基因工程乙肝病毒表面抗原纯化工艺研究

:1997-12-15,:1999-01-25王妍王群罗璇官桂范魏自力阎昆明(130062)HBsAg,,,DNAQ789A1000-3061(1999)02-0263-66(r-HBsAg)CHO,,CHOr-HBsAg,,,CHOr-HBsAg,,r-HBsAg,,,,,2[1]DNA,,1材料和方法11111CHO-C28:SCHO,r-HBsAg,[2]112CHO:5%DMEM,CHO,[3]113:ButylSSepharose6FastFlow,SephadexG-25,DEAESepharoseFastFlow,Sepharose4FastFlowPharmacia-LKB,100kDMillipore,r-HBsAg(RPHA)12121[4](HIC),(IEC),(GFC),[5]122:[6]50ng/dose(WHOcriterion)DNA:[7]100pg/dose(WHOcriterion)r-HBsAg:A1(OH)3,Balb/c,(-HBs),ED50()2实验结果213r-HBsAg,,Pharmacia15219994ChineseJournalofBiotechnologyVol.15No.2April1999,2,3::(NH4)2SO4KBr1KBr2KBr:()[8]:()KBr223HBsAg3CHO,HBsAg1128,50L3,3113HBsAg/mLHBsAg/mgHBsAg/%/%50000600100(NH4)2SO410005529281KBr23048080126d2KBr210438737GFC28037266211150000600100HIC100004872812194dIEC180003005031GFC200219036513550000600100HIC1000048080204dKBr2603846416GFC2202868478162121.Process;2.Process;3.ProcessHIC1,3GFC21:HBsAg,621%,,478%,,365%,,6d,4d,4d,HBsAg3HBsAg,IEC,31%,HIC20%264153r-HBsAgSDS-PAGE1.Marker;2.r-HBsAgsample;3.Thefinalproductofr-HBsAgfromprocess;4.Process;5.Process,,10%GFCGFC,16%(1)2,3GFCABC33GFCA(,),GFCB(HBsAg)GFCC()23HBsAg3HBsAg,SDS-PAGE23kD,27kD,30kD,45kD(3),,HBsAgPAGEHPLC,95%,99%,97%3HBsAg10g/mL,,1,WHO2DNA/mgHBsAg/mg/(ng/10g)DNA/(pg/10g)7779460011003200(NH4)2SO4708855.21010016001KBr28248.022110080012KBr19443.8293=80=200GFC4037.261208=10=507779460.011003200HIC175048.723650=100IEC4030.097250=25GFC2321.91235=6.5=6.25GFC=13=125,,DNA(2),3HBsAg(50ng/10g),,HBsAg65ng/10g,,10ng/10g13ng/10gCHO,DNAHBsAg(100pg/10g),,HBsAgDNA625pg/10g,125pg/10g,50pg/10g,,3HBsAg,HBsAg,,HBsAg,,,,3讨论3:,2652:,,HBsAg,,,,HBsAg,DNA,,HBsAg,HIC,IEC,GFC,HBsAg,,,HICIEC,,,,,HBsAg,IEC,,KBrIEC,SephadexG-25,,HBsAg,,,,,,HBsAg[1],,.,1995,8(1):56~59.[2],,.,1987,3(4):313~320.[3],,.,1993,9(2):163~165.[4].,:,1990.[5]Janchrister,Janson,LarsRyden.ProteinPurification,NewYork,VCHPublishers,1989.[6],.,:,1994.[7]BOEHRINGERMANNHEIMBIOCHEMICAL,NonradioactiveInSituHybridizationApplicationManual,1992.[8]MakonnenBelew,MeiYafang,LiBinetal.Bioseparation,1991,1:397~408.StudyonPurificationProcessofRecombinantHBsAgWangYanWangQunLuoXuanGuanGuifanWeiZiliYanKunming(ChangchunInstituteofBiologicalProducts,Changchun130062)AbstractRecombinantHBsAgwasexcretedandsecretedinculturingmammaliancelltransferredwithhepatitisBvirusSproteingene.Wehaveappliedandstudiedtwokindsofpurificationprocessestopurifyr-HBsAgfromcellculturesupernatant.Atthebaseofit,consideringthephysicochemicalpropertiesofr-HBsAg,wedevisedandcreatedthethirdpurificationprocessandbroughtbetterre-sult.TheresultshowedthatthefinalHBsAgproductsfromthreepurificationprocessesattainedthecriteriaregulatedbyWHOinpurityandimmunity.Theoperatingconditionandapplyingmeasureofthreeprocesseswerecertain,whilethefreelevelofimpuritiesandresidualcellularDNAinvariousstepswereanalyzedandcompared.Theseresearchindicatedthecharacterofeachpurificationporce-dureandrealizedthepurposeofadoptingpurificationprocessbasedonthefeatureoftargetprotein.KeywordsRecombinantHBsAg,purificationprocess,calfserum,DNA26615