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3432007JOURNALOFBEIJINGUNIVERSITYOFCHEMICALTECHNOLOGYVol.34,No.3200713(,100029):1pET28a(+),BL21,IPTG,,,,,DEAE2SepharoseFastFlow,260mgNi2Sepharose,SephadexG225,97%1,,97%1,82mg/L,1:1;;Ni2Sepharose;:Q512.7:2006211210:,1982,3E2mail:jingchunchen@hotmail.com1(T1)2070Gold2stein5(TF5),T[122],T128,3108,412,N,T1TF5101000[324],T1,:,,T1[526],,,T1,0156%110%T1,,,,[7]T1,,,,,,,,70%,,T1,T1(rhT1),1111,pET28a(+),,BL21112,Oxiod;22D(IPTG),Sigma;;1(),;;DEAE2SepharoseFastFlowNi2SepharoseSephadexG225,Pharmacia;,Sigma;JY9823D,;DYY25,;GUJS210C,;WFB2UV22000,;(HITACHI),113DNApET28a(+),BL21Sambrook[8]-20LB[8](100mg/L),3710h2YT[8](100mg/L),378h,IPTG1mmol/L,6h,114,100mmol/LTris2HCl,10mmol/L(EDTA),100mmol/LNaCl,pH714,,,430min,2mol/L100mmol/LTris2HCl,pH714,430min,pH714,[9]20mmol/LTris2HCl,pH714DEAE2SepharoseFastFlow(DEAE),280nm,0500mmol/LNaCl20mmol/LTris2HCl,pH714,200mmol/LNaCl1615%SDS2PAGE[10]115rhT1,150150mmol/LTris2HCl,1mmol/LCaCl2,011%Tween220,pH810,812hA(20mmol/LNa2HPO4,10mmol/L,015mol/LNa2Cl,pH714),ANi2Sepharose(Ni),,SephadexG225,280nm,rhT1116rhT1RP2HPLC:L27490,L27420;Dia2monsil,C18,id5m,416mm250mm;:30;A:011%TFA/;B:011%TFA/100%;:VA:VB=5050,1mL/min;:214nm;5L[11]2211rhT1IPTG,16g/L,,4g/L,,,DEAE,,200mmol/LNaCl,1;110cm10cm;110mL/min;:0500mmol/LNaCl1DEAE2SepharoseFastFlowFig.1DEAE2SepharoseFastFlowchromatograph212200mmol/LNaClSDS2PAGE,SDS2PAGE2M:a200mmol/LNaCl;b2SDS2PAGEFig.2SDS2PAGEanalysisoffusionprotein7200,719717,,,,,7923:1,,,2,200mmol/LNaClDEAE,,DEAErhT1,rhT1260mg/L213rhT1N(His)6,T1C,,(His)6,Ni,Ni,T1,,SephadexG225,,rhT133rhT1Fig.3MassspectrumofrhT13rhT130671179,(3065125,N),,NirhT1,,,SephadexG225,,rhT1,82mg214rhT15LrhT1()rhT1,RP2HPLC,44,rhT121409min,rhT121386min,,rhT197%,,rhT1,rhT1,,[12]rhT130mg/L10L,rhT1170mg/L4rhT1RP2HPLCFig.4RP2HPLCchromatogramofrhT1rhT1,T1,,,rhT128,,,,rhT1,,rhT1,3(1),rhT182mg/L,10LrhT1170mg/L(2)RP2HPLC,97%(3),,4d,rhT1:[1]KNUTSENAP,FREEMANJJ,MUELLERKR,etal.Thymosin21stimulatesmaturationofCD34+stemcellsintoCD3+4+cellsinaninvitrothymicepithelia8922007organcoculturemodel[J].InternationalJournalofIm2munopharmacology,1999,21(1):15-26.[2]GOLDSTEINAL,GUHAA,HARDYMA,etal.Purificationandbiologicalactivityofthymosin,ahor2moneofthethymusgland[J].ProceedingsoftheNatio2nalAcademyofSciencesoftheUnitedStatesofAmerica,1972,69(7):1800-1803.[3]GOLDSTEINAL,LOWTLK,MCDOOM,etal.Thymosin1:Isolationandsequenceanalysisofanim2munologicallyactivethymicpolypeptide[J].ProceedingsoftheNationalAcademyofSciencesoftheUnitedStatesofAmerica,1977,74(2):725-729.[4],,,.1[J].:,2004,40(1):49-57.[5]SUGAHARAS,ICHIDAT,YAMAGIWAS,etal.Thymosin21increasesintrahepaticNKTcellsandCTLsinpatientswithchronichepatitisB[J].HepatologyRe2search,2002,24(4):346-354.[6]SARUEM,OZDENN,TURKELN,etal.Long2termoutcomesofthymosin2alphalandinterferonalpha22bcom2binationtherapyinpatientswithhepatitisBeantigen(HBeAg)negativechronichepatitisB[J].JournalofPharmaceuticalSciences,2003,92(7):1386-1395.[7]BILLICHA.Thymosinalpha1.SciClonePharmaceuti2cals[J].CurrentOpinioninInvestigationalDrugs,2002,3(5):698-707.[8]J,DW.[M].:,2002.[9],.2a1[J].,2006,33(1):23-26.[10]F,R,RE,.[M].:,2001.[11],,.1[J].,2004,55(3):497-500.[12],,,.1[J].,2003,19(5):636-639.Asimpleandrapidprocessforpreparingrhthymosin1withtransgenicEscherichiacoliQINWeiTaoCHENJinChunLIDongDong(CollegeofLifeScienceandTechnology,BeijingUniversityofChemicalTechnology,Beijing100029,China)Abstract:Humanthymosin1,asmallhormonewithalengthof28aminoacids,isapowerfulantiviraldrugfortreatmentofHBV,SARSandvariouscancers.Afterconsiderationofthefeasibilityofproducinghumanthy2mosin1onalargescale,anover2expressionvectorhasbeenconstructedbasedontheplasmidpET28a(+).Af2terinductionbyisopropyl2D212thiogalactopyranoside(IPTG),theobservedyieldofcrudefusionproteinswasupto260mg/L.Inordertoobtainthepurifiedtargetproteins,threedifferentchromatographiccolumns,name2lyDEAE2SepharoseFF,Ni2chelating2SepharoseandSephadexG225,wereoperatedinseries.ThepurityoftheresultingrhThymosin1wasmorethan97%,asshownbyRP2HPLCanalysis.Finally,82mgofpurifiedrhThymosin1wasobtainedfroma1literculture.Keywords:rhthymosin1;Escherichiacoli;Ni2chelating2Sepharose;fusionproteins9923:1