Cloning, characterization, and expression analysis of orange-spotted grouper (Epinephelus coioides) ILF2 gene (EcILF2)
Cloning, characterization, and expression analysis of orange-spotted grouper (Epinephelus coioides) ILF2 gene (EcILF2)
复制标题
斜带石斑鱼 (Epinephelus coioides) ILF2 基因 (EcILF2) 的克隆、表征和表达分析
DOI：
10.1016/j.fsi.2010.11.015
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发表时间：
2011-01-01
影响因子：
4.7
通讯作者：
Qin, Qiwei
中科院分区：
文献类型：
--
作者：
Cui, Huachun;Yan, Yang;Qin, Qiwei
白细胞介素-2增强子结合因子2（IL-2 enhancer binding factor 2，IL-2）又称核因子45（nuclear factor 45，NF 45），在调节白细胞介素-2（IL-2）的表达中起重要作用。本研究克隆了一个新的ILF 2基因（命名为EcILF 2），并对其进行了功能鉴定。海菠萝全长EcILF 2 cDNA由1544 bp组成，编码387个氨基酸的多肽，与大西洋鲑鱼ILF 2的同一性为98%。EcILF 2基因组DNA由14个外显子和13个内含子组成，长度约为6.9kb。EcILF 2含有两个保守结构域，包括富含RGG的单链RNA结合结构域和DZF锌指核酸结合结构域。重组EcILF 2在大肠杆菌BL 21（DE 3）中表达并纯化，制备小鼠抗EcILF 2血清。亚细胞定位分析显示EcILF 2主要分布在细胞核内。实时定量PCR（RT-qPCR）分析显示EcILF 2广泛表达，在皮肤、肝脏、脑、头肾和脾脏中具有相对强的表达。在创伤弧菌、金黄色葡萄球菌、酿酒酵母和新加坡石斑鱼虹彩病毒（SGIV）刺激后，EcILF 2的表达差异上调。此外，EcILF 2能够激活不同细胞系中的人IL-2启动子，并促进人H9 T细胞中内源性IL-2的转录。这些结果表明EcILF 2可能参与石斑鱼对细菌和病毒病原体入侵的免疫应答。(C)2010爱思唯尔有限公司版权所有。
Interleukin-2 enhancer binding factor 2 (ILF2), also named as nuclear factor 45 (NF45), plays important roles in regulating interleukin-2 expression in mammals. In the present study, a novel ILF2 gene (designated EcILF2) was cloned and well characterized from orange-spotted grouper. Epinephelus coioides. The full-length EcILF2 cDNA is composed of 1544 bp and encodes a polypeptide of 387 amino acids with 98% identity to ILF2 of Atlantic salmon. The genomic DNA of EcILF2 consists of 14 exons and 13 introns, with a length of approximately 6.9 kb. EcILF2 contains two conserved domains including an RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain. Recombinant EcILF2 was expressed in Escherichia colt BL21 (DE3) and purified for mouse anti-EcILF2 serum preparation. Subcellular localization analysis revealed that EcILF2 was distributed predominantly in the nucleus. Realtime quantitative PCR (RT-qPCR) analysis revealed a broad expression of EcILF2, with a relative strong expression in skin, liver, brain, head kidney and spleen. The expression of EcILF2 was differentially up-regulated after stimulation with Vibrio vulnificus, Staphylococcus aureus, Saccharomyces cerevisiae and Singapore grouper iridovirus (SGIV). Furthermore, EcILF2 was able to activate human IL-2 promoter in different cell lines and promote the endogenous IL-2 transcription in human H9 T cells. These results suggest that EcILF2 is potentially involved in grouper immune responses to invasion of bacterial and viral pathogens. (C) 2010 Elsevier Ltd. All rights reserved.