中文摘要：

目的构建一种受肾癌G250启动子及肿瘤细胞p53突变双重调控的表达Ki67基因小干扰RNA（Ki67-E1B55kDsiRNA）的新型增殖腺病毒。方法将含有H1启动子的Ki67-siRNA表达框插入E1B55kD缺失增殖腺病毒载体pZD55中，构建pZD55-Ki67质粒。用G250启动子取代pZD55-Ki67的E1A启动子，构建双调控增殖腺病毒载体pZD—G250-Ki67。将pZD—G250-Ki67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）鉴定正确者即为条件增殖腺病毒ZD—G250-Ki67。鉴定、扩增、纯化、测病毒滴度。结果成功构建G250启动子调控E1B55kD蛋白编码基因缺失的表达Ki67-siRNA的双调控增殖型腺病毒ZD—G250-Ki67。病毒滴度为2×10^11PFU／ml。结论成功构建的ZD—G250-Ki67为利用Ki67-siRNA靶向肾癌治疗奠定基础。

英文摘要：

Objective To construct conditionally replicative adenovirus （CRAds） expressing short interference RNA targeting Ki67 gene （Ki67-siRNA） regulated by G250 promoter and p53-dysfunction of tumor cells. Methods The siRNA expression cassettes of Ki67 gene containing H1 promoter were produced by PCR from pSilencer3. 1-Ki67 and inserted into pZD55, an E1B 55kD-deficient adenovirus vector to construct pZD55-Ki67-siRNA successfully. The E1A promoter of pZD55-Ki67-siRNA was replaced by G250 promoter to construct pZD-G250-Ki67 successfully. The plasmid pZD-G250-Ki67 was transfected into 293 cells together with plasmid pBHGE3 to obtain the recombinant CRAds, ZD-G250- Ki67. The viral plaques appeared 9-12 days after infection. The recombinant adenoviruses were verified by PCR. Viruses were plaque purified, propagated on HEK293 ceils and functional PFU titers were determined by plaque assay on 293 ceils. Results CRAds expressing Ki67-siRNA gene regulated by G250 promoter and p53-dysfunction of tumor cells had been constructed successfully. Viral PFU titers were 2 × 10^11 PFU/ ml. Conclusion The CRAds constructed may be used for further investigation on Gene-Viro therapy of renal carcinoma.