SARS coronavirus S protein gene expression and monoclonal antibodies.docx

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1、SARScoronavirusSproteingeneexpressionandmonoclonalantibodiesSAKScoronavirusSproteingeneexpressionandmonoclonalantibodiesSARScoronavirusSproteingeneexpressionandmonoclonalantibodiesOf:PROCEEDINGSJohnson,XiaMei,MiaoFengqin,XieD,ZhangJianqiongAbstractObjective:ExpressionofSARS-CoVSproteinfragment,andpr

2、eparespecificmonoclonalantibody(11Ab).Methods:ProkaryoticexpressionofSARS-CoVSproteinfragment,CDNAwasamplifiedfromtheSARSvirusSprotein(Nterminal205to419aageneandsequencedcorrectly.ThisgenefragmentwasinsertedintoPQE30constructtherecombinantplasmidQE30Slm,therecombinantplasmidwastransformedintoE.coliM

3、15inducedHisSlmprotein,purifiedimmuneB1.B/cmicewerefused,screeningPreparationofspecificmAb.RESU1.TS:TheexpressedandpurifiedrecombinantproteinIlisSlm,getananti-HisSproteinmAblH2.IgsubgroupidentifiedasIgGla,lightchaintype,byWesternblottingdetection,mblU2withtheSproteinwasspecificresponse,butnocrossrea

4、ctionwiththeHisprotein.Conclusion:TherecombinantSproteinwassuccessfullyexpressedandpreparedspecificmAb,fortheearlydiagnosisofSRSCoVSproteinandfurtherstudythefunctionofthefoundation.Keywords:severeacuterespiratorysyndrome,SRScoronavirus,Sprotein,monoclonalantibodySevereacuterespiratorysyndrome(severe

5、acuterespiratorysyndrome,SRS)isinNovember2002anewoutbreakofinfectiousdisease,infectiousdisease,strong,shortlatency,rapid,andhighmortality12.AlthoughSRSepidemichasbeenbroughtundercontrol,butsti11thepossibi1ityofrecurrence.Therefore,thedevelopmentofprevention,diagnosisandtreatmentmethodsisimportant.SA

6、RSisolationandidentificationofpathogensthat,SARS-CoV(coronavirus)(SRSCoV)isanewcoronavirus.ThemainstructuralproteinsS,E,UandN,whereSproteinisatypeglycoprotein(genelength3768bp,encoding1255aminoacids),infunctioncanbedividedintoSlandS2:N-sideforthesub-unitsSI,constitutethematureballpartofthespikeprote

7、in,CterminaltosubunitS2,thehandlepartoftheformationofelongatedprocesses.Sproteinsinthevirusandhostcel1surfacereceptorandmediatestheprocessofthevirustoentercellsplayakeyrole.SRSCoVSproteinrecognitionreceptorsonhostcellsandothercoronavirusisnotthesame,itismainlythroughtheangiotensin-convertingenzyme2(

8、angiotensinconvertingenzyme2,ACE2)asitsfunctionalreceptors34.Wongetal4foundthatSI,12327aaand12481aanotwithACE2binding,buttheSI,318510aaofthe193aminoacidregioncanbeeffectivelycombinedwithCE2.Moreover,Sproteinisthemajorcoronavirusantigen,antibodiestothevirusonitandinmanysitesofSproteinwasconfirmedbyne

9、utralizingantibodiesinducemajorcomponentofprotectivevaccinesareusedtodevelopthebestpartsof57.ThisexperimentusesPCRmethodstoSARSviruscDNasatemplatetoamplifySlmprotein(Nterminal6131256aaofthegenefragmentexpressionofrecombinantproteinSlm,purifiedimmuneB1.AB/cmice,monoclonalantibodiespreparedtofortheear

10、lydetectionofSARSCoV,furtherstudythefunctionofSproteinbasis.1Materialsandmethods1.1Materials1.1.1SARSviruscDNAbytheBeijingGenomicsInstitute1.imited.1.1.2Strains,plasmidsE.coliM15,DH5andplasmidPQE30arestoredinourlaboratory.1.1.3Experimentalanimals,cellB1.B/cmicewereprovidedbytheUniversityAnimalCenter

11、,SP2/0mousemyelomacellsinourlaboratory.1.1.4ReagentExTaqDNpolymerasewaspurchasedfromTaKaRaCompany.BamH,HindwerepurchasedfromMBICompany,1.Bsolid,liquidmedium,Ampicilinpreparedbythelaboratory.DNApurificationkitwerepurchasedfromQiagenCompany,GeneRulerTM1kbDNAladderwaspurchasedfromMBICompany.BCAproteink

12、itwaspurchasedfromPierceCompany,goatanti-mouseantibodywaspurchasedfromZhongshanCompany,primersynthesisandsequencingbyShanghaiShenstealcompany.Freundcompleteandincompleteadjuvant,newborncalfserumandHRPgoatanti-mouseIgGwerepurchasedfromSigmaCompany,HATandHT,PEGwaspurchasedfromNanjingMedicalUniversity,

13、mouseIgsubtypedeterminationkit(Isotripkit)purchasedfromBoehringerMannheimCorporation.RPMI1640mediumpurchasedfromGibcoCompany.EC1.waschromogenicsubstratewerepurchasedfromPierceCompany.PVDFmembranewaspurchasedfromSchleichcramp;Schullcompany,forSlmproteindesignPCRprimersforthe5GCGGTCCGTGTTCGTGTCTCCTTC3

14、,downstreamprimeris5GCAAGCTTCACCCTGAAATCATCTGG3(restrictionsiteisunderlined).1.2Methods1.2.1SproteingenefragmentobtainedandtherecombinantplasmidtoSARSviruscDNasatemplate,usingspecificprimersbyPCRtoobtainSprotein(Nterminal205419aagenefragments,usingBamH,Hindrestrictionpoint,thetargetgenewasinsertedin

15、tovectorPQE30andtransformedintoE.coliDH5amplifiedandplasmidwasextracted,recombinantplasmidwasHindandBamIIrestrictionenzymedigestion,agarosegelelectrophoresisproductsweredigested.AfterdigestionIdentificationofthecorrectrecombinantplasmiddeliverycompanyShanghaiShensteallinessequenced.1.2.2Prokaryoticr

16、ecombinantproteinexpressionandpurificationoftherecombinantplasmidPQE30SlmintoE.coliM15,adding0.05mmol1.-IofIPTG,at30for3hinducedtheexpressionofrecombinantproteinexpressionproductsstainedbyCoomassiebrilliantblueobservations,tothesameconditions,alargenumberofinducedexpressionofrecombinantprotein,thecentrifugedbacterialpciletbysonication,accordingtotheinstructionswithaNicolumnpurifiedHisSlmprotein,usingSDSP.GEidentificationofpurificationc