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：Wehaveconfirmedefficientanti-tumoractivitiesoftheperipherallymphocytestransducedwithap185HER2-specificchimericT-cellreceptorgenebothinmurineandinhumaninourpreviousstudies.TofurthertestthefeasibilityofchimericT-cellreceptorinabonemarrowtransplantationmodel,wefirst,madetwomurinetumorcelllines:MT901andMCA-205,toexpresshumanp185HER2byretroviralgenetransduction.MurinebonemarrowcellswereretrovirallytransducedtoexpressthechimericT-cellreceptorandgene-modifiedbonemarrowcellsweretransplantedintolethallyirradiatedmouse.Sixmonthsposttransplantation,p185HER2-positivetumorcells:MT-901/HER2orMCA-205/HER2wassubcutaneouslyorintravenouslyinjectedtomakemousemodelssimulatingprimarybreastcancerorpulmonarymetastasis.Theinvivoanti-tumoreffectsweremonitoredbythesizeofthesubcutaneoustumororcountingthetumornodulesinthelungsafterIndiainkstaining.ThesizeofthesubcutaneoustumorwassignificantlyinhibitedandthenumberofpulmonarynodulesweresignificantlydecreasedinmouserecipientstransplantedwithchimericT-cellreceptormodifiedbonemarrowcellscomparedwiththecontrolgroup.Ourresultssuggesttheefficientinvivoanti-tumoractivitiesofchimericT-cellreceptorgenemodifiedbonemarrowcells.

简介：WehavedevelopedandtestedchimericT-cellreceptors(TCR)specificforp185HER2.Intheseexperiments,retroviralvectorsexpressingtheN297orN29ξreceptorswereconstructedinpRET6.AmphotropicviralproducercellswereestablishedintheGALV-basedPG13packagingcellline.Ficollpurifiedhumanperipheralbloodlymphocytes(PBL)werevitallytransducedusinganoptimizedprotocolincorporatingactivationwithimmobilizedanti-CD3/anti-CD28monoclonalantibodies,followedbyviralinfectioninthepresenceoffibronectinfragmentCH296.Transducedcellswereco-culturedwithhumantumorcelllinesthatoverexpress(SK-OV-3)orunderexpress(MCF7)p185HER2toassayforantigenspecificimmuneresponses.BothCD4^+andCD8^+T-cellstransducedwiththeN297orN29ξchTCRdemonstratedHER2-specificantigenresponses,asdeterminedbyreleaseofTh1likecytokines,andcellularcytotoxicityassays.OurresultssupportthefeasibilityofadoptiveimmunothempywithgeneticallymodifiedT-cellsexpressingachTCRspecificforp185HER2.

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