人ERRα基因小分子干扰RNA慢病毒载体构建及鉴定  

Construction and identification of a lentiviral vector harboring RNAi targeted on gene ERRα

作　　者：孙蓬明[1] 毛晓丹[1] 林芬[1] 蔡良知[1] 吴齐斌[1] 宋一一[1] 

机构地区：[1]福建省妇幼保健院妇科肿瘤研究室,福州350001

出　　处：《福建医药杂志》2011年第4期1-4,共4页Fujian Medical Journal

基　　金：国家自然科学基金项目(30600666);人事部2007年度留学人员科技活动择优资助项目[国人厅发(2007)170号];福建省科技计划重点项目(2008I0011)

摘　　要：目的构建并鉴定靶向人ERRα基因的小分子干扰RNA的慢病毒载体。方法针对ERRαmRNA设计了4条si RNA,并构建pGCSIL-GFP-siERRα慢病毒质粒,PCR扩增阳性克隆并测序鉴定。用pGCSIL-GFP-siERRα、pHelp-er1.0和pHelper2.0质粒共转染293T细胞包装产生慢病毒,测定病毒滴度。将慢病毒干扰RNA及含有ERRα过表达载体共转染293T细胞,Western-blot检测ERRα表达,观察蛋白表达抑制效果。结果 PCR和测序结果与设计的干扰序列一致,病毒滴度达2×109TU/ml。转染细胞中ERRα蛋白表达显著降低。结论成功构建高表达、高效率的人ERRα基因小分子干扰RNA慢病毒载体,为进一步研究ERRα在细胞核内转导中的作用机制和靶向ERRα治疗奠定基础。Objective To construct and identify a lentiviral vector harboring RNAi sequence targeting ERRα gene.Methods Four siRNA targeting the ERRα mRNA were designed and reconstructed,and named as the pGCSIL-GFP-siERRα lentivirus vectors,which contained U6 promoter and green fluorescent protein（GFP）.The positive clone was chosen and confirmed by PCR and DNA sequencing.293T cells were co-transfected with pGCSIL-GFP-siERRα,pHelper1.0 and pHelper2.0 for the virus stocks produced,the titer of the virus was tested.After lentivirus-shRNA and over-expression plasmid of ERRα were transfected into 293T cells,Western-blot were used to determine the expression of ERRα gene and the efficacy of RNA interference.Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human NSBP1 gene was successfully inserted into the lentiviral vector.The titer of the recombinant lentiviral vector as 2×109 TU/ml.The expression level of ERRα protein in transfected cells was significantly decreased after treated with lentivirus-shRNA.Conclusion The construction of the lentiviral vector of ERRα has been successfully prepared.The Lv-shRNA target on ERRα can help us to explore the potential role of it in signal pathway and provide a new molecular therapy.

关 键 词：雌激素受体相关受体α RNA干扰 重组载体 子宫内膜癌 

分 类 号：R346[医药卫生—基础医学]