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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 plays an important role in activities such as cell survival, cell proliferation, cell metabolism and apoptosis. Although Akt’s mechanism of action still has many unknown fields, as an important component of survival signal, Akt has become an important issue in molecular biology research in recent years. Objective: To construct the eukaryotic expression vector pDC316-Akt1 of Akt1 gene and observe its expression in 293 cells (human embryonic kidney blast cell). DESIGN, TIME AND SETTING: Observational experiments were performed at the Molecular Biology Laboratory, Chinese Academy of Military Medical Sciences, September 9, 2006. Material: pDC316 plasmid, provided by the company Zhengyang; DH5α, 293 cells for their own. Methods: The Akt1 DNA fragment was cloned from the total RNA of rat liver tissue by reverse transcription polymerase chain reaction (RT - PCR). After sequencing, the EcoRI and HindIII restriction sites were inserted into the eukaryotic expression vector pDC316 to construct eukaryotic expression vector of Akt1 Vector Akt-pDC316, lipofectamine mediated 293 cells. MAIN OUTCOME MEASURES: Akt1 expression was detected by Western blotting in 293 cells. Results: After sequencing, the constructed Akt1 amino acid sequence was completely consistent with the wild type Akt1 sequence. Akt-pDC316 was transfected into 293 cells, Western blot detection showed that the relative molecular mass of 55000 position Akt1 expression. Conclusion: The construction of eukaryotic expression vector of Akt1 gene can be fully expressed in 293 cells.