特异性抗细胞表面分子单链抗体的分离和鉴定

Isolation and identification of specific single chain antibodies against cell surface molecules

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Westernblot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34kD/22kD。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。

Objective ： Isolation and identification of single chain antibodies against surface molecules of hematopoietic progenitor cells via phage display antibody library. Methods ： An individual KG1 a^＋ 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 KGla 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.