抗菌肽dcd-antimicrobial peptide dcd.docxVIP

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抗菌肽dcd-antimicrobial peptide dcd.docx

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抗菌肽dcd-antimicrobial peptide dcd

ABSTRACTDermcidinisanewantibacterialpeptideidentifiedmostrecentlyfromhumansweatglands,andthegeneofDCD-1LisaderivantofDermcidinwhichhasabroadspectrumofactivitiesagainstGramnegativeandpositivebacteria.Theyarenewclassofantibacterialmedicineshavingenormouspotentialities.Thereforeithasgreateconomicvalueandtheoreticalsignificance.InthisresearchweobtainDCD-1Lbyoverlapextention,cloneandexpressitinPichiapastorisGS115,determinetheantifungalactivitysimply,constructtheexpressionvectorofrandommutantpopulationofDCD-1L,andtrytoscreenhighexpressingPichiarecombinantclones.Themethodisasfollow:(1)DNAfragmentsweredesignedaccordingtotheaminoacidsequenceanddifferencesincodonusage,obtainDCD-1LbyoverlapextentionandclonedintoexpressionvectorpPICZ?-A,Whichcontainsα-factorsignalsequence.Therecombinantplasmidwassequenced,digestedwithSacⅠandtransformedPichiapastorisGS115byelectroporation.ScreenanddeterminetheZeocin-resistantcolonies.UnderthecontrolofthepromoterAOX1(alcoholoxidase1),thepeptidewasexpressedinsuper-natantprotein.AnalyzethesupernatantsbyantibacterialactivityassayandCoomassie-stainedSDS.(2)ObtainDCD-1LwithhesequenceofrandommutationsbyoverlapextentionandclonedintoexpressionvectorpGAPZα-C,WhichcontainsGAPpromotorandα-factorsignalsequence.TherecombinantplasmidwasdigestedwithBspHⅠandtransformedPichiapastorisSMDII68byelectroporation.ScreenthehighexpressingPichiaStrainswithreplicaplatingfromrandommutantpopulationofDCD-1L.Theresultsareasfollows:TheDCD-1Landitsmutantgenewereobtained.TheDCD-1LgenewascorrectlyinsertedintothevevtorpPICZα-A.SequencinganalysisindicatedthatDCD-1Lwasidenticaltothesequencewanted.TransformedtherecombinantplasmidintoPichiapastorisGS115byelectroporation,theproteinbandabout6KDappearedonSDSgel.Throughdeferminationofinhibitingbacteriacircle,weobservedtheinhibitionofbacteriawithsupernatants.ThemurantofDCD-1LgenewasinsertedintothevevtorpGAPZα-C.TransformedtherecombinantplasmidintoPichiapastorisSMD1168byelectroporation.ResearchonscreeninghighexpressingPichiaStrainsisanongoin