多肽设计原理和方法PPT课件.ppt

多肽设计原则和方法1.多肽抗原的特点l含有的epitope数目少；l抗原性比融合蛋白弱；l产生的抗原特异性通常比融合蛋白强；l是一种半抗原，需要连接载体蛋白；2.多肽设计的原则lHydrophilicity,tobesuretheantigenisonthesurfaceoftheprotein.lAntigenicity,aminoacidprovidesgoodbonds.lN-orC-terminalpeptide,fortheyareexposedinmostproteins.lContainsMHCIIpreferredmotifs.SuchasD,Y,P,andFwhoappearsinMHCmoleculesmostfrequently.lNohomologywithotherproteinsbyblast.lPreferthecytoplasmicdomainforatransmembraneprotein,fortheyaremoreimmunogeniclAvoidglycosylationandphosphorylationsitesifpossiblelAvoidcystininthemiddlepartofthepeptide3.20种氨基酸的抗原性比较L0.75 K-0.05H0.65 P-0.05D0.31 R-0.07T0.21 W-0.11A0.12 C-0.12E0.06 N-0.14Y0.01 G-0.18V-0.01 M-0.38S-0.01 F-0.77Q-0.03 I-2.924.什么时候选用多肽做抗原l我们没有基因的抗原；l有基因，但是蛋白质和其他蛋白质有较强的同源性，或者蛋白质属于同源性较强的蛋白家族；l有基因，需要区分该基因编码的蛋白质加工后的不同亚型；l抗原是修饰蛋白质，包括磷酸化，乙酰基化蛋白质等5.案例1 我们没有的蛋白质lNM_004445，EPHB6lMATEGAAQLGNRVAGMVCSLWVLLLVSSVLALEEVLLDTTGETSEIGWLTYPPGGWDEVSVLDDQRRLTRTFEACHVAGAPPGTGQDNWLQTHFVERRGAQRAHIRLHFSVRACSSLGVSGGTCRETFTLYYRQAEEPDSPDSVSSWHLKRWTKVDTIAADESFPSSSSSSSSSSSAAWAVGPHGAGQRAGLQLNVKERSFGPLTQRGFYVAFQDTGACLALVAVRLFSYTCPAVLRSFASFPETQASGAGGASLVAAVGTCVAHAEPEEDGVGGQAGGSPPRLHCNGEGKWMVAVGGCRCQPGYQPARGDKACQACPRGLYKSSAGNAPCSPCPARSHAPNPAAPVCPCLEGFYRASSDPPEAPCTGPPSAPQELWFEVQGSALMLHWRLPRELGGRGDLLFNVVCKECEGRQEPASGGGGTCHRCRDEVHFDPRQRGLTESRVLVGGLRAHVPYILEVQAVNGVSELSPDPPQAAAINVSTSHEVPSAVPVVHQVSRASNSITVSWPQPDQTNGNILDYQLRYYDQAEDESHSFTLTSETNTATVTQLSPGHIYGFQVRARTAAGHGPYGGKVYFQTLPQGELSSQLPERLSLVIGSILGALAFLLLAAITVLAVVFQRKRRGTGYTEQLQQYSSPGLGVKYYIDPSTYEDPCQAIRELAREVDPAYIKIEEVIGTGSFGEVRQGRLQPRGRREQTVAIQALWAGGAESLQMTFLGRAAVLGQFQHPNILRLEGVVTKSRPLMVLTEFMELGPLDSFLRQREGQFSSLQLVAMQRGVAAAMQYLSSFAFVHRSLSAHSVLVNSHLVCKVARLGHSPQGPSCLLRWAAPEVIAHGKHTTSSDVWSFGILMWEVMSYGERPYWDMSEQEVLNAIEQEFRLPPPPGCPPGLHLLMLDTWQKDRARRPHFDQLVAAFDKMIRKPDTLQAGGDPGERPSQALLTPVALDFPCLDSPQAWLSAIGLECYQDNFSKFGLCTFSDVAQLSLEDLPALGITLAGHQKKLLHHIQLLQQHLRQQGSVEVl共1021aa，N-15aa为信号肽6.案例1 我们没有的蛋白质（2）lWDEVSVLDDQRRLTRTFEAC7.案例1 我们没有的蛋白质（3）lGENEID:2051EPHB6|EPHreceptorB6HomosapienslQuery1WDEVSVLDDQRRLTRTFEAC20lWDEVSVLDDQRRLTRTFEAClSbjct41WDEVSVLDDQRRLTRTFEAC60lGENEID:814CAMK4|calcium/calmodulin-dependentproteinkinaseIVlQuery1lWDEVS-VLDDQRRLTRTFEA19lWDEVSVLD+RLTTF+ASbjct69WDEVSLNAKDLVRKLIVLDPKKRLT-TFQA978.案例2 家族性蛋白的多肽设计（transgelin）lThetransgelinfamilyisagroupofproteinsthatbelongto22kdactin-relatedcorpninsuperfamily.lOfallthreeisoforms,transgelin1isthebestcharacterized.Transgelin1,alsoknownasSM22alpha,isaspecificmarkerfordifferentiatedsmoothmusclecells.lTransgelin2,alsoknownasSM22beta,isexpressedbybothsmoothmuscleandnon-smoothmusclecellsinatemporallyandspatiallyregulatedpattern.lTrangenlin3,alsoknownasNP25,isonlyfoundinhighlydifferentiatedneuronalcells.9.案例2家族性蛋白的多肽设计（transgelin）210.案例2 家族性蛋白的多肽设计（transgelin）3lTransgelin1:KYDEELEERLVEWIIVQClTransgelin2:QPGRENFQNWLKDGTVLC11.案例3 家族性蛋白的多肽设计 RAB3A,3B,3ClSequence2:NM_002866,RAB3A,memberRASoncogenefamilylSequence3:NM_002867,RAB3B,memberRASoncogenefamilylSequence4:NM_138453,RAB3C,memberRASoncogenefamily12.案例4 家族性蛋白的多肽设计actin对应的处理方法？13.案例5 同一蛋白质不同亚型的多肽设计 NFKB1lNFKB1,lNUCLEAR FACTOR KAPPA-B,SUBUNIT 1;NFKB1lAlternative titles;symbols lTRANSCRIPTION FACTOR NFKB1NUCLEAR FACTOR OF KAPPA LIGHT CHAIN GENE ENHANCER IN B CELLS 1NFKB p105,INCLUDEDNFKB p50,INCLUDED14.案例5 同一蛋白质不同亚型的多肽设计 NFKB1lThisgeneencodesa105kDproteinwhichcanundergocotranslationalprocessingbythe26Sproteasometoproducea50kDprotein.The105kDproteinisaRelprotein-specifictranscriptioninhibitorandthe50kDproteinisaDNAbindingsubunitoftheNF-kappa-B(NFKB)proteincomplex.l1433aminoacidsoffulllength968p105proteinformp50.15.案例5 同一蛋白质不同亚型的多肽设计 NFKB1lMAEDDPYLGRPEQMFHLDPSLTHTIFNPEVFQPQMALPTADGPYLQILEQPKQRGFRFRYVCEGPSHGGLPGASSEKNKKSYPQVKICNYVGPAKVIVQLVTNGKNIHLHAHSLVGKHCEDGICTVTAGPKDMVVGFANLGILHVTKKKVFETLEARMTEACIRGYNPGLLVHPDLAYLQAEGGGDRQLGDREKELIRQAALQQTKEMDLSVVRLMFTAFLPDSTGSFTRRLEPVVSDAIYDSKAPNASNLKIVRMDRTAGCVTGGEEIYLLCDKVQKDDIQIRFYEEEENGGVWEGFGDFSPTDVHRQFAIVFKTPKYKDINITKPASVFVQLRRKSDLETSEPKPFLYYPEIKDKEEVQRKRQKLMPNFSDSFGGGSGAGAGGGGMFGSGGGGGGTGSTGPGYSFPHYGFPTYGGITFHPGTTKSNAGMKHGTMDTESKKDPEGCDKSDDKNTVNLFGKVIETTEQDQEPSEATVGNGEVTLTYATGTKEESAGVQDNLFLEKAMQLAKRHANALFDYAVTGDVKMLLAVQRHLTAVQDENGDSVLHLAIIHLHSQLVRDLLEVTSGLISDDIINMRNDLYQTPLHLAVITKQEDVVEDLLRAGADLSLLDRLGNSVLHLAAKEGHDKVLSILLKHKKAALLLDHPNGDGLNAIHLAMMSNSLPCLLLLVAAGADVNAQEQKSGRTALHLAVEHDNISLAGCLLLEGDAHVDSTTYDGTTPLHIAAGRGSTRLAALLKAAGADPLVENFEPLYDLDDSWENAGEDEGVVPGTTPLDMATSWQVFDILNGKPYEPEFTSDDLLAQGDMKQLAEDVKLQLYKLLEIPDPDKNWATLAQKLGLGILNNAFRLSPAPSKTLMDNYEVSGGTVRELVEALRQMGYTEAIEVIQAASSPVKTTSQAHSLPLSPASTRQQIDELRDSDSVCDSGVETSFRKLSFTESLTSGASLLTLNKMPHDYGQEGPLEGKI16.案例5 同一蛋白质不同亚型的多肽设计 NFKB117.案例6 同一蛋白质不同亚型的多肽设计 CXCR3lTheCXCR3proteinbelongstoGprotein-coupledreceptorfamily.Severalalternativelysplicedtranscriptvariantsencodingdistinctisoformshavebeenreported.TheCXCR3Bproteincontains416aminoacidsandhasalongerNterminusthatdiffersfromtheoriginal368-aminoacidCXCR3protein,CXCR3A,inthefirst52residues.Thisantibody,60065-1-Ig,isspecificforCXCR3B,notbindingCXCR3A.18.案例6 同一蛋白质不同亚型的多肽设计 CXCR360065-2-Ig，45kdAbcam，41kd19.多肽抗体的生产流程及注意事项l确定待设计模板收集市场信息，组织特异性表达抗体和其它背景知识；lBlast比较蛋白质在整个（人）蛋白组的同源性l综合特异性，抗原性，膜蛋白定位等信息设计多肽l再次blast确定多肽的抗原性，特异性l输入predictedMW，targetedtissue，antigenintroduction等资料l输入westernbloting检测组织和IHC检测组织20.