重组人肝细胞生成素的纯化及活性研究3

ISSN100727626CN1123870öQChin.J.Biochem.Mol.Biol.2000,Vol.16,No.1,101105161200023(863210220820521)33Tel:[phone],Fax:[phone]E2mail:[e-mail],,19681,:1999204212,:1999208203333(,100850)(humanhepatopoietin,hHPO).hHPO(rhHPO),20%.,8molöL,rhHPO,SDS2PAGE15kD.rhHPONcDNA;rhHPO.,rhHPO.,,,Q789StudiesonPurificationandActivityofRecombinantHumanHepatopoietinWANGQing2ming,CHENHui2peng33,CHENJi2zhong,FANGuo2cai,WEIHan2dong,YANGXiao2ming,HEFu2chu(InstituteofRadiationMedicine,AcademyofMilitaryMedicalSciences,Beijing100850)AbstractHepatopoietin(HPO)isanovalcytokinewhichspecificallystimulateshepaticcellpro2liferation.Recombinanthumanhepatopoietin(rhHPO),overexpressedinE.coli,wasfoundtoaccumulateincytoplasticinclusionbodies.Afterwashing,theinclusionbodiesweredissolvedin8molöLurea.Underthedenaturedstates,rhHPOwaspurifiedbysize2exclusionchromatography,thenitwasrenaturedbydilutinganddialysis.TherenaturedrhHPOwasthenseparatedbyion2exchangechromatographywithcontinuousgradientofincreasingsodiumchlorideupto500mmolöL.ThefinalpurifiedrhHPOwasofhighpurity(96%)anditexhibitedthemassofmolecule15kDanalyzedbyreducedSDS2PAGE.Thesequenceof16aminoacidresiduesfromNH22terminusofthepurifiedrhHPOwasfoundtoconsistwiththatofrhHPOdeducedfromcD2NA.Thecompositionofaminoacidsoftheproteinwasalsoanalyzed,theresultalsoconsistedwellwiththetheoreticvaluesofaminoacidsofrhHPO.TherenaturedrhHPOcouldproducethesignificantincreaseof3H2TdRintoprimaryhepatocytesinculture,whichdemonstratedthattherhHPOmightbeusedinclinicaltrialforliverregenerationinthetreatmentofhepaticdisease.KeywordsHumanhepatopoietin,Inclusionbody,Renaturation,Chromatography.1975LeBrecque[1],(hepaticstimulatorsubstance,HSS).,HSS,HSS[25].1984Hagiya[6]HSS,(augmenterofliverregene2ration,ALR).,[7]cDNAALR(humanhepatopoietin,©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.)cDNA,,.,rhHPO.1111pBV2rhHPOöJM109.[8][9].pBV2rhHPOöJM1093034h,42,45h,,,SDS2PAGE,rhH2PO.112,50mmolöLTris2HCl1mmolöLEDTApH810110(WöV),1mgöml,42h,(650W),3,3min,2min,,10000g20min,.1molöLNaCl015%TritonX21004molöL,50mmolöLTris2HCl(pH810)8molöL,10000g20min,.113SephacrylS2100HR(Pharmacia),80cm,216cm.,50mmolöLTris2HCl(pH810)8molöL,,20ml,5mgöml.114SephacrylS2100HR(216cm80cm)(1mmolöL,011mmolöL20mmolöLTris2HCl,pH810)10,4.,20mmolöLTris2HCl(pH810)..115MonoQHR5ö5(Pharmacia)20mmolöLTris2HCl(pH810),,10ml,015mgöml,,NaCl,,SDS2PAGE.116rhHPOSDS2PAGE.15%.R250,rhHPO;,rhHPOrhHPO.117rhHPONEdman,rhHPON16.118rhHPOrhHPO:rhHPO1mg,6molöL1ml,,11024h,,,6300(Beck2man).,rhHPO.rhHPO1mg,412h,,,rhHPO.119rhHPO[10].10%10-9molöLRPMI1640,100Ll(2104)96,375%CO26h,rhHPO(520ngöml)TGF2A(20ngöml)EGF(20ngöml)RPMI1640,150Ll,48h,3HTdR015LCi,3h,,,.=cpmöcpm.2211rhHPOrhHPO,rhHPO20%.1molöLNaCl015%TritonX21004molöL,50mmolöLTris2HCl(pH810)8molöL,10000g20min,.8molöL,SephacrylS2100HR(Fig.1).,rhHPO.rhHPO.20116©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.öml,4,,20mmolöLTris2HCl(pH810).,015mgöml.MonoQHR5ö5(Fig.2).MonoQHR5ö520mmolöLTris2HCl(pH810),NaCl,0015molöLNaCl,ËSDS2PAGE(Fig.3),rhHPO96%.rhHPO,SephacrylS2100HRMonoQHR5ö5,rhHPO,rhHPO1819%(Table1).Fig.1GelfiltrationseparationofdenaturedrhHPOMedium:SephacrylS2100HR;Column:216cm80cm;Buffer:8molöLureain20mmolöLTris2HCl(pH810);Flowrate:115mlömin;Collection:10minötuberhHPOwaselutedinpeakÊTable1SummaryofpurificationofrhHPOStepTotalproteinömgYield(%)Inclusionbodies200Gelfiltration8040Renaturation753715Ion2exchangechromatography37181819Fig.2SeparationofrenaturedrhHPObyion2exchangechromatographyColumn:MonoQHR5ö5;BufferA:20mmolöLTris2HCl(pH810);BufferB:1molöLNaClin20mmolöLTris2HCl(pH810);Flowrate:110mlömin;Collection:5minötubePurerhHPOwaselutedinpeakËFig.3AssayofpurityofrhHPObyreducedSDS2PAGEM:Proteinsofthestantardmolecularweight;1:Inclusionbodieswashedbyvariousbuffers;2:rhHPOpurifiedbySephacrylS2100HRfiltration(peakÊinFig.1);3:rhHPOpurifiedbyMonoQHR5ö5ion2exchangechro2matography(peakËinFig.2)212rhHPOrhHPO,,N3011:©1994-2006ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(C).cDNArhHPON.rhHPO,6300(Beckman)rhHPO,Table2.Table2rhHPOrhHPO.Table2AminoacidcompositionofrhHPOAminoacidNumberofresiduespermoleculeValuesdeducedfromcDNAsequenceAspandAsn1419916Thr61237Ser41894GluandGln1719216Pro61318Gly91134Ala61276Val41503Ile11911Met01612Leu916810Tyr31173Phe51016His41606Lys71258Arg1014613Cys8Trp4,rhHPO.rhHPO3161,rhHPO41,.213rhHPOTable3,(),rhHPODNA,.20ngöml,rhHPODNA219,EGF(214),TGF2A(319).Table3EffectsofrhHPOonDNAsynthesisofprimaryrathepatocytesinvitroTreatmentcpmperwellcell(xs)StimulationindexSalinecontrol162567EGF39454202141)MouseTGF2A64603923191)rhHPO(5ngöml)156784019rhHPO(10ngöml)26731781162)rhHPO(15ngöml)37852372131)rhHPO(20ngöml)47505282191)1)P01012)P01053,.1975LeBrecque[1]HSS.,,HSS[25].1981,,.,HSS.rhHPO,,.,rhHPO,,rhHPO,rhH2PO.rhHPO.(References)1LaBrecqueDR,PeschLA.Preparationandpartialcharacteri2zationofhepaticregenerativestimulatorsubstance(HSS)fromratliver.J