抗转铁蛋白受体四价抗体tfr teab的构建 表达和鉴定-construction, expression and identification of tfr teab antibody against transferrin receptor tetravalent antibody.docxVIP

华中科技大学硕士学位论文分子量的大小为36kD，与理论值相符合；④纯化蛋白经梯度透析复性后，Native结果显示抗体蛋白以四聚体形式存在，分子量大小与TfR单克隆抗体和人IgG相近，约为144kD。FCM分析和免疫荧光显微镜观察，结果显示TfR-TeAb能与TfR表达阳性的HepG2细胞特异性结合且结合率与TfR-scFv相比有明显提高。结论①成功构建了TfR-TeAb原核表达载体；②目的蛋白在大肠杆菌BL21中以包涵体的形式表达；③复性后的TfR-TeAb能与TfR阳性细胞特异性结合，且亲和力较TfR-scFv有明显的提高。关键词转铁蛋白受体（TfR）；单链抗体（scFv）；四价抗体（TeAb）；p53四聚体寡聚化结构域华中科技大学硕士学位论文Construction,expressionandcharacterizationofatetravalentminiantibodyspecificallybindingtoTransferrinreceptor(TfR)Candidate:LiuYingSupervisor:AssociateProf.LeiPingProf.ShenGuanxinAbstractObjectives:Inordertoimprovethebindingaffinityofsinglechainantibody(scFv)toitstarget,aprokaryoticplasmidencodingthefusiongeneofanti-transferrinscFvandtetramerizationdomainofp53wasconstructedtoexpressatetravalentminiantibodyspecificallybindingtotransferrinreceptor(TfR-TeAb).Methods:ApairofprimersweredesignedwithintroductionofrestrictionendonucleasesitesofNcoⅠandEcoRⅠ.Theanti-TfR-scFvgenewasamplifiedfrompET28(a)-TfR-scFv-D4S×5byPCR.ThenthefragmentwasclonedintotheplasmidpET28a–HSA-p53toconstructtheprokaryoticexpressionvectorpET28(a)-TfR-TeAb.AftertherecombinantplasmidwastransformedintoE.coilBL21,positivecloneswereselectedandidentifiedbyrestrictionenzymedigestionandsequencing.ExpressionconditionswereoptimizedandtheexpressionstyleinE.coliwasanalyzed.Afterlargescaleexpression,theinclusionbodywerecollectedandpurifiedbyIMACunderdenaturingcondition.ThepuritywasanalyzedbySDSandwestern-blot.Forrefoldingtheprotein,thepuritywassubjectedtoastepwiseurea-removaldialysis.Thenatureproteinweightwasanalyzedbynondenaturingpolyacrylamidegelelectrophoresis(native)andthecell-bindingactivityoftheproteinwasanalyzedbyFCMandimmunoflourescence华中科技大学硕士学位论文microscopy.Results:1.DNAsequencingshowedthatanti-TfR-scFvgenewassuccessfullysubclonedintopET28a–HSA-p53andwasinframewiththedownstreamORFoftetramerizationdomainofp53.2.Afterfourhourexpression,theproteinwasaccumulatedasinclusionbody.Aspecialproteinbandwith36kDwasfoundinSDSandwesternblot,whichwasinconformitywiththetheoreticalvalue.3.NativePAGEsh