Preparation and Immunogenicity Evaluation of Recombinant Nanoparticles of African swine fever virus p34 Protein
ZHANG Rong1,2,3, RU Yi2, JIANG Cheng-Hui1,2,3, ZHANG Yue2, ZHAO Dong-Mei2, HAO Rong-Zeng2, LI Ya-Jun2,3, ZHENG Hai-Xue2, LIU Xue-Rong3, YANG Jin-Cai3, AN Lan-Fang3, WEI Yan-Ming1,*
1 College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China; 2 Lanzhou Veterinary Research Institute/State Key Laboratory for Animal Disease Control and Prevention, Chinese Academy of Agricultural Sciences, Lanzhou 730000, China; 3 China Agricultural Vet. Bio. Science and Technology Co., Ltd., Lanzhou 730046, China
Abstract：Ferritin nanoparticles are widely used in vaccine research due to their surface that can display multiple antigens, which can better promote cell uptake and antigen presentation, and thereby enhance immune efficacy. This study developed ferritin-based nanoparticles carrying the African swine fever virus (ASFV) p34 protein and evaluated its immunogenicity, aiming to provide an reference for the research on nanoparticle vaccines against ASFV. Initially, the gene sequences encoding the p34 protein and Spytag (ST) were cascaded and inserted into the pET-28a(+) vector to create the pET-p34-ST recombinant plasmid. The gene sequences encoding Spycatcher (SC) and ferritin (F) were cascaded and then inserted into the pET-28a(+) vector to produce the pET-SC-F recombinant plasmid. Both plasmids were expressed in Escherichia coli upon induction. Subsequently, after purifying the 2 recombinant proteins p34-ST and SC-F by affinity chromatography, they were conjugated in vitro to obtain the recombinant nanoparticle F-p34. The F-p34 was identified and morphologically characterized by SDS-PAGE, Western blot, dynamic light scattering (DLS) and transmission electron microscopy (TEM), and finally, the immunogenicity was evaluated by immunizing BALB/c mice (Mus musculus). The results showed that in this study, the prokaryotic expression vectors pET-28a-p34-ST and pET-28a-SC-F were successfully designed and constructed. Through expression, purification and coupling in vitro, recombinant nanoparticle F-p34, which was conjugated with ASFV p34 protein, was successfully prepared, and it could specifically react with ASFV positive serum. In vitro the characterization by DLS and TEM showed that the particle size of F-p34 was approximately 21 nm, and it had a uniform nanoparticle structure. After immunizing mice with F-p34, a specific immune response could be elicited in the body within 7 d. The antibody titer reached the highest level at 42 d and was significantly higher than that of the p34 immunization group (P<0.05). The proliferation level of spleen lymphocytes in the F-p34 immunization group and the secretion levels of cytokines interleukin 4 (IL-4), IL-10, IL-2 and interferon (IFN-γ) in serum were significantly higher than those in the p34 immunization group (P<0.05). In conclusion, this research successfully prepared nanoparticle antigen carrying the ASFV p34 protein with good immunogenicity, expanding the research perspectives of subunit vaccines for African swine fever.
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