冠狀病毒Coronavirusl論文-2010 Preparation and Characterization of Polyclonal Antibody Against Severe Acute Respiratory Syndrome-associated Corona.pdf

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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)wasampli?edbyPCR.ThegenewasclonedintoprokaryoticexpressionvectorpGEX-6P-1,resultinginarecombinantplasmidpGEX-SARS-S1.Subse-quently,pGEX-SARS-S1wastransformedintohostcellsBL21(DE3)pLysS,andtheexpressionoftheS1protei

3、nwasinducedbyisopropylb-D-thiogalactoside(IPTG).PolyclonalantibodyagainstSARS-CoVS1proteinwasgeneratedinarabbitimmunizedwiththepuri?edS1protein.ThereactivityoftheantibodytotheSARS-CoVS1proteinwascon?rmedbyWesternblotanalysis.ELISAindicatedthattheantibodyagainstSARS-CoVS1proteinhadnocrossrea

4、ctionwithS1proteinsoftransmissiblegastroenteritisvirus,aporcinecoronavirus,andinfectiousbronchitisvirus,anaviancoronavirus.TheSARS-CoVS1proteinanditsantibodyarevaluablereagentsforrelatedstudies.IntroductionSincetheviralSproteinisamainsurfaceantigenandfunctionalprotein,theavailabilityofSARS-

5、CoVSproteinSevereacuterespiratorysyndrome-associatedbene?tspreparationofviraldiagnosticreagentsanddevel-coronavirus(SARS-CoV)isclassi?edintothefamilyopmentofvaccines.Nevertheless,isolationofSproteinfromCoronaviridae,accordingtothesequenceandphygeneticthecrudevirusandarti?cialsynthesisoftheS

6、proteinmay(1)analysis.Coronavirusesarelarge,enveloped,andpositivehavesomeconcernsonthebio-safetyandproductioncost.Insensesingle-strandedRNAvirusesthatreplicateinthecyto-thisstudy,weconstructedarecombinantplasmidencoding(2)plasmofhostcells.TheSARS-CoVgenomecontains?vethemajorreceptor-binding

7、domainintheSprotein(desig-majoropenreadingframes(ORF)thatencodethereplicasenatedS1)ofSARS-CoV.ThehighlevelexpressionoftheS1polyprotein;thespike(S),envelope(E),andmembrane(M)proteinwasachievedinEscherichiacolisystem.Anti-S1anti-(3)glycoproteins;andthenucl