副溶血性弧菌基因敲除方法的建立及应用[1]

20116193ACTALABORATORIUMANIMALISSCIENTIASINICAJune2011Vol．19No．330930001、30900823、307711799732009CB522600。[e-mail]。[e-mail][e-mail]檵檵檵檵檵檵檵檵檵檵殝殝殝殝。1223122221221．21201302．1000713．102206【】。PCRpDS132S17λpirpDS132sacB。RIMD2210633ΔopaRΔtoxRΔaphA。。【】【】Q95-33【】A【】1005-4847201103-0188-05Doi10.3969/j．issn．1005-4847.2011.03.002Establishmentofasuicidevector-basedgeneknockoutmethodinstudiesofVibrioparahaemolyticusLIUXia12GAOHe23YANGLin12ZHANGYi-quan2TANYa-fang2GUOZhao-biao2HUANGXin-xiang1YANGRui-fu2ZHOUDong-sheng21.SchoolofMedicalTechnologyJiangsuUniversityZhenjiangJiangsu212013China2.StateKeyLaboratoryofPathogensandBiosecurityBeijingInstituteofMicrobiologyandEpidemiologyBeijing1000713.StateKeyLaboratoryforInfectiousDiseasePreventionandControlNationalInstituteforCommunicableDiseaseControlandPreventionChineseCenterforDiseaseControlandPreventionBeijing102206【Abstract】ObjectiveToestablishasuicidevector-basedgeneknockoutmethodinstudiesofVibrioparahaemo-lyticus．MethodsTheupstreamanddownstreamflankingDNAfragmentsoftargetgenewerefusedbyPCRandthenclonedintothesuicidevectorpDS132.TherecombinantplasmidwasintroducedintoE．colistrainSM17λpirandtrans-ferredintoV．parahaemolyticusbyconjugation．ThesacBgeneinpDS132wassucrose-sensitiveandusedtoscreenthede-letionmutants．ResultsTheopaRtoxRandaphAmullmutantsofV．parahaemolyticusweresuccessfullyconstructed．Eachtargetgenewasexactlydeletedandthemutantsconstructedwereunmarked．ConclusionsThegeneknockoutmethodisstablewithagoodefficiencyandcanbeusedforgenefunctionstudiesofV．parahaemolyticus．【Keywords】VibrioparahaemolyticusSuicidevectorHomologousrecombinationGeneknockoutVibrioparahaemolyticusVP。20116193ActaLabAnimSciSinJune2011Vol．19.No．3、、、、、1。。β-thermo-stabledirecthemolysinTDHKanagawaphenomenonKP。2-4。RIMD2210633O3K6KPTDH5。、、501962。。40%6。。。RIMD22106330opaR、toxRaphA。opaRpDS132。11.11.1.1RIMD2210633GS-PCR+tdh+KP+pDS132E．coliS17λpirCDC。Ap50μg/mL、Cm5μg/mL。1.1.2dNTPs、DNAExTaq、PfuTaKaRa、PCRQiagen、T4DNANEB。1.21.2.1RIMD2210633opaR400bp400bp1。RIMD2210633DNAVP-opaR-a/VP-opaR-bVP-opaR-c/VP-opaR-dPCRPCR30ngPfu1UExTaq2U0.15μmol/L50μL。94℃5min94℃40s60℃40s72℃1min3072℃5min。PCR。1.2.2PCR1∶1DNAPCRVP-opaR-a/VP-opaR-d。PCRPCR。1ΔopaRTab．1TheprimersusedinthisstudyPrimer5’-3’Sequence5’-3’RestrictionenzymecuttingsiteVP-opaR-aGTGACTGCAGACTGCCTTGGTAACGCTCTGpstIVP-opaR-bGTTCGTGTTCAAATCTGAGCTATCCATTTTCCTTGCCATTTG-VP-opaR-cCAAATGGCAAGGAAAATGGATAGCTCAGATTTGAACACGAAC-VP-opaR-dGTGAGCATGCATGGGCTGCATCAGGTCGsphIpDS132-FGTTTCTGTTGCATGGGCATAAAG－pDS132-RAACAAGCCAGGGATGTAACG－VP-opaR-RT-FTGTCTACCAACCGCACTAACC－VP-opaR-RT-RGCTCTTTCAACTCGGCTTCAC－。NoteBoldlettersprotectivebaseHorizontallinemarkedlettersrepresentrestrictionenzymecuttingsites．1.2.3sphIpstIpDS132。T44℃E．coliS17λpir37℃200r/min1h100μL98120116193ActaLabAnimSciSinJune2011Vol．19.No．31.5%LB34μg/mL37℃150r/min。PCRpDS132-F/pDS132-R、VP-opaR-a/pDS132-R、pDS132-F/VP-opaR-d。1.2.4E．coliS17λpirRIMD2210633LB37℃100r/minA6001.0。4℃3000g/min3min100μLLB。0.45μm1cm230μL。LB30℃6～8h。LBApRCmR30℃。1.2.510LB37℃100r/min1000150μL10%LB30℃。LB30℃LBLBPCRopaR-RT-F/opaR-RT-RVP-opaR-a/VP-opaR-d。1.2.6ΔopaR5mLLBD600nm1.21μLLB37℃。opaR1。22.1PCRVP-opaR-aVP-opaR-bopaR414bpVP-opaR-cVP-opaR-dopaR417bp831bp。1%2PCRPCR。2.2pDS132pstIsphI1ΔopaRFig.1FlowchartoftheΔopaRmutantconstruc-tion1.PCR2.3.M．DNA2opaRPCRNote1.ProductsoffusionPCR2.Upstreamflankingregion3.DownstreamflankingregionM．DNAmolecularmarkersFig.2AmplificationandfusionofflankingregionsofopaRDNA35630bps。PCRE．coliS17λpirpDS132pDS132-F/pDS132-R09120116193ActaLabAnimSciSinJune2011Vol．19.No．31.pDS132M．DNA3pDS132Note1.DigestedvectorDNAM．DNAmolecularmarkersFig.3pDS132DNAdigestedbypstIandsphIpDS132-F/VP-opaR-dVP-1.pDS132-F/pDS132-RpDS1322.pDS132-F/pDS132-R3.VP-opaR-a/pDS132-R4.pDS132-F/VP-opaR-dM．DNA4PCRNote1.pDS132-F/pDS132-Rasprimerpairandemptyvec-torastemplate2.pDS132-F/pDS132-Rasprimerpairandrecombinantvectorastemplate3.VP-opaR-a/pDS132-Rasprimerpairandrecombinantvectorastemplate4.pDS132-F/VP-opaR-dasprimerpairandrecombinantvectorastemplateFig.4PCRidentificationoftherecombinantvec-torsopaR-a/pDS132-R4。4-24-1PCR831bp。PCR。2.3E．coliS17λpir0.45μm。。2.4pDS132sacB10sacB。10%LBopaRopaR-RT-F/opaR-RT-RVP-opaR-a/VP-opaR-dPCRRIMD2210633DNApDS132。55A-1opaR5A-35B-35B-1615bpopaR。2.56ΔopaR7ΔopaR。62。3。sacBpDS132ΔopaR。sacBsacB。pDS132。sacB8。pDS132。pDS132LB0510152034μg/mL0μg/mL19120116193ActaLabAnimSciSinJune2011Vol．19.No．3M．DNA1.RIMD22106332.pDS1323.ΔopaR5opaRABNoteTheinternalprimersAandflankingprimersBofopaRwereusedrespectively．M．DNAmolecularmarkers1Wild-typestrainRIMD22106332.VectorpDS1323.ΔopaRFig.5PCRidentificationoftheopaRmutants5μg/mLpDS132E．coliS17λpir34μg/mL。。PCR。OPTR。OpaR。IPTGOpaRopaRΔopaR。OpaR。opaR9-11。ΔopaRopaRΔopaR。62。1YangLZhouDSLiuXMetal．Cold-inducedgeneexpres-sionprofilesofVibrioparahaemolyticusatime-courseanalysisJ．FEMSMicrobiolLett2009291150－58.2OnoTParkKSUetaMetal．Identificationofproteinssecre-tedviaVibrioparahaemolyticustypeIIIsecretionsystem1J．InfectImmun20067421032－1042.3Yang