Nanog基因RNA干扰慢病毒载体构建与鉴定  

Construction and identification of lentiviral vector of the RNA interference of human Nanog gene

作　　者：李冬雪 牛朝诗 鲍得俊 夏成雨 程传东 杨洋 李静[1] 

机构地区：[1]安徽医科大学附属省立医院神经外科脑功能与脑疾病安徽省重点实验室安徽省脑立体定向神经外科研究所,合肥230001

出　　处：《中华实验外科杂志》2014年第11期2450-2452,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81172407）;安徽医科大学校科学研究基金项目（2012xkj043）

摘　　要：目的 构建Nanog基因RNA干扰慢病毒载体,为后续进一步体内外研究干扰Nanog基因表达对胶质瘤细胞生物学活性的影响奠定实验基础.方法 按照RNA干扰序列设计原则结合慢病毒载体质粒pLKO.1-puro结构的特征设计合成shRNA序列,通过分子克隆技术构建Nanog基因RNA干扰慢病毒载体pLKO.1-Nanog-shRNA,同包装质粒pCMV-dR 8.2 dvpr和包膜质粒pCMV-VSV-G共同转染293T包装细胞,收集储存可表达Nanog特异性shRNA慢病毒载体的病毒颗粒并进行滴度测定.结果 构建的Nanog基因RNA干扰慢病毒载体pLKO.1-Nanog-shRNA经聚合酶链反应（PCR）鉴定和测序,结果与设计序列完全相符,慢病毒重组体构建成功,将pLKO.1-Nanog-shRNA、pCMV-dR 8.2dvpr和pCMV-VSV-G共同转染293T包装细胞后,收获病毒并测定病毒滴度为106 TU/ml.结论 成功构建了慢病毒RNA干扰载体pLKO.1-Nanog-shRNA,测序验证构建成功,并完成了病毒的包装、储存和滴度的测定,为后续进一步体内外研究干扰Nanog基因的表达对胶质瘤细胞生物学活性的影响奠定实验基础.Objective To construct a lentiviral vector of RNAi of Nanog,which lay a foundation to the further explore its role in biological behaviour of gliomas by lentivirus-mediated silencing Nanog.Methods The effective sequence of shRNA targeting Nanog gene was designed according to the Elbashir＇ s criteria and the RNAi vector rule.The complementary DNA containing both sense and antisense oligo DNA of targeting sequence of Nanog was synthesized and cloned into pLKO.1-puro vector,to construct the lentiviral vector which expressed shRNA,and it was confirmed by PCR identification and DNA sequencing.The pLKO.1-Nanog-shRNA,pCMV-dR 8.2 dvpr and pCMV-VSV-G were cotransfected into 293T incasing cells.Harvesting recombinant lentivirus and detecting viral titer that was able to express shRNA targeting Nanog gene.Results The recombinant lentiviral vector expressing shRNA targeting Nanog gene was successfully constructed and identified by polymerase chain reaction （PCR） and DNA sequencing.The recombinant lentivirus were harvested from supernatant of 293T cells with a viral titer of 106 TU/ml.Conclusion The lentivirus RNAi vector of Nanog was constructed successfully,which provide a basis for investigation of the role of Nanog and the mechanism of Nanog transcriptional regulation in gliomas in vitro and in vivo.

关 键 词：胶质瘤 NANOG 慢病毒载体 RNA干扰 

分 类 号：R346[医药卫生—基础医学]