应用抑制性消减杂交技术分析淋球菌染色体介导的耐药相关核苷酸序

DNADNA,5DNA,GenBank,5;;;CloningandidentificationofdifferentialgenomicgenesofantibioticresistantNeisseriagonorrhoeaebysup2pressionsubtractivehybridizationJIMing2chun,CHENHong2ju,YANHua,SHENHou2feng.DepartmentofMicrobiologyandImmunology,YangzhouUniversity,Yangzhou225001,ChinaCorrespondingauthor:JIMing2chun,E2mail:[e-mail]nethedifferentialgenesinantibioticresistenceNeisseriagonorrhoeae.MethodsUsingsuppressionsubtractivehybridizationconstruct,toconstructthelibrarywhichcontainsthediffer2entialgenesbetweenantibioticresistancestrainandstandardreferencestrainofNeisseriagonorrhoeae.Thentheantibioticresistancerelationalgeneswereclonedandanalyzed.ResultsAntibioticresistanceNeisseriagonor2rhoeaesubtractivelibrarywhichhashighsubtractiveefficiencywassuccessfullysetup.Theamplifiedlibrarycon2tained2500positiveclones.Sequenceanalysiswasperformedfor5clones.Allthefivesequenceswereunknownpreviously.ConclusionsThehighlyefficientDNAsubtractivelibrarymayestablishasolidfoundationforscreen2ingandcloningspecificantibioticresistancegenesofNeisseriagonorrhoeae.Thenoveldifferentialgenesmaypro2videanimportantclueforstudyingthemechanismofoccurrenceanddevelopmentofantibioticresistanceNeisseriagonorrhoeae.KeywordsNeisseriagonorrhoeae;Suppressionsubtractivehybridization;Antibioticresistance;Genes:225001:(E2mail:[e-mail]),1,:WHOA(WHO2A)RSM292C4HafizahYCRSM292C4,STD,2GC(Ox2iod,England)10%IsoVitaleX(BectonDickinson,Cockeysville,MD),37,6%CO224h(MIC)22,,11(MIC,g/ml)Table1.Bacterialstrainsandantibioticsminimumin2hibitiveconcentration(MIC,g/ml)WHO2ARSM292C4Penicillin0.0084.0Tetracycline0.254.0Ceftriaxone0.00050.004Ciprofloxacin0.01616.0Spectincmycin128256:PCR2selectbacterialgenomesub2tractionClontech,RsaRoche,pGEM2Teasy88320035235ChinJMicrobiolImmunol,May2003,Vol23,No.5©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.Promoga,MegaprimerDNAlabellingsystemsdCTP232PAmershamPharmaciaBiotechALFTMDNASequencer(adaptor)22Table2.AdaptorandprimersequencesSequencesAdaptor152CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT2332GGCCCGTCCA25Adaptor2R52CTAATACGACTCACTATAGGGCAGCCTGGTCGCGGCCGACCT2332GCCGGCTCCA25PCRprimer152CTAATACGACTCACTATAGGGC23Nestedprimer152TCGAGCGGCCGCCCGGGCAGGT23Nestedprimer2R52AGCGTGGTCGCGGCCGAGGT23DNA:CTAB/NaClDNA,DNA34:WHO2ADNA(driver),RSM292DNA(tester)(1)DNARsa2:DNA2g30Rsa40l,378h,MinElutekit,5l1.8l1.2lDNA,2,adaptor1adaptor2R,16(2)2:,2RSM292C4DNA1l2lRsaWHO2ADNA636h,2,DNA2l,63100l,687min,220(3)2PCR(PCR):1l2,50advantagecDNA,adaptor1adaptor2PCRprimer153,PCR1lPCR,1401l,50advantagecDNA,adaptor1adaptor2R(nestedprimer1nestedprimer2R)53PCR,PCRdCTP232P(Amersham)DNAtester2PCR,,RsatesterdriverDNASouthenblot,3,DNA:2lPCR1lpGEM2Teasy,4T4,42l50lTOP10F,,250lSOC,37300r/min1h,200l50ng/mlLB/X2gal/IPTG,3718hDNA:1mm,,,nestedprimer1nestedprimer2PCR,PCR2l,dCTP232PRsatesterdriverDNA,DotblotBLAST:,PharmaciaBiotechALFTMDNASequencer,pGEM2FpGEM2RGenBank()()11DNA:1,DNA,PCRA(NspA),,2%2380bpRSM292C4WHOWHO2ADNA0.56g/l0.53g/l,A260/A2802.0,1.0%DNA2.0kbRsaDNA,2.0%2.0kbDNA,21DNA:12RtesterdsDNA,,30PCR,2%,DNA350bp(2)31DNA:tester2PCR,RsatesterdriverDNASouthenblot(3)testerPCRtesterdriverDNA,tester98320035235ChinJMicrobiolImmunol,May2003,Vol23,No.5©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.PCRtesterDNA,1DNAFig1.AnalysisofNeisseriagonorrhoeaegenomicDNAA:PCRamplificationofNspAfromN.gonorrhoeaegenomicDNA.Lane1:RSM292C4strain;lane2:WHO2Astrain;laneM:marker(Roche);B:PurifyN.gonorrhoeaegenomicDNAofRSM292C4strain(lane1)andWHO2Astrain(lane2);laneM:marker(Roche);C:NeisseriagonorrhoeaegenomicDNAofRSM292C4(lane1)andWHO2A(lane2)afterRsadigestion;laneM:marker(Roche)2RsaDNAFig2.TheresultoftheadaptorligationefficiencyanalysisLane1:marker;lane2:PCRproductsobtainedusingtester121(adaptor12ligated)asthetemplateandPCRprimer141DNA:DNA,PCR,DNA2500,82%,96,,nestedPCR,100600bp,(4)51Dotblot:2PCRPCR,RsatesterdriverDNA,3SouthenblotFig3.AnalysisofsubtractionefficiencywithSouthernblotA:UsingunsubtractedsecondaryPCRproductsasprobestodeterminetheefficiencyofsubtration;B:UsingsubtractedsecondaryPCRproductsasprobestodeterminetheefficiencyofsubtration.Lane1:drivergenomicDNAafterRsadigestion;lane2:testergenomicDNAafterRsadigestion4Fig4.TheDNAfragmentsderivedfromindividualclonesdCTP-32P,96,5,32driverDNAtesterDNA5testerDNA,driverDNA,,RSM292C4DNA,5DNAFig5.DifferentialscreeningofasubtractedantibioticNeisseriagonorrhoeaeDNAlibrary09320035235ChinJMicrobiolImmunol,May2003,Vol23,No.5©1995-2004TsinghuaTongfangOpticalDiscCo.,Ltd.Allrightsreserved.61:5,Gen2Bank,BLAST,,5DNADNA:No.1(376bp):TTCGAGCGGCCGCCGGGCAGGTGATGTCTGGGCTGTTGGGAAAGGCAGACAGCGGTGTGCACTTTATCGTCAACGACCTGCGGTTGCCGCGTGCGCTGCTGGCGCTGCTGGTGGGCGGCGCGCTGGCAATATCCGGTTTGATTCTGCAAAGCATTGTGCGTAATCCGCTGGCTTCGCCGGATATTCTGGGGATTACCAGCGGCGCATCGGCAGCGGCAGTGTTCTTTCTCTCGTTTCTGGCGACGGCGATAAGCCAGCGCTGGCTCCCTGTAGCCGCGATGGGTGGGGCGTGGATCACAGCCGTCGCCATTTATCTGCTGGCCTGGAAACAAGGCGCATCGCCGCTGCGGCTAGTACCTCGGCCGCGACCACGCTANo.2(84bp):CGAATTGGGCCAGAGACGTCGCATGCATCA