1、抗SARS冠状病毒SI蛋白N端249至667的单克隆抗体的制抗SAKS冠状病毒S1.蛋白N端249至667的单克隆抗体的制备与鉴定作者：温坤车小燕东广州510282；1；梅亚波1（1；丘立文1：廖志勇1；袁国勇2：1第一军医高校珠江医院中心试验室，广2香港高校微生物学系，香港）摘要：目的在获得了具有免疫原性的SARS冠状病毒S1.蛋白片段的基础上，制备和鉴定特异性抗该段S1.蛋白单克隆抗体（mAb）.方法原核表达含S蛋白受体结合区的SARS冠状病毒S1.蛋白片段S1.c（N端249-667氨基酸残基），其免疫原性经SARS病人复原期血清鉴定后免疫BAI.B/C小鼠，按常规方法制备单克隆抗体，并
2、采纳E1.1.SA间接法、免疫荧光和免疫印迹进行筛选和鉴定。结果筛选出3株特异性针对SARS冠状病毒S1.蛋白N端249-667的mAb杂交瘤细胞株，IgC亚类鉴定1株为IgG1.,2株为1.gG2a,经免疫荧光鉴定与人冠状病毒株229E和0C43无交叉反应。结论获得3株抗SAKS冠状病毒S蛋白受体结合区特异性单克隆抗体，为建立新的SARS冠状病毒检测方法的和进一步探讨S蛋白的功能奠定了基础。关键词：SARS冠状病毒；单克隆抗体：刺突蛋白：受体结合区Preparationandcharacterizationofmonoc1.ona1.antibodiesagainstS1.domainatN
3、termina1.residues249to667ofSRS-associatedcoionavirusS1.proteinWENKun1.：MEIYa-bo1.；Q1.U1.i-wcn1.；1.IAOZhi-yong1.；YuenKwok-yung2;CHEXiao-yan1.!Centra1.1.aboratory,ZhujiangHospita1.,FirstMi1.itaryMedica1.University,Guangzhou510282,China;2DepartmentofMicrobio1.ogy,UniversityofHongKong,HongKong,China.Ab
4、stract:ObjectiveToprepareandcharacterizemonoc1.ona1.antibodies(mbs)againstS1.proteinofsevereacuterespirato-rysyndrome(SARS)-associatedcoronavirus(SARS-CoV).Methods6-His-taggedrecombinantfragmentatN-termina1.residues249to667ofSARS-CoVS1.proteininc1.udingSproteinreceptor-bindingdomainwasexpressedinE.c
5、o1.i.Theim-munogenicityofthisS1.domainwasidentifiedandusedtoimmunizeBA1.B/cmicefortheproductionofhybridomas.TheidentificationofthemAbsagainstthisS1.domainwasperformedusingindirectenzyme-1inkedimmunosorbentassay(E1.ISA),indirectimmunof1.uorescenceassay(IF)andWesternb1.otting,respective1.y.Resu1.tsThr
6、eehybridomasproducingmAbsspe-cifictotheS1.domainwasobtained,withare1.ativemo1.ecu1.armassof48500.Noneofthe3mAbswerereactivewithhumancoronaviruses229Eand0C43.TwooftheInAbSwereIgG2aisotype,andtheotherwasIgG1.Conc1.usionThisisthefirstre-portofnbsproducedagainstSproteinreceptor-bindingdomainofSARS-CoV.T
7、he3S1.-spccificmAbsmaybeusefu1.forfurtherstudyofthefunctionoftheSproteinandfordiagnosisofSARS-CoVinfection.Keywords:severeacuterespiratorysyndrome-associatedCOrOnaVirus;SARS-CoV;monoc1.ona1.antibody：spikeg1.ycoprotein;receptor-bindingdomainReceived:2004-01-03ThisworkissupportedastheKeyMedica1.Resear
8、chProjectforSARSPreventionsponsoredbytheMinistryofScienceandTechno1.ogyofChinaandbyGuangdongProvinceCorrespondingauthor:CHEXiao-yan,M.D.,ProfessorintheCentra1.1.aboratoryofZhujiangHospita1.,Te1.:86-20-61643592,Fax:86-20-61643592,E-mai1.:IinChe,chexiaoyanThetwoauthors,WENKunandMEIYa-bo,havemadeequa1.
9、contribu-tionstothiswork.nove1.coronavirushasbeenidentifiedasthema-jorcauseofseveracuterespiratorysyndrome(SARS)1.23,butthebio1.ogica1.functionsofthisSARS-associatedcoronavirus(SARS-CoV)remaincurrent1.ypoor1.ychar-acterized.Recentstudiesreportedthatangiotensin-con-vertingenzyme2(AEC2)cou1.dbeafuncti
10、ona1.receptorforSARS-CoVandthereceptor-bindingdomain(RBD)wasidentifiedintheN-termina1.residues17to274oftheS1.spikeproteinofthevirus4.SubsequentstudiesdemonstratedthattheRBDwas1.ocatedintheN-tcrmi-na1.residues303to5375orresidues318to5106.TheS1.proteinisa1.sofoundtobeatargetforinducingneu-tra1.izingan
11、tibodyresponsestoSARS-CoVinfection7.InspiteofthefactthatthefunctionofSproteinisnotfu1.1.yunderstood,S1.proteinseemstop1.ayakeyro1.eintheinitia1.virusinfection.Basedonthisunderstanding,themonoc1.ona1.antiodies(mbs)targetedatS1.domaincanbeapotentia1.1.yusefu1.too1.forthestudyofthefunc-tionofSproteinan
12、dforthediagnosisofSARS.Fur-thermore,theavai1.abi1.ityofb1.ockingantibodiesmaybemeaningfu1.inthetreatmentofSARS.Inthispaper,wereportthec1.oning,expressionandantigeniccharacterizationofthevariousfragmentsofS1.domainaswe1.1.asthepreparationandcharacter!zationofmAbsagainsttherecombinantproteinS1.cde-riv
13、edfromtheS1.proteincontainingtheN-termina1.residues249to667.MATERIA1.SANDMETHODSVirusandce1.11.inesHumancoronavirusstrains229E(No.VR740),0C43(No.VR759)andce1.1.1.inesVeroE6(No.CR1.-1586),MRC-5(No.CC1.-171),BS-C-I(No.CC1.-26)werea1.1.purchasedfromAmericanTypeCu1.tureCo1.1.ection(ATCC).P1.asmidsThep1.
14、asmidencodingS1.proteingene,pET_28b(+)/SI(402OO1.bp,HKU-39849),waskind1.yprovid-edbytheDepartmentofMicrobio1.ogy,UniversityofHongKong.TheexpressionvcctorPQE-30andM1.5strainofEscherichiaco1.iwerepurchasedfromQ1.A-GEN.ReagentsTherestrictionendonuc1.easesBamHIandHindIIIwerepurchasedfromNewEng1.andBio1.
15、abs,andKpn1.,PstI,ExTaq,dNTPandDN1.adderwerefromTaKaRa.NiresinwasobtainedfromQIAGEN.Freund,Sadju-vant,50%PEG1450so1.ution,HTandHTwerepur-chasedfromSigmaA1.drich.Goatanti-mouseIgM,IgG,IgG1.,IgG2a,IgG2bandIgG3peroxidaseconjugates,goatanti-humanIgGperoxidaseconjugates,goatan-ti-mouseIgGFITCconjugateand
16、aminoethy1.carbazo1.e(EC)sing1.eso1.utionweretheproductsofZYMED.RPMI1640cu1.turemediurnandfeta1.bovinoserumwerefromGibco-BR1.1.1.thechemica1.reagentsusedinthisstudywereofana1.ytica1.grade,andpreparedwithion-dep1.etedwater.Seraandanima1.sSerumsamp1.esofSARSpatientsforthisstudyweresero1.ogica1.1.yconf
17、irmedbyindirectimmunof1.uo-rescenceandenzyme-1inkedimmunosorbentassay(E1.ISA)8,andtheserafromhea1.thydonorswereusedascontro1.forthesameassay.Five6-week-o1.dfema1.eBA1.B/cmicewereprovidedbytheExperimenta1.Ani-ma1.CenterofFirstMi1.itaryMedica1.University.C1.oningS1.fragmentsintotheexpressionvectorandi
18、tsexpressionS1.geneencodingtheaminoacidresidues14to667ofthespikeproteinoftheSARS-CoVwasamp1.i-fiedbyPCRusingthefo1.1.owingprimers:5,-CGGGTCCAGTGACCTTGACCGGTGCACCAC-3,;5,-CGGGGTCCTTCGTTGAACTG-3,.TheS1.genewasdigestedwithBam1.1.IandKpnIand1.igatedintothecorrespondingrestrictionsitesinpQE-30p1.asmidtop
19、reparethepQE-30S1.Threegenefragmentsrespec-tivc1.yencodingtheaminoacidresidues14to248,249to445and219-667oftheS1.spikeproteinderivedfromS1.proteinwereobtainedbydigestionoftherecombi-nantp1.asmid(pQE-30S1.)withtherestrictionendonuc1.eases,a1.11igatedintothepQE-30p1.asmidaccordingtotheirrestrictionsite
20、sintheframeandupstreamoftheseriesof6histidineresiduesanddesignatedaspQE-30S1.a,pQE30S1.bandpQE30S1.crespective1.y.Thep1.asmidsweretransformedintocompetentM15ce1.1.sfo1.1.owingtheprotoco1.ofQIGENmanua1.Thestrainscontainingtherecombinantp1.asmidswerecu1.turedin1.uria-Bertanimediurn(1.Bmedium)withconti
21、nuousshakingat37Cti1.1.theODva1.ueofthemediumreached0.7,fo1.1.owedbyinductionwith1mmoI/1.isopropy1.-D-thioga1actopyranoside(IPTG)at30*Cfor4h.PurificationandWesternb1.otana1.ysisofS1.fragmentsThecu1.turedce1.1.swerecentrifugedandharvested.6-His-taggedrecombinantfragmentswerepurifiedus-ingNiresinunder
22、denaturingconditionaccordingtothemanufacturer,sinstructionsandana1.yzedbyWesternb1.otting.Thepurifiedproteinswereseparatedbysodi-umdodecy1.su1.fate(SDS)-po1.yacry1.amidege1.e1.ectro-phoresis(PAGE),ande1.ectrophoretica1.Iytransferredtopo1.yviny1.idenef1.uoride(PVDF)membraneswiththemethoddescribedprev
23、ious1.y9.Afterthetransfer,themembraneswereb1.ockedinPBS-Tcontaining7%de-fattedmi1.kfor2hatroomtemperatureandthenincu-batedrespective1.ywiththeserafromSARSpatientsandhea1.thydonorsatthedi1.utionof1:100intheb1.ockingbufferfor12hat4C.After6washes,themembraneswereincubatedwithperoxidaseconjugatetigoatan
24、ti-mouseIgGatthediIutionof1:500for1hatroomtemperature.ThemembraneswereincubatedwithAECsigna1.so1.utionfor10minatroomtemperatureaftersuf-ficientwashingandthereactionsterminatedwithdis-ti11.edwater.ImmunizationsThespecificimmunogenicityofthefragmentsdesignatedS1.cwascharacterizedbyWesternb1.ottingusi
25、ngtheserafromSARSpatientsandhea1.thydonorsrespec-tive1.y.Theimmunizationwasperformedasdescribedprevious1.y10.Brief1.y,5fema1.e6-weeko1.dB?1.B/cmicewereimmunizedintherearfootpadsandsubcuta-neoustissueswithpurifiedrecombinantS1.c(50gpro-teinpermouse)emu1.sifiedwithanequa1.vo1.umeofcomp1.eteFreund*sadj
26、uvantforthefirstinjectionandwithincomp1.eteFreund,sadjuvantforthefo1.1.owing3boosterinjections.Theserasamp1.edfromtheeyepitofthemiceafterthefourthimmunizationweretestedbyE1.ISAtoidentifythemousewiththestrongestresponsetoSic.Threedaysbeforefusionexperiment,theidenti-fiedmousewasinjectedwith100gS1.cpr
27、oteinintra-venousIywithouttheadjuvant.ProductionandscreeningofIiybridomasThe96-we1.1.cu1.turep1.ateswerepreparedforthefusionbyadding5104norma1.mouseSp1.enocytesinRPMI1640mediumsupp1.ementedwithhypoxanthine-aminopterin-1.hymidinemediumintoeachwe1.1onthedaybeforethefusion.Thep1.ateswereincubatedin5%C0
28、2at37C,andhybridomaswereinducedbyfusing1108immunizedmousesp1.enocytesand2107mye1.omace1.1.s(NS1:TCC)with50%PEG1450so1.ution.Thecu1.turesupernatantsofthehybridomace1.1.swerescreenedforantibodyproductionbyindirectE1.ISAWithrecombinantproteinand1.ysatesofthemutantstrainofSARS-CoVasthecoatingantigens,rc
29、spectivc-1.y.Positivehybridomac1.oneswerese1.ectedandc1.onedby1.imitingdi1.utionti1.1.thepositiveratereached100%.Thece1.11.ineswerethenfrozenandstoredin1.iquidni-trogen.DeterminationoftheimmunogIobu1.insubc1.assThemicrotiterp1.ateswerecoatedwithpurifiedS1.cprotein(100ng/we1.1.)for12handb1.ockedwith3
30、bovineseruma1.bumin.Thece1.1.cu1.turesupernatantwasaddedintoeachwe1.1.indup1.icate(1001.we1.1.)andin-cubatedat374Cfor1h,fo1.1.owedbytheadditionofgoatanti-mouseIgG,IgM,IgG1.,IgG2a,IgG2bandIgG3peroxidaseconjugates(di1.utedat1:1000,1001.we1.1.)andfurtherincubationat37,Cfor30min.Te-tramethyIbenzidinewa
31、susedasthesubstrate.Thereac-tionwasterminatedwith0.5moI/1.H2S04andtheab-sorbancemeasuredat450-nmwave1.engthinami-crop1.ateE1.ISAreader.Westernb1.otana1.ysisofmAbsTheexperimentwascarriedoutfo1.1.owingthesameprocedureasdescribedabove.PurifiedrecombinantS1.cproteinwasseparatedonSDS-po1.yacry1.amidege1.
32、sun-derreducedconditionandthege1.wastransferredtoPVDFmembranes.Themembraneswereincubatedwiththem?bfromhybridomacu1.turesupernatants.Af-terbeingwashedWithPBScontaining0.5%Tween-20,themembraneswereincubatedwithgoatanti-mouseIgGperoxidaseconjugate.AECchromogenwasusedforsigna1.detection.IFana1.ysisofthe
33、mbsVeroE6ce1.1.sinfectedwithSARS-CoV,MRC-5ce1.1.sinfectedwithhumancoronavirus229E,BS-C-1ce1.1.sinfectedwithhumancoronavirusOC43werehar-vestedandwashedtwiceinco1.dPBS.Thece1.1.sweredepositedon8-10we1.1.chambersiides,air-driedandfixedin30%methano1.and70%acetoneat-20Cfor10min.AfterbeingwashedwithPBS,th
34、es1.ideswereincubatedwithhybridomacu1.turesupernatantsat37,Cfor1handfurtherincubatedwithgoatanti-mouseFITCconjugateat37for30min.Thes1.ideswerestainedwith0.25%EvansB1.ue/PBSbufferandexaminedwitha1.eicaEc1.ipseepif1.uorescentmicroscope.RESU1.TSExpressionandpurificationofS1.fragmentsandantigeniccharact
35、erizationbyWesternb1.otana1.ysisExpressionof6-His-taggedrecombinantfragmentsderivedfromS1.proteinwereobservedinE.co1.iM15treatedwithIPTG.Expressionofcomp1.eteS1.proteinwasnotseeninE.co1.i.Thefragmentscontainingaminoacidresidues14to248,249to445and249to667oftheS1.protein,weresuccessfu1.1.yexpressedand
36、purified(Fig.1).Amongthethreefragments,designatedasS1.a,S1.bandS1.crespective1.y,on1.ythefragmentS1.creactedwiththeseraof12SARSpatientsinWesternb1.otting.prominentimmunoreactiveproteinbandofS1.c(withre1.ativemo1.ecu1.armassof48500)wasseenintheWesternb1.otting(Fig.2),whereastheS1.aandS1.bfrag-mentsfa
37、i1.edtoexhibitreactionwiththeseraoftheSARSpatients(datanotshown).Theseresu1.tssuggestthattheantigenicdomainis1.ocatedbetweenN-termina1.aminoacidresidues249to667oftheS1.spikeprotein.Fig.1SDS-PAGEana1.ysisofthe3purifiedS1.fragmentsPane1.1.ane1:Afragmentcontainingaminoacidresidues14to248ofS1.protein,de
38、signatedasSia：1.ane2:1.ow-moIecu1.armassproteinmarker;1.ane3:Afragmentcontainingaminoacidresidues249to445ofS1.protein,designatedasS1.b;Pane1.B1.anes1,2:Afragmentcontainingaminoacidresidues249-667ofS1.protein,designatedasSic;1.ane3:1.ow-mo1.ecu1.ar-massproteinmarkerProductionofmAbstoS1.proteinHybrido
39、masproducingantibodiesthatarespecifi-ca1.Iyreactivewiththeresidues249to667ofS1.proteinweresuccessfu1.1.yestab1.ishedfromSp1.enocytesofthemiceimmunizedwithpurifiedrecombinantS1.cprotein.FusionoftheSp1.enocytesofimmunizedmicewithNSTmye1.omace1.1.sproducedmorethan100hybridomace1.11ines,ofwhich3(A50,B30
40、andC31)werese1.ectedonthebasisoftheirstrongreactivitywiththeS1.cproteinasshownbyE1.ISAandIF.Theisotypesofthembs50,B30andC31wereIgG1.,IgG2aandIgG2a,re-spective1.y.Fig.2Westernb1.otana1.ysisofthe3purifiedS1.fragmentsPurifiedS1.cprotein(10gge1.)wasseparatedon10%po1.yacry1.amidege1.ande1.ectrob1.ottedon
41、toPVDFmembranes.Thestripswereincubatedwithcitherconva1.escentSARSpatientsseraorcontro1.sera;Pane1.:Thebandabout48500inre1.ativemo1.ecu1.armassshowsthatS1.cproteinisspecifica1.Iyrecognizedbytheserafrom12SARS-CoVinfectionpatients;Pane1.B:S1.cproteindoesnotreactwiththeserafrom12hea1.thsubjects.Characte
42、rizationofthem?bstoS1.proteinbyWesternb1.ottingandIFAsshowninFig.3,themAbsspecifica1.1.yboundtorecombinantS1.cprotein.Itcanbenotedthatthe11bsproducedasing1.ebandwiththeS1.cproteinatexact1.ythesame1.ocation(about48500inre1.ativemo1.ecu1.armass)asthereactionbandoftheserumfromconva1.es-centSARSpatient.
43、StrongreactionoftheantibodieswithSARS-CoV-infectedVeroE6ce1.1.swasobservedinindirectimmunof1.Uorescenceassay,withouttheirrecognitionofhumancoronavirusesOC43,229E(Fig.4),CCVorIBV(datanotshown).Theseresu1.tssuggestthatthemAbswerespecifictoSARS-CoVwithnocross-reactivityWithothercoronaviruses.Fig.3Weste
44、rnb1.otana1.ysisoftheMbsagaintpurifiedrecombinantS1.cproteinPurifiedS1.cproteinwasseparatedona10%po1.yacry1.amidege1.ande1.ectrob1.ottedontoaPVDFmembrane.Thestripswereincubatedwithdifferentanti-SIcproteinmonoc1.ona1.antibodiesfromIiybridomace1.1.cu1.turesupernatants.1.anCS1-3:DifferentmAbc1.ones(A50
45、B30,C31);1.aneS4,5:nti-histidineantibodyandmouseanti-S1.cpo1.yc1.ona1.serum;1.ancs6:Serumofaconva1.escentSARSpatient：1.anCS7,8:Negativecontro1.s(anti-humanIgMandanti-SARS-CoVrecombinantNproteinmAbsfromhybridomace1.1cu1.turesupernatants)DISCUSSIONTheSproteinisbc1.ievedtop1.ayacrucia1.ro1.einvira1.en
46、tryintothetargetce1.1.s1.1.RecentstudieshaveshownthattheS1.domainofSARS-CoVisCharaCter-izedasthebindingmo1.ecu1.eonthesurfaceoftheviruspartic1.es456.Inourstudy,wefoundthatantigenicdo-mainofS1.proteinis1.ocatedbetweenN-termina1.aminoacidresidues249667,inc1.udingtheS-proteinreceptor-bindingdomainatres
47、idues318-510.Theidentificationofthefunctiona1.S1.domainhasa1.1.owedustocarryoutfurtherstudyofthisvirus.Fig.4Immunof1.uorescenceofmAbC31toSARS-CoV-infectedVeroE6ce1.1.s(A,400),I1.uman-CoV229E-infectedMRC5ce1.1.s(B,200)and0C43-infectedBS-C1ce1.1.s(C,200),withnorma1.VeroE6ce1.1.s(D,200)usedasthenegativ
48、econtro1.Thece1.1.swerestainedwithhybridomace1.1cu1.turesupernatantsfo1.1.owedbygoatanti-mouseIgGFITCconjugate.Theresu1.tswereexaminedwithepifIuorescentmicroscope,showingspecificcombinationofthemAbswithSARS-CoVwithoutcross-reactionwithhuman-CoV.Inthisstudy,wehaveproducedandcharacterized3mAbsspecific
49、forS1.domainatresidues249667.Theisotypesofthe3MbswereidentifiedasIgG1.,IgG2aandIgG2arespective1.y,anda1.1.ofthemrecog-nizedS1.domainwithare1.ativemo1.ecu1.armassof48500.Indirectimmunof1.uorescenceassayshowedthatthemAbsreactedstrong1.ytoSARS-CoVTnfectedVeroE6ce1.1.s,withoutcross-reactionwithhumancorona-viruses0C43and229E.Sofartoourknow1.edgethisisthefirstreportofmb
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