人akt1基因真核表达载体的构建及表达

目的:构建带有flag标签的人akt1及豆蔻酰化akt1真核表达载体,并检测其在真核细胞中的表达及对细胞周期的影响.方法:应用RT-PCR的方法从人牙髓组织mRNA中扩增出akt1和myr-akt1基因,并在其C-末端带上含24bp的flag标签,克隆到pMD-18T载体并测序,再亚克隆至真核表达载体pcDNA3.1( ),酶切鉴定正确后采用脂质体法瞬时转染COS-7细胞,Western blot检测flag-akt1及flag-myr-akt1在细胞中的表达,同时流式细胞术观察细胞周期.结果:测序证实从人牙髓组织mRNA中扩增出的flag-akt1及flag-myr-akt1融合基因的序列以及读框全部正确;脂质体法转染COS-7后检测到预期目的蛋白的表达.流式细胞术的结果显示当AKT1过度表达以及过度活化时对COS-7细胞周期未产生明显影响.结论:成功构建了C-末端带flag标签的akt1及豆蔻酰化akt1真核表达载体,并使其在真核细胞COS-7中表达.但是AKT1对细胞周期的调控有待进一步研究.

Construction and expression of human akt1 gene

AIM: To construct the eukaryotic expressing vectors of human akt 1 and N-terminally myristoylation signal-attached akt 1 ( myr - akt 1) and to test their expression in African green monkey kidney cell line COS-7. METHODS: Human akt 1 and myr-akt 1 genes tagged with flag were amplified from human dental pulp mRNA by RT-PCR. The products were cloned into pMD-18T vector, sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1(+). The recombined vectors were then transfected into COS-7 cells with lipofectin to detect the expression of akt 1 and myr-akt 1 by Western blot. The cell cycle profiles of COS-7 cells were analyzed by flow cytometry (FCM). RESULTS: The eukaryotic expressing vectors containing human akt 1 and myr-akt 1 were successfully constructed and their expression in COS-7 cells could be detected. AKT1 overexpression and activation showed little effect on the cell cycle of COS-7 by cell cycle profiles. CONCLUSION: The eukaryotic expressing vectors containing human akt 1 and myr-akt 1 are constructed and they can express flag-akt 1 and flag-myr-akt 1 fused proteins correctly in COS-7 cells. But the regulation of cell cycle by AKT1 has to be further investigated.