SIRT1及其突变体T200I、E420K真核表达载体的构建及鉴定  

Construction and verification of SIRT1 and its mutant T200I, E420K eukaryotic expression vector

作　　者：安晓静[1] 崔敏[2] 张燕[3] 牟清海[3] 朱玉红[3] 金昌洙[1] 曹璋[3] 

机构地区：[1]滨州医学院人体解剖学教研室,256603 [2]滨州市人民医院小儿科,256600 [3]滨州医学院病理学教研室,256603

出　　处：《国际生物医学工程杂志》2014年第1期39-42,I0003,共5页International Journal of Biomedical Engineering

基　　金：山东省自然科学基金资助项目（ZR2011HQ025,ZR2012HL32）;滨州医学院科研启动基金项目（BY2010KYQD10）

摘　　要：目的 克隆沉默信息调节因子1（SIRTl）基因的全长cDNA,构建含有SIRT1基因及其突变体T200I、E420K的重组真核表达载体,为进一步研究SIRT1基因功能奠定基础.方法 采用RT-PCR方法扩增SIRT1基因的全长cDNA,扩增产物通过双酶切将全长cDNA克隆到真核表达载体pcDNA3.1（＋）,得到pcDNA3.1 （＋）-SIRT1重组质粒;同时采用定点突变法构建其突变体pcDNA3.1 （＋）-T200I和pcDNA3.1（＋）-E420K表达载体.重组质粒经酶切鉴定和DNA序列测定,筛选出重组成功的真核表达载体.结果 成功克隆了SIRT1基因全长cDNA,并成功构建了pcDNA3.1 （＋）-SIRT1及其突变体的真核表达载体;阳性重组质粒酶切后经测序比对鉴定,与预期序列完全相符,转染293T细胞后可以表达带有HIS标签的SIRT1蛋白.结论 此方法可成功构建重组质粒pcDNA3.1（＋）-SIRT1及其突变体pcDNA3.1（＋）-T200I、pcDNA3.1（＋）-E420K真核表达载体,为SIRT1基因及其突变体T200I、E420K的生物学功能研究提供了基因材料.Objective To clone silent information regulator 1 （SIRT1） gene full-length cDNA,construct recombinant eukaryotic expression vector containing SIRT1 gene and its mutant T200I,E420K,so as to lay the foundation for further research of SIRT1 gene function.Methods RT-PCR amplified SIRT1 gene full-length cDNA.PCR products were cloned into the eukaryotic expression vector pcDNA3.1 （＋） through double digestion and pcDNA3.1（＋）-SIRT1 recombinant plasmid was obtained.Meanwhile,site-directed mutagenesis was applied to build its mutant pcDNA3.1 （＋）-T200I and pcDNA3.1 （＋）-E420K expression vector.Recombinant plasmid was identified by enzyme digestion and DNA sequencing and the recombinant eukaryotic expression vector of success was screened out.Results SIRT1 gene full-length cDNA was successfully cloned,and pcDNA3.1 （＋）-SIRT1 eukaryotic expression vector and its mutant were also successfully constructed.Positive recombinant plasmid sequencing was compared after enzyme digestion,and it was completely consistent with the expected sequence.Transfected 293T cell line was established and HIS tagged SIRT1 protein was expressed.Conclusions We successfully constructed pcDNA3.1 （＋）-SIRT1 and its mutant pcDNA3.1 （＋）-T200I,pcDNA3.1 （＋）-E420K eukaryotic expression vector,which may provide genetic material for biological function study of SIRT1 gene and its mutant T200I,E420K.

关 键 词：沉默信息调节因子1 真核表达载体 重组质粒 

分 类 号：Q78[生物学—分子生物学]