抗原设计

PredictthelocationsofPredictthelocationsofepitopeepitopeforproteinsequenceforproteinsequenceAntigenAntigen的取得的取得1.1.ChimericChimericgeneproduct(geneproduct(如如GSTfusionprotein)GSTfusionprotein)2.2.Taggedgeneproduct(Taggedgeneproduct(如如HisHis--tagprotein)tagprotein)NativeproteinisjustnotavailableNativeproteinisjustnotavailable••TheproteinistoxicTheproteinistoxic••ProteinSolubilityProteinSolubility••ProteinPurificationProteinPurification••ProteinStabilityProteinStability3.Syntheticpeptide3.Syntheticpeptide，再予以接合，再予以接合carrierproteincarrierprotein（如（如KLH,BSAKLH,BSA））--簡便有效簡便有效EpitopeEpitope••蛋白質表面部分可以使免疫系統產生抗體蛋白質表面部分可以使免疫系統產生抗體的區域的區域::抗原的表位抗原的表位((epitopeepitope))••由由66－－1212氨基酸或碳水基團組成，它可以是氨基酸或碳水基團組成，它可以是由連續序列（蛋白質一級結構）組成或由由連續序列（蛋白質一級結構）組成或由不連續的蛋白質三維結構組成不連續的蛋白質三維結構組成TypesofPeptideEpitopeTypesofPeptideEpitopeLinearLinearBcellEpitopeBcellEpitopeTcellEpitopeTcellEpitopeAntibodyAntibodyoror““BcellBcell””EpitopeEpitopeConformationalConformationalNonNon--ConformationalConformationalClassIIClassIIMHCsMHCsProfessionalAntigenProfessionalAntigenPresentingcellsPresentingcellsForeignproteinsForeignproteins88--20aminoacids20aminoacidsClassIClassIMHCsMHCsallcellsallcellsForeignandselfproteinsForeignandselfproteins88--10aminoacids10aminoacidsDiscontinuousEpitopesDiscontinuousEpitopes••ConstitutiveresiduesarenonConstitutiveresiduesarenon--sequentialinthesequentialintheprimarysequence.primarysequence.••Highlyconformationaldependant.Highlyconformationaldependant.••Accountforapprox.90%ofAccountforapprox.90%ofepitopesepitopesonagivenantigenic(globular)protein.onagivenantigenic(globular)protein.LinearEpitopesLinearEpitopes••ConstitutiveresiduesaresequentialintheprimaryConstitutiveresiduesaresequentialintheprimarysequenceoftheprotein.sequenceoftheprotein.••FewerconformationalconstraintsonFewerconformationalconstraintsonAbAbrecognition.recognition.••CanbedefinedthroughELISAbasedCanbedefinedthroughELISAbasedepitopemappingtechniques(PEPSCAN).epitopemappingtechniques(PEPSCAN).AALLRLAVLAALLRLAVLALLRLAVLLALLRLAVLLLLRLAVLLPLLRLAVLLPLRLAVLLPLLRLAVLLPLRLAVLLPLARLAVLLPLAOverlappingpeptideOverlappingpeptidefragmentsofwholefragmentsofwholeproteinproteinLinearEpitopePredictionLinearEpitopePrediction••NotpossibletopredictdiscontinuousNotpossibletopredictdiscontinuousepitopesepitopesfromprimaryproteinsequence,fromprimaryproteinsequence,tertiarytertiarystructurerequiredstructurerequired..••一般來說蛋白質的一般來說蛋白質的NN端及端及CC端是很好的端是很好的EpitopesEpitopes區域區域••SignalSignalpeptidepeptideLinearLinearEpitopeEpitopePredictionPrediction1.親水性(Hydrophilicity)常用的有五種方法：Nozaki-Tanfordscale，Hopp-Woodsscale，Eisenbergscale，Kyte-Doolittlescale，HPLCscale。其中尤以Hopp-Woods最為有名。疏水性殘基一般埋在蛋白內部，而親水性殘基位於表面，因此蛋白的親水部位與蛋白抗原表位有密切的聯繫。Hopp-Woods是以殘基由有機相環境轉移到水相環境的自由能為依據計算各個氨基酸的親水性。現已明確，親水性部位與epitope並無很好的一致性，即高親水性部位不一定是epitope，epitope也不一定是親水性部位。2.可及性(Accessibility)如Janin可及性參數，指蛋白質抗原中氨基酸殘基被溶劑分子接觸的可能性。它反映了蛋白質抗原內、外各層殘基的分佈情況。3.抗原性(Antigenicity)對20個已研究得很透的蛋白質的69個連續位點的606個氨基酸統計分析，Welling建立了抗原性刻度。每個氨基酸用出現在抗原區的頻率描述，此頻率除以各氨基酸在所有蛋白質中的頻率就可推出此刻度值。疏水性氨基酸殘基對epitope形成亦有貢獻。缺點是其所用的數據有限。4.可塑性(Flexibility)指蛋白抗原構象不是剛性不變的，其多肽鏈骨架有一定程度的活動性，活動性強的氨基酸殘基即可塑性大的位點，易形成抗原表位。Karplas和Schulz基於已知結構的31個蛋白質的Cx的溫度β-factor，發展了一種預測蛋白質片段活動性的方法。5.二級結構預測(Secondarystructure)認為β轉角結構為凸出結構，多出現在蛋白質抗原表面，利於與抗體嵌合，較可能成為epitope。而α螺旋、β-strand結構規則不易形變，較難嵌和抗體，一般不作為抗原表位。可預測蛋白質β轉角的有Chou-Fasman，Garnier，Cohen等方法。其中各種方法預測的成功率均不超過65%。LinearLinearEpitopeEpitopePredictionPredictionDirectionsforthepredictionofDirectionsforthepredictionofAntigenicpeptidesAntigenicpeptides1.Antigenicpeptidesshouldbelocatedinsolventaccessibleregionsandcontainbothhydrophobicandhydrophilicresidues.Forproteinsofknown3DstructuresolventaccessibilitycanbedeterminedusingavarietyofprogramssuchasDSSP,NACESSorWHATIF,amongothers.AwebservertocalculatesolventaccessibilityusingWhatifisalsoavailablefollowingthislinkIfthe3Dstructureisnotknown,useanyofthefollowingwebserverstopredictaccessibilities:PHD,JPRED,PredAcc(c),ACCpro,2.PreferablyselectpeptideslyinginlongloopsconnectingSecondaryStructure(SS)motifs,avoidingpeptideslocatedinhelicalregions.ThiswillincreasetheoddsthattheAbrecognizesthenativeprotein.Forproteinwithknown3Dcoordinates,SScanbeobtainedfromthesequencelinkoftherelevantentryattheBrookhavendatabank.ThePDBsumserveralsoofferSSanalysisofpdbrecords.Whennostructureisavailablesecondarystructurepredictionscanbeobtainedfromanyofthefollowingservers:PHD,JPRED,PSI-PRED,NNSP,YASPIN,HYPROSPIIetc3.Whenpossible,choosepeptidesthatareintheN-andC-terminalregionoftheprotein.BecausetheN-andC-terminalregionsofproteinsareusuallysolventaccessibleandunstructured,Absagainstthoseregionsarealsolikelytorecognizethenativeprotein.4.ForcellsurfaceglycoproteinseliminatefrominitialpeptidesthosecontainingconsesussitesforN-glycosylation.N-glycosylationsitescanbedetectedusingScanprosite,orNetNGlycCase1Case11WS6=extendedstrand,=turn,=disulfidebond=alphahelix,=310helix,=pihelix,1WS6TheStructureofThermusthermphillusHB8hypotheticalproteinTTHA0928Release2006-02-07Resolution2.50[Å]Query=1WS6:A|PDBID|CHAIN|SEQUENCE(171letters)Scor