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1、HYBRIDOMAVolume29,Number6,2010ªMaryAnnLiebert,Inc.DOI:10.1089/hyb.2010.0044PreparationandCharacterizationofPolyclonalAntibodyAgainstSevereAcuteRespiratorySyndrome-associatedCoronavirusSpikeProteinChaoWangandXiaofengRenAtruncatedgene(designatedS1)encodingthereceptor-bindingdomain(RBD)inthesp
2、ike(S)proteinofsevereacuterespiratorysyndrome-associatedcoronavirus(SARS-CoV)wasamplifiedbyPCR.ThegenewasclonedintoprokaryoticexpressionvectorpGEX-6P-1,resultinginarecombinantplasmidpGEX-SARS-S1.Subse-quently,pGEX-SARS-S1wastransformedintohostcellsBL21(DE3)pLysS,andtheexpressionoftheS1protei
3、nwasinducedbyisopropylb-D-thiogalactoside(IPTG).PolyclonalantibodyagainstSARS-CoVS1proteinwasgeneratedinarabbitimmunizedwiththepurifiedS1protein.ThereactivityoftheantibodytotheSARS-CoVS1proteinwasconfirmedbyWesternblotanalysis.ELISAindicatedthattheantibodyagainstSARS-CoVS1proteinhadnocrossrea
4、ctionwithS1proteinsoftransmissiblegastroenteritisvirus,aporcinecoronavirus,andinfectiousbronchitisvirus,anaviancoronavirus.TheSARS-CoVS1proteinanditsantibodyarevaluablereagentsforrelatedstudies.IntroductionSincetheviralSproteinisamainsurfaceantigenandfunctionalprotein,theavailabilityofSARS-
5、CoVSproteinSevereacuterespiratorysyndrome-associatedbenefitspreparationofviraldiagnosticreagentsanddevel-coronavirus(SARS-CoV)isclassifiedintothefamilyopmentofvaccines.Nevertheless,isolationofSproteinfromCoronaviridae,accordingtothesequenceandphygeneticthecrudevirusandartificialsynthesisoftheS
6、proteinmay(1)analysis.Coronavirusesarelarge,enveloped,andpositivehavesomeconcernsonthebio-safetyandproductioncost.Insensesingle-strandedRNAvirusesthatreplicateinthecyto-thisstudy,weconstructedarecombinantplasmidencoding(2)plasmofhostcells.TheSARS-CoVgenomecontainsfivethemajorreceptor-binding
7、domainintheSprotein(desig-majoropenreadingframes(ORF)thatencodethereplicasenatedS1)ofSARS-CoV.ThehighlevelexpressionoftheS1polyprotein;thespike(S),envelope(E),andmembrane(M)proteinwasachievedinEscherichiacolisystem.Anti-S1anti-(3)glycoproteins;andthenucl
