Generation and immunogenicity assessment of ELPylated virus-like particles of porcine circovirus type 2

Fig. 1. Expression of ELP-Cap and Cap-His fusion proteins in E. coli. a, b The schematic structures of pELP-Cap and pET-Cap vectors. The coding sequences for ELP, His-tag, Cap protein of PCV2b and the VN epitopes of PCV2a, PCV2d and PCV2e are indicated. The expression of fusion sequence was under the control of T7 promoter (PT7). c, d SDS-PAGE analysis of ELP-Cap and Cap-His fusion protein expression. The expression of two fusion proteins was induced for 24 h at 20 °C with 0.2 mM IPTG. After sonication treatment, the total cell lysates of recombinant E. coli before IPTG induction (1), after IPTG induction (2), supernatants (3) and pellets (4) of centrifuged cell lysates were analyzed by 12% SDS-PAGE. M indicates protein molecular mass marker. The small arrows indicate ELP-Cap and Cap-His fusion proteins

Fig. 2. Purification of ELP-Cap and Cap-His fusion proteins. a The total cell lysate of pELP-Cap recombinant E. coli (1) and ELP-Cap fusion protein (2) purified by ITC. b Purification of ELP-Cap protein by ITC in the presence of different concentrations of Triton X-100. c Purification of ELP-Cap protein by ITC in the presence of different concentrations of urea. d The total cell lysate of pELP-Cap recombinant E. coli (1) and ELP-Cap protein (2) purified by ITC in the presence of 1% Triton X-100 and 0.5 M urea. e The total cell lysate of pET-Cap recombinant E. coli (1) and Cap-His fusion protein (2) purified by nickel affinity chromatography. All samples were analyzed by 12% SDS-PAGE

Fig. 3. Characterization of two different forms of PCV2 VLP. a Transmission electron microscopy of ELP-VLP and VLP. b Western blotting of ELP-VLP and VLP using pig anti-PCV2 serum as the first antibody and DeLyght800-labeled goat anti-pig IgG as the second antibody

Fig. 4. Comparison of the antibody responses after immunization with two different forms of VLPs. Mice were immunized two times with the same dose of ELP-VLP or VLP without use of adjuvant. a The serum samples were collected from the immunized and control mice at indicated time and analyzed for the Cap-specific IgG antibody. b The serum samples were collected from the immunized and control mice at 28 dpi and analyzed for PCV2 VN antibody. *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.001 as compared with the control group

Fig. 5. Comparison of cytokine responses after immunization with two different forms of VLPs. Three mice from each group were sacrificed at 28 dpi for splenocyte isolation. After 72-h stimulation with VLP or Con A, the splenocyte culture media were assayed for three cytokines using commercial ELISA kits. ** indicates p < 0.01 as compared with the control group

Fig. 6. Comparison of immunoprotection of two different forms of VLP against PCV2 challenge. After two-time immunization with ELP-VLP or VLP, three mice from each group were challenged with PCV2b at 28 dpi. The serum samples were collected from the immunized and control mice at indicated times and assayed for PCV2 DNA copies by quantitative PCR. * and ** indicate p < 0.05 and p < 0.01 as compared with the control group