应用Gibson Assembly法快速构建人组蛋白甲基化转移酶启动子荧光素酶报告载体

Construction of human enhancer of zeste homolog 2 promoter reporter vector using Gibson Assembly method

目的:快速构建人组蛋白甲基转移酶(enhancer of zeste homolog 2,EZH2)启动子荧光素酶报告载体并验证其活性。方法:因EZH2启动子富含GC,故应用Gibson Assembly方法设计分两段扩增,然后将片段一步法连入pGL3-Basic质粒,构建荧光素酶报告载体,进行酶切鉴定。与p RL-TK内参照质粒共转染C4-2细胞,双荧光素酶分析法检测启动子的活性。结果:经酶切鉴定证实成功构建了EZH2启动子报告载体,双荧光索酶报告基因检测结果显示构建的报告载体具有启动子活性。结论:快速成功构建了具有启动子活性的EZH2启动子荧光素酶报告载体,为进一步研究前列腺癌中EZH2表达调控机制提供必要的实验材料。

Objective： To construct a promoter reporter vector of enhancer of zeste homolog 2 （EZH2）.Methods： Since EZH2 promoter is GC rich, its sequence were divided into two segments to clone. Primers were designed according to the Gibson rule to amplify sequence of the two segments. Polymerase chain reaction was performed. PCR products were then recovered and cloned in to pGL3-Basic vector using Gibson Assembly method. The constructed pGL3-EZH2-promoter vector was then veriifed by dual luciferase assay in prostate cancer C4-2 cells.Results： hTe EZH2 promoter reporter vector pGL3-EZH2-Promoter was successfully constructed. Dual luciferase reporter assays showed that the reporter vector have promoter activity.Conclusion：EZH2 promoter reporter vector was constructed using a quick method.