應(yīng)用RNA干涉技術(shù)阻斷EBV潛伏期基因LMP1表達的實驗研究.pdf

1、青島大學碩士學位論文應(yīng)用RNA干涉技術(shù)阻斷EBV潛伏期基因LMP1表達的實驗研究姓名：殷凡申請學位級別：碩士專業(yè)：病原生物學指導教師：羅兵20060603RNAIntcrferenceforlnh_bjtionofEBVLatentMembraneGeneI—MPlExpressjonAbstJactobjectiVeTbconstnlctarecombinantretmviralvectorpsuPER—LMPlthatta唱etEB

2、Vlatentmembranepmteingenel(LMPl)andselectastableVimsproducingcellcloⅡe，toexPJoletheap叩tosisofEBVposi“VeRajjcell(LMP】)anertheta曙etgenewassikncedbyRNAiMethodsThe60ntsequencesencodedfortransc曲ingLMPlshRNAwereclonedintoaretr

3、0VimalVectorpsUPERretrowi血DNArecombinanttechniqueTherecombinantVectorwasidentmedby出eelec咀phoresisan“ysisofrestrictionenzymedigestionaIldDNAsequencingThepackagingceUPA317wastransfectedw“hrecombinantplas硼jdusingliposomebas

4、edtransfectionmethodandthestablevims—producingce兒cloncwasselectedbyusingG4l8mediumTheVimlsupemataⅡtwasusedtoinfecttargetcells，meexpressionofLMPlwastestedbyRTPCRandtheapoptosisofRajiceuswasteStedbyf10wcytomeⅡyR曬ul協(xié)Theresu

5、ltofDNAsequenciⅡgdemonstrated血at60bphadbeeninsertedinthee鼉pectedsiteandtheiIlsertionsequencewasexactlycorrectWhentherecOmbinaIltVectorwastransfectediⅡtot11epacka百ngceu，greeⅡfluorescentprotein(GFP)wasexpressedThe枷一G418pos

6、itiVecloⅡeswerealsoselectedaIldt|屺ycouldexcreterecombinantre廿0VinlsRT—PCRresultshowedmathi曲一emciencyinhibitionofLMPlmRNAwasachieVedbyretmVimsvectormediatedRNAiinR螄ceIis，aIldthe珊PIsilencinginducedRajicellsapoptosisbynowcy

7、tome町ConclI玨ionWbsucceededincoⅡst兀lctin窟arecombinamretroviralvectorpSUPER一ⅢPlandselectingastablevims—pmducingpackagingcelllineⅢP1expressioninRajicellswasspecificallysilencedbyrecombinantretrovimspSUPER一嘣P1，andthee骶ctofSp