重组人血清白蛋白在酵母中的表达及分析鉴定

2001-03-141964-100850rHSAPichiapastorisrHSAG418SDS-PAGEWesternBrodfordELISA3rHSAWesternrHSArHSA83.5g/L3.5g/LQ949.326.1A1000-5501200103-0202-03HighexpressionandanalysisofrecombinanthumanserumalbumininPichiapastorisCHENLinZHAOYan-HuaMAPingZHANGYa-DongBUFeng-RongInstituteofFieldTransfusionAcademyofMilitaryMedicalSciencesBeijing100850ChinaAbstractObjectiveTodevelopahigh-levelhumanserumalbuminHSAexpressionsystemusingPichiapastoris.MethodsTheexpressionvectorforHSAwhichwasconstructedinthislabwastransformedtoP.pastorisbyelectroporation.ThetransformantswereselectedbygrowthonG418andscreenedforHSApro-ductionbySDS-PAGEandWesternblot.TheHSAsecretionlevelswereestimatedbybiochemicalmethodelectrophoresisscanandBrodford'smethodandcompetitiveELISAmethod.ResultsTheWesternblotanal-ysisshowedthattherecombinantproductsasa67000Mrsecretionproteinreactedwithananti-HSAmono-clonalantibody.Byinducedexpressioninshakeflaskcultureforeightdaysabout3.5g/LofrHSAwasob-tainedfromZ162strainsupernatantofP.pastoris.ConclusionsAyeaststrainwasconstructedintroducinganexpressionvectorforHSAintotheP.pastorisgenome.Thestrainproducedasmuchas3.5gHSAperliterofshakeflaskculturemedium.KeywordrHSAserumallbuminPichiapastorisblottingWestern12HSA34rHSA56HSAPichiapastorisG41811.1GS115rHSApPIC9KHSAcDNA20220019253BullAcadMilMedSciSep2001Vol25No3HRP-IgGHSASigmaELISAHoeferBioRadAthermobioanalysisAmershamPhar-maciaBiotechBeckman1.21.2.1SalⅠrHSAGS115MD200µlYPD9630℃2d10µl190µlYPD961µlG4180.25～8mg/mlYPDBMGY30℃D6002～61/5～1/10BMMYSDS-PAGE1.2.2WesternSDS-PAGEHSA1.2.3rHSA3172pH4.23Brodford8595nmSDS-PAGEHSA22.1MDG418YPDG4181SDS-PAGE8mg/mlG418Z1621G418G418A.0.25mg/mlB.1mg/mlC.8mg/mlZ162SDS-PAGE67000HSA8d6d22Z162SDS-PAGE1.9740066200430003100020100144002.HSA3.4.Z1626d5.Z1628d6.2.2WesternZ162SDS-PAGEHSA670003HSA3Western1.662004300031000201002.3.2.3Z1622.3.143023.DZ1628dDrHSA3.3g/L4ELISAB1～6C1～6D1～65102550100200µg/mlB7～8C7～8200100B9C9B10～11C10～11Z1622001002.3.2D6300.275Z1628dD6300.3113.5g/L2.3.3BrodfordZ1628dD5950.38404.6g/LSDS-PAGErHSA83.038%rHSA3.8g/L39G418G418pH1BuckholzRGGleesonMA.YeastsystemsforthecommercialproductionofheterologousproteinsJ.Biotechnology19919111067-1072.2SchneiderSBuchertMHovensCM.Aninvitroassayofbeta-galactosidasefromyeastJ.Biotechniques1996206960-962.3FosterJF.ThealbuminstructurefunctionandusesM.Ox-fordPergamonPress1977.53-84.4PetersT.AllaboutalbuminbiochemistrygeneticsandmedicalapplicationsM.SanDiegoAcademicPress1996.2-12.5OhiHMiuraMHiramatsuRetal.Thepositiveandneg-ativecis-actingelementsformethanolregulationinthePichiapastorisAOX2geneJ.MolGenGenet19942435489-499.6CollinsSH.ProteinproductionbybiotechnologyM.LondonandNewYorkElsevierAppliedScience1990.61-77.7.J.1997183-A85-87.8.J.2000184350-352.9CereghinoJLCreggJM.HeterologousproteinexpressioninthemethylotrophicyeastPichiapastorisJ.FEMSMicrobiolRev200024145-66.201magneticfieldshealtheffectsandresearchneedsJ.Bio-electromagnetics19981911-19.2.J.C199727135-39.3.J.1994126454-456.4.3000MHzJ.199584262-264.5MerrittJHKielJLHurtWD.Considerationsforhumanex-posurestandardsforfast-rise-timehigh-peak-powerelectro-magneticpulsesJ.AviatSpaceEnvironMed1995666586-589.6.J.199422683-87.7AlbaneseRBlaschakJMedinaRetal.Ultrashortelectro-magneticsignalsbiophysicalquestionssafetyissuesandmed-icalopportunitiesJ.AviatSpaceEnvironMed1994655SupplA116-A120.40220019253