中文摘要：

目的构建hTERT启动子调控的表达β—actin、GAPDH基因小干扰RNA（siRNA）的条件增殖腺病毒。方法分别将β—actin—siRNA、GAPDH—siRNA的模板DNA克隆至质粒pGPH1／GFP／Neo，构建pGPH1／GFP／Neo—β-actin、pGPH1／GFP／Neo—GAPDH。以pGPH1／GFP／Neo-β—actin为模板，PCR扩增出β—actin—siRNA表达框并克隆进质粒pZHTERT，获得pZHTERT—β—actin；以pGPH1／GFP／Neo-GAPDH为模板，扩增出GAPDH表达框并克隆进质粒pZD55，获得pZD55-GAPDH。将pZHTERT—β—actin、pZD55-GAPDH重组，构建穿梭质粒pTD—GAPDH—β—actin，并与腺病毒右臂质粒pBHGE3共转染293细胞，9～12d后出现病毒空斑，提取重组腺病毒的DNA、聚合酶链反应（PCR）鉴定正确者即为双调控增殖腺病毒TD—GAPDH—β—actin。扩增、纯化、测病毒滴度。结果成功构建hTERT启动子调控的表达GAPDH、β—actin小干扰RNA的条件增殖腺病毒。病毒滴度为2×10^11PFU／ml。结论成功构建了TD—GAPDH—β—actin，为研究双基因小干扰RNA靶向治疗肿瘤奠定了基础。

英文摘要：

Objective To construct conditionally replicative adenovirus （CRAds） expressing short interference RNA （siRNA） targeting the β-actin and GAPDH genes with hTERT promoter regulation and control. Methods β-actin and GAPDH siRNA template DNA sequences were inserted into pGPH1/ GFP/Neo plasmid, respectively. Two pairs of specific primers were designed and used to get the expression cassettes of β-actin-siRNA and GAPDH-siRNA which were inserted into pZHTERT and pZD55 plasmid to construct the recombinant plasmid pZHTERT-β-actin and pZD55-GAPDH,respectively. The pZHTERT-β- actin plasmid was cut with Kpn I and Pvu [ to get the expression cassette of β-actin which was cloned into pZD55-GAPDH to form pTD-GAPDH-β-actin plasmid. The plasmid pTD-GAPDH-β-actin was transfected into 293 cells together with plasmid pBHGE3 to obtain the recombinant CRAds, TD-GAPDH-β-actin. The viral plaques appeared 9-12 days after infection. The extracted DNA of recombinant adenoviruses was verified by PCR. Viruses were plaque purified, propagated on HEK293 cells and purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. Results The conditionally replicative adenovirus expressing siRNA targeting the GAPDH and β-actin genes with hTERT promoter regulation and control had been constructed successfully. Viral PFU titers were 2 × 10^11 PFU/ml. Conclusion The conditionally replicative adenovirus will lay a good foundation for further study on cancer gene therapy.