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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:ToclarifytheroleofAPOBEC3G(A3G)incellulardefenseagainsthepatitisBvirus(HBV),theexpressionofA3GinnormalhumanliverandtheregulationoftheA3Gexpressioninhep-atomacellline(HuH-7)wereinvestigated.ExpressionlevelofAPOBEC3smRNAinhumanliverwasdeterminedbyRT-PCR.HuH-7andHepG2cellsweretreatedwithvariousconcentrationsofIFN-α(0U/ml,100U/ml,500U/ml,1000U/ml)for12h.ThemRNAlevelsweremeasuredbyaquantitativeRT-PCR,theresultswerenormalizedrelativetothespecimenswithoutIFN-αstimulation.TotalproteinofHuH-7cellstreatedwithvariousconcentrationsofIFN-αfor48hwassubjectedtoWesternblotanalysis.Forreportergeneassay,HuH-7cellsweretransfectedwiththereporterplasmidscontainingIRF-Esitesanditsmutantswithdifferentlengths.Thenthecellsweretreatedwithorwithout1200U/mlIFN-aforadditional12h(1000U/ml)after24hoftransfection,andthecelllysatewaspreparedandassayedforluciferaseactivity.ItwasfoundthatnormalhumanliverexpressedthemRNAofA3G.A3GmRNAexpressioninHuH-7andHepG2cellswereup-regulatedbyIFN-αstimulationinadose-depen-dentmanner.WesternblotanalysisindicatedthatA3GproteinexpressionwasalsoenhancedbyIFN-αstimulation.SequenceanalysisshowedtheexistenceofputativesitesofIFNregulatoryfactorelement(IRF-E)in5'regionofA3Ggeneupstreamtheinitiationcodon.IFN-αstimulationresultsin6-to8-foldincreaseinluciferaseactivityincellstransfectedwiththeplasmidcontainingIRF-Esitesofthe5'upstreamsequences,whereasluciferaseactivitydidnotchangeincellstransfectedwiththeplasmidcontainingmutantIRF-EsitesorwithoutIRF-Esites.Asaconclusion,A3Gareexpressedinnormalhumanliver.A3Gexpressionwasup-regulatedbyIFN-αstimulationinhepatomacellsandcouldbeinvolvedinhostdefensemechanismsagainstHBV.ERF-Esitein5'regionofAP0BEC3Ggeneupstreamtheinitiationcodonplaysanimportantroleinthisp