树突状细胞 (DC) 是启动和指导适应性免疫反应的抗原呈递细胞,因此在疫苗设计中至关重要。尽管靶向DC的疫苗引起了人们的关注,但对鸡的相关研究却很少。构建了一个高多样性 T7 噬菌体展示纳米抗体文库,用于对完整的鸡骨髓 DCs 进行生物淘选,以寻找 DC 特异性结合纳米抗体。经过三轮筛选,从125个随机选择的噬菌体克隆中鉴定出46个独特序列的噬菌体克隆。使用特异性测定法选择了几个结合 DC 的噬菌体克隆。Phage-54、-74、-16 和 -121 不仅与鸡 DCs 结合,而且与鸭和鹅 DCs 结合。在体外,共聚焦显微镜观察表明,与 T7-wt 相比,phage-54 和 phage-74 在 15 分钟内有效吸附到 DCs 上。然而,下拉试验没有检测到任何先前报道的鸡 DC 蛋白质,这些蛋白质可能与 phage-54 和 phage-74 上展示的纳米抗体相互作用。尽管如此,用 phage-54 和 phage-74 免疫的特定无病原体鸡显示出比 T7-wt 更高水平的抗 p10 抗体,表明纳米抗体介导的 DC 靶向增强了抗体产生。因此,本研究确定了两种禽类(鸡、鸭和鹅)DC特异性结合纳米抗体,可用于开发DC靶向疫苗。用 phage-54 和 phage-74 免疫的特定无病原体鸡显示出比 T7-wt 更高水平的抗 p10 抗体,表明纳米抗体介导的 DC 靶向增强了抗体产生。因此,本研究确定了两种禽类(鸡、鸭和鹅)DC特异性结合纳米抗体,可用于开发DC靶向疫苗。用 phage-54 和 phage-74 免疫的特定无病原体鸡显示出比 T7-wt 更高水平的抗 p10 抗体,表明纳米抗体介导的 DC 靶向增强了抗体产生。因此,本研究确定了两种禽类(鸡、鸭和鹅)DC特异性结合纳米抗体,可用于开发DC靶向疫苗。
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Construction of a T7 phage display nanobody library for bio-panning and identification of chicken dendritic cell-specific binding nanobodies
Dendritic cells (DCs) are the antigen-presenting cells that initiate and direct adaptive immune responses, and thus are critically important in vaccine design. Although DC-targeting vaccines have attracted attention, relevant studies on chicken are rare. A high diversity T7 phage display nanobody library was constructed for bio-panning of intact chicken bone marrow DCs to find DC-specific binding nanobodies. After three rounds of screening, 46 unique sequence phage clones were identified from 125 randomly selected phage clones. Several DC-binding phage clones were selected using the specificity assay. Phage-54, -74, -16 and -121 bound not only with chicken DCs, but also with duck and goose DCs. In vitro, confocal microscopy observation demonstrated that phage-54 and phage-74 efficiently adsorbed onto DCs within 15 min compared to T7-wt. The pull-down assay, however, did not detect any of the previously reported proteins for chicken DCs that could have interacted with the nanobodies displayed on phage-54 and phage-74. Nonetheless, Specified pathogen-free chickens immunized with phage-54 and phage-74 displayed higher levels of anti-p10 antibody than the T7-wt, indicating enhanced antibody production by nanobody mediated-DC targeting. Therefore, this study identified two avian (chicken, duck and goose) DC-specific binding nanobodies, which may be used for the development of DC-targeting vaccines.