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1、大腸癌噬菌體抗體庫(kù)的構(gòu)建及抗CEA抗體的篩選作者：南清振，高蕾，張振書(shū)，楊希山，肖冰，姜泊【摘要】目的構(gòu)建及初步鑒定大腸癌噬菌體抗體Fab呈現(xiàn)庫(kù)，篩選人CEA單克隆抗體，并進(jìn)行序列分析。方法分離大腸癌患者外周血淋巴細(xì)胞，提取淋巴細(xì)胞總RNA，逆轉(zhuǎn)錄成cDNA。用PCR擴(kuò)增人全套抗體基因片段，克隆于pComb3載體，再經(jīng)電穿孔轉(zhuǎn)化大腸桿菌XL1Blue菌株，形成噬菌體抗體庫(kù)。以固相化CEA抗原淘篩抗體庫(kù)，ELISA鑒定噬菌體抗體。其中一個(gè)陽(yáng)性克隆進(jìn)行測(cè)序。結(jié)果逆轉(zhuǎn)錄PCR分別擴(kuò)增出約680bp大小的k、A和Fd基因。PCR產(chǎn)物和載體經(jīng)純化、雙酶切后進(jìn)行連接轉(zhuǎn)化，成功地構(gòu)建了人源性Fab抗體基因
2、庫(kù)，庫(kù)容量達(dá)X107，F(xiàn)ab基因重組率為50%。以單抗捕獲的CEA抗原淘洗4輪，出現(xiàn)特異性富集，陽(yáng)性克隆經(jīng)直接ELISA和交叉反應(yīng)ELISA實(shí)驗(yàn)證實(shí)具有良好的抗CEA抗原特異性。DNA測(cè)序表明該抗體重鏈屬IgG亞類并含有一條IgL亞類的輕鏈。結(jié)論成功構(gòu)建了大腸癌患者自然致敏抗體Fab段噬菌體呈現(xiàn)庫(kù)，從中獲得可與CEA抗原結(jié)合的噬菌體抗體，由此為大腸癌早期診斷及基因治療提供了一種新的思路和方法?！娟P(guān)鍵詞】大腸腫瘤;噬菌體抗體庫(kù);Fab抗體;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
