分子伴侣疫苗抗肿瘤的研究进展

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-EⅡpcDNA3-GFP-EⅡ-w3各上含GFP的重组质粒，在Lipofectin介导下分别转染Hela和Bel7402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W estern blotting检测，利用GEL Doc2000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Western blotting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27kD 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Bel7402细胞中的表达量明显高于其在H ela细胞中的表达量。结果：HBV启动子调控下的GFP报告基因能够在肝癌Bel7402细胞中表达并具有一定的专一性。

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

Objective: To investigate the expression of GFP reporter geneunder the direction of HBV promoters in tumor cells. Methods: GFPs65T and EⅡmCMV promoter(enhancer) of HBV were subcloned to mammalian expression vector pcDNA3 by recombinant DNA technology. Three recombinant plasmids(pcDNA3-GFP，pcDNA3-GFP-EⅡ and pcDNA3-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 pcDNA3 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.