二硫鍵穩(wěn)定單鏈抗體B3（ds-scFv）靶向超抗原SEA的制備及活性鑒定.pdf

1、西北農(nóng)林科技大學(xué)碩士學(xué)位論文二硫鍵穩(wěn)定單鏈抗體B3（dsscFv）靶向超抗原SEA的制備及活性鑒定姓名：王建麗申請學(xué)位級別：碩士專業(yè)：生物化學(xué)與分子生物學(xué)指導(dǎo)教師：王保莉姜永強(qiáng)20050601PREPAR蘆口IoNANDE、廠ALUATION0FDISULFIDE—S11ABLILIZEDSINGLECHAINANTIBODYB3(DSSCFV)TARGETEDSEAABSTRACT確econceptionofsuperantigen(

2、SAgslwascreatedbyWhitein1989TheyareagroupofproteinmoleculalsencodedbybacterialsorviRISSAgsbindasunprocessedproteinstelativelyinvaliantregionsofMHCclassIIantigenswhichalefoundmainlyonprofessionalantigenpresentingcells(APe

3、s)Onceboundto刪CIreelIsSAgscartactivateTlymphocytesexpressingpaldculalV峨ableregiop選(vS)oftheTcullreceptor(TCR)鼻chainandthenumberoftheactivatedTlymphocytesis100010000foldscomparestocommonantigens111eCTLsactivatedbysuperant

4、igenhassignificantcytotoxiceffectagainsttulnorcells，thereforeaspotentTlymphocytesactivatorsnperantigenmaydeveloptoanewtypeofantitumorimmunomoleculalSEAi8theproductofstaphylococcusoureUsitisoneofdifferenttypesofsuperantig

5、ens，andtlleresearchonitisdeeperthanothersrecentyears。Onthenegativeside，thecytotoxiceffectmediatedbytheTlymphocyteswhichisactivatedbySEAalsohascytotoxicityagainstthenormalcellswhenusedtothecarcinomacellsandhasnoeffecton姍C(jī)

6、IrcarcinomaeelIsBecauseofthereasonsmentionedabeveutilizinggeneticengineeringantibodytodirectedSEAtoR。ifflertissuesenddecreasethenegativeeffectsismoreandmorenecessaryInordertoimprovetllestabilityofthesinglechainantibodyfS

7、cFv)soastoachievebettertargetedeffect，apairofinterchaindisulfidebondwasintroducedtoB3ScFvandformeddisulfidestablesinglechainamibedy(B3dsseFv)inthispaper111eVHandVLfragmentsofthemAbB3wereligatcdbyoveflapPCR，thePCRproductw

8、asclonedtothepET22bexpressi’onvector；thentheSEAfragmentwasinsertedintotheB3dsscF“vpET22bexpressionvectorwhichwnsdigestedbythesamerestrictione1：lzyrflesneexpressionplasmidwasidentifiedbyrestrictionendonuclea∞sdigesti∞andt

9、ansfonnedintoEcoliBL21(DE3)followedbyIPTGinducion；meinclusionbodywaspurifiedthroughSP—SephalosecationexchangecolumnafterdenatudngandrefoldingTbebindingandcytotoxicabilityofthepurifiedproductswasexaminedbycellELISAandNonR

10、adioactiveCellProliferationAssayseparatelyn圮stabilityassaywasperformedbyincubatingtheproteinsampleat37℃foraweekandatdifferenttemperaturesfor30minAsaresulttheexpressionvectorB3dsscFvSEA(D227A)pETwascons掃uctedSuccessfullya

11、ndtheexpressionproductexistsmainlyintheinclusionbody，amountingto33％ofthetotleproteinTherefoldingproductmmainsgoodantigenbindingabilityandhascylotoxiceffeetonH■29coloncarcinomacells111estabilityassayshowedthat也eresultingp

12、roteinwasstableat37℃IIlconclusionthisgencticallyengineeredB3dsscFvSEA(D227A)fusionproteinhasbiftmctionoftunlortargetingandrunleteellkillingandpromisestobeaneffectivereagentfortHnlortargetedimmunotherapyKEYWORDS：B3monoclo