抗体噬菌体展示技术.ppt

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1、AntibodyPhageDisplayMeilingXiong20180629ContentsIntroductionofAbphageDisplayTechnologyAbFormatsforPhageDisplayAbLibrariesConstructionPhageAbSelectionMethods&StrategiesPhageAbScreeningApplicationsInvitroAffinityMaturationExpression&PurificationofPhageAbFragment

2、sIntroductionofPhageDisplayTechnologyTheFfbacteriophagestructureIntroductionofPhageDisplayTechnologyTheschemeofphagemidvectorIGregion:intergenicregion,usuallycontainsthepackingsequenceandreplicationoriginofminusandplusstrandsMoleculartag:tofacilitatelibraryscre

3、eningandforproteinanalysisRestrictionenzymerecognitionsites:usefulforDNArecombinationandgenemanipulation;multiplecloningsites(MCS)Coatprotein:PIII(largerprotein,lessthan5copies,)PVIII(morethan5copies,decreasedlength)AmbercodonTAG:supEstrains(glutamicacidcodon),

4、non-suppressorstrains(stopcodon)ProteasecleavagesitePromoterSignalpeptides:phageproteintranslocation,crucialfordisplaylevelSelectivemarker:forselectionofinfectedhostcellsIntroductionofPhageDisplayTechnologyNonlyticfilamentousphageisthemostoftenusedforphagedispl

5、ay,primarilytheM13andFdstrains.Proteinstobeselectedareinfusedtoallfivecoatproteins,withpIIIandpVIIImostcommonlyused.pIIIproteinisessentialforinfectionofbacteriaHelperphage:wild-typepIIIhelperphageandspecialhelperphageAntigenimmobilizedonmagneticbeads,polystryre

6、nesurfaces,oroncolumns,orisusedinsolutionasbiotinylatedantigenandlatercapturedbyimmobilizedstreptavidinAdvantagesofPhageDisplayforRecombinantAntibodySelectionMoreefficientlythanthroughconventionalhybridomasystem.Cheapertoproducerecombinantantibodiesusingbacteri

7、a,ratherthanmammaliancellline.Easiertomaintainandgrowbacterialculturesforrecombinantantibodyproduction.Bypassimmunizationinantibodyselection.Bypasstheuseofanimalcellsforproductionofantibodies.Producingthecombinatoriallibrary(ideallywith108to109members)offunctio

8、nalantibodiestogeneratealargerrepertoireofantibodiesthanthoseavailablethroughconventionalhybridomatechnology.Easyisolationandexpressionoftheclonedgeneinabacterialhost.Excell