Aβ-,1-42-寡聚體特異性scFv的篩選、鑒定和親和力成熟的初步研究.pdf

1、內(nèi)蒙古農(nóng)業(yè)大學(xué)博士學(xué)位論文Aβ寡聚體特異性scFv的篩選、鑒定和親和力成熟的初步研究姓名：包福祥申請(qǐng)學(xué)位級(jí)別：博士專業(yè)：基礎(chǔ)獸醫(yī)學(xué)指導(dǎo)教師：曹貴方何金生20090401Selection，IdentificationandpreliminarystudyofAffinityMaturationofscFvSpecificAgainstA1342OligomersAbstractAIzheimer’Sdisease(AD)iSthemos

2、tprevalentneurodegeneratiVediseaseIntheelderThediseaseischaracterizedbymemoryimpairmentandcognitiveobstacleThecurrenttreatmentsofADprovideonlysymptomaticreliefandarepracticall)inefficientNowantiA13antibodieshavebeensugge

3、stedasapotentialtherapeuticstrategyforthetreatmentofADGeneticengineeringmadeitpossibletOgenerateantibodywithoutconstantregionwhichistermedscFv(singlechainfragmentvariable)orFab(Antigenbindingfragment)ScFvisasmallerfuncti

4、onalunitremainingantigenicspecificityandaffinityComparedwithotherantibodies，scFvhaSmanyadvantages，smallersize，reducedimmunogenicity，sgongerpenetratingpower，rapidlocalizationandbeingeaSilyclearedupfromthenormaltissueMeanw

5、hile，scFvCanbeproducedinlargescaleinbacteriaoryeastandmanipulatedb’geneticengineeringThereforedevelopingscFvisofgreatsignificanceintheareasofADdiagnosisandtherapyToconstructthehumansinglechainFv(scFv)antibodylibrarybypha

6、gedisplaytechnologyandobtainthespecificscFvs(single—chainfragmentvariable)againstsolubleAp142(Amyloidbeta)oligomersbyscreeningthehumanscFvlibrary，peripheralbloodlymphocytes(PBL)wereseparatedfromtenhealthydonors，andthetot

7、alRNAWaSextractedfromthePBLThevariableheavy(VH)andvariablelight(VL)geneswereamplifiedbyRTPCRandthenthescFvwasobtainedthroughSOEPCRThescFvfragmentswereclonedintothevectorpCANTAB5EandelectroporatedintocompetentEcoliTGlcell

8、sandthenascFvphagedisplaylibrarycontaining25x10’clonesWaSgainedTherecombinantphagemidswererescuedbyreinfectionofhelperphageMI3K07RecombinantphagesspecificforApI42oligomerswereenrichedafterfourroundsofbiopanningandtheanti

9、gen—positivecloneswereselectedfromtheenrichedclonesbyphageELISAPositivecloneB19WaSusedtoinfectEcoliHB2151toexpresssolublescFvThesolublescFvantibodieswereexpressedsuccessfully，anddisplayedspecificbindingtOAp142trimerandpr

10、otofiberbySDSPAGEandWesternblotanalysisThebindingaffinityofsolublescFvantibodiestoA136420ligomersWaS910。6mol／LForthepurposeofaffinitymaturation，theVHandVLgenefiagmentswereclonedintothepComb3C／cFabvector，andtransformedtOc