草鱼IL-10单克隆抗体的制备与鉴定

白细胞介素10(Interleukin-10, IL-10)参与机体免疫应答调节, 协同其他细胞因子维持免疫系统稳态。为探究草鱼(Ctenopharyngodon idella)IL-10的细胞来源, 研究利用大肠杆菌(Escherichia coli, E. coli)表达系统制备了高纯度的草鱼IL-10重组蛋白, 免疫小鼠制备单克隆抗体后通过免疫印迹、激光共聚焦和流式细胞术对抗体进行分析。结果表明, 获得的单克隆抗体不仅能特异识别大肠杆菌表达的CiIL-10重组蛋白, 而且能识别HEK293细胞中表达的真核IL-10重组蛋白。脂多糖(Lipopolysaccharide, LPS)和草鱼白细胞介素1β(IL-1β)刺激草鱼性腺细胞系(GCO)36h的免疫荧光染色分析表明, 草鱼IL-10单克隆抗体能与草鱼内源IL-10结合, 但IL-10阳性细胞数量在LPS处理前后无明显变化。该研究成功制备了高纯度CiIL-10重组蛋白, 获得了特异性好的高质量单克隆抗体, 为研究草鱼ILC2和Th2细胞的增殖、分化和功能奠定了基础。

PRODUCTION AND CHARACTERISATION OF IL-10 MONOCLONAL ANTIBODIES IN GRASS CARP (CTENOPHARYNGODON IDELLA)

Interleukin (IL) 10 is synthesised and secreted mainly by type 2 innate lymphoid cells (ILC2) and T helper 2 cells (Th2) and regulates the growth, proliferation and differentiation of immune cells such as T cells, B cells, monocytes/macrophages and dendritic cells. It plays an important role in regulating immune response and maintaining immune homeostasis by synergizing with other cytokines. In this study, a plasmid pET-21d-CiIL-10 was constructed for expression of the recombinant protein in the Escherichia coli Rosetta (DE3) cells. After induction with IPTG, the recombinant protein of grass carp (Ctenopharyngodon idella, Ci) IL-10 was found in the inclusion bodies. After denaturation with 6M guanidine hydrochloride and refolding, the CiIL-10 protein was purified by size exclusion chromatography and analyzed by SDS-PAGE. The results showed that a single band of the CiIL-10 protein was visualized, confirming that the protein was pure. The recombinant CiIL-10 protein was then used to immunize mice to generate monoclonal antibodies. Four monoclonal antibodies were obtained, among which two could recognize the CiIL-10 protein expressed in the HEK293 cells. One monoclonal antibody was selected for purification and further analyzed by confocal microscopy and flow cytometry. It was shown that the CiIL-10 monoclonal antibody could react with the recombinant CiIL-10 protein expressed in the E. coli cells and HEK293 cells. In addition, confocal microscopic analysis revealed that the monoclonal antibody could detect the native CiIL-10 protein in the grass carp ovary (GCO) cells treated with PBS, lipopolysaccharide (LPS) or CiIL-1β for 36h. Interestingly, compared to the control group (PBS), the numbers of CiIL-10 producing cells remained largely unchanged after stimulation with LPS or CiIL-1β. In summary, high purity of the recombinant CiIL-10 protein was obtained from the E. coli cells and the CiIL-10 monoclonal antibody generated is highly specific. It is expected that the CiIL-10 monoclonal antibody will have great potential to be used in detection of IL-10 in grass carp by Western blotting, enzyme linked immunosorbent assay (ELISA), flow cytometry and confocal microscopy.