Introduction Porcine circovirus (PCV) is a small non-enveloped virus belonging to the Circovirus genus of the Circoviridae family. established ELISA method has good sensitivity, specificity, repeatability and clinical applicability. These results demonstrate the successful expression of the PCV3 recombinant Cap protein and the preparation of recombinant Cap protein VLPs, which can be utilized for the preparation of subunit vaccines. In the mean time, the established I-ELISA method lays a foundation for the development of the commercial PCV3 serological antibody detection kit. Keywords: porcine circovirus type 3 (PCV3), capsid protein (Cap), virus-like particles (VLPs), enzyme-linked immunosorbent assay (ELISA) 1. Introduction Porcine circovirus (PCV) is usually a small non-enveloped virus belonging to the Circovirus genus of the Circoviridae family. Its virion diameter is about 14 to 17 nm, making it one of the smallest animal viruses known so far [1]. PCV includes four genotypes, namely PCV1, PCV2, PCV3 and PCV4, which are very comparable in structure and composition [2]. PCV1 was first discovered in PK-15 cells in 1974 and is not pathogenic [3]. PCV2, a major pathogen, was discovered in Canada in 1995 [4]. PCV4 was first discovered in Hunan Province, China in 2019, and its contamination can cause severe clinical symptoms such as respiratory disease and diarrhea in pigs, but its pathogenic mechanism remains unclear [5]. PCV3 was also discovered in the United States in 2016, and this viral disease can cause porcine dermatitis and nephropathy syndrome (PDNS), sow abortion, piglet heart and multi-system inflammation, and has been widely found around the world [6]. Much like other circoviruses, the full length of the PCV3 genome is usually 2000 bp and contains three inverted open reading frames, ORF1, ORF2 and ORF3. The full length of ORF1 encoding replicase protein (Rep) is usually 896 bp and encoding 296 aa. The full length of ORF3 is usually 693 bp, the code is usually 230 aa and the specific function is not obvious. ORF2 encodes nucleocapsid protein (Cap), which has a full length of 645 bp and encodes 214 aa. It is the only structural protein of PCV3 and can self-assemble into virus-like particles (VLP) that are morphographically much like PCV3 virions. The genome of PCV3 has been sequenced, but the homology with other porcine circoviruses is usually Atractylenolide III low, and the sequence homology with PCV2 is only 37% to 40%. There have been no reports of cross-protection against PCV3 with a commercially available PCV2 vaccine. The VLP has no viral genetic material, cannot replicate and does not have the ability to infect, with a similar spatial three-dimensional structure of natural computer virus particles, which can Atractylenolide III effectively induce an immune protection effect in the organism [7]. At the same time, VLPs are more suitable for the development of serological diagnostic assessments because of their higher security and ease of use. An enzyme-linked immunosorbent assay (ELISA) based on VLPs is usually widely used to measure antibodies or neutralizing epitopes. For example, PCV2 VLPs are used as coated antigens so that serum-neutralizing antibodies can be detected in the ELISA [8]. Therefore, CLU the PCV3 Cap protein has become an ideal target for the in vitro expression and development of subunit vaccines and related detection methods. Pro-inflammatory cytokines play a key role in the development and maintenance of inflammation and can lead to multiple organ damage. Previous studies have shown that PCV3 contamination up-regulates pro-inflammatory cytokines in pigs [9]. Moreover, the PCV3-mediated clinical symptoms of illness and tissue damage may be caused by the high levels of pro-inflammatory cytokines. Recent studies have also confirmed that PCV3 contamination does cause an inflammatory response, in which IFN-, IL-6, IL-8, TNF- and other cytokines are significantly increased after contamination [10]. In this study, we successfully obtained the truncated expression of the recombinant Cap protein through the prokaryotic expression system, and successfully put together it into VLPs in vitro. Antibody levels and interferon (IFN-), IL-4, IL-2 and T cell subsets were evaluated by immunizing mice to understand the potential application of the recombinant Atractylenolide III Cap protein as a candidate vaccine based on VLP. This study also developed a highly specific,.