Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, Jiaushi, Ilan 262, Taiwan.
Fish Shellfish Immunol. 2012 Jun;32(6):947-57. doi: 10.1016/j.fsi.2012.01.023. Epub 2012 Mar 1.
Immunostimulatory effects of the oral administration of the recombinant epinecidin-1 protein from BL21 Escherichia coli (containing the pET28a-epinecidin-1-dsRed plasmid) were studied in grouper (Epinephelus coioides) and zebrafish (Danio rerio). For this purpose, fish were fed diets for 30 days containing the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) at different bacterial numbers (10(4), 10(6), 10(8), and 10(10) colony-forming units (cfu) of BL21 E. coli in 50 ml of LB medium) mixed with 50 g of eel powder as fodder. After 30 days of feeding, immune-related gene expressions for bacterial-infection responses and disease resistance against Vibrio vulnificus (204) were determined. The V. vulnificus (204) injected into the fish abdominal cavity mimicked gram-negative bacterial infections in culture ponds. Experimental results assessed whether the recombinant epinecidin-1 protein from BL21 E. coli (containing the pET28a-epinecidin-1-dsRed plasmid) has up- (or down-) regulation immune-related genes expression. Results indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement significantly enhanced expressions several immune-related genes such as tumor necrosis factor (TNF)-1 in grouper and Toll-like receptor (TLR)4, interleukin (IL)-1β, nitric oxide synthase (NOS)2, and nuclear factor (NF)-κB in zebrafish. After being challenged with V. vulnificus (204) for 24, 48, 72, or 96 h, the percentage mortality was significantly reduced in treated fish, which indicated that the recombinant epinecidin-1 protein from BL21 E. coli administered as a feed supplement could bring about downregulation of TNF-1 expression and functioned like an antagonist for binding TLR4, which reduced the signal transduction pathway for inhibiting TNF and IL-1β expressions while reducing binding of the transcription factor, NF-κB, to TNF and the IL-1β promoter region. The experimental results indicated that dietary intake of the recombinant epinecidin-1 protein from BL21 E. coli modulated immune-related gene expressions and disease resistance of grouper and zebrafish after a V. vulnificus (204) infection.
从 BL21 大肠杆菌（含 pET28a-epinecidin-1-dsRed 质粒）中口服重组鱼抗菌肽 1 蛋白对军曹鱼（Epinephelus coioides）和斑马鱼（Danio rerio）的免疫刺激作用进行了研究。为此，将鱼用含有 BL21 大肠杆菌（含 pET28a-epinecidin-1-dsRed 质粒）的重组鱼抗菌肽 1 蛋白的饮食喂养 30 天，不同细菌数量（BL21 大肠杆菌 50mlLB 培养基中的 10(4)、10(6)、10(8)和 10(10)个菌落形成单位（cfu））混合 50 克鳗鱼粉作为饲料。喂养 30 天后，测定了对细菌感染反应和对创伤弧菌（204）的疾病抵抗力的免疫相关基因表达。将注射到鱼腹腔中的创伤弧菌（204）模拟了养殖池塘中的革兰氏阴性细菌感染。实验结果评估了 BL21 大肠杆菌（含 pET28a-epinecidin-1-dsRed 质粒）中的重组鱼抗菌肽 1 蛋白是否具有上调（或下调）免疫相关基因表达的作用。结果表明，作为饲料补充剂的 BL21 大肠杆菌的重组鱼抗菌肽 1 蛋白显著增强了几种免疫相关基因的表达，如军曹鱼中的肿瘤坏死因子（TNF）-1和斑马鱼中的 Toll 样受体（TLR）4、白细胞介素（IL）-1β、一氧化氮合酶（NOS）2 和核因子（NF）-κB。在感染创伤弧菌（204）24、48、72 或 96 小时后，经处理的鱼的死亡率显著降低，表明 BL21 大肠杆菌中的重组鱼抗菌肽 1 蛋白作为饲料补充剂可以下调 TNF-1 的表达，并起到与 TLR4 结合的拮抗剂的作用，从而减少了抑制 TNF 和 IL-1β 表达的信号转导途径，同时减少了转录因子 NF-κB 与 TNF 和 IL-1β 启动子区域的结合。实验结果表明，饮食摄入 BL21 大肠杆菌的重组鱼抗菌肽 1 蛋白可调节军曹鱼和斑马鱼的免疫相关基因表达和抗病能力，在感染创伤弧菌（204）后。
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