SARS coronavirus S protein gene expression and monoclonal antibodies.docx

SARScoronavirusSproteingeneexpressionandmonoclonalantibodiesSAKScoronavirusSproteingeneexpressionandmonoclonalantibodiesSARScoronavirusSproteingeneexpressionandmonoclonalantibodiesOf:PROCEEDINGSJohnson,XiaMei,MiaoFengqin,XieD,ZhangJianqiongAbstractObjective:ExpressionofSARS-CoVSproteinfragment,andpreparespecificmonoclonalantibody(11Ab).Methods:ProkaryoticexpressionofSARS-CoVSproteinfragment,CDNAwasamplifiedfromtheSARSvirusSprotein(Nterminal205to419aageneandsequencedcorrectly.ThisgenefragmentwasinsertedintoPQE30constructtherecombinantplasmidQE30Slm,therecombinantplasmidwastransformedintoE.coliM15inducedHisSlmprotein,purifiedimmuneB1.B/cmicewerefused,screeningPreparationofspecificmAb.RESU1.TS:TheexpressedandpurifiedrecombinantproteinIlisSlm,getananti-HisSproteinmAblH2.IgsubgroupidentifiedasIgGla,lightchaintype,byWesternblottingdetection,mblU2withtheSproteinwasspecificresponse,butnocrossreactionwiththeHisprotein.Conclusion:TherecombinantSproteinwassuccessfullyexpressedandpreparedspecificmAb,fortheearlydiagnosisofSRSCoVSproteinandfurtherstudythefunctionofthefoundation.Keywords:severeacuterespiratorysyndrome,SRScoronavirus,Sprotein,monoclonalantibodySevereacuterespiratorysyndrome(severeacuterespiratorysyndrome,SRS)isinNovember2002anewoutbreakofinfectiousdisease,infectiousdisease,strong,shortlatency,rapid,andhighmortality12.AlthoughSRSepidemichasbeenbroughtundercontrol,butsti11thepossibi1ityofrecurrence.Therefore,thedevelopmentofprevention,diagnosisandtreatmentmethodsisimportant.SARSisolationandidentificationofpathogensthat,SARS-CoV(coronavirus)(SRSCoV)isanewcoronavirus.ThemainstructuralproteinsS,E,UandN,whereSproteinisatypeglycoprotein(genelength3768bp,encoding1255aminoacids),infunctioncanbedividedintoSlandS2:N-sideforthesub-unitsSI,constitutethematureballpartofthespikeprotein,CterminaltosubunitS2,thehandlepartoftheformationofelongatedprocesses.Sproteinsinthevirusandhostcel1surfacereceptorandmediatestheprocessofthevirustoentercellsplayakeyrole.SRSCoVSproteinrecognitionreceptorsonhostcellsandothercoronavirusisnotthesame,itismainlythroughtheangiotensin-convertingenzyme2(angiotensinconvertingenzyme2,ACE2)asitsfunctionalreceptors34.Wongetal4foundthatSI,12'327aaand12481aanotwithACE2binding,buttheSI,318'510aaofthe193aminoacidregioncanbeeffectivelycombinedwithCE2.Moreover,Sproteinisthemajorcoronavirusantigen,antibodiestothevirusonitandinmanysitesofSproteinwasconfirmedbyneutralizingantibodiesinducemajorcomponentofprotectivevaccinesareusedtodevelopthebestpartsof57.ThisexperimentusesPCRmethodstoSARSviruscDNasatemplatetoamplifySlmprotein(Nterminal6131256aaofthegenefragmentexpressionofrecombinantproteinSlm,purifiedimmuneB1.AB/cmice,monoclonalantibodiespreparedtofortheearlydetectionofSARSCoV,furtherstudythefunctionofSproteinbasis.1Materialsandmethods1.1Materials1.1.1SARSviruscDNAbytheBeijingGenomicsInstitute1.imited.1.1.2Strains,plasmidsE.coliM15,DH5andplasmidPQE30arestoredinourlaboratory.1.1.3Experimentalanimals,cellB1.B/cmicewereprovidedbytheUniversityAnimalCenter,SP2/0mousemyelomacellsinourlaboratory.1.1.4ReagentExTaqDNpolymerasewaspurchasedfromTaKaRaCompany.BamH,HindwerepurchasedfromMBICompany,1.Bsolid,liquidmedium,Ampicilinpreparedbythelaboratory.DNApurificationkitwerepurchasedfromQiagenCompany,GeneRulerTM1kbDNAladderwaspurchasedfromMBICompany.BCAproteinkitwaspurchasedfromPierceCompany,goatanti-mouseantibodywaspurchasedfromZhongshanCompany,primersynthesisandsequencingbyShanghaiShenstealcompany.Freundcompleteandincompleteadjuvant,newborncalfserumandHRPgoatanti-mouseIgGwerepurchasedfromSigmaCompany,HATandHT,PEGwaspurchasedfromNanjingMedicalUniversity,mouseIgsubtypedeterminationkit(Isotripkit)purchasedfromBoehringerMannheimCorporation.RPMI1640mediumpurchasedfromGibcoCompany.EC1.waschromogenicsubstratewerepurchasedfromPierceCompany.PVDFmembranewaspurchasedfromSchleichcramp;Schullcompany,forSlmproteindesignPCRprimersforthe5GCGGTCCGTGTTCGTGTCTCCTTC3,downstreamprimeris5GCAAGCTTCACCCTGAAATCATCTGG3(restrictionsiteisunderlined).1.2Methods1.2.1SproteingenefragmentobtainedandtherecombinantplasmidtoSARSviruscDNasatemplate,usingspecificprimersbyPCRtoobtainSprotein(Nterminal205419aagenefragments,usingBamH,Hindrestrictionpoint,thetargetgenewasinsertedintovectorPQE30andtransformedintoE.coliDH5amplifiedandplasmidwasextracted,recombinantplasmidwasHindandBamIIrestrictionenzymedigestion,agarosegelelectrophoresisproductsweredigested.AfterdigestionIdentificationofthecorrectrecombinantplasmiddeliverycompanyShanghaiShensteallinessequenced.1.2.2ProkaryoticrecombinantproteinexpressionandpurificationoftherecombinantplasmidPQE30SlmintoE.coliM15,adding0.05mmol1.-IofIPTG,at30for3hinducedtheexpressionofrecombinantproteinexpressionproductsstainedbyCoomassiebrilliantblueobservations,tothesameconditions,alargenumberofinducedexpressionofrecombinantprotein,thecentrifugedbacterialpciletbysonication,accordingtotheinstructionswithaNicolumnpurifiedHisSlmprotein,usingSDSP.GEidentificationofpurificationc