【摘要】 目的:在HEK293细胞中获得Sirt3全长基因,构建其高效真核表达载体。方法:提取人HEK293细胞总RNA,反转录合成c DNA,以特异性引物扩增Sirt3全长基因并克隆入pc DNA3.1载体,筛选阳性克隆进行序列测定后,鉴定其表达情况。结果:PCR扩增得到特异性的约1000 bp的片段,序列测定结果表明与国外已发表的序列完全一致;将构建的pc DNA3.1-Sirt3质粒转入AGS和SGC-7901细胞,检测到Sirt3的m RNA和蛋白表达水平均提高。结论:构建获得Sirt3基因高效真核表达载体,将用于Sirt3的功能研究。更多还原

【Abstract】 Objective: To clone the Sirt3 full length gene from human HEK293 cells and construct its eukaryot-ic expression vector. Methods: Total RNA was extracted from cultured human HEK293, and the Sirt3 c DNA frag-ment was obtained by RT-PCR with two specific gene primers, and then it was inserted into pc DNA3.1 vector toconstruct the expression vector. Results: The 1000 bp specific fragment was obtained, and the sequence of whichwas consistent with published details. After the plasmid pc DNA3.1-Sirt3 was transformed into AGS and SGC-7901 cells, the increased m RNA and protein level of Sir3 were all detected. Conclusion: The Sirt3 expression vectorwill be used in the further studies of the roles of Sirt3.更多还原