PCV cap proteins fused with calreticulin expressed into polymers in Escherichia coli with high immunogenicity in mice

Fig. 1. The schematic structure (a) and SDS-PAGE (b) of four recombinant Cap-CRT fusion proteins. a Fragments were used for constructing the recombinant proteins. Blue squares represent completely cap of PCV2 and truncated calreticulin. GGGGS in grey are linkers between cap and calreticulin. Each fragment is encoded by BamHI and XhoI, respectively. b Solubility of rP4C, rC4P, rP5F and rF5P induced by IPTG at 37 °C. M: protein ladder; Lane 1,3,5,7: precipitate of pET-28a-rP4C/rC4P/rV5P/rF5P; Lane 2,4,6,8: supernatant of pET-28a-rP4C/rC4P/rV5P/rF5P

Fig. 2. Purification and identification of rF5P. SDS-PAGE (a), Western-Blot (b) and Size-exclusion chromatography (c) of rF5P. M: protein ladder; Lane 1: lysate of rF5P; Lane 2: supernatant after settling the Ni-NTA resin by gravity; Lane 3: supernatant after washing resin; Lane 4: fraction after eluting (purified rF5P); Lane 5: the first peak of flow through by Superdex 200 pg (enriched rF5P); Lane 6: the third peak

Fig. 3. Characterization of rF5P. a Negative staining electron microscopy of rF5P, bar size, 100 nm. b Dynamic light scattering result of rF5P

Fig. 4. Antigenic characterizations of rF5P using swine clinical positive sera (a), anti-PCV2 mAbs 6A4 (b) and anti-His mAbs (c) by ELISA, and the results are expressed as mean OD value ± SEM, the statistical significance differences between each group was analyzed by two-way ANOVA statistical analysis, *P < 0.05, **P < 0.01, ***P < 0.001, ns represented not significant

Fig. 5. Detection of PCV2-specific immune responses in mice. Groups of mice (n = 6) were immunized with 30 μg and 15 μg of rF5P, commercial inactivated Circovac® vaccine (Merial), subunit vaccine Ingelvac CircoFLEX® (Boehringer Ingelheim) and PBS in injection, Blood samples were collected for PCV2-speicific IgG titers(a) and virus neutralization antibody (b). Titers of antibodies are expressed as mean ± SEM. Different letters (a, b, and c) indicate statistically significant difference (P < 0.05) among groups

Fig. 6. Analysis of cytokines secreted by lymphocyte of mice. Lymphocytes isolated from the spleen of mice at 56 dpi were stimulated with PCV2 strain DF-1 for 72 h, the supernatants were collected to detect the concentrations of cytokine of IFN-γ (a), IL-10 (b), IL-18 (c), TNF-ɑ (d), GM-CSF (e) by ELISA method and the T-lymphocyte proliferation (f) . Date are shown as mean ± SEM, statistical differences between each group was measured by one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ns represented not significant

Fig. 7. Protection from PCV2 strain DF-1 challenge in mice. All of the mice were challenged with 100 μL of 106.5 TCID50/mL of the PCV2 strain DF-1 at 56 dpi and examined for 28 days. Spleens were isolated and the genomes were extracted to measure the content of PCV2 using quantitative real-time PCR. Date are shown as mean ± SEM, statistical differences between each group was measured by one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ns represented not significant