摘要
皮肤黏液是鱼类免疫防御的第一道防线。大弹涂鱼皮肤黏液对其免疫防御、渗透压维持以及适应水陆生活等方面具有重要意义。为深入了解大弹涂鱼皮肤黏液的蛋白质分子组成,对其皮肤黏液开展了抑菌活性分析和蛋白质组学分析。通过电刺激法收集大弹涂鱼皮肤黏液,采用打孔法比较了皮肤黏液和血清的抑菌活性差异;进一步利用鳗弧菌对大弹涂鱼进行诱导,采用生长曲线抑制法分析和比较了诱导前后皮肤黏液的抑菌活性差异。利用Shotgun质谱技术,结合大弹涂鱼皮肤转录组数据库,对大弹涂鱼皮肤黏液开展了蛋白质组学分析;进一步利用String软件对所鉴定的蛋白质开展了蛋白质相互作用预测。大弹涂鱼皮肤黏液具有广谱抑菌活性,鳗弧菌诱导后的大弹涂鱼皮肤黏液与诱导前相比,对部分革兰氏阴性菌的抑菌活性明显上升,但对其他菌株的抑菌活性差异不明显。从大弹涂鱼皮肤黏液中共鉴定各类蛋白质分子97种,基本分子组成与其他硬骨鱼类皮肤黏液蛋白相似,但也有少数蛋白如泛素蛋白、胸腺素蛋白等未在其他鱼类皮肤黏液中报道。此外,其他鱼类中所鉴定到的部分蛋白如热休克蛋白、抗菌肽等在大弹涂鱼皮肤黏液中未能鉴定到。大弹涂鱼皮肤黏液中已鉴定的蛋白多数具有与免疫功能相关的结构域,且存在一个以肌动蛋白为核心的相互作用网络。大弹涂鱼皮肤黏液具有明显的广谱抑菌活性,其蛋白质组成与其他鱼类皮肤黏液具有一定的相似性。本研究为深入了解大弹涂鱼皮肤黏液的分子多样性及功能机制奠定了基础。
Fish skin mucus serves as the first line of defense against pathogens and external stressors. The mudskipper Boleophthalmus pectinirostris inhabit intertidal mudflats containing abundant and diverse microbial population, thus, the skin together with the mucus of B. pectinirostris are very important for the immune defense,osmotic pressure maintenance and adaptation to amphibian life. For exploring the proteomic profile of the skin mucus and understanding the molecular mechanism of B. pectinirostris adaptation to amphibious environments,the antibacterial activity was determined by agar diffusion plate method for the mucus and the serum samples. In addition, a growth curve inhibition method was used to compare the antibacterial activities of B. pectinirostris mucus before and after vibrio induction. Furthermore, the proteomic profile of natural B. pectinirostris mucus was identified by Shotgun mass spectrometry technology combining with skin transcriptome searching. The interaction network analysis of the identified proteins from mucus was performed by String software. Skin mucus was collected from B. pectinirostris after electrical stimulation. The mucus of B. pectinirostris showed a broad spectrum of antimicrobial activity more than serum of the same species, indicating that the mucus has a stronger antimicrobial activity than the serum. After vibrio induction, the antibacterial activity of mucus was slightly stronger for some gram-negative bacteria than that of un-induced mucus. A total of 97 proteins were identified from natural mucus of B. pectinirostris with a similar results from studies of other fish species, including actins,keratins, apolipoproteins, transferrins, calmodulins, ubiquitins, pentraxins, and various enzymes. However, some proteins, such as ubiquitin-like proteins and thymosin, were identified first from fish mucus. The identified proteins can be clustered into structural proteins, enzymes, material transport related proteins, immune proteins,and other proteins. Most of these proteins are known to be involved in immune and/or stress responses. Protein interaction analysis showed strong interactions among the identified proteins, such as actin, myosin, cofilin,filamin, apolipoprotein, transferrin, calmodulin, and superoxide dismutase. The proteomic profile established in this study could not only provide knowledge on the routes involved in mucosal innate immunity, but also put forward a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.
引文
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