细胞核增殖抗原启动子调控荷载双基因RNA干扰的条件增殖腺病毒的构建  

Construction of conditionally replicative adenovirus expressing proliferation cell nuclear antigen siRNA, human telomerase reverse transcriptase siRNA with proliferation cell nuclear antigen pro- moter regulation

作　　者：陈仁富[1] 程伟松[1] 程乾 李连涛 方琳 刘俊杰[1] 温儒民[1] 李望[1] 郑骏年 

机构地区：[1]徐州医学院附属医院泌尿外科,江苏221002 [2]江苏省肿瘤生物治疗重点实验室

出　　处：《中华实验外科杂志》2012年第10期1974-1976,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30972976）;江苏省自然基金资助项目（BK2010177、BK2009091）

摘　　要：目的构建细胞核增殖抗原（Ki-67）启动子调控表达Ki．67基因和人端粒酶逆转录酶（hTERT）基因小干扰RNA（siRNA）的条件增殖腺病毒。方法（1）合成hTERT—siRNA插入pSilenc—er3．1，构建pShTERTsiRNA质粒。（2）分别酶切Ki-67启动子质粒pZKi-67、Ki-67．siRNA质粒pZD55-Ki-67-siRNA，获得Ki-67启动子片段和Ki-67．siRNA片段并连接，构建质粒pZKi-67-ZD-Ki-67-siRNA。（3）以pShTERT—siRNA为模板，PCR获得hTERT—siRNA及H1启动子片段，克隆进pZKi-67-ZD—Ki-67-siRNA，构建质粒pZKi-67-ZD—Ki-67-siRNA．hTERT．siRNA。（4）将其与腺病毒骨架质粒pBHGE3共转染293细胞，9～12d后出现病毒空斑。提取重组腺病毒DNA，行聚合酶链反应（PCR）鉴定。结果病毒ZKi-67-ZD—Ki-67-siRNA—hTERT—siRNA构建成功，其包含Ki-67启动子、Ki-67-siRNA、hTERT—siRNA片段。结论成功构建Ki-67启动子调控荷载Ki-67、hTERT双基因RNA干扰的条件增殖腺病毒，为研究靶向肿瘤治疗奠定基础。Objective To construct conditionally replicative adenovirus （CRAds） expressing proliferation cell nuclear antigen （Ki-67） siRNA and human telomerase reverse transcriptase （hTERT） siRNA with Ki-67 promoter regulation. Methods （ 1 ） The hTERT siRNA sequence was synthesized and cloned into pSilencer3.1, resulting in the plasmid pShTERT siRNA. （2） Both pZKi-67 and pZD55-Ki-67 siRNA plasmid were digested with Xba I/EcoRV, the fragment containing Ki-67 promoter and the fragment con- taining Ki-67siRNA were connected, resulting in the plasmid pZKi-67-ZD-Ki-67siRNA. （3） The hTER- TsiRNA was amplified from pShTERT siRNA and cloned into pZKi-67-ZD-Ki-67siRNA, resulting in the plasmid pZKi-67-ZD-Ki-67siRNA-hTERT siRNA. （4） pZKi-67-ZD-Ki-67 siRNA-hTERT siRNA was mixed with adenovirus packaging plasmid pBHGE3 and then co-transfected into HEK293 cells. The viral plaques appeared 9-12 days after infection./To verify the sequences of ZKi-67-ZD-Ki-67siRNA-hTERTsiR- NA, DNA was extracted from purified viru^esr and analysed by polymerase chain reaction （PCR）. Results Confirmed by PCR assay and sequence! assay, a dual-RNA interferenced oncolytic adenovirus, which carried the Ki-67-siRNA and hTERT-siRNA and synchronously was regulated by both Ki-67 promoter was constructed. Conclusion We successfully constructed a dual-RNA interferenced oncolytic adenovirus and provide a new platform for cancer gene therapy.

关 键 词：条件增殖腺病毒 细胞核增殖抗原启动子 细胞核增殖抗原基因 人端粒酶逆转录酶基因 RNA干扰 

分 类 号：R3[医药卫生—基础医学]