小鼠抗人PD-1单克隆抗体的制备及其生物学活性评价

发布时间：2018-05-18 15:39

本文选题：程序性死亡受体 + 单克隆抗体　；参考：《免疫学杂志》2017年04期

【摘要】：目的制备鼠抗人PD-1单克隆抗体,并对其生物阻断活性进行评价。方法利用重组人源PD-1/PDCD1蛋白作为免疫蛋白,免疫接种Balb/c小鼠,分离并提取小鼠脾淋巴细胞,然后与小鼠骨髓瘤细胞SP2/0细胞融合培养,经过多次克隆化培养筛选出稳定分泌鼠抗人PD-1单抗的杂交瘤细胞株。鉴定单抗Ig G类别及亚类,并分析其特异性和细胞毒性;建立Hep G2细胞和Jurkat细胞共同培养体系,ELISA法检测PD-1单抗对Jurkat细胞分泌抗肿瘤细胞因子的影响,MTT法分析PD-1单抗对Jurkat细胞杀伤Hep G2细胞能力的影响。结果获得了1株新的小鼠抗人PD-1单克隆抗体,命名为2H7。单抗2H7能够特异性识别T细胞表面的PD-1蛋白;高浓度长时间处理对Jurkat细胞有一定毒性作用;低浓度单抗2H7可阻断PD-1/PD-L1信号通路,使Jurkat细胞对Hep G2细胞杀伤能力显著增强(P0.01),且Jurkat细胞分泌的IL-2和IFN-γ等细胞因子水平显著高于对照组(P0.01)。结论成功制备了1株具有良好生物阻断活性的小鼠抗人PD-1单克隆抗体2H7,该抗体能够显著提高T细胞对肝癌细胞Hep G2的杀伤能力。
[Abstract]:Objective to prepare mouse anti-human PD-1 monoclonal antibody and evaluate its biological blocking activity. Methods Recombinant human PD-1/PDCD1 protein was used as immune protein to immunize Balb/c mice. Spleen lymphocytes of mice were isolated and extracted, and then fused with SP2/0 cells of mouse myeloma cells. The hybridoma cell lines secreting murine anti-human PD-1 McAb stably were screened by multiple clonal culture. The specific and cytotoxic characteristics of McAb Ig G were identified. A co-culture system of Hep G2 cells and Jurkat cells was established to detect the effect of PD-1 monoclonal antibody on the secretion of anti-tumor cytokines in Jurkat cells by Elisa. The effect of PD-1 monoclonal antibody on the ability of Jurkat cells to kill Hep G2 cells was analyzed by MTT assay. Results A new mouse monoclonal antibody against human PD-1 was obtained and named as 2H 7. Monoclonal antibody 2H7 can specifically recognize PD-1 protein on T cell surface, high concentration of 2H7 has toxic effect on Jurkat cell, and low concentration of 2H7 can block PD-1/PD-L1 signal pathway. The cytotoxicity of Jurkat cells to Hep G2 cells was significantly enhanced, and the levels of cytokines such as IL-2 and IFN- 纬 secreted by Jurkat cells were significantly higher than those of control cells. Conclusion A mouse anti-human PD-1 monoclonal antibody 2H7 with good bioblocking activity was successfully prepared. The antibody can significantly enhance the cytotoxicity of T cells to Hep G2 cells.
【作者单位】：第三军医大学大坪医院野战外科研究所药学部;
【基金】：国家自然基金(81273608) 重庆市基础科学与前沿技术研究重点项目(CSTC2015jcyj BX0018) 第三军医大学科技成果转化基金培育项目(2015XZH19)
【分类号】：R392.1

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