Sirt3过表达载体的构建及表达

Construction of Sirt3 Eukaryotic Expression Vector

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。

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.