Construction and verification of SIRT1 and its mutant T200I, E420K eukaryotic expression vector
10.3760/cma.j.issn.1673-4181.2014.01.009
- VernacularTitle:SIRT1及其突变体T200I、E420K真核表达载体的构建及鉴定
- Author:
Xiaojing AN
;
Min CUI
;
Yan ZHANG
;
Qinghai MU
;
Yuhong ZHU
;
Changzhu JIN
;
Zhang CAO
- Publication Type:Journal Article
- Keywords:
Silent information regulator 1;
Eukaryotic expression vector;
Recombinant plasmid
- From:International Journal of Biomedical Engineering
2014;37(1):39-42,后插3
- CountryChina
- Language:Chinese
-
Abstract:
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.
