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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: To screen the phage antibody library using KG1a cells (myeloid leukemia cell lines) to obtain single-chain antibody (ScFv) that can bind KG1a cells with specific specificity, clone its gene and study the distribution of its corresponding antigens on different cell surfaces. Methods A complete library of KG1a cells was screened for a ScFv phage antibody library of mouse spleen cells immunized with KG1a cells and screened for 4 rounds. The binding reaction of 96 clones with KG1a cells was confirmed by ELISA. Three clones were selected from the clones and further analyzed by immunofluorescence staining. The distributions of their respective antigens on different cell surfaces were analyzed by flow cytometry. The two cloned ScFv genes were sequenced. Results The results of flow cytometry analysis showed that the antigens recognized by the three clones were found on the surface of different leukemia cells, but the distribution was different, but there was a slight difference between the three clones. Two of the clones showed DNA sequence analysis Its nucleotide sequence and amino acid sequence are different, are mouse immunoglobulin same variable region gene family and subgroup, the heavy chain variable region (VH) is a subgroup Ⅱ, family Ⅵ, light chain variable region (Vκ) belongs to subgroup Ⅲ, family Ⅸ. Conclusion Screening of phage antibody library with intact cells can effectively isolate antibody fragments of cell surface molecules and their genes. This method has good application value for screening and obtaining antibodies that are difficult to separate and purify and unknown cell surface molecules. Using KG1a cells Screening the resulting antibodies and their genes is of great importance for the further study and discovery of leukemia cell surface molecules.