Sirt2基因的克隆及其在HEK293中的表达  

Cloning and expression analysis of Sirt2 in HEK293 cells

作　　者：王涛[1] 王伟[1] 徐清[1] 关路媛[1] 张斌[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2009年第11期970-972,共3页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(3077042030871239)

摘　　要：目的:克隆大鼠Sirt2基因,构建其真核表达载体并在HEK293细胞中表达。方法:利用RT-PCR从大鼠脑组织总RNA中扩增出包含Sirt2编码区的cDNA片段,产物纯化后T-A克隆连接至pMD20-T载体。以此为模板,将该基因编码区克隆入真核表达载体pcDNA3.1myc-his(-)中,转染HEK293细胞检测其表达。结果:测序证实所克隆的Sirt2编码区cDNA正确地插入pcDNA3.1myc-his(-)中,经免疫荧光检测证实其在HEK293细胞中得到表达。结论:成功克隆了大鼠Sirt2 cDNA,构建了其真核表达载体,并在HEK293细胞中得到有效表达,为进一步研究大鼠Sirt2的功能奠定了基础。AIM： To construct eukaryotic expression vector of Sirt2, and detect its expression in HEK293 cells. METHODS： Total RNA was isolated from brain tissue of adult SD rat, A 1130 bp fragment containing the coding region of Sirt2 was amplified by RT-PCR and the resulting PCR product was subcloned into PMD20-T vector and sequenced. Coding region of Sirt2 was generated with PCR by using the PMD20-T-Sirt2 as template, the amplified PCR fragment was inserted into the EcoR Ⅰand Hind Ⅲ sites of the pcDNA3.1 myc-his （ - ）A expression vector, and the sequence was confirmed by DNA sequencing. The expression of new construct pcDNA3.1 myc-his（ - ）A-Sirt2 in HEK293 cells was detected by immunofluorescence. RESULTS： The full length coding region of Sirt2 was obtained and confirmed by sequencing, the expression of Sirt2 was detected successfully in HEK293 cells. CONCLUSION： The eukaryotic expression vector of Sirt2 has been successfully constructed, which will provide a useful tool for designing an in-depth investigation of the role of Sirt2.

关 键 词：Sirt2 真核表达 克隆 CDNA 

分 类 号：Q786[生物学—分子生物学]