College of Wildlife Resources, Northeast Forestry University, Harbin 150040, PR China.
College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, PR China.
Gene. 2014 Jul 1;544(1):49-55. doi: 10.1016/j.gene.2014.04.036. Epub 2014 Apr 21.
Interferon-α (IFN-α) genes have been cloned from a variety of animals, but information regarding crane IFN-α has not been reported to date. In this study, we cloned a full-length Red-crowned Crane interferon-α (crIFN-α) gene sequence consisting of a 486bp partial 5' UTR, 741bp complete ORF and 559bp partial 3' UTR. This gene encodes a protein of 246 amino acids and shares 60 to 80% identity with avian IFN-α and less than 45% identity with mammalian IFN-α. The expression of crIFN-α with an N-terminal His-tag was investigated in Escherichia coli, and the protein was purified on a nickel column. To obtain activated proteins, crIFN-α inclusion bodies were renatured by dialysis. In vitro cytopathic inhibition assays indicated that the recombinant crIFN-α could inhibit the replication of vesicular stomatitis virus in chicken fibroblasts. These antiviral activities were abrogated by rabbit anti-crIFN-α antibodies in vitro. In addition, an immunofluorescence assay indicated that crIFN-α could be expressed in chicken fibroblasts and was primarily located in the cytoplasm. Taken together, our results suggest that the crIFN-α gene may play an important role in inhibiting the replication of viruses.
干扰素-α（IFN-α）基因已从多种动物中克隆出来，但迄今为止尚未有关于鹤类 IFN-α的信息报道。在本研究中，我们克隆了一个全长的丹顶鹤干扰素-α（crIFN-α）基因序列，该序列包括 486bp 的部分 5'UTR、741bp 的完整 ORF 和 559bp 的部分 3'UTR。该基因编码一个 246 个氨基酸的蛋白质，与禽源 IFN-α的同源性为 60%~80%，与哺乳动物 IFN-α的同源性小于 45%。我们在大肠杆菌中对带有 N 端 His 标签的 crIFN-α进行了表达研究，并通过镍柱对其进行了纯化。为了获得激活的蛋白，我们通过透析对 crIFN-α包涵体进行复性。体外细胞病变抑制试验表明，重组 crIFN-α可抑制鸡成纤维细胞中水泡性口炎病毒的复制。在体外，兔抗 crIFN-α 抗体可使这些抗病毒活性丧失。此外，免疫荧光试验表明 crIFN-α可在鸡成纤维细胞中表达，并主要定位于细胞质中。综上所述，我们的研究结果表明，crIFN-α 基因可能在抑制病毒复制中发挥重要作用。
