Sirt2基因的克隆及其在HEK293中的表达

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.

Cloning and expression analysis of Sirt2 in HEK293 cells

AIM:To construct eukaryotic expression vector of Sirt2 and detect its expression in HEK293 cells. METHODS: Total RNA was isolated from brain tissue of a-dult SD rat. A 1 130 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 se-quenced. 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 I and Hind Ⅲ sites of the pcDNA3. 1myc-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.