EZH2基因RNAi慢病毒载体的包装及鉴定-论文.pdf

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1、·21O·国际生物医学工程杂志2014年8月第37卷第4期IntJBiomedEng，August2014，Vo1．37，No．4·论著·EZH2基因RNAi慢病毒载体的包装及鉴定陈青青杨冰赵秀娟马芹刘超梁绍平闵琦吴少华孟歆怿王玺王华庆张会来【摘要】目的外周T细胞淋巴瘤(PTCLs)源于成熟的胸腺后淋巴细胞，是一类少见的生物学行为及临床表现均高度异质性的疾病，且预后差。包装zeste基因增强子同源物2(EZH2)RNA干扰(RNAi)慢病毒载体，可为今后相关实验研究奠定基础。方法靶向EZH2基因的短发夹RNA(EZH2一shRN

2、A)质粒转化感受态细菌，筛选阳性克隆，经测序鉴定正确后通过脂质体将慢病毒三质粒系统共转染293T细胞，进行慢病毒包装。病毒载体感染Hut78细胞后，观察感染效率，并用反转录定量PCR(RT—qPCR)和WesternBlot检测EZH2mRNA和蛋白表达水平。结果成功包装了慢病毒表达载体，对Hut78细胞的感染效率在95％以上。RT—qPCR和WesternBlot检测结果显示，EZH2基因在靶细胞中的表达水平降低。结论成功包装并鉴定了EZH2基因RNAi慢病毒表达载体。【关键词】外周T细胞淋巴瘤；zeste基因增强子同源物2；

3、慢病毒载体；RNA干扰中图分类号：R318；Q78文献标识码：A文章编号：1673—4181(2014)04—0210—05PackageandidentificationofalentiviralvectorofRNAinterferencecontainingEZH2geneChenQingqing，YangBing,ZhaoXiujuan,MaQin,LiuChao，LiangShaoping,MinQi,WuShaohua，MengXinyi,WangXi，WangHuaqing,ZhangHuilai．Departme

4、ntoflymphoma,TianjinMedicalUniversityCancerInstituteandHospital，ClinicalMedicalResearchCenterofNationalCancer,TianjinKeyLaboratoryofCancerPreventionandTherapy,Tianjin300060,China【Abstract】ObjectivePeripheralT—celllymphomas(PTCLs)originatedfrommature，postthymicTcellsa

5、rearareheterogeneousgroupofclinicallyaggressivenon—Hodgkinlymphoma(NHL)withadismalprognosis．ThisstudywasaimedtopackageandidentifyalentiviralvectorofRNAinterference(RNAi)targetingenhancerofzestehomolog2(EZH2)，wouldlaythefoundationforthefuturestudy．MethodsAftertransfor

6、mationintocompetentE．colibacteria，thecandidatecloneswereidentifiedbyDNAsequencing．TheEZH2一shorthairpinRNA(shRNA)plasmidandthetwopackagingplasmidswereCO—transfectedintothehumanembryonickidney293Tcellsbylipofectamine2000toproducethelentiviralvector．TheHut78cellswereinf

7、ectedwiththelentiviralvectorobtainedandthetransfectioneficiencywasassessedunderthefluorescentmicroscope．ThentheexpressionofEZH2inthetransfectedcellswasdeterminedbyRT—qPCRandWesternBlot．ResultsThelentiviralRNAivectorfortheEZH2genewaspackagedsuccessfully．S~ongredfluore

8、scencewasobservedintheHut78cellsunderthefluorescentmicroscopeafterCO-transfectionofthecellswiththe3plasmidsofthelentiviralvector．Th