【摘要】 目的构建表达肿瘤增殖基因Ki67小干扰RNA(Ki67-siRNA)的选择性增殖溶瘤腺病毒。方法设计有小发夹结构的Ki67-siRNA对应模板DNA序列,煺火处理后克隆至pCA13质粒,构建重组质粒pCA13-Ki67。用BglⅡ从pCA13-Ki67酶切出包含CMV启动子及Ki67-siRNA模板DNA序列的表达框,将此表达框克隆进选择性增殖腺病毒质粒pZD55,构建重组质粒pZD55- Ki67。将pZD55-Ki67与含有腺病毒右臂的质粒pBHGE3共转染293细胞,9～12 d后出现病毒空斑。提取重组腺病毒的DNA,聚合酶链反应(PCR)鉴定正确者即为选择性增殖溶瘤腺病毒ZD55- Ki67。大量扩增,氯化铯梯度离心纯化,测病毒滴度。结果成功构建了表达Ki67-siRNA的选择性增殖溶瘤腺病毒。结论成功构建的ZD55-Ki67为利用Ki67-siRNA靶向肿瘤治疗奠定了基础。更多还原

【Abstract】 Objective To construct replication-selective oncolytic adenovirus(CRAds)expressing short interference RNA(siRNA)targeting Ki67 gene(Ki67-siRNA).Methods Ki67 siRNA template DNA sequences were designed and synthesized.The annealed siRNA template was inserted into pCA13 plasmid to construct the recombinant plasmid(pCA13-Ki67).It was identified that vector had been con- strutted successfully.The pCA13-Ki67 plasmid was cut with BglⅡto get the expression cassette of Ki67- siRNA,and then the expression cassette was cloned into pZD55 which has been cut with BglⅡand de- phosphated to form pZD55-Ki67 plasmid.It was identified that the pZD55-Ki67 had been constructed successfully.The plasmid pZD55-Ki67 was transfected into 293 cells together with plasmid pBHGE3 to abtain the recombinant CRAds,ZD55-Ki67.The viral plaques appeared 9-12 days after infection.The DNA was extracted from recombinant adenoviruses and verified by PCR.Viruses were plaque purified, propagated on HEK293 cells and purified by CsCl gradient according to standard techniques,and func- tional PFU titers were determined by plaque assay on 293 cells.Results CRAds expressing siRNA tar- geting Ki67 gene had been constructed successfully.Conclusion CRAds expressing Ki67-siRNA may be used for further investigation of Gene-Viro therapy of cancer.更多还原