具有G-CSF抗原结合活性的三个重组噬菌体单链抗体基因的筛选及序列测定

Screening and sequencing of ScFv genes of three recombined phages with G-CSF binding activity

利用固相化GCSF亲和吸附法,从已构建的抗rhGCSF全套单链抗体(ScFv)噬菌体表面展示文库中,筛选出含有特异抗体片段的噬菌体颗粒。结合直接感染法和酸性洗脱液洗脱法,进行4轮亲和富集筛选,并通过ELISA法结合双酶切鉴定,共筛选出9个具有GCSF结合活性的完整的重组噬菌粒克隆。对其中3个阳性克隆进行了ScFv基因全序列测定,获得6个抗体可变区基因的全序列。计算机分析表明,它们均为新发现的小鼠免疫球蛋白可变区基因。

UsingbindingtosolidphaseG CSFfollowedbyelutionandre infection ,wescreendedoutspecificrecombinedScFv phagemidclonesfromconstructedphagedisplaylibrarycontaininganti rhG CSFrepertoireScFvgene .Afterthroughfourroundsofpanning,nineG CSF bindingcloneswereidentifiedwithELISAandEcoRⅠ /HindⅢrestictionanalysis.These quencesofthreepositiverecombinedScFvphagemidclonesweredeterminedbySanger’smethod .ThreeVHgenesandthreeVL geneswereobtainedandcomputeranalysisconfirmedthattheirsequencesbelongedtothemouseimmunoglobulinV region genes.TheyappearedtobenewgenesbycomparisonwiththepublisheddatainEMBLGeneBank .