Abstract: 【Objective】 This study aimed to design and develop the multi-epitope nanoparticle vaccine against gB and gD proteins of Feline herpesvirus type 1 (FHV-1), and provide a theoretical basis for antigen screening and optimization of FHV-1 multi-epitope nanoparticle vaccines. 【Method】 In this study, a variety of bioinformatics tools were used to analyze the physicochemical properties, transmembrane structure, N-glycosylation site, secondary structure, B cell epitope, immunogenicity and conservation of FHV-1 gB and gD proteins. The recombinant plasmid pET20b-FBDF was constructed and transformed into Escherichia coli BL21 (DE3) competent cells to induce expression. The recombinant protein FBDF were purified by Ni column affinity chromatography and dialyed concentration treatment, and then identified by SDS-PAGE, the protein content was determined by BCA. The morphology and particle size of self-assembled nanoparticles were observed by transmission electron microscope (TEM) and dynamic light scattering (DLS). The prepared immunogens were immunized subcutaneously in BALB/c mice to evaluate their immunogenicity. 【Result】 Multiple amino acid domains in FHV-1 gB and gD proteins exhibited excellent hydrophilicity, antigenicity, structural flexibility, and surface accessibility characteristics. FHV-1 gB and gD proteins had 11 and 5 potential glycosylation sites, respectively. The secondary structures of FHV-1 gB and gD proteins were primarily composed of alpha helix, beta sheet, beta turns, and random coil, with random coil constituting the predominant structural component. And FHV-1 gB and gD proteins produced 17 and 6 B-cell linear epitopes, respectively. Three dominant antigenic epitopes with high antigenicity and conservation were screend， which were gB₁: 197－208, gB₂: 314－325 and gD: 221－232, respectively. The epitope was connected with Ferritin by flexible linker (GGGGS). After codon optimization, the recombinant plasmid pET20b-FBDF was constructed. SDS-PAGE results showed that the recombinant protein FBDF was successfully expressed at 30 ℃, 0.5 mmol/L IPTG for 16 h. The results of TEM and DLS showed that the purified FBDF protein could be self-assembled into nanoparticles with a diameter of 16.55 nm. The results of mouse immunization showed that serum antibody titers peaked after 14 days of triple immunization, and the antibody titers in FBDF+Al(OH)₃, FBDF+CpG and FRCPV groups were 1∶680 000, 1∶15 600 and 1∶3 500, respectively, which were significantly higher than those in PBS and Ferritin groups (P＜0.05). Vaccination with FBDF could induce mice to produce higher levels of interferon-γ (IFN-γ), interleukin-4 (IL-4), and neutralizing antibodies, with neutralizing antibody levels up to 1∶2⁵. 【Conclusion】 The multi-epitope nanoparticle vaccine FBDF constructed in this study had good immunogenicity and could induce humoral and cellular immune responses in BALB/c mice， which laid a foundation for the research of FHV-1 multi-epitope vaccine.

Key words: Feline herpesvirus (FHV); gB protein; gD protein; antigen epitope prediction; multi-epitope vaccine