人CTGF基因启动子荧光素酶报告基因的构建及活性鉴定  

Construction and activity identification of luciferase reporter containing human CTGF gene promoter

作　　者：朱国念[1] 钟礼春 吴思思[1] 田鑫 汤小菊[2] ZHU Guonian;ZHONG Lichun;WU Sisi;TIAN Xin;TANG Xiaoju(Research Core Facility,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,P.R.China;Department of Respiratory and Critical Care Medicine,West China Hospital,Sichuan University,Chengdu,Sichuan 610041,P.R.China)

机构地区：[1]四川大学华西医院公共实验技术中心,成都610041 [2]四川大学华西医院呼吸与危重症医学科,成都610041

出　　处：《华西医学》2020年第1期22-27,共6页West China Medical Journal

基　　金：国家自然科学基金(81800087);四川省科学技术厅计划项目(2017SZ0129)

摘　　要：目的构建人结缔组织生长因子(connective tissue growth factor,CTGF)基因启动子荧光素酶报告基因的真核表达载体。方法设计特异性引物,通过聚合酶链反应实验扩增人基因组中CTGF启动子区域的DNA(-835/+214),通过分子克隆方式将人CTGF启动子的序列插入到pGL3.0-Basic载体,通过单克隆菌落聚合酶链反应、质粒双酶切鉴定、测序等方法对其进行阳性质粒的筛选和鉴定;然后使用转染试剂Lipofectamine 3000将pGL3.0-Basic-CTGF分别转染于人胚肾细胞293T、人气道上皮细胞HBE和人肺泡上皮细胞A549,通过测定荧光素酶活性鉴定其功能。结果序列比对结果为99.50%,提示pGL3.0-Basic-CTGF载体构建成功。将pGL3.0-Basic-CTGF分别转染于293T、HBE和A549细胞48 h后检测,在293T、HBE和A549细胞中均有启动子活性,分别为2.416、0.027、0.121(P<0.01);而且在A549细胞中荧光素酶的活性明显高于HBE细胞组,差异有统计学意义(P<0.01)。结论人pGL3.0-Basic-CTGF荧光素酶系统构建成功,且在气道上皮细胞HBE和肺泡上皮细胞A549中均有启动子活性,可为后续转录调控研究提供工具。Objective To construct a luciferase reporter fusion containing the human connective tissue growth factor(CTGF)gene promoter.Methods The promoter region of the human CTGF gene(-835/+214)was amplified by polymerase chain reaction(PCR)using specially-designed primers,and subsequently cloned into the pGL3.0-Basic vector.Following screening and verification by single colony PCR,double digestion,and sequencing,the resulting pGL3.0-BasicCTGF was used to transfect the human embryonic kidney cells 293 T,human bronchial epithelial cells HBE and human lung epithelial cells A549,and its function in each cell line was determined by luciferase assay.Results Sequence alignment showed 99.5%identity,suggesting successful construction of the pGL3.0-Basic-CTGF reporter fusion.Promoter activities were detected 48 hours after transfection of pGL3.0-Basic-CTGF into the 293 T,HBE,and A549 cells,and the promoter activities were 2.416,0.027,and 0.121,respectively(P<0.01).Moreover,the luciferase activity in the A549 cells was statistically higher than that in the HBE cells(P<0.01).Conclusions The human pGL3.0-Basic-CTGF luciferase reporter fusion has been successfully constructed.The construct exhibits promoter activity in the bronchial epithelial cells HBE and the lung epithelial cells A549,and can therefore serve as a useful tool for future research in transcriptional regulation.

关 键 词：CTGF基因 启动子 荧光素酶 报告基因 肺纤维化 

分 类 号：Q78[生物学—分子生物学]