简介:Inordertoanalyzethesequencesoftheinternaltranscribedspacer(ITS)includingthe5.8SribosomalDNA(rDNA)ofcommondermatophytes,soastoobtainarapidandaccuratemethodtoidentifythespeciesofdermatophytesandtoestablishthephylogenetictreeofthesespeciestounderstandtheirrelationship,16strainsofdermatophyteswerecollectedandpreliminarilyidentifiedbymorphologicalcharacteristics.GeneralprimersforfungiITS1andITS4wereusedtoamplifytheITSrDNAofeachstrainswithPCR.ThePCRproductsafterpurificationweresequenceddirectlyandwereanalyzedthroughinternet.Intheresults,11strainswereidentifiedbymeansofmorphologicalfeatures,amongwhich5strainswereTrichophyton,5strainswereMicrosporumand1wasEpidermaphyton,whichwasconsistentwiththeresultsbymolecularbiology.Inthe5unidentifiablestrains,1strainwasprovedtobeChrysosporiumbymolecularbiology.Thesestrainsstudiedcouldbedividedinto3differentclassesasindicatedintheanalysisofthephylogenetictreeofthesequencesinITS,whichwerequitedifferentfromthoseofmorphologicalclassification.ItisevidentfromtheaboveobservationsthatthemolecularmethodofanalysisontheITSsequencesisarapid,highlysensitiveandaccurateapproachforthedetectionofdematophytespecies,however,itstillexhibitssomelimitationsneedingthesupplementationwithmorphologicalidentification.
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:AThepurposeofthisinvestigationwastostudythetherapeuticeffectofLamivudineonHBVDNAinperipheralbloodmononuclearcells(PBMC)andserum,andthelevelofcytokinesinserumofthepatientswithchronichepatitisB.Thepatientsweredividedintotwogroups(A=47,B=34),andtreatedbyLamivudine,routinemedicine,respectively.ThelevelsofHBV-DNAinPBMCandserumandcytokineswerealldetectedbeforeandaftertreatment.AfterthetreatmentofLamivndinefor36weeks,thetotalconversionnegativeratesofHBV-DNAinPBMCandserumofthepatientstreatedwithLamivudinewere55.32%(26/47)and61.70%(29/47),respectively.ThetotalnegativeconversionratesofHBV-DNAinPBMCandserumofthepatientstreatedbyroutinemedicinewere26.47%(9/34)and32.35%(11/34),respectively.TherewassignificantdifferencebetweenLamivudinegroupandroutinemedicinegroup(P<0.01).ThenegativeconversionratesofHBeAginserumofthepatientswere46.81%(22/47)and68.09%(32/47)attheendof24weeksand36weeks,andwerehigherthanthoseofroutinemedicinegroup(P<0.05andP<0.01).Thelevelsofalanineaminotransferase(ALT),aspartateaminotransferase(AST),ALT/ASTinserumofthepatientsafterbeingtreatedbyLamivudine,routinemedicineweredown-regulatedto(30.1±9.6)U/ml,(32.3±10.7)U/ml,0.9±0.1and(48.4±10.7)U/ml,(44.7±11.0)U/ml,1.1±0.2.Aftertheanalysisofvariance,thehighsignificantdifferencewasobviousbetweenthetwogroups(P<0.01).ItwasduetothehighlevelsofIL-6,IL-8andTNF-αinchronichepatitisBwhichcouldbedown-regulatedto(250.5±33.3)pg/ml,(153.4±22.2)pg/ml,(232.6±21.2)pg/mlbyLamivudine,whichwasmoreobviousthanthatofroutinemedicine(P<0.01).LamivudinehashightherapeuticeffectonthetreatmentofHBVDNAinPBMCandserumandhasbettertherapeuticeffectthanthatofroutinetherapy.Lamivudinemayalsohavehigherdown-regulatedinflammatoryinfiltrationandsecretioninlocalsitecaused
简介:TheaimofthisstudyistoinvestigatethefeasibilityandmechanismofhIL-2-preSDNAvaccineaspreventionandtherapeuticapproachagainstHepatitisB.EukaryonexpressionvectorinvolvinghIL-2andpreSgenewasconstructedwithrecombinanttechniqueandtransferredintonormalBALB/cmiceandHBVtransgenicmice(Tg-Mice)respectively.Tnenaseriesofdetectionwereperformed:detectionofanti-preS2,HBsantibodyandHBsAginBALB/cmiceandTg-micewithELISA,quantificationofHBVDNAcopiesinHBVTg-miceserumwithreal-timePCR,determinationofhepatitisdegreewithimmunopathologicalHEstaininganddetectionofliverfunction.Anti-preS1canbedetectedat4^th,6^thand10^thweekininoculatedBALB/cmice.Injectionwithgenegungainedanadvantageovermuscularandsubcutaneousinjectionsinceitacquiredjust1/10inoculationquantity(10μg/mouse).HighestexpressionofIgG2aat4^thweeksuggestedThl-mediatedimmuneresponse,whichfacilitatedHBVcleaning.OfallinoculatedHBVTg-mice,80%ofthemshowedanfi-preS2,HBsantibodypositiveandHBVDNAdecreased,and20%showednegativeforHBsAg.HEstainingtohepatictissueshowedobviousinfiltrationofinflammatorycells,swellingandgranulardegenerationofhepatocytes.Inourstudy,IL-2-preSDNAvaccinewhichcanprovokethehumoralandcellularimmuneresponseandbreaktheimmunetolerancesupportsthedesignationandconstructionofnewvaccineagainstHBVandspecificimmuneremedyforHBVcontinuousinfection.
简介:【摘要】目的:讨论乙肝病毒性肝炎患者临床医学检验应用探究。方法:选取我院治疗的乙肝病毒性肝炎的患者50例,均实行两对半和HBV-DNA定量检测,回顾性分析检测结果,选取的患者均需要使用两对半临床医学检验,采集患者的清晨空腹状态下静脉血4毫升,将血液放入离心机中,使用每分钟3000转,10分钟后,将血清分离出来,放在4摄氏度的条件下保存。结果:所有患者经临床检测诊断结果均为乙肝病毒性肝炎,其中小三阳患者占18例(36.0%),大三阳患者占13例(26.0%),其它类型占19例(38.0%),三组之间的差异性较为显著(P
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
简介:TherRNAgeneticlocusisfoundinallprokaryoticorganisms,andishighlyconservative,althoughitsrelativelystablevariationsarefoundfrequentlyindifferentbacteria.Theutilityofthislocusasataxonomicandphylogenetictoolhasbeenreportedwidely.Thisstudy,aimedat16SrRNAgene(16SrDNA)andwiththehelpofbiomolecularmethods,attemptedtoachievethegoalofrapididentificationofcommonpathogensInthisstudy,333clinicalisolatedpathogenicbacteriawerecollected。TwopairsofprimerswerechosenandlabeledwithdifferentfluorescentdyesandthenusedtoamplifythegenomicDNAextractedfrombacteria.ThePCRproductswerethendetectedbycapillaryelectrophoresis-singlestrandconformationpolymorphism(CE-SSCP).Inordertopursuehigherresolutionandpeak-separationeffect,ahighefficientseparatingmedium,linerpolyacrylamidedel(LPA),wasputtouseinthisstudy.Finally,everybacteriacolonygenerateddistinctpatternsfromeachother,whichwereeasilytobeusedforidentification.TheseresultsindicatedthatPCR-CE-SSCPwasarapididentificationmethodforbacterialidentification,withtheaspectsofhighefficiencyandhighprecision.Comparedwithtraditionalmethod,thistechnologyisofgreatutilityforclinicaluseespeciallyforitshighsensitivity.