抗猪PD-1单克隆抗体的制备与其生物学特性鉴定

Preparation of the anti-porcine PD-1 monoclonal antibodies and identification of their biological characteristics

制备猪程序性死亡因子-1(PD-1)单克隆抗体,鉴定其生物学特性。以表达纯化的猪程序性死亡因子-1(Programmed death-1,PD-1)胞外区重组蛋白为抗原,免疫BALB/c小鼠,用细胞杂交瘤技术将免疫BALB/c小鼠脾细胞与小鼠骨髓瘤NS0细胞融合,经多次克隆化培养,筛选分泌特异性抗猪PD-1单克隆抗体的杂交瘤细胞株。结果:获得1株可稳定分泌抗猪PD-1单克隆抗体的杂交瘤细胞株2B5,Ig亚型为Ig G1,细胞上清和腹水效价分别为1∶1×212和1∶2.048×105,酶联免疫吸附试验(Enzyme linked immunosorbent assay,ELISA)和Western-Blot结果表明,该株单抗能特异性识别重组猪PD-1胞外区蛋白,流式细胞术结果表明,该单抗可以结合猪外周血单核细胞上PD-1蛋白。结论:成功制备了1株分泌抗猪PD-1单抗的杂交瘤细胞株2B5,为研究猪PD-1/PD-L1通路在猪传染性疾病中的致病机制提供了有力的检测工具。

To prepare anti - porcine PD - 1 monoclonal antibody and identify its biological characteristics. BALB/c mice wereimmunized with the extracellular domain recombinant protein of porcine PD - 1. The splenocytes of immunized BALB/c mice werefused with NSO cells by hybridoma technique for obtaining mAb against PD - 1. A hybridoma cell line that secreting anti - PD - 1mAb was successfully obtained, and was named as 2B5. The mAb was IgG1 subtype. The titers of hybridoma cell supernatant and as-cites were 1 ： 1 - 212 and 1 ： 2. 048 x 10s , respectively. Results of ELISA and Western - blot showed that the mAb specifically recog-nized the extracellular domain of recombinant PD - 1 protein. Flow cytometry showed that the mAb effectively bound to the PD - 1protein on Peripheral blood mononuclear cell （PBMC） of pigs. A hybridoma cell line 2B5 secreting mAb against porcine PD - 1 pro-tein was successfully prepared, which provides a powerful detection tool for further studying the mechanism of porcine PD - 1/PD -L1 pathway in porcine infectious diseases,