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.