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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:CytoehromeP450norgenewasclonedintotheexpressionvectorpET-28toyieldtherecombinantexpressionplasmidpET-P450nor,whichcoulddirectthesynthesisofaeukaryoticderivedproteininEscherichiacoliBL21.Thevectorallowsoverproductionandsingle-steppurificationof(His)6-taggedcytoehromeP450norbythefacifitationofmetal(Ni^2+)chelateafl]nitychromatography.TheexpressionlevelofcytoehromeP450norwashighat30℃afterIPTGinduction.SDSPAGEandWesternblotanalysisshowedaspecificband(about43kDa).TheoverproducedcytochromeP450norwaspurifiedtoeleetrophoretichomogeneitywithin2.5handabout20.8mgpurifiedproteinwasobtainedfrom2Lcellculture.TheproliferationofSSMC-7721celllinecouldbeinhibitedbycytoehromeP450nor.Rabbitpolyclonalantibodies(titerover64000)wereproducedagainstrecombinantcytoehromeP450norandprovedtobeveryusefulforimmunoblottingstudy.AvailabilityofthisexpressionsystemandspecificantibodiesshouldfacilitatecharacterizationoftheroleofcytochromeP450norinthemetabolismofNO.
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简介:ThisstudywasaimedtoobservetheexpressionofP70S6kinase(P70S6K)inoralaciniccellcarcinoma.PT0S6kinaseexpressionwasexaminedbymeansofWestern-blottestandActivityas-say.Specimenswerefrom30casesoforalaciniccellcarcinomaand15casesofnormaloraltissuewereusedascontrols.StatisticalanalysissoftwareSPSS10.0wasusedforttesttodeterminetherelationshipbetweengeneexpressionandclinicalfeatures.TheexpressionlevelofP70S6Kincreasedobviouslyinoralaciniccellcarcinomatissue(P<0.01).ActivityassaywasthesameastheWestemblottest(P<0.01).P70S6Kexpressionlevelandactivityplayedanimportantroleinthedevelopmentoforalaciniccellcarcinoma.Inconclusion,P70S6Kisamplifiedandoverexpressedinoralaciniccellcarci-nomatissue,whichsuggestsapotentialoncogenicfunction.P70S6KandotherpossibletargetsofmTORcontributesignificantlytotumordevelopmentandthatinhibitionoftheseproteinsmaybethera-peuticforcancerpatients.OverexpressionofP70S6Kmaybeinvolvedinthepathogenesisoforalacin-iccellcarcinoma.
简介:Toinvestigatetheroleofnegative-regulatoryfactorsA20,IRF-4andTRAF4ofthetoll-likereceptor(TLR)signalpathwaysinimmunologicalpathogenesisofKawasakidisease(KD),48childrenwithKawasakidisease,16childrenwithinfectiousdisease(ID)and16age-matchedhealthychildrenwerestudied.Reverse-transcriptionPCR(RT-PCR)andreal-timePCRwereusedtoevaluatetheexpres-sionlevelsofnegative-regulatoryandeffectivefactorsintoll-likereceptor4(TLR4)signalpathwaysandproinflammatoryfactorsinperipheralbloodmonocyte/macrophage(MC).Inthisstudy,expressionlevelsofTLR4,MD-2,MyD88,IRAK-4,TRAF6,TAK1,andTAB2mRNAinKDgroupweredetectedtobeelevatedsignificantlyduringacutephaseofKD.Transcriptionlevelsofnegative-regulatoryfactorsA20,IRF-4andTRAF4mRNAinKDorIDpatientsincreasedremarkably.However,expressionsofIRF-4andTRAF4inKDpatientsweredetectedtobelowerthanthatinIDpatients,exceptthattran-scriptionlevelsofA20werefoundtobehigherthanthatinIDpatients.Simultaneously,expressionsofproinflammatorycytokinessuchasL-1β,IL-6andTNF-αinKDpatientsweresignificantlyelevatedcom-paredwiththoseinIDpatients.Furthermore,itwasfoundthatstimulationoflipopolysaccharide(LPS)remarkablyup-regulatedtheexpressionsofnegative-regulatoryfactorsA20,IRF-4andTRAF4inKDpa-tientsorhealthyvolunteers.ThemRNAlevelsofallthethreefactorsinKDpatientswerefoundtobelowerthanthatinthelatter.Inaddition,transcriptionlevelsofIRF-4andTRAF4inKDpatientswithcoronaryarterylesion(KD-CAL~+)weredetectedtobelowerthanthoseinKDpatientswithoutcoronaryarterylesion(KD-CAL~-)duringacutephase,whilethatofA20inKD-CAL~+groupwerelowerthanthatinthelatter.AndthelevelsofexpressionsofproinflammatorycytokinesinKD-CAL~+groupwerefoundtobehigherthanthoseinKD-CAL-group(P<0.01).Thesefindingssuggestthataberrantexpressionofnegative-regulatoryfactorsofTLRssignalpathwaysmaybeinvolved