D2蛋白酶酶学生物活性研究及ELISA方法建立

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1、EnzymaticPropertiesofD2ProteaseandEstablishmentELISAforDetectingD2Abstractobjeetive：1．ToisolateandpurifytheserinealkalineproteasefromtheD2fermentation．2．ToinvestigatetheeffectofmetalionsandlaundrydetergentontheenzymaticpropertiesandstabilityofD2protease．3．ToestablishtheELISAforofD2quantitati

2、veanalysisproteasebypreparingthepolyclonalantibody．4．TotheaminoacidofD2investigatesequenceprotease．Methods：1．Theproteasewaspurifiedbyastepwiseprocedureincludingcentrifugation，gelsiccationandsize—exclusionchromatography,andidentifiedbySDS-PAGE．2．Theproteaseactivityandstabilitywasinvestigatedb

3、yaddingthementalionsca2+，cu2+，M92+'K+andcommercialdetergents．3．ThedoubleantibodysandwichELISAandindirectELISAforquantitativeanalysisofD2wereestablishedtheantiseraproteasebyagainstD2proteaseobtainedfromtherabbitsandguineapigs．meansofof4．Byelectrosprayionization—quadrupole-timefight—massspectr

4、ometry(ESI—Q-TOF—MS)，aminoacidsequenceofpurifiedD2proteasewasdeduced．Results：1．Thethree—steppurificationprotocolresultedina3．0-foldpurificationwith39．1％activityrecovery．2．EnzymaticactivityisenhancedbytlleadditionofmentalionssuchasCa2+,Cu2+,K+，Mg+．nestabilityandactivityofD2proteasehaveasignif

5、icantactivationwith5mMCaCl2．Theproteasecarlbestableinthecommercialdetergents．3．IndoubleantibodysandwichELISA，theoptimalworkingconcentrationsofcoatinganddetectionantibodieswerel：4000andl：5000respectively．ThelinearrangeanddetectionlimitfordeterminationofD2were28-1180p∥L，4．6p∥Lrespectively．Ther

6、ecoveryratesofD2rangedfrom92．44％～105．26％．ThedevelopedmethodshowedgoodrelationshiptothatbyBradfordmethod．InindirectELISA，tllelineardetectionrangeWasfrom18to1160rtg／L．Thecoincidenceratewas95．5％betweenthismethodandagardiffusionreaction．5．BymeansofESI—Q-TOF—MS，theaminoacidsequenceofD2wasdetermin

7、edasQVAWATGETAAPK，FAVQSTFK,LGYVSPPALVLADDNAAAR，QLNLNVTQLOCGVFSGK,AHGFLPLTK．APSATGGSALYPLEFVVGK。ConcIusion：TheELISAtechniquewasestablishedsuccessfully,ithaslaiedthefoundationforthestudyofenzymologyandbiology．TheconfirmationofD2Nterminalsequenceprovi