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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。
OBJECTIVE: To clone rat Sirt2 gene and construct its eukaryotic expression vector and express it in HEK293 cells. Methods: The cDNA fragment containing the Sirt2 coding region was amplified by RT-PCR from rat brain total RNA. The purified T-A clone was ligated into pMD20-T vector. Using this as a template, the coding region of the gene was cloned into the eukaryotic expression vector pcDNA3.1myc-his (-) and transfected into HEK293 cells to detect its expression. Results: Sequencing confirmed that the cloned Sirt2 coding region was correctly inserted into pcDNA3.1myc-his (-) and confirmed by immunofluorescence in HEK293 cells. CONCLUSION: The rat Sirt2 cDNA was cloned successfully and its eukaryotic expression vector was constructed and expressed efficiently in HEK293 cells, which laid the foundation for further study on the function of Sirt2 in rats.