应用酵母双杂交技术筛选人白细胞中与NS5ATP9蛋白结合蛋白

WorldChinJDigestol2004April;12(4):828-831ISSN1009-3079CN14-1260/RPOBox2345Beijing100023,ChinaFax:[phone]Email:[e-mail]•VIRALHEPATITIS•ScreeningandcloningofgenescodingforleukocyteproteinsinteractingwithNS5ATP9byyeast-twohybridtechniqueQiangLi,Yao-DongLiang,JunCheng,LinWang,JianZhang,QingShao,MingLiu,Ming-LiangChengQiangLi,Yao-DongLiang,JunCheng,LinWang,JianZhang,QingShao,MingLiu,GeneTherapyResearchCenter,InstituteofInfectiousDiseases,The302HospitalofPLA,Beijing100039,ChinaMing-LiangCheng,TheFirstAffiliatedHospital,GuiyangMedicalCollege,Guiyang550004,GuizhouProvince,ChinaSupportedbyGrantsfromtheNationalNaturalScientificFoundation,No.C03011402,No.C30070689,No.C39970674,No.C39900130;andthe9.5ResearchandTechniqueFoundationofPLA,No.98D063;andtheLaunchingFoundationforStudentStudyingAbroadofPLA,No.98H038;andthe10.5YouthResearchandTechniqueFoundationofPLA,No.01Q138;andthe10.5ResearchandTechniqueFoundationofPLA,No.01MB135Correspondenceto:Dr.JunCheng,GeneTherapyResearchCenter,InstituteofInfectiousDiseases,The302HospitalofPLA,100XisihuanzhongRoad,Beijing100039,[e-mail]:2003-11-13Accepted:2003-12-16AbstractAIM:ToinvestigatethebiologicalfunctionsofNS5ATP9,andtoscreenproteinsinleukocytesinteractingNS5ATP9proteinbyyeast-twohybrid.METHODS:TheNS5ATP9genewasamplifiedbypoly-merasechainreaction(PCR)andNS5ATP9baitplasmidwasconstructedbyusingyeast-twohybridsystem3,andtheyeastAH109wasthentransformed.ThetransformedyeastmatedwithyeastY187containingleukocytescDNAlibraryplasmidin2×YPDAmedium.Diploidyeastwasplatedonsyntheticdropoutnutrientmedium(SD/-Trp-Leu-His-Ade)andsyntheticdropoutnutrientmedium(SD/-Trp-Leu-His-Ade)containingX-α-galforselectingtwotimesandscreening.AfterextractingandsequencingofplasmidDNAfrombluecolonies,weunderwentanalysisbybioinformatics.RESULTS:Fortysixcoloniesweresequenced,amongwhichthirteencolonieswereHomosapiensimmunoglobulinlightchain,tenubiquitin,twoferritinheavychain,elevenHomosapiensrearrangedimmunoglobulinlambdalightchain,one14-3-3familyprotein,oneMeningococcusPorAprotein,threeRNApolymeraseIII,onetobaccomitogenactivatedproteinkinase,twocytochromeP450II,oneSLIT2protein,andonedependent-proteinkinasecatalylicsubunit.CONCLUSION:GenesofNS5ATP9interactingproteinsinleukocytesaresuccessfullyclonedandtheresultsbringsomenewcluesforstudyingthebiologicalfunctionsofNS5ATP9andassociatedproteins.LiQ,LiangYD,ChengJ,WangL,ZhangJ,ShaoQ,LiuM,ChengML.ScreeningandcloningofgenescodingforleukocyteproteinsinteractingwithNS5ATP9byyeast-twohybridtechnique.ShijieHuarenXiaohuaZazhi2004;12(4):828-831ααααααααα829M2000677663797178771000bp750bp1ShiratoriY,ShiinaS,ImamuraM,KatoN,KanaiF,OkudairaT,TerataniT,TohgoG,TodaN,OhashiM.CharacteristicdifferenceofhepatocellularcarcinomabetweenhepatitisB-andC-viralinfectioninJapan.Hepatology1995;22:1883-18852LiK,WangL,ChengJ,ZhangLX,DuanHJ,LuYY,YangJZ,LiuY,HongY,XiaXB,WangG,DongJ,LiL,ZhongYW,ChenJM.Screeningandcloningofgeneofhepatocyteprotein1interactionwithHCVcoreprotein.ShijieHuarenXiaohuaZazhi2001;9:1379-13833LiK,WangL,ChengJ,LuYY,ZhangLX,LiuY,DuanHJ.ExpressionofNS2geneofhepatitisCvirusfromyeasttwohybrid‘bait’vectorinyeast.ShijieHuarenXiaohuaZazhi2002;10:129-1324WangXZ,JiangXR,ChenXC,ChenZX,LiD,LinJY,TaoQM.SeekproteinwhichcaninteractwithhepatitisBvirusXpro-teinfromhumanlivercDNAlibrarybyyeasttwo-hybridsystem.WorldJGastroenterol2002;8:95-985NagpalS,GhosnCR,ChandraratnaRA.Identificationofnuclearreceptorinteractingproteinsusingyeasttwo-hybridtechnology.MethodsMolBiol2001;176:359-3766SerebriiskiiIG,TobyGG,FinleyRLJr,GolemisEA.Genomicanalysisutilizingtheyeasttwo-hybridsystem.MethodsMolBiol2001;175:415-4547GietzRD,WoodsRA.Screeningforprotein-proteininterac-tionsintheyeasttwo-hybridsystem.MethodsMolBiol2002;185:471-4868ZhenZ.Progressinproteomics.ShengwuGongchengXuebao2001;17:491-4939YaffeMB,RittingerK,VoliniaS,CaronPR,AitkenA,LeffersH,GamblinSJ,SmerdonSJ,CantleyLC.Thestructuralbasisfor14-3-3:phosphopeptidebindingspecificity.Cell1997;91:961-97110ConklinDS,GalaktionovK,BeachD.14-3-3proteinsassoci-atewithcdc25phosphatases.ProcNatlAcadSciUSA1995;92:7892-289611LeffersH,MadsenP,RasmussenHH,HonoreB,AndersenAH,WalbumE,VandekerckhoveJ,CelisJE.Molecularclon-ingandexpressionofthetransformationsensitiveepithelialmarkerstratifin.AmemberofaproteinfamilythathasbeeninvolvedintheproteinkinaseCsignalingpathway.JMolBiol1993;231:982-99812YuT,RobbVA,SinghV,GutmannDH,NewshamIF.The4.1/ezrin/radixin/moesindomainoftheDAL-1/Protein4.1Btumoursuppressorinteractswith14-3-3proteins.BiochemJ2002;365:783-789830ISSN1009-3079CN14-1260/R13O’KellyI,ButlerMH,ZilberbergN,GoldsteinSA.Forwardtransport.14-3-3bindingovercomesretentioninendoplas-micreticulumbydibasicsignals.Cell2002;111:577-58814LiY,InokiK,YeungR,GuanKL.RegulationofTSC2by14-3-3binding.JBiolChem2002;277:44593-4459615ShumwaySD,LiY,XiongY.14-3-3betabindstoandnegativelyregulatesthetuberoussclerosiscomplex2(TSC2)tumorsup-pressorgeneproduct,tuberin.JBiolChem2003;278:2089-209216LiY,InokiK,Va