减蛋综合征病毒五邻体蛋白与宿主蛋白GABARAPL1和RPSA的相互作用

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密级：：论文编号中国农业科学院学位论文减蛋综合征病毒五邻体蛋白与宿主蛋白ＧＡＢＡＲＡＰＬ１和ＲＰＳＡ的相互作用ＩｎｔｅｒａｃｔｉｏｎｏｆＥＤｒｏＳｎｄｒｏｍｅＶｉｒｕｓＰｅｎｔｏｎＰｒｏｔｅｉｎｗｉｔｈｇｇｐｙＤｕｃｋＨｏｓｔＰｒｏｔｅｉｎｓＧＡＢＡＲＡＰＬ１ａｎｄＲＰＳＡ博士研究生：ＺｈｅｎｅｙＭａｋａｙ指导教师：李刚教授申请学位类别：农学博士专业：预防兽医学研究方向：病毒与宿主蛋白相互作用培养单位：北京畜牧兽医研究所研究生院２０１７年１１月 密级:论文编号:中国农业科学院学位论文减蛋综合征病毒五邻体蛋白与宿主蛋白GABARAPL1和RPSA的相互作用InteractionofEggDropSyndromeVirusPentonProteinwithDuckHostProteinsGABARAPL1andRPSA博士研究生：ZheneyMakay指导教师：李刚教授申请学位类别：农学博士专业：预防兽医学研究方向：病毒与宿主蛋白相互作用培养单位：北京畜牧兽医研究所研究生院2017年11月 Secrecy:No.ChineseAcademyofAgriculturalSciencesDissertationInteractionofEggDropSyndromeVirusPentonProteinwithDuckHostProteinsGABARAPL1andRPSAPhD.Candidate：ZheneyMakaySupervisor：Prof.LiGangMajor：PreventiveVeterinaryMedicineSpecialty：Virus-HostproteininteractionsNovember2017 独创性声明本人声明所呈交的论文是我个人在导师指导下进行的研宄工作及取得的研宄成果。尽我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人己经发表或撰写过的研究成果，也不包含为获得中国农业科学院或其它教育机构的学位或证书而使用过的材料一。与我同工作的同志对本研宂所做的任何贡献均已在论文中作了明确的说明并表示了谢意。ＤｅｃｌａｒａｔｉｏｎｏｆＯｒｉｇｉｎａｌｉｔｙＩｈｅｒｅｂｄｅｃｌａｒｅｔｈａｔｔｈｉｓｔｈｅｓｉｓｗａｓｃｏｍｐｏｓｅｄａｎｄｏｒｉｇｉｎａｔｅｄｅｎｔｉｒｅｌｂｍｙｓｅｌｆｕｎｄｅｒｔｈｅｙｙｙｄａｎｃｅｏｆｍｓｕｅｒｖｉ，Ｔｔｈｅｂｅｓｔｍｋｎｏｗｌｅｄｅｉｎａｄｄｉｔｉｏｎｒｍａｔｉｏｎｇｕｉｙｐｓｏｒｏｏｆｙｇ，ｔｏｍｆｏｄｅｒｉｖｅｄｆｒｏｍｔｈｅｕｂｌｉｓｈｅｄａｎｄｕｎｐｕｂｌｉｓｈｅｄｗｏｒｋｓｏｆｏｔｈｅｒｓｔｈａｔｈａｓｂｅｅｎｐａｃｋｎｏｗｌｅｄｅｄｉｎｔｈｅｔｅｘｔａｎｄａｌｉｓｔｏｆｒｅｆｅｒｅｎｃｅｓｉｓｇｖｅｎｉｎｔｈｅｂｉｂｌｉｏｒａｈｔｈｅｇｉｇｐｙ，ｔｈｅｓｉｓｄｏｅｓｎｏｔｃｏｎｔａｉｎａｎｙｏｔｈｅｒｐｕｂｌｉｓｈｅｄｏｒｕｎｐｕｂｌｉｓｈｅｄｒｅｓｅａｒｃｈｗｏｒｋｂｙｏｔｈｅｒｓ，ｏｒａｎｙｍａｔｅｒｉａｌｓｆｏｒａｎｏｔｈｅｒｄｅｒｅｅｏｒｄｉｐｌｏｍａｆｒｏｍｔｈｅＣｈｉｎｅｓｅＡｃａｄｅｍｙｏｆＡｇｒｉｃｕｌｔｕｒａｌｇＳｃｉｅｎｃｅｓａｎｄｏｔｈｅｒｅｄｕｃａｔｉｏｎａｌｉｎｓｔｉｔｕｔｉｏｎｓ．Ｔｈｅｗｏｒｋｓｃｏｎｔｒｉｂｕｔｅｄｂｏｔｈｅｒｃｏｌｌｅａｕｅｓｗｙｇｈａｖｅｂｅｅｎｓｔａｔｅｄａｎｄａｃｋｎｏｌｅｄｇｅｄ，关于论文使用授权的声明业本人完全了解送中国农业科学院有关保留、使用学位论文的规定，即：中国农科学院有权保留交论文的复印件和磁盘，允许论文被查阅和借阋，可以采用影方ＥＰ、缩印或扫描等复制手段保存、汇编学位论文。同意中国农业科学院可以用不同式在不同媒体上发表、传播学位论文的全部或部分内容。ＡｕｔｈｏｒｉｚｅｄＵｓｅＡｒｅｅｍｅｎｔｇＩｆｕｌｌｕｎｄｅｒｓｔａｎｄｔｈｅｒｅｕｌａｔｉｏｎｓｃｏｎｃｅｒｎｉｎｒｅｓｅｒｖａｔｉｏｎａｎｄｕｓａｅｏｆｔｈｅｔｈｅｓｉｓｉｎｔｈｅｙｇｇｇＣｈｉｎｅｓｅＡｃａｄｅｍｏｆＡｒｉｃｕｌｔｕｒａｌＳｃｉｅｎｃｅｓ：ＣＡＡＳｒｅｔａｉｎｓｔｈｅｒｉｈｔｔｏｋｅｅｔｈｅｃｏｉｅｓａｎｄｙｇｎｇｐｐｄｉｓｋｓｏｆｔｈｅｔｈｅｓｉｓ，ａｌｌｏｗｉｔｔｏｂｅａｃｃｅｓｓｅｄａｎｄｂｏｒｒｏｗｅｄ，ａｄｃｏｍｏｓｅｉｔｂｈｏｔｏｃｏａｎｄｐｙｐｐｙｓｃａｎ．ＣＡＡＳｃａｎａｌｓｏｄｉｓｓｅｍｉｎａｔｅａｎｄｕｂｌｉｓｈｔｈｅｆｕｌｌａｎｄａｒｔｏｆｔｈｅｔｈｅｓｉｓｉｎｄｉｆｆｅｒｅｎｔｎｐｐｗａｓａｄｏｎｄｉｆｆｅｒｅｎｔｍｅｄｉａ．ｙ’学生签名／Ｓｔｕｄｅｎｔｓｓｉｎａｔｕｒｅ：边Ｄａｔｅ：＾２〇（１ｇ’＾导师签名／Ｓｕｅｒｖｉｓｏｒｓｓｉｇｎａｔｕｒｅ：Ｄａｔｅ：ｐ－＜／２１ 摘要减蛋综合征病毒（EggDropSyndromevirus，EDSV）是直径约为70-80nm的无包膜二十面体核衣壳的双链DNA病毒。它属于腺病毒科腺胸腺病毒属。EDSV于1976年首次在荷兰报道，当时从产蛋鸡中分离出该病毒。EDSV主要导致产蛋突然严重下降，以及外观健康的产蛋鸡产生无壳，薄壳，变色或变形的鸡蛋。EDSV基因组含有33,213个碱基对。它也被称为鸭腺病毒1（DAdV-1），鸭腺病毒A型，腺病毒127。六邻体、纤维蛋白和五邻体（penton）是EDSV的主要衣壳蛋白。五邻体蛋白在腺病毒入侵宿主细胞中起主要作用。腺病毒五邻体基质可以与细胞膜整合素结合并促进病毒入侵到细胞中。EDSV蛋白可能与其他腺病毒penton蛋白在病毒复制中具有相同的功能，而不仅仅是结构蛋白。因此，本研究的目的是表达五邻体，研究五邻体在病毒复制中的功能，并鉴定与五邻体相互作用的宿主细胞蛋白。为了找到EDSV五邻体蛋白的受体或与其有相互作用的配体，应用酵母双杂交（Y2H）方法进行筛选。选择10种蛋白作为潜在的蛋白相互作用候选物。为了确定相互作用，使用酵母回补实验来消除误差。结果表明10种蛋白质中有4种蛋白质即鸭GABA（A）受体相关蛋白1（GABARAPL1），鸭蛋白质核糖体蛋白SA（RPSA），鸭蛋白整联蛋白α4亚基（ITGA4）,鸭肌动蛋白-β（ACTB）与EDSV五邻体蛋白有相互作用的配体。我们选择4种蛋白中的两种蛋白(GABARAPL1和RPSA）验证了它们与五邻体蛋白之间的相互作用，使用原核和真核表达系统来表达猎物蛋白和诱饵蛋白，然后通过体外GSTpull-down实验验证其相互作用。结果表明GABARAPL1和RPSA与五邻体蛋白具有直接相互作用。在腺病毒的诊断中，常使用基于核酸的技术，例如通过PCR方法体外扩增病毒DNA。为了检测由EDSV引起的感染，我们建立了实时荧光环介导等温扩增技术（RealAmp）。该方法与PCR相比具有一些优点，包括灵敏性和特异性以及能够通过荧光信号观察在一个平台上同时进行扩增和检测。检测感染样品（尿囊液和细胞培养物）的EDSV，直接使用RealAmp方法，无需提取病毒DNA。RealAmp成功用于直接检测样品中EDSV纤维蛋白基因。RealAmp能在15-30分钟内扩增病毒DNA。灵敏度可达26fg/μl，该方法特异性强，与其他DNA病毒无交叉反应。关键词：EDSV,五邻体,RPSA,GABARAPL1，实时荧光LAMPI AbstractTheeggdropsyndromevirus(EDSV)isastrandedDNAviruswithnon-envelopedicosahedralnucleocapsidofapproximately70–80nmindiameter.ItbelongstogenusAtadenovirusunderthefamilyAdenoviridae.EDSVwasfirstreportedintheNetherlandsin1976,whenadenoviruseswereisolatedfromlayingchickensTheEDSVgenomecontains33,213basepairs.Ithasalsobeenknownasduckadenovirus1(DAdV-1),duckadenovirusA,adenovirus127.EDSVprimarilycausesasuddenseveredropineggproductionaswellastheproductionofshell-less,thin-shelled,discolored,ormisshapeneggsinapparentlyhealthylayingbirds.Thehexon,fiberandpentonarethemaincapsidproteinsofEDSV.Adenoviruspentonbaseproteinhasmainroleinvirusinternalizationintothehostcell.Theadenoviruspentonbasecanbindtocellmembraneintergrinsandsupportthevirustointernalizeintocell.EDSVpentonproteinmayhavesamefunctionswithotheradenoviruspentonproteininviralreplicationratherthanjustbeingastructuralprotein.Therefore,theobjectiveofthisstudyistocharacterizepenton,investigatethefunctionsofpentoninviralreplicationandidentifythecellularproteinsinteractingwithpenton.TofindreceptorsorinteractionpartnersofEDSVpentongene,theyeasttwo-hybrid(Y2H)screeningwascarriedout.Totalof10proteinswereselectedasthepotentialproteininteractioncandidates.Todeterminethepositiveinteractions,co-transformationanalysiswasusedtoeliminatethefalsepositives.Among10preyproteins,fourproteinswereselectedasthepositives.TheanasplatyrhynchosGABA(A)receptor-associatedproteinlike1(GABARAPL1),anasplatyrhynchosribosomalproteinSA(RPSA),anasplatyrhynchosintegrinsubunitalpha4(ITGA4)andanasplatyrhynchosactinbeta(ACTB)werefoundtobepositivelyinteractedwithpentonintheY2Hassay.Toverifytheinteractionbetweenpentonandselectedproteins,thepreyandbaitproteinswereexpressedusingbacterialandmammalianexpressionplasmids.FinallytheexpressedproteinswereconfirmedbyinvitroGSTpull-downsystem.Aftertheconfirmationassay,thepentonproteinofEDSVandbothpreycandidateproteins(GABARAPL1andRPSA)haveinteractions.Indiagnosisofavianadenoviruses,thenucleicacidbasedtechnologieshavebeenused,suchasinvitroamplificationofDNAbythePCRmethod.TodetectinfectioncausedbytheEDSV,thereal-timefluorescenceloop-mediatedisothermalamplification(RealAmp)isoneoftheLAMPmethodswithsomeadvantagesascomparisonwithPCR,includingsensitiveandspecificaswellascapableofperformingboththeamplificationanddetectionbyfluorescenceinoneplatform.TodetecttheEDSVfrominfectedsamples(allantoicfluidandcellculture)weutilizedRealAmpmethoddirectlywithoutviralDNAextraction.RealAmpwassuccessfullyemployedtodetectEDSVfibergenedirectlybyusingdilutedsamples.RealAmpwasamplifiedviralDNAinshorttimespanof15–30minutes.Thesensitivitywasaslowas(26fg/μl)andspecificforEDSVfiberwithindifferentDNAviruses.II Keywords:EDSV,Penton,RPSA,GABARAPL1,RealAmpIII TABLEOFCONTENTSCHAPTERILITERATUREREVIEW............................................................................11.1ADENOVIRUS.................................................................................................................11.1.1Adenovirusclassification.......................................................................................11.2ATADENOVIRUS.............................................................................................................21.3EGGDROPSYNDROMEVIRUS(EDSV)...........................................................................31.3.1Genomeorganizationandreplication...................................................................41.3.2Adsorptionandentry.............................................................................................81.3.3Pathogenesis........................................................................................................101.3.4TransmissionofEDSV.........................................................................................111.3.5Diagnosisofinfection..........................................................................................121.3.6Preventionandcontrol........................................................................................131.4ADENOVIRUSVECTORS................................................................................................141.5PROTEIN-PROTEININTERACTION.................................................................................141.5.1Yeast-Twohybridsystem......................................................................................161.5.1.1MatchmakerGAL4Two-hybridsystem............................................................171.5.1.2Reportergenesandthreedifferentbindingsites..............................................181.5.2GST-pulldownassay...........................................................................................191.6OBJECTIVES.................................................................................................................21CHAPTERIIREAL-TIMEFLUORESCENCELOOP-MEDIATEDISOTHERMALAMPLIFICATIONASSAYFORDIRECTDETECTIONOFEGGDROPSYNDROMEVIRUS........................................................................................................222.1INTRODUCTION............................................................................................................222.2MATERIALSANDMETHOD...........................................................................................232.2.1Chemicalsandreagents.......................................................................................232.2.2Viruses..................................................................................................................242.2.3InoculationofembryonatedduckeggswiththeEDSV.......................................242.2.4Cellcultureandvirusinoculation.......................................................................242.2.5ViralDNAextraction...........................................................................................252.2.6DesignofprimersfortheRealAmpandPCR.....................................................252.2.7RealAmpassay.....................................................................................................25IV 2.2.8SpecificityandsensitivityoftheRealAmpassay.................................................262.2.9ConventionalPCR...............................................................................................262.3RESULTS......................................................................................................................272.3.1RealAmpofEDSVDNA.......................................................................................282.3.2DirectRealAmpassay..........................................................................................292.3.3SpecificityoftheRealAmpmethod......................................................................312.3.4SensitivityoftheRealAmpassay.........................................................................32CHAPTERIIIYEASTTWO-HYBRIDSCREENING.................................................343.1INTRODUCTION............................................................................................................343.2MATERIALSANDMETHODS.........................................................................................353.2.1Preparationofduckembryosfibroblastcellculture...........................................353.2.2TotalRNAextractionfromduckembryofibroblastcellculture..........................363.2.3First-StrandcDNASynthesis...............................................................................373.2.4TheligationofcDNAintopGADT7vector.........................................................383.2.5PreparationofY187andY2HGoldcompetentyeastcells..................................383.2.6TransformationofcompetentY187yeastcellswithduckcDNAlibraryplasmids......................................................................................................................................393.3CONSTRUCTIONOFPGBKT7-PENTONBAITFUSIONVECTOR.......................................403.3.1PCRamplificationofpentongene.......................................................................403.3.1.1DNAextraction.................................................................................................403.3.1.2Primerdesign...................................................................................................403.3.1.3PCR...................................................................................................................413.3.2GelpurificationofPCRproduct..........................................................................413.3.3LigationofpentongeneintopMD19Tsimplevector..........................................423.3.4TransformationofpMD19T-pentonintoE.colicompetentcells.........................423.3.4.1Preparationofmediaandsolutions.................................................................423.3.4.2PreparationofE.coliDH5αcompetentcells...................................................433.3.4.3TransformationofligationmixtureintoE.coli................................................433.3.5PCRforpositivecolonies....................................................................................443.3.6Plasmidextraction...............................................................................................443.3.7InsertionofpentongeneintopGBKT7baitvector..............................................443.3.7.1Restrictionenzymedigestion............................................................................44V 3.3.7.2LigationofpentonintopGBKT7baitvector....................................................453.3.8TransformationofpGBKT7-pentonplasmidintoY2HGoldcells.......................453.3.8.1YeastcolonyPCR..............................................................................................463.4YEASTTWO‐HYBRIDSCREENINGUSINGYEASTMATING............................................463.4.1Yeastplasmidextraction......................................................................................483.4.2PCRanalysistoeliminateduplicateclones........................................................483.5CONFIRMATIONOFPOSITIVEINTERACTIONS................................................................483.5.1Co-transformation...............................................................................................483.6RESULTS......................................................................................................................493.6.1CharacterizationofcDNAlibrary.......................................................................493.6.2ConstructionofpGBKT7-pentonbaitfusionplasmid.........................................513.6.3VerificationofpGBKT7-pentonexpressioninY2HGoldcells............................533.6.4Identificationofinteractionpartnersbylibraryscreening.................................553.6.5Confirmationofpositiveinteractorsbyco-transformationintoY2HGoldcells573.6.6Characterizationofpreycandidateinteractionpartners....................................593.6.6.1AnasplatyrhynchosGABAtypeAreceptorassociatedproteinlike1(GABARAPL1)..............................................................................................................593.6.6.2AnasplatyrhynchosribosomalproteinSA(RPSA)or40Sribosomalprotein.603.6.6.3Anasplatyrhynchosintegrinsubunitalpha4(ITGA4)gene............................613.6.6.4Anasplatyrhynchosactinbeta(ACTB)............................................................63CHAPTERIVCONFIRMATIONOFTHEINTERACTIONBETWEENBAITPROTEIN(PGBKT7:PENTON)ANDPREYPROTEINS(GABARAPL1ANDRPSA).................................................................................................................................644.1INTRODUCTION............................................................................................................644.2MATERIALSANDMETHODS..........................................................................................664.2.1ConstructionofbaitandpreyfusionvectorsforGSTpull-downassay..............664.2.1.1PCRamplificationofbaitandpreygenes........................................................664.2.1.2DNAligationintovectorsandbacterialtransformation..................................674.2.2ExpressionofBaitfusionprotein.........................................................................674.2.2.1ExpressionofpGEX-6P-1:pentoninBL21(DE3)cells..................................684.2.3Expressionofbaitandpreyfusionproteinsineukaryoticcells..........................684.2.3.1Cellculturepreparationandtransfection........................................................68VI 4.2.4Detectionofproteinexpressioninmammaliancelllines....................................694.2.4.1Preparationofcelllysates................................................................................694.2.4.2Preparationofrunning,transferandblockingbuffers....................................704.2.4.3SDSpolyacrylamidegelelectrophoresis..........................................................704.2.4.4Westernblotting................................................................................................714.3GSTPULL-DOWNASSAY.............................................................................................714.3.1Purificationofbaitfusionprotein.......................................................................714.3.2GSTpull-downprocedure....................................................................................724.4RESULTS......................................................................................................................734.4.1ConstructionofpGEX-6P-1:pentonplasmid.....................................................734.4.2Constructionofpreyplasmids.............................................................................744.4.3Expressionofbaitproteininbacterialcells........................................................754.4.4Expressionofbaitandpreyproteinsinmammaliancelllines............................764.4.5GSTpulldownassay...........................................................................................80CHAPTERVDISCUSSION............................................................................................83CONCLUSION..................................................................................................................88REFERENCES..................................................................................................................89ACKNOWLEDGMENT.................................................................................................100ADDITIONALMATERIALS........................................................................................103VII ABBREVIATIONSAdAdenovirusADActivatingdomainCARCoxsackieandadenovirusreceptorCDSCodingdomainsequenceDBPDNAbindingproteinGFPGreenfluorescentproteinGSTGlutathione-S-TransferasebpBasepairCPECytopathiceffectsDMEMDulbecco’sminimalessentialmediumDAdV-1Duckadenovirus1DVEVDuckviralenteritisvirusDCVDuckcircovirusEEarlyEDSEggdropsyndromeEDSVEggdropsyndromevirusFAdV-1FowlAdenovirus1FBSFetalbovineserumFPVFowlpoxvirusGSTGlutathioneS-transferaseMDVMarek’sdiseasevirusHAHaemagglutinationHAdVsHumanadenovirusesITRsInvertedterminalrepeatskDaKiloDaltonLLateLAMPLoopmediatedisothermalamplificationORFsOpenreadingframesPBSPhosphate-bufferedsalinePCRPolymerasechainreactionVIII PMSFPhenylmethylsulfonylfluoridePolPolymerasePPIProtein-proteininteractionpTPPre-terminalproteinqPCRQuantitativepolymerasechainreaction(PCR)RERestrictionendonucleaseRealAmpReal-timefluorescenceloopmediatedisothermalamplificationRGDArginine-glycine-asparticacidRPSARibosomalproteinSART-PCRReal-timepolymerasechainreactionSDS-PAGESodiumdodecylsulphatepolyacrylamidegelelectrophoresisSV40Simianvirus40TBSTTris-bufferedsalinewithtween-20TBSTris-bufferedsalineTPTerminalproteinY2HYeasttwohybridIX 中国农业科学院博士学位论文ChapterⅠCHAPTERILiteraturereview1.1AdenovirusAdenoviruses(Ads)havebeencharacterizedextensivelysincetheirinitialdescriptionintheearly1950s(HillemanandWerneretal.,1954,Rowe,Huebneretal.,1953).Internationalcommitteeontaxonomyofviruseshasbeenidentified,morethan100adenoviruseswhichcaninfectabroadrangeofmammal,birds,reptile,amphibianandfishhosts(Russelletal.,2009).AdenovirusesarebelongingtothefamilyAdenoviridae,dsDNAviruseswithnon-envelopedicosahedralparticlesof70-90nmindiameter.Theicosahedralparticlesconsistingofthreemajorproteins;hexon(II),pentonbase(III)andaknobbedfiber(IV).Thevirusgenomeisalinear,double-strandedDNAwithaterminalprotein(TP)attachedcovalentlytothe5’termini(Boulanger,Lemayetal.,1979,Rekosh,Russelletal.,1977).Thesizeofadenovirusgenomesfullysequencedtodaterangesbetween26,163and48,395bp,withITRsof36to371bp.Someofadenovirusproteinsarecommontoalladenoviruses,includingstructuralproteinshexon,pentonbase,pVIII,pVI,IIIaandnon-structuralprotein100Kand52K.Also,theyhavedifferencesinproteinsencodedbyEarly(E)region1,E3andE4regionamongmembersofdifferentadenovirusgenera(Davison,Benkőetal.,2003,Pitcovski,Mualemetal.,1998).Terminalproteincovalentlybindstotheendsoftheviralgenome,whichplaysaroleininitiatingviralgenomereplication.1.1.1AdenovirusclassificationAdenovirusesdividedintofiveofficialgenera:twogenera(MastadenovirusandAviadenovirus)originatefrommammalsorbirds,respectively,andtheothertwogenera(AtadenovirusandSiadenovirus)haveabroaderrangeofhosts.Theonlyconfirmedfish1 中国农业科学院博士学位论文ChapterⅠadenovirusfallsintothefifthclade(Ichtadenovirus)(Fig.1.1).Withineachgenus,virusesaregroupedintospecies(B,Metal.,2012,Davison,Benkőetal.,2003,Harrachetal.,2008).Figure1.1PhylogenetictreeofadenovirusesbasedonmaximumlikelihoodanalysisofavailableDNApolymeraseaminoacidsequences.(HarrachB.(2014)Adenoviruses:GeneralFeatureswithauthor’spermission).1.2AtadenovirusMembersofAtadenoviruscaninfectavarietyofhostspecies,includingmammals,birdsandscaledreptiles(Benkó,Eloetal.,2002).Thegenomesizeofsequencedisolatesrangesfrom29,576(ovineadenovirus7,OAdV-7)to33,213bp(duckadenovirus1,DAdV-1)withITRsof46(OAdV-7)to118bp(snakeadenovirus1,SnAdV-1).Forruminant,marsupialandavianatadenoviruses,theG+CcontentoftheDNAislowandvariesbetween33.6(OAdV-7)and43.0%(DAdV-1).ThecorrespondinghighA+Tcontentwasdeemedsufficientlycharacteristictobeusedasthebasisofthenameofthegenus,2 中国农业科学院博士学位论文ChapterⅠthoughatadenovirusesoriginatingfromscaledreptileshaveanon-biasednucleotidecomposition(Benkó,Eloetal.,2002,Harrachetal.,2008,Hess,Blöckeretal.,1997).Atadenovirusesareclassifiedinto5species:BovineadenovirusD,DuckadenovirusA,OvineadenovirusD,PossumadenovirusandSnakeadenovirusA(VMRIbasedonICTVdecisions,2011)(Table1.1inadditionalsection).Figure1.2Schematicillustrationofthegenomeorganizationofatadenovirus(Ovineadenovirus).Blackarrowsdepictgenesconservedineverygenus,grayarrowsshowgenespresentinmorethanonegenusandgreenarrowsshowgenus-specificgenes.ReprintedfromHarrachB.(2014)(Adenoviruses:GeneralFeatures)withauthor’spermission.1.3Eggdropsyndromevirus(EDSV)ThespeciesDuckadenovirusAincludesonesinglemember,Duckadenovirus1(DAdV-1),whichistheofficialnameoftheEggdropsyndrome1976virus.Eggdropsyndromeisaviraldisease,causedbytheeggdropsyndromeviruswhichisamemberofgenusAtadenovirusunderthefamilyAdenoviridae.EDSVwasfirstreportedintheNetherlandsin1976,whenadenoviruseswereisolatedfromlayingchickens(VanEck,Davelaaretal.,1976).Ithasalsobeenknownasduckadenovirus1(DAdV-1),duck3 中国农业科学院博士学位论文ChapterⅠadenovirusA,adenovirus127,andegg-drop-syndrome-76(EDS-76)virus(B,Metal.,2012).TheEDSVvirioniscomposedofdouble-strandedDNAandanon-envelopedicosahedralnucleocapsidofapproximately70–80nmindiameter.TheEDSVgenome6contains33,213basepairsandhasamolecularweightof21.9x10Da(Hess,Blöckeretal.,1997).EDSVprimarilycausesasuddenseveredropineggproductionaswellastheproductionofshell-less,thin-shelled,discolored,ormisshapeneggsinapparentlyhealthylayingbirds(VanEck,Davelaaretal.,1976).DucksandgeesearethenaturalhostsoftheEDSV,howeverthevirusdirectlyinfectschickens,resultinginmajoreconomiclossesbecauseofitsdirecteffectsoneggproduction(Hafezetal.,2011,Mcferran,Mccrackenetal.,1978).LaterareportdemonstratedthehighprevalenceofEDSVinwildanddomesticducks.AlsoEDSVfromducksshowedvariableadaptabilityinchickens(Cha,Kangetal.,2013).1.3.1GenomeorganizationandreplicationTheadenovirusreplicationcyclecanbedividedintofivestages:adsorptionandentry,activationofearlygenes,DNAreplication,andactivationoflategenes,assemblyandrelease.Adenovirusgenomecarriesfourearlytranscriptionregions(E1-E4),twointermediategenes(IVa2andpIX)andonemajorlateunitthatistranscribedandalternativelyslicedintofivegroupsoflatemRNA(L1-5).AllofthesegenesaretranscribedbyRNApolymeraseII(Harrachetal.,2008).Whenadenovirusenterstothenucleus,theearlyregionscanstarttoexpress,whichencodemostlynon-structuralproteinsthatfacilitateviralreplication.ProteinsinvolveinthisprocessareE1A,E1BandE4regionsproteins.TheE2regiongenesarerequiredfor4 中国农业科学院博士学位论文ChapterⅠviralDNAreplication.Adenoviruscanfinishreplicationbeforetheinfectedcelliseliminatedbythehostimmuneresponse.ProductsencodedbyE3andVARNAgenesareassociatedwiththisprocess(Leppardetal.,2008,Russelletal.,2009).EDSVgenomeincludedthefollowinggenes:100kDaprotein,52kDaprotein,completegenome,coreprotein,DNApolymerase,DNA-bindingprotein,E1Bgene,E4protein,endopeptidase,fiberprotein,hexonprotein,IIIaprotein,IVa2protein,largeT-antigen,muprotein,pentonprotein,pVIprotein,pVIIprotein,pVIIIprotein,repeatregion,smallt-antigen,terminalproteinandvirus-associatedRNA.EDSVhasnoE1Aregion.Inadenovirusinfectedcells,E1Btranscriptionalunitencodestwoproteins,E1B-55KandE1B-19K,whichareinvolvedinpreventingapoptosis.TheE1B-55Kinhibitsp53-dependentapoptosisbydirectlybindingtoanddegradingp53(Li,Zhaoetal.,2011).Ineggdropsyndromevirusgenome,thefirstrightwardlytranscribedORFoftheupperstrandwithmorethan100aastartsatposition1175andstopsat1651.Togetherwiththefollowingproteinof393aa,thesetwoORFsrepresentprobablytheE1Bregion.ApossibleE1Bpromoterislocatedatposition996–1010andthetranscriptsterminateattheAATAAApolyadenylationsignalat2909–2914.Thesmall(17.5kDa)E1BproteinshowsonlyweakhomologytoE1Bproteinsofotheradenoviruses.TheEDSVgenomehasnopIXgene,whichinhumanadenovirusesfollowstheE1Bregion(Hess,Blöckeretal.,1997).TheE2regioncomprisesasetofproteinsinvolvedinadenovirusDNAreplication.ThesearetheDNA-bindingprotein(DBP),thepreterminalprotein(pTP),andtheDNApolymerase(Pol),transportedintothenucleusvianuclearlocalizationsignals(NLS)(Russelletal.,2009).InEDSVgenometheE2ORFsencodingDNAbindingprotein,DNApolymerase(pol),pre-terminalprotein(pTP)andIVaⅡgenes.TheEDSVDBPislocalizedbetweennt18,005and19,168,proteinconsistingof378aais151aa.Abasic5 中国农业科学院博士学位论文ChapterⅠsequencewhichishomologoustotheNLSsequenceoftheSV40largeTantigenislocatedinthisC-terminalextensionoftheEDSV(Hess,Blöckeretal.,1997,Zeng,Jinetal.,1998).InEDSVgenomesequence,thefibergenestartsonly45nucleotidesafterthepVIIIgeneandnoknownE3regioncanbeidentified(Hess,Blöckeretal.,1997).TheORFlocalizedimmediatelyafterthefiberproteincodesforaproteinof34.4kDa.ThisproteinhassignificanthomologytotheE434kDaofotheradenoviruses.InadenovirusestheE1,E3andmajorlateunitsaretranscribedfromtheforwardstrand,whileE2,E4andIVa2aretranscribedfromthereversestrand(B,Metal.,2012,Hess,Blöckeretal.,1997).EDSVL4transcripts;apossiblepolyadenylationsignalforthe52Kproteingeneislocatedatmapposition11,639.ProteinIIIageneterminatesprobablyatposition14,539togetherwiththeL2pentonbaseandcoreproteinsgenes.L3andL5transcriptscanbefoundatpositions18,219and24,638,respectively(Hess,Blöckeretal.,1997).Comparisonoftheaminoacidsequenceofthe100KproteinsfromhumanadenovirusesandfowladenovirusesofgroupIrevealedahomologyfrom32.3%to34.4%.Remarkably,EDSV100Kproteinshareshighhomology56.4%onaminoacidlevelwithovineadenovirus(Li,Jietal.,1998).ThemajorproteinsofeggdropsyndromevirusChinesestrainAAVgenomewasreportedwhichis32838bpinlength.Thegenesformajorviralproteins;52/55K,Ⅲa,pentonbase,PⅦ,pX,pⅥ,hexon,endopeptidase(EP),100K,pⅧ,andfiberarelocatedinthemiddleofthegenome(Zeng,Jinetal.,1998).Theadenovirusespentonbaseassemblesintoanon-covalentcomplex,thepentoncapsomer,withthehomotrimericfiber.ThepresenceofanexposedRGDmotifwithinthehypervariableloopregionofthepentonbaseofvariousadenovirusspeciesfacilitatesthe6 中国农业科学院博士学位论文ChapterⅠengagementofcellsurfaceintegrinsandithasalsobeensuggestedthatmultipleintegrinreceptorsbindeachpentonbase,promotingintegrinclusteringandtriggeringefficientvirioninternalization(Chiu,Mathiasetal.,1999).Wickhamandcolleaguesfirstdescribedsuccessfulinvitroretargetingofrecombinantpentonbaseproteininvitro,bysubstitutionoftheRGDsiteforanLDV-containingpeptidemotif,whichmediatedbindingtoα4β1integrin(Wickham,Carrionetal.,1995).ThenucleotidesequenceandlocationofthepentonbaseoftheEDSVwasdetermined.Thepentonbasegeneislocatedat34.8-38.8mapunits,thecodingsequencehasalengthof1359bpandencodesapolypeptideof452aminoacidswithamolecularweightof51,105Da(Hess,Blöckeretal.,1997).ThepentonbaseproteinlackstheintegrinbindingmotifsRGD(Arg-Gly-Asp)andLDV(Leu-Asp-Val)(Rohn,Prusasetal.,1997).TheITRsofEDSVare53nucleotides(nt)whichareveryshort,andstartswithdC.TheAT-richregionintheEDSITRsincludesthefirst39bpandcontains66.6%AT.TheATTAATAAsequenceinEDSITRscouldprobablybeanequivalentsequencemotif(Hess,Blöckeretal.,1997).EDSVdiffersinitsmorphologyfromotheradenovirusesbyhavingonlyonefiberperpentonbaseandabletoagglutinateerythrocytesofchickens,ducks,geese,turkeys,andpigeons(Adair,Mcferranetal.,1979,Fitrawati,Wibowoetal.,2015).EDSVhasauniquegenomeregionatthefarrightendwithsevenuncharacterizedORFsthatmaybehost-specificinfunction.ORF5andORF6arerelatedtoeachother.ThisuniqueregionofEDSValsocontainsaVARNAgenethatissupposedlyhomologoustothatofFAdV-1.EDSVisalsoassociatedwithaspecificdiseaseofhensthatischaracterizedgloballybysharpdecreasesineggproduction(Harrachetal.,2008,Russelletal.,2009).7 中国农业科学院博士学位论文ChapterⅠFigure1.3Genomeorganizationofeggdropsyndromevirus.Openreadingframesoftheupper,rightwardtranscribedstrandareindicatedontopandforthecomplementary,leftwardtranscribedstrandatthebottom.FilledboxesindicateunassignedORFs>100aa.Abbreviations:EP-endoproteinase;DBP-DNAbindingprotein;pTP-precursorterminalprotein;POL-DNApolymerase.Datawastakenfrom(Hess,Blöckeretal.,1997).1.3.2AdsorptionandentryThebeginningoftheadenoviruslifecycleistherecognitionofthetargetcell.Thisrecognitionismediatedbythefiberprotein.Thisinitialcontactisalsothecriticalfactorfortheadenovirusspecifictissuetropism.Theattachmentofadenovirusanduptakeintocellsareseparatebutcooperativeeventsthatresultfromtheinteractionofdistinctviralcoatproteinswithareceptorforattachmentandalphavintegrinreceptorsforinternalization(Wickham,Mathiasetal.,1993).Thefiberproteinofdifferentadenovirusescanbindtothedifferentcellsurfacereceptors.Themostpopularandfirstreceptoriscoxsakievirusandadenovirusreceptor(CAR),thatwascharacterizedwasforadenovirustype2and5in1997(Bergelson,Cunninghametal.,1997).8 中国农业科学院博士学位论文ChapterⅠTheprocessofvirusentryviaendocytosisiscomplex,involvinghundredsofcellularproteins.Manyvirusesgainentryintotargetcellsbytriggeringtheinitiationofanumberofendocyticpathways,includingclathrin-mediatedendocytosis(Spoden,Freitagetal.,2008),EDSVentrytocellisviareceptor-mediatedendocytosis.FollowingpartiallyuncoatedcapsidistransportedtothenuclearsurfacewheretheDNAentersthenucleus.TranscriptionofmRNAsoccursintemporalphases,usinghostRNApolymeraseII(Mishoe,Bradyetal.,1984).Proteinscanbeproducedafterinfectionbymodifyingthehostcellenvironment,andprovideessentialDNAreplicationfunctions,includingDNApolymerase.Oncereplicationhasbegun,thelateproteinsareproduced,andthenthenewvirusparticlesareassembled.Thedetailedunderstandingofadenovirusreplicationisbeingusedtodevelopthisvirusasagenedeliveryvector(Harrachetal.,2008,Hess,Blöckeretal.,1997,Leppardetal.,2008).Moreover,astudyreportedthattheEDSVentersduckembryofibroblastcellsthroughclathrin-mediatedendocytosisfollowedbyapH-dependentstep,whichissimilartothemechanismofentryofhumanadenovirustypes2and5(Huang,Danetal.,2015).Although,clathrin-mediatedendocytosisofadenovirusisthemostcharacterizedentrypathway,macropinocytosisentryhasbeendescribedaswell.MacropinocytosisleadstovacuoleformationatthecellularperipheryandhasbeenreportedforspeciesC(type2and5)(Wang,Huangetal.,1998).ThereisnoreportaboutEDSVreceptorsorvirus-hostcellbindingpartners.9 中国农业科学院博士学位论文ChapterⅠFigure1.4Adenovirusattachment,endocytosisandtransporttonucleus.Figureadoptedfrom(Coughlan,Albaetal.,2010).1.3.3PathogenesisWhentheEDSVaffectedtothebirdsespeciallybreedingflocks,thesymptomscanbeseenwithcolorchangesoneggshells,laterthin-shelledorshell-lesseggsoccur.Furthermore,eggswithroughandsandpaperlikesurfaceareproduced.Therateofabnormaleggshellscanamountupto20%,andoftenadecreaseinlayingperformanceupto50%appears.Individualsnormallydonotshowmanysymptoms(Mcferran,Mccrackenetal.,1978,VanEck,Davelaaretal.,1976).Inothercases,respiratorydiseaseswere10 中国农业科学院博士学位论文ChapterⅠreportedfromgoslingsindifferentcountries(Cha,Kangetal.,2013,Ivanics,Palyaetal.,2001).Afterexperimentaloralinfectionofchickens,EDSVreproducesinthenasalmucosaandthenspreadsthroughviremiaintothelymphoidtissue,followedbyaffectionoftheoviduct(McConnellAdair&Smyth,2008).EDSVsderivedfromduckshavevariouspathogenicitiesinlayinghens.Keyaminoacidcandidatesmighthavealteredtheiraffinitytotropismoflayinghens,causingdifferencepathogenicities(Min,Chaetal.,2017).Adenovirus-likeparticlesweredetectedbyelectronmicroscopy,whenanoutbreakofsevereacuterespiratorydiseasecharacterisedbytracheitisandbronchitisinyounggoslingsonalarge-scalegoosefarminHungary.Amphophilicintranuclearinclusionbodiesinthesuperficialepithelialcellsofthetracheaandbronchirevealedfromhistologicalexaminations.Thevirusisolatedfromthetracheaandthelungswasidentifiedaseggdropsyndromevirusbyserologicalandgenomicexamination.Theclinicalandpathologicalsignswerereproducedbyintratrachealadministrationofthevirusisolatetoday-oldgoslings,freeofEDSVantibodies.TheEDSVcaninvolveinsevererespiratorydiseaseofgeese(Eva,Vilmosetal.,2002,Ivanics,Palyaetal.,2001).1.3.4TransmissionofEDSVEDSVcanbetransmittedbyverticallyandhorizontally.Verticaltransmissionfromhentoeggisthemostimportantsourceofinfection.ThesporadicformofEDSoutbreaksresultsfromwatercontaminatedbyduck,gooseorwildbirdfaeces(Mcferran,Mccrackenetal.,1978).Horizontaltransmissionspreadviafaeceswithintheflockandbetweenflocksduetocontaminatedeggsoccuranendemictypeofthedisease.Viralcolonizationoftheuterineglandsleadstodysfunctionofeggshellgeneration(Ecketal.,1980,Mcferran,Mccrackenetal.,1978).TheinfectedeggmaybeimportantinthelateralspreadofEDS11 中国农业科学院博士学位论文ChapterⅠvirus,andthatattentionshouldbegiventotheriskfromre-usedeggtrays(SmythandAdairetal.,1988).EDSValsocanbetransmittedtonewhatchedgoslings.In2001,theinfectiousagentresponsibleforEDSwasconfirmedtobetheEDSVviaviralisolation,serologicalandgenomicexaminations,andexperimentalinfectionof1-day-oldgoslings(Ivanics,Palyaetal.,2001).In2007,anEDSoutbreakoccurredinturkeybreederflocksresultinginsignificantdecreaseinoveralleggproduction(BidinandLojkicetal.,2007).ArecentstudyreportedthattheEDSVcancausesevereacuterespiratorysymptomsin10-day-oldpekingducklings(Cha,Kangetal.,2013).1.3.5DiagnosisofinfectionEDSVcanbeisolatedinembryonatedduckorgooseeggs,andincellcultures.Susceptiblecelllinesincludeduckandchickembryoliver,duckkidney,andfibroblastcells(Gutter,Fingerutetal.,2008,Mcferran,Mccrackenetal.,1978).Asimple,user-friendly,andrapidmethodtodetectthepresenceofantibodiestoeggdropsyndrome76(EDS-76)virusinchickenserabasedonanimmunofiltration(flow-through)testwasdeveloped(Raj,Thiagarajanetal.,2007).Viralantigenscanbedetectedwithserologictestsincludehemagglutinationinhibitionusingfowlredbloodcells(RBC),enzyme-linkedimmunosorbentassay(ELISA),serumneutralizationandpolymerasechainreaction(PCR)(Banani,Sehatetal.,2007,Begum,Chowdhuryetal.,2013,Raj,Thiagarajanetal.,2007).TovalidatedetectingEDSantibodies,adiagnostickit(ELI-EDS)wasdevelopedbasedontheindirectELISAtechnique,usingasingledilutionofseratobetested(Botuş,Popaetal.,2009).EDSVgenewasfirstamplifiedbyhexongene-basedPCRcoupledwithrestrictionenzyme(RE)analysisofthePCRproducthasenableddifferentiationoffowladenovirusesfromEDSvirus(RaueandHessetal.,1998).12 中国农业科学院博士学位论文ChapterⅠAsasensitivetestingtool,afluorescentrealtimequantitativePCR(FQRTPCR)assayforrapiddetectionofeggdropsyndromeviruswasestablishedwithseveraladvantagesoverconventionalPCRmethodsincludingalowerriskofcontamination,thepossibilityofeasierquantificationandsavingoftime(Zhenyuan,Wenchaoetal.,2012).Anewreal-timepolymerasechainreaction(RT-PCR)assaytodetectduckadenovirusADNAwasdevelopedforapplicationtosamplesfromSwisspoultryflocks(Schybli,Sigristetal.,2014).1.3.6PreventionandcontrolThepreventioncanbecarriedoutbydisinfectingeggtraysandequipmentineachfarmhelptocontroltheendemicform.Alsoseparatingchickensfromotherbirds,andpotentiallyfromcontaminatedwater.Inactivatedvaccinesalso,successfullypreventandcontrolthedisease.Thebasicbreedingstock,andmanyflocks,manycommercialeggproducersroutinelyvaccinateflocksusingacommercialinactivatedvaccinewhichisveryeffectiveincontrollingdiseasewhenadministeredcorrectly(Guptaetal.,2007).Recombinanteggdropsyndromesubunitvaccineoffersanalternativetoviruspropagationinduckeggs.Astudyhasshownthattwovaccineswiththerecombinantfiberknobsproteinelicitedhighneutralizingantibodytitersthatpersistedformorethan50weeksoflay(Gutter,Fingerutetal.,2008).TheimmunizationoflayinghenshadnoapparentadverseeffectsoneggproductionandeffectivelypreventedclinicalsymptomsofEDSwhenthechickenswerechallengedwithpathogenicEDSV.ThisstudyhasdemonstratedthattheknobdomainlackingtheshaftsequencebutfusedtoatrimericcoiledcoilisapromisingcandidatesubunitvaccinefortheprophylaxisofEDSinchickens(Harakuni,Andohetal.,2016).13 中国农业科学院博士学位论文ChapterⅠ1.4AdenovirusvectorsAdenovirusescaninfectawiderangeofcelltypesandtissuesinbothdividingandnon-dividingcells.Thischaracteristic,togetherwiththeirrelativeeaseofpreparationandpurification,hasledtotheirextensiveuseasgenevectors.Adenoviralvectorsarewidelyemployedagainstinfectiousdiseasesorcancers.Vectorscanbeutilizedfor:(i)cancertherapytodelivergenesthatwillleadtotumorsuppressionandelimination;(ii)genetherapy,i.e.todelivergenestotissuestoaugmentdefectivegenes;(iii)supplementarytherapytodelivergenes,expressionofwhichwillcombatdiseaseprocesses(Russelletal.,2009).Adenovirusvectorscanbeusedastheexpressionvectorsaswell,inresearchfieldtoexpresstargetgenesinmammaliancelllines.Forthepreventionandcontrolofemerginginfectiousdiseasesorcancerdiseases,todevelopmentthebiologicalcharacteristicsofadenovirus(Ad)areimportantforlatestdevelopmentofAdvector-basedstrategies(ChaoandZhouetal.,2016).High-throughputapproachtoidentifyproteinsinvolvedinfunctionaladenovirushostinteractionsinvitroandinvivo.TheabilityoftheseproteinstobindtoAdcanbeconfirmedusinginvitroproteinbindingassaysaswellasinfectioncompetitionassays.Theapproachofusingastructuralproteinisuniversallyappliedforavarietyofviralandnonviralpathogensandcanrevealhostcellfactorscriticalinviralinfection,immuneevasion,andtissuespecificity.Thisinformationisalsoaprerequisitetoassessinvivosafetyandefficacyofadenovirusbasedgenetransfervectors(Gaggar,Shayakhmetovetal.,2005).1.5Protein-proteininteractionInthelate19thandthe20thcenturies,thestudyofproteininteractionsstartedslowlyandevolvedconsiderably,togetherwithconceptualandtechnologicalprogressindifferent14 中国农业科学院博士学位论文ChapterⅠareasofresearch(BraunandGingrasetal.,2012),Tostudytheprotein–proteininteractions(PPIs),numberofmethodshavebeendevelopedovertheyears.Thetypicalprotein–proteininteractionstudyusuallystartswithaninitialscreenfornovelbindingpartners(Berggard,Linseetal.,2007).Protein-proteininteractionshandleawiderangeofbiologicalprocesses,includingcell-to-cellinteractionsandmetabolicanddevelopmentalcontrol.NowPPIisbecomingoneofthemajorobjectivesofsystembiology.(BraunandGingrasetal.,2012,OfranandRostetal.,2003).Theanalysisofprotein-proteininteractionsisfundamentaltotheunderstandingofcellularorganization,processes,andfunctions.Recentlarge-scaleinvestigationsofprotein-proteininteractionsusingsuchtechniquesastwo-hybridsystems,massspectrometry,andproteinmicroarrayshaveenrichedtheavailableproteininteractiondataandfacilitatedtheconstructionofintegratedprotein-proteininteractionnetworks(Berggard,Linseetal.,2007,Zhangetal.,2009).Protein-proteininteractiondetectionmethodsarecategoricallyclassifiedintothreetypes:invitro,invivo,andinsilicomethods.Ininvitrotechniques,isperformedinacontrolledenvironmentoutsidealivingorganism.TheinvitromethodsinPPIdetectionaretandemaffinitypurification,affinitychromatography,co-immunoprecipitation,proteinarrays,proteinfragmentcomplementation,phagedisplay,X-raycrystallography,andNMRspectroscopy.Ininvivotechniques,agivenprocedureisperformedonthewholelivingorganismitself(Rao,Srinivasetal.,2014).TheinvivomethodsinPPIdetectionareyeasttwo-hybrid(Y2H,Y3H)andsyntheticlethality.Insilicotechniquesareperformedonacomputerorviacomputersimulation.TheinsilicomethodsinPPIdetectionaresequence-basedapproaches,structure-basedapproaches,chromosomeproximity,genefusion,insilico2hybrid,mirrortree,phylogenetictree,andgeneexpression-based15 中国农业科学院博士学位论文ChapterⅠapproaches(Rao,Srinivasetal.,2014).ThecompleteanalysisofPPIscanenablebetterunderstandingofcellularorganization,processes,andfunctions.TheotherapplicationsofPPInetworkincludebiologicalindispensabilityanalysisassessingthedrugabilityofmoleculartargetsfromnetworktopology(Zhangetal.,2009),estimationofinteractionsreliability(Bader,Chaudhurietal.,2004),predictdomain-domaininteractionsfromaprotein-proteininteractionnetwork,predictionofproteininteractions,detectionofproteinsinvolvedindiseasepathways(Guimarães,Jothietal.,2006,Rhodes,Tomlinsetal.,2005).1.5.1Yeast-TwohybridsystemBuddingyeast(Saccharomycescerevisiae)isapopularmodelorganismforvirusresearch.Yeastsaresinglecelleukaryoticorganisms.Manyofthefundamentalcellularfunctionssuchascellcycleregulationorprogramedcelldeatharehighlyconservedfromyeaststohighereukaryotes(ZhaoandZhaoetal.,2017).Analysesofinteractionofviralproteinswithcellularproteinshavenotonlyhelpedindefiningtheroleofcellularproteinsinvirusreplicationcyclebutalsotheirroleincellularprocesses.Viralinfectionsaremediatedbyseveralprotein-proteininteractions,whichcanberepresentedasnetworks.Apartfromgainingandlosinginteractionsviarewiringmechanisms,virus-hostproteinnetworksalsoevolveviageneduplication,conservation,horizontalgenetransferandmolecularmimicry(BritoandPinneyetal.,2017).AlsotheapplicationsofY2Harebroad,entailingfieldssuchasdrugdiscovery,clinicaltrialsforhumandiseaseincludingcancerandneurodegenerativedisease,andextendevenintosyntheticbiologyapplicationsandcellularengineering(LopezandMukhtaretal.,2017).NumberofY2Hstudieswassuccessfullyemployedtoidentifybacterial,viral16 中国农业科学院博士学位论文ChapterⅠproteininteractionswiththeirhostinhuman,animalandplantdisease(JanikandSchlinketal.,2017,Levin,Ballesteretal.,2017,Zhang,Panetal.,2017).1.5.1.1MatchmakerGAL4Two-hybridsystemTheMatchmakerGAL4twohybridsystemsareforidentifyingandinvestigatingprotein-proteininteractionsinvivousingtheyeasttwohybridassays.Today,theidentificationofproteinsthatinteractwithaproteinofinterestisafocusofintensiveresearchandisanessentialelementoftherapidlygrowingfieldofproteomics.Yeasttwo-hybridassayisatooltostudyproteininteractionsinvivo.GAL4-basedassay,forexample,usesyeasttranscriptionfactorGAL4fordetectionofproteininteractionsbytranscriptionalactivation.Sometranscriptionfactors(suchasGAL4)possessacharacteristicphenomenonthatthetransactivationfunctioncanberestoredwhenthefactor'sDNA-bindingdomain(DBD)anditstranscription-activationdomain(AD)arebroughttogetherbytwointeracting,heterologousproteins(FieldsandSongetal.,1989).TheGAL4-yeasttwo-hybridassayusestwoexpressionvectors,oneusesGAL4-DBDandtheotherusesGAL4-AD(Fig.1.5).DNAsequencesencodingthetwoproteinsofinterest(oraproteinandacomplementaryDNAlibrary)canbeclonedintheGAL4-DBDandGAL4-ADvectorstoformthebaitandthetargetoftheinteractiontrap,respectively(Osmanetal.,2004).IntheMatchmakergoldyeasttwohybridsystem(Clontech,Ca),(Cat.No.630489)whenthebaitfusionandlibrary(prey)proteinsareinteractedinyeastcell,theDNA-BDandADsareactivatedthereportergenes(AUR1-C,ADE2,andHIS3andMEL1)17 中国农业科学院博士学位论文ChapterⅠFigure1.5Thetwo-hybridprinciple.Twoproteinsareexpressedseparately,one(abaitprotein)fusedtotheGal4DNA-bindingdomain(BD)andtheother(apreyprotein)fusedtotheGal4transciptionalactivationdomain(AD).InY2HGoldYeastStrain,activationofthereporters(AUR1-C,ADE2,HIS3,andMEL1)onlyoccursinacellthatcontainsproteinswhichinteractandbindtotheGal4-responsivepromoter(ClontechLaboratories,Inc.www.clontech.com).1.5.1.2ReportergenesandthreedifferentbindingsitesTherearefewintegratedreportergenesunderthecontrolofthreedistinctGal4-responsivepromoters(Figure1.6)inClontech’sY2HGoldYeastStrain,whichareusedtodetecttwo-hybridinteractions.Y2HGoldisunabletosynthesizeandgrowonmedialackofhistidine(HIS3),adenine(ADE2)essentialaminoacids.Whenbaitandpreyproteinsinteract,Gal4-responsiveHis3expressionspermitsthecelltobiosynthesizehistidineandAde2expressionisactivationallowtocellsgrowonselectivemediawithouttheseaminoacids.MEL-1encodesa-galactosidase,anenzymeoccurringnaturallyinmanyyeaststrains.Asaresultoftwo-hybridinteractions,a-galactosidase(MEL1)isexpressedandsecretedbytheyeastcells.YeastcoloniesthatexpressMel1turnblueinthepresenceof18 中国农业科学院博士学位论文ChapterⅠthechromagenicsubstrateX-a-Gal.ThreepromoterscontrollingthefourreportergenesAUR1-C,HIS3,ADE2,andMEL1inY2HGoldareunrelatedexceptfortheshortproteinbindingsitesintheUASregionthatarespecificallyboundbytheGal4DNA-BD.Thus,libraryproteinsthatinteractwithunrelatedsequencesflankingorwithintheUAS(i.e.,falsepositives)areautomaticallyscreenedout.Figure1.6ReportergeneconstructsinMatchmakeryeaststrains.InY2HGold,theHIS3,ADE2,andMEL1/AUR1-CreportergenesareunderthecontrolofthreecompletelyheterologousGal4-responsivepromoterelements-G1,G2,andM1,respectively.Theprotein-bindingsiteswithinthepromotersaredifferent,althougheachisrelatedtothe17-merconsensussequencerecognizedbyGal4.1.5.2GST-pulldownassayTheGlutathione-S-transferase(GST)fusionproteinshavehadawiderangeofapplicationsfortheidentificationofprotein-proteininteractions.Thepull-downmethodisfundamentallysimilartoimmunoprecipitation.TheGSTpull-downisanaffinitycaptureof19 中国农业科学院博士学位论文ChapterⅠoneormoreproteins(eitherdefinedorunknown)insolutionbyitsinteractionwiththeGSTfusionprobeproteinandsubsequentisolationofthecomplexbycollectionoftheinteractingproteinsthroughthebindingofGSTtoglutathione-coupledbeads(Einarson,Pugachevaetal.,2007).Aproteincanbecovalentlycoupledtoamatrixsuchassepharoseundercontrolledconditionsandusedtoselectligandproteinsthatbindandareretainedfromanappropriateextract.Mostproteinspassthroughsuchcolumnsorarereadilywashedoffunderlow-saltconditions.Proteinsthatareretainedcanthenbeelutedbyhigh-saltsolutions,cofactors,chaotropicsolvents(Rao,Srinivasetal.,2014,Ratneretal.,1974).ThemostcommonsuchfusioncontainsglutathioneS-transferase(GST),whichcanbepurifiedonglutathione-agarosecolumns(SmithandJohnsonetal.,1988).Theproteinscanbefractionatedbypolyacrylamidegelelectrophoresis(PAGE)transferredtoanitrocellulosemembrane,andidentifiedbytheirabilitytobindaproteinandcanbeanalyzedwithoutanypurification.Awidevarietyofplasmidvectorsarecommerciallyavailablefortheproductionoffusionproteinsinbacterialcells.MostarealsodesignedtoincorporateatagthatallowsaffinitypurificationoftheexpressedfusionproteinfrombacterialcellextractssuchaspGEXexpressionsystems(Jin,Yuanetal.,2017).ThemostcommonlyusedvectorsareincorporateaportionoftheGSTenzymethatisabletobindtoimmobilizedglutathione(RebayandFehonetal.,2009).MostoftheGSTpulldownassaywereusedfortovalidatetheproteininteractionwhichpreviouslyidentifiedwithY2Hsystem.20 中国农业科学院博士学位论文ChapterⅠ1.6ObjectivesTheaimsofthisstudyweretodetecttheEDSVbyusingreal-timefluorescenceloop-mediatedisothermalamplificationassay(RealAmp),andtofindouttheeggdropsyndromevirusreceptorsoridentifytheinteractionpartnersofpentonproteinfromhostcellproteins.ThepresentstudywasthereforedesignedtobetterdefinetheinteractionofEDSVwithspecificproteinsofduckfibroblastcellsinvitroandinvivo.Withintheseaims,thefollowingobjectiveshavebeenundertaken:1.ToutilizetheRealAmpfordirectdetectionofeggdropsyndromevirus2.ToidentifytheduckhostproteinswhichhaveinteractionwithpentonproteinofEDSVbyY2Hscreening3.ToconfirminteractedproteinsofduckandEDSVpentonusingGSTpull-downassay21 中国农业科学院博士学位论文ChapterIICHAPTERIIReal-timefluorescenceloop-mediatedisothermalamplificationassayfordirectdetectionofeggdropsyndromevirus2.1IntroductionIndiagnosisofEDSV,severalserologicalandmolecularmethodsusedforEDSVdetectionviralantigensorDNA(Adair,Toddetal.,1986).Thediagnosisofavianadenoviruseshasmainlybeendoneatamolecularlevel.SeveralPCRstudieshavebeenpublished,fordiagnosisofallavianadenovirusesthatarerelevanceforthepoultrypractice(Hessetal.,2000,Raj,Sivakumaretal.,2001).Forexample,nucleicacidbasedtechnologies,suchasinvitroamplificationofDNAbythePCRmethod,havebeenusedcommonlytodemonstrateinfectioncausedbytheEDSV(Begum,Chowdhuryetal.,2013).Real-timeloop-mediatedisothermalamplificationisaDNAamplificationmethodwithsomeadvantagesascomparisonwithPCRmethods,includingimprovedsensitivityandspecificity,aswellastimeefficiency(Nagamine,Haseetal.,2002).Loop-mediatedisothermalamplification(LAMP)isamethodwhichcanamplifyDNAunderisothermalconditions.ItwasfirstdevelopedbytheJapaneseresearchers,theLAMPemploysaDNApolymeraseandasetoffourspeciallydesignedprimersthatrecognizeatotalofsixdistinctsequencesonthetargetDNA(Notomi,Okayamaetal.,2000).Later,LAMPwassupplementedbyusingadditionalprimers,termedloopprimerswhichprimestranddisplacementDNAsynthesis.Moreover,LAMPhassomeadvantagesascomparisonwithPCRmethods,includingimprovedsensitivityandspecificity,aswellastimeefficiency(Nagamine,Haseetal.,2002).SinceLAMPwaspublished,arangeofLAMPmethodshavebeendeveloped,real-timefluorescenceloop-mediatedisothermalamplification(RealAmp)isoneofthem,whichattemptedtoimprovethemethodfor22 中国农业科学院博士学位论文ChapterIIdiagnosisbyusingasimpleandportabledevicecapableofperformingboththeamplificationanddetectionbyfluorescenceinoneplatform(Lucchi,Demasetal.,2010).Currently,severaldifferentLAMPandRealAmpmethodshavebeenutilizedtodetectbacterialandviralinfectiousdiseasesandagentsinhumans,animalsandplants,includingavianinfluenza(Imai,Aietal.,2006),middleeastrespiratorysyndrome(Bhadra,Jiangetal.,2015),Mycoplasmabovis(Yumiko,Ryokoetal.,2016),Malaria(Lucchi,Demasetal.,2010,Ocker,Prompunjaietal.,2016),Plasmodiumspp.Parasites(Lucchi,Ljoljeetal.,2016),ShigellaandSalmonella(Yi,Yanetal.,2015)andFusariumoxysporumf.sp.cubense(Li,Benjinetal.,2013).VariousLAMPmethodshavebeensuccessfullyemployedforDNAamplificationusingDNAtemplatesextractedfromthesamples.ThepurposeofthiscurrentstudywastouseRealAmpmethodforrapiddetectionoftheEDSVindiversesamplesavailable,withoutpriorneedofnucleicacidextraction.Therefore,weinfectedbothduckembryosandduckfibroblastcellculturewithEDSV,thentheviralsampleswerecollectedandemployedtotheassaydirectlybyserialdilutions.2.2MaterialsandMethod2.2.1ChemicalsandreagentsEnzymesincludingBst2.0DNApolymeraseandBsrGI-HFwereobtainedfromNewEnglandBiolabs(NEB,USA).PrimersforRealAmpandPCR(oligonucleotides)wereobtainedfromHuada(Beijing,China)andsuspendedindeionizedwaterwithfinalconcentrationof10μMandstoredat–20°C.TheconcentrationsofeachDNAsuspensionusedinthisstudyweremeasuredbyNanoVuePlusspectrophotometer(GEHealthcare,USA).23 中国农业科学院博士学位论文ChapterII2.2.2VirusesFowlpoxvirus(FPV),duckviralenteritisvirus(DVEV),duckcircovirus(DCV)orMarek’sdiseasevirus(MDV)usedinthisstudywerekeptintissueculturesupernatant,at–80°C.2.2.3InoculationofembryonatedduckeggswiththeEDSVForvirusinoculation9-day-oldembryonatedduckeggswereobtainedfromBeijingDayinghongguangDuckFarm,andincubatedfor2daysinanincubator(DHP-9052).EDSVusedinthisexperimentwasisolatedin1992(Li,Zhengetal.,1994),andkeptintheAnimalDiseaseControlLaboratoryofIAS,CAAS.Theviruswasdilutedwithsterile1×PBSataratioof1/100,followedbyinoculationoftheallantoicsacofembryonatedduckeggswiththeviraldilution(200μl).Theeggswereincubatedat37°Candexaminedthtwiceeachday.Afterthe6dayofincubation,theeggswerechilledat4°Cfor4h,andthentheallantoicfluidwascollectedfromeachembryoandstoredat–80°C.Thehaemagglutination(HA)titerofEDSVincollectedallantoicfluidwaslog212.2.2.4CellcultureandvirusinoculationDuckfibroblastcellcultureswerepreparedfrom11-day-oldduckembryo.TheculturemediumcontainsDulbecco’smodifiedEagle’smedium(DMEM,Gibco,Shanghai,China)supplementedwith10%fetalbovineserum(FBS,Gibco,USA)and1%gentamycin2(Sigma-Aldrich).Duckembryofibroblastcellsgrownin75cmflaskswereinoculatedwith1mLofdiluted(1:100)EDSVcollectedfromallantoicfluid(HAlog212),followedbyincubationat37°Cwith5%CO2supplyfor1h.Theviralinoculumwasremovedfromthecelllayer,andreplacedwith10mLoffreshDMEMsupplementedwith2%FBSand1%24 中国农业科学院博士学位论文ChapterIIgentamycin,followedbyincubationfor48h.PBSwasusedasanegativecontrol,withoutviralinoculumsinthecontrollabeledflask.Thecellswereexamineddailyforanycytopathiceffects(CPE).Infectedcellculturefluidwasharvestedafter46hofincubationandthenusedforRealAmpanalysis.TheHAtiterofEDSVinharvestedcellculturesupernatantwaslog29.2.2.5ViralDNAextractionViralDNAwasisolatedfromtheallantoicfluidcollectedfrominfectedduckembryosandthesupernatantofduckembryofibroblastsculturedcells.TotalnucleicacidswereTMextractedusingtheAxyPrepBodyFluidviralDNA/RNAMiniprepKit(Axygen,USA)accordingtothemanufacturer’sinstructionsandstoredat–20°C.2.2.6DesignofprimersfortheRealAmpandPCRSixspecificRealAmpprimers(F3,B3,FIP,BIP,LFandLB)weredesignedusingPrimerExplorerV5software(http://primerexplorer.jp/lampv5e/index.html.EikenChemicalCo.Ltd.,Tokyo,Japan)basedonthesequenceoffibergeneoftheEDSV(GenBankaccessionNo.CAA70814.1).PCRprimersweredesignedusingOligo7PrimerAnalysissoftware(MolecularBiologyInsights,Inc.USA).2.2.7RealAmpassayRealAmpreactionswereperformedusingEDSVDNA,allantoicfluidandcellculture–1–6supernatant.TheviralDNAwasdilutedfrom10to10priortouse.TheallantoicfluidandcellculturesupernatantswereuseddirectlytothereactionwithoutviralDNA–1–5extractionbydilutingthesamples(10to10).The25μlofreactionincludes1μloftemplate(DNAandvirusliquids),2.5μlof10Xisothermalbuffer(BioLabs,USA;20mM25 中国农业科学院博士学位论文ChapterIITris–HCl,50mMKCl,10mM(NH4)2SO4,2mMMgSO4,0.1%Tween20,pH8.8),1μlofdNTPs(10mM),4μlofbetaine(1M),1μlofFIP(40μM)andBIP(40μM),1μlofF3(10μM)andB3(10μM),1μlofLF(20μM)andLB(20μM),1μlofBst2.0DNApolymerase(8000units),9μlofddH2Oand0.5μlofEvaGreen(10×)(Invitrogen,Carlsbad,CA).Reactionswerecarriedoutat65°Cfor45–50min.ThegraphoffluorescenceunitsandtimewasplottedusinganESE-QuantTubeScanner(Qiagen,Germany).Thegraphshowsthefluorescenceinmillivolts(mV)onthey-axisandtimeinminutesonthex-axis.ResultscanbereadinrealtimeusingTubeScannerStudiosoftware(http://tube-scanner-studio.software.informer.com/2.7/).2.2.8SpecificityandsensitivityoftheRealAmpassayAdditionalDNAviruses(FPV,DVEV,DCVandMDV)wereassessedforspecificityoftheRealAmp.TovalidatetheexpectedEDSVtargetedRealAmpamplicon,2μloftheRealAmpproductwasdigestedwith1μlofrestrictionenzymeBsrGI-HF,2.5μlofCut-Smart®buffer,and19.5μlofddH2O.Thereactionmixturewasincubatedat37°Cfor2handseparatedbyelectrophoresisina1.5%agarosegel.–1–6SensitivityoftheRealAmpassaywastestedusing10-foldserialdilutions(10to10)offiber-pMD19TplasmidDNA(26ng/μl)followedbyconventionalPCRmethod.Thesensitivitywascalculatedusingthefollowingformulae:23-9(6.02×10)×(ng/μl×10)/(DNAlength×660)=copies/μlInformulae:ng/μl-concentrationoffinaldilutedDNAtemplateDNAlength-totallengthofDNAusedinassay(containstargetsequence)2.2.9ConventionalPCRTwosetsofPCRreactionswereperformedforsensitivityoftheassay.Onesetof26 中国农业科学院博士学位论文ChapterIIreactionusedtheRealAmpouterprimers(F3andB3),whiletheothersetofreactionusedthefiberPCRprimers(Table2.1).EachPCRreactionwasconductedwithatotalreactionvolumeof25μl,whichcontain12.5μlof2×EasyTaqPCRMix(Transgene;0.2mMofeachdNTP,1.5mMMgCl2),1μloftheforwardprimer(10μM),1μlofthereverseprimer(10μM),9.5μlofddH2Oand1μloftemplate.ThePCRprogramconsistedofaninitialdenaturationstepat94°Cfor4min,followedby30cyclesofdenaturationat94°Cfor30s,primerannealingat58°Cfor30s,extensionat72°Cfor2min(fibergeneprimers),30sfor(F3andB3primers)andafinalextensionstepat72°Cfor10min.PCRproductswereanalyzedbyelectrophoresison1.0%agarosegelandphotographedunderUVlight.2.3ResultsPrimersweredesignedusingthefulllengthsequenceoffibergene.TheRealAmpandPCRprimerswereshowninTable2.1andlocationofRealAmpsequencesinfibergeneisshowninFig2.1.Table2.1RealAmpandPCRprimers.MethodPrimerLength(bp)Sequence(5’-3’)fiberF320AAAGGTTGCAGGGTATGTGTfiberB318TAATGGCATTGGCCGCAAfiberFIP42TTCCCCCCGTAAACCAATACCCGTTGGTGGRealAmpGCTTGTACATGGfiberBIP42TGTCCTTTTAGTGCTCGCGACCTCACCACTCCACACTACTGGfiberLF25CGCAGTAGCTTTAATCTGAATGGTCfiberLB20CCACTGCTAACCTGTCAGGCPCRFiber-F20ATGAAGCGACTACGGTTGGAFiber-R26CTACTGTGCTCCAACATATGTAAAGNote:FdenotesPCRforwardprimerandRdenotesreverseprimer27 中国农业科学院博士学位论文ChapterIIFigure2.1LocationofRealAmpprimersinfibergene2.3.1RealAmpofEDSVDNAInthisassay,theEDSVDNAwassuccessfullyamplifiedfromdilutedviralDNAsamplesextractedfrominfectedallantoicfluidandcellculturesupernatantswithin45min(Fig.2.2aandb).Noamplificationwasobtainedfromnon-infectedcontrolsamples.Ladder-likeDNAbandswereobtainedfromRealAmpproductsseparatedina1.5%agarosegel(Fig.2.2candd).28 中国农业科学院博士学位论文ChapterIIFigure2.2RealAmpusingviralDNAisolatedfrominfectedallantoicfluidandcellsuspension.(a)RealAmpofviralDNAextractedfromallantoicfluid;(b)RealAmpofviralDNAfrominfectedduckfibroblastcellculturesupernatantusedwithserialdilutions.For–1–6bothreactions,tube1–6DNAsampledilutions(10to10);tube7forpositive(EDSVDNA)andtube8negative(non-infected)controls.(c)and(d)1.5%agarosegel–1–6electrophoresisresultsof(a)and(b),lane1–6,sampledilutions(10to10);lane7positivecontrol;lane8negativecontrol;laneM-Trans2KplusDNAmarker.2.3.2DirectRealAmpassayByusinginfectedallantoicfluidandcellculturesupernatants,aswellasundilutedsamplesdirectlytotheassay,wesuccessfullyamplifiedEDSVDNA.Analysisofeachsamplewascarriedoutthreetimesindependently.TheresultsobtainedweresimilartothoseobtainedbyusingEDSVnucleicacids(Fig.2.2).Agraphoffluorescenceunitsandtimewasproducedforallsampledilutions.Fortheallantoicfluidsamples,alldilutions29 中国农业科学院博士学位论文ChapterII–1–5from10to10showednormalamplificationbeganafter15minofincubationat65°C.However,amplificationoftheundilutedsamplebeganafter40min(Fig.2.3a).Serial–1–4dilutedcellculturesupernatants(10to10)andundilutedsampleprovidedresultsafter–520–30minofscanningwhereasthe10dilutionandnon-infected(asanegativecontrol)samplesprovidednoamplification(Fig.2.3b).TheRealAmpproductsfrombothsampleswereseparatedin1.5%agarosegel(Figure2.3candd).Figure2.3DirectRealAmpforallantoicfluidandcellculturetesting.(a)Infectedallantoicfluid;(b)cellculturesupernatantwithserial10-folddilutions,tube1–5weresample–1–5dilutions(10to10);tube6undilutedsample;tube7positive(EDSVDNA)andtube8negativecontrol.(c)and(d)1.5%Agarosegelelectrophoresisresultsof(a)and(b),lanes–1–51–5,sampledilutions(10to10);lane6undilutedsamples;lane7positiveandlane8negativecontrols.M-Trans2KplusDNAmarker.30 中国农业科学院博士学位论文ChapterII2.3.3SpecificityoftheRealAmpmethodWeusedseveralpoultrydiseaseviruses(FPV,DVEV,DCVandMDV)forspecificity.TheresultsshowedthattheRealAmpcouldamplifyanddifferentiateEDSVgenewithinotherDNAviruses(Fig.2.4aandb).TheamplifiedproductwasdigestedwithBsrGI-HF,whichresultsinthedigestionoftargetregionoutfromatotalamplifiedDNA.ThetargetRealAmpregionwascleavedbyspecificenzymewhichislocatedintheEDSVfibergene,thenseparatedona1.5%agarosegelandshownin(Fig.2.4c).Figure2.4Specificitytestofthereal-timefluorescenceloopmediatedisothermalamplificationassayforthedetectionofEDSV.(a)specificityofRealAmpamongdifferentvirusstrains;tube1negativecontrol(ddH2O);tube2FPV;tube3DVEV;tube4DCV;tube5MDV;tube6EDSVandtube7-positivecontrol(EDSVDNA).(b)1.5%agarosegelelectrophoresisofRealAmpproduct;lane1negativecontrol;lane2FPV;lane3DVEV;lane4DCV;lane5MDV;lane6EDSVandlane7positivecontrol.(c)Validationof31 中国农业科学院博士学位论文ChapterIIspecificity.Lane1digestedRealAmpproduct;lane2undigestedRealAmpproduct;M-Trans2KplusDNAmarker.M-Trans2KplusDNAmarker.2.3.4SensitivityoftheRealAmpassayThesensitivityoftheRealAmpassaywascomparedwithconventionalPCRandexaminedbyanalyzingtheproductsgeneratedfromserialdilutions(2.6ng,260pg,26pg,2.6pg,260fg,and26fgpermicroliter)offiber-pMD19TplasmidDNA.Thesensitivityof365theRealAmpwas5.9×10copies/μlwhileconventionalPCRwas5.9×10and5.9×10copies/μl.TheprocedurewasmonitoredusinganESE-QuantTubeScanner(Fig.2.5a).RealAmpproductswereassessedby1.5%agarosegelelectrophoresis(Fig.2.5b).PCRproductswereseparatedby1.0%agarosegelelectrophoresis(Fig.2.5candd).32 中国农业科学院博士学位论文ChapterIIFigure2.5SensitivityoftheRealAmpandPCR;(a)AmplifiedfiberDNAbyRealAmp–1–6–1–2–3(10to10);tube1-10(2.6ng/μl);tube2-10(260pg/μl);tube3-10(26pg/μl);tube–4–5–64-10(2.6pg/μl);tube5-10(260fg/μl);tube6-10(26fg/μl);tube7positive(EDSVDNA)andtube8negativecontrol(ddH2O).(b)1.5%agarosegelelectrophoresisresultofRealAmpamplicon.(c)and(d)DeterminationofthedetectionlimitofthePCRwithfibergenespecificPCRprimersandouterprimersF3andB3(Table2.1).BothPCRproducts–1–2wereseparatedin1.0%agarosegel.Lane1-10(2.6ng/μl);lane2-10(260pg/μl);lane–3–4–5–63-10(26pg/μl);lane4-10(2.6pg/μl);lane5-10(260fg/μl);lane6-10(26fg/μl);lane7positivecontrolandlane8negativecontrol.Lane-MTrans2KplusDNAmarker.33 中国农业科学院博士学位论文ChapterⅢCHAPTERIIIYeastTwo-HybridScreening3.1IntroductionTheinteractionsbetweenviruspathogensandhostsincludeexploitationofhostcellsforviralreplicationbyusingmanycellularresources.Thecellulareventsaremodulatedbyfunctionalinteractionsbetweenspecificproteins.Asingleviralproteincaninteractwithdifferentpartnersinthehostcell.Identifyingthenewinteractionsbetweenvirusesandtheirhostsishelpstocharacterizethevirusgenes,andtheirfunctions,andmolecularpathwaysinthecell.InformationofgenomeandpathogenicmechanismsuniquetoEDSVislimited.Furthermore,theidentificationofEDSVpentonproteininteractionswithunknownhostcellproteinsmaydemonstratethenewandunexpectedrolesofpentonproteinduringtheEDSVinfectionandreplicationcycle.Severaltechniquesareavailabletoanalyzeprotein–proteininteractions.Biochemicalapproaches,suchasco-immunoprecipitationandaffinitychromatographyfollowedbymassspectrometry,havebeenwidelyusedwithsuccess,althoughtheidentificationofinteractors.Theyeasttwo-hybridsystem(Y2H)isapowerfulmethodtoidentifybinaryprotein–proteininteractionsinvivo.Theprincipleoftheassayreliedonmajordiscoveriesontranscriptioninitiation(BrentandPtashneetal.,1985).ItwasfirstdescribedusingtheGAL4transcriptionfactorforyeastS.cerevisiea,(FieldsandSongetal.,1989).Thistechniqueallowsrapididentificationofseveralputativeinteractingproteinsforagivenproteinofinterest,enablingalsotheisolationofthecDNAassociatedwiththeinteractingpeptides(Chien,Barteletal.,1991)Inparticular,thetranscriptionfactorGAL4iscomposedoftwodomains,theDNA-bindingdomain(DBD)andactivationdomain(AD).Inthetwo-hybridsystem,aDNA-bindingdomainisfusedtoaproteingenerallycalledbait34 中国农业科学院博士学位论文ChapterⅢforwhichonewantstofindinteractingpartner.AtranscriptionalactivationdomainisthenfusedtooneormoreORFs(preys)(FieldsandSongetal.,1989).TheY2Hassayhasbeenusedforvirusanditshostprotein-proteininteractionsindifferentviruspathogens,suchasdetectingprotein-proteininteractionsinvesicularstomatitisvirususingacytoplasmicyeasttwohybridsystem(Moerdykschauwecker,Destephanisetal.,2011),alsoinhumanviraldiseases(Geng,Yangetal.,2013),Vacciniavirus-hostproteininteractions(Zhang,Villaetal.,2009)andgenome-scalescreensforintraviralandvirus-hostproteininteractions(BailerandHaasetal.,2009),TheY2Hassaycanbeutilizedforhigh-throughputscreeningofsmall-moleculeinhibitorsofPPIsandtoidentifydomainsthatstabilizePPIs.Inthischapter,thecDNAfrompurifiedduckfibroblastcellswasinsertedintopGADT7-SfiIvectorforconstructingtheyeasttwo-hybridcDNAlibrary.EDSVpentonwasclonedintothepGBKT7vector(pGBKT7:penton)andconsideredasbaitplasmidafterevaluatingtheexpression,auto-activationandtoxicitytestsintheyeaststrainY2HGold.Thepositivescreeningplasmidswerefurtherconfirmedviaco-transformingpositivebaitandpreyplasmidsintoyeaststrainY2HGold.SequencesofpositivepreyswereanalyzedusingBLAST,UniProtandGeneOntology.Theresultofthischapterwillprovideimportantinformationaboutpentongeneanditsunknownpartnersinthehostcell.3.2MaterialsandMethods3.2.1PreparationofduckembryosfibroblastcellcultureThefollowinginstrumentswerepreparedforduckfibroblastcellculturepreparation;2sterileforceps,scissors,sterilepetridishes,sterile75-cmflasks,10%FBS,DMEM,sterile0.25%trypsininsalineA,5-mland10-mlpipettes,1×PBS,gloves,filter,35 中国农业科学院博士学位论文ChapterⅢchloroform,isopropylalcohol,75%ethanol(inDEPC-treatedwater),RNase-freewaterpolypropylenemicrocentrifugetubes,waterbath(55–60°C).Inordertoprepareduckembryofibroblastcellculture,11day-oldduckembryoswereused.Surfaceoftheeggovertheairsacwasdisinfectedwith75%ethanol.Thentheshellovertheairsacwasremovedwithforceps.Usingthesterileforceps,theentireembryowasremovedfromtheeggtoasterilepetridish.Theembryowasdecapitated,evisceratedandmincedcarcassintoverysmallfragments.Thefragmentswerewashedthreetimeswith10mlofsterilePBS.Then10mlofsterilewarmtrypsinsolutionwasaddedtofragments,incubated5to10minutes.Thetrypsin-cellsuspensionwasaddedintoa15-mlcentrifugetubeandcentrifugedat2000rpmfor10minutes.Thesupernatantwasdiscardedandpooledcellpelletwassuspendedinatotalof5mlofDMEMandcentrifugedat2000rpmfor10minutes.50mlof10%FBSwasaddedintothepelletandthesuspensionwas2filtered.Then10mlofcellsuspensionwereaddedintoper75cmcellcultureflask,placedoat37CCO2for48hours.3.2.2TotalRNAextractionfromduckembryofibroblastcellcultureInordertototalRNAextraction,duckembryofibroblastcellcultureswerelysedusingappropriatevolumeofTRIzol®Reagentdependingonwelltype(100µlfor96-wellplateto500µlfor6-wellplate).Thenthecellswerehomogenizedfor5minutesatroomtemperaturetopermitcompletedissociationofthenucleoproteincomplex.Total0.2®mLofchloroformwasaddedtoper1mLofTRIzolReagent,thetubesweremixedvigorouslybyhandfor6-8times.Thesampleswereincubatedfor2–3minutesatroomtemperatureandcentrifugedat12,000×gfor15minutesat4°C.Theaqueousphaseofthesamplewasaddedintonew1.5mltubes.0.5mlof100%isopropanolwasaddedtothe®aqueousphase,forper1mLofTRIzolReagent.Sampleswereincubatedatroom36 中国农业科学院博士学位论文ChapterⅢtemperaturefor10minutesandccentrifugedat12,000×gfor10minutesat4°C.SupernatantswereremovedandtheRNApelletswerewashedwith1mLof75%ethanol®per1mLofTRIzolReagent.Thesampleswerevortexedbriefly,thencentrifugedat7500×gfor5minutesat4°C.TheRNApelletswereairdriedfor10minutesandthen,theRNApelletswereresuspendin40μlofRNase-freewaterandstoredat–80°Cuntiluse.3.2.3First-StrandcDNASynthesisSingle-strandcDNAsynthesiswascarriedoutusingSMARTcDNAsynthesiskit(Clonetech,Dalian,China).MouselivertotalRNAwasusedasacontrol.Thefollowingreagentswerecombinedinseparatemicrocentrifugetubes:2µlRNA1µl3’In-FusionSMARTERCDSPrimer(12μM)1.5µldeionizedH2O4.5µlTotalVolumeThemixturewasincubatedat72°Cinahot-lidthermalcyclerfor3minandthenthetemperaturewasreducedto42°Cfor2min.Duringthisincubationtime,mastermixwaspreparedbycombiningthefollowingreagentsintheordershown:2.0µl5×First-StrandBuffer0.25µlDTT(100mM)1.0µldNTPMix(10mM)1.0µlSMARTerVOligonucleotide(12μM)0.25µlRNaseInhibitor1.0µlSMARTScribeReverseTranscriptase5.5µlTotalVolumeaddedperreactionThecontentsofthetubes(4.5µl+5.5µl)weremixedbygentlypipetting,andspinthetubesbrieflytocollectthecontentsatthebottom.Thetubewasincubatedat42°Cfor90min.Heatedat68°Cfor10mintoterminatefirst-strandcDNAsynthesis.37 中国农业科学院博士学位论文ChapterⅢFurther,theamplifiedcDNAwaspurifiedusingTaKaRaMiniBESTDNAFragmentPurificationKit,andelutedindH2O.ThenthecDNAwasdigestedwithrestrictionendonucleaseSfiIandpurifiedwithCHROMASPIN-1000-TE.Theshortfragmentswereremovedduringthepurification.OnemicroliterofcDNAwassubjectedto1.5%agarosegelelectrophoresis.3.2.4TheligationofcDNAintopGADT7vectorThepGADT7-SfiIvectorwasligatedwiththeappropriatepost-columncDNA,andincubatedat12°C,overnightusingtheDNAligationkit.TheobtainedligationsolutionwaspurifiedtoobtainaprimarycDNAlibrary.DetectionofprimarylibrarystoragecapacityhasdonebytransformationasmallamountofprimarylibrarysolutionintoDH5αelectrocompetentcells.AppropriateamountoftheculturewasplatedonampicillinresistantLBplate,incubatedat37°Covernight.Theprimarylibrarycapacitywascalculatedusingthenumberofcoloniesgrowingontheplate.TheinsertionofcDNAwasconfirmedbyPCRusingspecificprimersofpGADT7vector(Table3.1).Table3.1PreyvectorprimersPrimernameSequence(5’-3’)pGADT7-FGGAGTACCCATACGACGTACCpGADT7-RTATCTACGATTCATCTGCAGC3.2.5PreparationofY187andY2HGoldcompetentyeastcellsTheyeast(Saccharomycescerevisiae)strainsY187andY2HGoldwereusedforpreparationyeastcompetentcells.Competentcellwerepreparedusingyeasttransformationsystem2kit.Onefreshcolonywasinoculated(diameter2–3mm)into3mlYPDAmediumandincubatedat30°Cshakingat250rpmfor12h.5µlofthefreshculturewastransferred38 中国农业科学院博士学位论文ChapterⅢto50mlofYPDAina250mlflask.IncubatedshakinguntiltheOD600reaches0.3.Thecellswerecentrifugedat700gfor5minatroomtemperature.Supernatantwasdiscardedandthepelletwasresuspendedin100mloffreshYPDA.Incubatedat30°CuntiltheOD600reaches0.4–0.5(3–5h).Thentheculturewasdividedintotwo50mlsterileFalconconicaltubescentrifugedat700gfor5minatroomtemperature.Eachpelletwaswashedin30mlsterile,deionizedH20.Pelletswereresuspendedin1.5mlof1.1xTE/LiAcandtransferredtotworespective1.5mltubesfollowedbycentrifugationathighspeedfor15sec.Finally,eachpelletwasresuspendedin600µlof1.1xTE/LiAcandplacedoniceuntiluse.3.2.6TransformationofcompetentY187yeastcellswithduckcDNAlibraryplasmidsY187yeastcompetentcellswerefreshlypreparedpriortotransformation.ThecDNAlibrarywastransformedusinglibrary-scaleyeasttransformationmethodbycombiningthefollowingreagentsinpre-chilled,sterile15mltube:15µgofplasmidDNA,20µlofyeastmakercarrierDNA(denatured),600µlofcompetentcellsand2.5mlofPEG/LiAcbygentlymixing.Afterincubatedat30°Cfor45min,160µlofDMSOwasaddedandplacedthetubesina42°Cwaterbathfor20min.Thecellswerecentrifugedat700×g5min.Thenthepelletswereresuspendin3mlofYPDPlusmedium,andincubatedat30°Cfor90minwithshaking.Finally,thecellswerecentrifugedandpelletswereresuspendedin15mlof0.9%(w/v)NaClsolution.Thetransformationsolutionwasplatedon150mmSD/-Leuplateswithampicillin.AfterthreedaysincubationthecoloniesgrownontheplateswerecollectedbyaddingYPDAyeastgrowthmedium.ThecDNAlibrarydensitywascountedusinghemocytometerunderthemicroscope.ThecDNAlibraryculturewasthenaliquotedintoindividual1mlvolumesinto2mltubesandstoredat-80°C.39 中国农业科学院博士学位论文ChapterⅢ3.3ConstructionofpGBKT7-pentonbaitfusionvectorTodetecttheprotein-proteininteractions,abaitplasmidwasconstructedusingfulllengthofEDSVpentongenecodingdomainsequenceintopGBKT7fusionvector.3.3.1PCRamplificationofpentongene3.3.1.1DNAextractionTheDNAextractionwasbasedontheclassicalphenol/chloroformextractionmethod.Atotal250µlofsamplewasaddedto250µlofphenol/chloroform/isoamylalcohol(pH7.5-7.8)and25µlof3Msodiumacetate(pH5),vortexedfor1minandincubatedonicefor15min.Themixturewascentrifugedfor10minat12,000rpmatroomtemperature.Theaqueouslayerwastransferredtoa1.5mltubecontaining250µlofisopropanolandincubatedat-20°Cfor5min.TheDNAwasprecipitatedbycentrifugation(5min,12,000rpmat4°C)andthepelletresuspendedin500µlof70%ethanol,followedbycentrifugationfor2minat13,000rpm.Thepelletwasair-driedinahalf-opentubeforatleast10min,suspendedin20µlofddH2Obypipetting,andstoredat–20°C.3.3.1.2PrimerdesignApairofprimerswasdesignedusingcodingdomainsequenceofEDSVpentongene(Genbankaccessionnumber>Y09598.1)byPrimer3KplusonlineprimerdesignsoftwarewithappropriaterestrictionenzymesitesofpGBKT7inMCS.PrimersareshowninTable3.2.40 中国农业科学院博士学位论文ChapterⅢTable3.2PentongeneprimersforpGBKT7vectorNameofprimerPrimersequence(5’-3’)lengthEcoRI/penton2FCGGAATTCATGGAGTCTTTTGTGCCGCC28SalI/penton2RACGCGTCGACTTATTGCAAAGTAGCGCTAGAAATC353.3.1.3PCREachPCRreactionwasconductedwithatotalreactionvolumeof50μl,whichcontain25μlof2×EasyTaqPCRMix(Transgene;0.2mMofeachdNTP,1.5mMMgCl2),1μloftheforwardprimer(10μM),1μlofthereverseprimer(10μM),21μlofddH2Oand2μloftemplate.ThePCRprogramconsistedofaninitialdenaturationstepat95°Cfor3min,followedby34cyclesofdenaturationat95°Cfor30s,primerannealingat59°Cfor30s,extensionat72°Cfor1minandafinalextensionstepat72°Cfor7min.PCRproductswereanalyzedbyelectrophoresison1.0%agarosegelandphotographedunderUVlight.3.3.2GelpurificationofPCRproductTheamplifiedPCRproductwascutfromagarosegelandpurifiedusingOMEGAgelextractionkit(Cat№D2501-02).Theappropriatevolumeofthegelslicewasdeterminedbyweighinginaclean1.5mlmicrocentrifugetube.Appropriatevolumeofbindingbuffer(XP2)wasaddedandincubatedat60°Cfor7minutesoruntilthegelhascompletelymeltedbyshakingthetubeevery2-3minutes.AHiBind®DNAMiniColumnwasinsertedina2mlcollectiontube.ThenthemeltedDNAagarosesolutionwasaddedtothecolumnandcentrifugedat10,000×gfor1minuteatroomtemperature.300µlofbindingbuffer(XP2)wasaddedtothecolumnandcentrifugedatmaximumspeedfor1minute.Thecolumnwaswashedtwo41 中国农业科学院博士学位论文ChapterⅢtimeswith700μlofSPWwashbufferwithmaximumspeedcentrifugation.Theemptycolumnmatrixwasdriedfor2minutesatmaximumspeedandtransferredintoaclean1.5mlmicrocentrifugetube.DNAwaselutedusingdeionizedwaterdirectlytothecenterofthecolumnmembraneandstoredat-20°C.3.3.3LigationofpentongeneintopMD19TsimplevectorPentongenewasligatedintopMD19TvectorbyaddingfollowingreagentsinaPCRtube:VectorDNA(pMD19Tsimple)1µlPCRproduct4µlSolutionI5µlTheligationmixturewasincubatedat16°Cfor30min.3.3.4TransformationofpMD19T-pentonintoE.colicompetentcellsThepMD19T-pentonplasmidwastransformedintoDH5αchemicallycompetentcellsusingcommonbacterialtransformationmethod.3.3.4.1PreparationofmediaandsolutionsLuria-Bertani(LB)media1L:10goftryptone,5gofyeastextract,and10gofNaClweredissolvedin800mlwateranddH2Oaddedupto1L,autoclavedat121°Cfor15min.1MCaCl21L:111gofCaCl2wasdissolvedin1LofdH2Oandsterilizedthrougha0.22µmfilter.100mlliterof0.1MCaCl2waspreparedbydiluting10mlof1MCaCl2in90mlofdH2O.Finalsolutionwassterilizedthrougha0.22µmfilter.50%Glycerol(500ml):Toprepare50mlof50%glycerol,50mlofglycerolwasmixedwith50mlofdH2Oandautoclavedat115°Cfor15min.42 中国农业科学院博士学位论文ChapterⅢToprepare0.5Lof0.1MCaCl2+15%glycerol,50mlof1MCaCl2wasmixedwith150mlof50%glycerol,and300mlofdH2Ounderthesterilecondition.LBplates:7gofagarwasaddedtofreshlyprepared500mlofLBmediaandautoclavedat121°Cfor15min.Theadditionofantibioticswasaddedafterthemediumtemperaturewascooledto55-65°Cpriortopouring.3.3.4.2PreparationofE.coliDH5αcompetentcellsAfreshcolonywasinoculatedin10mlLBmedia(withoutantibiotic)andincubatedovernightat37°C.5mlofovernightDH5aculturewastransferredinto500mlLBmediain3Lflask.Theflaskwasincubatedat37°C(200rpm),untilOD600=0.4(2-3h).Thecellswereplacedonicefor20mins,centrifugedat4°Cfor10minat3,000×g,andresuspendedin30mlofcold0.1MCaCl2.Thesuspendedcellstransferredinto50mlpolypropylenefalcontubes,andincubateonicefor30mins,centrifugedat4°Cfor10minsat3,000×g.Thepelletwasresuspendedin8mlcold0.1MCaCl2containing15%oglycerol,100µlofcompetentcellswastransferredinto1.5mltubes,andstoredat-80C.3.3.4.3TransformationofligationmixtureintoE.coliThe10μlofligationmixturewasmixedwith50μlthawedcompetentDH5αcellsandincubatedonicefor30minutes.Aheat-shockof42°Cwasgiveninawaterbathfor90secfollowedbyincubationonicefor2minutes.Cellswererecoveredbyincubationat37°Cin500mlLBmedia(withoutantibiotic)foronehourshakingat200rpm.200μloftheculturewasplatedonLB-agarplateswiththeappropriateantibiotic.Theplateswereincubatedovernightinadryincubatorat37°C.43 中国农业科学院博士学位论文ChapterⅢ3.3.5PCRforpositivecoloniesPCRwascarriedouttocheckthepositivecoloniesforsuccessfulligationandtransformation.Positivecolonieswereinoculatedinto1.5mltubeswith1mlofLB,at37°Cfor8h.ThenthecultureswerecheckedbyPCR.PCREasyTaqmix10µlPrimerforward1µlPrimerreverses1µlddH2O7µlGrowingculture1µlPCRreactioncondition:95°C3min,30cycles(95°C30sec,59°C30sec,72°C1min)72°C7min,4°C.PCRproductswereanalyzedbyelectrophoresison1.0%agarosegelandphotographedunderUVlight.3.3.6PlasmidextractionAfterPCR,thepositivecolonieswereinoculatedinto5mlofLBmedium,andincubatedat37°Covernightforplasmidextraction.TheplasmidswereextractedusingOMEGAPlasmidMiniKit,accordingtomanufacturer’sinstructions.3.3.7InsertionofpentongeneintopGBKT7baitvector3.3.7.1RestrictionenzymedigestionThepentongenewascleavedfrompMD19TplasmidbydigestingwithEcoRIandSalIrestrictionenzymes(Table3.3),mixturewasincubatedat37°Cfor2h,andtheDNAwas44 中国农业科学院博士学位论文ChapterⅢpurifiedusingOMEGAgelextractionkit(insection3.3.2).Table3.3RestrictionenzymedigestionofpMD19T-pentonplasmid;SamplesReagentspMD19T-pentonpGBKT7vector(170ng/µl)(100ng/µl)-6µl310XBufferH2µl2EcoRI1µl1SalI1µl1ddH2O10µl13µlTotalvolume20µl20µlNote:Enzymes,buffersandvectorsarefrom(Takara,Dalian,China).3.3.7.2LigationofpentonintopGBKT7baitvectorForDNAligation,thefollowingreagentsweremixedinaPCRtube:8µl(32ng/µl)ofREdigestedpenton,7µl(12ng/µl)ofREdigestedpGBKT7vector,2.5µlofT4ligationbuffer,1µlofT4DNALigase(Takara,Japan)and6.5µlofddH2O.Thesamplewasincubatedat4°Covernightandfollowedbybacterialtransformationandplasmidextractionsteps.3.3.8TransformationofpGBKT7-pentonplasmidintoY2HGoldcellsThepositivepGBKT7-pentonplasmidswereextractedfromE.colicellusingOMEGAminiplasmidextractionkit,andstoredat-20°C.Y2HGoldyeastcompetentcellswerefreshlypreparedpriortotransformationusingyeasttransformationsystem2(Clontech,Canada).ThepGBKT7-pentonplasmidandcontrolplasmids(pGBKT7-53,pGBKT7-lamandpGBKT7-empty)weretransformedusingsmall-scaleyeasttransformationmethodbycombiningthefollowingreagentsinfourpre-chilled,sterile1.5mltubes:100ngofplasmid45 中国农业科学院博士学位论文ChapterⅢDNA,5µlofYeastmakercarrierDNA(denatured),50µlofcompetentcellsand500µlofPEG/LiAcbygentlymixing.Afterincubatedat30°Cfor30min,20µlofDMSOwasaddedandplacedthetubesina42°Cwaterbathfor15min.Thecellswerecentrifugedathighspeed30sec.ThenthepelletswereresuspendinYPDPlusmedium,andincubatedat30°Cfor30minwithshakingevery10minonce.Finally,thecellswerecentrifugedandpelletswereresuspendedin0.9%(w/v)NaClsolution.100µlof1/10and1/100dilutionsoftransformedcellswereplatedontofollowingplates:SD/–Trpplates=SDOSD/–Trp/X-a-Gal=SDO/XSD/-Trp/-His/-Adeplates3.3.8.1YeastcolonyPCRYeastcolonyPCRwasappliedtoverifythepositivetransformations,forthatthecolonieswereinoculatedintoYPDAyeastmediumandincubatedat30°Cfor20hwithshakingat250rpm.ThentheplasmidswereextractedusingOMEGAyeastplasmidextractionkit(OMEGA,USA).3.4Yeasttwo‐hybridscreeningusingyeastmatingForyeasttwo-hybridscreening,aconcentratedbaitculturewasmixedwith1mlofduckcDNAlibrary(seesection3.2.6)andincubatedat30°Cwithslowlyshakingat50rpm,formorethan20hours.Theconcentratedbaitculturewaspreparedasfollows:Onefresh,large(2–3mm)colonyofbaitstrainwasinoculatedinto50mlofSD/–Trpliquidmediumina250mlflask.Theflaskwasincubatedat30°Cwithshakingat250rpmuntiltheOD600reaches0.8(16–20h).Theyeastcellswerecentrifugedat1,000×gfor5min,andthesupernatantwasdiscarded.Thepelletwasresuspendedtoacelldensity46 中国农业科学院博士学位论文ChapterⅢ8of>1x10cellspermlinSD/–Trp(4–5ml).Thecellsdensitywascountedusingahemocytometer.Thebaitandlibrarystrainswerecombinedasfollows:onemilliliteroflibraryaliquotwasthawedinaroomtemperaturewaterbath.Fortitteringon100mmSD/–Leuagarplates,10µloflibraryaliquotwasremoved.Aftercompletelythawed,thecDNAlibrarywascombinedwith4–5mlbaitstraininasterile2Lflaskcontaining45mlof2xYPDAliquidmedium(with50µg/mlkanamycin).Cellsfromthelibraryvialwererinsedtwicewith1ml2×YPDAandaddtothe2literflask.Theflaskwasincubatedat30°Cfor20–24hr,slowlyshaking(30–50rpm).After20hmatingtime,adropoftheculturewascheckedunderaphasecontrastmicroscope(40X).Whenthezygoteswerepresent,thecellswerepelletedbycentrifugationat1,000gfor10min.Meanwhile,the2Lflaskwasrinsedtwicewith50mlof0.5xYPDAfreshmedium(with50µg/mlkanamycin),andusedtoresuspendthepelletedcells.Thenthecellswerecentrifugedat1,000×gfor10min.anddiscardthesupernatant.Thepelletedcellswereresuspendedin10mlof0.5XYPDA/Kanliquidmedium.Fromthematedculture,100µlof1/10,1/100,1/1,000,and1/10,000dilutionswerespreadedoneachofthe100mmagarplatesandincubatedat30°Cfor3–5days.SD/–TrpSD/–LeuSD/–Leu/–Trp(DDO)The200µlofremainderculturewasplatedonper150mmDDO/X(nearly50plates).Theplateswereincubatedat30°Cfor3–5days.Numbersofscreenedcloneswerecalculatedusingformulabelow:NumberofScreenedClones=cfu/mlofdiploidsxresuspensionvolume(ml)ThebluecoloniesgrownonDDO/Xplatesweretransferredtohigherstringency47 中国农业科学院博士学位论文ChapterⅢQDO/Xagarplatesusingthesterileyellowpipettetips.AlltheQDO/XpositiveinteractionswerefurtheranalyzedbyyeastplasmidextractionandPCR.Matingefficiencycanbecalculated(percentageofdiploids)asbelow:No.ofcfu/mlofdiploids/No.ofcfu/mloflimitingpartner×100%=0%Diploids3.4.1YeastplasmidextractionAllpositivecoloniesgrownonQDO/XplateswereinoculatedinQDObrothandincubatedat30°Cfor24h.Thentheplasmidswereextractedusingyeastplasmidextractionkit(OMEGA,USA)accordingtothemanufacturer’sinstructions.3.4.2PCRanalysistoeliminateduplicateclonesThecandidatepreyplasmidsweretestedbyPCRamplificationusingpreyvectorprimers,andanalyzedon1%agarosegel/EtBr.Thepresenceofmorethanonebandiscommon,ithasindicatesthatmorethanonepreyplasmidispresentingincell.AllPCRpositivesinglebandsweresequencedusingT7primersbyGENWIZECompany,China.3.5Confirmationofpositiveinteractions3.5.1Co-transformationTodistinguishfalsepositiveinteractions,total10candidatepreyplasmidswereco-transformedwithbaitplasmid(pGBKT7-penton)intoY2HGoldcells,alongwithpositiveandnegativecontrols,accordingtoyeasttransformationsystem2manual’sinstructions(CatNos.630439).ThenthetransformantswerespreadedonQDO/X-α-galplates.48 中国农业科学院博士学位论文ChapterⅢ3.6Results3.6.1CharacterizationofcDNAlibraryForthecDNAconstruction,totalRNAwasisolatedfromduckfibroblastcellsusingTRIzolreagent(Invitrogen,USA)accordingtothemanufacturer’sinstructions.ThehighqualityofRNAisthemainkeytopreparehighqualityofcDNAlibrary.TheratiooftheA260/A280fortotalRNAisshownin(Table3.4).TheRNAqualitywascheckedbyelectrophoresingof300ngRNAin1.5%agarosegel,theRNAbandsin(Fig.3.1)shownthatRNAqualitywaspureandmorestabletomakecDNA.ThecDNAwassuccessfullysynthesizedfrommRNAusingSMARTcDNAlibraryconstructionkitandAdvantage2PCRkit(Clontech,USA)accordingtotheinstructions(Fig3.2).Table3.4ThequalityoftotalRNASamplenameOD260/280OD260/230Duckfibroblastcell1.972.08Figure3.1QualityoftotalRNAFig.3.2SynthesizedcDNAM1:LambdaEcoT14IdigestM1:LambdaEcoT14IdigestM2：250bpDNALadderM2：250bpDNALadderLane1:TotalRNALane1:cDNA49 中国农业科学院博士学位论文ChapterⅢThecDNAlibrarywasanalyzedbyPCRamplificationusingpGADT7-forwardand6pGADT7-reverseprimers.Theprimarylibrarycapacitywas>about1.5×10cfuandtheaverageinsertsizewas≥1.0kb(Fig3.3)Figure3.3cDNApositivecoloniesrandomlyselectedfromplatesM1:LambdaEcoT14Idigest(Takara,Dalian,China);M2:250bpDNALaddermarker(Takara,Dalian,China).50 中国农业科学院博士学位论文ChapterⅢ3.6.2ConstructionofpGBKT7-pentonbaitfusionplasmidInordertoconstructthebaitfusionvectorwithpentonprotein,weamplifiedthepentongenebyPCR,usinggenespecificpentonforwardandreverseprimers(Fig3.4AandTable2).ThefulllengthofamplifiedpentongenewasligatedintopMD19Tsimplevector(Fig3.4B).ThepositiverecombinantplasmidswereidentifiedbyPCR.Figure3.4Amplificationofpentongeneandcloning.(A)PCRamplifiedpenton;lane1-Pentongene(1395bp);lane2-negativecontrol.(B)CloningofpentonintopMD19Tvector;lane1-8pMD19T-pentontransformationcolonies;lane9-positivecontrol(EDSVDNA);lane10-negativecontrol.ThepMD19T-pentonrecombinantplasmidwasfurtherdigestedwithEcoRIandSalIrestrictionenzymes(Fig.3.5B),andtheninsertedintopGBKT7baitfusionvector.TheligationandtransformationresultsweretestedbyPCR.ThefullcodingdomainsequenceofEDSVpentongenewassuccessfullyinsertedintopGBKT7vector,andtheresultsareshownin(Fig3.5A).51 中国农业科学院博士学位论文ChapterⅢFig.3.5TransformationresultofpGBKT7-pentonplasmid(A);lane1-12randomlyselectedcolonies;lane-13positivecontrol;lane-14negativecontrol.(B)RestrictionenzymedigestionofpMD19T-pentonplasmid;M-Trans2KplusDNAmarker.Further,thepositivebaitplasmidwasextractedandverifiedbyPCRamplificationandrestrictionenzymedigestionwithEcoRIandSalI(Fig.3.6).Figure3.6Verificationofbaitplasmid.(A)PCRamplificationofpGBKT7-pentonplasmid;lane1,2-pGBKT7-penton;lane-3positivecontrolandlane-4negativecontrol.(B)MapofpGBKT7baitvector;(C)RestrictionenzymedigestionofpGBKT7-pentonplasmid;lane1-Trans15KplusDNAmarker;M-Trans2KplusDNAmarker.52 中国农业科学院博士学位论文ChapterⅢ3.6.3VerificationofpGBKT7-pentonexpressioninY2HGoldcellsTherecombinantbaitfusionplasmid(pGBKT7-pentonandcontrolplasmids(pGBKT7-53andpGBKT7-lam)weretransformedintoY2HGoldyeastcells,andtheexpressionofbaitfusionproteincheckedbyPCRusingpGBKT7vector’sforwardandreverseprimers.ThetoxicityofpGBKT7-pentoninyeastcellsweredetectedpositiveresultsonSD/Trpalongwithcontrolplasmids.ThesizeofpGBKT7-pentonyeastcolonieswassamewithcontrolcolonies(Fig3.7A).TheAuto-activationisdefinedasdetectablebait-dependentreportergeneactivationintheabsenceofanypreyinteractionpartner.TheautoactivationofpGBKT7-pentonwastestedbyplatingtheyeastcellsonSD/-Trp/X-α-Gal(Fig.3.7B)Fig.3.7ThetoxicityandautoactivationofpGBKT7-pentonfusionprotein;(A)SD/-Trpplate,(B)SD/-Trp/Xplate,1-pGBKT7-penton;2-pGBKT7-lam;3-pGBKT7-empty;4-pGBKT7-53onSD/-TrpandSD/-Trp/XplatesTheself-activationtestingresultsindicatedthatbaithasnoautoactivationintheabsenceofpreyproteins(Fig.3.8).Thebaitandcontrolplasmidsweredidn’tgrowonSD–Trp/–His/–Ade,SD/Trp/-Leu(DDO)andSD/-Ade/-His/-Trp/-Leuplates.53 中国农业科学院博士学位论文ChapterⅢFigure3.8AutoactivationtestofpGBKT7-pentononhigh-stringencyplates;(A)DDOplate,(B)QDOplate,(C)SD/-Trp/-His/-Adeplate;1-pGBKT7-penton;2-pGBKT7-lam;3-pGBKT7-empty;4-pGBKT7-53onSD/-TrpandSD/-Trp/Xplates.Afterthetransformedcellsgrownonappropriateselectivemedium,thePCRwascarriedouttotestthepresenceofthepGBKT7-pentonplasmidandothercontrolplasmidsinyeastcellsusingpGBKT7forwardandreverseprimers.Forthat,thecolonieswererandomlyselectedandusedtothePCR.AllthecoloniesusedinPCRweregavebandson1%agarosegel(Fig.3.9).Figure3.9PCRamplificationofpGBKT7-pentonfusionproteinandcontrolvectors;lane1-pGBKT7-penton;lane2-pGBKT7-53;lane3-pGBKT7-lam;lane4-pGBKT7-empty;lane5-positivecontrol;lane6-negativecontrol;M-Trans2KplusDNAmarker.54 中国农业科学院博士学位论文ChapterⅢ3.6.4IdentificationofinteractionpartnersbylibraryscreeningInthelibraryscreen,alibraryofpreyproteinswastestedforinteractionswithpGBKT7-pentonbaitprotein.Thebaitproteinwasexpressedinoneyeaststrain(Y2HGold)andthepreywasexpressedinanotheryeaststrain(Y187).Inthescreening,thetwostrainswerematedsothatthetwoproteinswereexpressedintheresultingdiploidcell.ThediploidswereplatedoninteractionselectivemediumplateswhereonlyyeastcellshavingbaitandpreycangrowonDDOselectiveplates(Fig3.10A).Thelibraryscreeningyielded208bluecoloniesontheSD/-Leu/-Trp/X-α-Galmediumafter3daysofgrowthat30°C.Thenumberwasreducedto145byeliminatingfalse-positiveinteractorsonQDO/X-α-Galselectivemedia(Fig.3.11).Thepositivepreyproteinswereidentifiedbyisolatingthepreyplasmids,byPCRamplificationoftheinsert,andsequencing,thepositiveinteractionswereshownin(Fig.3.10Band3.12).Figure3.10LibrarymatingzygotesandpositivecoloniesbyPCR;(A)diploids(zygotes);(B)Positivecoloniesfrommating;lane1-5pGBKT7-penton;lane6-positivecontrol(pGBKT7vector);lane7-negativecontrol.Lane9-13positivepreyplasmids;lane14-pGADT7vector;lane15-negativecontrol;M-Trans2KplusDNAmarker.55 中国农业科学院博士学位论文ChapterⅢFigure3.11PutativepositivecoloniesgrownonQDOplatesfromyeasttwohybridscreening.Figure3.12PCRpositivepreycolonies;lane1-21coloniesfromQDOplatesaftermating;lane22-positivecontrol(pGADT7-emptyvector);lane23-negativecontrol;M-Trans2KplusDNAmarker(Transgene,China).56 中国农业科学院博士学位论文ChapterⅢ3.6.5Confirmationofpositiveinteractorsbyco-transformationintoY2HGoldcellsTheplasmidsfromyeastwereextractedusingyeastplasmidextractionkit(OMEGA,USA).PreyplasmidswererescuedbytransformationtheplasmidsintoE.colicellsandplatingonampicillintreatedLBplates.Thesequencesofthe145PCRpositivepreyinsertswereusedforBLASTsearchesoftheNCBIdatabase.Falsepositiveswereeliminatedbytheco-transformationofpositivepreyandpGBKT7-pentonbaitplasmidsintoY2HGoldyeastcells.ThepGBKT7-pentonbaitproteinyieldedpositivebluecolonieswiththepreyproteins:GABAtypeAreceptorassociatedproteinlike1(GABARAPL1)transcriptvariantX1,ribosomalproteinSA(RPSA),integrinsubunitalpha4(ITGA4)andactinbeta(ACTB),onSD/-Trp/-Leu/-Ade/-His/X-α-Gal(QDO/X),butnoco-expressionhappenedoncontrolsemptyprey+pGBKT7-pentonandpGBKT7empty+duckcDNAlibraryplasmids(Fig.3.13).57 中国农业科学院博士学位论文ChapterⅢFigure3.13Co-transformationofpreyplasmidswithpentonprotein;Controlplates1-positivecontrol(pGBKT7-53+pGADT7-T);2-negativecontrol(PGBKT7-lam+pGADT7-T);3-penton+pGADT7emptyvector;4-pGBKT7emptyvector+pGADT7emptyvector.58 中国农业科学院博士学位论文ChapterⅢ3.6.6CharacterizationofpreycandidateinteractionpartnersFourpreyproteinsweregivenpositiveresultsbyco-transformationinY2HGoldinyeastcellwithEDSVpentonprotein.3.6.6.1AnasplatyrhynchosGABAtypeAreceptorassociatedproteinlike1(GABARAPL1)TheGABAtypeAreceptorassociatedproteinlike1(GABARAPL1)(>XP_005012734.1)islocatedinmitochondrionandautophagosomeincell(Fig.3.14).Themolecularfunctionsaretatproteinbindingandubiquitinproteinligasebinding.Proteinlengthisabout152aa,ithassimilaritieswithsomeotherorganismproteins(Table3.5inadditionalsection).Figure3.14SubcellularlocalizationofGABARAPL1proteinfrom(UniProtgeneontologyannotation,http://www.uniprot.org/).59 中国农业科学院博士学位论文ChapterⅢ3.6.6.2AnasplatyrhynchosribosomalproteinSA(RPSA)or40SribosomalproteinRibosomalproteinSA(RPSA)or40Sribosomalprotein(>XP_005016997.1)isacellsurfacereceptorforlaminin,andplaysaroleincelladhesiontothebasementmembraneandintheconsequentactivationofsignalingtransductionpathways.Italsomayplayaroleincellfatedeterminationandtissuemorphogenesis.Molecularfunctionsarelamininbinding,lamininreceptoractivity,structuralconstituentofribosome.Theproteinislocatedincellmembraneandnucleus(Fig3.15).Thesizeofproteinis296aa,theproteinsimilaritieswithsomeotherorganismshaveshownin(Table3.6inadditionalsection).Figure3.15Subcellularlocalizationof40Sribosomalproteinfrom(UniProtgeneontologyannotation,http://www.uniprot.org/).60 中国农业科学院博士学位论文ChapterⅢ3.6.6.3Anasplatyrhynchosintegrinsubunitalpha4(ITGA4)geneAnasplatyrhynchosaIntegrinalpha-4isoformX2(>XP_012952316.1)isamemberoftheintegrinalphachainfamilyofproteins.Integrinsareheterodimericintegralmembraneproteinscomposedofanalphachainandabetachainthatfunctionincellsurfaceadhesionandsignaling.Theencodedpreproproteinisproteolyticallyprocessedtogeneratelightandheavychainsthatcomprisethealpha4subunit.TheITGA4isabigproteinwith921aawith102.8kDamolecularmassandlocatedonplasmamembraneandcellsurface(Fig3.16).ThemolecularfunctionsoftheproteinarecelladhesionmoleculebindingandC-X3-Cchemokinebinding.SomesimilarproteinswerefoundbyUniprotGoannotation(Table3.7inadditionalsection).Itcanalsobeattendtothefollowingbiologicalprocessincell:1.Cell-matrixadhesioninvolvedinameboidalcellmigration2.Endodermalcelldifferentiation3.Heterotypiccell-celladhesion4.Integrin-mediatedsignalingpathway5.Negativeregulationofproteinhomodimerizationactivity6.Receptorclustering7.Substrateadhesion-dependentcellspreading61 中国农业科学院博士学位论文ChapterⅢFigure3.16Subcellularlocalizationofintegrinsubunitalpha4from(UniProtgeneontologyannotation,http://www.uniprot.org/).62 中国农业科学院博士学位论文ChapterⅢ3.6.6.4Anasplatyrhynchosactinbeta(ACTB)Actinbeta(NP_001297350.1)isanATPbindingprotein,belongstotheactinfamily,locatedincellmembraneandcytoskeleton.Theproteinlengthisabout375aawith41.7kDamolecularmass.ACTBhassimilaritieswithsomeotherorganisms(Table3.8inadditionalsection).Figure3.17Subcellularlocalizationofactinbeta(ACTB)from(UniProtgeneontologyannotation,http://www.uniprot.org/).63 中国农业科学院博士学位论文ChapterⅣCHAPTERIVConfirmationoftheinteractionbetweenbaitprotein(pGBKT7:penton)andpreyproteins(GABARAPL1andRPSA)4.1IntroductionYeasttwohybridsystemhasbeenwidelyusedtoidentifyprotein-proteininteractions;however,thepredictedinteractionpartnershavetobeconfirmedusingdifferentinvitromethods.GSTpull-downassayisaninvitromethodusedtodetermineaphysicalinteractionbetweenaGSTtaggedprobeproteintoanunknownoraknowntargetprotein.TheGSTtaggedproteinshavevarietyofapplicationsincludingpurificationoffusionproteins(SmithandJohnsonetal.,1988).TheprocedureinvolvesincubationoftheGSTfusionproteinimmobilizedonglutathione-agarosebeadswiththetotalcelllysate.GSTpull-downassayisalsoimportanttechniqueforbothconfirmingtheprotein-proteininteractionpredictedbyotherresearchtechniquesincludingco-immunoprecipitation,yeasttwohybridetc.,aswellasforscreeningofnovelunknownproteininteractions(SambrookandRusselletal.,2004).Pull-downassaysareakindofaffinitypurificationthatnotonlyenhancetheefficiencyofproteinpurificationbutalsocanbeusedsimultaneouslytoperformapull-down,orco-purification,ofpotentialbindingpartners.Also,Itcanbedetectaninteractionbetweentwopurifiedbacterialproteinsorbetweenbacterialandeukaryoticproteinscanbedetected(Louche,Salcedoetal.,2017).ProteinexpressioninE.coliiseasytohandle，acost-effectiveandhigh-levelproductionofheterologousproteins.Widecollectionofexpressionvectorswithwell-definedandstrongpromotersisavailable.ThisincludesthetranscriptionoftherecombinantDNAtomessengerRNA(mRNA),thetranslationofmRNAintopolypeptidechains,whichareultimatelyfoldedintofunctionalproteinsandmaybetargetedtospecificsubcellularorextracellularlocations.64 中国农业科学院博士学位论文ChapterⅣInourstudy,thebaitfusionproteinwasexpressedinE.colicellsandthepreyfusionproteinswereexpressedinmammaliancelllines.ThenthebaitproteinwaspurifiedusingGSTagarosebeadsandincubatedwithpreycelllysates.ThecomplexrecoveredfromthebeadswereresolvedbySDS-PAGEandfollowedbywesternblotting.Inthechapter3wehavepredictedtwopreyproteinswhichhaveinteractionactivitywithpentonproteinofEDSV.InthischapterwehaveconfirmedtheproteininteractionsinvitrobyGSTpulldownassay.Ourresultwillsustaintoimprovetheresearchactivityinstudyoftropismandproteinfunctionsofeggdropsyndromevirus,alsoimproveitsroleinadenovirusvectordevelopment.Fig.4.1MechanismofGSTpull-downassayfromwebsite(http://www.bioon.com.cn/server/show_product.asp?id=15232)65 中国农业科学院博士学位论文ChapterⅣ4.2Materialsandmethods4.2.1ConstructionofbaitandpreyfusionvectorsforGSTpull-downassayForconstructionofbaitandpreyfusionvectors,thefullcodingdomainsequencesofgeneswereamplifiedbyPCR.Thepenton(bait)genewasfirstlyligatedintopMD19T,sequenced,thenthecorrectgenesequencewascleavedusingappropriaterestrictionenzymes,andinsertedintopGEX-6p-1andpEGPF-C1expressionvectors.Moreover,toconstructthepreyfusionvectors(GABARAPL1andRPSA);thefullcodingsequencesofbothgeneswereamplifiedfromduckcDNAlibrarybyPCR.TheamplifiedsequenceswereclonedintopMD19Tsimplevector,andsequencedforsuccessfulcloning.AfterBLASTconfirmation,thesequenceswererestrictionenzymedigestedfrompMD19TandligatedintopEGFP-C1mammalianexpressionvector.4.2.1.1PCRamplificationofbaitandpreygenesToamplifythebaitandpreygenes,thetotal50µlofPCRreactionswerecarriedoutforpergene.PCREasyTaqMix25µlForwardprimer2µlReverseprimer2µlddH2O20µlTemplate1µl66 中国农业科学院博士学位论文ChapterⅣPCRreactionconditions:58-60°C30sec(bait),72°C90sec95°C3min,35cycles(95°C30sec,)72°C7min.63-65°C30sec(prey),72°C50secPCRproductswereanalyzedbyelectrophoresison1.0%agarosegelandphotographedunderUVlight.4.2.1.2DNAligationintovectorsandbacterialtransformationTheampliconsforbaitandpreyswereligatedtopMD19Tcloningvector.ThesequenceswereconfirmedbysequencingandBLASTanalysis.ThenthebaitwasinsertedintopGEX-6P-1andpEGFP-C1expressionvectorsandpreyproteinswereinsertedintopEGFP-C1respectively.TheligationsweretransformedintoDH5αE.colichemicallycompetentcellsusingmethodinsection3.3.4.3.ThepositiverecombinantswerecheckedbyPCRamplification,aswellasrestrictionenzymedigestion.4.2.2ExpressionofBaitfusionproteinTheexpressioncanbedescribedasatranscriptionandtranslationofagene.Aparticularhostisrequiredtoexpressaparticulargene.TheseexpressionsystemsincludeE.coli,baculovirus-mediatedinsectcellexpression,yeast,andseveralmammalianbasedsystems.Thedifferentvectorsystemsthattransferthegeneintomammaliancellslikeplasmidbasedexpressionvectors,adenovirusvectors,vacciniavectors,retroviralvectorandbaculovirusasvectors(Verma,Boletietal.,1998).Thenucleotidesequencesfortheproteinofinterestcouldbeclonedintoahigh67 中国农业科学院博士学位论文ChapterⅣcopy-numberplasmidcontainingthelacpromoter,whichisthentransformedintothebacteriumE.coli.TheIPTGactivatesthelacpromoterandcausesthebacteriatoexpresstheproteinofinterest.ThebaitfusionproteinpGEX-6P-1:pentonwasexpressedinbacterialcellsandpEGFP-C1:pentonwasexpressedineukaryoticcellsaswell.4.2.2.1ExpressionofpGEX-6P-1:pentoninBL21(DE3)cellsBL21(DE3)bacteriacontainingplasmids(pGEX-6P-1:pentonandpGEX-6P-1empty)-70°Cstock,)inoculatedinto3mlofLBmediumcontainingtheappropriateantibiotic.Thecellswereincubatedwithshakingat37°Covernight.Nextday,300mLofLBmediumcontainstheappropriateantibioticwithall5mloftheovernightculture.TheOD600wasmeasuredandincubatedwithshakingat37°CuntiltheOD600reaches0.8for3h.After3hincubation,0.5mlofIPTG(1M)wasaddedtotheculture.Thetimeforinductionofthetargetproteinwasfor16hat21-22°C.Thecellculturewasplacedonicefor10minutesandharvestedbycentrifugationat6000rpmfor10minutesat4°C.8.Thenthepelletswereresuspendedwith40mlofPBSandultrasonicated.Thecellswerecentrifugedat12,000×gfor20minat4°C.ThefusionproteinwaspurifiedusingGSTagarosebeads.4.2.3Expressionofbaitandpreyfusionproteinsineukaryoticcells4.2.3.1CellculturepreparationandtransfectionThemammaliancellsallowexpressingtheglycosylatedormembraneproteinsthatcannotbeexpressedusingyeastorprokaryoticcells.The293TandDF1cellcultureswerepreparedwithmediumcontainsDulbecco’smodifiedEagle’smedium(DMEM,Gibco,Shanghai,China)supplementedwith10%fetalbovineserum(FBS,Gibco,USA)and1%gentamycin(Sigma-Aldrich)inappropriatecellcultureflasks.Thecellswereincubatedat37°Cwith5%CO2supplyfor24h.68 中国农业科学院博士学位论文ChapterⅣThepEGFP-C1:penton,pEGFP-C1:GABARAPL1,pEGFP-C1:RPSArecombinantplasmidsandpEGFP-C1emptyplasmidweretransfectedinto293T,VeroandDF1celllinesusingJetPrimetransfectionreagent(Polyplus-transfection,France).Per2µgofplasmidDNAwasdilutedinto200µljetPRIME®bufferandmixedbyvortexing.Thenthe4µlofjetPRIME®reagentwasaddedandvortexedfor10sec.Themixturewasincubatedfor10minatroomtemperature.200µloftransfectionmixturewasaddedontothecells(6wellplates)inserumcontainingmedium,anddistributedevenly.Theplatesweregentlyrockedbackandforthandfromsidetoside.Thegrowthmediumwasreplacedafter4h,andplateswerereturnedtotheincubator.Thegreenflorescenceprotein(GFP)canbeseenundertheLeicamicroscopy,iftherecombinantproteinsexpressincells.Transfectedcellswerecheckedafter24hand48hposttransfection.4.2.4Detectionofproteinexpressioninmammaliancelllines4.2.4.1PreparationofcelllysatesCelllysateswerepreparedusingRIPAlysisbuffer(CWBIO,China),themaincomponentsofRIPAlysisbufferare50mMTris(pH7.4),150mMNaCl,1%TritonX-100,1%sodiumdeoxycholate,0.1%SDSandsodiumorthovanadate,sodiumfluoride,EDTA,leupeptin.Thecellculturemediumwasdiscardedandthecellsrinsedwithpre-cooledPBS.ThentheappropriateamountofRIPAlysisbuffercontaining1%ofproteaseinhibitorcocktailandPMSF(100mM)wereaddedtothelysisbufferwithin2-3minutesbeforeuse(RIPAlysisbufferusage:100mm(500-1000μl),60mm(250-500μl)6-wellplate(200-400μl),24-wellplate(100-200μl),96-wellplate(50-100μl)).Usingthepipettethelysatesweretransferredtoanewcentrifugetubeandincubatedonicefor20minutesfortoallowsufficientlysisofthecells.Thenthecelllysateswerecentrifugedat14,000×gfor10minutes.Thesupernatantcanbeusedforfurtheranalysis.69 中国农业科学院博士学位论文ChapterⅣ4.2.4.2Preparationofrunning,transferandblockingbuffersToprepare1literofrunningandtransferbuffers(1×),thechemicalsweredissolvedin600-800mlofwaterandadditionalwateraddedupto1Lvolume.Primaryandthesecondaryantibodiesweredilutedin5%milkpriortouse.SDSPAGErunningbuffer(1×)Glycine14.4gTrisbase3.03gSDS1.008gTransferbuffer(1×)Trisbase(3.03g)Glycine(14.4g)Methanol(100%)200mLTBST(1×)Tris2.42gNaCl8.76gTween-2010mlBlockingbuffer(5%milk)100mlofTBST+5gdrymilk4.2.4.3SDSpolyacrylamidegelelectrophoresisTheSDSpolyacrylamidegelswerepreparedusingSDSpolyacrylamidekit(CWBIO,China)accordingtothemanufacturer’sinstructions.PriortorunSDSPAGE,the20%of2×SDSloadingbufferwasaddedtothesample,andthesampleswereboiledfor5minat100°C.Thenthetubeswerecentrifugedat12000×gfor5minat4°C.Thesupernatantswereloadedin10%-12%proteingels,at80Vinrunningbufferfor20minutesuntilthe70 中国农业科学院博士学位论文ChapterⅣproteinshaveenteredtotheresolvinggel.Thevoltagewasincreasedto120Vfor1hand15minuntilproteinsweretotallyseparated.TheSDS-PAGEgelswerestainedwithcoomassiebrilliantbluetovisualizetheproteinbands.Forwesternblotanalysis,theloadedproteinsweretransferredtothePVDF(Millipore,USA)membrane.4.2.4.4WesternblottingInasandwich,thegelsfrom(section4.2.4.3)wereplacedbetweenthetwoWhatmanpaperswithPVDFmembrane,andthesandwicheswereclosed.Sampleswererunincoldtransferbufferwithcoldpackfor1hourat300mA.ThemembraneswerecarefullyremovedfromsandwichandimmerseinTBSTfor5minutesandblockedforovernightat4°C,inblockingbuffer(5%milk).Primaryantibodiesweredilutedinblockingbufferattheappropriateconcentration,andmembraneswereincubatedfor1houratroomtemperature.ThemembraneswerewashedwithTBSTfor5min,for3times.Thesecondaryantibodywasdilutedattheappropriateconcentration.Themembraneswereincubatedfor1h,insecondaryantibody.Finally,themembraneswerewashed3timesinevery5minuteswithwashbuffer(TBST),proteinbandsweredetectedbyenhancedHRP-DABsubstratechromogenkit(PA110).4.3GSTpull-downassay4.3.1PurificationofbaitfusionproteinTheproteinspGEX-6P-1:pentonandGSTwerepurifiedafterexpressionatlowertemperature(21-22°C)asinsection4.2.2.1.Cellswereharvestedbycentrifugationat3000×gfor10minat4°C.Thepelletswereresuspendedincold1×PBS(3mlofcold1×PBSrequiredfor50mlofcellculturemedium),andcentrifuged.Thepelletswerefreezedfor171 中国农业科学院博士学位论文ChapterⅣhourat-80°C.Thenthethecellswerethawedoniceandresuspendedwithice-cold1×PBS.1%PMSFandproteaseinhibitorcocktailwereaddedtopermillilitersuspension.Thecellsweresonicatedoniceuntilthesamplewasnolongerviscousandcrushcompletely.ThepreparedclarifiedcelllysatecontainingGSTfusionproteinandGSTproteinswereaddedtothecolumncontainingGSTbeadswithaflowratecontrolof10-15cm/h.Theproteinswereelutedwithappropriatevolumeof10mMglutathioneeluate(0.154gReducedGlutathionein50ml50mMTris-HCl,pH8)andstoredat–80°C.4.3.2GSTpull-downprocedureCelllysateswerepreparedusingGSTpulldownlysisbuffer(Thermo,USA).Fortopreclearthecelllysatefromnonspecificinteractionproteins,theappropriatevolumeofcelllysates(0.5-1mlgoodstartingpoint)wereincubatedwith50μlofglutathione-Sepharosebeads,and25μgofGSTproteinfor2hat4°Cwithendoverendmixing.Centrifugedat13,000rpmfor30sec,thenthepreclearedsupernatantwasusedtotheinteraction.Equalvolumeofsupernatantwasaddedtotwo1.5mltubes,andincubatedwithpGEX-6P-1:pentonandGSTprotein(10μg).Thetubeswereincubatedfor2hat4°C,andcentrifugedat13,000rpmfor30sec.Thenthebeadswerewashedwith1mlofice-coldlysisbufferfor4times.50μlofelutionbuffer(20mMreducedglutathionein50mMTris-HCl,pH8.0)addedtothebeadsandincubatedatroomtemperaturefor5min,elutedsampleswereboiledwithequalvolumeof2×SDSgel-loadingbufferat100°Cfor5min.Theloadedsamplesin12%proteingelthentransferredtoPVDF(Millipore,USA)membraneandfollowedbywesternblotting.72 中国农业科学院博士学位论文ChapterⅣ4.4Results4.4.1ConstructionofpGEX-6P-1:pentonplasmidThecodingdomainsequenceofEDSVpentongene(1359bp)wasamplifiedbyPCR(Fig4.1A)andligatedintopMD19Tsimplevector.TherecombinantbaitplasmidpMD19T:pentonandtheemptypGEX-6P-1vectorweredigestedwithEcoRIandSalI(Fig.4.1B)andthepentongenewassuccessfullyligatedintopGEX-6P-1bacterialexpressionvector;thepositiverecombinantplasmidwasverifiedbyPCRamplificationoftargetsequence,andBLASTanalysis(Fig.4.2).Figure4.1ConstructionofpGEX-6P-1:pentonplasmid;(A)Pentongeneamplification;lane1-penton(1359bp);lane2-negativecontrol.(B)Restrictionenzymedigestion;lane1-pGEX-6P-1;lane2-digestedpentonfrompMD19Tvector;M-Trans2Kplusmarker73 中国农业科学院博士学位论文ChapterⅣFigure4.2PCRpositivecoloniesfrombacterialtransformationofpGEX-6P-1:pentonplasmids.Lane1-10randomselectedcolonies;lane11-positivecontrol;lane12-negativecontrol.4.4.2ConstructionofpreyplasmidsThefullcodingdomainsequenceofpreyproteinswereamplifiedbyPCR(4.3AandB)PCRresultswereanalyzedin1%agarosegel.Figure4.3AmplificationofpreygenesbyPCR.(A)GABARAPL1gene;lane1-GABARAPL1;lane2-positivecontrol;lane3-negativecontrol.(B)RPSAgene;lane1-RPSA;lane2-positivecontrol;lane3-negativecontrol.74 中国农业科学院博士学位论文ChapterⅣThecorrectnucleotidesequenceofpreyproteinswereefficientlyclonedintopEGFP-C1mammalianexpressionvector,cloningpositiveresultswereconfirmedbyPCRusinggenespecificprimersofpreyproteins(Fig.4.4).Figure4.4PCRamplificationofrecombinantplasmidcontainingnucleotidesequenceofpreyproteins;(A)GABARAPL1:pEGFP-C1;lane1,2,3-GABARAPL1;lane4-positivecontrol;lane5-negativecontrol.(B)RPSA:pEGFP-C1;lane1,2,3-RPSA;lane4-positivecontrol;lane5-negativecontrol.4.4.3ExpressionofbaitproteininbacterialcellsProteinexpressionhasbeenwidelyusedsincefromthefirsthumanproteinobtainedinE.colicell(Itakura,Hiroseetal.,1977).E.coliisasuitablehostforexpressingproteinsfromprokaryotesandeukaryotes.ThepentonproteinwasexpressedwellinBL21(DE3)cell,thesizeofpentonproteinis51kDa.Theresultofbaitproteinexpressionisshownin(Fig.4.5).75 中国农业科学院博士学位论文ChapterⅣFigure4.5ExpressionofpentonproteininBL21(DE3)bacterialcells.M-Proteinmarker;lane1,2-pGEX-6P-1:penton.4.4.4ExpressionofbaitandpreyproteinsinmammaliancelllinesTheapplicationofthemammaliancellproteinexpressionhasbecomethedominantrecombinantproteinproductionsysteminlasttwodecades.Inthepresentmammaliancelllinesareusedtoexpressthegenetogetmoreefficientproteinshavinghighbiologicalactivities(Khanetal.,2013).Recombinantproteinsareprimarilyproducedinmammaliancellsbecausethenecessaryandoftencomplextasksofproperassembly,folding,andpost-translationalmodifications(PTMs)canbeonlyefficientlycarriedoutwithmammaliancellmachinery(Wuest,Harcumetal.,2012).Thebaitfusionproteinwasexpressedin293TandDF1celllines.Thepentonfusionproteinshowedhighexpressionin293Tcellline(Fig.4.6).Theexpressionofbaitfusionproteinwasverifiedbywesternblottingusinganti-GSTmousemonoclonalantibody(Fig.4.7and4.8).76 中国农业科学院博士学位论文ChapterⅣFig4.6Expressionofpentonproteinin293TandDF1cellsfor24h;(A)pEGFP-C1:pentongene;(B)pEGFP-C1empty;(C)pEGFP-C1:pentongene;(D)pEGFP-C1empty.77 中国农业科学院博士学位论文ChapterⅣFig.4.7ExpressionofpentongeneinDF1cellFig.4.8ExpressionofpentongeneLane1-pentonprotein(51kDa)in293TcellLane2-pEGFP-C1vector(29kDa)Lane1,2-pentonprotein(51kDa)M-ProteinMarkerLane3-pEGFP-C1vector(29kDa)M-ProteinMarkerThepreyfusionproteinGABARAPL1wasexpressedinVeroandRPSAwasexpressedin293Tcelllines(Fig.4.9).Theexpressionofpreyfusionproteinsweredetectedbywesternblottingtechnique(Fig.4.10).Theanti-GFPmousemonoclonalantibody(CellSignaling,USA)wasusedforprimaryantibody.78 中国农业科学院博士学位论文ChapterⅣFigure4.9ExpressionofpreyproteinspEGFP-C1:GABARAPL1andpEGFP-C1:RPSAinVeroand293Tcells;(A)RPSA;(B)pEGFP-C1empty;(C)GABARAPL1;(D)pEGFP-C1empty.79 中国农业科学院博士学位论文ChapterⅣFigure4.10ExpressionofpreyproteinsinVero(GABARAPL1)and293T(RPSA)cells.Lane1-RPSAprotein;lane2-GABARAPL1protein;lane3-pEGFP-C1empty;M–proteinmarker.4.4.5GSTpulldownassayTheinteractionsbetweenpentonfusionandGABARAPL1,RPSAwereconfirmedusingGST-pulldownassay.Forthattheappropriatevolumeofpreyproteins(preclearedcelllysates)wereincubatedwithpGEX-6P-1:pentonandGSTprotein(10μg)at4°Cfor2-4h,andcentrifugedat13,000rpmfor30sec(supernatantcanbestoredandloadedforcontrol).Thenthebeadswerewashedwith1mlofice-coldwashbuffer(TBS+GSTpulldownlysisbuffer)forfourtimes.Theelutedproteinswereboiledwithequalvolumeof2×SDSgel-loadingbuffer.Theloadedsamplesin12%proteingelthentransferredtoPVDF(Millipore,USA)membraneandfollowedbywesternblotting.InteractionsofEDSVpentonproteinwithpreyproteinsareshownin(Fig.4.11and4.12).80 中国农业科学院博士学位论文ChapterⅣFigure4.11Invitroprotein-proteininteractionsbetweenpentonandGABARAPL1;(A)InteractionbetweenpentonandGABARAPL1withGSTantibody;(B)Protein-proteininteractionbetweenPentonandGABARAPL1withGFPantibody.81 中国农业科学院博士学位论文ChapterⅣFigure4.12Invitroprotein-proteininteractionsofpentonandRPSAbyGSTpull-down.(A)InteractionofpentonandRPSAwithanti-GSTantibody.(B)InteractionofpentonandRPSAwithGFPantibody.82 中国农业科学院博士学位论文CHAPTERVCHAPTERVDISCUSSIONDiagnosisofEDShasbeendoneatthemolecularlevelinmanyvirusinfectedcountries.Thepreviouscompletenucleotidesequencestudiesofeggdropsyndromevirus,allowedthefuturedevelopmentofthePCRassaysfortheEDSVdetection(Hess,Blöckeretal.,1997).SincethenthehexonbasedPCRtechniquewasusedtodetectanddifferentiatetheeggdropsyndromevirusfromfowladenoviruses(RaueandHessetal.,1998).TheconventionalPCRandRT-PCRmethodshavebeenemployedinpreviousstudiestodiagnosetheEDSVinfectionasaspecificandsensitivemethodascomparedtotheserologicalmethods(Banani,Sehatetal.,2007,Schyblietal.,2010).LaterthefluorescentrealtimequantitativePCR(FQRTPCR)assaywasdevelopedforrapiddetectionofhexonproteingeneofeggdropsyndromevirus(Zhen-Yuan,Gangetal.,2012).In2014anewquantitativereal-timepolymerasechain(qRT-PCR)reactionassaywasusedtodetectEDSVDNAinthetestsamples(Schybli,Sigristetal.,2014).Currently,a151bpfragmentoftheEDSVstrain127pentongenewasamplifiedbyPCRwith100%nucleotideidentityandconfirmedbyqRT-PCR(Huang,Tanetal.,2015).However,totestlargenumberofsamples,theqRT-PCR,FQRTPCRandreal-timePCR(RT-PCR)methodsarequiteexpensiveascomparetoRealAmpmethod.Also,theinstrumentswhichrequiredcompletingtheassaysarefewerinnumberorunavailableinmostofplaces.TheresultsofdirectRealAmpclearlyindicatethesuperiorityoftheRealAmpassayindetectionoftheEDSV.Inordertofacilitateimprovedvirusdetection,dilutedsamplesofallantoicfluid,andcellculturesupernatantswereusedinthedirectRealAmpassayandtheresultshaveshowedthatourmethodwassuccessfullyidentifiedtheEDSVfrombothsamples(Fig.2.3aandb).TheRealAmpcannotgiveaclearandrapidresult,whenthe83 中国农业科学院博士学位论文CHAPTERVsamplewashighlyconcentrated(undilutedsample);theproblemliesinthequantityofprimertobemuchdispersedondifferentandmanyDNApieces.Sonocyclinghappened,aself-limitingprocess(Fig.2.3a).Inourstudy,RealAmpcouldamplifiedtheEDSVfibergenebyusingdilutedDNAsamplesaslowas26fg/μl,in30min,whilethePCRwasaround26pg/μl–2.6pg/μlin1hand30min(Fig.2.5).MostoftheamplificationmethodsareneedtousepriorpreparedDNA.Becauseofitssensitiveandspecificability,thedirectRealAmpmethodcanbesuccessfullyusedwithoutpriorrequirementofviralDNA.ThepentonproteinofEDSVis1359bpinlengthandencodes452aminoacids.ThemolecularfunctionofEDSVpentongeneisunknownduringvirusinfectioncycle.Arangeofpentagonscancontributetheinvasionandinternalizationofadenovirusthroughtheactionoftheirarginine-glycine-asparticacid(RGD)orLeu-Asp-Val(LDV)motifsoncellsurfaceintegrins.Recentstudieshaveshownthatwhentheadenovirusfibrinistooshort,thepentoncaneffecttothecombinationoftheadenovirusandthehost.EDSVpentonproteinantigenstructureandfunctionisnotveryclear,thestudyofstructuralproteinsmainlyconcentratedinthehexonprotein,pentonproteinactivityontheaspectofthestudyhasnotbeenreported.Inprotein-proteininteractionsscreening,theyeasttwo-hybridsystemisoneofthemostpopular,costeffectiveinvivogeneticapproach.Awell-characterized,high-qualitycDNAlibraryexpressinghostproteinsiscrucialforstudyinghost-virusinteractionsusingayeasttwo-hybridsystem.Inourcurrentstudy,weconstructedahigh-qualityyeasttwo-hybridcDNAlibrary(prey)usingRNAisolatedfromduckembryofibroblastcellsandpGBKT7-penton(bait)vectors(Fig.3.3).Aftertheastrongyeasttwohybridscreeningofthebothbaitandpreyplasmidsinyeastcells,wefoundthatamolecularinteractionoccurredbetweenthepentonproteinofEDSVandcellularGABARAPL1,RPSA,ACTBandITGA4(Fig.3.13).84 中国农业科学院博士学位论文CHAPTERVFromthesefourcandidateproteins,theinteractionsofGABARAPL1andRPSAwithEDSVpentonproteinwasfurtherconfirmedbyGSTpull-downassay.GSTpulldownresultswereshowedthatthebothpreyproteinsGABARAPL1andRPSAhaveinteractionswithEDSVpentonprotein(Fig.4.11and4.12).Insubsequentinternalizationtohostcells,theadenoviruscapsidproteinsfiberandpentonbasearerespectivelyimportantforbindingtoitsreceptorinadenovirusinfectedcells.Theabundanceandaccessibilityofaprimaryvirusreceptorarecriticalfactorsthatimpactthesusceptibilityofahostcelltovirusinfection(Sharma,Martisetal.,2016).Numbersofstudieswerepublishedaboutadenoviruspentonproteins,itsfunctioninvirusinternalizationandreplicationasoneofthemainstructuralproteins.Theadenovirusesentryoccursbyattachmentviathefiberknobtodifferentreceptorsonthesurfaceofsusceptiblecells,andsubsequentinternalizationviainteractionbetweenthepentonbaseandcellularαvintegrins(B,Metal.,2012).Areviewwasalsohighlightedtheknowledgeofadenovirusdisassemblyandnucleartransportgleanedfromstructural,biophysicalandfunctionalanalysesofadenovirusinteractionswithsolubleandmembrane-associatedhostmolecules(NemerowandStewartetal.,2016).ThesequenceanalysisrevealedthepresenceofaLDVmotifintheHEVpentonbaseaminoacidsequencesimilartomostofthehumanadenoviruses.LDVmotifonthefibronectinhasbeenshowntointeractwiththealpha4beta1integrinsoncells,whichincludeslymphocytesandmonocytes.ThepresenceofLDVmotifinthepentonbaseofHEVimplicatestheinvolvementofalpha4beta1integrinsintheviralinternalizationintohostcells(Suresh,Cyretal.,1995).Thepentonbaseintegrininternalizationreceptorinteractiontriggersleadtovirusinternalizationviaclathrin-mediatedendocytosisandmicropinocytosis.Absentexpression85 中国农业科学院博士学位论文CHAPTERVofcellsurfaceintegrinsmaybetheprimaryreasonfortheinabilityofAdenovirus5(Ad5)basedvectorstotransducecellsoflymphocyticoriginandsomemyeloidcellsbutthisisnottrueforallhematopoieticorigincells.ForefficientuseofAd5-basedtherapeuticvectorsincancersoflymphocyticorigin,itisimportanttoaddressthedefectsincellsurfaceintegrins(Agarwal,Gammonetal.,2017).Theendosomalyticactivityofthevirusisattributedtothepenton,asstudiesshowthatthepentonundergoesaconformationalchangeatlowpH,exposinghydrophobicregionsthatbindnonionicdetergents(Medinakauweetal.,2003).ThecriticalroleofinteractionsbetweenadenoviruspentonRGDmotifsandαvβ5integrinsforefficientvirusinfectionhasbeendemonstratedinstudieswithAdsinwhichRGDhasbeendeletedormutatedablockingantibodyagainstthepentoninhibitsendosomeescape.AtlowpH,theviruspentonisboundtointegrins,whichenablesescapetothecytosol(Bai,Harfeetal.,1993).InadenovirusestheinteractionbetweenpentonRGDsequenceandintegrinhasbeenprovedtobecriticalforbothinternalizationandendosomeescape(Shayakhmetov,Eberlyetal.,2005).AstudyalsodemonstratestheroleofpentonRGDmotifsinfacilitatingtheendosomeescapestepofvirusinfectionandindicatesthatpenton-integrininteractionsareinvolvedininternalizationofcapsid-chimericCD46-interactingAdswithlongfibershafts(Shayakhmetov,Eberlyetal.,2005).Aftertheinitialinteractionoftheviruswiththefiberreceptor,entryofthevirusproceedsviaclathrin-mediatedendocytosis.Theadenovirusattachmentanduptakeintocellsareseparatebutcooperativeeventsthatresultfromtheinteractionofdistinctviralcoatproteinswithinteractionoftheviruspentonbaseproteinwithαvintegrinspromotesvirusinternalization(Wickham,Mathiasetal.,1993).Theprogressofthevirusthroughtheendosomesandintothecellcytoplasmisnormallymediatedbyclathrinandthecoatedpitpathway(Wang,Huangetal.,1998).Theblocking86 中国农业科学院博士学位论文CHAPTERVadenovirusentrybyDAV-1anti-pentonbaseantibodyagainstseveraldifferentadenovirusstudyshowedthatthefulllengthmonoclonalantibody(mAb)ortheantigen-binding(Fab)fragmentsfromDAV-1blockedpentonbasebindingtoA549cells(Stewart,Chiuetal.,1997).Thepentonbasecapsomers,incooperationwithfibers,havebeenfoundtoinducestrongimmunologicalreactionsinpatientsreceivingrecombinantAdingenetherapyprotocols(SmithandScottetal.,1993,Stevenson,Rollenceetal.,1995).Thefunctionofadenoviruspentongeneisresponsibleforintegrinmediatedendocystosisduringthrvirusinternalizationintocellmembraneusingtheirmotifsarginine-glycine-asparticacidandLeu-Asp-Val.HoweverastudywasreportedthatEDSVpentonbaseproteinlackstheintegrinbindingmotifsRGD(Arg-Gly-Asp)andLDV(Leu-Asp-Val)(Rohn,Prusasetal.,1997).ThefunctionofEDSVpentongeneisnotclear.DuringtheEDSVinfection,theeggdropsyndromevirusmediatedbyclathrin-mediatedendocytosis.Thefurtherinvestigationwillneedtoverifythefunctionofthesepreyproteins(GABARAPL1andRPSA)inEDSVinfection.87 中国农业科学院博士学位论文ConclusionConclusionTheRealAmpisamoresensitive,rapidandcosteffectivetechniquethatcanbesuccessfullyemployedtodetectEDSVfibergenebyusingdilutedsamplesaslowas(26fg/μL),withinshorttimespanof15–30minutes.ThiscosteffectivetechniqueascomparedtoRT-PCRandsensitiveascomparedtoconventionalPCRholdspotentialroletodetectthepresenceofotherDNAvirusesorDNAinvarietyoffieldsamples,suchasswabextractsortissuehomogenatesoforgansforprimaryandspecificdetection.TheinteractionbetweenEDSVpentonandfourduckhostcellproteinswereidentifiedbyY2Hscreening(GABARAPL1,RPSA,ACTBandITGA4).TheinteractionofGABARAPL1andRPSAwerefurtherconfirmedbyGSTpulldownassay.ThisisthefirststudytoidentifythereceptororinteractionpartnersofEDSVpentongene,theresultsmayleadtofindouttheroleofpentongeneinEDSVinfection.Furtherthesefindingsneedtoverifyagain,tovalidatethestrengthofinteractionbynewandefficientmethods.Suchasconfocalmicroscopyfortodetecttheco-localizationsofbaitandpreyproteinsinthehostcell.Also,thevirusblockingassaysusingappropriateantibodiesofpreyproteins.88 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中国农业科学院博士学位论文AcknowledgmentAcknowledgementFirstandforemost,IwouldliketothankmysupervisorProfGangLiforgivingmethisopportunitytostudyandworkinhislaboratoryandpursuemygraduatedegree.Thankyouforallhisinvaluableguidance,advice,supportandgivingmeagoldenopportunitytoworkunderhissupervisioncriticalsupport,adviceandencouragement.Besidesmysupervisor,IwouldliketothanktoProfCuiandProfZhufortheirkindlyvaluableguidance.MyheartiestappreciationalsogoestomyChinesefriendandlabmates:LingnaZhao,WeiLiuandWeidongLin,whogavemeencouragement,cooperation,friendshipallthetimewheneverIhavedifficultyinmylifeinChina.Iwouldalsoliketothankallofthemembersofourlab:CunLiu,JingWang,ChangLiu,QiLiu,XiuboLi,JiaYangLifortheircooperationandhelp.IwouldalsoliketoespeciallythankstohonorableteachersDrZhao,DrShi,DrHongJia,DrXiaoyuGuo,ShaohuaHouandTingXin,andneighborlabstudents:QiangHou,XiukunCui,MingLi,XixiWang,ShanZhang,MeirongChen,TianqiSong,foralltheirkindness,cooperation,supportandhelp.Ireallywanttoexpressmydeeplythankstomycountrymatesmyclosefriends:TussipkhanDilnur,SagynbayGulinur,BolatDegdar,NurbekSamal,ShalgynbayevaAydanaandYerenAydingulwhogavemeencouragement,endlesssupport,friendshipandwarmlyloveallthetimewheneverIhavedifficultyinmyworkorlife.IwouldalsoliketothanktoChineseGovernmentofScholarshipConsulforgivingmeabigopportunitytostudyinChina.IwouldliketoshowmygreatestgratitudetoMs.ZhangYanhua,ZhangYoujinandMs.GracefromlnternationalcooperationstudentofficeofGSCAASfortheirvaluable,indispensablehelpandinspirationduringrnystudytimeofChina.100 中国农业科学院博士学位论文AcknowledgmentIwouldliketodedicatethisworktomybelovedgrandmother,parents,brothersandsisterswhogavegreatlyencouragementandsupporttocompletemydecision.Iwouldliketoexpressmyenormousindebtednessandprofoundgratitudetomybelovedparentsfortheirencouragement,kindness,andmoralsupportthankyoudearmyMomandDad.Finally,IexpressmyhumblethankstoallwhohelpeddirectlyandindirectlyformystudyinChina.Ihavereceivedkindhelp,supportfromseveralpeoplefortheachievementandcomplementofPhDdegree.Withoutsuchkindsupport,itwouldnotbecompletemywork.101 中国农业科学院博士学位论文AuthorBiographyAuthorBiographyPERSONALDATAFamilyname:ZheneyNationality:KazakhstanGivenname:MakayDateofBirth:Jun10,1989Sex:FemaleE-mail:Maritalstatus:SingleAddress:EDUCATION2013-2017:AppearedforPhDinPreventiveVeterinaryMedicine,InstituteofAnimalScience,CAAS,China.2011-2013:MasterofVeterinaryScience(VeterinaryMedicine),FacultyofVeterinary,KazakhNationalAgrarianUniversity,Kazakhstan.2007-2011:BachelorofBiotechnology(Biotechnology)FacultyofVeterinaryandBiotechnology,KazakhNationalAgrarianUniversity,Kazakhstan.PUBLICATIONANDPRESENTATIONMakayZheney,ZhambulKaziyev,GulmiraKassenova,LingnaZhaO,WeiLiu,LinLiangandGangLi.Real-timefluorescenceloop-mediatedisothermalamplificationassayfordirectdetectionofeggdropsyndromevirus(underreview).MakayZheney,ZhambulKaziyev,YlgekbaevaGulnaz,LingnaZhao,WeiLiu,andGangLi.InteractionofEggDropSyndromeVirusPentonProteinwithDuckHostProteinsGABARAPL1andRPSA(inpreparation).PRESENTATIONMakayZheney.IdentificationofbirdspeciesusingfeathersbyDNAbarcodingtechnique.TheBranchofAnimalInformation,ChineseAssociationofAnimalScienceandVeterinarythMedicine.The10Symposiumproceedings,Beijing2015.102 中国农业科学院博士学位论文AdditionalmaterialsAdditionalmaterialsTable1.1ListofspeciesinthegenusAtadenovirus.NameofSpeciesNameoftypesSequenceaccessionAssignednumbersabbreviationsBovineadenovirus4[AF036092=NC_002685](BAdV-4)Bovineadenovirus5[AF207658](BAdV-5)BovineadenovirusDBovineadenovirus8[AF238233](BAdV-8)Bovineadenovirus[AF238880](BAdV-Rus)strainRusDuckadenovirusADuckadenovirus1[Y09598=AC_000004](DAdV-1)OvineadenovirusDGoatadenovirus1[AF207660](GAdV-1)Ovineadenovirus7[U40839=NC_004037](OAdV-7)PossumadenovirusPossumadenovirus[AF338822](PoAdV-1)1SnakeadenovirusASnakeadenovirus1[DQ106414=NC_009989](SnAdV-1)103 中国农业科学院博士学位论文AdditionalmaterialsTable3.5SimilarproteinstoGABAtypeareceptorassociatedproteinlike1.Name/GeneDescriptionLocationAliasesIDGABAtypeAreceptorChromosome12,APG8-LIKE,GABARAPL1associatedproteinlike1NC_000012.12APG8L,ATG8,ID:23710[Homosapiens(human)](10212841..10223128)ATG8B,ATG8L,GEC1gamma-aminobutyricacidChromosome6,3110025G09Rik,Gabarapl1(GABA)ANC_000072.69130422N19Rik,ID:57436receptor-associated(129533192..129542331)AI196471,protein-like1[MusApg8l,Atg8l,musculus(housemouse)]GECI,MNCb-0091GABAtypeAreceptorChromosome4,Gec1Gabarapl1associatedproteinlike1NC_005103.4ID:689161[Rattusnorvegicus(163293724..163302866)(Norwayrat)]GABAtypeAreceptorChromosome5,GABARAPL1associatedproteinlike1AC_000162.1ID:338472[Bostaurus(cattle)](100205182..100215034,complement)GABAtypeAreceptorChromosome11,EGK_03360GABARAPL1associatedproteinlike1NC_027903.1ID:717640[Macacamulatta(Rhesus(10565058..10575844)monkey)]GABAtypeAreceptorChromosome1,GABARAPL3GABARAPL1associatedproteinlike1NC_006088.4ID:769118[Gallusgallus(chicken)](78391075..78401001,complement)GABA(A)Chromosome7,apg8l,atg8,gabarapl1receptor-associatedNC_030683.1atg8l,gec1ID:595040proteinlike1[Xenopus(10488959..10500650,tropicalis(tropicalclawedcomplement)frog)]GABAtypeAreceptorGEC-1,GEC1Gabarapl1associatedproteinlike1ID:[Caviaporcellus100135524(domesticguineapig)]GABAtypeAreceptorChromosome12,GABARAPL1associatedproteinlike1NC_006479.4ID:737951[Pantroglodytes(10646861..10657168)(chimpanzee)]104 中国农业科学院博士学位论文AdditionalmaterialsTable3.6SimilarproteinstoAnasplatyrhynchos40SribosomalproteinSA.Name/GeneDescriptionLocationAliasesIDRpsaribosomalproteinSAChromosome3,37LRP,67LR,ID:3921[HomosapiensNC_000003.12ICAS,LAMBR,(human)](39406689..39412542)LAMR1,LBP,LBP/p40,LRP,LRP/LR,NEM/1CHD4,SA,lamR,p40RpsaribosomalproteinSAChromosome9,67kDa,67lr,ID:16785[Musmusculus(houseNC_000075.6AL022858,Lamr,mouse)](120127766..120132369)Lamr1,Lamrl1,MLR,P40,P40-3,P40-8RpsaribosomalproteinSAChromosome8,Lamr1ID:29236[RattusnorvegicusNC_005107.4(Norwayrat)](128806053..128809987)rpsaribosomalproteinSAChromosome6,lamr1,zgc:55831,ID:394027[DaniorerioNC_007117.7zgc:77824(zebrafish)](7713342..7720332,complement)RPSAribosomalproteinSAChromosome22,LAMR1ID:281898[Bostaurus(cattle)]AC_000179.1(12728175..12741658)RpsaribosomalproteinSAI79_013226,37lrp,ID:[Cricetulusgriseus67lr,Lamr,Lamr1,100689045(Chinesehamster)]Lbp/p40,Lrp/lrRPSAribosomalproteinSAChromosome13,LAMR1ID:641351[Susscrofa(pig)]NC_010455.5(24072551..24081536)RPSAribosomalproteinSAChromosome2,37LRP,67LR,ID:395181[GallusgallusNC_006089.4DMRT1,LBP/p40,(chicken)](44510172..44514919,LRP/LRP58,lamR,complement)RPSARPSAribosomalproteinSAChromosome19,LamR1ID:[Ovisaries(sheep)]NC_019476.2100125628(12624508..12636310)105 中国农业科学院博士学位论文AdditionalmaterialsTable3.7SimilarproteinstoAnasplatyrhynchos,Integrinsubunitalpha4.SimilarproteinsOrganismsUPI0005213787Pygoscelisadeliae(Adeliepenguin)UPI000522B1BECharadriusvociferus(Killdeer)(Aegialitisvocifera)UPI000678C895Haliaeetusalbicilla(White-tailedsea-eagle)UPI0008470144Falcoperegrinus(Peregrinefalcon)UPI000510A6CFManacusvitellinus(golden-collaredmanakin)UPI000523578DPelecanuscrispus(Dalmatianpelican)R0LC44Nestornotabilis(Kea)A0A093QSJ5Coliusstriatus(Speckledmousebird)Table3.8SimilarproteinstoAnasplatyrhynchosactinbeta(ACTB).SimilarproteinsOrganismsP02572Drosophilamelanogaster(Fruitfly)B4QCQ5Drosophilasimulans(Fruitfly)B3N3G9Drosophilaerecta(Fruitfly)B4II24Drosophilasechellia(Fruitfly)B4MPR6Drosophilawillistoni(Fruitfly)A0A1W4UNW6Drosophilaficusphila(Fruitfly)L9KVG3Tupaiachinensis(Chinesetreeshrew)P41339Limuluspolyphemus(Atlantichorseshoecrab)UPI0006B10654Physarumpolycephalum(Slimemold)P02576Escherichiacoli106