Enhancement of immune proteins expression in skin mucus of Japanese flounder Paralicthys olivaceus upon feeding a diet supplemented with high concentration of ascorbic acid
Enhancement of immune proteins expression in skin mucus of Japanese flounder Paralicthys olivaceus upon feeding a diet supplemented with high concentration of ascorbic acid
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DOI：
10.1016/j.fsi.2021.04.009
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发表时间：
2021-04-20
影响因子：
4.7
通讯作者：
Mano, Nobuhiro
中科院分区：
文献类型：
--
作者：
Mori, Misato;Ito, Tasuku;Mano, Nobuhiro
为了寻找添加高浓度抗坏血酸饲料的牙鲆皮肤粘液中的免疫防御蛋白，我们进行了2D-PAGE，并比较了添加商品饲料的对照组和添加高浓度抗坏血酸（2，000 mg/kg）饲料7天的抗坏血酸添加组（阿萨组）的蛋白质解析模式。结果显示，阿萨组有多种蛋白质表达明显增加。其中，6个区域，显示了显着的升高，蛋白质鉴定使用LC-MS/MS分析和Mascot数据库搜索。鉴定出6种蛋白质，即血清转铁蛋白（Sero）、转铁蛋白（Trans）、暖温适应相关的65 kDa蛋白（Wap 65）、补体成分C3（C3）、血红蛋白β-A链（Hisp）和载脂蛋白A-1（Apo）。定量RT-PCR分析显示，阿萨A组表皮中Hbss mRNA水平较对照组明显升高（11.6倍）;两组之间Sero/Trans、Wap 65、C3和Apo水平无明显差异。与对照组相比，阿萨组肝脏中wap 65和c3的mRNA水平以及肾脏中Apo的mRNA水平均显著升高。在分泌型免疫球蛋白M（IgM）和溶菌酶（lyz）的情况下，IgM在表皮、鳃、肾、脾和肠中的mRNA水平没有差异，lyz在表皮、鳃、脾和肠中的mRNA水平没有差异。原位杂交结果证实阿萨组表皮组织中Hsp 7 mRNA水平升高。本研究为阿萨激活鱼类皮肤粘膜组织天然免疫防御系统提供了新的证据。
To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbss) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbss in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbss mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.