Antitumor effects of virus vectors that induces multisubstrate deoxyribonucleoside kinase of Drosoph

• 作 者: Xiao-Shen Dong Xin-YuZheng
• 机 构: Department of Surgical Oncology,Department of Breast Surgery,the First Affiliated Hospital,China Med
• 出 处: 22nd Asia Pacific Cancer Conference(第22届亚太抗癌大会)
• 发表日期: 2013年7期

Objective: To test the new system of suicide gene/analogs, we constructed novel gene-viral vector system with Dm-dNK, and tested the replication ability and oncolytic activity of Dm-dNK in cancer cell lines in vitro and in vivo.Methods: Synthetic DNA sequence Dm-dNK was released and ligated into plasmid pENTER12, which contains multiple clone sites, to generate pENTER12-dNK.After pENTER12-dNKmu and PPE3-ccdB recombination, these genes were transfected into 293 cells together with SG502.Nine to 14 days later,the viral plaques were obtained.Afterward, the recombinant adenovirus was extracted and verified via PCR.Enzyme assays were performed with protein extract and [3H]-labeled substrates.To estimate Dm-dNK expression, RNA from human breast cancer cell lines (MDA-MB-231, MCF-7) and human embryonic lung fibroblasts and epithelial cells (MRC-5, WI38),with SG500-CMV-DNK infection, was purified.These cells were then subjected to RT-PCR.Afterward, we tested cell cytotoxicity using MTT assay.Finally, we performed in vivo tumor-killing assay by animal experiments.