简介:Mycoplasmagenitaliumisthemaincausativeagentfornon-gonococcalandnon-chlamydialurethritis.P32istheputativesurface-exposedmembraneproteinofM.genitaliumandithassubstaintialidentityinaminoacidsequencewithadhesinproteinP30fromM.pnewnoniae.SinceM.pneumoniaemutantslackingP30proteinisdefectiveincytadherence,P32proteinhasbeenproposedtobeanessentialadhesinimplicatedintheadherenceofM.genitaliumtohostcells.TheprokaryoticexpressionvectorpET-30(+)/p32wasconstructedinthepresentstudy,andtherecombinantproteinwasexpressedinE.coliandpurifiedunderdenaturingcondition.Asdemonstratedbytheimmunoblottinganalysis,therecombinantproteincouldreactwithrabbitantiseraagainstM.genitalium,andadherenceinhibitionassayswerepetformedwithantiseraagainstthisrecombinantprotein.ItwasdemonstratedthatP32proteinapperaredtobeanadhesionproteinofM.genitalium,thusprovidingtheexperimentalbasisforbetterunderstandingofthepathogenesisofM.genitaliuminfectionandforthedevelopmentoftherelatedvaccinesagainsttheinfection.
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:ExposureofnaivemurineCD4^+TlymphocytestosuperantigensuchasstaphylococcalenterotoxinB(SEB)inducesastrongproliferativeresponse.ProlongedexposureorsubsequentrestimulationoftherespondingTcellpopulationwithSEBleadstotheapoptoticeventsofactivation-inducedcelldeath(AICD).ThesignalingmechanismresponsiblefortheAICDisatargetofintensiveinvestigation.However,theprecisedownstreamsignahngpathwaysofSEB-inducedAICDremainsunclear.Ourresultshereshowthatthesequentialactivationofcaspase-1/ICE-hkeandcaspase-3/CPP32-hkecysteineproteasesprobablyplaysaroleinthesignalingtransductionofSEB-inducedAICD,butcaspase-3/CPP32-hkeproteasesactivationdoesnotdependoncaspase-1-likeproteasesactivation.HerbimycinA,aspecificinhibitorofproteintyresinekinases,inhibitcaspase-3/CPP32-1ikecysteineproteasesactivation.However,itdoesnotpreventDNAfragmentationofCD4^+TcellsapoptosisinducedbySEB.TheseresultsindicatethatproteintyrosinekinasespathwayisprobablyinvolvedinthesignalingtransductionofCD4^+TcellsapoptosisinducedbySEBand“crosstalks”withthepathwayofcaspase-3/CPP32-1ikeproteasesactivation.