幽门螺杆菌烷基过氧化氢还原酶基因克隆 表达及多克隆抗体制备

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1、郑州大学2009届硕士学位论文英文摘要Clone一exPressionandPolyelonalantibodyPreParationof石殆疵obacterPytoriahPCgenePostgraduate:YanqingLiSuPe川sor:ProLGuangcaiDuanAbstractAhP(Alkylh川roPerexidereductase)15onememberofthePeroxydaseProtein几mily，whieh1523kD.TheseProteinsPlayanimPortantroleindecomPositionofh对rogenPerexideandhy

2、drog，Peroxidederivate.灿pC15eomPosedof汕PFandA冲C.Inreeentstudy，M/angdiseoveredthatmutantstraindefeetiveinalkylhydroPeroxidereductase(AhPC)weremoresensitivetooxidativestresseonditionsthanwildtyPecells.AndthisabilitytooxidativestressdireetlyimPactthelivingofH.Pytoriinthestomachofmiee.AhPCgenesequencesW

3、ereobtainedbyPCRmethod.ConstnjetionofahPCProkaryotieexPressionveetorwasea币edoutthroughdesignofsuitablesites.UnderoPtimizedinduetioneonditionthePositivecloneseffieientlyexPressedfusionProtein.ThefusionProteinwerePurifiedbyNiionaffinitychromatography.Polyelonalantibodieswereobtainedininllnunlzedrabbi

4、ts.InthefuturestudieswewillfurtherexaminehowtheeagAgeneimPactonAhPCProtein.MethodS1.AeeordingtothemultiPleeloningsiteofveetorPET30a(+)，thePrimersweredesignedbyusingPrernier5.0software.筋介1sitewasintrodueedintheuPstreamPrimer，and双01sitewasintroducedinthedownstrearnPrimer.2.Thefull一lengthahPCwaseloned

5、byPCRfromHP27.TheamPlifiedDNAfraglllentswerefirstligatedintoPMD18一TveetorthenintoPET-30aveetorandlasttransformedintoE.eolistainBL2l.ThePositiveeloneswerescreenedoutbyPCR.3.ThePositiveeloneswereoPtimizatedbyadjustinginducedeoneenirationofIPTG111尸，郑州大学2009届硕士学位论文英文摘要andtheinductiontime.4.Inclusionbod

6、ywasdissolvedbyusingurea.ThefusionProteinswereP而fiedbyNiionaffinitychromatograPhv.5.RabbitswereinununlzedwithfusionProteintogetherwithFreund，5adjuvant，阴dwereinnnunizedthereaftereveryaweekwithatotalof4times.6.AntibodytitersweredetectedbyELISA.Results1.PCRProduetshowedthatahPCgeneeonsistsof594bP.llwa

7、selonedintoPMD18TVeCtof.2.TheProk田下otieexPressionveetorofahPCwassuecessfullyeonstructed.ThesequencehomogenuitywasuPto99%.3.ThefusioncouldbeexPressedeffeeientlywhenitwasinducedbyIPTGandthequantitywasuPtomaxi