实用生物医学实验技术 病毒制备与转染课件

病毒制备与转染病毒制备与转染Virus Production and Transduction实用生物医学实验技术实用生物医学实验技术病毒制备与转染VirusProductionandT病毒的定义Avirus(thewordisfromtheLatinvirusreferringtopoisonandnoxioussubstances)isasmallinfectiousagentthatreplicatesonlyinsidethelivingcellsofotherorganisms.Virusescaninfectalltypesoflifeforms,fromanimalsandplantstobacteriaandarcheaSARS病毒艾滋病病毒致病之毒素，极其微小，结构简单、以寄生、复制进行繁殖的一类非细胞型微生物病毒的定义Avirus(thewordisfroVirusparticles(knownasvirions)consistoftwoorthreeparts:i)thegeneticmaterialmadefromeitherDNAorRNA,longmoleculesthatcarrygeneticinformation;ii)aproteincoatthatprotectsthesegenesiii)anenvelopeoflipidsthatsurroundstheproteincoatwhentheyareoutsideacell.病毒的组成Virusparticles(knownasviri病毒的特点病毒能增殖、遗传和演化，因而具有生命最基本的特征，其主要特点是：形体极其微小，一般都能通过细菌滤器，必须在电子显微镜下才能观察没有细胞构造，其主要成分仅为核酸（DNA或RNA）和蛋白质两种既无产能酶系，也无蛋白质和核酸合成酶系，只能利用宿主活细胞内现成代谢系统合成自身的核酸和蛋白质成分。以核酸和蛋白质等“元件”的装配实现其大量繁殖。在离体条件下，能以无生命的生物大分子状态存在，并长期保持其侵染活力。有些病毒的核酸还能整合到宿主的基因组中，并诱发潜伏性感染。病毒的特点病毒能增殖、遗传和演化，因而具有生命最基本的特征，病毒的分类有不同类别病毒共上百万个，目前有详细了解的只有约5000个从遗传物质分类：DNA病毒、RNA病毒、蛋白质病毒（如：朊病毒）ThevastmajorityofviruseshaveRNAgenomes从病毒结构分类：真病毒（Euvirus，简称病毒）和亚病毒（Subvirus，比病毒更为简单，仅具有某种核酸不具有蛋白质，或仅具有蛋白质而不具有核酸，包括类病毒、拟病毒、朊病毒）从寄主类型分类：噬菌体（细菌病毒）、植物病毒（如烟草花叶病毒）、动物病毒（如禽流感病毒、天花病毒、HIV等）从性质来分：温和病毒（HIV）、烈性病毒（狂犬病毒）。病毒的分类有不同类别病毒共上百万个，目前有详细了解的只有约病毒的生命周期病毒的生命周期Virusesprovidesimplesystemsthatcanbeusedtomanipulateandinvestigatethefunctionsofcells.Viruseshavebeenusefulinthestudyofgeneticsandhelpedourunderstandingofthebasicmechanismsofmoleculargenetics,suchasDNAreplication,transcription,RNAprocessing,translation,proteintransport,andimmunology.Mostlyintheformofviralvectors:introducingforeignDNAsintocellsandforgenetherapyandindustrialproductionofvaccinesBiologicalweapons病毒在生物医学中的应用Virusesprovidesimplesystem逆转录病毒（Retrovirus）腺病毒（Adenovirus）腺相关病毒（Adeno-associatedvirus）仙台病毒（SendaiVirus）慢病毒（Lentivirus）生物医学研究中常用的病毒表达系统逆转录病毒（Retrovirus）生物医学研究中常用的病毒逆转录病毒（Retrovirus）Aretrovirusisasingle-strandedRNAvirusthatstoresitsnucleicacidintheformofanmRNAgenome,100nminsizeRetro-reverse,RNA-DNA-RNA-proteinReversetranscribe-integrate-transcribe-translate-package1.freevirus,2.attach,3.endocytosis,4.RNAtoDNA,5.integrate,6.dormant,7.transcribetomRNA,8.translate,9.packageintonewvirusandexit逆转录病毒（Retrovirus）Aretrovirus三个核心基因：gag-编码病毒的核心蛋白，起保护作用；pol-编码逆转录酶；env-编码病毒的被膜糖蛋白逆转录病毒分类：（1）单嗜性逆转录病毒（ecotropicretrovirus），只感染小鼠和少数几个品种的大鼠；（2）兼嗜性逆转录病毒（amphotropicretrovius），能感染小鼠的细胞，也能感染其他种属动物的细胞；用得较多（3）异嗜性逆转录病毒（xenotropicretrovirus），能感染多种动物细胞，但不能感染小鼠细胞。（4）全嗜性逆转录病毒（pantropicretrovirus）三个核心基因：gag-编码病毒的核心蛋白，起保护作用；po逆转录病毒表达系统1.PackagingCells,e.g.Phoenixcells(anadenovirusAd5-transformedhumanembryonickidneycellline293T,transfectedwithtwoMMLVpackaginggeneconstructs:CMV-Env-PolyA,andRSV-Gag/Pol-Tyt2-PolyA.)Usepassagenumber20,90%areachievableformostmitoticcelltypesThecopynumberpercellcanbeeasilycontrolledbyvaryingthemultiplicityofinfection(MOI)逆转录病毒表达系统Retroviralvectorsa腺病毒（Adenovirus）Medium-sized(90100nm),nonenveloped(withoutanouterlipidbilayer)viruseswithdoublestrandedDNAgenome,thelargestnonenvelopedviruses.Inhumans,thereare57acceptedhumanadenovirusserotypes(HAdV-1to57)insevenspecies(HumanadenovirusAtoG)腺病毒（Adenovirus）Medium-sized(AdenovirusespossessalineardsDNAgenomeandareabletoreplicateinthenucleusofvertebratecellsusingthehostsreplicationmachinery.Adenovirus life cycle1.freevirus,2.attach,bybindingtoCD46ortheCoxsackie/AdenovirusReceptor(CAR)3.Entrywithinanendosome4.ViralDNAissubsequentlyreleasedandenterthenucleus5.transcribetomRNA,6.translate,7.packageintonewvirusandexitAdenovirusespossessalinear腺病毒基因组包含早期表达的与腺病毒复制相关的E1E4基因和晚期表达的与腺病毒颗粒组装相关的L1L5基因腺病毒基因组Twophases:anearlyandalatephaseTheearlygenesareresponsibleforexpressingmainlynon-structural,regulatoryproteins.Oncetheearlygeneshaveliberatedadequatevirusproteins,replicationmachinery,andreplicationsubstrates,replicationoftheadenovirusgenomecanoccur.Thelatephaseoftheadenoviruslifecycleisfocusedonproducingsufficientquantitiesofstructuralproteintopackallthegeneticmaterial腺病毒基因组包含早期表达的与腺病毒复制相关的E1E4基因和1.Achoiceofadenoviralvectorsthatallowhighlyefficientgenerationofarecombinantadenoviruscontainingthegeneofinterestunderthecontrolofapromoterofchoice.Thevectoralsocontainstheelementsrequiredtoallowpackagingoftheexpressionconstructintovirions(e.g.5and3ITRs,encapsidationsignal,adenovirallategenes)2.Anoptimizedcellline,e.g.293A,thecelllinecontainsastablyintegratedcopyoftheE1genethatsuppliestheE1proteins(E1aandE1b)requiredtogeneraterecombinantadenovirus.Theflatmorphologyofthecellsmakesthetiteringprocedureeasier.3.Transfectionreagent,e.g.lipofectamine2000,30004.Medium,DMEMwith10%FBSand1xpenicillin-streptomycin5.Re-infectcellline腺病毒表达系统1.AchoiceofadenoviralvectTherateofinfectionandthesubsequentyieldofrecombinantproteinaretypicallyquitehigh,betterthanretroviralsystemThepermissivehostcellrangeisverywide.Thevirushasbeenusedtoinfectmanymammaliancelltypes(bothreplicativeandnon-replicative)forhighexpressionoftherecombinantprotein.Adenovirusesareespeciallyusefulininfectingcelllinesthathavelowtransfectionefficiencywithliposome.DoesnotintegrateintothehostchromosomesodoesnotactivateorinactivatehostgenesExpressionistransientinnature,aslongastheviralgenomeisnotdegraded,1-2weeksdependingonthecelltype.Longerexpressioncanbeobservedinslowdividingcellssuchasneurons.腺病毒表达系统的特点Therateofinfectionandthe腺相关病毒(AAV)AAVwasdiscoveredin1965asacontaminantofadenovirus(Ad)preparations，11AAVserotypeshavebeendescribed.Smallvirusesinfectshumansandsomeotherprimatespecies,non-enveloped,approximately22nmAco-infectinghelpervirusisusuallyrequiredforaproductiveinfectiontooccur腺相关病毒(AAV)AAVwasdiscoveredTheAAVgenomeisbuiltofsingle-strandedDNA(ssDNA),whichisabout4.7KBThegenomecomprises：i)invertedterminalrepeats(ITRs)atbothendsoftheDNAstrand,requiredforefficientmultiplicationoftheAAVgenomeandintegrationoftheAAVDNAintothehostcellgenome(19thchromosomeinhumans)ii)twoopenreadingframes(ORFs):repandcapcapencodedscapsidproteinVP1,VP2andVP3,whichinteracttogethertoformacapsidi)repencodesfourRepproteins,Rep78,Rep68,Rep52,andRep40,requiredforreplicationofDNAandintegrationintochromosome.Infectbothdividingandnon-dividingcells,stablyintegrateintothehostcellgenomeataspecificsite(designatedAAVS1)inthehumanchromosome19腺相关病毒结构腺相关病毒结构腺相关病毒基因组Schematic Map of AAV Genome 腺相关病毒基因组SchematicMapofAAVG腺相关病毒1.attach2.receptor-mediatedendocytosis（heparansulfateproteoglycan）3.endosomaltrafficking4.escapefromthelateendosome5.translocationtothenucleus6.uncoating7.formationofdouble-strandedDNAreplicativeformoftheAAVgenome8.expressionofrepgenes9.genomereplication10.expressionofcapgenes,synthesisofprogenyssDNAparticles11.assemblyofcompletevirions12.releasefromtheinfectedcell.腺相关病毒1.attachAAVcausesaverymildimmuneresponse,notcurrentlyknowntocausedisease-makeAAVaveryattractivecandidateforcreatingviralvectorsforgenetherapyAAVcaninfectbothdividingandquiescentcellsDevelopmentofAAVsasgenetherapyvectors,however,haseliminatedthisintegrativecapacitybyremovaloftherepandcapfromtheDNAofthevector.Thecloningcapacityofthevectorisrelativelylimitedandmosttherapeuticgenesrequirethecompletereplacementoftheviruss4.8kilobasegenome,cloningcapacity4.5KB腺相关病毒表达系统AAVcausesaverymildimmuneAAVHelper-FreeSystem,eliminatingtherequirementforlivehelpervirustheAAVHelper-FreeSystemprovidesasaferandmoreconvenientgenedeliverysystemMostoftheadenovirusgeneproductsrequiredfortheproductionofinfectiveAAVparticlesaresuppliedonanadditionalplasmid(e.g.CellBiolabspHelperplasmidscontainsE2A,E4,andVARNAgenes)thatisco-transfectedintocellswithhumanAAVvectorDNATheremainingadenoviralgeneproductissuppliedbythe293hostcells,whichstablyexpresstheadenovirusE1gene.腺相关病毒表达系统AAVHelper-FreeSystem,elimiAAV Helper-Free SystemAAVHelper-FreeSystemAAV-DJAAVserotypes(AAV1-AAV11)differbroadlyintransductionefficaciesandtissuetropisms.DNAfamilyshufflingtechnologywasusedtocreateacomplexlibraryofhybridcapsidsfromeightdifferentwild-typevirusesStringentselectionofAAVvariantsonhumanlivercellsandwithhumananti-AAVantiseraresultinAAV-DJ(aclonenamedafterthefirsttwoauthors)-superiorinvitrotransductionefficaciesincomparisonwithanyotherwildtypeserotypes.(upto100,000-foldbetterthanAAV-8orAAV-9,alsosubstantiallybetterthanAAV-2.AAV-DJAAVserotypes(AAV1-AAV1.Onedaybeforetransfection,platesufficient293cellsor293AAVcellstoachieve70-80%confluenceonthedayoftransfection.2.CotransfectcellswithpAAVExpressionvector,pAAV-DJandpHelper,ratioofvectorsat1:1:1Calcium Phosphate transfection method is preferred for AAV production.3.48-72hoursaftertransfection,harvestcells.4.Centrifugethecellsuspension.RemovethesupernatantandresuspendthecellpelletinDMEMorsterilePBS.5.Freezeandthawthecellsuspensionfourtimesbyplacingitalternatelyinadryice/ethanolbathandawaterbathof37C.Removecelldebrisbycentrifugationat10,000gfor10minandcollectthesupernatantasAAVcrudelysate.6.AAVcrudelysatecanbeuseddirectlyorpurified/concentratedifneeded.Forlongtermstorage,storesupernatantat-80Cinaliquots.腺相关病毒制备1.OnedaybeforetransfectionSeV(mouseparainfluenzavirustype1,hemagglutinatingvirusofJapan(HVJ)isasingle-strandRNAvirus,sphericalshapeandanaveragediameterof260nmASeVvirionconsistsofthenucleocapsid(genomicRNAcomplexedwithNP,PandLproteins),anenvelope(alipidbilayerwithFandHNproteins,responsibleforinsertintocellmembrane)andamatrix(Mprotein)connectingthenucleocapsidandenvelopeFirstisolationinthe1950sinJapan仙台病毒（Sendai Virus）SeV(mouseparainfluenzaviruSeVisresponsibleforahighlytransmissiblerespiratorytractinfectioninmice,hamsters,guineapigs,andratsthroughbothairanddirectcontactroutesSeVisneithertumorigenicnorpathogenictohumansLowspeciesandcellspecificity-infectmostcelltypesDonotintegrateintohostgenomeInducetransientbutverystronggeneexpression仙台病毒的特点SeVisresponsibleforahighAPPLICATION OF SeV VECTORS IN GENE THERAPYRecombinantSeVvectorcaninducestrongex-geneexpressioninthecardiovascularsystem,retinalepithelium,hepatocytes,colonicepithelium,neurons,dendriticcellsandinhumanhematopoieticstemcells.ThisremarkablywidehostrangepartlydependsonthefactthattheprimarySeVreceptor,sialicacid,isdistributeduniversallyamonganimalcellsSeVvectorsrelyfortheirgeneexpressiononlyonvirus-encodedRNApolymeraseandtubulin,aubiquitousconservedcytoskeletalproteinReplication-defectiveSeVvectorsexpressingFGF2havebeendevelopedfortreatmentofcriticallimbischemiaApplicationsofSeVvectorstocancergenetherapyhavebeeninvestigatedatthepreclinicalstageSeVinducessyncytia,usedtomergeamonoclonalBcell,exposedtoachosenantigen,andamyelomatumorcelltoproducehybridomasAPPLICATIONOFSeVVECTORSINTheSeVvectorstandsuniqueamongothervectorsystemsingenomicreprogrammingbecauseitcanexpressthereprogramminggeneswithoutchromosomalintegration.SeVvectorscanreprogramthenucleiofvarioushumancells,includingCD34+cordbloodcells,activatedTlymphocytesandmonocytesHighefficiencyApplication of SeV in Cellular ReprogrammingTheSeVvectorstandsuniqueLentivirus(lente-,Latinforslow)isamemberofRetrovirusfamily,characterizedbyalongincubationperiod.Thevirionsaresphericaland80100nmindiameter,enveloped,withtinyspikes(about8nm)LentivirusescandeliverasignificantamountofviralRNAintotheDNAofthehostcellandbeingabletoinfectnon-dividingcells.LentivirusexpressionsystemisausefulresearchtooltointroduceageneproductorshRNAintocellsoranimalmodelsforprolongedexpression慢病毒(Lentivirus)Lentivirus(lente-,Latinfor慢病毒结构Genomess-RNA,threemaingenescodingfortheviralproteinsintheorder:5-gag-pol-env-3andtworegulatorygenes,tatandrevStructuralProteinThelentiviralproteomeconsistsoffivemajorstructuralproteins:Gp120glycosylatedsurfaceenvelopeproteinSU,encodedbytheviralgeneenv.Largest120KDa.Gp41glycosylatedtransmembraneenvelopeproteinTM,alsoencodedbytheviralgeneenv.2ndlargest41KDa.P24non-glycosylatedcapsidproteinCA,encodedbytheviralgenegag.3rdlargest24KDa.P17non-glycosylatedmatrixproteinMA,alsoencodedbygag.4thlargest17KDa.Non-glycosylatedcapsidproteinNC,alsoencodedbygag.5thlargest7-11KDa.慢病毒结构GenomeStructuralProtein慢病毒表达系统Lentivirusvectorbasedonthehumanimmunodeficiencyvirus-1(HIV-1)hasbecomeapromisingvectorforgenetransferstudies.Theadvantageousfeatureoflentivirusvectoristheabilityofgenetransferandintegrationintodividingandnon-dividingcells.ThepseudotypedenvelopewithvesicularstomatitisvirusenvelopeG(VSV-G)proteinbroadensthetargetcellrange.Lentiviralvectorshavebeenshowntodelivergenestoneurons,lymphocytesandmacrophages,celltypesthatpreviousretrovirusvectorscouldnotbeused.Lentiviralvectorshavealsoproventobeeffectiveintransducingbrain,liver,muscle,andretinainvivowithouttoxicityorimmuneresponses.LentivirussystemiswidelyusedtointegratesiRNAefficientlyinawidevarietyofcelllinesandprimarycellsbothinvitroandinvivo.慢病毒表达系统Lentivirusvectorbase慢病毒表达系统慢病毒表达系统第二代慢病毒表达系统atransferplasmid,asinglepackagingplasmid,andanenvelopeplasmid第三代，4质粒系统，更安全，课后阅读第二代慢病毒表达系统atransferplasmid,慢病毒的制备Forproducinglentiviralparticles,youtypicallyneed4components:Alentiviralvectoror“transfervector”containingtheshRNAortransgeneandtheflankingLTRsApackagingvectororsetofpackagingplasmidsAnenvelopevectorPackagingcells:293Tor293FT来源于SV40大T抗原转化的人胚肾细胞stablyexpresstheneomycinresistancegene慢病毒的制备Forproducinglentiviral成功制备慢病毒的要点1.PackagingCells,细胞质量要高，尽量用早代的细胞，冻存一批早代细胞以备后用，最佳转染密度70-80%2.LentiplasmidDNA,最大DNA载荷12KB，质粒抽提，纯度，浓度，比例：Lenti:psPAX2:pMD2.G=12:8:43.Transfectionreagent,e.g.CaCl2,lipofectamine2000,3000，争取转染效率达到最佳，最好每次用GFP质粒做参照4.Medium,不能对细胞生长有负作用，FBS，双抗等5.Polybrene1000 x(4mg/mL)inPBS,stocksterilefiltered6.Testvirusbytransductionof293cellsorothercells成功制备慢病毒的要点1.PackagingCells,慢病毒的浓缩1.超速离心法2.利用蛋白质浓缩离心管3.PEG(polyethyleneglycol)沉淀浓缩法，亲水性很高，在溶液里会吸收大量水分，减少病毒颗粒之间的距离，使病毒聚集在一起，提高病毒的相对浓度，达到沉淀浓缩的目的慢病毒的浓缩1.超速离心法滴度测定DETERMINATIONOFTOTALVECTORCONCENTRATIONUSINGANTI-p24IMMUNOASSAYBIOLOGICALTITRATIONOFLENTIVECTORSUSINGFLOWCYTOMETRY-Thismethodcanonlybeusedtotiterstocksofvectorsthatcarryatransgenewhichiseasilymonitoredbyflowcytometry,suchasGFPBIOLOGICALTITRATIONOFLENTIVECTORSBYQUANTITATIVEPCR(QPCR)-gag滴度测定DETERMINATIONOFTOTALVECMultiplicity of Infection(MOI)Multiplicityofinfection(MOI)isafrequentlyusedterminvirologywhichreferstothenumberofvirionsthatareaddedpercellduringinfection.Ifonemillionvirionsareaddedtoonemillioncells,theMOIis1.Iftenmillionvirionsareadded,theMOIis10HighMOIisusedwhentheexperimentrequiresthateverycellinthecultureisinfected.Bycontrast,lowMOIisusedwhenmultiplecyclesofinfectionarerequired.MultiplicityofInfection(MOI慢病毒的保存1.新鲜的病毒上清或浓缩病毒效果最佳2.可以分装成小管，保存于-80度冰箱3.避免频繁的freeze/thaw，每一次冻化，病毒感染效率大幅下降4.-80保存半年左右慢病毒的保存1.新鲜的病毒上清或浓缩病毒效果最佳Infection:Appliestosituationswhereviralreplicationoccursandinfectiousviralprogenyaregenerated.Onlycelllinesthatstablyexpressvirusreplicationelementscanbeinfected.Transduction:Appliestosituationswherenoviralreplicationoccursandnoinfectiousviralprogenyaregenerated.Mammaliancelllinesthatdonotexpressreplicationelementsaretransduced.Transfection:istheprocessofdeliberatelyintroducingnucleicacidsintocells,oftenusedfornon-viralmethodsineukaryoticcellsInfection and TransductionInfection:Appliestosituatio操作病毒时应注意防护注意防护P2practices：Opentubesalwaysbehandledinthelaminarflowhood.Tubescanbetakenoutofthelaminarflowonlywhentheyareclosed,andthattheybesprayedwith75%ethanol.Allsolidwasteandplasticwaremustbediscardedinatrashbininthelaminarflowhoodsandallliquidsmustbeaspiratedintoaliquidwastebottlecontainingfreshconcentratedbleach.Whenfull,bagsareclosedinsidethelaminarflowhood,thenautoclaved.Whenfull,andatleast10minafterneutralizationwithfreshbleach,theliquidwastebottlecanbeemptiedintoaregularsink.Incaseofamajorspillofvector-containingliquid,absorbliquidwithpapertowelsandneutralizewithfreshconcentratedbleachpriortodisposal.Incasethereisaleakinbuckets,removethetubesinthehood,fillthebucketswith75%ethanol,andinvertthemseveraltimes.Leaveunderthehoodfor20min.Discardthe75%ethanolunderthehood.操作病毒时应注意防护P2practices：Assigned Readinghttp:/www.addgene.org/lentiviral/packaging/http:/www.addgene.org/lentiviral/protocols-resources/#protocolsAssignedReadinghttp:/www.add