鞍带石斑鱼isg15基因cDNA克隆及其在虹彩病毒感染下的表达分析

Molecular Characterization and Expression Analyses of the Viral Infection of the Interferon-Stimulated Gene 15(isg15)in Epinephelus lanceolatus

本研究利用PCR和RACE技术首次获得了鞍带石斑鱼(Epinephelus lanceolatus)干扰素刺激基因15(interferon stimulated gene 15,isg15)全长序列。isg15基因序列长为910 bp,包含一个468 bp的开放阅读框,可编码155个氨基酸,预测分子量为17.09 kDa,理论等电点为9.33。保守结构域分析显示,鞍带石斑鱼ISG15蛋白包含2个类似泛素结构域,且C末端具有高度保守的“Leu Arg Leu Arg Gly Gly(LRLRGG)”结构域。序列分析和系统发育分析显示,鞍带石斑鱼与斜带石斑鱼(E.coioides)isg15的相似性最高,为88.24%,与大菱鲆(Scophthalmus maximus)和大黄鱼(Larimichthys crocea)isg15的相似性分别为61.18%和60.59%。采用实时荧光定量PCR技术检测了鞍带石斑鱼健康组织中的isg15表达,以及虹彩病毒(iridovirus)感染后不同时间脾脏和肾脏中的isg15表达变化。结果显示,isg15在血液中的表达量最高,在肝脏、肾脏等组织中的表达量较高。经虹彩病毒感染后,isg15在脾脏和肾脏中的表达显著升高,在72 h到达最高水平,说明isg15在鞍带石斑鱼防御虹彩病毒的过程中发挥着重要作用。对鞍带石斑鱼isg15基因的分析和表达模式的研究,有助于进一步了解isg15基因在硬骨鱼体内的抗病毒调控机制,为鞍带石斑鱼抗病分子育种提供基础数据。

This study aimed to explore the pathogenic mechanism of the iridescent virus and provide the theory basis for clinical diagnosis and treatment in Epinephelus lanceolatus.The full-length E.lanceolatus interferon-stimulated gene 15(isg15)was obtained by PCR and rapid amplification of cDNA ends(RACE).The full-length cDNA of isg15 was 910 bp,including an open reading frame(ORF)of 468 bp,which encoded a polypeptide of 155 amino acids.The molecular mass of the deduced amino acid sequence was 17.09 kDa,with an estimated pI of 9.33.Conserved domain analysis revealed that the isg15 protein of E.lanceolatus contained two ubiquitin-like domains,and the C-terminal had a highly conserved motif of"Leu Arg Leu Arg Gly Gly(LRLRGG)".Sequence and phylogenetic analyses showed that the amino acid sequence of isg15 in E.lanceolatus had the closest identity to Epinephelus coioides,with a similarity of approximately 88.24%.In addition,the homologous similarity of the E.lanceolatus isg15 with Scophthalmus maximus and Larimichthys crocea was 61.18%and 60.59%,respectively.Real-time quantitative PCR was used to detect the expression patterns of the isg15 gene in the healthy tissues of E.lanceolatus and the changes in the spleen and kidney at different times after infection with the iridovirus.The results showed that the isg15 gene was mainly expressed in the blood of E.lanceolatus and was highly expressed in various immune-related tissues,such as the liver and kidney.Upon induction with iridovirus,isg15 gene expression was up-regulated in the kidney and spleen,and reached a peak at 72 h,indicating that isg15 may play an important role in the immune response of E.lanceolatus against viral infection.In this study,the analyses and expression patterns of isg15 in E.lanceolatus were studied,which is helpful to further understand the antiviral regulatory mechanism of isg15 in teleosts,and provides a theoretical basis for the disease-resistant molecular breeding of E.lanceolatus.