rAd-DKK1-shRNA表达载体的构建.pdfVIP

生堡生塑医堂工程塞盍2Q13生!Q旦筮!!鲞篁§期￡hiⅡ』旦iQ堡趟堑磐：Q!!!b丛2Q13，y!!：19：盟!：§ ·生物技术· rAd—DKKl一shRNA表达载体的构建 李哲海张霄雁刘国栋呼和武宇赤 marrow．derived stem 【摘要】 目的 筛选构建针对骨髓间充质干细胞bone cells， mesenchymal BMSC)的小干扰RNA(shRNA)腺病毒载体。方法根据shRNA靶位点的选择原则，从TRCshRNA数 据库及AmbionshRNA数据库中择优选取3条DKKIshRNA序列，通过DKKl构建过表达载体，筛选出 最佳干扰序列，体外同源重组构建DKKl．shRNA腺病毒表达载体，经酶切、PCR鉴定、测序。结果成 通过RT．PCR与Western印迹检测DKKl的表达，结果显示pYr—1．1一DKKl—shRNA一153为最佳干扰序 列。结论成功构建了针对BMSC的小干扰RNA(shRNA)腺病毒载体，为后续靶向目的基因的实验研 究提供了依据。 【关键词】基因表达； 构建；质粒；DKKl蛋白 vectorsLI ConstructionofrAd．DKKl．shltNA Zhe—hai，ZHANG expression Xiao—yan，LIUGuo—dong， HUHe，WU Medical of Mongolia Yu—chi．DepartmentOrthopedics，孤e豫irdAffiliatedHospital，Inner 014010，China University，Baotou Correspondingauthor：WU玩一chi，E—maif：wyc2290@rip．sina．corn Toscreenandconstructthesmall RNA(shRNA)adenovirus 【Abstract】 interfering Objective stem Basedonthe of vectorofbonemarrow—derived cells(BMSC)．Methods mesenchymal principle DKK1 shRNA werechosenfromtheshRNAdatabaseand shRNAsitesselection，three target sequences AmbionshRNAdatabase．The wasscreenedconstructionofDKKl over— optimalinterferingsequence by vectorandidentifiedrestriction and viaconstructionof expression by analysis，PCRsequenced homologus DKK1一shRNAadenovirnsvectorinvitro．Results recombination Expressionvectors，includingpYr一1．1．