重组人脑乙酰胆碱酯酶的基因表达和生化毒理学性质

ISSN100727626CN1123870PQChineseJournalofBiochemistryandMolecularBiology2002218(1):44491),2),1),1)3(1),100850;2)0,100039)cDNApcDNA3.1,pcDNA2AChE293,rhAChE.rhAChEAChE.rhAChEKm137molPL;;huperzineAeserine(IC5021510-8molPL11010-7molPL);HI26(10-4molPL)sarin(10-6molPL10-7molPL)rhAChE,4h86%97%.rhAChE3,.,,Q55,Q78GeneExpressionandBiotoxicologicalPropertiesofRecombinantHumanBrainAcetylcholinesteraseLIQian1),LIUWei2),LIFeng2zhen1),SUNMan2ji1)3(1)BeijingInstituteofPharmacologyandToxicology,AcademyofMilitaryMedicalSciences,Beijing100850,China;2)ClinicalLaboratory,304HospitalofChinesePeoplesLiberationArmy,Beijing100039,China)AbstractTheencodingsequenceofhumanbrainacetylcholinesterasewassubclonedintoaneukaryoticex2pressionvectorpcDNA3.1andthentransfectedintohumanembryonickidneycellline293forexpressionofrecombinanthumanacetylcholinesterase.Thebiotoxicologicalpropertiesofrecombinantacetylcholinesteraseandnativeacetylcholinesterasewereverymuchalike.TheKmvalueofrhAChEwas137molPL;rhAChEex2hibitedtheexcesssubstrateinhibition.InhibitorhuperzineAandeserinecouldinhibitrhAChEwithIC50of21510-8molPLand110-7molPLrespectively.HI26(10-4molPL)reactivatedthesarin(10-6molPLand10-7molPL)2inhibitedrhAChEwithreactivationratesof86%and97%.TherhAChEcouldstandrepeatedfreezingandthawingforthreetimeswithoutlossofenzymeactivity.Keywordshumanbrainacetylcholinesterase,biotoxicologicalproperties,eukaryoticexpression:2001203208,:2001206204(962Z2016)3Tel:[phone],Fax:[phone]E2mail:[e-mail],,1970,,Received:March8,2001;Accepted:June4,2001Supportedbythe9thFive2Years2PlanMedicalResearchKeyProgramofPLA(No.962Z2016)3CorrespondingauthorTel:[phone],Fax:[phone]E2mail:[e-mail](acetylcholinesterase,AChE),Ach,.1990Soreq[1]AChE,AChE.Velan[2]Kronman[3]AChE,.AChE.AChEAChEAChE,AChE.AChEcDNACMV2E6,pcDNA2AChE,AChE,.©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(+)Invitrogen,.112293,.113XbaHindT4DNAPromega;DNA(Wiz2ardRplusminiprepDNApurificationsystem),Promeag;DNA,.114Sigma;5,52222(DTNB)HI26sarin.IgG(11000),IgM(11000);AChEAChE,.115DNA.116293371824h,50%80%,.A(12gDNA100l)B(225l100l).AB,1545min,DNA2,018ml,.23,DNA2.3710h,1ml2,814h..2472h.117DTNBAChE[4]PB(1P15molPL,pH712)180l,10mmolPL90l,251h,1molPLHCl30l,20min0103%DT2NB600l,300l96,414nm.118[5]SDS2PAGE,,.(25mmolPLTris,10%)15min,(25mmolPLTris,011molPLGly,10%),(013molPLTris,10%).323222,118mAPcm21h.,1%BSA1h.AChE1502h,TBST3,5min.HRPIgM11001h,TBST3,6mgDAB9ml011molPLTris2HCl,pH7161mlCoCl2(0103%),10lH2O2.119[6],9%,215%.50mmolPLTris,011molPLGly,110.100V.,(01065molPLPBS,pH610,5mmolPL,3mmolPLCuSO4,015molPLKFe(CN)3,0105%),3730min.1110[7]PBS2,4%P014%,15min.PBS,(01065molPLPBSpH610,5mmolPL,3mmolPLCuSO4,015mmolPLKFe(CN)3,0105%),372h.2211pcDNA2AChE21111pcDNA2AChECMV2E6AChEcDNA,AChE,AChEcDNACMV2E65Hind3Xba,pcDNA311541:©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(Fig.1).Km,rhAChELinewaver2BurkFig.1,Km137molPL.Fig.1KineticparametersofrhAChE:Lineweaver2Burkdouble2reciprocalplotKmwascalculatedfromtheLineweaver2Burkplot21212rhAChE,,,.AChE.,,AChE.010530mmolPL,DTNBrhAChE.,293rhAChE,Fig.2.21213HuperzineAeserinerhAChE10-5molPLeserine.HuperzineAAChE.20lPBeserinehuperzine180l,11.371h,,,Fig.2SubstrateinhibitioncurveofrhAChErhAChEwasassayedwiththeindicatedconcentrationsofacetylthio2cholineAChE.eserinehuperzineA100%,(Fig.3).huperzineAIC5021510-8molPL,eserineIC5011010-7molPL1Fig.3EffectsofselectiveinhibitorsonrhAChEactivityrhAChEwasassayedinthepresenceofeserine(),andhuperzineA(Ó)attheindicatedconcentrations.Acetylthiocholine(10mmolPL)wasthesubstrateinallcases21214HI26sarinrhAChE,AChE,011molPL(pH714)PBsarin211ml,sarin10-5molPL,371h100l,AChE,.2ml2,1ml,1800lPBsarin,1600lHI26200lPB(HI26110-4molPL),HI26sarin.PB,6418©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.=(EIR(EPER-EI)P(E-EI)100%HI26sarinrhAChE.(Table1)10-6molPL10-7molPLsarinrhAChEHI26(10-4molPL,37pH714),86%97%(Ta2ble1).Table1Reactivationofsarin2inhibitedrhAChEbyHI26SarinconcentrationEnzymeactivityEEIEIRERReactivation(%)byHI2610-6molPL01810010420117701018019070104511016011168610-7molPL018100104201187010210199101092110160111697€xs,n=3E:normalenzymeactivity;EI:inhibited2rhAChEactivity;EIR:inhibited2rhAChEreactivatedbyHI26;ER:HI26control21215rhAChE293,-203,30l.,rhAChE,3,(Table2).Table2StabilityofenzymeactivityofrhAChEduringrepeatedfreezing2thawingStoreat4Freezing2thawingOnceTwiceThrice0169501022017500101301746010090178601030€xs,n=321216rhAChE6ml,,PEG200001ml,011molPLPB(pH714).20lSDS2PAGE,rhAChE,.PVDF,AChEFig.4Western2blottingofrecombinantrhAChEA:ProteinmarkerB:Concentratedcellsupernatantoftransfected293cellshH11,Western2(Fig.4).rhAChE,67kD.21217293,,3730min.Fig.5.rhAChE242.Fig.5NondenaturinggelstainedforAChEenzymeactivityA,B,C:PurifiedtorpedoAChED,E:rhAChE21218pcDNA2AChE293,72h,293.Fig.6.