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1、全人源抗肝癌噬菌體單鏈抗體篩選及鑒定作者：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1.0X108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
2、對HepG2呈強陽性反應(yīng)，而與QSG7701等人正常細胞系呈弱陽性或不反應(yīng)。對克隆SA3進行免疫細胞化學鑒定，結(jié)果與ELISA一致。免疫組織化學鑒定表明SA3與肝癌組織和肝組織陽性率的差別有統(tǒng)計學意義。結(jié)論：構(gòu)建了庫容量達1X108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向治療奠定了基礎(chǔ)?！娟P(guān)鍵詞】EE病毒轉(zhuǎn)化噬菌體抗體庫肝腫瘤單鏈抗體[Abstract]AIM：Toconstruetafullyhumanantihepatomasi
3、nglechainphageantibodylibrary,selecttheantihepatomascFvfromitandidentifyitscharacteristics.METHODS：AfullyhumanscFvdisplayingphagelibrarywasconstructedbyusingphageantibodylibrarytechniqueincombinationwithinvitroimmunizationandEBVtransformation.Thelibrarywassu
4、bjectedtothreeroundsofpositiveandnegativecellpanningandenrichmentandthenitwasselectedbyELISA.Thebindingspecificityofphageantibodieswithhepatomacarcinomacellswasconfirmedbyimmunohistochemistry.RESULTS：AfterscFvgenesbeingclonedintovectorFuse5andtransfonnedinto
5、E.coliMCI061viaelectroporation,aphageantibodylibrarycontaining1.0X108TU(transducedunit)wasobtainedthroughtetracyclineresistantscreening.Afterpanning,enrichmentandtestingbyELISA,3phageantibodyclonesreactingmorestronglytoHepG2thanQSG7701werepickedoutof2798clon
6、es.Oneclone,SA3,wasfurtheranalysedafterDNAsequencing?TheresuItsofimmunohistochemistrywithculturedcellsweresimilartothoseofELISA?SA3reactedspecificallytohepatomacellsinmosthumanhepatomatissuesectionsbutinfewhumannormallivertissuesections.Thedistinctionofposit
7、iveratesisofagreatstatisticalsignificance?CONCLUSION：AfullyhumanantihepatomascFvfusionphagelibraryhasbeenconstrueted.ELISAandimmunohistochemistryresuItsconformSA3specificallybindwithhepatomacarcinomacells.ThescFvfragmentagainsthepatomamaybefurtherdevelopedan
8、dappliedinclinicaldiagnosisandtherapyoflivercancer.[Keywords]EBVimmortalization；phageantibodylibrary；hepatoma；scFv肝癌是一種惡性程度極高的腫瘤，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，發(fā)病隱匿，發(fā)現(xiàn)時多數(shù)已處于中晚期,喪失了
