腺病毒载体说明

BDAdeno-X™ExpressionSystem1UserManualCat.No.631513orK1650-1PT3414-1(PR3X481)Published28October2003BDBiosciencesClontech™ExpressionSystem1UserManualTableofContentsI.Introduction&ProtocolOverview5II.ListofComponents8III.AdditionalMaterialsRequired9IV.Safety&HandlingofAdenoviruses11V.BDAdeno-X™ExpressionSystemOverview12VI.CellCultureGuidelines14A.GeneralConsiderations14B.MaintainingHEK293CellsinCulture14C.PreparingFrozenCulturesofHEK293Cells15VII.PilotExperiment15VIII.ConstructingRecombinantpShuttle216A.ProducingandStoringpShuttle2PlasmidDNA16B.CloningYourGeneorDNAFragmentintopShuttle216C.PI-SceI/I-CeuIDigestionofRecombinantpShuttle2PlasmidDNA18D.ExtractionwithPhenol:Chloroform:IsoamylAlcohol19IX.ConstructingRecombinantAdenoviralDNA20A.SubcloningYourExpressionCassetteintotheBDAdeno-X™Genome20B.SwaIDigestionofNon-RecombinantBDAdeno-X™DNA21C.TransformingE.coliwithRecombinantBDAdeno-X™DNA22D.Mini-ScalePurificationofRecombinantBDAdeno-X™PlasmidDNA23E.AnalyzingPutativeRecombinantBDAdeno-X™DNA24X.ProducingRecombinantAdenovirus26A.PreparingRecombinantBDAdeno-X™DNAforTransfection26B.TransfectingHEK293CellswithPacI-DigestedBDAdeno-X™DNA28C.AmplifyingRecombinantAdenovirus:29PreparingHigh-TiterStocksD.EvaluatingRecombinantVirus:ConfirmationofConstruct30ProtocolNo.PT3414-1™ExpressionSystem1UserManualNote:Theviralsupernatantsproducedbytransfecting293cellswithrecombi-nantpAdeno-XViralDNAcould,dependingonyourDNAinsert,containpotentiallyhazardousrecombinantvirus.Duecautionmustbeexercisedintheproductionandhandlingofrecombinantadenovirus.Theuserisstronglyadvisednottocreateadenovirusescapableofexpressingknownoncogenes.AppropriateNIH,regional,andinstitutionalguidelinesapply,aswellasguide-linesspecifictoothercountries.NIHguidelinesrequirethatadenoviralproduc-tionandtransductionbeperformedinaBiosafetyLevel2facility.Formoreinformation,seeappropriateHHSpublications.SectionIVinthisUserManualcontainsabriefdescriptionofBiosafetyLevel2aswellasothergeneralinformationandprecautions.NoticetoPurchaserThisproductisintendedtobeusedforresearchpurposesonly.Itisnottobeusedfordrugordiagnosticpurposesnorisitintendedforhumanuse.BDBiosciencesClontechproductsmaynotberesold,modifiedforresale,orusedtomanufacturecommercialproductswithoutwrittenapprovalofBDBiosciencesClontech.ThisproductiscoveredunderU.S.PatentNo.6,303,362.NucleoBond®andNucleoSpin®areregisteredtrademarksofMachery-NagelGmbH&Co.BD,BDLogoandallothertrademarksarethepropertyofBecton,DickinsonandCompany.©2003BDTableofContentscontinuedXI.InfectingTargetCellswithAdenovirus31A.InfectingTargetCells31B.Analyzingβ-galactosidaseExpressioninInfectedCells31XII.TroubleshootingGuide32XIII.References36XIV.RelatedProducts40AppendixA:VectorInformation41AppendixB:TypicalResultsofaRestrictionAnalysis43AppendixC:PlaquePurificationProtocol44AppendixD:DeterminingAdenoviralTiter45BDBiosciencesClontech™ExpressionSystem1UserManualListofFiguresFigure1.ConstructingrecombinantadenoviruswiththeBDAdeno-X™ExpressionSystem.6Figure2.OverviewoftheBDAdeno-X™ExpressionSystemprotocol.13Figure3.Producingrecombinantadenovirus27Figure4.PlasmidmapandmultiplecloningsiteofpShuttle241Figure5.PlasmidmapofpAdeno-X42Figure6.RestrictionanalysisofrecombinantpAdeno-XViralDNA43Figure7.Determiningadenoviraltiterwiththeend-pointdilutionassay47ListofTablesTableI.Adenovirus-MediatedGeneTransferinNon-HumanSpecies7TableII.PI-SceI/I-CeuIDouble-DigestionofRecombinantpShuttle2PlasmidDNA18TableIII.LigatingExpressionCassettestoBDAdeno-X™DNA20TableIV.SwaIDigestionofLigationReactionProducts21TableV.RestrictionanalysisofrecombinantpAdeno-XDNA25TableVI.PacIdigestionofRecombinantpAdeno-XDNA26TableofContentscontinuedProtocolNo.PT3414-1™ExpressionSystem1UserManualI.Introduction&ProtocolOverviewTheBDAdeno-X™ExpressionSystem1providesanefficientmethodforconstructingrecombinantadenovirus.Ourprocedureusesconventionalinvitroligation(nothomologusrecombination)toincorporateamammalianexpressioncassetteintoareplication-incompetent(∆E1/∆E3)humanadenoviraltype5(Ad5)genome.Thisapproach,originallydevelopedbyMizuguchi&Kay(1998,1999),enablesyoutoproducerecombinantadenovirusinlessthanthreeweeks.ConstructingrecombinantadenoviruswiththeBDAdeno-X™System1Theassemblyandproductionofrecombinantadenovirusiscompletedinthreestages(Figure1).First,amammalianexpressioncassetteismadebycloningyourgeneofinterestintopShuttle2.AfteramplificationinE.coli,theexpressioncassetteisexcisedfrompShuttle2andligatedtoBDAdeno-XViralDNA(theadenoviralgenome).BecausepShuttle2andBDAdeno-XViralDNAcarrydifferentantibioticselectionmarkers,youdonotneedtopurifytheexpressioncassettefragmentforligationwithBDAdeno-X.Inthefinalstage,therecombi-nantBDAdeno-Xvectorispackagedintoinfectiousadenovirusbytransfectinghumanembryonickidney(HEK)293cells.Recombi