重叠延伸PCR法构建VPS4B基因定点突变真核表达载体  

Site-directed mutagenesis of VPS4B gene based on overlap extension PCR and construction of eukaryotic expression vectors

作　　者：王维鹏[1,2] 夏剑波[1,2] 李磊[1] 郝友华[1] 杨东亮[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院临床免疫研究室,武汉430030 [2]湖北省妇幼保健院检验科,武汉430070

出　　处：《临床检验杂志》2011年第1期57-59,共3页Chinese Journal of Clinical Laboratory Science

基　　金：国家"十一五"传染病防治重大专项资助项目(2008ZX10003-011)

摘　　要：目的构建含液泡蛋白质分选因子4B(VPS4B)基因定点突变的真核表达载体。方法用RT-PCR法从HuH-7细胞中扩增VPS4B基因,并克隆到真核载体pXF3H上。采用重叠延伸PCR定点突变技术,构建K180Q和E235Q两种突变质粒,经DNA测序确证定点突变的结果,再将VPS4B及两种突变载体转染HepG2细胞,western blot检测融合蛋白表达。结果克隆了VPS4基因,构建了真核载体pXF3H-VPS4B,经重叠延伸PCR得到突变体。DNA测序结果显示,编码180位氨基酸的538～540位碱基由AAG突变为CAG;编码235位氨基酸的703～705位碱基由GAA突变为CAA,其他碱基均无突变。VPS4B及两种突变载体转染HepG2细胞可检出HA-VPS4B融合蛋白表达。结论成功构建了VPS4以及K180Q和E235Q两种突变的真核表达载体。Objective To construct eukaryotic expression vectors of vacuolar protein sorting 4B（VPS4B） gene with site-directed mutagenesis.Methods VPS4B gene was amplified by RT-PCR from Huh7 cells and cloned into the eukaryotic vector pXF3H.Site-directed mutagenesis method based on overlap extension PCR was used to construct two dominant negative（DN） mutants,K180Q and E235Q.The point mutation was verified by DNA sequencing.The mammalian hepatoma cell HepG2 was transfected with VPS4B and the 2 mutants.The expression of the fused proteins was determined by Western blot.Results VPS4B gene was cloned and the eukaryotic vector pXF3H-VPS4B was constructed.The mutants were obtained by overlap extension PCR.DNA sequencing showed that AAG at 538-540 bp site,which encoded the 180-amino acid,was changed to CAG,and in another mutant site,GAA at 703-705 bp site,which encoded the 235-amino acid,was changed to CAA.No mutation in other base pair sites of the recombinant plasmids.The fusion protein HA-VPS4B was detectable by Western blot in HepG2 cells transfected with the 2 mutant vectors.Conclusion The eukaryotic expression vectors for VPS4B and its two DN mutants K180Q and E235Q were successfully implemented.

关 键 词：细胞液泡蛋白质分选因子4B 重叠延伸PCR 定点突变 真核表达载体 

分 类 号：R971.1[医药卫生—药品]