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* 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, 南大学生物工程研究所，广州　510632EGR1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 signaling pathways. Key words: EGR1 promoter；overlapping PCR ；site mutant EGR1(early growth response 1)属于即刻早期基因家族中的一员 [1]，位于人类染色体5q31，由533个氨基酸组成的分子量为75kD的蛋白质[2]。EGR1在不同细胞类型或刺激下，可结合到目的基因DNA上起到激活转录作用，促进细胞的分化、生长、生长抑制和凋亡[3]。有研究报道，EGR1启动子上有多种转录因子结合位点，包括血清反应元件SREs、AP-1结合位点、cAMP调控元件CREs和SP1结合位点等[4]。Ahmed[5]发现，EGR1作为癌症基因治疗中的应用主要有两个方面：第一，EGR1启动子含有空间和时间表达的信息，可通过离子辐射控制具有凋亡和自杀功能的基因的表达来调控EGR1的表达