fth1在b ovis 019株侵染小鼠巨噬细胞中的功能研究-study on the function of ft h1 in murine macrophages infected by b ovis 019 strain.docx

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fth1在b ovis 019株侵染小鼠巨噬细胞中的功能研究-study on the function of ft h1 in murine macrophages infected by b ovis 019 strain.docx

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fth1在b ovis 019株侵染小鼠巨噬细胞中的功能研究-study on the function of ft h1 in murine macrophages infected by b ovis 019 strain

AbstractObject：BrucellosisisazoonticdiseasecausedbymembersofthegenusBrucella.Brucellamayparasiteinmacrophageandinhibitcellapoptosisatlong-term,whichcausesmany organsdamageandchronicinfectionforhumanandlivestock.Itisveryimportanttoexploretherelationbetweenbrucella’sintracellullarmechanismandchronicinfection.Theresultsofcurrentetiologicstudyshowthatoutermembraneproteins(OMPs)andtypeⅣsecretionsystem(TFSS)proteins areputative virulentfactorsbecausetheseelementsplayanimportantroleinbrucella’sinvasion,survivalandmultiplication.Thefunctionstudyofmacrophagetargetproteinsinteractingwithbrucellavirulentfactorsbecomesmolecularbasisofbrucella intracellullarparasitemechanism.Thisstudyisusefultoscreenspecificcandidatedrugstobrucellosis.Methods:(1)Toverificateferritinheavychain1(FTH1)interactedwithbrucellaTFSSVirB5 proteinbyco-immunoprecipitation(Co-IP).(2)ThetargetgenesincludingFTH1,16SrRNA, GAPDHandapoptosis-relatedgeneswerecloned,whoseStdandmeltcurveweremade.(3)ThreepositiveandonenegativepSIREN-siRNAstargetedtoFTH1weretransfectedintomouseRAW264.7macrophageswithTurboFectTMinvitroTransfectionReagent.TheoptimalpSIREN-siRNAwerescreenedbydetectingtheexpressionofFTH1byReal-timePCR. (4)TheoptimalpSIREN-siRNAwastransfectedintomouseRAW264.7macrophages.After48hthetransfectedmacrophageswereinfectedwithB.ovis019strain[0]for4handtheexpressionsof16SrRNAandapoptosis-relatedgeneswerequantitatedandcomparedby Real-timePCR.(5)Themorphologic change ofRAW264.7 macrophagesinfected withB.ovis 019strainbeforeRNAiandafterRNAiwereobservedbyelectronmicroscope.(6)ThecellculturefluidsupernatantofMouseRAW264.7macrophagestransfectedwithoptimalpSIREN-siRNAandinfectedwithB.ovis019strainfor15min,30min,1hand4hwasdetectedby ELISA.Results:(1)TheresultsofCo-IPshowedthatVirB5interactedwithFTH1.(2)TheeleventargetgenesincludingFTH1,16SrRNA,GAPDHandapoptosis-relatedgeneswereclonedandtheirStdvalueweremade.(3)ThreepositivesiRNAplasmidsweresuccessfullytransfectedintomouseRAW264.7macrophages.TheoptimalpSIREN-Cwasscreenedan