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目的 为深入探究非洲猪瘟病毒(African swine fever virus,ASFV)CP312R蛋白的生物学特性，通过2株与CP312R蛋白发生特异性反应的单克隆抗体，识别出该蛋白的一个线性抗原表位，通过合成多肽与ASF阳性临床猪血清反应，确认阳性血清中含有该线性抗原表位的抗体。方法 通过原核表达一系列CP312R蛋白截短体多肽，使用此前制备的两株单克隆抗体进行Western blot试验，鉴定出单克隆抗体结合位点。进一步诱导表达截短重叠蛋白，与两株单克隆抗体进行Western blot反应，鉴定该蛋白的线性抗原表位。继而合成包含该抗原表位的多肽，与ASF临床阳性猪血清进行ELISA反应。结果 初步鉴定出这两株单克隆抗体结合位点为117GLTVKKNEQG126氨基酸，进而通过截短重叠蛋白鉴定出CP312R蛋白1个核心线性抗原表位，即118LTVK121。对ASFV不同基因型比对发现，该位置在ASFV Ⅰ/Ⅱ型重组毒株和基因Ⅰ型、Ⅱ型、Ⅳ型、Ⅸ型、ⅩⅩ型、ⅩⅫ型中高度保守。包含该抗原表位的多肽与ASF临床阳性猪血清进行ELISA反应，显示血清中含有该抗原表位的抗体，证明猪感染ASFV后可以产生针对该抗原表位的抗体。结论 上述研究结果有助于了解ASFV CP312R的抗原表位，为开发更精准的ASF血清学诊断方法和设计基于CP312R蛋白的疫苗候选抗原提供了理论依据。
Abstract：Objective To further characterize the biological properties of the African swine fever virus(ASFV) CP312R protein, two monoclonal antibodies specifically recognizing CP312R were used to identify a linear antigenic epitope. Synthetic peptides were subsequently evaluated for reactivity with ASFV-positive clinical swine sera to confirm the presence of antibodies targeting this epitope.Methods A series of truncated CP312R protein fragments were expressed in a prokaryotic expression system. Western blot analysis using two previously generated monoclonal antibodies was performed to preliminarily map the antibody-binding regions. Overlapping truncated proteins were then expressed and analyzed by Western blot to precisely identify the linear antigenic epitope. A synthetic peptide containing the identified epitope was further subjected to enzyme-linked immunosorbent assay(ELISA) using ASFV-positive clinical swine sera. Results The binding region recognized by both monoclonal antibodies was initially identified mapped to amino acids 117 – 126(GLTVKKNEQG) of the CP312R protein. Further analysis using overlapping truncated proteins identified a single linear antigenic epitope located at residues 118 – 121(LTVK). Sequence alignment across different ASFV genotypes demonstrated that this epitope is highly conserved among genotype Ⅰ/Ⅱ type recombinant strains and genotypes Ⅰ, Ⅱ, Ⅳ, Ⅸ, ⅩⅩ, and ⅩⅫ. ELISA results showed that synthetic peptides containing this epitope reacted positively with ASFV-positive clinical swine sera, indicating that pigs infected with ASFV can generate antibodies targeting this linear epitope. Conclusion These findings contribute to a better understanding of the antigenic structure of the ASFV CP312R protein and provide a theoretical basis for the development of more precise serological diagnostic assays and the design of CP312R-based vaccine candidate antigens.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250350
中图分类号:S852.651
引用信息:
[1]张龚蕊,司安琪,刘田雨,等.非洲猪瘟病毒CP312R蛋白线性抗原表位鉴定[J].病毒学报,2026,42(01):115-125.DOI:10.13242/j.cnki.bingduxuebao.250350.
基金信息:
国家重点研发计划（项目号：2022YFD1800500）,题目：重大外来动物疫病阻断与防控技术研发项目~~