Abstract：
In the field of aquaculture, the enhancement of animal health and disease prevention is progressively being tackled using alternatives to antibiotics, including vaccines and probiotics. This study was designed to evaluate the potential of a recombinant Bacillus methylotrophicus, engineered to express the outer membrane channel protein TolC of Aeromonas hydrophila AH3 and the green fluorescent protein GFP, as an oral vaccine. Initially, the genes encoding tolC and GFP were cloned into a prokaryotic expression system, and anti-TolC mouse antiserum was generated. Subsequently, the tolC gene was subcloned into a modified pMDGFP plasmid, which was transformed into B. methylotrophicus WM-1 for protein expression. The recombinant B. methylotrophicus BmT was then administered to grass carp via co-feeding, and its efficacy as an oral vaccine was assessed. Our findings demonstrated successful expression of the 55 kDa TolC and 28 kDa GFP proteins, and the preparation of polyclonal antibodies with high specificity. The BmT exhibited stable expression of the GFP-TolC fusion protein and excellent genetic stability. Following oral immunization, significant elevations were observed in serum-specific IgM levels and the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), superoxide dismutase (SOD), and lysozyme (LZM) in grass carp. Concurrently, significant upregulation of immune-related genes, including IFN-I, IL-10, IL-1β, TNF-α, and IgT, was noted in the intestines, head kidney, and spleen of the grass carp. Colonization tests further revealed that the BmT persisted in the gut of immunized fish even after a fasting period of 7 days. Notably, oral administration of BmT enhanced the survival rate of grass carp following A. hydrophila infection. These results suggest that the oral BmT vaccine developed in this study holds promise for future applications in aquaculture.
摘要：
在水产养殖领域,加强动物健康和疾病预防正在逐步解决使用抗生素的替代品，包括疫苗和益生菌。本研究旨在评估重组嗜甲基芽孢杆菌的潜力，工程表达嗜水气单胞菌AH3的外膜通道蛋白TolC和绿色荧光蛋白GFP，作为口服疫苗。最初，将编码tolC和GFP的基因克隆到原核表达系统中，并产生抗TolC小鼠抗血清。随后，将tolC基因亚克隆到修饰的pMDGFP质粒中，将其转化为甲基营养芽孢杆菌WM-1用于蛋白质表达。然后通过共同饲喂将重组的甲基营养芽孢杆菌BmT施用于草鱼，并评估了其作为口服疫苗的功效。我们的发现证明了55kDaTolC和28kDaGFP蛋白的成功表达，制备了具有高特异性的多克隆抗体。BmT表现出GFP-tolC融合蛋白的稳定表达和优异的遗传稳定性。口服免疫后，血清特异性IgM水平和酸性磷酸酶（ACP）活性显着升高，碱性磷酸酶(AKP),超氧化物歧化酶（SOD），草鱼中的溶菌酶（LZM）。同时,免疫相关基因的显著上调，包括IFN-I，IL-10，IL-1β，TNF-α,还有IgT,在肠道中被注意到，头肾,和草鱼的脾脏。定植测试进一步显示，即使在7天的禁食期后，BmT仍在免疫鱼的肠道中持续存在。值得注意的是,口服BmT可提高嗜水菌感染后草鱼的存活率。这些结果表明，本研究中开发的口服BmT疫苗有望在水产养殖中应用。
