Ooi Ei Lin, Verjan Noel, Hirono Ikuo, Nochi Tomonori, Kondo Hidehiro, Aoki Takashi, Kiyono Hiroshi, Yuki Yoshikazu
Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Fish Shellfish Immunol. 2008 May;24(5):506-13. doi: 10.1016/j.fsi.2007.10.004. Epub 2007 Oct 25.
Type I (alpha/beta) interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins that mediate an antiviral state in uninfected cells. Two Atlantic salmon (Salmo salar) IFN-alpha (SasaIFN-alpha1 & 2) genes have previously been cloned and both were found to contain a putative N-linked glycosylation site. Recombinant SasaIFN-alpha1 (rSasaIFN-alpha1) produced in eukaryotic systems has repeatedly been shown to confer antiviral properties. However, different IFN-alpha subtypes may exhibit differential antiviral activities and be subject to glycosylation. To evaluate the potential therapeutic impact of a rSasaIFN-alpha, the mature form of the SasaIFN-alpha2 protein was produced in a high-level Escherichia coli expression system. Expression of the rSasaIFN-alpha2 was detected by SDS-PAGE and Western blot, and its identity was confirmed by mass spectrometry. In the homologous Chinook salmon embryonic (CHSE-214) cell line, the rSasaIFN-alpha2 incited early expression of the IFN-induced Mx protein and exhibited high antiviral activity of about 2.8 x 10(6) U mg(-1) against infectious pancreatic necrosis virus (IPNV). Conversely, antiviral protection by rSasaIFN-alpha2 was not observed in a heterologous Japanese flounder embryo (HINAE) cell line. Hence, a biologically active form of rSasaIFN-alpha2 was successfully produced using a glycosylation-deficient prokaryotic system and purified to homogeneity, suggesting that glycosylation is not required for its antiviral activity.
I型（α/β）干扰素（IFN）是一类细胞因子，可刺激众多蛋白质的表达，这些蛋白质在未感染的细胞中介导抗病毒状态。此前已克隆出两个大西洋鲑（Salmo salar）IFN-α（SasaIFN-α1和2）基因，发现它们都含有一个推定的N-连接糖基化位点。在真核系统中产生的重组SasaIFN-α1（rSasaIFN-α1）已多次显示具有抗病毒特性。然而，不同的IFN-α亚型可能表现出不同的抗病毒活性，并且会发生糖基化。为了评估rSasaIFN-α的潜在治疗效果，在高水平的大肠杆菌表达系统中产生了SasaIFN-α2蛋白的成熟形式。通过SDS-PAGE和蛋白质印迹检测rSasaIFN-α2的表达，并通过质谱法确认其身份。在同源的奇努克鲑胚胎（CHSE-214）细胞系中，rSasaIFN-α2诱导了IFN诱导的Mx蛋白的早期表达，并对传染性胰腺坏死病毒（IPNV）表现出约2.8×10⁶U mg⁻¹的高抗病毒活性。相反，在异源的日本牙鲆胚胎（HINAE）细胞系中未观察到rSasaIFN-α2的抗病毒保护作用。因此，使用糖基化缺陷的原核系统成功产生了具有生物活性的rSasaIFN-α2形式，并将其纯化至同质，这表明其抗病毒活性不需要糖基化。
