重叠PCR构建EGR1启动子中Elk1结合位点突变载体.doc

ELK1结合位点突变的EGR1启动子载体构建和活性测定 梁旭竞1 ，张惠华2，熊毅2，陈小佳2*通讯作者 通讯作者: 陈小佳? 电话：(020Email: tchenxj@ 1.暨南大学附属第一医院感染科2.基因工程药物国家工程研究中心, 广东省生物工程药物重点实验室, 暨南大学生物工程研究所，广州　510632 摘要 目的 构建EGR1启动子片段中ELK1结合位点突变的载体并测定其活性。方法 以已构建的人EGR1启动子载体为模板，在引物上引入突变碱基，重叠PCR扩增获得突变启动子序列，T载体亚克隆入pGL3-basic荧光表达载体，获得重组突变载体pGL3-EGR1mt。用Lipofectamine 2000共转染293A细胞，化学发光法检测荧光素酶活性。结果 经酶切、PCR及测序鉴定显示，成功构建了pGL3-EGR1mt载体，应用双荧光报告系统检测显示转染了重组突变载体的细胞的荧光素酶活性明显低于未突变组，说明ELK1位点突变成功抑制了启动子的活性。结论 成功构建了pGL3-EGR1mt载体，为进一步研究EGR1对下游信号通路的影响提供了实验基础。 关键词 :EGR1启动子；重叠PCR ；点突变 中图分类号：Q78 Construction and activity determination of EGR1 promoter vector with ELK1 binding site mutation Liang Xu-Jing1, Zhang Hui-hua2, Xiong Yi2, Chen Xiao-Jia2 （1Department of Infectious Diseases, the First Affiliated Hospital of Jinan University, Guangzhou 510630；2 National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, Bioengineering Institute of Jinan ,University, Guangzhou 510632, China） Abstract Objective To construct EGR1 promoter report vector with ELK1 binding site mutation and evaluate its activity. Methods The EGR1 promoter sequence with ELK1 binding site mutation was obtained by overlap PCR assay using point mutated primers. pGL3-EGR1mt was obtained by which the targeted sequence was subcloned into pGL3-basic vector through T-vector. Then the activities of fluorescence were detected by chemiluminescence method after the vectors were cotransfected into the 293A cells with Lipofectamine 2000. Results The pGL3-EGR1mt plasmid was constructed successfully by restriction enzyme digestion, PCR and the analysis of nucleotide sequence. The activities of fluorescence in the group of pGL3-EGR1mt were lower than that of pGL3-EGR1 by double fluorescent reporter system, which implied that mutation of ELK1 inhibited promoter activity. Conclusion The pGL3-EGR1mt plasmid was constructed successfully, which provide an experimental basis for further study of the effect of EGR1 on downstream signa