机构地区:[1]湖南农业大学,长沙410000 [2]汉寿县畜牧水产事务中心,湖南常德415900
出 处:《淡水渔业》2025年第2期62-73,共12页Freshwater Fisheries
基 金:湖南省自然科学基金面上项目(2022JJ30293)。
摘 要:草鱼(Ctenopharyngodon idellus)为鲤科草鱼属的鱼类,在我国淡水养殖业中占据举足轻重的地位。然而草鱼呼肠弧病毒(Grass carp reovirus,GCRV)导致的出血病是养殖业面临的一大挑战,因此深入探究GCRV的致病机制及防控手段至关重要。Viperin是一种干扰素诱导的病毒抑制蛋白,主要参与机体天然免疫机制,但其在草鱼中的研究尚且不足。为研究草鱼Viperin是否参与抗GCRV的免疫机制,本研究采用快速扩增cDNA末端技术(Rapid Amplification of cDNA Ends,RACE)克隆出草鱼Viperin基因的全长cDNA序列,并用生物信息学方法分析其结构特点和进化关系。结果表明,草鱼Viperin与青鱼Viperin在氨基酸序列上具有高度同源性。为分析Viperin在草鱼不同组织中的表达变化,对草鱼腹腔内注射GCRV、Poly(I∶C)和LPS,RT-PCR结果显示,在GCRV或Poly(I∶C)刺激下,各组织Viperin表达都出现先升后降趋势。而LPS刺激下,头肾及肾脏组织中Viperin表达量总体下降。Viperin在不同组织呈现出的不同表达提示该蛋白可能参与草鱼对多种外来刺激的响应过程,且其表达变化可能与组织功能特异性相关。过表达Viperin的草鱼肾细胞(Grass carp kidney,CIK)中GCRV外衣壳蛋白VP7的表达显著下降。说明草鱼Viperin的表达水平直接影响病毒的复制和感染能力。这些结果为深入研究草鱼Viperin蛋白在抗GCRV的免疫调控机制和遗传育种中的作用提供重要的理论依据。The grass carp is a freshwater fish belonging to the Cyprinidae family and a core species in China's freshwater aquaculture industry.However,the high lethality of haemorrhagic disease caused by grass carp reovirus(GCRV)represents a significant challenge to the sustainable development of freshwater aquaculture.It is,therefore,of particular importance to identify an effective response to GCRV.Viperin is an interferon-induced viral inhibitory protein that plays an important role in natural immunity.However,its role in grass carp immunity remains poorly understood.The full-length cDNA sequence of the grass carp Viperin gene was cloned using the rapid amplification of cDNA ends(RACE)technique to ascertain whether grass carp Viperin played a role in the immune response to GCRV.The structural features,evolutionary relationship and gene covariance comparison of the grass carp Viperin gene were analysed using bioinformatics methods.Furthermore,fluorescence quantitative PCR was employed to examine the expression of grass carp viperin in normal tissues and in relevant immune tissues of grass carp subjected to GCRV,Poly(I∶C),and LPS challenge.The expression vector of pEGFP-N1-Flag-Viperin was constructed to investigate the preliminary function of Viperin against grass carp reovirus in CIK cells.The results demonstrated that the full-length cDNA of the grass carp Viperin gene was 1885 bp,comprising 29 bp of the 5′-end non-coding region(UTR),776 bp of the 3′UTR,and 1080 bp of the open reading frame(ORF),which encoded 359 amino acids.The phylogenetic tree demonstrated that the amino acid sequence of grass carp Viperin grouped with fish Viperin and exhibited high similarity and homology with cyprinid fish.The grass carp Viperin gene was expressed in 10 tissues of healthy grass carp,including liver,spleen,kidney,head kidney,gill,skin,muscle,intestine,brain and heart.The expression was higher in the gill and spleen,followed by the brain and heart.GCRV,poly(I∶C)and LPS were injected intraperitoneally into the fish to observ
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