Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service-U.S. Department of Agriculture, Beltsville, MD 20705, USA.
Comp Immunol Microbiol Infect Dis. 2011 May;34(3):273-9. doi: 10.1016/j.cimid.2011.01.003. Epub 2011 Feb 21.
This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD80 (chCD80). A recombinant plasmid containing a chCD80/horse IgG4 fusion gene was constructed and expressed in CHO cells to produce recombinant chCD80/IgG4 protein. Chicken CD80 was purified from the chCD80/IgG4 fusion protein following enterokinase digestion, and used to immunize BALB/c mice, resulting in 158 hybridomas that produced mAbs against chCD80. Three mAbs with high binding specificity for recombinant chCD80/IgG4-transfected CHO cells were identified by flow cytometry, and one of these (#112) was selected for further characterization. Immunoprecipitation of CD80/IgG4-CHO cell extract, or lipopolysaccharide (LPS)-treated monocytes identified 35.0kDa proteins. Immunohistochemical analysis revealed chCD80-expressing cells exclusively in the bursal follicles at the outer portion of the cortex, and throughout the red pulp and the outer boundary of the white pulp in the spleen. By immunofluorescence microscopy, chCD80 was observed on intestinal dendritic cells. LPS treatment of bursa or spleen monocytes for 24 or 48h increased chCD80 expression. Finally, addition of chCD80 mAb to Con A-stimulated spleen cells inhibited the expression of major histocompatibility complex class II antigens and IL-2-driven proliferation of lymphoblast cells. In summary, these chCD80 mAbs will serve as valuable immunological reagents for basic and applied poultry immunology research.
本研究旨在开发和鉴定针对鸡 CD80（chCD80）的鼠源单克隆抗体（mAbs）。构建了含有 chCD80/马 IgG4 融合基因的重组质粒，并在 CHO 细胞中表达，以产生重组 chCD80/IgG4 蛋白。用肠激酶消化 chCD80/IgG4 融合蛋白，从其中纯化鸡 CD80，并用于免疫 BALB/c 小鼠，得到了 158 株产生针对 chCD80 的杂交瘤。通过流式细胞术鉴定出 3 株对重组 chCD80/IgG4 转染 CHO 细胞具有高结合特异性的 mAbs，其中一株（#112）被选中进行进一步鉴定。免疫沉淀 CD80/IgG4-CHO 细胞提取物或脂多糖（LPS）处理的单核细胞鉴定出 35.0kDa 的蛋白。免疫组织化学分析显示 chCD80 仅在外皮质的囊状滤泡中表达，并在脾脏的红髓和白髓的外边界表达。通过免疫荧光显微镜观察到肠树突状细胞上表达 chCD80。LPS 处理囊或脾单核细胞 24 或 48 小时可增加 chCD80 的表达。最后，将 chCD80 mAb 添加到 Con A 刺激的脾细胞中可抑制主要组织相容性复合体 II 抗原的表达和 IL-2 驱动的淋巴母细胞增殖。总之，这些 chCD80 mAbs 将成为基础和应用家禽免疫学研究的有价值的免疫试剂。
