鸡PD-1单克隆抗体的制备及生物学功能研究

Study on Preparation and Biological Function of Chicken PD-1 Monoclonal Antibody

试验旨在筛选并制备鸡PD-1单克隆抗体,对该单克隆抗体的免疫学特性、结合活性及其对鸡PD-1/PDL1信号通路激活的阻断作用进行初步研究。运用杂交瘤细胞融合技术筛选杂交瘤细胞株,采用ELISA方法、Ig抗体亚型鉴定试剂盒、Western blotting鉴定抗体的免疫学特性,利用间接免疫荧光技术及流式细胞术检测筛选单抗与鸡PBMCs的结合情况,应用该单抗与IBDV感染7d后的鸡PBMC细胞作用,利用实时荧光定量PCR技术检测IL-2、IL-6和IFN-γ等细胞因子的表达情况。结果显示,试验获得1株特异、稳定分泌鸡PD-1单克隆抗体的杂交瘤细胞株,命名为PD-1-D05。该单克隆抗体的亚型属于IgG1,杂交瘤细胞培养上清和腹水的效价分别为1∶211和1∶2.048×105。ELISA和Western blotting检测结果表明,PD-1-D05单抗能与免疫原发生特异性反应,与pET-28a(+)、Rosetta菌株蛋白提取液上清及无关蛋白无交叉反应。间接免疫荧光及流式细胞术检测结果显示,PD-1-D05单克隆抗体能与鸡PBMC特异性结合,且IBDV感染7d后的PBMC经单抗处理后,IL-2表达量显著升高(P<0.05),IFN-γ转录水平显著下降(P<0.05),IL-6表达水平较IBDV攻毒组细胞虽有所下降,但并无统计学差异(P>0.05)。结果表明,试验成功筛选并制备了能够稳定分泌鸡PD-1单克隆抗体的细胞株,所获得的PD-1单克隆抗体具有良好的免疫学特性,该单抗能够特异性识别鸡PD-1分子并与鸡PBMC细胞特异结合,并在一定程度上恢复由于IBDV感染导致的PD-1/PD-L1信号通路激活引发的免疫调节相关细胞因子IL-2、IFN-γ的异常表达。

This study was aimed to screen and prepare a monoclonal antibody against chicken PD-1,and preliminarily study the immunological characteristics,binding activity of the monoclonal antibody and its blocking effect on the activation of the chicken PD-1/PD-L1 signaling pathway.The hybridism cell lines was obtained using hybridism technique,the immunological activity of antibody was identified by ELISA method,the biological activity of Ig antibody subtype identification kit and Western blotting.The combination of corresponding monoclonal antibody with the PBMCswas detected by indirect immunofluorescence technique and flow cytometry.Furthermore,the monoclonal antibody was used to treat chicken PBMC cells after IBDV infected for 7 days,and the expressions of IL-2,IL-6 and IFN-γ were detected by Real-time quantitative PCR.The results showed that a hybridism cell line with specific and stable secretion of chicken PD-1 monoclonal antibody was obtained and named PD-1-D05.The subtype of monoclonal antibody belonged to IgG1,and the titers of hybridism cell culture supernatant and ascites were 1∶211 and 1∶2.048×105,respectively.ELISA and Western blotting results showed that PD-1-D05 monoclonal antibody reacted specifically with immunogens,no cross reaction was found between PD-1-D05 and pET-28 a(+),Rosetta strain protein extract supernatant and unrelated proteins.The indirect immunofluorescence and flow cytometry results showed that the obtained PD-1-D05 monoclonal antibody could specifically combine with the chicken PBMC,the expression of IL-2 significantly increased after PBMC treatment with PD-1-D05 after 7 days of IBDV infection(P<0.05),the expression of IFN-γsignificantly decreased(P<0.05),the expression of IL-6 decreased compared with IBDV attack group,but there was no statistical difference(P>0.05).The results suggested that it successfully screened and prepared a cell line that secretes the monoclonal antibody to the extracellular domain of chicken PD-1 and obtained a PD-1 monoclonal antibody with good immunological activity.This monoclonal antibody could specifically identify the PD-1 molecule and combine the PBMCs.Moreover,it could also effectively inhibit and restore immune regulation related cytokines caused by activation of PD-1/PD-L1 signaling pathways induced by IBDV infection,such as the abnormal expression of IL-2 and IFN-γ.