作　　者： ; ; ; ; ;

机构地区： 中国科学院

出　　处： 《水产学报》 2013年第7期1095-1105,共11页

摘　　要： 新加坡石斑鱼虹彩病毒(Singapore grouper iridovirus,SGIV)是导致石斑鱼养殖产业严重经济损失的主要病毒病原之一。SGIV是大分子DNA病毒,包含162个基因开放阅读框,其中ORF050是一个肿瘤坏死因子受体类似物,可能在SGIV的免疫逃避中发挥作用。本研究克隆了SGIV ORF050基因,并构建了全长基因的真核表达重组质粒和四个半胱氨酸富集结构域(CRD)分别缺失的突变体。RT-PCR和药物抑制实验结果表明,SGIV ORF050是病毒的一个立即早期基因。亚细胞定位结果表明,该基因在细胞质内均匀地弥散性分布,并在细胞核周围聚集;第一个CRD缺失后,基因的定位发生明显的变化,即呈点状分布在胞质中,推测第一个CRD对其功能有影响。在过表达SGIV ORF050的鱼类细胞中观察SGIV感染引起的CPE,发现与对照相比没有明显区别;荧光定量PCR检测SGIV主要衣壳蛋白MCP的转录表达水平,也没有明显变化,提示该基因对SGIV在宿主细胞内的复制增殖可能没有影响。荧光定量PCR检测过表达ORF050的细胞在SGIV感染后宿主TNF/TNFR的转录水平,结果显示在感染10 h后TNF1、TNF2和TNFR2的表达量升高了2～3倍,而TNFR1的表达量没有明显变化,说明SGIV可能通过ORF050来调节细胞TNF和TNFR的表达,从而逃避宿主的免疫攻击。 Singapore grouper iridovirus （SGIV）is a major viral pathogen which can result in heavy economic losses in grouper aquaculture. SGIV is a large DNA virus containing 162 open reading frames （ORFs）, among which the ORF050, a tumor necrosis factor receptor like gene, may play possible roles in the virus immune evasion. In this study, the gene of SGIV ORF050 was cloned, and the recombinant vectors containing full length gene or the four Cysteine-rich Domain （CRD）deletion mutants were constructed, respectively. RT-PCR and drug inhibition assay demonstrated that SGIV ORF050 is an immediate-early（IE） gene. Intracellular localization revealed that SIGV ORF050 is distributed in the cytoplasm evenly and gathered around the nucleus. The localization changed when the first CRD was deleted, which presented punctated distribution in the cytoplasm. The cytopathic effect （CPE）was no changed significantly in SGIV infected ORF050-expressing fish cells compared with control ceils. In addition, real-time quantitative PCR （qRT-PCR） analysis showed that there was not significant change of SGIV MCP gene transcription in ORF050-expressing fish cells, suggesting that ORF050 might not have much effect on SGIV replication. Overexpressing ORF050 could enhance grouper TNF and TNFR transcription levels in SGIV infected fish cells ,revealing that SGIV may regulate the host TNF/TNFR signaling pathway to evade the host immune attack via ORF050.

关 键 词： 新加坡石斑鱼虹彩病毒 肿瘤坏死因子受体 分子特征 免疫逃避

领　　域： [农业科学] [农业科学]