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
| 作者: |
1朱进,
1单玉喜,
1阳东荣
1 苏州大学附属第二医院泌尿外科，江苏 苏州 215004 |
| 通讯: |
阳东荣
Email: doc_ydr@163.com |
| DOI: | 10.3978/j.issn.2095-6959.2016.12.015 |
| 基金: | 国家自然科学基金, 81472776 教育部博士学科点专项科研基金, 20133201110016 江苏省自然科学基 金, BK20161222 苏州市科技计划项目, SYS201629 |
摘要
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pRL-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。
关键词：
Gibson Assembly
报告载体
人组蛋白甲基转移酶
启动子活性
Construction of human enhancer of zeste homolog 2 promoter reporter vector using Gibson Assembly method
CorrespondingAuthor: YANG Dongrong Email: doc_ydr@163.com
DOI: 10.3978/j.issn.2095-6959.2016.12.015
Abstract
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 verified by dual luciferase assay in prostate cancer C4-2 cells. Results: The 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.