全人源抗肝癌噬菌體單鏈抗體的篩選與鑒定

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1、全人源抗肝癌噬菌體單鏈抗體的篩選與鑒定作者：黃建,肖艷,童永清,郭鋒杰,周國華,李躍輝,胡錦躍,李官成【摘要】　　目的:構(gòu)建人源抗肝癌噬菌體單鏈抗體庫,從中篩選抗肝癌單鏈抗體（scFv）,并對其特異性進行鑒定。方法:采用體外致敏法和EBV轉(zhuǎn)化技術(shù)聯(lián)合噬菌體展示技術(shù)構(gòu)建噬菌體抗體庫。對初級抗體庫進行親和富集及ELISA篩選,獲得的陽性克隆進行免疫組化鑒定并測序。結(jié)果:scFv基因與載體連接后得到庫容量為1.0×108的初級噬菌體抗體庫。對抗體庫進行3輪正負淘選和富集后,隨機挑取2798個克隆進行ELISA,發(fā)現(xiàn)3個克隆對HepG2呈強陽性反應(yīng),而與QSG

2、7701等人正常細胞系呈弱陽性或不反應(yīng)。對克隆SA3進行免疫細胞化學(xué)鑒定,結(jié)果與ELISA一致。免疫組織化學(xué)鑒定表明SA3與肝癌組織和肝組織陽性率的差別有統(tǒng)計學(xué)意義。結(jié)論:構(gòu)建了庫容量達1×108的全人源抗肝癌噬菌體抗體庫。通過淘選富集、ELISA和免疫組織化學(xué)鑒定獲得特異性較強的噬菌體克隆,為肝癌的臨床診斷及導(dǎo)向治療奠定了基礎(chǔ)?！娟P(guān)鍵詞】EB病毒轉(zhuǎn)化噬菌體抗體庫肝腫瘤單鏈抗體　　[Abstract]AIM:Toconstructafullyhumanantihepatomasinglechainphageantibodylibrary,sele

3、cttheanti13hepatomascFvfromitandidentifyitscharacteristics.METHODS:AfullyhumanscFvdisplayingphagelibrarywasconstructedbyusingphageantibodylibrarytechniqueincombinationwithinvitroimmunizationandEBVtransformation.Thelibrarywassubjectedtothreeroundsofpositiveandnegativecellpannin

4、gandenrichmentandthenitwasselectedbyELISA.Thebindingspecificityofphageantibodieswithhepatomacarcinomacellswasconfirmedbyimmunohistochemistry.RESULTS:AfterscFvgenesbeingclonedintovectorFuse5andtransformedintoE.coliMC1061viaelectroporation,aphageantibodylibrarycontaining1.0×108TU(

5、transducedunit)wasobtainedthroughtetracyclineresistantscreening.Afterpanning,enrichmentandtestingbyELISA,3phageantibodyclonesreactingmorestronglytoHepG2thanQSG7701werepickedoutof2798clones.Oneclone,SA3,wasfurtheranalysedafterDNAsequencing.Theresultsofimmunohistochemistrywithcu

6、lturedcellsweresimilartothoseofELISA.SA3reactedspecificallytohepatomacellsinmosthumanhepatomatissuesectionsbutinfewhumannormallivertissuesections.Thedistinctionofpositiveratesisofagreatstatisticalsignificance.CONCLUSION:AfullyhumanantihepatomascFvfusionphagelibraryhasbeenconstr

7、ucted.ELISAand13immunohistochemistryresultsconformSA3specificallybindwithhepatomacarcinomacells.ThescFvfragmentagainsthepatomamaybefurtherdevelopedandappliedinclinicaldiagnosisandtherapyoflivercancer.　　[Keywords]EBVimmortalization;phageantibodylibrary;hepatoma;scFv　　肝癌是一種惡性程度極高的

8、腫瘤,是我國和亞洲地區(qū)的常見病和多發(fā)病,病死率居惡性腫瘤的第3位[1]。肝癌90%以上為原發(fā)性