利用日本血吸虫重组信号蛋白14-using schistosoma japonicum recombinant signal protein 14.docxVIP

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2018-05-29 发布于上海
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利用日本血吸虫重组信号蛋白14-using schistosoma japonicum recombinant signal protein 14.docx

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利用日本血吸虫重组信号蛋白14-using schistosoma japonicum recombinant signal protein 14

AbstractObjective:TopreparerecombinantSchistosomajaponicum(Chinesestrain)signalingprotein14-3-3andtheantibodiesagainstrSj14-3-3(monoclonalantibodiesandpolyclonalantibodies).ToevaluatethediagnosticvalueofrSj14-3-3anditsantibodies(monoclonalantibodiesandpolyclonalantibodies)inthediagnosisofschistosomiasis.Methods:rSj14-3-3proteinwaspreparedfromPET28a/E.coliBL21byIPTGinductionandpurifiedinscalebyHis-BindPurificationKit.Balb/cmicewereimmunizedwithpurifiedrSj14-3-3protein.Theanti-rSjl4-3-3monoclonalantibodywasobtainedbyusinghybridomatechnique.Characterizationoftheantibodieswasperformedbyindirectenzyme-linkedimmunosorbentassayandWesternblottinganalysis.RabbitswereimmunizedwiththerSj14-3-3proteintopreparepolyclonalantibodiesagainstrSj14-3-3．Thetiterofthepolyclonalantibodieswasdetectedbyindirectenzyme-linkedimmunosorbentassay.SpecificantibodiestorSj14-3-3proteininseraofpatientswithacuteandchronicschistosomiasisandnormalpeopleweredetectedwithpurifiedrSj14-3-3antigenbyindirectenzyme-linkedimmunoadsordentassay.Simultaneously,thecirculatingSj14-3-3antigenwastestedwithanti-Sj14-3-3polyclonal-monoclonalantibodybymonoclonalantibody(HRP-1D4)sandwichELISA.Results:HighPurifiedrSjl4-3-3proteinandthehybridomacellline1D4secretingmonoclonalantibodies(McAbs)againstrSj14-3-3wereobtained.ThesubclassofMcAbswasfoundtobeIgG1.Thetiterofperitonealexudateswasl:1×107.ThepositiverateofrSj14-3-3ELISAintestingspecificantibodiesfrom30provedcaseswithacuteandchronicschistosomiasiswere100%and96.7%.ThepositiverateofsandwichELISAintestingcirculatingantigenfrom30provedcaseswithacuteandchronicschistosomiasiswere73.3%and53.3%.Nofalsepositivereaetionwasdetectedin50normalcontrolswiththistwomethods.Conclusions:Theindirectenzyme-linkedimmunoadsordentassayindetectspecificantibodiesshowedhighersensitivityandspecificityforthediagnosisofacuteandchronicschistosomiasis,rSj14-3-3antigencanbeproductedstandardlybygeneengineeringtechnique,suggestingthatthedetectionofanti-rSj14-3-3antibodiesinseraofpatientsinfectedwithS.jap