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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。
Objective To construct a selectively proliferating oncolytic adenovirus expressing Ki67 siRNA (Ki67-siRNA). Methods The corresponding template DNA sequence of Ki67-siRNA with small hairpin structure was designed and cloned into pCA13 plasmid after fire treatment. The recombinant plasmid pCA13-Ki67 was constructed. The expression cassette containing CMV promoter and Ki67-siRNA template DNA was digested with BglII from pCA13-Ki67. The expression cassette was cloned into the recombinant adenovirus plasmid pZD55 to construct the recombinant plasmid pZD55- Ki67. 293 cells were co-transfected with pZD55-Ki67 and plasmid pBHGE3 containing the right arm of adenovirus, and viral plaques appeared after 9 to 12 days. DNA extracted recombinant adenovirus, polymerase chain reaction (PCR) identification is the correct selection of oncolytic adenovirus ZD55- Ki67. A large number of amplification, cesium chloride gradient centrifugation, virus titer. Results The selective proliferation oncolytic adenovirus expressing Ki67-siRNA was successfully constructed. Conclusion The successful construction of ZD55-Ki67 lays a foundation for targeting Ki67-siRNA targeting tumors.