{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T13:17:13Z","timestamp":1780406233487,"version":"3.54.1"},"reference-count":33,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T00:00:00Z","timestamp":1780012800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2022YFD1800902"],"award-info":[{"award-number":["2022YFD1800902"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Porcine circovirus type 3 (PCV3) capsid protein (Cap) is a key antigen for immunological studies and vaccine development. Different optimized PCV3 ORF2 sequences were used to construct six baculovirus transfer plasmids, with the pOET1.1-based design yielding the highest Cap level. Cap expression was confirmed by Western blot, IPMA and IFA. Recombinant baculovirus amplification was optimized, achieving the highest titer at an MOI of 0.1 with a 72 h harvest to 107.5TCID50\/0.1 mL, while maximal Cap production was obtained at an MOI of 0.1 with a 96 h harvest. PCV3 Cap virus-like particles (VLPs) were purified by sucrose density-gradient ultracentrifugation and cation-exchange chromatography, and TEM revealed spherical particles of approximately 17\u201320 nm. In mice, VLP immunization induced increasing antigen-specific IgG from day 14. Immunization increased both IgG1 and IgG2a without a significant difference, and post-immunization serum specifically recognized PCV3-positive passaged PK-15 cells in an indirect immunofluorescence assay. In splenic lymphocytes, IFN-\u03b3, TNF-\u03b1, IL-4, and IL-10 mRNA levels were significantly upregulated (p &lt; 0.01). Moreover, pig challenge data supported the protective potential of PCV3 Cap VLPs in the natural host. In our study, Cap assembled into VLPs and induced immune responses, providing a basis for PCV3 subunit vaccine development.<\/jats:p>","DOI":"10.3390\/ijms27114930","type":"journal-article","created":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T12:45:08Z","timestamp":1780404308000},"page":"4930","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Immunogenicity Analysis of PCV3 Capsid Highly Expressed Using Baculovirus"],"prefix":"10.3390","volume":"27","author":[{"given":"Baoge","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Bacteriology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lumen","family":"Chao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bacteriology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuchen","family":"Cai","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bacteriology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yufeng","family":"Li","sequence":"additional","affiliation":[{"name":"Key Laboratory of Bacteriology, Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1080\/22221751.2019.1613176","article-title":"Pcv3-Associated Disease in the United States Swine Herd","volume":"8","author":"Arruda","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"BSR20201109","DOI":"10.1042\/BSR20201109","article-title":"Structural Insight into the Type-Specific Epitope of Porcine Circovirus Type 3","volume":"40","author":"Bi","year":"2020","journal-title":"Biosci. Rep."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1186\/s13568-023-01652-6","article-title":"Exploring the Surface Epitope and Nuclear Localization Analysis of Porcine Circovirus Type 3 Capsid Protein","volume":"13","author":"Chang","year":"2023","journal-title":"AMB Express"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"4329","DOI":"10.1007\/s12033-024-01334-z","article-title":"Design of a Golden Gate Cloning Plasmid for the Generation of a Chimeric Virus-Like Particle-Based Subunit Vaccine against Porcine Circovirus Type 2","volume":"67","author":"Rodriguez","year":"2025","journal-title":"Mol. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1186\/s13568-020-01163-8","article-title":"High Repetitive Arginine in the Anterior of Pcv3 Capsid Protein is a Severe Obstacle for Its Expression in E. coli","volume":"10","author":"Liu","year":"2020","journal-title":"AMB Express"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1186\/s12951-022-01710-4","article-title":"A Modular and Self-Adjuvanted Multivalent Vaccine Platform Based on Porcine Circovirus Virus-Like Nanoparticles","volume":"20","author":"Liu","year":"2022","journal-title":"J. Nanobiotechnology"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1501","DOI":"10.1007\/s00253-012-4015-2","article-title":"Characterization of Porcine Circovirus Type 2 (Pcv2) Capsid Particle Assembly and Its Application to Virus-Like Particle Vaccine Development","volume":"95","author":"Wu","year":"2012","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"10415","DOI":"10.1007\/s00253-015-7000-8","article-title":"The Application of Virus-Like Particles as Vaccines and Biological Vehicles","volume":"99","author":"Yan","year":"2015","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Cao, X., Huang, M., Wang, Y., Chen, Y., Yang, H., and Quan, F. (2023). Immunogenicity Analysis of Pcv3 Recombinant Capsid Protein Virus-like Particles and Their Application in Antibodies Detection. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241210377"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1016\/j.vaccine.2020.12.013","article-title":"Evaluation of Novel Recombinant Porcine Circovirus Type 2d (Pcv2d) Vaccine in Pigs Naturally Infected with Pcv2d","volume":"39","author":"Kim","year":"2021","journal-title":"Vaccine"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6307","DOI":"10.1016\/j.vaccine.2012.07.082","article-title":"Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Capsid Protein of Pcv2","volume":"30","author":"Lin","year":"2012","journal-title":"Vaccine"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1975","DOI":"10.1016\/j.vaccine.2020.01.013","article-title":"Porcine Circovirus Type 2a or 2b Based Experimental Vaccines Provide Protection against Pcv2d\/Porcine Parvovirus 2 Co-Challenge","volume":"38","author":"Opriessnig","year":"2020","journal-title":"Vaccine"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Rakibuzzaman, A., Kolyvushko, O., Singh, G., Nara, P., Pineyro, P., Leclerc, E., Pillatzki, A., and Ramamoorthy, S. (2020). Targeted Alteration of Antibody-Based Immunodominance Enhances the Heterosubtypic Immunity of an Experimental Pcv2 Vaccine. Vaccines, 8.","DOI":"10.3390\/vaccines8030506"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"254","DOI":"10.1111\/j.1348-0421.2011.00311.x","article-title":"A Candidate Inactivated Chimeric Vaccine Pcv1-2 Constructed Based on Pcv1 and Pcv2 Isolates Originating in China and Its Evaluation in Conventional Pigs in Regard to Protective Efficacy against Pcv2 Infection","volume":"55","author":"Xujie","year":"2011","journal-title":"Microbiol. Immunol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"600","DOI":"10.1089\/vim.2015.0021","article-title":"Correlates of Protection Following Vaccination with Inactivated Porcine Circovirus 2 Vaccines","volume":"28","author":"Zanotti","year":"2015","journal-title":"Viral Immunol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Kim, K., Choi, K., Shin, M., and Hahn, T.W. (2023). A Porcine Circovirus Type 2d-Based Virus-Like Particle Vaccine Induces Humoral and Cellular Immune Responses and Effectively Protects Pigs against Pcv2d Challenge. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1334968"},{"key":"ref_17","first-page":"891","article-title":"Human Papillomavirus Type16- L1 Vlp Production in Insect Cells","volume":"16","author":"Abdoli","year":"2013","journal-title":"Iran. J. Basic Med. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1586\/erv.12.151","article-title":"Virus-Like Particles for the Prevention of Human Papillomavirus-Associated Malignancies","volume":"12","author":"Wang","year":"2013","journal-title":"Expert Rev. Vaccines"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.biologicals.2009.02.014","article-title":"Flublok, a Next Generation Influenza Vaccine Manufactured in Insect Cells","volume":"37","author":"Cox","year":"2009","journal-title":"Biologicals"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1007\/s40265-013-0103-6","article-title":"Recombinant Trivalent Influenza Vaccine (Flublok\u00ae): A Review of Its Use in the Prevention of Seasonal Influenza in Adults","volume":"73","author":"Yang","year":"2013","journal-title":"Drugs"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"159","DOI":"10.1016\/j.pep.2015.03.004","article-title":"Positional Effects of Fusion Partners on the Yield and Solubility of Mbp Fusion Proteins","volume":"110","author":"Keefe","year":"2015","journal-title":"Protein Expr. Purif."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1007\/978-1-0716-3710-4_26","article-title":"Gene Stacking and Stoichiometric Expression of Er-Targeted Constructs Using \u201c2a\u201d Self-Cleaving Peptides","volume":"Volume 2772","author":"Fricker","year":"2024","journal-title":"The Plant Endoplasmic Reticulum: Methods and Protocols; Methods in Molecular Biology"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Ge, M., Ren, J., Xie, Y.L., Zhao, D., Fan, F.C., Song, X.Q., Li, M.X., and Xiao, C.T. (2021). Prevalence and Genetic Analysis of Porcine Circovirus 3 in China from 2019 to 2020. Front. Vet. Sci., 8.","DOI":"10.3389\/fvets.2021.773912"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Li, Y., Yu, P., Bao, Y., Ren, Y., Zhao, S., and Zhang, X. (2023). Production of Virus-Like Particles of Porcine Circovirus 2 in Baculovirus Expression System and Its Application for Antibody Detection. BMC Vet. Res., 19.","DOI":"10.1186\/s12917-023-03648-7"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"101228","DOI":"10.1016\/j.omtm.2024.101228","article-title":"A Robust and Flexible Baculovirus-Insect Cell System for Aav Vector Production with Improved Yield, Capsid Ratios and Potency","volume":"32","author":"Marwidi","year":"2024","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"134955","DOI":"10.1016\/j.ijbiomac.2024.134955","article-title":"Efficacy Comparison in Cap Vlps of Pcv2 and Pcv3 as Swine Vaccine Vehicle","volume":"278","author":"Qiu","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1007\/978-1-0716-3961-0_12","article-title":"Production of Recombinant Viral Antigens Using the Baculovirus-Insect Cell Expression System","volume":"Volume 2829","author":"Shivhare","year":"2024","journal-title":"Baculovirus: Methods and Protocols; Methods in Molecular Biology"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Wang, J., Lei, B., Zhang, W., Li, L., Ji, J., Liu, M., Zhao, K., and Yuan, W. (2024). Preparation of Monoclonal Antibodies against the Capsid Protein and Development of an Epitope-Blocking Enzyme-Linked Immunosorbent Assay for Detection of the Antibody against Porcine Circovirus 3. Animals, 14.","DOI":"10.3390\/ani14020235"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"933","DOI":"10.1007\/s00705-012-1565-7","article-title":"Characterization and Immunogenicity of Norovirus Capsid-Derived Virus-Like Particles Purified by Anion Exchange Chromatography","volume":"158","author":"Huhti","year":"2013","journal-title":"Arch. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1923","DOI":"10.1002\/elps.202200270","article-title":"Purification of a Recombinant Oncolytic Virus from Clarified Cell Culture Media by Anion Exchange Monolith Chromatography","volume":"44","author":"Rogerson","year":"2023","journal-title":"Electrophoresis"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/j.jchromb.2015.04.004","article-title":"Purification of Recombinant Virus-Like Particles of Porcine Circovirus Type 2 Capsid Protein Using Ion-Exchange Monolith Chromatography","volume":"991","author":"Zaveckas","year":"2015","journal-title":"J. Chromatogr. B"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e0034125","DOI":"10.1128\/jvi.00341-25","article-title":"Rescue of Naive Porcine Circovirus Type 3 and Its Pathogenesis in Cd Pigs","volume":"99","author":"Zhang","year":"2025","journal-title":"J. Virol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"267","DOI":"10.1007\/978-1-0716-3961-0_20","article-title":"TCID50 Assay: A Simple Method to Determine Baculovirus Titer","volume":"Volume 2829","author":"Zhang","year":"2024","journal-title":"Baculovirus: Methods and Protocols; Methods in Molecular Biology"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/4930\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,2]],"date-time":"2026-06-02T12:55:34Z","timestamp":1780404934000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/11\/4930"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,29]]},"references-count":33,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["ijms27114930"],"URL":"https:\/\/doi.org\/10.3390\/ijms27114930","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,29]]}}}