pGL3-Basic-COX-2-promoter报告基因重组质粒的构建及其功能鉴定  

Construction of pGL3-Basic-COX-2-promoter reporter gene vector and detection of its function

作　　者：李琦[1] 周利红[1] 王炎[1] 孙珏[1] 高虹[1] 范忠泽[1] 

机构地区：[1]上海中医药大学附属普陀医院肿瘤科,上海市200062

出　　处：《世界华人消化杂志》2008年第31期3498-3504,共7页World Chinese Journal of Digestology

基　　金：国家自然科学基金资助项目;No.30600844;上海市教委第五期重点学科资助项目;No.J50304~~

摘　　要：目的:构建COX-2基因启动子荧光素酶报告基因重组质粒pGL3-Basic-COX-2-promoter,并进行功能鉴定.方法:根据已知人的COX-2基因启动子序列设计两端引物,扩增人基因组DNA中的COX-2启动子.载体质粒pGL3-Basic和COX-2启动子分别用限制性内切酶HindⅢ和BglⅡ双酶切,将COX-2基因启动子插入到pGL3-Basic报告载体中.构建的pGL3-Basic-COX-2-promoter重组质粒和内参质粒pRL-SV40瞬时共转染胃癌MKN45细胞,加入100倍细胞数量的Hpylori共培养不同时间后,检测双荧光素酶的活性.结果:成功构建COX-2基因启动子重组质粒pGL3-Basic-COX-2-promoter,质粒测序及酶切结果完全正确.瞬时转染实验显示,COX-2启动子在MKN45细胞中的转录表达随时间的变化而升高,转染后40h的双报告基因活性是转染后8h的3.5倍(P<0.01);加入Hpylori共培养后,同时间点的荧光素酶活性明显升高(P<0.05或0.01),转染后40h的活性是转染后8h的5倍(P<0.01).结论:pGL3-Basic-COX-2-promoter在胃癌MKN45细胞中能被转录激活,加入Hpylori刺激后COX-2活性显著增强,pGL3-Basic-COX-2-promoter可以用来鉴定和检测细胞中COX-2的水平.AIM： To construct COX-2 promoter recombined luciferase reporter gene vector, and to detect its function. METHODS： Primers were designed based on human COX-2 gene promoter, then COX-2 promoter from human genome DNA was replicated, pGL3-Basic Vector. and COX-2 promoter were digested with restriction enzymes Hind Ⅲand Bgl ⅡI separately, then COX-2 promoter was inserted into pGL3-Basic Vector. The recombinant vector pGL3-Basic-COX-2-promoter was transiently co-transfected into MKN45 cells with control vector pRL-SV40 respectively, twelve hours later, cells were treated with H pylori 100 times amount of cells for different length of time, then the activity of dual luciferase was detected. RESULTS： COX-2 promoter recombined luciferase reporter gene vector was constructed successfully, and the result of sequencing and double digesting of recombined plasmid were completely correct. The experiment of transient transfection showed that the expression of COX-2 promoter in MKN45 cells was increased with time. Activity of dual-luciferase after trans- fection for 40 h was 3.5 folds of that for 8 h （P 〈 0.05）. When H pylori for co-culture, activity was added to medium of dual-luciferase was increased up markedly compared with groups that were not added with H pylori （P 〈 0.05 or 0.01）, and it was 5 folds after transfection for 40 h than 8 h （P 〈 0.01）. CONCLUSION： pGL3-Basic-COX-2-promoter can be transcribed and activated in MKN45 cells, and activity of COX-2 promoter increases obviously after H pylori addition, pGL3-Basic-COX-2- promoter can be used to identify and detect the level of COX-2 in cells.

关 键 词：环氧合酶2 启动子 质粒 幽门螺杆菌 双荧光素酶 

分 类 号：R735.2[医药卫生—肿瘤]