报告基因能够在肝癌Bel740 2细胞中表达并具有一定的专一性。Objective: To investigate the expression of GFP reporter gene under the direction of HBV promoters in tumor cells. Methods: GFP s65T and EⅡmCMV promoter(enhancer) of HBV were subcloned to mammalian expression vector pcDNA 3 by recombinant DNA technology. Three recombinant plasmids(pcDNA 3-GFP,pcDNA 3-GFP-EⅡ and pcDNA 3-GFP-EⅡ-w)were constructed. They were transfected into Hela and Bel7402 cells by Lipofectin and selected by G418 respectively, after amplification of the positive cell clones, they were used for fluorescence and Western blotting detection, GFP was quantitatively analysed by GEL Doc2000 digital image systems. Results: The correction of the recombinant plasmids was confirmed by restriction analysis and electrophorosis and positive cell clones were obtained through stable selection and GFP was within the value of background in parent and pcDNA 3 transfected Hela and Bel7402 cells and GFP protein of 27 kD existed in all GFP transfected cells. The expression of GFP was lower in Hela-GFP-EⅡ and Hela-GFP-EⅡ-w than that of Hela-GFP, but the expression of GFP in Bel-GFP-EⅡ-w was much higher than that of Hela-GFP-EⅡ-w. Conclusion: GFP reporter gene under the direction of HBV promoter(enhancer) could be expressed in tumor cells and be expressed definitely and specifically in HCC Bel7402 cell.

关 键 词：基因转染 GFP HBV 肝肿瘤 启动子 基因调控 

分 类 号：R735.7[医药卫生—肿瘤]