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目的:制备抗脯氨酸富含区缺失的人FOXP3(△PRD-hFOXP3)的单克隆抗体(mAb),为进一步研究人FOXP3各片段功能奠定基础。方法:以纯化的△PRD-hFOXP3重组蛋白为抗原免疫BALB/c小鼠,取免疫小鼠的脾细胞和骨髓瘤细胞Sp2/0进行常规融合,经筛选及克隆化建立2株分泌抗△PRD-hFOXP3的mAb杂交瘤细胞株;利用核型分析、间接ELISA及Western blot分别鉴定了杂交瘤的细胞核型、抗体的效价与特异性。结果:筛选到2株可稳定分泌抗△PRD-hFOXP3的mAb的细胞株,分别命名为1A2和3A11;2株抗体均为IgG1,腹水经ELISA方法测定效价均可达1∶105;Western blot显示这2株杂交瘤分泌的mAb均可与△PRD-hFOXP3蛋白特异性地结合。结论:成功获得2株能稳定分泌特异性抗△PRD-hFOXP3的mAb的杂交瘤细胞株,可用于人FOXP3的进一步研究及相关试剂的研发等。
OBJECTIVE: To prepare monoclonal antibodies (mAbs) against human prolyl-rich-deficient human FOXP3 (△ PRD-hFOXP3) and to lay the foundation for further study on the function of human FOXP3 fragments. Methods: BALB / c mice were immunized with the purified recombinant protein of △ PRD-hFOXP3 as antigen. Sp2 / 0 cells of the immunized mice were fused with myeloma cells Sp2 / 0. After screening and cloning, two secreting anti-△ PRD -hFOXP3 mAb hybridoma cell line; karyotype analysis, indirect ELISA and Western blot were identified hybridoma cell karyotype, antibody titer and specificity. Results: Two strains of mAbs secreting △ PRD-hFOXP3 were screened and named as 1A2 and 3A11 respectively. The two antibodies were all IgG1. The ascites titer was 1: 105 by ELISA. Western blot The mAbs showing the secretion of these two hybridomas all showed specific binding to? PRD-hFOXP3 protein. CONCLUSION: Two hybridoma cell lines that can stably secrete monoclonal antibodies against △ PRD-hFOXP3 were successfully obtained and could be used for further research on human FOXP3 and related reagents.