人14-3-3σ基因启动子荧光素酶报告基因载体的构建及其转录活性分析  

Constructions of luciferase reporter gene vectors for human 14-3-3σ gene promoter and analysis of its transcriptional activity

作　　者：甘露[1] 黄明元[2] 郭进强[1] 万为人[1] 罗炳德[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院预防医学实验教学中心,广州510515 [2]广东医学院公共卫生学院劳动卫生与环境卫生教研室

出　　处：《现代预防医学》2013年第1期108-111,共4页Modern Preventive Medicine

基　　金：国家自然科学基金(31070687);广东省自然科学基金(9451051501002535)

摘　　要：目的构建人14-3-3σ基因启动子的荧光素酶报告基因载体,探讨该启动子的转录活性。方法运用UCSC软件对人14-3-3σ基因5′端2000bp进行启动子特征分析和转录因子结合位点分析。克隆14-3-3σ基因5′端5个不同长度的启动子片段,与荧光素酶报告基因载体pGL3-Basic重组,获得5个不同长度的14-3-3σ启动子萤光素酶报告基因载体,分别转染HeLa细胞,通过双荧光素酶活性分析实验进行转录活性分析。将转录因子AP-2α的过表达质粒pCMV-Myc/AP-2α分别与以上不同长度的14-3-3σ启动子萤光素酶报告基因载体共转染HeLa细胞,通过双荧光素酶活性分析实验检测AP-2α对14-3-3σ启动子转录活性的影响。结果序列分析发现人14-3-3σ启动子基因5′端1349bp的区域内存在多个潜在的AP-2α结合位点,成功构建了5个不同长度的14-3-3σ启动子报告基因载体。双荧光素酶活性分析显示5个启动子片段均有转录活性,AP-2α可增强14-3-3σ启动子转录活性。结论成功构建了14-3-3σ启动子的报告基因载体,为进一步研究AP-2α对14-3-3σ的调控奠定了基础。OBJECTIVE To construct the luciferase reporter gene vectors for human 14-3-3σ gene promoter and analyze its transcriptional activity. METHODS The 2 000 bp DNA sequence at the human 14-3-3σ gene 5 end was analyzed by the UCSC software for the promoter character and the transcription factor binding sites. Five different length 14-3-3σ promoter fragments were cloned into luciferase reporter gene vector to product five re-constructors. Then the five re-constructors were transfected into HeLa cells respectively, the dual-luciferase analysis was performed to detect the transcriptional activity of 14-3-3σ pro- moter. The AP-2α overexpression vector pCMV-Myc/AP-2α was co-transfected into HeLa cells, and the effect of AP-2α on the transcriptional activity of 14-3-3σ promoter was determined by the dual-luciferase analysis. RESULTS There were multiple potential AP-2α binding sites in the 1 349 bp region at the human 14-3-3σ gene 5 end. Five luciferase reporter gene vectors containing five different length 14-3-3σ promoters were constructed. The dual-luciferase analysis results showed all of the five promoter fragments had the transcriptional activity, and the AP-2α protein could enhance the transcriptional activity of the 14- 3-3g promoter. CONCLUSION The luciferase reporter gene vectors with the 14-3-3σ promoters are constructed successfully. This research provides an important basis for the further study on the regulation of 14-3-3σ gene by AP-2α.

关 键 词：人14—3—3σ基因 启动子 基因序列分析 转录活性分析 

分 类 号：R394-3[医药卫生—医学遗传学]