Department of Bioscience, College of Life Science and Food Engineering, Nanchang University, Nanchang 330031, China.
Gene. 2012 Aug 1;504(1):31-40. doi: 10.1016/j.gene.2012.04.091. Epub 2012 May 8.
Type I interferons and interferon regulatory factor 7 (IRF7), which are crucial for innate immunity against viral infection, have been identified in many teleost fishes in recent years. In this study, the complete genomic sequence of grass carp (Ctenopharyngodon idella) type I interferon (termed CiIFN) (GU139255) and the full-length IRF7 cDNA sequence of grass carp (termed CiIRF7) (GQ141741) were cloned and characterized. CiIFN consists of 3368 bp, retaining the characteristic 5-exon/4-intron gene organization in fish type I IFNs. The CiIFN spans 5 exons and encodes a polypeptide of 180 amino acids, with the first 22 amino acids representing a putative signal peptide. The CiIFN promoter sequence was found to be 760 bp, which can be divided into a proximal region (from -1 to -140 bp) and a distal region (from -400 to -700 bp). The cDNA of CiIRF7 was found to be 1808 bp in full length, with an ORF of 1293 bp that encodes a putative protein of 430 amino acids. The putative amino acid sequence of CiIRF7 possesses a DNA-binding domain (DBD) in the N-terminal region. Real-time PCR analysis revealed that CiIFN displayed a low constitutive expression in all the tissues tested. After stimulation by polyinosinic:polycytidylic acid (Poly I:C), the expression of CiIFN was significantly up-regulated in most tissues of grass carp, with a relatively strong expression in spleen, kidney and intestine. The recombinant polypeptides of CiIRF7 and CiIRF7-nDBD were analyzed in gel mobility shift assays, along with the PCR amplification products of the proximal region (CiIFNP2), the distal region (CiIFNP6) and the full-length (CiIFNP7) of CiIFN promoter sequence. The results revealed that CiIRF7 could bind to the distal region as well as to the proximal region of CiIFN promoter sequence in vitro. Subsequently, the CiIFNPs (CiIFNP7/2/6) were cloned into pGL3-Basic vectors and CiIRF7 was subcloned into pcDNA3.1 vectors, then pGL3-CiIFNPs were separately transiently transfected or co-transfected with pcDNA3.1-CiIRF7 into the mouse myeloma cell lines (MMCL) SP2/0 and the grass carp kidney cell lines (CIK), and the impact of CiIRF7 on CiIFN promoter activity was measured by luciferase assays in the transfected cells. These results demonstrated that CiIRF7 acted as a positive regulator on the transcription of CiIFN.
近年来，已在多种鱼类中发现了对病毒感染的固有免疫至关重要的 I 型干扰素 (IFN) 和干扰素调节因子 7 (IRF7)。在这项研究中，克隆并鉴定了草鱼 (Ctenopharyngodon idella) I 型干扰素 (称为 CiIFN) (GU139255) 的完整基因组序列和草鱼 IRF7 cDNA 全长序列 (称为 CiIRF7) (GQ141741)。CiIFN 由 3368 bp 组成，保留了鱼类 I 型 IFNs 中特征性的 5 个外显子/4 个内含子基因组织。CiIFN 跨越 5 个外显子，编码 180 个氨基酸的多肽，前 22 个氨基酸代表一个假定的信号肽。CiIFN 启动子序列长 760 bp，可分为近端区域 (从 -1 到 -140 bp) 和远端区域 (从 -400 到 -700 bp)。CiIRF7 的 cDNA 全长 1808 bp，其 ORF 为 1293 bp，编码 430 个氨基酸的假定蛋白。CiIRF7 的假定氨基酸序列在 N 端区域具有 DNA 结合域 (DBD)。实时 PCR 分析显示，CiIFN 在所有测试组织中均表现出低组成型表达。在用多聚肌苷酸：聚胞苷酸 (Poly I:C) 刺激后，CiIFN 在草鱼大多数组织中的表达显著上调，在脾脏、肾脏和肠道中表达较强。在凝胶迁移率变动分析中分析了 CiIRF7 和 CiIRF7-nDBD 的重组多肽，以及 CiIFN 启动子序列的近端区域 (CiIFNP2)、远端区域 (CiIFNP6) 和全长 (CiIFNP7) 的 PCR 扩增产物。结果表明，CiIRF7 可以在体外与 CiIFN 启动子序列的远端区域以及近端区域结合。随后，将 CiIFNPs (CiIFNP7/2/6) 克隆到 pGL3-Basic 载体中，将 CiIRF7 亚克隆到 pcDNA3.1 载体中，然后将 pGL3-CiIFNPs 分别瞬时转染或共转染到小鼠骨髓瘤细胞系 (MMCL) SP2/0 和草鱼肾细胞系 (CIK) 中，并通过荧光素酶测定法测量 CiIRF7 对 CiIFN 启动子活性的影响转染细胞。这些结果表明 CiIRF7 作为 CiIFN 转录的正调节剂发挥作用。
