AKT1S1-pBABE重组逆转录病毒载体的构建及其稳定过表达细胞系的筛选

Construction of Recombinant Retroviral Vector AKT1S1-pBABE and Screening of Stable Overexpression Cell-line

AKT Substrate 1 (AKT1S1)又名富含脯氨酸AKT1底物蛋白(proline-rich AKT1 substrate of 40 kDa,PRAS40)。作为经典的AKT1激酶底物,其在肿瘤生成过程中发挥着重要功能。为研究AKT1S1具体的相互作用蛋白以及作用通路,通过逆转录的方法获得了来自293T细胞的cDNA库。通过基因克隆的方法,成功克隆到AKT1S1基因,并将该基因插入至重组逆转录病毒载体pBABE-Flag质粒中,构建了Flag-AKT1S1-pBABE质粒。在辅助质粒的帮助下,在p293T细胞中包装病毒,侵染293T细胞,使用嘌呤霉素筛选目标细胞,经过蛋白质免疫印迹试验鉴定,获得了Flag-AKT1S1-pBABE稳定过表达细胞系,为下一步开展AKT1S1相互作用蛋白的质谱鉴定及其在肿瘤发生中的研究打下坚实基础。

AKT Substrate 1(AKT1 S1) is also known as proline-rich AKT1 substrate of 40 kDa(PRAS40). As a classical AKT kinase substrate, it plays an important role in tumorigenesis. The cDNA library from 293 T cells was obtained by reverse transcription to study the protein-protein interaction and pathway of AKT1 S1. The AKT1 S1 gene was successfully cloned by molecular cloning and inserted into the recombinant retroviral vector pBABE-Flag plasmid to construct Flag-AKT1 S1-pBABE plasmid. With the help of auxiliary plasmid, the virus was packaged in p293 T cells and infected 293 T cells. Puromycin was used to screen target cells. The stable expression cell line Flag-AKT1 S1-pBABE was obtained and identified by Western blot, which laid a solid foundation for the research on AKT1 S1 interacting protein identification and its oncogenesis studies.