Preparation and Epitope Analysis of Monoclonal Antibody against African Swine Fever Virus D205R Protein

doi:10.15933/j.cnki.1004-3268.2025.02.014

Abstract

Abstract: African swine fever（ASF）is a lethal infectious disease，and few vaccines or drugs were available to prevent or control ASF to date.In order to provide important materials for the diagnosis of African swine fever virus（ASFV），the ASFV recombinant D205R protein was expressed by E.coli system，a monoclonal antibody（mAb）against D205R protein was prepared，and antigenic epitope of mAb was identified.The results showed that the pET32a⁃D205R expression plasmid was successfully constructed，and the recombinant D205R protein with a size of about 44 ku was purified.Western blot assay showed that the recombinant D205R protein reacted specifically with ASFV⁃positive serum and had good immunogenicity.By hybridoma cell fusion and screening，mAb 19A5 was obtained. Western blot and indirect immunofluorescence assays showed that mAb 19A5 specifically recognized eukaryotically expressed recombinant D205R protein，and detected wild⁃type D205R protein.Alanine scanning identified 167⁃SDPPVVWLGGRPGD⁃180 as the antigenic epitope of mAb 19A5，and S167，W173，L174，G175，P178，and D180 were the key amino acids that binded to mAb 19A5.Homologous and structural analysis revealed that the antigenic epitope was highly conserved and located on the surface of the protein，which was a linear epitope.In conclusion，mAb 19A5 was successfully prepared and the antigenic epitope recognized by mAb 19A5 was identified.

Key words: African swine fever virus, D205R protein, Monoclonal antibody, Antigenic epitope

摘要： 非洲猪瘟（ASF）是一种致命的传染病，迄今为止，还没有开发出有效的疫苗或药物用于预防或控制ASF。为提供诊断非洲猪瘟病毒（ASFV）的重要材料，利用大肠杆菌系统表达ASFV重组D205R蛋白，制备D205R蛋白的单克隆抗体（mAb），鉴定mAb识别的抗原表位。结果显示，成功构建pET32a-D205R表达质粒，并纯化大小约为44 ku的重组D205R蛋白。蛋白质免疫印迹（Western blot）检测结果表明，重组D205R蛋白与ASFV阳性血清发生特异性反应，有良好的免疫原性。利用杂交瘤细胞融合、筛选的方法，得到mAb 19A5。Western blot和间接免疫荧光试验（IFA）检测结果表明，mAb 19A5能特异性识别真核表达的重组D205R 蛋白，并能检测到野生型D205R 蛋白。用丙氨酸扫描法鉴定出167-SDPPVVWLGGRPGD-180 是mAb 19A5 的抗原表位，S167、W173、L174、G175、P178 和D180 是与mAb19A5结合的关键氨基酸。保守性和结构分析表明，抗原表位高度保守，且位于蛋白质表面，是线性表位。综上，成功制备mAb 19A5，并鉴定了mAb 19A5识别的抗原表位。

关键词: 非洲猪瘟病毒, D205R蛋白, 单克隆抗体, 抗原表位

CLC Number:

S855.3

LIN Zhizhao, ZHAO Yanyan, REN Haojie, SHI Saiyan, HAN Shichong, HE Wenrui, WAN Bo, ZHANG Yuhang, ZHANG Gaiping. Preparation and Epitope Analysis of Monoclonal Antibody against African Swine Fever Virus D205R Protein[J]. Journal of Henan Agricultural Sciences, 2025, 54(2): 124-130.

林志钊, 赵燕燕, 任豪杰, 史赛燕, 韩世充, 何文瑞, 万博, 张雨杭, 张改平. 非洲猪瘟病毒D205R蛋白单克隆抗体的制备和抗原表位鉴定[J]. 河南农业科学, 2025, 54(2): 124-130.

Figures/Tables 6

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