重组含HSV-TK基因腺病毒的构建及对肿瘤细胞的杀伤作用

CONSTRUCTURE OF A RECOMBINANT ADENOVIRUS VECTOR WITH HSV-TK AND IT'S KILLER EFFECT ON TUMOR CELLS IN VITRO

构建含CMV启动子和HSV-TK基因的复制缺陷型重组腺病毒载体(AdCMVTK),用PCR及Southern杂交筛选鉴定阳性空斑,证实AdCMVTK含HSV-TK基因。纯化的Ad-CMVTK滴度达10^(12)pfu/ml。GCV对经Ad-CMVTK感染(M.O.I.=100)的HeLa等3种肿瘤细胞的IC50<4μmol/L,同时观察到明显的旁杀伤效应。AdCMVTK/GCV系统的建立为肿瘤基因治疗的研究提供了新的手段。

High-titer replication-defective recombinant adenovirus expressing HSV-TK gene was constructed. Firstly, shuttle plasmid pAdCMVTK containing HSV-TK gene and CMV promoter was constructed and then recombinated with right arm of adenovirus DNA. Secondly, the positive plaques containing recombinant adenovirus were identified and selected out by PCR and Southern blotting after infection into human embryo kindny 293 cells. The titer of recombinant adenovirus AdCMVTK was determined by plaque forming assay and it was as high as 1012 pfu/ml. Tumor cells were infected with AdCMVTK and then treated with GCV. Cytotoxic effects were assayed with MTT method. HeLa, A549 and LoVo cells infected with AdCMVTK (M. O. I. =100) became sensitive to the prodrug GCV, with IC50 less than 4 μmol/L. Significant bystander effect was observed. Results here show that the AdCMVTK/GCV system might be potential in the gene therapy for cancer.