系列截短和缺失的hTERT启动子报告载体的构建及活性验证  

Construction of series reporter plasmids with truncated and deleted human telomerase reverse transcriptase promoter

作　　者：栾芳[1,2] 刘华[1,3] 刘斌[4] 马春红[1] 

机构地区：[1]山东大学医学院免疫学研究所,济南250012 [2]山东大学附属省立医院检验科 [3]山东大学附属济南市中心医院中心实验室 [4]山东大学附属省立医院医学工程二科

出　　处：《中华微生物学和免疫学杂志》2012年第4期309-314,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金青年科学基金项目（81101484）;山东省优秀中青年科学家科研奖励基金（BS2011SW035）

摘　　要：目的构建系列截短及缺失的hTERT启动子荧光素酶报告载体并验证其活性。方法PCR扩增系列截短及缺失的hTERT启动子基因，克隆入pGL3-Basic质粒，构建荧光素酶报告载体。分别与pRL—TK内参照质粒共转染HepG2和COS-7细胞，48h后裂解细胞，双荧光素酶分析法检测启动子的活性。结果成功构建系列截短及缺失的hTERT启动子报告载体，分别命名为pGL3B-895、pGL3B-371、pGL3B-DELS2、pGL3B-349、pGL3B-329、pGL3B-318、pGL3B-306。双荧光素酶报告基因检测结果显示在肝癌细胞和工程细胞中上述报告载体均具有启动子活性。结论成功构建具有启动子活性的系列截短及缺失的hTERT启动子荧光素酶报告载体，为进一步探讨肝癌发生中人端粒酶逆转录酶表达调控机制提供必要的实验材料。Objective To construct series of reporter plasmids with truncated and deleted hTERT promoter. Methods Gene fragments of hTERT promoter was amplified by PCR and cloned into pGL3-Basic to construct luciferase reporter vectors. Dual luciferase assays were performed with cell lysates of HepG2 and COS-7 cells cotransfected with hTERT promoter reporter plasmids and pRL-TK. Results Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed and respectively named pGL3B-895, pGL3B-371, pGL3B-DELS2, pGL3B-349, pGL3B-329, pGL3B-318, pGL3B- 306. Dual luciferase reporter assays showed that all the reporter vectors have promoter activity both in HepG2 and COS-7. Conclusion Series of luciferase reporter plasmids with truncated and deleted hTERT promoter were successfully constructed, and their promoter activity were verified. These plasmids provide necessary experimental materials for further investigation of regulation of hTERT during hepatocarcinoma development.

关 键 词：人端粒酶逆转录酶 报告载体 启动子活性 

分 类 号：R346[医药卫生—基础医学]