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Q503A10045503(2007)0213303DNA殷玉和1孙博1郭丽姝2所丹3孔维1DNADNADNA95%,58%,DNA,,DNA;;LargescalePurificationofPlasmidDNAbyUltrafiltrationwithHollowFiberFilterandHydrophobicChromatographyYINYuhe,SUNBo,GUOLishu,etal(CollegeofLifeScience,JilinUniversity,Changchun130012,China)AbstractObjectiveTodevelopaprocedureforlargescalepurificationofplasmidDNA.MethodsPurifyplasmidDNAbyaprocedureconsistingofalkalinelysis,ultrafiltrationwithhollowfiberfilter,hydrophobicchromatographyandmolecularsievechromatography.ResultsThepurifiedplasmidDNAreachedapurityofmorethan95%andmettherequirementsforplasmidDNAformedicaluse.Theyieldoftheprocedurewas58%.ConclusionThedevelopedpurificationprocedurewassimpleandofhighyield.ItlaidafoundationoflargescalepurificationofDNAvaccine.KeywordsPlasmidDNA;Ultrafiltration;Hydrophobicchromatography,DNADNADNA,DNA,10001[1]DNA,DNA,,,,DNA[2],DNA1DH5;DGPEi,CMVHIV1,13000bp:1(130012);2(130012);3(130012).:,Emai:lweikong@mai.ljlu.edu.cn2PhenylSepharose6FastFlow()Sepharose4FastFlow()Amersham;3(BIOFLO5000NewBrunswickScientificUSA)Quicksand(AmershamSweden)4DH5,,37,pH70,2L/min,DO50%,16h2700g30min,5100g,800mlTE(50mmol/LTrisHC,l10mmol/LEDTApH80),,1600ml(0155mol/LNaOH,1%SDS),,5min800ml(3mol/LKAcpH55),,10min12000g20min,10m,2800ml6DNA61:Quicksand(300000)13320072202ChinJBiologicalsFebruary2007,Vo1.20No.201~03MPa,300m,l10(100mmol/LTris,10mmol/LEDTApH80),25mol/L62:5[25mol/L(NH4)2SO4,100mmol/LTris,10mmol/LEDTApH80](xk50/30),,15ml/min,[3]63:2(01mmol/LPBpH70)(xk50/100),20%,10ml/min,7DNA71:,08%,,,DNA72:DNABCA(Pierce)100l100lBCA,3730min,562nm73:DNA(2000)1DNA11,,,,RNA,DNA,112,2,1DNA,2,22DNA21,95%,322DNADNA,11:alkalinelysis;2:ultrafiltration;3:HIC;4:SEC;M:1kbDNAmarker.1Fig1ElectrophoreticprofileofpurifiedplasmidDNA2Fig2PurificationofplasmidDNAbyHIC3DNAFig3ScanningofelectrophoreticprofileofplasmidDNA13420072202ChinJBiologicalsFebruary2007,Vo1.20No.21DNATab1ControltestonpurifiedplasmidDNAIndexAlkalinelysisUltrafiltrationHICSEC84796858Yield(mg)948685Recovery(%)11468995Purity(%)27008033NDResidualProtein(g/mgDNA)110020055Endotoxin(EU/mgDNA)DNA,[4]DNA,RNA,,,,,RNA,RNADNA,,,,10RNA[5]DNA,DNA,DNA,DNA,,QIAGEN100EU/mg,,,DNA10EU/mg,,,DNA,,,,,,[6],,DNA,,20%,DNA,,DNARNADNA,:90%;DNA0002g/g,5g;0001g/g,5g;DNA;001EU/g,20EU;DNA;A260/A280,175DNA,(RNA)(),DNA,,[1]FerreiraGN,MonteiroGA,PrazeresDM,eta.lDownstreamprocessingofplasmidDNAforgenetherapyandDNAvaccineapplications.TrendsBiotechno,l2000,18(9):380388.[2]PratherJK,SagarS,MurphyJ,eta.lIndustrialscaleproductionofplasmidDNAforvaccineandgenetherapy:plasmiddesign,production,andpurification.EnzymeMicrobialTechno,l2003,33(19):865883.[3]DiogoMM,RibeirSC,MonteiroGA,eta.lProduction,purificationandanalysisofanexperimentalDNAvaccineagainstrabies.JGeneMed,2001,3(6):577584.[4]KahnD,ButlerM,CohenD,eta.lPurificationofplasmidDNAbytangentialflowfiltration.BiotechnolBioeng,2000,69(1):101106.[5]EonDuvalA,MacDuffRH,FisherCA,eta.lRemovalofRNAimpuritiesbytangentialflowfiltrationinanRNasefreeplasmidDNApurificationprocess.AnalyticalBiochem,2003,316(1):6673.[6]DiogoMM,RibeiroSC,QueirozJA,eta.lScaleupofhydrophobicinteractionchromatographyforthepurificationofaDNAvaccineagainstrabies.BiotechnolLett,2000,22(17):13971400.(:20060324)13520072202ChinJBiologicalsFebruary2007,Vo1.20No.2