特异性抗细胞表面分子单链抗体分离及鉴定.docVIP

特异性抗细胞表面分子单链抗体分离及鉴定 　 作者：许静 吴立华 徐洁 刘俊霞 孟磊 何一心 宋增璇 【摘要】 目的：从抗KG1a细胞的噬菌体展示文库中分离和鉴定抗造血干/祖细胞表面分子单链抗体（scFv），克隆其基因并研究其对应抗原在不同细胞表面的分布。方法：用完整的KG1a细胞吸附法富集文库3轮，再用CD44单克隆抗体为引导分子进行引导选择，分离单克隆后用间接免疫荧光法鉴别其细胞结合特异性。对具有不同细胞结合特异性的克隆进行DNA序列分析，用一级结构不同的单链抗体作免疫印迹鉴别抗原分子量。结果：间接免疫荧光结果显示，64个阳性克隆分别识别不同的细胞系，分属3种细胞结合谱，DNA序列测定结果证明C95、H1两个克隆具有独特的一级结构，利用Western blot成功测定它们特异性识别KG1a细胞膜蛋白的表观分子量分别为34 kD/22 kD。结论：成功建立了分离和鉴定抗细胞表面分子phagescFv单克隆的方法，并从抗KG1a细胞的噬菌体展示文库中分离出2个抗造血干/祖细胞的特异性克隆。 【关键词】 噬菌体展示 引导选择 表面分子 单链抗体 　　Isolation and identification of specific single chain antibodies against cell surface molecules 　　［Abstract］Objective:Isolation and identification of single chain antibodies against surface molecules of hematopoietic progenitor cells via phage display antibody library.Methods:An individual KG1a+ phagescFv library for the binding specificity to eight hematopoietic progenitor cell lines by indirect immunoflurorescein were identified. Then,the monoclonality of the scFvs was proved by testing specific binding to antigen proteins by Western blot.Results:64 KG1a+ phagescFvs were identified. They showed three kinds of cell binding repertoire. The unique primary structures of two phagescFv clones were proved by DNA sequence. Their recombinant scFvSA fusion proteins bound to the single band of proteins from KG1a cells with molecular weights of 34 kD and 22 kD,respectively.Conclusion:Reliable methods to identify phagescFv clones against cell surface molecules have been established and obtained two unique phage-scFv clones against hematopoietic progenitor cell surface molecules from an antiKG1a cell phagescFv library. ［Key words］Phage display;Pathfinder selection;Surface molecule;ScFv 　　免疫球蛋白的可变区是抗体识别和结合抗原的功能区，其分子量只有全抗体的1/5左右，由于分子量小、组织穿透力强，因此由抗体可变区构成的单链抗体（Single chain antibody fragments, scFv）具有体内良好的应用前景，也是构建成各种双功能抗体的基本单位[1]。近年快速发展的噬菌体表面展示抗体技术日益成为制备单克隆抗体的有效手段，如何从包含众多非特异性和未知特异性的文库中分离特异性单克隆phagescFv，涉及到筛选策略选择和多项技术的应用。本研究的目的在于寻找新的有功能的抗造血干/祖细胞表面分子的抗体，并通过所获得的抗体分离抗原分子及其基因，我们探索了从自建的噬菌体抗体库中分离和鉴定特异性单克