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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)andpurifiedbyasingle-stepaffinitychro-matographyonaNi-NTAcolumn.BALB/cmic
4、ewereimmunizedwiththepurifiedrecombinantproteinstoprepareMAbsbyhybridomatechnique.ThereactivityandspecificityoftheMAbswereanalyzedbyELISAandWesternblotanalysis.SevenMAbsagainstN1andtwoMAbsagainstN2wereobtained.Inthepresentstudy,recombinantSARS-CoVNproteinwasexpressedandpurifiedan
5、dninespecificMAbsagainstSARS-CoVNproteinwereobtainedsuccessfully.Thispanelofanti-NMAbsmaybeusedasatoolforrapidandspecificdiagnosisofSARS-CoV.(6)Introductiondesigned;however,PCRmethodsneedspecialdevicesandhighqualificationswithsomewhatcomplexprotocolsandSevereacuterespiratorysyndr
6、omecoronavirusmaybringfalse-positiveresultsforcontamination.Aspecific(SARSCoV)causedaworld-wideepidemicin2002andantibodyorantigendetectiontestwouldbetechnologically2003,andinfectedmorethan8000humans,withafatalityratesimplerandlessexpensive.Inthisstudy,weexpressedand(1)ofabout10
7、%.Althoughtherehavebeennorecentout-purifiedthefragmentsoftheNproteinofSARS-CoVandbreaks,thedevelopmentofadiagnostictestforspecificandproducedMAbsagainstthisprotein.ThecharacteristicsoftheearlydetectionofSARSCoVremainsofhighimportance.antibodieswerefurtherstudiedbyWesternblotanal
8、ysis.TheSARS-CoVisasingle-strandedplus-senseRNAvirus.Itsgener
