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为制备抗猪程序性死亡因子配体1(PD-L1)的单克隆抗体(MAb)并鉴定其免疫学特性,本研究将猪PD-L1基因的外膜区利用原核表达载体pET32a进行表达,以纯化的表达蛋白免疫BALB/c小鼠,利用常规淋巴细胞杂交瘤技术进行细胞融合,以表达PD-L1-Fc蛋白的293T细胞作为抗原,采用间接免疫荧光试验筛选杂交瘤培养上清,获得两株稳定分泌抗猪PD-L1 MAb的杂交瘤细胞系。MAb亚型鉴定均为IgG1型,轻链为κ链。Western blot试验表明这两株MAb均可以特异性识别PD-L1蛋白。流式细胞检测结果显示这两株MAb可以识别表达于293T细胞膜表面的PD-L1分子,但不能阻断PD-L1分子与其受体PD-1的结合。这两株MAb的获得为研究PD-1/PD-L1通路与猪的某些感染性疾病发展进程的关系提供了必要的检测工具。
In order to prepare the monoclonal antibody (MAb) against porcine PD-L1 and identify its immunological properties, the outer membrane of porcine PD-L1 gene was expressed in prokaryotic expression vector pET32a, BALB / c mice were immunized with the purified protein, and the cells were fused by conventional lymphocyte hybridoma technique to express 293T cells expressing PD-L1-Fc protein as an antigen. The hybridoma culture supernatants were screened by indirect immunofluorescence assay to obtain Two hybridoma cell lines stably secreting anti-porcine PD-L1 MAb. MAb subtypes were identified as IgG1 type, the light chain kappa chain. Western blot showed that both MAbs could specifically recognize PD-L1 protein. Flow cytometry showed that these two MAbs could recognize PD-L1 molecules expressed on the surface of 293T cell membrane, but could not block the binding of PD-L1 molecules to their receptor PD-1. The availability of these two MAbs provided the necessary testing tools for studying the relationship between the PD-1 / PD-L1 pathway and the development of certain infectious diseases in pigs.