Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60

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.图2：兔源单核细胞源性树突状细胞（raMoDCs）的典型形态与分子表型。(A) raMoDCs的形态学观察。(B) raMoDCs的免疫染色分析。(C) 流式细胞术分析raMoDCs表面标志物的表达情况。(D) 流式细胞术评估raMoDCs对异硫氰酸荧光素（FITC）标记葡聚糖的吞噬能力。数据以阳性细胞群体的百分占比呈现。 图3：酶联免疫吸附试验（ELISA）分析噬菌体与raMoDCs的结合能力。(A) 12肽的序列信息。(B) 细胞ELISA法评估最后一轮生物淘选获得的8个噬菌体克隆对raMoDCs的结合选择性。以未展示任何外源肽段的M13野生型噬菌体作为阴性对照。数据以三次独立实验的平均值±标准差（SD）形式呈现。同一时间点下，不同字母（a与b、a与c、b与c）代表组间具有显著性差异（p < 0.01）。 图4：树突状细胞靶向肽对raMoDCs的结合能力。(A) 荧光显微镜图像，展示FITC标记的HS、KC1、MY肽及阴性对照（NC）肽与raMoDCs的结合情况。(B) 流式细胞术分析各肽段对raMoDCs的结合能力。红色荧光标记表达CD86的兔源树突状细胞，绿色荧光代表FITC标记的肽段，蓝色荧光代表经4',6-二脒基-2-苯基吲哚（DAPI）染色的细胞核。所有显微图像的像素均进行了统一的亮度、对比度或色彩平衡调整。 图5：罗伊氏乳杆菌（L. reuteri）表达蛋白的表征。(B) 通过蛋白质免疫印迹（Western Blotting）验证罗伊氏乳杆菌中VP60蛋白的表达。(C) 荧光显微镜可视化罗伊氏乳杆菌的表达蛋白。 图6：本研究通过扫描电子显微镜，以及分析raMoDCs在重组罗伊氏乳杆菌与脂多糖（LPS）刺激下的Toll样受体及细胞因子mRNA表达水平，评估了raMoDCs识别并捕获重组罗伊氏乳杆菌的能力。(A) 扫描电子显微镜图像，展示raMoDCs在30、60、120分钟时捕获重组罗伊氏乳杆菌的形态变化。(B) 重组罗伊氏乳杆菌与LPS刺激raMoDCs 12小时，以未接受刺激的raMoDCs作为空白对照。同一时间点下，不同字母（a与b、a与c、b与c）代表组间具有显著性差异（P < 0.01）。 图7：抗兔出血症病毒（RHDV）特异性IgG/分泌型IgA（sIgA）抗体水平检测及攻毒后家兔存活率统计。(A) 经磷酸盐缓冲液（PBS）与重组罗伊氏乳杆菌口服免疫的家兔体内特异性抗RHDV IgG与sIgA抗体水平：以RHDV为包被抗原，通过ELISA法检测免疫家兔血清中的特异性抗RHDV IgG抗体；以RHDV为包被抗原，通过ELISA法检测粪便与鼻腔分泌物中的特异性抗RHDV分泌型IgA抗体水平。(B) 攻毒后家兔的存活率。(C) 感染致死家兔的肝脏、脾脏、肺脏及肾脏中的RHDV载量。同一时间点下，不同字母（a与b、a与c、b与c）代表组间具有显著性差异（p < 0.01）。