错配修复基因hMLH1启动子萤光素酶报告基因载体的构建与鉴定  

Construction and identification of luciferase reporter gene vector containing hMLH1 promoter

作　　者：卢俊宇[1,2] 金鹏[1,3] 高巍[1] 陆晓娟[1] 王亚婷[1] 盛剑秋[1] 

机构地区：[1]北京军区总医院消化内科,北京100700 [2]第三军医大学,重庆400038 [3]北京协和医学院基础医学研究所医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2012年第4期338-341,共4页Basic and Clinical Medicine

基　　金：国家自然科学基金(81072041);医学分子生物学国家重点实验室专项经费(2060204)

摘　　要：目的构建含hMLH1启动子片段的萤光素酶报告基因载体,并检测其在雌激素诱导下的转录活性。方法以正常人基因组DNA为模板,PCR扩增hMLH1启动子片段(-1 953/+53),插入萤光素酶报告基因载体pGL3-Basic,将重组质粒转入HEK293细胞,检测在有和无雌激素诱导下萤光素酶的活性变化。结果酶切和测序结果证实重组萤光素酶报告基因载体pGL3-Promoter1-luc构建成功。转染pGL3-Promoter1-luc后,雌激素诱导的萤光素酶活性为7.45±0.81,显著高于无水乙醇组的3.28±0.19(n=3,P<0.001)。结论 hMLH1启动子片段存在与雌激素相关的调控序列。Objective To construct luciferase reporter gene vector containing human MLH1 promoter and to assay the transcriptional activity of hMLH1 promoter induced by E2.Methods hMLH1 promoter（-1 953/＋53）were amplified from the genomic DNA of human by PCR and cloned into luciferase reporter gene vector,pGL3-Basic.The recombined vector was transfected into HEK293 cells,and the activity of the luciferase was determined after simulation by E2.Results The results of restriction enzyme digestion and sequencing indicated that the recombinant vector pGL3-Promoter1-luc was successfully constructed.After transcription of pGL3-Promoter1-luc,the activity fold of the luciferase was 7.45±0.81 induced by E2,which is significantly higher than 3.28±0.19 without E2（n=3,P0.001）.Conclusions The hMLH1 promoter contains the regulatory sequence associated with E2.

关 键 词：错配修复基因 HMLH1 启动子 雌激素 双萤光素酶报告基因 

分 类 号：R346[医药卫生—基础医学]