IRF-1 acts as a positive regulator in the transcription of grass carp (Ctenopharyngodon idella) IFN gene.
IRF-1 acts as a positive regulator in the transcription of grass carp (Ctenopharyngodon idella) IFN gene.
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DOI：
10.1016/j.fsi.2013.03.358
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发表时间：
2013-06
影响因子：
4.7
通讯作者：
Q. Lai;Gang Lin;Meisheng Ma;Shenghe Huang;Wen Li;Dongming Li;Meihui Gu;Huiling Mao;Chengyu Hu-C
中科院分区：
文献类型：
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作者：
Q. Lai;Gang Lin;Meisheng Ma;Shenghe Huang;Wen Li;Dongming Li;Meihui Gu;Huiling Mao;Chengyu Hu-C
众所周知，干扰素调节因子（IRFs）在调节病毒感染的先天免疫反应中起着至关重要的作用。本研究对草鱼（Ctenopharyngodon idella）的IRF-1基因（简称CiIRF-1）进行了克隆和鉴定。CiIRF-1全长3150bp，包含9个外显子和8个内含子。CiIRF-1启动子序列长度为558 bp。CiIRF-1 cDNA全序列的最大开放阅读框（ORF）为870 bp，编码289个氨基酸的多肽。假设的CiIRF-1具有一个保守的n端DBD (113 aa)，并包含5个保守的色氨酸残基。系统发育关系分析表明，CiIRF-1与其他硬骨鱼和哺乳动物的对应基因高度同源。CiIRF-1的组成水平较低，但在Poly I:C或重组草鱼（C. idella） IFN （rCiIFN）刺激后，在所有6个测试组织中均显著上调，尤其是在脾脏和鳃中。重组CiIRF-1在BL21大肠杆菌中表达，用Ni-NTA His-Bind Resin亲和层析纯化表达蛋白。从草鱼I型IFN （CiIFN）基因GU139255中扩增出3个不同的启动子序列片段。这些片段包括近端区域（CiIFNP2）、远端区域（CiIFNP6）和CiIFN启动子序列的全长（CiIFNP7）。凝胶迁移率转移法用于分析ciiff -1和CiIFN启动子序列之间的相互作用。结果表明，CiIRF-1能在体外高亲和力地结合CiIFN启动子。随后构建pGL3-CiIFNPs和pcDNA3.1-CiIRF-1重组质粒，瞬时共转染C. idella kidney （CIK）细胞。通过荧光素酶测定CiIFN启动子序列对CiIFN -1的影响。这些结果表明，CiIRF-1在草鱼IFN基因的转录中起正调节作用。
Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. In the present study, the IRF-1 gene of grass carp (Ctenopharyngodon idella) (termed CiIRF-1) was cloned and characterized. The complete genomic sequence of CiIRF-1 was 3150 bp in length and comprised 9 exons and 8 introns. The CiIRF-1 promoter sequence was 558 bp in length. The largest open reading frame (ORF) of the full CiIRF-1 cDNA sequence was 870 bp, and encoded a polypeptide of 289 amino acids. The putative CiIRF-1 was characterized by a conserved N-terminal DBD (113 aa), and included a signature of five conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-1 was highly homologous to the counterparts of other teleosts and mammalians. CiIRF-1 was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly I:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all 6 tested tissues, especially in spleen and gill. The recombinant CiIRF-1 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. Three different fragments of promoter sequences from grass carp type I IFN (CiIFN) gene (GU139255) were amplified. These fragments included the proximal region (CiIFNP2), the distal region (CiIFNP6), and the full length of CiIFN promoter sequences (CiIFNP7). Gel mobility shift assays were employed to analyze the interaction between CiIRF-1 and CiIFN promoter sequences. The results revealed that CiIRF-1 could bind to CiIFN promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1 were constructed and transiently co-transfected into C. idella kidney (CIK) cells. The impact of CiIRF-1 on CiIFN promoter sequences were measured by luciferase assays. These results demonstrated that CiIRF-1 acts as a positive regulator in the transcription of grass carp IFN gene.