肝细胞永生化逆转录病毒载体pLTSN的构建

Construction of retroviral vector pLTSN for hepatocyte immortalization

目的构建含猿猴病毒40大T抗原基因(SV40T)的逆转录病毒永生化载体pLTSN,为肝细胞永生化奠定基础。方法以质粒pUC19-SV40T为模板,高保真PCR扩增SV40T,双粘端连接法将其克隆到pLXSN的EcoRⅠ和BamHⅠ位点之间,通过菌落PCR和酶切法筛选、鉴定阳性克隆并经测序验证。结果用菌落PCR和酶切法随机筛选的10个菌落中9个为阳性,阳性克隆经质粒DNA测序分析确证。结论成功构建了逆转录病毒永生化载体pLTSN。

[Objective] To construct a retroviral vector pLTSN containing simian virus large T antigen gene (SV40T) for hepatocyte immortalization. [Methods] SV40T gene was amplified by high fidelity polymerase chain reaction (PCR) using the plasmid pUC19-SV40T as the template and cloned into the EcoRⅠand BamHⅠsites of the retroviral vector pLXSN. The positive recombinant clones were screened and identified by PCR using colonies directly as templates, and by restriction endonuclease digestion analysis. [Results] Among the ten colonies randomly screened, nine proved positive, and one of them was verified by plasmid DNA sequencing. [Conclusions] The retroviral vector pLTSN containing SV40T is constructed successfully.