全人源抗肝癌噬菌体单链抗体的筛选与鉴定

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1、全人源抗肝癌噬菌体单链抗体的筛选与鉴定作者：黄建,肖艳,童永清,郭锋杰,周国华,李跃辉,胡锦跃,李官成【摘要】　　目的:构建人源抗肝癌噬菌体单链抗体库,从中筛选抗肝癌单链抗体（scFv）,并对其特异性进行鉴定。方法:采用体外致敏法和EBV转化技术联合噬菌体展示技术构建噬菌体抗体库。对初级抗体库进行亲和富集及ELISA筛选,获得的阳性克隆进行免疫组化鉴定并测序。结果:scFv基因与载体连接后得到库容量为1.0×108的初级噬菌体抗体库。对抗体库进行3轮正负淘选和富集后,随机挑取2798个克隆进行ELISA,发现3个克隆对HepG2呈强阳性反应,而与QSG

2、7701等人正常细胞系呈弱阳性或不反应。对克隆SA3进行免疫细胞化学鉴定,结果与ELISA一致。免疫组织化学鉴定表明SA3与肝癌组织和肝组织阳性率的差别有统计学意义。结论:构建了库容量达1×108的全人源抗肝癌噬菌体抗体库。通过淘选富集、ELISA和免疫组织化学鉴定获得特异性较强的噬菌体克隆,为肝癌的临床诊断及导向治疗奠定了基础。【关键词】EB病毒转化噬菌体抗体库肝肿瘤单链抗体　　[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　　肝癌是一种恶性程度极高的

8、肿瘤,是我国和亚洲地区的常见病和多发病,病死率居恶性肿瘤的第3位[1]。肝癌90%以上为原发性