HRE/CMV启动子的克隆和缺氧调控活性的测定  

Cloning of HRE/CMV promoters and determination of hypoxia regulatory activities

作　　者：杨洁[1] 杜德伟[1] 李占亭[1] 贾林涛[2] 赵晶[3] 许彦鸣[3] 杨安钢[2] 孙脊峰[1] 

机构地区：[1]第四军医大学唐都医院肾脏内科,陕西西安710038 [2]第四军医大学基础部免疫学教研室,陕西西安710032 [3]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032

出　　处：《中国病理生理杂志》2007年第12期2432-2435,共4页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No39670283);陕西省自然科学基金资助项目(No2003C2052)

摘　　要：目的:将具有缺氧调控作用的多种缺氧反应元件(HRE)与CMV启动子组合,以获得缺氧应答启动子。将荧光素酶报告基因置于缺氧应答启动子的下游,通过检测荧光素酶活性的方法,方便地研究缺氧应答启动子缺氧诱导的作用。方法:合成多种HRE核苷酸序列,并设立HRE突变对照,克隆入pCI-neo中,构建HRE/CMV启动子。扩增HRE/CMV序列,定向亚克隆入pGL3-Basic中,获得HRE/CMV荧光素酶报告载体。瞬时转染HeLa细胞,在0.1%O2环境下培养细胞,并设立正常氧分压环境对照。检测荧光素酶的相对活性。结果:成功构建了HRE/CMV荧光素酶报告载体,其中mPGK-HRE/CMV载体表现出很好的缺氧诱导作用和高水平的表达。结论:mPGK-HRE/CMV启动子能够有效地进行缺氧诱导和调控工作。AIM： To obtain various hypoxia response promoters by combining CMV promoter with diverse hypoxia response element （ HRE ） and to identify the potency of hypoxia response promoters which were put upstream of firefly luciferase reporter gene and switched firefly luciferase reporter gene on and off based upon sensing tissue hypoxia by means of detecting relative luciferase activity. METHODS： Diverse HRE genes and mutation control were synthesized and cloned into pCI -neo vectors to construct HRE/CMV promoters. Amplified HRE/CMV promoter genes were then subcloned into pGL3 -Basic vectors to obtain HRE/CMV luciferase reporter vectors. The vectors were transiently transfected into HaLa cells, respectively. To characterize the transcriptional activation capacity of HRE under hypoxia, the normoxic and hypoxic responsiveness of luciferase reporter genes were examined. RESULTS： HRE/CMV luciferase reporter vectors were con- structed successfully. Of them, mPGK - HRE/CMV vector confered good induction and high maximum level of expression. CONCLUSION ： mPGK - HRE/CMV promoter is effective in induction and regulation.

关 键 词：缺氧反应元件 荧光素酶 基因 报告 

分 类 号：R692[医药卫生—泌尿科学]