禽4型腺病毒单克隆抗体的制备、鉴定与初步应用

doi:10.11843/j.issn.0366-6964.2025.05.033

摘要/Abstract

摘要：

本研究旨在制备禽4型腺病毒(fowl adenovirus type 4，FAdV-4)的单克隆抗体，鉴定其特异性结合位点，并评估其在免疫组化(immunohistochemistry，IHC)和免疫沉淀反应中的应用价值。使用纯化的FAdV-4-AH-F41株全病毒免疫小鼠，通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)方法筛选出一株阳性杂交瘤细胞并进行亚克隆，并对所制备的单克隆抗体进行效价检测。利用间接免疫荧光(indirect immunofluorescence assay, IFA)与蛋白免疫印迹(Western blot)方法鉴定单克隆抗体生物学特性，构建了pCold TF-Hexon、pET-32a(+)-Fiber1、pCold TF-Penton、pCold TF-Fiber2的原核表达载体，并通过Western blot初步鉴定所得单克隆抗体的特异性结合位点，在检测出抗体的亚型后，将其初步应用于IHC和免疫沉淀试验。结果表明本试验获得1株稳定分泌抗Fiber1蛋白的单克隆抗体，并将其命名为5C7。该抗体效价为1∶102 400，亚型为IgG-2b，表现出良好的生物学特性。在IHC中，能够观察到明显的病变特征；在免疫沉淀试验中，5C7可作为捕获抗体，与病毒感染细胞中的Fiber1蛋白特异性结合。结果提示，本试验通过全病毒粒子免疫小鼠所制备的单克隆抗体能够特异性识别Fiber1蛋白，并在IHC和免疫沉淀反应中显示出良好的应用前景，为FAdV-4实验室病理诊断方法的建立和Fiber1蛋白的功能研究奠定了基础。

关键词: 血清4型禽腺病毒, 单克隆抗体, 抗体鉴定, Fiber1蛋白, IHC, 免疫共沉淀

Abstract:

The aim of this study was to prepare a monoclonal antibody to avian adenovirus type 4 (FAdV-4), to characterize its specific binding site, and to evaluate its value for use in immunohistochemistry (IHC) and immunoprecipitation reactions. Mice were immunized with purified FAdV-4-AH-F41 whole virus, a positive hybridoma cell line was screened and subcloned by indirect enzyme linked immunosorbent assay (ELISA), and the prepared monoclonal antibody was tested for potency. Indirect immunofluorescence assay (IFA) and protein immunoblotting (Western blot) were used to characterize the biological properties of the monoclonal antibodies. pCold TF-Hexon, pET-32a(+)-Fiber1, pCold TF-Penton, pCold TF-Fiber2 prokaryotic expression vectors, and the specific binding sites of the resulting monoclonal antibodies were preliminarily characterized by Western blot, and after the subtypes of the antibodies were detected, they were initially applied to IHC and immunoprecipitation experiments. The results showed that a monoclonal antibody strain with stable secretion of anti-Fiber1 protein was obtained in this experiment, and it was named 5C7. The antibody had a potency of 1∶102 400, and its subtype was IgG-2b, which showed good biological properties. In IHC, obvious lesion characteristics could be observed; in immunoprecipitation experiments, 5C7 could act as a capture antibody and bind specifically to the Fiber1 protein in virus-infected cells. The results suggest that the monoclonal antibody prepared in this experiment by immunizing mice with whole virus particles can specifically recognize the Fiber1 protein and shows good application prospects in IHC and immunoprecipitation reactions, which lays the foundation for the establishment of laboratory pathological diagnosis methods of FAdV-4 and the functional study of Fiber1 protein.

Key words: serotype 4 of fowl adenovirus, monoclonal antibody, antibody identification, fiber 1 protein, IHC, immunoprecipitation

中图分类号:

S852.659.1

张宇, 程凡玉, 俞赵荣, 邵颖, 魏宁波, 陈芳芳, 王振宇, 宋祥军, 涂健, 祁克宗. 禽4型腺病毒单克隆抗体的制备、鉴定与初步应用[J]. 畜牧兽医学报, 2025, 56(5): 2364-2371.

ZHANG Yu, CHENG Fanyu, YU Zhaorong, SHAO Ying, WEI Ningbo, CHEN Fangfang, WANG Zhenyu, SONG Xiangjun, TU Jian, QI Kezong*. Preparation, Identification, and Preliminary Application of Monoclonal Antibodies against Fowl Adenovirus Serotype 4[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2364-2371.

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包被抗原 Coated antigen	血清稀释度Serum dilution degree
包被抗原 Coated antigen	800	1 600	3 200	6 400	12 800	25 600	51 200	102 400	204 800
P	1.11	1.04	0.73	0.54	0.38	0.27	0.18	0.13	0.10
N	0.07	0.06	0.05	0.06	0.05	0.05	0.06	0.06	0.05
P/N	15.07	17.33	14.16	8.78	8.21	5.78	3.10	2.14	1.89

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