大肠癌噬菌体抗体库的构建及抗cea抗体的筛选.doc

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1、大肠癌噬菌体抗体库的构建及抗CEA抗体的筛选作者：南清振，高蕾，张振书，杨希山，肖冰，姜泊【摘要】目的构建及初步鉴定大肠癌噬菌体抗体Fab呈现库，筛选人CEA单克隆抗体，并进行序列分析。方法分离大肠癌患者外周血淋巴细胞，提取淋巴细胞总RNA，逆转录成cDNA。用PCR扩增人全套抗体基因片段，克隆于pComb3载体，再经电穿孔转化大肠杆菌XL1Blue菌株，形成噬菌体抗体库。以固相化CEA抗原淘筛抗体库，ELISA鉴定噬菌体抗体。其中一个阳性克隆进行测序。结果逆转录PCR分别扩增出约680bp大小的k、A和Fd基因。PCR产物和载体经纯化、双酶切后进行连接转化，成功地构建了人源性Fab抗体基因

2、库，库容量达X107，Fab基因重组率为50%。以单抗捕获的CEA抗原淘洗4轮，出现特异性富集，阳性克隆经直接ELISA和交叉反应ELISA实验证实具有良好的抗CEA抗原特异性。DNA测序表明该抗体重链属IgG亚类并含有一条IgL亚类的轻链。结论成功构建了大肠癌患者自然致敏抗体Fab段噬菌体呈现库，从中获得可与CEA抗原结合的噬菌体抗体，由此为大肠癌早期诊断及基因治疗提供了一种新的思路和方法。【关键词】大肠肿瘤;噬菌体抗体库;Fab抗体;胚抗原Abstract:ObjectiveToconstruettheFabphagedisplayantibodylibraryofcolorectalc

3、ancersoastoscreencareinoembryonicantigen(CEA)phageantibodyfromtheconstructedphagedisplayThetotalcellRNAwasextractedfromacolorectalcancerpatient1speripherallymphocytes,andreversetranscribedintocDNA.ThesecDNAwereamplifiedtothelightandtheheavychainDNAbyPCRusingrelevantprimers,andthenclonedintotheexpre

4、ssionpComb3vector.ThroughtransformedintoXL1BlueEscherichiacolibyelectroporation,thephagedisplayantibody1ibrarywasconstructedsuccessively.Afterthat,CEAantigenwascoatedtopantheconstructedlibraryinordertoselectantigenbinders,andtheselectedphageantibodieswereassayedbyELISAanalysis.Onepositiveclonewasan

5、alyzedbyDNAsequenced.ResultsTheamplifiedfragmentsofk,入andFdDNAwereabout680bp.TheamplificationproductswereclonedintotheexpressionpComb3vectorandwereexpresrichiacoli.AhumanFasconstructedwithaicacyofabout50%(kserted).UsingcoatesedinXLlBlueEscheabantibodylibrarywsizeofX107andaneff入andFdDNAwereal1indCEA

6、antigentopanthephagedisplayantibds,thephageantibodody1ibraryfourrouniesshowedenrichmentspecifically.ThepositivecloneshadgoodantiCEAspecificitywhichverifiedbyELISAdirectlyandcrossreaction.Throunepositiveclone,itchainbelongedtoIgGghtchaintoIgLsubvaesucceedinconstrucayantibodieslibrartybyacolorectalca

7、nwealsosucceedinobtghDNAsequencingofoshowedthatitsheavysubvarietyanditsliriety.ConclusionWtingaFabphagedisplywithnaturalimmunicerpatient.Fromit,ainingtheantibodywhichhasthebindingactivitytoCEAantigen.Keywor