靶向 Pokemon 基因 shRNA 重组质粒的构建及其在 CNE2细胞的筛选.pdfVIP

9·94 · 刘 斐，等 靶向Pokemon基因shRNA重组质粒的构建及其在CNE2细胞的筛选 靶向Pokemon基因 shRNA重组质粒的构建及其在 CNE2细胞的筛选 刘 斐，焦 伟，蓝 娇，肖瑞平 Ｃｏｎｓｔｒｕｃｔｉｏｎ ｏｆ ｓｈｏｒｔ ｈａｉｒｐｉｎ ＲＮＡ ｒｅｃｏｍｂｉｎａｎｔ ｐｌａｓｍｉｄｓ ｔａｒｇｅｔｉｎｇ ｈｕｍａｎ Ｐｏｋｅｍｏｎ ｇｅｎｅ ａｎｄ ｓｃｒｅｅｎｉｎｇ ｉｎ ＣＮＥ2 ｃｅｌｌｓ LiuFei，JiaoWei，LanJiao，XiaoRuiPing ResearchCenterofMedicalSciences，thePeoplesHospitalofGuangxiZhuangAutonomousRegion，GuangxiNanning530021，China. 【Abstract】 Objective：ToconstructthreeshorthairPinRNA （shRNA）recombinantPlasmidstargetinghuman PokemongeneandscreenaPlasmidwhichcanblockthePokemonexPressionatmaximaldegreeinnasoPharyngeal carcinoma（NPC）CNE2 cells.Methods：ThreeshRNAsweredesignedaccordingtothecodingsequencesofthe Pokemongene.TheshRNAsequenceswereinsertedintoPRNAT-U6.1/Neoduringrestrictionenzyme，connection， transformationandDNAsequencing.TherecombinantPlasmidsandblankPlasmidweretransfectedintoCNE2cells usingLiPofectamine2000.TheexPressionofPokemonmRNAandProteinwasdetectedbyrealtimePolymerasechain reaction（PCR）andWesternblot，thetransfectedblankPlasmidCNE2wasusedasanegativecontrol，theuntreated CNE2wasusedasablankcontrol.Results：TheresultsofDNAsequencingshowedshRNAfragmentswereaccordance withthedesignedsequences.ComParingwiththenegativecontrol，thePokemonmRNAandProteinexPressionlevelsof theshRNAgrouPsweredown-regulated.ThePokemonresultsofshRNA2grouPwaslowest.Conclusion：Construc- tionandscreeningshRNArecombinantPlasmidstargetinghumanPokemongenecanProvideaneffectivetoolforfur- therstudyingtheroleofPokemongeneinoccu enceanddeveloPmentofNPC. 【Keywords】Pokemon；shRNA；construction；screening；CNE2 ModernOncology2014，22（05）：0994-0997 【摘要】 目的：构建3个靶向Pokemon基因的短发夹RNA（shRNA）真核表达载体，在鼻咽癌细胞株CNE2 筛选出干扰效果最佳的载体。方法：依据 shRNA设计原则，针对 PokemonmRNA序列设计并化学合成3对 RNA干扰序列，经酶切、连接和转化，插入PRNAT-U6.1/Neo质粒，经DNA测序验证。脂质体介导shRNA重 组质粒及空白质粒转染 CNE2 细胞，荧光定量PCR 和Wes