大鼠Akt1基因真核表达载体构建及其在293细胞中的表达(英文)

Construction of eukaryotic expression vector for rat Akt1 gene and its expression in 293 cells

背景:Akt在细胞存活、细胞增殖、细胞代谢和细胞凋亡等活动中扮演着重要角色。虽然Akt的作用机制目前尚有很多的未知领域,但其作为生存信号的一个重要组件,已成为近年分子生物研究中的一个重要课题。目的:构建Akt1基因真核表达载体pDC316-Akt1,观察其在293细胞(人胚肾母细胞)中的表达。设计、时间及地点:观察性实验,于2006-09/12在解放军军事医学科学院分子生物实验室完成。材料:pDC316质粒,由本元正阳公司提供;DH5α、293细胞为自备。方法:采用反转录-聚合酶链反应方法自大鼠肝组织总RNA中克隆Akt1DNA片段,测序鉴定正确后,插入真核表达载体pDC316的EcoRⅠ、HindⅢ的酶切位点,构建Akt1真核表达载体Akt-pDC316,脂质体介导法转染293细胞。主要观察指标:蛋白免疫印迹检测转染293细胞中Akt1的表达。结果:经测序鉴定,构建的Akt1氨基酸序列与野生型Akt1序列完全相符。将Akt-pDC316转入293细胞,蛋白免疫印迹检测可见相对分子质量55000位置有Akt1的表达。结论:构建Akt1基因真核表达载体在293细胞中可以充分的表达。

BACKGROUND： Akt gene plays an important role in cell survival, proliferation, metabolism, and apoptosis. Although mechanism of Akt gene still remains unclear, it, a major element of survival signal, has aroused much attention in the domain of molecular biological research. OBJECTIVE： To construct eukaryotic expression vector pDC316-Akt1 for Aktl gene and to ob.qerve the expression in 293 cells. DESIGN, TIME AND SETTING： An observational study was performed at Molecular Biological Laboratory of Academy of Military Medical Sciences of Chinese PLA between September and December 2006. MATERIALS： pCD316 plasmid was provided by Benyuan Zhengyang Company; DH5o and 293 cells were reserved in our laboratory. METHODS： Aktl DNA segment was cloned from total RNA of hepatic tissue using RT-PCR and inserted into eukaryotic expressing vector pDC316 between EcoRI and Hind III sites after sequencing. And then, 293 cells were transfected with the recombinant by liposome. MAIN OUTCOME MEASURES： Aktl gene content in transfected 293 cells using Western blotting. RESULTS： Sequencing test indicated that Aktl amino acid sequence was in accordance with wild Aktl sequence. Akt-pDC316 was transcripted in 293 cells, and Aktl protein with relative molecular weight of 55 000 was highly expressed in 293 cells when assayed using Western blotting. CONCLUSION： Eukaryotic expression vector for Aktl gene highly expresses in 293 cells.