抗KG1a细胞噬菌体展示ScFv的筛选及鉴定

Screening and identification of anti-KG1a cell ScFvs from a phage display antibody library

目的 应用KG1a细胞 (髓系白血病细胞系 )筛选噬菌体抗体库 ,获得能以一定特异性结合KG1a细胞的单链抗体 (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细胞筛选一个经KG1a细胞免疫小鼠脾细胞的ScFv噬菌体抗体库 ,重复筛选 4轮 ;细胞ELISA鉴定了其中 96个克隆与KG1a细胞的结合反应 ;从 2 6个KG1a细胞ELISA阳性克隆中挑选了 3个克隆 ,进一步用免疫荧光染色、流式细胞仪分析其各自对应抗原在不同细胞表面的分布 ,并对其中2个克隆的ScFv编码基因进行序列测定。结果 流式细胞仪分析结果显示 ,3个克隆所识别的抗原均存在于不同白血病细胞表面 ,但比例分布不同 ,而 3个克隆之间又略有差异 ,其中 2个克隆的DNA序列测定结果显示其核苷酸序列和氨基酸顺序有所不同 ,均属小鼠免疫球蛋白同一可变区基因家族和亚群 ,重链可变区 (VH)属亚群Ⅱ、家族Ⅵ ,轻链可变区 (Vκ)属亚群Ⅲ、家族Ⅸ。结论 用完整细胞筛选噬菌体抗体库可有效分离细胞表面分子的抗体片段及其基因 ,这种方法对于筛选和获得此类难以分离纯化和未知的细胞表面分子的抗体具有良好的应用价值 ;用KG1a细胞筛选所得的抗体及其基因对于进一步研究和发现白血病细胞表面分子具有重要意义。

Objective Screening and cloning of anti KG1a cell single chain antibodies (ScFvs) from phage display antibody library. Methods A ScFv phage display library from the spleen cells of mice immunized with KG1a cells (acute myelogenous leukemia cell line) was selected by using the immunizing cells. After 4 rounds of panning, 96 individual clones of phage ScFvs were tested by cell ELISA. Three out of the 26 positive clones them were further checked for the distribution of their antigens on different cells by using immunofluorescence flow cytometry. Results Flow cytometry assay showed different distribution of the antigens on different leukemia cells. DNA sequences and deduced amino acid sequences of the two clones analyzed both belong to the same mouse Ig V region subgroup and family: The V H belongs to subgroup Ⅱand family Ⅵ, the Vκ belongs to subgroup Ⅲ and family Ⅸ. Conclusion Intact cells can be used effectively to screen, isolate and clone ScFv genes against cell surface molecules which are usually hard to isolate and purify. The anti KG1a ScFvs we obtained are promising for further studying and isolating the cell surface molecules of leukemia cells.