siRNA表达载体的构建及其表达.pdfVIP

维普资讯 第 19卷 5期 中 国 病 毒 学 19 (5)：510—513 2004年 10月 VIROLOGICA SINICA October2004 siRNA表达载体的构建及其表达 张建林，邬开朗，张 雪，朱 应，李 雁，赵伟光料，吴建国 f武汉大学生命科学院病毒学教育部重点实验室，湖北武汉 430072) ConstructionandExpressionofVectorsExpressingsiRNA ZHANGJian—lin，WU Kai—lang，ZHANGXue，ZHUYing，LiYan，ZHAOWei—guang ，WUJian—guo rCollegeOfLifeSciences，WuhanUniversity~KeyLaboratoryoftheMinistryofEducationforVirology,Wuhan430072，ChinaJ Abstract：TherfagmentofplasmidpCMV-tag一2BexcludingCytomegalovirus(CMV)promotersequence wasobtainedbyrestrictionenzymedigestion．APairofp~dmersWassynthesizedtoamplifyarfagment ofhteH1promoteraccordingtoreference．TherfagmentofH1promoterwasamplifiedandinserted intohterfagmentofplasmidpCMV-tag一2B excludingCMV promotersequenceandplasmidpGEM一 1lfz，respectively．TherecombinantplasmidswerenamedpCH1nadpGH1．Apairofprimers，which includedsmallinterferingRNA(siRNA)targetsiteformRNA ofEGFPgene．wassynhtesizedand annealed invitro，nadtheninsertde intohtecorrespondingvectorspCH1andpGH1．In addition。hte plasmidpEGFP—N3expressingEGFPproteinwastransfectedintolivertumorlineBel一7402alone．orin combinationwithhteplasmidsproducingsiRNA todegrademRNAofEGFPgene．Theexpressionlevel ofEGFPproteinwasviewedunderhtemicroscope ．Th eresultsindicatedhtathteplasmidsincluding siRNA targetsiteformRNA ofEGFPgeneproduced siRNAmoleculra na ddegradedmRNA ofEGFP gene．Th evector,PCH andpGH．willbeusedtostudypreventionofvirusinfection． Keywords：siRNA；Expressingvector；Construction；Identification 摘要：本研究用限制性内切酶消化质粒pCMV-tag一2B，除去巨细胞病毒 (Cytomegaiovirus，CMV)启动子核苷酸 序列，剩下的核苷酸序列作为构建表达 siRNA (SmallinterferingRNA，siRNA)载体的前体。依据文献提供的扩 增 H1RNA启动子核苷酸序列的引物序列合成一对引物，以带有 H1RNA启动子序列的质粒 DNA为模板扩增 HIRNA启动子序列，插入