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ID:15170903
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时间:2018-08-01
《大肠癌噬菌体抗体库的构建及抗cea抗体的筛选》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
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κ、λ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
2、人源性Fab抗体基因库,库容量达2.1×107,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;癌胚抗原22 Abstract:ObjectiveToconstructtheFabphagedisplaya
3、ntibodylibraryofcolorectalcancersoastoscreencarcinoembryonicantigen(CEA)phageantibodyfromtheconstructedphagedisplaylibrary.MethodsThetotalcellRNAwasextractedfromacolorectalcancerpatient'speripherallymphocytes,andreversetranscribedintocDNA.ThesecDNAwereamplifiedtothelightandtheheavychainDN
4、AbyPCRusingrelevantprimers,andthenclonedintotheexpressionpComb3vector.ThroughtransformedintoXL1BlueEscherichiacolibyelectroporation,thephagedisplayantibodylibrarywasconstructedsuccessively.Afterthat,CEAantigenwascoatedtopantheconstructedlibraryinordertoselectantigenbinders,andtheselectedp
5、hageantibodieswereassayedbyELISAanalysis.OnepositiveclonewasanalyzedbyDNAsequenced.ResultsTheamplifiedfragmentsofκ,λandFdDNAwereabout680bp.TheamplificationproductswereclonedintotheexpressionpComb3vectorandwereexpressedinXL1BlueEscherichiacoli.AhumanFabantibodylibrarywasconstructedwithasiz
6、eof2.1×107andanefficacyofabout50%(κ,λandFdDNAwereallinserted).UsingcoatedCEAantigentopanthephagedisplayantibodylibraryfourrounds,thephageantibodiesshowedenrichmentspecifically.ThepositivecloneshadgoodantiCEA22specificitywhichverifiedbyELISAdirectlyandcrossreaction.ThroughDNAsequencingofo
7、nepositiveclone,itshowedthatitsheavychainbelongedtoIgGsubvarietyanditslightchaintoIgLsubvariety.ConclusionWesucceedinconstructingaFabphagedisplayantibodieslibrarywithnaturalimmunitybyacolorectalcancerpatient.Fromit,wealsosucceedinobtainingtheantibodywhichhasth
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