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1、JournalofHuazhongUniversityofScienceandTechnologyEMedSei~24(5):414-416,2004414ExpressionandPurificationofSARSCoronavirusMembraneProteinDAIWuxing(j~.~~.)~,LEIMingjun('g'~B-g)~,WUShaoting(~v9"N.)2,CHENZhihao(I~$#ff~)t,LIANGIAang(@r~,PANHuirong(.~N~)I,QINLi(g,~)~,GAOShitong(N4-N)2,YUANShishan(.~4
2、:t:~-g)2,ZHANGRenli(~4:~t'])e~DepartmentofBiochemistryandMolecularBiology,SchoolofBasicMedicalScience,TongjiMedicalCollege,Huang-:hongUniversityofScienceandTechnology,Wuhan430040,ChinaeShenzhenMunicipalCenterforDiseaseControlandPrevention,Shenzhe*z518020,ChinaSummary:Toconstructarecombinantpla
3、smidPet23a-M,thegeneencodingsevereacuterespirato-rysyndrome(SARS)coronavirusmembraneproteinwasamplifiedbyRT-PCRandclonedintotheexpressionplasmidPet23a.Resultsofrestrictionendonucleaseanalysis,PCRdetectionandDNAsequencinganalysisrevealedthattheclonedDNAsequencewasthesameasthatreported.There-com
4、binantsweretransformedintoEscherichiacoli(E.Coil)BL21(DE3)andinducedbyIsopropyl-13-D-thiogalactopyranoside(IPTG).Theexpressionof27kD(1kD=0.9921ku)proteinwasde-tectedbySDS-PAGEandpuredbymetalchelatedchromatography.ResultsofWestern-blotshowedthatthisexpressedproteincouldreactwithantibodiesinsera
5、ofSARSpatientsduringconvales-cence.ThisprovidedthebasisforthefurtherstudyonSARSvirusvaccineanddiagnosticagents.Keywords:SARS;membraneprotein;geneexpression;proteinpurification;Western-blotAnovelcoronavirus(SARS-CoV)wasdiscov-Ltd.PlasmidDNAisolationkitwasobtainedfromeredinassociationwithcasesof
6、severeacutere-ShanghaiDNABiotechnologiesCo,Ltd.Isopro-spiratorysyndrome(SARS).Thesequenceofthepyl-~3-D-thiogalactopyranoside(IPTG),SDS-Na,completegnomeofSARS-CoVwasdeter-EDTA-Na2,Trisbase,Ampicillinwerepurchasedmined:1'8.ItisnotcloselyrelatedtoanyofthefromShanghaiSangonBiologicalEngineering&-p
7、reviouslycharacterizedcoronavirus.TheanalysisTechnologyServiceCo,Ltd.Sheepanti-humanofopenreadingframesofSARS-CoVshowedthatIgGlabeledbyhorseradishperoxidasewaspur-thereweresimilarmajorstructuralproteinsthatchasedfromPierceCompany(USA).S
