Evaluation of Immunoprotective Efficacy of Recombinant Pseudomonas plecoglossicida TolC Protein(rPpTolC)in Zebrafish

Pseudomonas plecoglossicida infection induces rapid mass mortality in fish; the outer membrane protein TolC from Gram-negative bacteria typically exhibits potent immunogenicity, and this study focuses on the immunoprotective efficacy of TolC from this specific pathogen in zebrafish(Danio rerio). The PptolC gene was successfully cloned, and its B-cell antigenic epitopes and sequence conservation were analyzed. Zebrafish were immunized with rPpTolC to assess its capacity to induce non-specific immune responses and immunoglobulin production, as well as its protective efficacy against challenges with heterologous pathogens. Results showed that PpTolC encodes a 457-amino acid protein with high sequence conservation and five high-confidence B-cell antigenic epitopes. Zebrafish were immunized with rPpTolC to assess its capacity to induce non-specific immune responses(such as myd88, tnf-α and tgf-β1)and immunoglobulin production(IgM, IgD, IgZ)in gill and intestinal tissues. Following separate challenges with P. plecoglossicida and Vibrio parahaemolyticus(each at 2×LD50), immunized zebrafish exhibited survival rates exceeding 60% against both pathogens individually. Concurrently, immunized fish showed significantly reduced intestinal ROS and MPO levels, alongside decreased pathogen loads in respective challenge groups. Collectively, rPpTolC demonstrates robust cross-protective immunity in zebrafish and represents a promising candidate for subunit vaccine development.

杀舌鳎假单胞菌（Pseudomonas plecoglossicida）感染可引发鱼类急性大批量死亡；革兰氏阴性菌外膜蛋白TolC通常具备较强免疫原性，本研究聚焦于该病原菌来源的TolC在斑马鱼（Danio rerio）中的免疫保护效力。研究成功克隆了PptolC基因，并对其B细胞抗原表位与序列保守性展开分析。通过重组PpTolC（rPpTolC）免疫斑马鱼，评估其诱导非特异性免疫应答、免疫球蛋白生成的能力，以及针对异源病原体攻击的保护效果。结果显示，PpTolC编码一条含457个氨基酸的蛋白质，序列保守性较高，且包含5个高可信度B细胞抗原表位。实验采用rPpTolC免疫斑马鱼后，检测其鳃与肠道组织中的非特异性免疫应答（如髓系分化因子88（MyD88）、肿瘤坏死因子-α（TNF-α）及转化生长因子-β1（TGF-β1））与免疫球蛋白生成（免疫球蛋白M（IgM）、免疫球蛋白D（IgD）、免疫球蛋白Z（IgZ））水平。分别以杀舌鳎假单胞菌与副溶血性弧菌（均为2倍半数致死量（2×LD₅₀）剂量）进行攻击后，免疫组斑马鱼对两种病原体单独攻击的存活率均超过60%。与此同时，免疫组鱼类的肠道活性氧（ROS）与髓过氧化物酶（MPO）水平显著降低，对应攻击组的病原体载量也有所下降。综上，rPpTolC在斑马鱼中展现出优异的交叉保护性免疫效果，是一种极具开发潜力的亚单位疫苗候选分子。