Lee Sung Hyen, Lillehoj Hyun S, Jang Seung I, Baldwin Cynthia, Tompkins Dannielle, Wagner Bettina, Parcells Mark, Del Cacho Emilio, Hong Yeong Ho, Min Wongi, Lillehoj Erik P
Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA.
Vet Immunol Immunopathol. 2011 Dec 15;144(3-4):396-404. doi: 10.1016/j.vetimm.2011.08.001. Epub 2011 Aug 9.
This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD25 (chCD25), the alpha chain of the interleukin-2 (IL-2) receptor. A recombinant chimeric chCD25/IgG4 fusion protein was expressed in Chinese hamster ovary (CHO) cells and isolated from spent cell culture medium by protein G affinity chromatography. Purified chCD25 protein was used to immunize mice, from which 54 stable hybridomas secreting chCD25 mAbs were produced. Two mAbs, chCD25-32 and chCD25-54, with high binding affinity for chCD25-expressing CHO cells were selected for further characterization. By flow cytometry, both mAbs detected cells in the spleen, bursa of Fabricius, intestinal duodenum, and immunostained established chicken T cell, B cell, and macrophage cell lines. Both mAbs reacted with a 55 kDa protein on Western blots of lysates from concanavalin A (Con A)-stimulated spleen mononuclear cells. Intraperitoneal injection of chickens with bacterial lipopolysaccharide increased the percentage of chCD25(+) spleen cells by approximately 4-fold compared with untreated animals. In vitro stimulation of spleen cells with Con A increased the percentage of chCD25(+) cells by up to 50-fold compared with cells treated with medium alone. Finally, the chCD25-32 mAb suppressed IL-2-driven spleen cell proliferation and reduced IL-2-induced nitric oxide production. These mAbs may be useful for future investigation of chicken regulatory T cells.
本研究旨在开发并鉴定针对鸡白细胞介素-2（IL-2）受体α链鸡CD25（chCD25）的小鼠单克隆抗体（mAb）。重组嵌合chCD25/IgG4融合蛋白在中国仓鼠卵巢（CHO）细胞中表达，并通过蛋白G亲和层析从细胞培养液中分离出来。纯化的chCD25蛋白用于免疫小鼠，从中产生了54个分泌chCD25 mAb的稳定杂交瘤。选择了两个对表达chCD25的CHO细胞具有高结合亲和力的mAb，chCD25-32和chCD25-54进行进一步鉴定。通过流式细胞术，这两种mAb均检测到脾脏、法氏囊、十二指肠中的细胞，并对已建立的鸡T细胞、B细胞和巨噬细胞系进行了免疫染色。这两种mAb在伴刀豆球蛋白A（Con A）刺激的脾单核细胞裂解物的蛋白质印迹上与一种55 kDa的蛋白质发生反应。与未处理的动物相比，给鸡腹腔注射细菌脂多糖可使chCD25(+)脾细胞的百分比增加约4倍。与仅用培养基处理的细胞相比，用Con A体外刺激脾细胞可使chCD25(+)细胞的百分比增加多达50倍。最后，chCD25-32 mAb抑制了IL-2驱动的脾细胞增殖，并减少了IL-2诱导的一氧化氮产生。这些mAb可能对未来鸡调节性T细胞的研究有用。
