CYP7A1基因启动子克隆及萤光素酶报告基因载体构建  

CYP7A1 Gene Promoter Clone and Luciferase Reporter Gene Vector Construction

作　　者：张照研 王宇光 高月 ZHANG Zhao-Yan;WANG Yu-Guang;GAO Yue(College of Life Science and Bioengineering,Beijing University of Technology,Bejing 100124;Department of Pharmaceutical Sciences,Beijing Institute of Radiation Medicine,Beijing 100850,China)

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]军事科学院军事医学研究院辐射医学研究所,北京100850

出　　处：《生物技术通讯》2018年第5期647-652,共6页Letters in Biotechnology

基　　金：国家重大科技专项(2015ZX09501004-003-003);中医药行业科研专项(201507004)

摘　　要：目的:克隆细胞色素P450 7A1(CYP7A1)基因启动子,构建以CYP7A1基因启动子为启动序列的双萤光素酶报告基因系统并分析其活性,为研究CYP7A1基因转录调控提供有效筛选工具。方法:采用PCR方法克隆CYP7A1基因启动子序列,连接到pUC57载体中,双酶切后连接至萤光素酶报告质粒pGL3-Basic上,构建重组萤光素酶报告质粒pGL3-CYP7A1-promoter-luc。结果:重组质粒经双酶切和测序,证实构建正确;pGL3-CYP7A1-promoter-luc质粒2、3转染HepG2细胞均具有显著性启动子活性,pGL3-CYP7A1-promoter-luc2转染24和48 h后经检测分别为对照组(pGL3-basic空载体)的13.1±2.8倍(P<0.001)和23.3±2.9倍(P<0.001);pGL3-CYP7A1-promoter-luc3转染24、48 h后,荧光响应值分别是对照组的8.4±1.6倍(P<0.001)和22.1±1.9倍(P<0.01),呈现强启动子活性。结论:构建了CYP7A1基因启动子萤光素酶报告基因重组质粒pGL3-CYP7A1-promoter-luc,为后续深入研究药物对其调控作用机制奠定了基础。Objective： To clone the cytochrome P450 7A1（CYP7A1） gene promoter and construct a dual luciferase reporter gene system with the CYP7A1 gene promoter as the promoter sequence and analyze its activity, providing an effective tool for studying the transcriptional regulation of CYP7A1 gene. Methods： The CYP7A1 gene promoter sequence was cloned by PCR and ligated into pUC57 vector. After double digestion, it was ligated into lucif- erase reporter plasmid pGL3-Basic to construct recombinant luciferase reporter plasmid pGL3-CYP7A1-promoterluc. Results： The recombinant plasmid was confirmed by double restriction enzyme digestion and sequencing. The pGL3-CYP7A1-promoterluc plasmid 2,3 transfected HepG2 cells had significant promoter activity. After transfection for 24 and 48 h, pGL3-CYP7A1-promoter-luc2 plasmid was tested as control group（pGL3-basic empty vector） 13.1±2.8 times（P〈0.001）, 23.3±2.9 times（P〈0.001）; after 24 and 48 h of pGL3-CYP7Al-promoter-luc3 plasmid transfection, the fluorescence response values were 8.4±1.6 times（P〈0.001） and 22.1±1.9 times（P〈0.01） of control group, these results showed that this two plasmid exhibited strong promoter activity. Conclusion： The CYPTA1 gene promoter luciferase reporter gene recombinant plasmid pGL3-CYP7A1-promoter-luc was obtained, which laid a foundation for further research on the mechanism of its regulation.

关 键 词：细胞色素P4507A1(CYP7A1) 报告基因法 载体构建 

分 类 号：Q78[生物学—分子生物学]