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。

Although African swine fever (ASF) has been prevalent for more than a century, it remains the number one swine disease that seriously endangers the global pig industry, and there is no effective means of prevention and treatment (Wang et al. 2023). Due to its enormous economic and social impact, it is listed as a notifiable animal disease by the World Organization for Animal Health (Costard et al. 2013). ASF has been present in sub-Saharan Africa since its first discovery in Kenya. During the 1950s and 1970s, it spread to Western Europe and Latin America, but most were quickly eradicated. In 2007, it spread to Russia and its neighboring Eastern European countries (Pejsak et al. 2014; Martinez-Lopez et al. 2015), and in 2018, it was first transmitted to China and quickly spread to other Asian countries and regions, posing a serious threat to global pork production (Dixon et al. 2020).

African swine fever virus (ASFV), the causative agent of ASF, is an icosahedral nucleocytoplasmic large DNA virus belonging to the genus Asfivirus in the family Asfarviridae (Alonso et al. 2018). To date, more than 150 proteins encoded by ASFV have been identified; however, the functions of most of them remain unknown. The structure of ASFV virion is extremely complex, consisting of five layers, namely outer envelope, capsid, inner envelope, core shell, and nucleoid (Liu et al. 2019). Among them, the core shell is mainly composed of two polyproteins, pp62 and pp220. Both play an important role in the correct assembly of ASFV core shell and viral replication (Andrés et al. 1997; Suárez et al. 2010). The pp62, encoded by the CP530R gene, is located in the viral factory and can be proteolytically cleaved into mature proteins p35 and p15 by the ASFV cysteine protease pS273R in the late stage of infection (Simón-Mateo et al. 1997; Andrés et al. 2002; Jia et al. 2017). The pp62 derivatives p35 and p15 are important structural proteins of ASFV, which participate in the formation of icosahedral capsid structure. Moreover, pp62 was demonstrated to be a late protein that is incredibly immunogenic and is highly conserved among different ASFV strains (Simón-Mateo et al. 1997). Considering the important properties of pp62 and its critical role in the ASFV replication cycle, development of monoclonal antibodies (mAbs) against pp62 can provide valuable tools for the diagnosis and basic research of ASFV.

In this study, multiple bioinformatics analyses of pp62 showed that its 1−180 amino acid (aa) region has good antigenicity and solubility, and contains abundant antigenic epitopes (Appendix A). Therefore, this region was prokarytically expressed as a His-tagged fusion protein (designated pp62_(1−180aa)-His) by constructing a recombinant plasmid pET-28a(+)-pp62_(1−180aa)-His, transforming it into E. coli Rosette (DE3) cells and then inducing with isopropyl β-D-1-thiogalactopyranoside. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a protein band of ~21 kDa was detected in the supernatant of transformed cell lysates (Appendix B), and its size was consistent with the theoretical calculation. The soluble pp62_(1−180aa)-His was affinity-purified from the supernatants of bacterial lysates using Ni-NTA agarose under native conditions. SDS-PAGE analysis indicated that the purity of purified pp62_(1−180aa)-His was about 95%, and western blot (WB) analysis further revealed that the protein could be recognized by mouse anti-His-tag mAb and swine ASFV antiserum (Fig. 1-A). The purified pp62_(1−180aa)-His was used to immunize 6-week-old BALB/c mice subcutaneously using 60 μg protein emulsified in Freund's complete adjuvant, followed by two booster immunizations with 30 μg protein emulsified in Freund's incomplete adjuvant at two-week interval. Splenocytes from the immunized mice were fused with SP2/0 cells. By means of indirect ELISA screening combined with three rounds of subcloning, two monoclonal hybridoma cell clones (4B5-19-7 and 4C5-11-6) stably secreting anti-pp62 mAbs were obtained, and then intraperitoneally injected into BALB/c mice to produce ascites. The titers of 4B5-19-7 and 4C5-11-6 in ascites were 1:204800 and 1:409600, respectively, as determined by indirect ELISA (Appendix C). The isotypes of mAbs 4B5-19-7 and 4C5-11-6 were determined to be IgG2b/κ and IgG1/κ, respectively (Appendix D). Furthermore, RT-PCR was performed to amplify the heavy- and light-chain variable regions of each mAb using the RNA extracted from hybridoma cells as the template and with the specific primers (Appendix E, F). The amplicons were sequenced and the results were submitted to the GenBank database under accession numbers PQ037594−PQ037597 (Table 1). The corresponding aa sequences were annotated using the Kabat Antibody Numbering Scheme online tool to show the complementarity-determining regions (CDR), including CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The results showed that aa sequences of the heavy-chain CDR regions of pp62 mAbs are different from each other, while their light-chain CDR region aa sequences are completely identical (Table 1). Next, the reaction of mAbs with pp62 protein was analyzed by an indirect immunofluorescence assay (IFA), which showed that both mAbs react well with the natural pp62 protein in ASFV-infected WSL cells (Fig. 1-B). However, the two mAbs showed no cross reactivity with other important porcine pathogens, such as porcine reproductive and respiratory syndrome virus and Getah virus (Appendix G), indicating that they have good specificity. WB analysis further demonstrated that pp62 mAbs could recognize a ~62 kDa protein band in ASFV-infected WSL cells (Fig. 1-C), which matches the theoretical molecular weight of the natural pp62. Considering that the protein denaturation condition was used when analyzing the reactivity of mAbs with pp62 by WB, it was concluded that the epitopes recognized by two mAbs are linear. Notably, the expression of pp62 was detectable from 12 h post-infection onward, which is consistent with the previous reports that pp62 is a late protein (Andrés et al. 2002; Simón-Mateo et al. 1997).

To our knowledge, there are very few reports on preparation of mAbs against the ASFV pp62 protein. Bai and colleagues prepared eighteen mAbs against pp62, of which fifteen recognize p15 and the remaining three recognize p35 (Bai et al. 2020). In addition, Chu et al. generated four mAbs against pp62, of which two recognize pp62 and the remaining recognize p15 and p35, respectively (Chu et al. 2022). Interestingly, when using WB to analyze the reaction of pp62 mAbs with the total proteins of ASFV-infected WSL cells, we found that in addition to producing a clear protein band at ~62 kDa, the 4B5-19-7 also produced a clear band at ~15 kDa, while 4C5-11-6 at ~35 kDa (Fig. 1-D). This suggests that the epitopes recognized by pp62 mAbs should be located at different positions in pp62. Previous studies have shown that pp62 needs to be proteolytically cleaved into two mature structural proteins, p15 and p35, when assembled into the core shell in the late stage of ASFV infection (Simón-Mateo et al. 1997; Andrés et al. 2002; Jia et al. 2017). The cleavage of pp62 has been demonstrated to be mediated by the ASFV pS273R, with cleavage sites located at G158-G159 and G463-G464 (Appendix H). The resultant p15 and p35 are composed of aa at positions 1−158 and 159−462 of pp62, respectively (Simón-Mateo et al. 1997; Suárez et al. 2010). To verify whether 4B5-19-7 and 4C5-11-6 recognize p15 and p35, respectively, the eukaryotic recombinant plasmids pCMV-HA-p15 and pCMV-HA-p35 constructed with the primers shown in Appendix F were individually transfected into WSL cells, which were then analyzed by IFA. The results showed that 4B5-19-7 only reacted with p15, while 4C5-11-6 only reacted with p35 (Appendix I). This indicates that pp62 mAbs recognize different antigenic epitopes.

To identify the epitopes recognized by pp62 mAbs, a series of recombinant prokaryotic plasmids respectively expressing GST-tagged pp62_(1−180aa) and 19 additional truncated pp62_(1−180aa) were constructed (Appendix F, J). Fig. 1-E shows that only the truncated proteins containing 95–105 aa (SYTGVKLEVEK) were able to react with both 4B5-19-7 and GST polyclonal antibody, while those lacking this domain could not. This indicate that the epitope recognized by 4B5-19-7 is located at 95–105 aa of pp62. Similarly, the epitope recognized by 4C5-11-6 was shown to be located at 171–180 aa (YTPRTRIAIE). As illustrated in Appendix H, the epitopes recognized by mAbs 4B5-19-7 and 4C5-11-6 are located exactly within the aa range of p15 and p35.

To analyze whether the epitopes recognized by pp62 mAbs are conserved, a total of 36 pp62 aa sequences of representative ASFV strains of different genotypes currently available in the GenBank database were downloaded and analyzed. These ASFV strains used for analysis originate from 21 countries in Africa, Europe, and Asia, involving 12 genotypes. The results showed that the pp62 is highly conserved among ASFV strains of different genotypes, and the epitopes recognized by pp62 mAbs are more conserved, without any mutations in the aa regions where the epitopes are located (Fig. 1-F).

In summary, we successfully prepared two mAbs that can specifically recognize the ASFV pp62. Among them, 4B5-19-7 and 4C5-11-6 can further recognize the p15 and p35 of pp62 cleavage products, respectively. The epitopes recognized by 4B5-19-7 and 4C5-11-6 are highly conserved and located at amino acids 95−105 (SYTGVKLEVEK) and 171−180 (YTPRTRIAIE) of pp62, respectively. The mAbs provide valuable materials for pp62 functional study and development of improved serological diagnostic agents and assays.