病毒纯化与分析课件

ViruspurificationandanalysisusingOptiPrep™-competitivemediaSucroseGlycerolCaesiumchlorideSOLUTIONHANDLINGDensitysolutionspreparedbydilutingOptiPrep™withculturemediumoranybalancedsaltsolution(ApplicationSheetV01)Sterilesolution

CsCl/sucrosesolutionsrequirelengthypreparationtimesandsterilizationCsClisexpensiveCsClPROBLEMS

Biglossesinviralinfectivity;lowrecoveries;highparticle:infectivityratiosSolutionshaveveryhighosmolality:viruseslosewater;highvirusdensity;high[CsCl]requiredforgradientsHighlytoxictocells:mustbedialyzedpriortore-infectionofcellsSolutionsionicandcorrosive:samplesmustbedialyzedbeforeelectrophoresisorHPLCSUCROSEPROBLEMS(I)Solutionsarehyperosmotic:

virusdensityinsucroseusuallyhigherthaniniodixanolSolutionsareviscous:slowsedimentationofvirusparticlesHighlytoxictocells:mustbedialyzedpriortore-infectionofcellsOPTIPREP–NOPROBLEMSSolutionsareisoosmotic:virusdensityislowSolutionshavelowviscosity:rapidsedimentationofvirusparticlesIsnon-toxictocellsHaslittleornoeffectonvirusinfectivityCellscanbere-infectedandelectrophoresisandHPLCperformedwithoutdialysisVirusbandingdensityVirusCsClSucroseIodixanolEpstein-Barr1.261.14SemlikiForest1.261.181.18Poliovirus1.341.261.22Measles1.211.181.1615%iodixanolin1MNaCl25%iodixanol40%iodixanol54%iodixanolVirusfluidproteinsadenovirusrAAV350,000g1hrAAVindiscontinuousgradient(V04)ZolotukhinS.etal(1999)GeneTherapy,6,973-985rAAVincontinuousgradient(V04)Hermans,W.T.J.M.C.etal(1999)HumanGeneTherapy,10,1885-1891Opti-PrepVirusfluidBGradientMasterGradientMasterprofilesfrom10%and40%iodixanolat80°and20rpm:effectoftimeDensity(g/ml)FractionNumber135791.071.091.111.132min2.5min3.0min3Formatsforseparationofparticlesaccording

totheirdensity123rAAVincontinuousgradient(V04)

Hermans,W.T.J.M.C.etal(1999)HumanGeneTherapy,10,1885-1891348000g3hrAAVmembranesOptiPrepVirusfluidComparisonofrAAVinCsClandOptiPrep™Pre-gradientGradient%Recov’ryVP/ICU(NH4)2SO4CsCl0.1-60250-108(NH4)2SO4OptiPrep26-120264-1333Cellufine-SO4OptiPrep25-91385-600DatafromHermans,WTJMCetal(1999)HumanGeneTherapy,10,1885-1891PurificationofHIV-1(V06)Dettenhoffer,M&Yu,X-F(1999)J.Virol.,731460-1467250,000gfor1.5h6-18%iodixanolMicrovesiclesVifgeneSolubleproteinsRT(cpmx10-5/ml)Useofself-generatediodixanolgradientsinverticalrotorsforviruspurificationandanalysis1345250,000g1h350,000g1-3hViruspurificationflow-chart–withoutviruspelletingPurificationofHerpesvirusinself-generated25%iodixanolgradient(V05)FractionNumberLoginfectivityDensity(g/ml)13579111315012345611.051.11.151.21.251.3InfectivityDensityFractionnumberOD(PERT)x10-2Density(g/ml)13579111315171905101520253011.051.11.151.21.25MS1533MS1874DensityPurificationofretrovirus:self-generatedgradient(V09)Moller-LarsenA&Christensen,T.(1998)J.VirolMeth.,73,151-161OptiPrep™ApplicationSheetsfor:AdenovirusEbolavirusFoamyvirusHerpessimplexvirusHIV-1HumanpapillomavirusLassavirusMurineleukemiavirusMurineoncornavirusNorwalkvirusPolyomavirusrAAVRabiesvirusRoussarcomavirusRetrovirusesSemlikiForestvirusSARSCoronavirusVacciniavirusPublicationsdatabaseonvirusesOptiPrep(sinc