草鱼感染GCRV后血液中淋巴细胞变化及BCL10表达分析

LYMPHOCYTES AND BCL10 EXPRESSION IN GRASS CARP(CTENOPHARYNGODON IDELLA)AFTER THE INFECTION OF GCRV

为了研究草鱼BCL10基因在草鱼出血病中的应答机制,文章克隆了BCL10基因,并利用生物信息学、荧光定量和血涂片等技术对其进行了分析。生物信息学结果显示,BCL10基因开放阅读框为738 bp,编码245个氨基酸。实时荧光定量PCR结果显示,感染病毒后草鱼体内BCL10表达量持续上调,在肝胰腺和中肾中第4天达到峰值,第7天表达量开始下调。血涂片显微镜观察发现了血液中淋巴细胞在感染病毒后第1到第4天下降,第7天时上升。肾脏的组织病理学观察也发现中肾中肾小管上皮细胞第1到第7天逐渐空泡化,脱落坏死。以上结果表明,BCL10基因参与了草鱼应对草鱼呼肠孤病毒(GCRV)入侵的免疫应答。

Grass carp(Ctenopharyngodon idella)is an essential freshwater aquaculture species in the world.The pro-duction of grass carp will be affected by grass carp hemorrhagic disease.The pathogen of grass carp hemorrhagic di-sease is grass carp reovirus(GCRV).Innate immune system is considered an essential defense for fish against a broad spectrum of pathogens.After the infection of the virus,the innate immune system can protect the host against the inva-sion.One of the innate immune response cell components is lymphocytes.Lymphocytes exist in abundance in the blood.Another typical immune response is inflammation inside the body.Many inflammation researches focus on the Toll-like receptors(TLRs)and Toll-interacting genes,but BCL10 also seems to be of great importance as the positive regulator.It has the function to activate the NF-kappaB pathway and then induce inflammation response.Besides,BCL10 also can be the termination of immune cell signaling.Our research found that GCRV infection reduced the number of the lymphocytes on day 1 and day 4 and then in-creased it on day 7,indicating the participation of lymphocytes in the fish immune defense.Besides,the vascular de-generation and exfoliation and necrosis of cells in trunk kidney indicated damaged blood circulatory system.Bioin-formatics analysis showed that the open reading frame of the BCL10 was 738 bp,encoding 245 amino acids.Phyloge-netic tree analysis showed that grass carp BCL10 has a good clustering relationship with the orthologs of other bony fish species.With the Real-time qPCR result,we found that after the infection of GCRV,BCL10 expression on the first day(D1)up-regulated in the hepatopancreas and gill compared to the control group.In contrast,blood cells and trunk kidney were significantly up-regulated.On the fourth day(D4),the expression of the BCL10 significantly up-regulated in all organs,but on the 7th day(D7),BCL10 expression was down-regulated in all organs compared to the control group.We speculated that as the immune organs in immune response,blood and kidney will respond to the infection of GCRV.This infection will activate BCL10,and then inflammation response happens to defend against the invading of GCRV.All the organs of grass carp were attacked by GCRV in D4,BCL10 gene significantly up-regulated which may induce inflammation to inhibit the virus proliferation.With the massive increase of virus,the inflammation inside the fish body loses control,BCL10 gene can not regulate the inflammation any more.Our study first observed the changes in lymphocytes number and morphology in the blood of grass carp after the infection GCRV and studied the changes in the expression of the B-cell apoptosis-related gene BCL10.However,re-search on BCL10 in grass carp hemorrhagic disease is still relatively lacking.This study provides an essential basis for subsequent studies of BCL10 gene function.It gives clues to the relevant mechanism of BCL10 in the innate immune response,which may help the future disease resistance breeding research of grass carp.