Immunogenicity evaluation of MS2 phage-mediated chimeric nanoparticle displaying an immunodominant B cell epitope of foot-and-mouth disease virus

Figures

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  Figure 1. Schematic drawing of constructing process for recombinant plasmids. (A and C) Schematic diagram of recombinant plasmid pCP and p-(EP131-160)3. (B) Strategy to construct recombinant plasmid pCP-EP131-160.

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  Figure 2. Amplification of target gene and identification of recombinant plasmids. (A) PCR amplification products analysis using agarose gel. Lane 1, DL2000 DNA ladder; Lane 2, Mature protein (AP) and coat protein (CP) genes of MS2 showing 1,644 bp amplicon; Lane 3, Upstream fragment showing 1,260 bp amplicon; Lane 4, Downstream fragment showing 487 bp amplicon; Lane 5, The whole fragment (AP + CP + EP131-160) showing 1,734 bp amplicon. (B) The identification of recombinant plasmids by digestion with restriction enzymes. Lane 1, DL2000 DNA ladder; Lane 2–3, Recombinant plasmids pCP and pCP-EP131-160 were digested using restriction enzymes BamH I and Hind III, respectively; Lane 4, Recombinant plasmid p-(EP131-160)3 was digested by Nde I and Xol I.

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  Figure 3. Expression and purification of recombinant proteins. (A) SDS-PAGE analysis of the solubility of three recombinant proteins. Lane 1, protein ladder; Lane 2–3, level of coat protein of MS2 expressed in soluble and insoluble fractions, respectively; Lane 4–5, level of chimeric protein expressed in soluble and insoluble fractions, respectively; Lane 6–7, level of tandem repeat peptide epitopes (TRE) expressed in soluble and insoluble fractions, respectively. As shown, coat protein of MS2, chimeric protein and TRE were detected with a molecule weight near 16 kDa, 18 kDa and 10 kDa, respectively. (B) Western blot analysis of recombinant proteins with anti-FMDV VP1 G-H loop monoclonal antibody. Lane 1–7, the order is consistent with SDS-PAGE (A). (C) SDS-PAGE analysis of the purity of three recombinant proteins. Lane 1, protein ladder; Lane 2, purified coat protein of MS2; Lane 3, purified chimeric protein; Lane 4, purified TRE.

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  Figure 4. Immune-activity analysis of recombinant proteins. Dot-ELISA immune assay with guinea pig anti-FMDV hyper-immune serum. (B) Dot-ELISA immune assay with anti-FMDV VP1 G-H loop monoclonal antibody. 1, Inactivated FMDV; 2, purified coat protein of MS2; 3, purified chimeric nanoparticles; 4, purified TRE.

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  Figure 5. Physical characterization of chimeric nanoparticles (CNPs). (A) Transmission electron microscope (TEM) image of negative staining CNPs. The scale is 100 nm. (B) Dynamic light scattering results of CNPs.

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  Figure 6. CNPs specific antibody levels in mice. Groups of mice were immunized with 15 µg chimeric nanoparticles, 30 µg tandem repeat peptide epitopes, commercialized synthetic peptide vaccine (more than 7.5 µg) and PBS. Sera were collected at every week after the first immunization for antibody levels until the eighth week. Date are presented as the mean ± SEM. Statistical differences were indicated by asterisks (* P < 0.05, **** P < 0.0001).

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  Figure 7. T-lymphocyte proliferation in mice. Splenic Lymphocytes were isolated at 56 days post vaccination and stimulated with inactivated FMDV and concanavalin (CoA), respectively. Lymphocyte proliferation was analyzed using the WST-8 colorimetric assay. The stimulation index (SI) means the ratio of stimulated sample to unstimulated sample at OD450 nm. Statistical differences were indicated by asterisk (* P < 0.05, ** P < 0.01, *** P < 0.001).

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  Figure 8. Analysis of cytokines secreted by lymphocyte of mice. Splenic Lymphocytes were stimulated with inactivated FMDV for 48 h. The culture supernatants were collected and tested by ELISA. (A–C) are the concentrations (pg/mL) of IFN-γ, IL-2 and IL-4 in the supernatants, respectively. Data are shown as mean ± SEM. Statistical differences were indicated by asterisk (* P < 0.05, ** P < 0.01, *** P < 0.001).