Preparation and characterization of monoclonal antibodies against the pp62 protein of African swine fever virus

doi:10.1016/j.jia.2024.11.020

Journal of Integrative Agriculture

2025, Vol. 24

Issue (6): 2443-2447 DOI: 10.1016/j.jia.2024.11.020

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Preparation and characterization of monoclonal antibodies against the pp62 protein of African swine fever virus

Zhiyong Xiang^{1, 2}, Huan Ye^{1, 2}, Peng Gao^{1, 2}, Lei Zhou^{1, 2}, Xinna Ge^{1, 2}, Xin Guo^{1, 2}, Jun Han^{1, 2}, Yongning Zhang^{1, 2#}, Hanchun Yang^{1, 2#}

¹National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

²Key Laboratory of Animal Epidemiology of Ministry of Agriculture and Rural Affairs, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China

Highlights
● Two mAbs (4B5-19-7 and 4C5-11-6) specifically recognizing the ASFV polyprotein pp62 were prepared, which can further recognize its mature proteins p15 and p35, respectively.
● Heavy-chain CDR sequences of the two mAbs were different, while their light-chain CDR sequences were identical.
● The epitopes recognized by 4B5-19-7 and 4C5-11-6 were located at amino acids 95–105 and 171–180 of pp62, respectively, and were highly conserved among ASFV strains of different genotypes.

Abstract
References

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摘要

非洲猪瘟病毒（ASFV）是一种核质大DNA病毒，其基因组庞大、编码蛋白众多以及病毒结构复杂。作为ASFV核心壳的主要成分之一，多聚蛋白pp62不仅对ASFV的组装至关重要，而且具有良好的抗原性和诊断潜力。因此，制备针对pp62蛋白的单克隆抗体（简称单抗）不仅有利于其功能研究，也有利于开发性能更好的ASFV检测方法。本研究通过原核表达pp62蛋白的截短体（1−180个氨基酸）作为免疫原免疫BALB/c小鼠，成功制备了两株单抗4B5-19-7和4C5-11-6。间接免疫荧光试验表明，两株单抗不仅可以识别重组的pp62蛋白，还可以识别ASFV感染的野猪肺细胞（WSL）中的天然pp62蛋白。通过变性Western blot分析进一步发现，两株单抗识别的抗原表位为线性表位。有趣的是，除了识别全长pp62多聚蛋白外，4B5-19-7和4C5-11-6还能够分别识别pp62的成熟蛋白p15和p35。单抗亚型鉴定结果表明，4B5-19-7和4C5-11-6分别为IgG2b/κ和IgG1/κ。重链和轻链可变区测序表明，两株单抗的重链互补决定区（CDR）序列完全不同，而它们的轻链CDR序列完全相同。通过构建pp62₍₁₋₁₈₀₎蛋白的系列截短体，借助Western blot检测两株单抗以及GST标签抗体与各截短体的反应情况，结果表明，4B5-19-7识别95−105位氨基酸（SYTGVKLEVEK），4C5-11-6识别171−180位氨基酸（YTPRTRIAI）。通过分析GenBank数据库中目前可用的不同基因型ASFV代表性毒株的pp62蛋白的氨基酸序列，发现两株单抗所识别的抗原表位高度保守。pp62蛋白单抗的成功制备不仅有助于该蛋白的功能研究，而且为将来研发检测性能更佳的ASFV检测方法提供了宝贵材料。

Received: 22 July 2024 Online: 06 November 2024 Accepted: 08 October 2024

Fund:

This study was supported by the National Key Research and Development Program of China (2021YFD1800100) and the earmarked fund for China Agriculture Research System (CARS-35).

About author: Zhiyong Xiang, E-mail: m18468166738@163.com; #Correspondence Yongning Zhang, Tel: +86-10-62734924, E-mail: zhangyongning@cau.edu.cn; Hanchun Yang, Tel: +86-10-62731296, E-mail: yanghanchun1@cau.edu.cn

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Cite this article:

Zhiyong Xiang, Huan Ye, Peng Gao, Lei Zhou, Xinna Ge, Xin Guo, Jun Han, Yongning Zhang, Hanchun Yang. 2025. Preparation and characterization of monoclonal antibodies against the pp62 protein of African swine fever virus. Journal of Integrative Agriculture, 24(6): 2443-2447.

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