PCNA蛋白突变体的真核表达载体构建及鉴定  

Construction and identification of eukaryotic expression vector for mutant PCNA

作　　者：刘小会[1] 淡松松[1] 秦焕焕[1] 高学娟[1] 刘朗夏[1] 

机构地区：[1]暨南大学生命与健康工程研究院,广州510632

出　　处：《重庆医学》2012年第12期1194-1196,共3页Chongqing medicine

基　　金：国家973重点基础研究发展计划资助项目(2011CB910701);国家自然科学基金青年科学基金资助项目(31000628);暨南大学引进优秀人才科研启动基金资助项目(50624001);广东省高等学校高层次人才资助项目(50524006)

摘　　要：目的构建带FLAG标签的细胞增殖核抗原(PCNA)蛋白Y211A、Y211D突变型重组质粒,真核表达及鉴定。方法以FLAG-PCNA野生型基因为模板,运用PCR定点突变技术扩增出带突变位点的基因序列,将其插入真核表达载体pCMV-N-FLAG,进行双酶切实验和测序验证。然后,利用脂质体将重组质粒转染至293T细胞,Western blotting鉴定融合蛋白的表达。结果 FLAG-PCNA(Y211A)、FLAG-PCNA(Y211D)重组质粒测序结果正确,获得PCNA蛋白突变体。结论成功构建了带FLAG标签的PCNA蛋白Y211A、Y211D真核表达载体,验证了突变型PCNA融合蛋白的表达,为PCNA蛋白功能的深入研究奠定了基础。Objective To construct and express FLAG-PCNA gene mutant in 293T cells.Methods Mutant FLAG-PCNA cDNA was amplified by PCR site-directed mutagenesis with FLAG-PCNA gene as template,and inserted into the eukaryotic expression vector pCMV-N-FLAG.The recombinant vector was then transfected into human embryonic kidney（HEK） 293T cells.The cells were harvested after 48h to identify the expression of plasmids by western blot.Results Sequences of recombinant plasmid were correct,and the mutants of PCNA were obtained.Conclusion Eukaryotic plasmids of mutant PCNA were obtained,and the expression of mutant PCNA protein was identified,which lay a foundation for further research of PCNA protein.

关 键 词：PCNA蛋白 突变体 真核表达 

分 类 号：Q78[生物学—分子生物学]