重叠延伸PCR法构建小鼠Sumf1基因的定点突变真核表达载体  

Site-directed Mutagenesis of Sumf 1 Gene of Mouse Based on Overlap Extension PCR and Construction of Eukaryotic Expression Vector

作　　者：帅勇[1] 胡兴旺[1] 易朵[1] 王成[1] 赵晓蒙[1] 周畅[1] 

机构地区：[1]湖南师范大学生命科学学院蛋白质化学与发育生物学教育部重点实验室,中国湖南长沙410081

出　　处：《生命科学研究》2009年第5期430-436,共7页Life Science Research

基　　金：国家自然科学基金资助项目(30500258);湖南省教育厅科研资助项目(09B059)

摘　　要：前期通过染色质免疫沉淀技术（chromatin immunoprecipitation assay，CHIP）筛选出了转录因子activator protein．2 alpha（AP-2α）的靶基因Sumf1．采用重叠延伸PCR定点诱变技术，对AP．2α在Sumf1内含子片段上的两个结合位点的碱基进行定点突变，并构建定点突变表达载体．DNA测序表明，Sumf1内含子片段103—111bp，411～419bp两处的碱基已分别由GCCGTCAGG突变为GAAGTCCTG，由GCCTCTAGG突变为GGATCTCTG，成功实现定点诱变，为进一步研究AP-2α对基因Sumf1的表达调控奠定了基础．Through ChIP experiment, Sumfl was screened out as one of the target genes of transcription factor activator protein-2 alpha（AP-2α）. Site-directed mutagenesis method based on overlap extension PCR was used to introduce mutations in the two sites which were AP-2α binding sites in the Sumfl intron fragment, and the site-directed mutagenesis eukaryotic expression vector were constructed. DNA sequencing showed that GCCGTCAGG of 103-111 bp sites had been changed into GAATGTCGTG and GCCTCTAGG of 411-419 bp sites had been changed into GGATCTGTG from mutagenesis. Site-directed mutagenesis was successfully implemented and laid the foundation for further study of AP-2α regulation of the expression of Sumf1.

关 键 词：转录因子AP-2α 硫酸酯酶修饰因子Sumf1 重叠延伸PCR 定点诱变 

分 类 号：Q782[生物学—分子生物学] Q78