禽腺病毒血清4型Fiber2蛋白单克隆抗体制备及其抗原表位鉴定

doi:10.11843/j.issn.0366-6964.2021.07.022

摘要/Abstract

摘要： 旨在制备禽腺病毒血清4型（FAdV-4）纤突蛋白（Fiber2）的特异性单克隆抗体（MAb），本研究将原核表达的可溶性重组蛋白NusA-Fiber2作为免疫原免疫BALB/c雌鼠，筛选获得3株能稳定分泌抗FAdV-4 Fiber2蛋白MAb的杂交瘤细胞株2G5、2G8、4C2，取细胞株2G5制备腹水并纯化，利用间接免疫荧光试验（immunofluorescence assay，IFA）和Western blot鉴定该单抗的特异性。用制备的单抗包被酶标板，通过一系列的优化，建立了FAdV-4 Fiber2双抗夹心ELISA检测方法。通过逐步截短分析鉴定出单克隆抗体识别的抗原表位区域。结果表明：成功获取3株单克隆细胞株2G5、2G8、4C2。MAb 2G5可与原核表达纯化的Fiber2蛋白及FAdV-4特异性反应。建立的双抗夹心ELISA检测方法具有很好的特异性、灵敏性和重复性。该MAb识别的表位序列是N端aa1—33。本研究成功制备了具有良好Western blot和IFA反应原性的单克隆抗体，为Fiber2蛋白功能研究和FAdV-4新型表位疫苗商品化研发奠定了基础。

关键词: 禽腺病毒血清4型, Fiber2蛋白, 单克隆抗体, 双抗夹心ELISA, 表位鉴定

Abstract: To prepare monoclonal antibodies specific to FAdV-4 Fiber2 protein, this study used NusA-Fiber2, a soluble recombinant protein expressed in prokaryotes, as an immunogen to immunize BALB/c female mice. Three hybridomas (2G5, 2G8 and 4C2) secreting monoclonal antibodies (MAb) against FAdV-4 Fiber2 protein were selected. Ascites were prepared from cell line 2G5 and purified. The specificity of the monoclonal antibody was determined by indirect immunofluorescence assay and Western blot. The prepared monoclonal antibody was used to coat the enzyme plate. Through a series of optimization, the detection method of FAdV-4 Fiber2 double antibody sandwich ELISA was established. The epitopes recognized by monoclonal antibodies were identified by progressively truncating Fiber2 protein. Results were as follows:Three hybridomas 2G5, 2G8 and 4C2 were successfully obtained. IFA and Western blot results showed that MAb 2G5 could react specifically with purified Fiber2 protein and FAdV-4 virus. The results showed that the sandwich ELISA was specific, sensitive and reproducible. Our results revealed that the antigenic epitope required for reactivity with the 2G5 was N-terminal aa1-33. In this study, monoclonal antibodies with good reactivity of Western blot and IFA were successfully prepared. This provides experimental data and theoretical basis for the development of rapid serological diagnosis and commercial vaccine against FAdV-4.

Key words: fowl adenovirus serotype 4 (FAdV-4), Fiber2 protein, monoclonal antibodies, sandwich ELISA, epitope identification

中图分类号:

S852.659.1

郭瑞珍, 苏冰倩, 王棋, 王一, 于朋伟, 孟洁洁, 齐艳丽, 杨国宇, 褚贝贝. 禽腺病毒血清4型Fiber2蛋白单克隆抗体制备及其抗原表位鉴定[J]. 畜牧兽医学报, 2021, 52(7): 2000-2012.

GUO Ruizhen, SU Bingqian, WANG Qi, WANG Yi, YU Pengwei, MENG Jiejie, QI Yanli, YANG Guoyu, CHU Beibei. Preparation of the Monoclonal Antibody against the Serotype 4 Fowl Adenovirus Fiber2 Protein and Identification of the Antigenic Epitope[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 2000-2012.

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