冠状病毒Coronavirusl论文-2016 [Springer Protocols Handbooks] Animal Coronaviruses __ Recombinant Turkey Coronavirus Nucleocapsid Protein Expresse.pdf

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1、Chapter4RecombinantTurkeyCoronavirusNucleocapsidProteinExpressedinEscherichiacoliChienChangLoa,ChingChingWu,andTsangLongLinAbstractExpressionandpuriÞcationofturkeycoronavirus(TCoV)nucleocapsid(N)proteinfromaprokaryoticexpressionsystemashistidine-taggedfusionproteinarepresentedinthischa

2、pter.Expressionofhistidine-taggedfusionNproteinwithamolecularmassof57kDaisinducedwithisopropylβ-D-1-thiogalactopyranoside(IPTG).TheexpressedNproteininclusionbodyisextractedandpuriÞedbychromatographyonnickel-agarosecolumntonearhomogeneity.Theproteinrecoverycanbe10mgfrom100mlofbacterialc

3、ulture.ThepuriÞedNproteinisasuperiorsourceofTCoVantigenforantibody-captureELISAfordetectionofantibodiestoTCoV.KeywordsCoronavirus,Nucleocapsidprotein,Recombinantprotein,Cloning,Proteinexpression1IntroductionTurkeycoronavirus(TCoV)isthecauseofanacuteandhighlycontagiousentericdiseaseaffe

4、ctingturkeysofallages.Thediseaseisseverein1-to4-week-oldturkeypoults[1].Turkeyßocksthatrecoverfromnaturalorexperimentalcoronaviralenteritismaydeveloplifelongimmunity[2].TCoVhasbeenrecognizedasanimportantpathogenofyoungturkeys.TCoVinfectioncausessig-niÞcanteconomiclossesintheturkeyindus

5、tryduetopoorfeedconversionandunevengrowth.OutbreaksofTCoVenteritisinturkeypoultsremainasathreattotheturkeyindustry.Inordertorapidlydiagnoseandeffectivelycontrolturkeycoronaviralenteritis,developmentofanantibody-captureenzyme-linkedimmunosorbentassay(ELISA)fordetectingantibodiestoTCoVis

6、essential.DevelopmentofELISAfordetectionofTCoVinfectionrequireslargeamountsofTCoVantigen.Molecularclon-ingandexpressionofTCoVNproteinwerecarriedoutforprepa-rationoflargequantitiesofhighlypuriÞedviralproteins.Coronavirusisanenvelopedandpositive-strandedRNAvirusthatpossessesthreemajorstr

7、ucturalproteinsincludingaLeyiWang(ed.),AnimalCoronaviruses,SpringerProtocolsHandbooks,DOI10.1007/978-1-4939-3414-0_4,©SpringerScience+BusinessMediaNewYork20163334ChienChangLoaetal.predominantphosphorylatednucleocapsid(N)protein,peplo-mericglycoprotein,andspike(S)proteinthatmakesupthe