人表面活性物质蛋白B基因启动子荧光素酶报告基因重组质粒的构建及活性检测  

作　　者：王席娟[1] 蔡保欢[1] 李文斌[1] 刘伟[1] 王靓[1] 常立文[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030

出　　处：《中国医师杂志》2013年第6期725-728,共4页Journal of Chinese Physician

基　　金：国家自然科学基金（81170001）;国家自然科学基金青年项目（81000261）

摘　　要：目的构建人表面活性物质蛋白-B（SP-B）基因启动子荧光素酶报告基因重组质粒，并分析其在H441细胞中的转录活性。方法（1）以人基因组DNA为模板，PCR扩增SP-B启动子（-218／＋435bp）序列片段，并通过TA克隆方法连接到pGM-T载体上，筛选阳性克隆将目的片段进行测序。测序鉴定正确后的目的片段被克隆到荧光素酶表达载体pGL3-Basic上构建pGL3-basic-SP-B-promoter重组质粒，酶切及测序鉴定重组质粒；（2）将构建的pGL3-basic-SP-B-promoter重组质粒酶切改造，sP-B启动子克隆进pGL4．17载体构建pGL4．17-SP-B-promoter重组质粒；经酶切及基因测序确认无误后，采用脂质体法将构建的两种重组质粒分别和内参质粒pRL-TK同时转染H441细胞，检测双荧光素酶活性。结果构建的重组质粒经酶切及测序鉴定完全正确，转染H441细胞后，检测细胞裂解液萤火虫荧光素酶（firefly）及海肾荧光素酶（renilla）活性，两者比值间接反映启动子活性，pGL3-basic／pGL4．17-SP-B-promoter重组质粒的firefly／reniUa比值（2．8±1．1、66．5±3．8）较空载体质粒pGL3-basic、pGIA．17（0．2±0．1，4．3±0．4）均明显升高（t=4．182，27．419，P=0．000）。pGL4．17重组质粒firefly／renilla比值明显高于pGL3-basic重组质粒，其差异有统计学意义（t=27．712，P=0．000）。结论成功构建具有sP-B基因转录活性的pGL3-basie／pGIA．17-SP-B-promoter重组质粒，oGL4．17萤组质粒荧光素酶活性高，为后续研究SP-B基因转录调控奠定了理论基础。Objective To construct human surfactant protein B (SP-B) gene promoter luciferase reporter plasmids and detect their transcriptional activities in H441 cells.Methods (1)The fragment of SP-B promoter (-218/+ 435 bp) was acquired from human genome DNA by polymerase chain reaction (PCR) amplification and then was inserted into pGM-T vector by the T4 DNA ligase.The vector was transfected into TOP10 E.coli.The positive clone was identified by DNA sequencing.The identified target SP-B promoter sequence was cloned into pGL3-basic vector to construct the recombinant vector pGL3-basic-SP-B-promoter and was identified by enzyme digestion and sequencing; (2)The pGL3-basic-SP-B-promoter vector was converted into pGL4.17-SP-B-promoter vector through enzyme digestion.The identified recombinant vectors and control plasmid pRL-TK were transfected into H441 cells by lipofectamine 2000,and luciferase assays was performed using the dual-luciferase reporter assay system.Results The sequences of SP-B promoter in the recombinant luciferase reporter plasmids were consistent with the one published on Genebank.The firefly/renilla luciferase activity ratio of pGL3-basic/pGL4.17-SP-B-promoter vector (2.8 ± 1.1,66.5±3.8) was significantly higher than pGL3-Basic,pGL4.17 control vector (0.2 ±0.1,4.3 ±0.4) with statistical significance (t =4.182,27.419,P =0.000),respectively.The SP-B promoter activity of pGL4.17-SP-B-promoter vector was significantly higher than pGL3-basic-SP-B-promoter vector (t =27.712,P =0.000).Conclusions The pGL3-basic/pGL4.17-SP-B-promoter vectors are successfully constructed with SP-B promoter activity in H441 cells and pGL4.17-SP-B-promoter vector is the better choice for further study with higher luciferase activity.

关 键 词：肺表面活性剂 遗传学 启动区(遗传学) 萤光素酶类 遗传学 重组 遗传 转录 遗传 质粒 遗传学 

分 类 号：R363[医药卫生—病理学]