抗猪PD-1和PD-L1蛋白单克隆抗体的制备及鉴定

发布时间：2018-06-25 06:17

本文选题：猪 + PD-1　；参考：《郑州大学》2017年硕士论文

【摘要】：程序性死亡因子-1(programmed death-1,PD-1)是参与机体免疫负调控的重要分子,其配体为PD-L1和PD-L2。PD-1和PD-L1表达量升高引起机体免疫抑制或免疫功能损伤是多种病毒感染的重要致病机制。本实验室前期研究表明,猪感染猪圆环病毒2型和猪瘟病毒后,诱导PD-1和PD-L1过表达,激活PD-1/PD-L1通路,造成机体免疫功能损伤。抗体阻断PD-1/PD-L通路可以恢复或逆转机体免疫力,因此,制备抗猪PD-1和PD-L1单克隆抗体,为阻断猪PD-1/PD-L通路提供有效方法途径。本研究的免疫原是表达纯化的猪PD-1和PD-L1胞外区重组蛋白,BALB/c小鼠作为免疫动物,用细胞融合技术融合免疫BALB/c鼠脾细胞和鼠骨髓瘤细胞NS0,用ELISA法多次筛选和克隆化,旨在筛选抗猪PD-1和PD-L1蛋白的单克隆抗体,并鉴定其生物学特性。研究结果显示,2株单抗_(SW)PD1-2B5和_(SW)PDL1-3B5能分别特异性识别猪重组PD-1和PD-L1蛋白,抗体Ig亚型均为IgG1,PD-1杂交瘤细胞上清和腹水效价分别为1:1×212和1:2.048×105,PD-L1杂交瘤细胞上清和腹水效价分别为1:1×210和1:1.024×105。获得的单克隆抗体_(SW)PD1-2B5和_(SW)PDL1-3B5能分别与相应的重组猪PD-1和PD-L1蛋白反应,且都不与鸡PD-1、Rosetta上清和pET-32a空载体蛋白反应,具有良好的特异性。连续3个月冻存、复苏3次,2B5和3B5杂交瘤细胞株均能稳定分泌抗体_(SW)PD1-2B5和_(SW)PDL1-3B5,培养细胞上清抗体效价分别为1:212和1:210。流式细胞术结果表明2株单抗能分别与猪PBMC有效结合。成功制备抗猪PD-1和PD-L1单克隆抗体,为在蛋白水平研究PD-1、PD-L1表达水平与疾病的关系以及应用抗体阻断PD-1/PD-L1通路在猪持续性病毒感染中的作用奠定了基础。
[Abstract]:Programmed death-1 (PD-1) is an important molecule involved in the negative regulation of immune system. Its ligands PD-L1, PD-L2.PD-1 and PD-L1 increase the expression of PD-L1, PD-L2.PD-1 and PD-L1, which lead to immune suppression or damage of immune function, which is an important pathogenetic mechanism of multiple viral infections. Previous studies in our laboratory showed that pigs infected with porcine circovirus type 2 and swine fever virus induced PD-1 and PD-L1 overexpression and activated PD-1 / PD-L1 pathway, resulting in damage of immune function. Antibody blocking PD-1 / PD-L pathway can restore or reverse the immune system. Therefore, the preparation of monoclonal antibodies against porcine PD-1 and PD-L1 provides an effective way to block the PD-1 / PD-L pathway. The immunogen of this study was the purified porcine PD-1 and PD-L1 extracellular domain recombinant protein BALBP-c mice as immunized animals. The spleen cells and myeloma cells NS0 of BALBr / c mice were immunized by cell fusion technique and screened and cloned by Elisa. The aim of this study was to screen monoclonal antibodies against porcine PD-1 and PD-L1 proteins and to identify their biological characteristics. The results showed that McAbs _ (SW) PD1-2B5 and _ (SW) PDL1-3B5 could specifically identify porcine recombinant PD-1 and PD-L1 proteins respectively. The titers of the supernatant and ascitic fluid of IgG 1pPD-1 hybridoma cells were 1:1 脳 212 and 1: 2.048 脳 105 respectively, and the ascites titers were 1:1 脳 210 and 11.024 脳 105respectively. The obtained monoclonal antibodies _ (SW) PD1-2B5 and _ (SW) PDL1-3B5 reacted with the corresponding recombinant porcine PD-1 and PD-L1 proteins, respectively, and did not react with the chicken PD-1 Rosetta supernatant and pET-32a empty carrier protein. After cryopreservation for 3 months, the resuscitation of 2B5 and 3B5 hybridoma cell lines could stably secrete antibody _ (SW) PD1-2B5 and _ (SW) PDL1-3B5. The antibody titers of supernatant of cultured cells were 1: 212 and 1: 210, respectively. The results of flow cytometry showed that the two McAbs could bind to porcine PBMC effectively. Monoclonal antibodies against porcine PD-1 and PD-L1 were successfully prepared, which laid a foundation for studying the relationship between PD-1pPD-L1 expression level and disease at protein level and blocking PD-1 / PD-L1 pathway in porcine persistent virus infection.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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