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[目的]构建稳定表达ePNP(E.coliPNP)的细胞模型,为肿瘤的基因治疗提供实验基础。[方法]提取大肠杆菌E.coliK12总DNA,PCR方法克隆大肠杆菌ePNP基因,构建逆转录病毒载体质粒pMSCV-ANGPTL4,鉴定测序。脂质体法将三种逆转录病毒载体pMSCV-ANGPTL4、pVSV、pGAG-POL共转染293-EBNA包装细胞,获得高滴度病毒并感染MDA-MB435细胞。荧光显微镜与RT-PCR检测MDA-MB435细胞ePNP基因的表达。[结果]所克隆的ePNP基因与文献报道一致;成功构建了pMSCV/ePNP重组逆转录病毒载体;ePNP基因在MDA-MB435细胞中获得稳定表达。[结论]pMSCV/ePNP自杀基因载体的成功构建及其表达为ePNP应用于肿瘤基因治疗奠定了基础。
[Objective] To construct a cell model stably expressing ePNP (E. coliPNP) and provide the experimental basis for the gene therapy of tumors. [Method] The total DNA of E. coli K12 was extracted. The ePNP gene of E. coli was cloned by PCR. The retroviral vector pMSCV-ANGPTL4 was constructed and sequenced. Three retroviral vectors, pMSCV-ANGPTL4, pVSV and pGAG-POL, were cotransfected into 293-EBNA packaging cells by liposome method to obtain high-titer virus and to infect MDA-MB435 cells. The expression of ePNP gene in MDA-MB435 cells was detected by fluorescence microscopy and RT-PCR. [Result] The cloned ePNP gene was consistent with that reported in the literature. The pMSCV / ePNP recombinant retroviral vector was successfully constructed and the ePNP gene was stably expressed in MDA-MB435 cells. [Conclusion] The successful construction of pMSCV / ePNP suicide gene vector and its expression laid the foundation for the application of ePNP in tumor gene therapy.