抗菌肽天蚕素B基因及其串联体在毕赤酵母中的表达

B王秀青1,张素芳2,曹瑞兵1,周斌1,陈溥言1*(1.,210095;2.,116023):(SOE)B,NKex2B3,Kex2,BpPICZA,Sac,SMD1168,TricineSDSPAGE,,,:B;;;;;(SOE):Q784:A:10002030(2007)03012004AntibacterialpeptideCecropinBanditstandemgeneexpressedinPichiapastorisWANGXiuqing1,ZHANGSufang2,CAORuibing1,ZHOUBin1,CHENPuyan1*(1.KeyLaboratoryofAnimalDiseaseDiagnosisandImmunology,MinistryofAgriculture,NanjingAgriculturalUniversity,Nanjing210095,China;2.DalianInstituteofChemicalPhysics,ChineseAcademyofSciences,Dalian116023,China)Abstrac:tCecropinBgenewasachievedthroughthegenesplicingbyoverlapextension(SOE).EspeciallyaKex2signalcleavagesitewasfusedinNendoftheantibacterialpeptidegene.CecropinBgenewassubclonedandligatedtandem.ThemodifiedCecropinBanditstandemgenesclonedintothepPICZAvectortoconstructtherecombinantexpressionvectors.TherecombinantexpressionvectorswerelinearizedbySacandtransformedintoPichiapastorisSMD1168strainbyelectroporation.Thepositivecloneswerescreenedandthosecloneswerefermentedinflask.TricineSDSPAGEshowedthattheCecropinBproteincouldbesecretedintothecultureleadingbyfactorfrompPICZA.Antibacterialactivitiesandheatstabilitywerealsofound.Keywords:CecropinB;antibacterialpeptide;Pichiapastoris;expression;antibacterialactivity;splicingbyoverlapextension(SOE)(antibacterialpeptide,ABP)(cecropins),37~39,4000,B(CecropinB)[1],[2],,[3],,B,,,,,1材料与方法11111质粒菌株CowanIK88:20060328:(BE2006364);(20050307023):,*:,,,Emai:laid@njaueducn2007,30(3):120~123JournalofNanjingAgriculturalUniversity(Pichiapastoris)SMD1168(His/Mut+)pPICZAInvitrogen112主要试剂DNAT4DNAligasedNTPTaKaRa,Tricine,SDSPAGE(C6210)Sigma,ZeocinInvitrogen,SDSPAGE,DDpeptoneY(yeastnitrogenbase,YNB)BBI,Invitrogen12BGenBankB,,DNAStarPrmierpremierB,NKex2,BN3F1F2F3,220,B:F1:5cctctcgagaaaagaaagtggaaggtcttcaagaagatcgaaaagatgggtcgtaac3;F2:5gatagctggaccagccttgacgataccgttacggatgttacgacccatcttttc3;F3:5gctggtccagctatcgctgtcctaggtgaagctaaggctctacaatgatctagatagact3(SOE)[4],B53XhoXba;1,,,5AOX,B,496bpP1:5gtctccacattgtatgcttc3;P2:5ctgtgcaggaacttgat3SOE,F1F2F3PCRSOE,(touchdown,TD)PCRTDPCR(50L):10PCRBuffer(Mg2+free)5L,MgCl2(25mmol!L-1)3L,dNTP(10mmol!L-1)1L,F1F2F3(40pmol!L-1)2L,ExTaqTM05L,385L,,TDPCRPCR:94∀2min;TDPCR:94∀30s,65∀50∀,1min,05∀,72∀1min,3050∀,52∀15;72∀6minTDPCR10L,15%,13BB,6B,53XhoEcoRBamHXba4N,5Kex26:P3:5accctcgagaaaagaaaagtggaag3;P4:5actggatccttgtagagccttag3;P5:5ttaggatccaaaagaaaagtggaag3;P6:5tgggaattcttgtagagccttag3;P7:5tgggaattcaaaagaaaagtggaag3;P8:5ccgtctagatcattgtagagcct314BPCRpPICZAXhoXba,XhoEcoRBamHXbaT4DNA,DH5,[5]PCRXho/Xba,[6]PCRInvitrogenpPICZACB,pPICZA3CB15SMD1168(His/Mut+)Mut+SMD1168(His/Mut+)(80L)SacpPICZACBpPICZA3CB(5g),02cm(Bio-Rad);5min,15kV25F200!5ms,1mL1mol!L-1;200LYPDS,30∀PichiaExpressionKitPCR,##PCR[7],P1P2,PCR:94∀5min;94∀45s,48∀45s,72∀45s,25;72∀6minpPICZACB496bppPICZA3CB35AOX,PCRPCR:94∀5min;94∀1min,55∀1min,72∀1min,30;72∀10min12kb!121!3:B165mLBMGY,30∀230r!min-122hD6003~6;3000r!min-12min,25mLBMMY,28∀250r!min-160h,24h1%()72h,5000r!min-110min,,17TricineSDSPAGE[8]18B[9],CowanK88(),(D600=02~03)200L55∀LB25mL;(5mm),20L,37∀8~12hpPICZA,Amp22结果与分析21,B123bp,Kex2,,B,322BTrisTricine,B(1)23(2),BEcoliDH5346mm,5LAmp(25g!mL-1),BAmp287mm1BTricineSDSPAGEFig1TricineSDSPAGEoftheCecropinBM1,M2.Molecularweightmarkersforproteins;1,2.pPICZACBSupernatantofpPICZACB;3.pPICZA3XCBSupernatantofpPICZA3CB;4.CK2BFig2AntibacterialactivityofCecropinBa.CecropinBagainstStaphylococcusaureus:1,2,4.CK;3,5.CecropinB;6.BPolycecropinB;A.Amp(25g!mL-1)b.K88CecropinBagainstEcoli(K88):1.CecropinB;2,5,6.CK;3,4:BPolycecropinB;A.Amp(25g!mL-1)c.DH5CecropinBagainstEcoliDH5:1,2.CecropinB;3,5,6.CK;4.BPolycecropinB;A.Amp(25g!mL-1)d.CecropinBagainstBacillussubtilis:1,3.CecropinB;2,4,6.CK;5.BPolycecropinB;A.Amp(25g!mL-1)!122!303讨论B,,B,TAG1CAA,∀,,,CB,,,,,[10-13],,,,,Kex2,,,,,,,3,,Kex2:[1],,.[J].,1999,19(5):1417[2]HultmarkD,EngstromA,BennichH,eta.lInsectimmunity:isolationandstructureofCecropinDandfourminorantibacterialcomponentsfromCecropiapupae[J].EurJBiochem,1982,127(1):207217[3],,,.CecropinB[J].,1999,6(4):193197[4]HortonRM,HuntHD,HoSN,eta.lEngineeringhybridgenewithouttheuseofrestrictionenzymes:genesplicingbyoverlapextension[J].Gene,1989,15,77(1):6168[5]J,EF,.[M].2.,,.:,1987[6],赟,蔡梅红,等.Magainin和CecAmil抗菌肽基因的密码子优化及在毕赤酵母中的高效表达[J].生物工程杂志,2004,24(7):9397[7],,,.PCRDNA[J].,2003,31(5):270272[8]SchaggerH,vonJagowG.Tricinesodiumdodecylsulfatepolyacrylamidegelelectrophoresisfortheseparationofproteinsintherangefrom1to100kDa[J].AnalyticalBiochemistry,1987,166(2):368379[9],,.[M].:,2006:108121[10]HiguchiR,KrummelB,SaikiRK.AgeneralmethodofinvitropreparationandspecificmutagenesisofDNAfragments:studyofproteinandDNAinteraction[J].NucleicAcidsResearch,1988,16(15):73517367[11]DavisGT,BedzykWD,VossEW,eta.lSinglechainantibodyencodinggenes:onestepconstructionandexpressionineukaryoticcells[J].Biotechnology,1991,88(9):37923796[12]KainKC,OrlandiPA,LanarDE.Universalpromoterforgeneexpressionwithoutcloning:expressionPCR[J].Biotechniques,1991,10(3):366374[13]CrucianiRA,BarkerJL,ZasloffM,eta.lAntibioticmagaininsexertcytolyticactivityagainsttransformedcelllinesthroughchannelformation[J].Pr