Lee Sung Hyen, Lillehoj Hyun S, Jang Seung I, Lee Kyung Woo, Baldwin Cynthia, Tompkins Dannielle, Wagner Bettina, Del Cacho Emilio, Lillehoj Erik P, Hong Yeong Ho
Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705, USA.
Vet Immunol Immunopathol. 2012 Jan 15;145(1-2):527-33. doi: 10.1016/j.vetimm.2011.11.020. Epub 2011 Dec 3.
This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.
本研究旨在开发并鉴定抗鸡CD83（chCD83）的小鼠单克隆抗体（mAb），chCD83是一种膜结合糖蛋白，属于免疫球蛋白超家族，主要表达于成熟树突状细胞（DC）。含chCD83胞外区的重组chCD83/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。chCD83蛋白的胞外区经纯化后用于免疫小鼠。进行细胞融合，从中筛选出342个杂交瘤，以获得抗chCD83的mAb。两种mAb，chCD83-159和chCD83-227，对chCD83转染的CHO细胞染色比例最高，被选作进一步鉴定。通过流式细胞术，这两种mAb均与鸡巨噬细胞系HD11发生反应。这两种mAb在脂多糖刺激的脾单核细胞或未刺激的HD11细胞裂解物的Western印迹中也识别出一条单一的53 kDa蛋白。鸡二级淋巴器官的免疫染色鉴定出chCD83(+)细胞，其形态和亚组织定位特性与哺乳动物DC相当。与单独用培养基处理的细胞相比，用伴刀豆球蛋白A（Con A）体外刺激脾单核细胞可降低chCD83(+)细胞的比例。有趣的是，与用同型匹配的阴性对照mAb处理的细胞相比，在chCD83-227 mAb存在下用Con A处理的脾细胞中MHCII(+)细胞的比例降低。这些chCD83 mAb可能有助于未来对鸡免疫细胞成熟及作用机制的研究。
