Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60
Description
FIGURE 2: Typical Morphology and Molecular Phenotype of raMoDCs (A) Morphology of raMoDCs. (B) Immunostaining of raMoDCs. (C) Flow cytometry analysis showing the expression of surface markers on raMoDCs . (D) Flow cytometry assessment of the phagocytic ability of raMoDCs toward FITC-labeled glucans. The data reflect % abundance for positive populations. FIGURE 3: Analysis of Phage Binding Ability to raMoDCs by ELISA. (A) Sequences of 12-mer peptides. (B) Cell-ELISA assessing the binding selectivity to raMoDCs of eight phage clones from the last round of biopanning. M13 wild-type phage without any displayed peptide served as a negative control. Data are presented as the mean ± SD of three independent experiments. Significant differences are denoted by different letters (a vs. b, a vs. c, b vs. c) at the same time point (p < 0.01).FIGURE 4: Binding Ability of DC-Targeting Peptides to raMoDCs. (A) Fluorescence microscopy images showing the binding of FITC-labeled HS, KC1, MY, and negative control (NC) peptides to raMoDCs. (B) Flow cytometry analysis of the binding ability of the peptides to raMoDCs. Red indicates rabbit DCs expressing CD86, green represents FITC-labeled peptides, and blue represents nuclei stained with DAPI. Changes in brightness, contrast, or color balance were applied uniformly to all pixels in the microscopy image.FIGURE 5: Characterization of Expressed Proteins in L. reuteri. (B) Verification of expressed VP60 protein in L. reuteri through western blotting. (C) Visualization of expressed proteins in Lactobacillus via fluorescence microscopy. FIGURE 6: The ability of raMoDCs to recognize and capture recombinant Lactobacillus was evaluated using scanning electron microscopy and the analysis of toll-like receptor and cytokine mRNA levels in raMoDCs in response to recombinant Lactobacillus and LPS stimulation. (A) Scanning electron microscopy images showing the morphology of raMoDCs capturing recombinant Lactobacillus at 30, 60, 120 min. (B) RaMoDCs were stimulated by recombinant Lactobacillus and LPS for 12 h. Unstimulated raMoDCs were used as a control. Different letters (a vs. b, a vs. c, b vs. c) indicate significant differences (P < 0.01) at the same time point.FIGURE 7: Detection of anti-RHDV-specific IgG/sIgA antibody levels and rabbit survival after challenge. (A) Specific anti-RHDV IgG and sIgA antibody levels in rabbits orally immunized with PBS and recombinant Lactobacillus. Measurement of a specific anti-RHDV IgG antibody in the antisera from immunized rabbits by ELISA using RHDV as the coating antigen. Measurement of specific anti-RHDV SIgA antibody levels in the feces and nasal cavity by ELISA using RHDV as the coating antigen. (B) Rabbit survival after challenge. (C) RHDV load in the liver, spleen, lungs, and kidneys of infected dead rabbits. Different letters (a vs. b, a vs. c, b vs. c) indicate significant differences (p < 0.01) at the same time point.