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1、HYBRIDOMAVolume30,Number5,2011aMaryAnnLiebert,Inc.DOI:10.1089/hyb.2011.0028SARSCoronavirusNucleocapsidProteinMonoclonalAntibodiesDevelopedUsingaProkaryoticExpressedProtein1111221JuanZhang,DingWang,YueLi,QingZhao,AilongHuang,JianZheng,andWeixianChenImmunologicaldetectionofvirus
2、esandtheircomponentsusingmonoclonalantibodies(MAbs)isapowerfuldiagnosticmethod.HerewereportadetailedmethodfortheestablishmentofMAbsagainstsevereacuterespiratorysyndromecoronavirus(SARS-CoV).Toexpressandpurifythenucleocapsidprotein(Nprotein)ofSARS-CoVandgenerateMAbsagainsttheNp
3、rotein,geneencodingNproteinwasseparatedintotwopartsaccordingtothepredictionofepitopesandclonedintopET32a(+),respectively.ExpressionofthetargetproteinswereinducedbyMisopropyl-b-thio-D-galactopyranoside(IPTG)andpuri?edbyasingle-stepaf?nitychro-matographyonaNi-NTAcolumn.BALB/cmic
4、ewereimmunizedwiththepuri?edrecombinantproteinstoprepareMAbsbyhybridomatechnique.Thereactivityandspeci?cityoftheMAbswereanalyzedbyELISAandWesternblotanalysis.SevenMAbsagainstN1andtwoMAbsagainstN2wereobtained.Inthepresentstudy,recombinantSARS-CoVNproteinwasexpressedandpuri?edan
5、dninespeci?cMAbsagainstSARS-CoVNproteinwereobtainedsuccessfully.Thispanelofanti-NMAbsmaybeusedasatoolforrapidandspeci?cdiagnosisofSARS-CoV.(6)Introductiondesigned;however,PCRmethodsneedspecialdevicesandhighquali?cationswithsomewhatcomplexprotocolsandSevereacuterespiratorysyndr
6、omecoronavirusmaybringfalse-positiveresultsforcontamination.Aspeci?c(SARSCoV)causedaworld-wideepidemicin2002andantibodyorantigendetectiontestwouldbetechnologically2003,andinfectedmorethan8000humans,withafatalityratesimplerandlessexpensive.Inthisstudy,weexpressedand(1)ofabout10
7、%.Althoughtherehavebeennorecentout-puri?edthefragmentsoftheNproteinofSARS-CoVandbreaks,thedevelopmentofadiagnostictestforspeci?candproducedMAbsagainstthisprotein.ThecharacteristicsoftheearlydetectionofSARSCoVremainsofhighimportance.antibodieswerefurtherstudiedbyWesternblotanal
8、ysis.TheSARS-CoVisasingle-strandedplus-senseRNAvirus.Itsgener
