Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan.
Doctoral Degree Program in Marine Biotechnology, The College of Life Sciences, National Taiwan Ocean University, Keelung 20224, Taiwan.
Int J Mol Sci. 2020 Feb 21;21(4):1465. doi: 10.3390/ijms21041465.
Nervous necrosis virus (NNV) results in high mortality rates of infected marine fish worldwide. Interferons (IFNs) are cytokines in vertebrates that suppress viral replication and regulate immune responses. Heterologous overexpression of fish IFN in bacteria could be problematic because of protein solubility and loss of function due to protein misfolding. In this study, a protein model of the IFN-α of was built based on comparative modeling. In addition, PelB and SacB signal peptides were fused to the N-terminus of IFN-α for overexpression of soluble, secreted IFN in (E-IFN) and (B-IFN). Cytotoxicity tests indicated that neither recombinant grouper IFN-α were cytotoxic to a grouper head kidney cell line (GK). The GK cells stimulated with E-IFN and B-IFN exhibited elevated expression of antiviral Mx genes when compared with the control group. The NNV challenge experiments demonstrated that GK cells pretreated or co-treated with E-IFN and B-IFN individually had three times the cell survival rates of untreated cells, indicating the cytoprotective ability of our recombinant IFNs. These data provide a protocol for the production of soluble, secreted, and functional grouper IFN of high purity, which may be applied to aquaculture fisheries for antiviral infection.
神经坏死病毒（NNV）导致全球感染海洋鱼类的高死亡率。干扰素（IFNs）是脊椎动物中的细胞因子，可抑制病毒复制并调节免疫反应。由于蛋白质溶解度和功能丧失导致蛋白质错误折叠，在细菌中异源过表达鱼类 IFN 可能会出现问题。在这项研究中，基于比较建模构建了 的 IFN-α蛋白模型。此外，PelB 和 SacB 信号肽被融合到 IFN-α的 N 端，用于在 （E-IFN）和 （B-IFN）中过表达可溶性、分泌型 IFN。细胞毒性试验表明，重组石斑鱼 IFN-α对石斑鱼头肾细胞系（GK）均无细胞毒性。与对照组相比，用 E-IFN 和 B-IFN 刺激的 GK 细胞显示抗病毒 Mx 基因的表达水平升高。NNV 攻毒实验表明，单独用 E-IFN 和 B-IFN 预处理或共同处理的 GK 细胞的细胞存活率是未处理细胞的三倍，表明我们的重组 IFNs 具有细胞保护能力。这些数据提供了一种生产高纯度可溶性、分泌型和功能齐全的石斑鱼 IFN 的方案，可应用于抗病毒感染的水产养殖渔业。
