携带PTEN基因的重组腺病毒表达载体构建的研究

构建携带抑癌基因PTEN(Phosphatase and temin homolog deleted on chromosome ten)的重组腺病毒表达裁体,为研究PTEN的功能和作用机制奠定基础.采用RT-PCR法从大鼠海马神经元扩增目的基因PTEN,克隆人含绿色荧光蛋白(Green fluorescence protein),GFP基因的pAdTrack-CMV穿梭质粒,在含有腺病毒骨架质粒pAdEasy-1的BJ5183大肠杆菌内进行同源重组;获得重组腺病毒质粒,经Pacl线性化后,转染AD293细胞.结果表明,感染腺病毒载体的AD293细胞表达GFP基因,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathic effect,CPE),经PCR对传代的Ad-PTEN分析证实得到目的基因.成功构建了携带PTEN基因的腺病毒表达载体,为研究PTEN的功能和作用机制奠定了基础.

Construction of Recombinant Adenovirus Expression Vector Containing PTEN Gene

To construct a recombinant adenovirus expression vector containing an anti-oncogene,PTEN gene was amplified form the hippocampus of rats by RT-PCR,and cloned into shuttle plasmid pAdTrack-CMV,which containing green fluorescence protein GFP gene. The shuttle plasmid was transformed into BJ5183,which had already contained adenovirus backbone vector pAdEasy-1 to achieve the homologous recombination and btain recombinant adenovirus genome plasmid as well. This recombinant construct was subsequently linearlized with PacI and transfected into AD293 cells. Results showed that, GFP gene was expressed in recombinant adenovirus infected AD293 cells. Cytopathic effect (CPE) was found in GFP expression,which was extending and brightening as time going on. Therefore the PTEN recombinant adenovirus expression vector was constructed successfully,and the foundation for the study on function and mechanism was established.