N-terminal truncation of the foot-and-mouth disease virus VP0 protein promotes soluble co-expression and particle assembly stability of viral capsid proteins in Escherichia coli

Figures

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  Fig. 1. Structural analysis of VP0 N-terminal domain interactions with the VP1 and VP3 subunits. The ribbon diagrams depict the VP0 (green), VP1 (yellow), and VP3 (red) subunits, with critical interaction interfaces highlighted in the surface representation. (A) Molecular interface between VP0 N-terminal residues 15–20 and adjacent VP1/VP3 domains. (B) Conformational coupling of VP0 residues 27–33 with the VP1 β-barrel structure. (C) Stabilizing interactions mediated by VP0 residues 34–40 at the VP1‒VP3 subunit junction

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  Fig. 2. Engineering a co-expression system for VP0 truncation mutants with the VP3 and VP1 structural subunits

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  Fig. 3. Expression optimization and purification profiling of N-terminally truncated VP0 complexes with VP3/VP1 structural subunits. (A) Induction analysis of ternary complex expression. Left: SDS-PAGE analysis; Right: Western blot analysis. To clearly delineate protein differences, E. coli lysate supernatants containing SUMO fusion proteins were enzymatically processed with SUMO protease (5 units, 4 °C, 12 h), followed by SDS-PAGE analysis using 12% resolving gels with Coomassie blue staining. Lane M: protein marker (10–250 kDa); Lane 1: negative control (empty vector-transformed BL21(DE3)); Lanes 2–6: soluble fractions of 031 (wild-type VP0), 031-VP0 dN5, 031-VP0 dN10, 031-VP0 dN15, and 031-VP0 dN20 truncation mutants co-expressed with VP3/VP1. (B) SDS-PAGE analysis of the VLPs after SUMO removal. Lane M: protein marker (10–250 kDa); Lane 1: negative control (empty vector transformed into E. coli BL21 (DE3) purified); Lanes 2–6: VLPs of 031, 031-VP0 dN5, 031-VP0 dN10, 031-VP0 dN15, and 031-VP0 dN20. (C) Quantitative yield comparison. Bar graph comparing total protein yields (mg/L) between wild-type and truncation variants. The data represent the means ± SD (n = 3 biological…

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  Fig. 4. Multidimensional characterization of VLPs assembly. (A) Purification and analysis of VLPs by sucrose gradient ultracentrifugation. The purified VLPs were loaded onto a 10%–30% (w/v) sucrose gradient. After centrifugation, 22 fractions were collected. Samples from each fraction were assayed by spectrophotometry. (B) High-performance size exclusion chromatography profiling. Elution profiles of VLPs on a TSK G4000 SWXL (300 × 7.8 mm, I.D., pore size 45 nm) column (Tosoh, Japan) in phase buffer (25 mM phosphate buffer, 500 mM NaCl, pH 7.4) at a 0.5 mL/min flow rate. The absorbance was monitored at 280 nm. The aggregates were eluted at 10 min, and monodisperse VLPs were eluted at 15.4 min. The positive control sample was prepared by breaking the emulsion and concentrating a commercially available inactivated serotype O FMDV vaccine. (C) Hydrodynamic diameter analysis. Dynamic light scattering measurements were performed using a Nano ZSE instrument (Malvern Instruments, UK) with an intensity-weighted size distribution (25 °C, 3 measurements/sample). (D) Negative-stain transmission electron microscopy images of uranyl acetate-stained VLPs. Scale bar: 100 nm. The diameters of the 031,…

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  Fig. 5. Maintenance of the neutralizing antigen site of the VLPs. F21-64 recognized the linear epitope and F21-58, F21-41, and F21-18 recognized the conformational epitopes

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  Fig. 6. Systemic immunogenicity evaluation of VLPs vaccine candidates in pigs. (A) Schematic of the immunization protocol. Pigs were divided into three groups (n = 5 per group). Negative control: inoculated with PBS. Positive control: immunized with inactivated FMDV vaccine. The experimental group was immunized with 100 µg of VLPs per dose emulsified in ISA 206 adjuvant (SEPPIC) via intramuscular injection. (B) FMDV-specific responses in pigs. Serum samples were collected before immunization (day 0) and weekly until 6 weeks postimmunization. FMDV-specific antibody responses were measured using LPBE. (C) Neutralizing antibody responses in pigs. Titers of neutralizing antibodies of experimental groups on day 28 after the immunization. (D) and (E) ELISpot assays of FMDV serotype O-specific IFN-γ after immunization with VLPs. (D) Representative IFN-γ ELISpot wells for the PBS, inactivated serotype O vaccine, and VLPs. The numbers in the upper left corners indicate counted spots of IFN-γ-producing cells per 3 × 105 responder PBMCs at 24 h after stimulation. (E) FMDV serotype O-specific IFN-γ of vaccine-immunized pigs. The PBMCs were collected at 28 dpi. The data are expressed as the means ±…