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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

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简介：Toexploretheroleofnuclearfactor-κB(NF-κB)inthesignalpathwayofproteinkinaseC(PKC)regulatingtheproliferationandapoptosisofTlymphocytesinasthma.Tlymphocyteswereisolatedfromtheasthmaticmodelofguineapigsandtheasthmaticpatients.EithertheTcellsstimulatedwithPMAaloneorthosestimulatedwithPMAtogetherwithpyrrolidinedithiocarbamate(PDTC)wereincubatedfor1and24h.TheproliferationofandthepresenceofNF-κBinthecellsincubatedfor1hwereobservedbyMTTandimmunohistochemicalstaining,respectivelyAndthecellsincubatedfor24hwereobservedfortheapoptosisbyTUNEL.Alltheassayswereparalleledwithcontrols,andallthedatawereanalyzedstatisticallywiththesoftwareSAS.ThepercentageofcellsofnuclearpositivestainingofNF-scBandtheproliferationofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithPMAweresignificantlyhigherthanthoseofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithoutPMArespectively(P<0.01)andthoseofTlymphocytesfromnormalcontrolguineapigsandnormalcontrolpersonsstimulatedwithPMArespectively(P<0.01),andweresignificantlyreducedbyPDTC(P<0.01).TheapoptosisindexofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithPMAweresignificantlylowerthanthoseofTlymphocytesfromasthmaticguineapigsandasthmaticpatientsstimulatedwithoutPMArespectively(P<0.01)andthoseofTlymphocytesfromnormalcontrolguineapigsandnormalcontrolpersonsstimulatedwithPMArespectively(P<0.01),andweresignificantlyinducedbyPDTC(P<0.01).ThereweregoodpositivecorrelationbetweenthepercentageofcellsofnuclearstainingofNF-κBofTlymphocytesandtheproliferationofTlymphocytes(r=0.51-0.72,P<0.001),andalsogoodnegativecorrelationbetweenthepercentageofcellsofnuclearstainingofNF-scBandtheapoptosisindexofTlymphocytes(r=-0.55-0.71,P