Surface Display of Antigen Protein VP8* of Porcine Rotavirus on Bacillus Subtilis Spores Using CotB as a Fusion Partner

Figure 1. Electrophoretogram of the target gene. (A) PCR identification of recombinant Escherichia coli; M: DL2000; lane 1: E. coli (pET-32a-VP8*); lane 2: E. coli Rosetta (DE3). (B) PCR identification of pET-32a-VP8*; M: DL15000; lane 1: the products of pET-32a-VP8* by double-restriction-enzyme digestion.

Figure 2. Western blot analysis: Specificity of VP8* protein detected by antirotavirus antiserum. M: protein marks; lane 1: crude cell extract E. coli expressing recombinant VP8* proteins; lane 2: protein expressed with the empty pET32a plasmid vector.

Figure 3. Map of the shuttle vector pDG364-CotB-VP8*. CotB and VP8* genes were integrated in the gene fusion on the pDG364 vector.

Figure 4. Agarose gel electrophoresis analysis of double-restriction-enzyme digested plasmid pDG364-CotB-VP8*. M: DNA marker; lane 1–4: products from recombinant plasmid digested with Xba I, BamH I/EcoR I, BamH I/Hind III, and Hind III/EcoR I.

Figure 5. Starch hydrolysis test. (A) Bacillus subtilis 168 and recombinant B. subtilis BV cultivate on medium containing 1% starch. (B) The plate stained with iodine.

Figure 6. PCR analysis with different primer pairs. M: DNA marker. The PCR primers were as follows: (1) VP8*-F and VP8*-R; (2) amyE-F and amyE-R; (3) amyE-F and VP8*-R; (4) VP8*-F and amyE-R. The PCR template is as follows: (WT) wild-type (B. subtilis 168); (BV) recombinant B. subtilis BV.

Figure 7. Western blot analysis of spore coat protein. M: TureColor prestained protein marker; lane 1: the spore coat protein of B. subtilis 168; lane 2: the spore coat protein of recombinant B. subtilis BV.

Figure 8. Immunofluorescence microscopy analysis: (A,B) recombinant B. subtilis BV; (C,D) B. subtilis 168; (A,C) bright-field images; (B,D) immunofluorescent images (10 × 100).

Figure 9. Recombinant B. subtilis BV spores induced specific mucosal and systemic immune responses following oral administration. The control group (A) was fed with a basal diet. B and D were fed a diet mixed with B. subtilis 168 and recombinant B. subtilis BV, respectively. C and E were orally immunized by gavage with B. subtilis 168 and recombinant B. subtilis BV. The feces and serum collected from mice on days 0, 14, 21, 28, and 35 were analyzed for rotavirus-specific (a) IgA and (b) IgG (the value of OD450nm (Optical density at 450 nm) was calculated) separately by ELISA. *, p < 0.05; **, p < 0.01. The error bars represent standard deviations.