含HSVtk基因重组逆转录病毒包装细胞PA317/TK的建立

Construction of the package cells( PA317/TK) with recombinant retroviral vector containing HSVtk gene

目的 :研究肿瘤的自杀基因疗法 ,建立含单纯疱疹病毒胸苷激酶 (HSVtk)基因重组逆转录病毒包装细胞PA317/TK。 方法 :应用脂质体转移方法将逆转录病毒载体GINaTK导入PA317,经G4 18筛选阳性克隆细胞后分别用PCR和扫描电镜检测HSVtk基因整合和病毒颗粒分泌情况。 结果 :逆转录病毒载体GINaTK导入PA317,G4 18后筛选得到阳性克隆细胞 ,命名为PA317/TK。PCR检测PA317/TK细胞出现一特异性 4 0 4bp阳性条带 ,而PA317细胞没有扩增出相应条带。PA317/TK细胞经扫描电镜检测见其表面有颗粒状突起 ,其周围有许多呈团状的病毒颗粒。证实含HSVtk基因重组逆转录病毒存在于包装细胞中。 结论

Objectives:In order to study suicide gene therapy on tumor, we constructed the package cell PA317/TK with recombinant retroviral vector containing HSVtk gene. Methods:GINaTK retroviral vector containing HSVtk gene was transduced into PA317 retroviral packaging cell by lipofectin,PA317 cells transfected with HSVtk gene were then screened by G418 medium,and positive clones were respectively measured with PCR and with scan electron microscrope. Results: PA317 cells were transfected successfully with HSVtk gene by lipofectin(named PA317/TK).HSVtk gene integrated into PA317 cells was further identified with PCR.Package cells contain recombinant retrovirus particulate with HSVtk gene with scan electron microscrope. Conclusions: The package cells( PA317/TK) with recombinant retroviral vector containing HSVtk gene were successfully constructed.