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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 luciferase reporter gene promoter of human 14-3-3σ gene and explore its transcriptional activity. Methods The promoter characteristics and transcription factor binding sites of 5 ’end of human 14-3-3σ gene were analyzed by UCSC software. Five different lengths of promoter fragment from the 5 ’end of 14-3-3σ gene were cloned and recombined with luciferase reporter gene vector pGL3-Basic to obtain 5 different lengths of 14-3-3σ promoter luciferase reporter gene The vector was transfected into HeLa cells respectively and transcriptional activity analysis was performed by dual luciferase activity assay. The over-expression plasmid pCMV-Myc / AP-2α of transcription factor AP-2α was cotransfected into HeLa cells with the above-mentioned 14-3-3σ promoter luciferase reporter vectors of different lengths, respectively, and analyzed by dual luciferase activity The effect of AP-2α on the transcriptional activity of 14-3-3σ promoter was tested. Results Sequence analysis revealed that there were multiple potential AP-2α binding sites in the 1349bp region of the 5 ’end of human 14-3-3σ promoter gene. Five 14-3-3σ promoter reporter vectors with different lengths were successfully constructed . Dual luciferase activity assay showed that all of the five promoter fragments had transcriptional activity, and AP-2α enhanced the transcriptional activity of 14-3-3σ promoter. Conclusion The reporter vector of 14-3-3σ promoter was successfully constructed, which lays the foundation for the further study on the regulation of 14-3-3σ by AP-2α.