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:TheaimofthisstudyistoinvestigatecyclinE,pl6inkdaandki67aspossiblediagnosticbiomarkersforcervicalpreneoplasiausingcervicalexfoliated-cellspecimens,andevaluatethesignificanceforscreeningpatientsathighriskofdevelopingcervicalcarcinoma.TheexpressionofcyclinE,pl6inkdaandki67wasexaminatedin78cervicalexfoliatedepithelialspecimensdiagnosedasatypicalsquamouscellsofundeterminedsignificance(ASCUS)(12cases),cervicalintraepithelialneoplasia(CIN)oftype1(17cases),CIN2_3(38cases)andinvasivecarcinoma(11cases)usingimmunohistochemicalanalysis,andsimultaneously,theDNAstatusofhumanpapillomavims(HPY)type16/18wasdetectedbypolymerasechainreaction(PCR)usingtypespecificprimers,cyclinE,pl6inkdaandki67werealloverexpressedinCINsandinvasivecarcinoma,comparedwithlittleexpressioninASCUS(P<0.005).OverexpressionofcyclinEwasobservedinCIN1(94.1%,X^2=21.16,P<0.01),andp16inkdaandki67wereoverexpressedininvasivecarcinoma(100%and90.9%respectively).Thedegreeofpl6inkdaandki67expressioncorrelatedwellwiththatofepitheliallesions(P<0.005).HPV16/18infectionwasassessedinC1Nsandinvasivecarcinomasamples,andrevealedasignificantrelationshipwiththedegreeofcervicalepitheliallession.Theexpressionlevelofpl6inkdaandki67seemedmorecloselyassociatedwithHPVI6infectionthanthatofcyclinE(rs=1.0vsrs=0.4).Only1caseinCINIanddcasesinCIN2-3ofHPV18positivesamplesweredetected.Thereforenostatisticalsignificancewasfoundbystatisticalanalysis.OverexpressionofcyclinE,pl6inkdaandki67inCINsandinvasivecarcinomacellsdemonstratesthepotentialuseofcyclinE,pl6inkdaandki67asdiagnosticbiomarkersforHPV-relatedcervicalneoplasticlesions.Inaddition,thistechniquecanbeusedforscreeningpatientsathighriskofdevelopingcervicalcarcinoma.
简介:ThegenomeoftheenterohemorrhagicEscherichiacoliO157:H7EDL933contains177“O”-islands(OIs).TostudytheirpotentialcontributiontotheO157-specificpathogenicity,wesurveyedthedistributionof22OIsbyPCRandDNAhybridizationin17isolatesofShigatoxinproducing(Stx-positive)E.coliO157:H7,andcomparedwiththeirdistributionin21isolatesofStx-negativeE.coliO157and21isolatesofnon-O157entericpathogens.Fourteenof22OIswerepresentinnon-O157entericpathogensanalyzed.Eightof22OIswerefoundonlyinthe17Shigatoxin-(Stx)positiveE.coliO157:H7isolates,buttheywereabsentfromthe21Stx-negativeE.coliO157:NMandO157Hundisolatestested.Amongthe8OIs,onlyOI43orOI48wereexclusivelydetectedinStx-positiveE.coliO157:H7,absentfromneitherofStx-negativeE.coliO157andnon-O157entericpathogens,suchasSalmonella,ShigeUa,Citrobacter,Vibriocholera,enteropathogen-icE.coli(EPEC),enteroadherentE.coli(EAEC),enteroinvasiveE.coli(E1EC)andenterotoxingenicE.coli(ETEC).TheOI43andOI48are83kbinsizeandidenticalinDNAsequences,whichencodegenesforurease,telluriteresistanceandadherence.ByanalyzingtheirjunctiongeneswithPCRandDNAhybridization,wefoundthat21ChineseisolateshaveOI48only.However,for7Japanesepatientisolates,4haveOI43and3haveOI48;forAmericanisolates,2havebothofO143andOI48,2haveOI48only.ThesedataconfirmedthehighlyplasticityofthepathogenicE.coligenome.TheuniquepresenceofOI43/OI48inStx-positiveE.coli0157:H7denotesitscriticalroleinthepathogenicityspecifictothispathogen.