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From modular vaccine production to protein assembly on nanoparticles, the SpyCatcher/SpyTag system provides a convenient plug-and-display procedure. Here, we established a general-purpose immunoaffinity chromatography (IAC) method for SpyTagged proteins (Spy&IAC). SpyTags are displayed on the surface of nanoparticles to induce high-affinity monoclonal antibodies, allowing the specific capture of the target protein. Taking the key core antigenic regions of two coronaviruses that are currently more threatened in the field of human and animal diseases, the nucleocapsid (N) protein of SARS-CoV-2 and the COE protein of porcine epidemic diarrhea virus (PEDV) as model proteins, a purification model with SpyTag at the N-terminal or C-terminal expressed in E. coli or mammalian cells was constructed. After the efficient elution of Spy&IAC, the final yield of several proteins is about 3.5–15 mg/L culture, and the protein purity is above 90 %. Purification also preserves the assembly function and immunogenicity of the protein to support subsequent modular assembly and immunization programs. This strategy provides a general tool for the efficient purification of SpyTagged proteins from different expression sources and different tag positions, enabling the production of modular vaccines at lower cost and in a shorter time, which will prepare the public health field for potential pandemic threats. [Display omitted] • The high-affinity monoclonal antibody was prepared by the multivalent display of nanoparticles. • An immunoaffinity chromatography method was established for the first time for the purification of SpyTagged proteins. • The assembly function and immunogenicity of elution SpyTagged protein were retained. [ABSTRACT FROM AUTHOR]

African swine fever (ASF) caused by African swine fever virus (ASFV) is becoming a serious threat to the swine industry worldwide. CD2v is a key pathogenic factor of ASFV and the protective antigen with low immunogenicity, whereas viral protein-based nanoparticles have advantages of precise assembly and high immunogenicity. In this study, the CD2v protein fused with Norovirus (NoV) P particle assembled into nanoparticle for improved immunogenicity. Then, CD2v protein nanoparticle and monomer CD2v protein were expressed in HEK293F cells. The former induced higher levels of antibodies, and thus highly potent monoclonal antibodies (mAbs) were generated and characterized. The highest antibody titration of mAb 10A3 reached 1:2048000, and mAb 2E9 had the highest inhibition percent of 84% when competed with ASFV positive serum. Meanwhile, all mAbs reacted specifically with the denatured CD2v protein, and the linear epitope with the location of amino acids 28th to 51st of CD2v extracellular domain sequence was identified. In summary, this study produced a highly immunogenic CD2v protein and generated high-titer mAbs, the precise location of linear epitope on the CD2v was further determined. These findings may provide a powerful help for etiology and serological detection of ASFV. • Highly immunogenic CD2v protein nanoparticle was obtained. • High-titer mAbs induced by CD2v protein nanoparticle were generated, and mAb 10A3 had the highest antibody titration of 1:2048000. • The linear epitope from the 28th to the 51st position of amino acids locating in the CD2v extracellular domain sequence was first reported. [ABSTRACT FROM AUTHOR]