简介：ToobtaintherecombinantsolubleproteinoftheextracellularfragmentofhumanTRAILgeneandtoidentifyitsfunctionpreliminarily,thisgenefragmentwasamplifiedfromperipheralbloodmononu-clearcells(PBMC)byRT-PCRandclonedintovectorpGEM-T-Easyforsequenceanalysis.Theexpres-sionvectorpET-30a/TRAILwasthenconstructedbyDNArecombinationmethodwithaHis-taggeneatthefrontoftheTRAILfragment,andtherecombinantproteinwasexpressedinE.coliBL21(DE3).Meanwhile,theexpressedtargetproteinwaspurifiedwithNi-NTAchromatographycolumnandidentifiedbySDS-PAGEandWesternblotting.TheproliferationinhibitionactivityofTRAIL-HiswasdetectedbyMTTassay.PIstainingandWright-GiemsastainingwereusedtodetectthepresenceoftheTRAIL-in-ducedcellapoptosis.ItwasdemonstratedthatthetargetproteinexpressedinE.coliBL21showedthesamerelativemolecularmassasthattheproteinexpectedandcouldberecognizedbyboththeanti-TRAILpolyclonalantibodyandanti-Hismonoclonalantibody.Inaddition,thisproteincouldalsoinhibitprolif-erationofhumanlymphomacelllineJurkatcellsorinduceapoptosisofthiscellline.ItisapparentthatarecombinantsolubleTRAILproteinwithbiologicalactivityisobtainedandthisprospectivestudycanlaysolidfoundationforfurtherresearchonthebiologicalactivityandapplicationintheanti-tumortherapy.

简介：Inordertoanalyzethenucleoprotein(NP)geneofCrimean-Congohemorrhagicfevervirus(CCHFV),viralRNAwasamplifiedbyRT-PCRbyusingtheproof-readingDNApolymerasetoproducethecompleteNPgene.ThePCRproductwassequenced,analyzedforphylogenesisandclonedintotheexpressionvectorpE132aandtherecombinantplasmidexpressedinE.coilBL-21withhighyield.Theprimarilypurifiedfusedprotein.wasusedtocoatELISAplatesforthedetectantibodies.ItwasfoundthesimilaritiesbetweenNPgeneofBA88166andotherXHFVsinnucleotidelevelandaminoacidcontentswereverysignificant,andtheNPgeneofBA88166encodedanucleoproteinwith482aminoacidandadeducedmolecularweight(MW)of54kDa.Westernblotassayshowedthatthefusionproteinexpressedinbacteriapossessedgoodantigenicity.TheresultswithELISAforthedetectionofthehumanandanimalseracollectedinendemicareaswerefoundtobeingoodaccordancetotheclinicaldiagnosis.ItconcludedthattherelationsofNPgenesofXHFVBA88166andotherXHFVsappearedtobeevolutionallyclose.Themethodologiesestablishedinthisstudywereaccurate,specific,rapidandreproduciblefortheclinicalexaminationsandepidemiologicalsurvey.

简介：ThegeneclustercfaABCED′ofenterotoxigenicEscherichiacoli,encodingthefimbriaewhichiscalledcolonizationfactorantigenⅠ(CFA/Ⅰ),locatedonaplasmid.ItispositivelyregulatedbycfaR,amemberoftheAraCfamily,andthecfaD′generegion,whichislocateddownstreamofcfaEandishomologoustocfaR,hadbeendescribedasatruncatedcrypticgene.InthepresentstudyweobservedthattheCFA/Ⅰfimbriaesubunit,cfaB,wasexpressedinloweramountbythecfoABCED′clonepNTP513inhostE.coliHB101.TheexpressionofCFA/ⅠdiminishedbydeletionofcfaD′generegionfrompNTP513,andwasrestoredbyacquisitionofcfaD′intrans.Furthermore,CFA/ⅠexpressionbycfaD′deletionmutant,thecfaABCEclone,wasremarkablyincreasedbythepresenceofCFA/ⅠintransinatopoisomeraseAdeficientstrainofE.coliDM800.ThesedatasuggestthatcfaD′regionisafunctionalregionofgene,thatregulatestheCFA/ⅠexpressionwithcfaRbyunknownmechanism.

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简介：Tosupportthescientificbasisforrapididentificationofpathogenicbacteriaandotherstud-ies,thesequencesofhsp60geneinmajor34speciesof16genusofpathogenicbacteriaweresearchoutinGenBankandaproperpairofuniversaldegenerateprimerwasdesignedbymeansofthemolecu-larbiologicalsoftwawePrimer5.0andOligo6.0.ThisprimerwasthenusedinthePCRamplification,andthehsp60genefragmentsoftheselectedpathogenicbacteriacouldbeamplifiedusingthisdegener-ateprimer.Bywayofbioinformationalanalysis,theconservation,variationandtheinterspeciesphylo-geneticrelationsofthehsp60genesequencewereanalysed.Fromtheresultsofthecomparativestudyonsequences,itwasdemonstratedthatthehsp60genewascharacterizedbyconservationandvaria-tion,inwhichtheconservedandmutantregionsco-existedandseparatelydistributedwithmanysmallmutantregionsdistributedamongtheconservedregions,justlikethemosaic.Thephylogenetictreeamongdifferentpathogenicbacteriadrawnfromthehsp60geneanalysiswasprovedtobeconsistentwiththosefrom16SrRNAand23SrRNA.Itisconcludedthatthesequencedistributionofhsp60genewouldprovideasolidbasisfortherapididentificationofpathogenicbacteriaandthedevelopmentofadiagnosticmicroarray.

简介：TheaimofthisstudyistofurtherunderstandthegenotypeofHantavirus(HV)fromperipheralbloodofpatientswithhemorrhagicfeverwithrenalsyndrome(HFRS)andtheepidemiologicalsignificanceofthisdiseaseinHeilongjiangprovinceinrecentyears.Thirty-oneserumsamplesofclinicallydiagnosedpatientswithHFRSwereexaminedbyRT-PCRtodecidethegeneticsubtype.Onthebasisofinfectionseason,theserumsamplesweredividedintotwogroups:winter(Nov,2003—Feb,2004),springandsummer(April,2004—Sep,2004).Furtheranalysiswasperformedincombinationwithclinicalsymptoms.Itwasfoundthatamongthetotal31samples,22weresero-positive.Among14serumsamplesinwinter,8weresero-positive,ofwhich5caseswereoftypeⅠ(Hantaanvirus,HTNV)and3oftypeⅡ(Seoulvirus,SEOV).Among17samplesinspringandsummer,14weresero-positive,ofwhich5caseswereoftypeⅠand9oftypeⅡ.SoitconcludesthatbothofthetwotypesofHantavinisexistinHeilongjiang.ThetypeⅠisthemainpathogenofHFRSinwinter,andtypeⅡisthemaininspringandsummer.

简介：Inthepastdecade,usesofantisepticsanddisinfectantsinhospitalsandotherhealthcarecentersarerathercommon,butthechancetodevelopresistancetoantisepticsanddisinfectantsisalsoincreased.Acinetobacterbaumanniiisoneoftheopportunisticbacteriainvolvinginthenosocomialinfection.Inthepresentstudy,thecorrelationoftheantisepticresistanceinA.baumanniiandtheantisepticresistancegeneqacEΔ1wasinvestigatedbymeansofdeterminationofMICs.Meanwhile,theMICsofglutaraldehyde,chlorhexidine,benzalkoniumbromide,iodophorandtrichloroisocyanurateto80clinicalisolatesofA.baumanniiweredetectedbytubedilutionassayandtheresistancegenesintI1andqacEΔ1intheseisolateswereamplifiedbyPCRandverifiedbyDNAsequencer.ItwasfoundthattheMIC50forthese5antisepticstestedwere32,8,8,4and1μg/mlrespectively,andthedetectionratesofintI1andqacEΔ1genewere60.0%and77.6%respectively.Inaddition,55%ofthe80isolatessimultaneouslypossessedbothintllandqacEΔ1gene,andthepercentageofantisepticresistanceofA.baumanniicarringbothgenestobenzalkoniumbromidewerehigherthanthatwithoutthesetwogenes,however,therewasnosignificantdifferencebetweenintllandqacEΔ1gene.Theresultinbactericidalefficiencyassayindicatedthatchlorhexidinecouldstillproducerapidandstrongbactericidaleffectatconcentrationof1MICafter10minexposure.TheseresultssuggestthattheantisepticresistanceofA.baumanniitovariousantisepticsiscorrelatedwiththepresenceoftheantisepticresistancegenesqacEΔ1inbacteria,thuswarningthattheincreaseoftheantisepticresistanceshouldnotbeignoredandtherelativehighconcentrationorprolongedapplicationtimeisrequiredtoachieveasufficientbactericidaleffect.

简介：BiofilmformationplaysamajorroleinthepathogenesisofnosocomialinfectionscausedbyStaphylococcusepidermidis(S.epidermidis).Ithasbeensuggestedthatproteinencodedbythefoe(fibrinogenbindingprotein)geneofS.epidermidisenhancesbacterialadherencetomedicaldevicesandbiofilmformationbybindingtohostfibrinogen(Fg).Inthisstudy,a1.7kbfoegenefragmentwasamplifiedin111of115strainsofS.epidermidischnicalisolatesusingPCR.Contrarytoexpectations,only14strainsshowedmarginallyincreasedadherencetoFg-coatedpolystyrenewellscomparedwithBSAcoatedwells.Quantitativereal-timePCRrevealednostatisticallysignificantdifferenceinFbeexpressionbetweenFgbindingstrainsandFgnon-bindingstrains.Fttahermore,inthepresenceofsolubleFg,S.epidermdisbiofilmformationdecreasedinadose-dependentmanner.Incontrast,theStaphylococcusaureus(S.aureus)strainCowanIandother5S.aureusclinicalisolatesshowedasubstantialincreaseinbothadherenceandbiofilmformationinthepresenceofFg.TheresuitssuggestthatinS.epidermidisthefoegenemaynotbeassociatedwithbacterialadherenceandbiofilmformation.

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简介：ToestablisharapididentificationmethodforcommonpathogenicbacteriaonthebasisofmolecularbiologyandtoconstructapreliminaryPolymeraseChainReaction-CapillaryElectrophoresis-RestrictionFragmentLengthPolymorphism(PCR-CE-RFLP)databaseofbacteriaisolatedfromclinicalspecimensfrequently,183strainscollectedfromclinicalsamplesbelongingto12generaand19specieswhosebiochemicalcharacterizationscorrespondedtothetypicaloneswereexamined.ThegenomicDNAswereamplifiedbytwopairsoffluorescencelabeledprimersaimingat16SrRNAgeneand16S-23SrRNAspacerregiongenerespectivelyatthesametime.PCRproductswerethendigestedbyrestrictionendonucleaseHaeⅢin-completelybeforetakingcapillaryelectrophoresis.TheresultswiththePCR-CE-RFLPpatternsof16SrRNAgeneswerejustalikewithinsomegenera,butwhenitcomesto16S-23SrRNAspacerregiongenes,eachbacteriumshowedauniquepattern,whichcanbedistinguishedfromeachothereasily.ItseemsthatPCR-CE-RFLPpatternsof16SrRNAgenecouldonlybeusedtoclassifythebacteriaintofamilylevel,whereasthedataof16S-23SrRNAspacerregiongenecouldbeutilizedtoidentifythewholemicroorganismsaspreciselyasthespecieslevel.Inspiteofthedataofthespacerregiongenealonecanbesufficientlytoverifythewholebacteria,weinsistthatthe16SrRNAgenecouldbeofsomeassistantincasethatthereshouldbelotsoffamiliesofbacteria,inwhichsomesimilarones,withthesameRFLPdataof16S-23SrRNAspacerregiongene,maycoexist.ThisstudyprovesthattheutilityofPCR-CE-RFLPisaconvenient,rapidmethodtoidentifypathogenicbacteria,andisalsoaquickdiagnosismeasureforapplicationtoclinicaluse.

简介：ToconstructanexpressionvectorcontainingtheE1glycoproteingeneofrubellavirusforthestudyontheeffectofmutationoftheE1geneglycoproteinandtheanalysisofphylogeneticdifferencesofsequences,thegeneencodingtheE1envelopeglycoproteinwasamplifiedfromrubellavirus,JinanstrainJR23,byRT-PCRandligatedintoPMD-18Tvector.TheclonesthatcarriedtheE1genewereidentifiedafteramprselectionandanalysisofrestrictionenzymedigestion.AftersequencingthisgenewasanalyzedbyDanstarandWinstarprograms,andthemapofphylogenetictreewasdrawn.ThecloneofE1glycoproteinwasthusconstructed.ItwasfoundthatthesequencedifferencesbetweenJR23strainandtheTCRBstrainfromJapanandthosebetweenJR23strainandThomasstrainofEnglandwererathersmallwithdifferencevaluesof0.9%and1.2%respectively.YetthosebetweenJR23strainandBRD2strainfromBeijingandthosebetweenJR23strainandXG379strainfromHongKongwerecomparativelylargerwithdifferencevaluesof7.6%and7.3%respectively.ThesequenceofJR23strainwithotherstrainswaslessthan3%excepttheNCstrain(3.7%).ItconcludesthattheconstructionofE1glycoproteingeneoffersanapproachtostudytherelationshipbetweenstructuresandfunctionsofE1geneanditsgeneproducts.Inthephylogenetictree,itshowsthattherearesignificantdifferencesinthesequencesofrubellavirusisolatedinChina,andthismightbehelpfultodevelopaneffectivesubunitvaccine.

简介：Theimmuneefficiencyofarecombinantadenovirustype5withtype35fibercontainingHIV-1gaggene(rAd5/F35-mod.gag)wasinvestigatedinBALB/cmice,inwhichtherAd5/F35-mod.gagwasfirstlyidentifiedwithPCR,thentransfectedto293cellsandtheinvitroexpressionlevelofGagproteinwasdeterminedbyWesternblottingandindirectimmuno-fluorescentassay.MicewereimmunizedwithintramuscularinjectionsofrAd5/F35-mod.gag,rAd5-mod.gagorDNAandwereboostedafter3weeks.Totesttheeffectofpre-existinganti-viralimmunityonimmunization,micewerealsoinjectedwithAd5-GFPvectorandthenimmunized4and7weekslaterwithAd5/F35-mod.gagvector.TheP24-specificIgGantibodyinseraofimmunizedmicewasdeterminedbyELISAandthespecificcytotoxicTlymphocyte(CTL)responsewasassayedbyintracellularcytokinestaining.ItwasdemonstratedthattherAd5/F35-mod.gagvectorcouldexpressefficientlytheHIVGagproteinin293cellsinvitroandinducestrongHIV-specificimmuneresponsesinvivo.ThestrongestCTLandserumIgGresponseoccurredwhenmicewereimmunizedtwicewithinjectionofrAd5/F35alone,buttheanti-Ad5antibodyafterprimaryinfectionwithadenoviruscouldinhibitthespecificimmuneresponsesinducedbyrAd5/F35vector.ItisconcludedthatsingleimmunizationwithrecombinantadenovirusrAd5/F35-mod.gagcaninducespecificCTLandserumIgGantibodyresponsesinmice,buttheimmunogenicityofrAd5/F35iscomparablyweakerthanthatofrAd5.

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简介：ThepurposeofthisstudywastoconstructaneukaryoticDNAvectorencodingamultipleepitopeantigen(MFC)ofhepatitisCvirus(HCV)andahepatitisBsurfaceantigen(HBsAg),andexploretheeffectofHBsAggeneontheimmunityofHCVmultiple-epitopeDNAconstructinvitroandinvivoinmice.AnHCVDNAvector(pVAX1-HBs-MFC)wasconstructedbyfusingHBsAggenetotheNterminalofanHCVmultiple-epitopeantigengene.ThepVAX1-HBs-MFCwastransfectedintoHEK293TcellsanditsexpressionwasmeasuredbyELISAandWesternblotting.BALB/cmicewereintramuscularlyimmunizedwiththepVAX1-HBs-MFC,andanELISAapproachwasappliedtodeterminethespecificantibodytitersandsubtypesinthemouseserum.Thecross-reactivityoftheantibodieswasalsocheckedwithtwosynthesizedHCVhypervariableregion1(HVR1)peptides.TheIFN-γproductionandcellproliferationofthemousespleencellswereevaluatedbyELISAandMTS(3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium,innersalt)assays,respectively.TheexpressionofpVAX1-HBs-MFCwasdetectableinthetransfectedHEK293Tcells.TheserumantibodyresponsewaseffectivelyelicitedinBALB/cmiceinjectedwithpVAX1-HBs-MFC.ThehighesttiterofantibodyagainstHCV(MFC)was1:1280,andtheratioofIgG2a/IgG1was1.50±0.12atthefifthweekafterfirstimmunization.Moreover,thecollectedmouseserumantibodyhadtheabilitytocross-reactwiththetwosynthesizedHCVHVR1peptides.ThestimulationindexofthemousesplenocytestoMFCwas1.79±0.07,andtheIFN-γlevelwas287±6pg/mlatweek21afterfirstimmunization.ThehighesttiteroftheantibodyincontrolBALB/cmiceimmunizedwithpVAX1-MFCwas1:320,andtheratioofIgG2a/IgG1was1.33±0.11atweek5post-immunization.Furthermore,thestimulationindexofthemousesplenocytescellstoMFCwas1.52+0.06,andtheIFN-γlevelwas225±9.3pg/mlatweek21post-immunization.TheHBsAggenecanenhancetheeffectsofanHCVmultiple-epitope

简介：Toinvestigatetheexistenceofthemajoroutermembranepmtein(MOMP)geneLip132in15dominantChinesestrainsof15semgroupsofLeptospirairaerrogansand2intemationalstrainsof2semgroupsofLeptospirabiflexa,andtocloneandconstructtheexpressionsystemaswellastoidentifytherecombinantpmteins,genomicDNAsfromstrainsofleptospirawerepreparedbymutinephenol-chloroformmethod,andthefragmentsoftheLipL32genewiththewholelengthfromthestrainswereamplifiedwithhighfidelityPCR.Thetargetamplificationproductswere,sequencedafterT-Acloning,andtheexpressionsystemforthegenesweretherebyconstructed,ExpressionoftherecombinantproteinswasidentifiedbyusingSDSPAGEafterinductionwithIPTGatdifferentdosages.WesternblotassayswithrabbitantiserumagainstthewholecellofTR/PatocⅠofLeptospiraandimmunizedserumwithrMOMPswereusedtodeterminetheimmunoreactivityandimmunogenicityoftherecombinantproteins.Microscopicagglutinationtestwasusedtodeterminethecross-agglutinationtitresinrabbitseraimmunizedwithrMOMPs,andthecelladherencemodelofLeptospirawasusedtoexaminetheblockingeffectsofrabbitantiseraagainsttheserMOMPs.ItwasfoundthattheLipL32genecouldbefoundinallthe17strainsofLeptospiramentionedabovewithtwodifferentgenotypes,i.e.LipL32/1andLipL32/2.AmountsofexpressionsofrMOMP1andrMOMP2afterIPTGaccountedfor40%and10%ofthetotalbacterialpmteinsrespectively.BothrMOMP1andrMOMP2couldcombinewiththerab-bitantiserumagainstleptospiralTR/PatocⅠ,andcouldinducetheproductionofagglutinationantibodiestothese17strainsofLeptospirawith1:2to1:64MATtitres.Therabbitanti-rMOMP1andanti-MOMP2antibodiesat1:2to1:16dilutionscouldefficientlyblockadherenceofLeptospira.ItconcludesthatalltheLeptospiratestedinthepresentstudypossessLipL32/1orLipL32/2genes,andtheconstructedexpressionsystemcanexpresstherMOMP1andrMOMP2.

简介：TherelationshipbetweenembBmutationofMycobacteriumtuberculosisandethambutol(EMB)resistanceoftheclinicalisolatesoftuberculouspatientsinChinawasinvestigatedbyreversedotblothybridization(RDBH)inadditiontoevaluatingtheclinicalvaluewithapplicationofPCR-RDBHtechniquetodetectEMBresistance.Inthepresentstudy,thegenotypesofthe258bpfragmentsofembBgenesfrom196clinicalisolatesofM.tuberculosiswereanalysedwithRDBHandDNAsequencing.Itwasdemonstratedthat60outof91phenotypicallyEMB-resistantisolates(65.9%)showed5typesofmissensemutationsatcodon306ofembBgene,resultinginthereplacementoftheMetresidueofthewildtypestrainwithVal,IleorLeuresidues.Inthesemutations,theGTPmutation(38/91,41.8%)andtheATAmutation(16/91,17.6%)werethemostencounteredgenotypes.TheembBmutationatcodon306couldalsobefoundin69isolatesofphenotypicallyEMB-sensitivebutresistanttootheranti-tuberculousdrugs,butnosuchgenemutationcouldbefoundin36strainsofdrug-sensitiveisolates.Meanwhile,theconcordancewiththeresultsofDNAsequencingforonewide-typeprobeand5probesforspecificmutationswas100%.ItwasconcludedthattheEMB-resistanceoccurringinmostM.tuberculosisisduetoappearanceofembBmutationatcodon306,andthePCR-RDBHassaywasprovedtobearapid,simpleandreliablemethodforthedetectionofgenemutations,whichmightbeagoodalternativeforthedrug-resistancescreening.

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简介：Theaimofthisstudywastoanalyzethepointmutationoftheexon1atcodon54ofthemannose(ormannan)-bindinglectin(MBL)geneinhealthyindividualsofChineseHansandMongolianpopulation,andtofindoutanyassociationbetweentheplasmalevelsofMBLandthegenemutationfrequencyinbethgroupsofindividuals.Bloodsampleswerecollectedrandomlyfrom56healthyindividualsofChineseHansand37Mongolian.ThedetectionofthepointmutationsoftheMBLgenewasperformedbypolymerasechainreaction-restrictionfragmentlengthpolymorphism(PCR-RFLP)anddetectionsforplasmalevelsofMBLweredeterminedbyusingMBLELISAkits.AMBLPCRmethodofassaywasestablishedwithhighspecificity,andgoodreproducibility.ByoptimizingthePCRcondition,theoptimalannealingtemperaturewas55℃,andthelowestdetec-tionlimitwas160pg.Nobandswerefoundinnon-specificitysamples(HAV,HBV,HCVandTB),andthesequencesofPCRproductswerethesameastheexpectedones.AlsoaMBLPCR-RFIPwasestablished.Uponelectrophoresisofthedigestedproductsin3%agarosegel,therewere3patterns:inwhich2bandscorrespondtomoleculeweight232bpand93bp;1band,correspondstomoleculeweight325bpand3bandscorrespondtomoleculeweight325bp,232bpand93bp,respectively.Threebandsof325bp,232bpand93bpofpointmutationswerefoundatcedon54ofMBLcedinggene.FrequenciesinhealthyHanandMongolianpopulationwere0.2321and0.1757respectively.TheaverageplasmaMBLconcentrationwas1998.750μg/L,withSDof1505.152in56healthyHanpopulationand2525.676μg/L,withSDof1955.188in37Mongolian.AnegativecorrelationbetweenMBLconcentrationandgenemutationfrequencywasfoundinhealthyHanpopulation.Frequencyofpointmutationwas1.00whentheMBLconcentrationswerebelow100μg/L;frequencyofpointmutationwas0.4524whentheconcentrationwas100μg/Lto1000μg/L;andthefrequencyofpointmutationwas0.0156whentheconcentrationwaso