Long Scott, Wilson Melanie, Bengten Eva, Bryan Locke, Clem L W, Miller N W, Chinchar V G
Department of Microbiology, University of Mississippi Medical Center, Jackson, MS 39216, USA.
Dev Comp Immunol. 2004 Feb;28(2):97-111. doi: 10.1016/s0145-305x(03)00122-8.
Despite considerable advances in our understanding of teleost immunity, relatively few cytokine genes, including those for interferon (IFN), have been identified at the molecular level. In contrast, numerous studies have shown that following virus infection or exposure to double-stranded RNA, fish or fish cells produce a soluble factor that is functionally similar to mammalian IFN. A putative catfish (CF) IFN cDNA was identified by BLASTX screening of a catfish EST library generated from a mixed lymphocyte culture enriched for NK-like cells. Consistent with its designation as a putative cytokine cDNA, the 3' non-translated region contained multiple copies of an RNA instability motif. Analysis of the deduced amino acid sequence of CF IFN showed low levels of identity/similarity to a panel of mammalian and avian IFN proteins, and markedly higher similarity to a recently identified zebrafish IFN. To determine if the identified cDNA encoded CF IFN, expression was monitored following infection of channel catfish ovary (CCO) cells with UV-inactivated catfish reovirus or exposure to double-stranded RNA, treatments which induce IFN or IFN-like activity in catfish and other species. In both cases, upregulation of putative CF IFN mRNA was detected. Moreover, upregulation of CF IFN mRNA was accompanied by the appearance of an antiviral factor in the culture medium. To confirm these results, recombinant CF IFN was synthesized in COS-7 cells and shown to have antiviral activity in CCO cells. Collectively, these results argue strongly that the identified catfish cDNA is an IFN homolog.
尽管我们对硬骨鱼免疫的理解有了相当大的进展,但在分子水平上鉴定出的细胞因子基因相对较少,包括干扰素(IFN)相关的基因。相比之下,大量研究表明,在病毒感染或暴露于双链RNA后,鱼类或鱼细胞会产生一种功能上类似于哺乳动物IFN的可溶性因子。通过对从富含NK样细胞的混合淋巴细胞培养物中产生的鲶鱼EST文库进行BLASTX筛选,鉴定出一个推定的鲶鱼(CF)IFN cDNA。与其作为推定细胞因子cDNA的命名一致,其3'非翻译区包含多个RNA不稳定基序的拷贝。对CF IFN推导的氨基酸序列分析表明,它与一组哺乳动物和鸟类IFN蛋白的同一性/相似性较低,而与最近鉴定的斑马鱼IFN的相似性明显更高。为了确定鉴定出的cDNA是否编码CF IFN,在用紫外线灭活的鲶鱼呼肠孤病毒感染鲶鱼卵巢(CCO)细胞或暴露于双链RNA后监测其表达,这些处理可在鲶鱼和其他物种中诱导IFN或IFN样活性。在这两种情况下,均检测到推定的CF IFN mRNA上调。此外,CF IFN mRNA的上调伴随着培养基中抗病毒因子的出现。为了证实这些结果,在COS-7细胞中合成了重组CF IFN,并证明其在CCO细胞中具有抗病毒活性。总体而言,这些结果有力地表明,鉴定出的鲶鱼cDNA是一种IFN同源物。