sars冠状病毒核衣壳蛋白对细胞色素氧化酶cyp450cyp4f3a)酶活性影响的研究.docx

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1、sars冠状病毒核衣壳蛋白对细胞色素氧化酶cyp450cyp4f3a)酶活性影响的探讨3ABSTRACTTheeffectsofSAKS-CoVnucIeocapsidproteinonthecatalyticactivityofCytochromeP450(CYP4F3)AbstractNucleocapsidproteinisoneofthemajorstructuralproteinsofSARS-CoV,WhichisknowntobindviralRNAtoformthehelicalnucIeocapsid.Also,NucleocapsidproteinofSARSCoVisth

2、emajorimmunogenicantigenwhichwascapableofgeneratingstronghumoralandcellularimmuneresponses,suggestinganimportantapplicationofSARSNproteininSARSdiagnosisandprevention.TheSAKSNproteinwasshowntoplayimportantrolessuchasregulationofSARS-CoVRNAsynthesisandnucleocapsidformation,disassemblyoftheviralcorebyb

3、indingtohumanCyclophilinA,andactivationofP-1signaltransductionpathway,whichinterferenceshostcelldivisionandinducesapoptosisinCOS-Icellsintheabsenceofgrowthfactors.OurpreviousworkalsodemonstratedthattheNproteinmustbeimportantlyinvolvedintheprocessofSARS-CoVinfection.Todelineatethemolecularapproachbyw

4、hichNproteinwasinvolvedinthepathology,fulllengthSRSCoVNproteinwasemployedasbaitproteintoscreenacDNA1ibraryfromhumanfetal1iverintheyeasttwohybridizationsystem.TheresultsindicatedthatSARS-CoVNproteinmayinteractwithhumanCytochromeP450famiIy4isoform3(CYP4F3).Inthisstudy,invitro,theenzymaticreactionSySIC

5、mandassaymethodofkineticconstantwasestablished.Weprovedthatthe-hydroxylaseactivityofCYP4F3AcanbesignificantlyinhibitedbybindingwithSARS-CoVNproteinthroughaseriesofion-changechromatography,molecularsievegelfiItrationchromatography,immunoprecipitation,westernblotandE1.ISAassay,andthendegradationofthesubstrate(1.TB4,animportantinflammatoryfactor)wasgreatlysuppressed.Furthermore,TheKmvalues(Michaelisconstant)ofCYP4F3Adeterminatedby1.ineweaver-Burkplottinghavechangedfrom2.5mol1.to10.2moI1.,comparedtotheKmvalueofCYP4F3,(4.82mol1.)whenequivalence229ENproteinwasinvolvedin.