简介:TostudytheexpressioncharacteristicofJapaneseencephalitisvirus(JEV)prMEandEproteinsandtheefficacyofDNAimmunizationbydifferentrecombinantplasmidscontainingJEVprME(2001bp)andE(1500bp)genes,tworecombinants(pJMEandpJE)containingJEVprMEandEgenesfusedwithFLAGwereconstructedandthentransfectedintoHepG2andCOS-1cellsbylipnsomefusion.TheexpressionfeatureofFLAG-prME(about72kDa)andFLAG-E(about54kDa)proteinsintransfectedcellswereanalyzedbyWesternblotandtwoantibodysystems(anti-FLAGandanti-E).BALB/cmicewereimmunizedwith100μgoftwokindsofrecombinantsbyintramuscularinjection,andJEVJaGAr-01strains(10^5PFU/100μl)weregiventoBALB/cmicebyintraperionealinjection3wkaftertwiceDNAimmunizationbyalethalviruschallenge.BALB/cmicewereobservedfor21daysafterchallenge.80%plaquereductionneutralizationtestwasperformedtotitrateneutralizationantibodybeforeandafterviralchallenge.ItwasfoundthattheexpressionofproteinsassociatedwithpJMEandpJEwasdeterminedintransfectedcellswithanti-FLAGandanewproteinof11kDawasdetectedinHepG2andCOS-1cellstransfectedwithpJME.OnlyE(53kDa)proteinwasidentifiedastransfectedwithpJMEusingantiE.HigherlevelofneutralizationantibodiesandtheefficacyofprotectiveimmunitywereinducedwithpJMEimmunization,andweresimilartothoseinducedbyinactivatedJapaneseencephalitisvaccine,butwerebetterthanthoseinducedwithpJE.ItconcludesthattheexpressionlevelfromprMtoEproteinsofJEVisdifferentinvitro,andtheinvitroexpressionefficiencyofpJMEwasbetterthanthatofpiE.FLAG-prMEproteinexpressedbypJMEcouldbecleavedbypeptidasefromhost.TheefficacyofDNAimmunizationiscorrelatedtotheexpressioncharacterizationofrelatedproteinsexpressedinvitro.
简介:ToconstructanexpressionvectorcontainingtheE1glycoproteingeneofrubellavirusforthestudyontheeffectofmutationoftheE1geneglycoproteinandtheanalysisofphylogeneticdifferencesofsequences,thegeneencodingtheE1envelopeglycoproteinwasamplifiedfromrubellavirus,JinanstrainJR23,byRT-PCRandligatedintoPMD-18Tvector.TheclonesthatcarriedtheE1genewereidentifiedafteramprselectionandanalysisofrestrictionenzymedigestion.AftersequencingthisgenewasanalyzedbyDanstarandWinstarprograms,andthemapofphylogenetictreewasdrawn.ThecloneofE1glycoproteinwasthusconstructed.ItwasfoundthatthesequencedifferencesbetweenJR23strainandtheTCRBstrainfromJapanandthosebetweenJR23strainandThomasstrainofEnglandwererathersmallwithdifferencevaluesof0.9%and1.2%respectively.YetthosebetweenJR23strainandBRD2strainfromBeijingandthosebetweenJR23strainandXG379strainfromHongKongwerecomparativelylargerwithdifferencevaluesof7.6%and7.3%respectively.ThesequenceofJR23strainwithotherstrainswaslessthan3%excepttheNCstrain(3.7%).ItconcludesthattheconstructionofE1glycoproteingeneoffersanapproachtostudytherelationshipbetweenstructuresandfunctionsofE1geneanditsgeneproducts.Inthephylogenetictree,itshowsthattherearesignificantdifferencesinthesequencesofrubellavirusisolatedinChina,andthismightbehelpfultodevelopaneffectivesubunitvaccine.
简介:摘要 目的 比较前交叉韧带(anterior cruciate ligament,ACL)重建术中股骨侧I.D.E.A.L.定位和解剖定位两种方式的短期临床效果。方法:选取2019年10月至2020年10月我院行ACL重建手术的43名患者。随机分为两组:股骨侧I.D.E.A.L.定位21例、解剖定位22例,均选择单束重建。比较两组术前术后12月Lachman试验、轴移试验阳性率;术后3月、6月、12月IKDC评分、Lysholm评分及术后6月重建移植物与胫骨平台夹角。结果:两组术后Lachman试验、轴移试验阳性率较术前显著降低(
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