石斑鱼半胱氨酸蛋白酶抑制剂B（CystatinB）在虹彩病毒SGIV感染中的作用及机制研究

Grouper, Epinephelus spp., one of the major species being maricultured in China, are high-priced and popular seafood fish. However, with the rapid production and development of fish worldwide, fish diseases have become severe and threaten the fish industry in recent years. Singapore grouper iridovirus (SGIV) is a major viral pathogen causing high mortality and huge economic losses in grouper cultures. So far, the recognition mechanisms of host cells to SGIV infection remain poorly understood. Our previous studies indicated that grouper Cathepsin B participated in the infection of SGIV. Cystatin B is a nature intracellular cysteine proteinase inhibitor which is a tightly-binding reversible inhibitor of Cathepsin B. In this study, we try to clone the grouper Cystatin B gene on the basis of EST screened from grouper splenic transcriptome library. Then the effects and mechanisms of grouper Cystatin B on SGIV induced apoptosis and virus replication will be investigated through over-expression, RNAi, flow cytometry, Co-IP, confocal microscopy, etc. The results will be beneficial to further understanding the mechanisms of immune responses in fish during virus infection, and ultimately development of new control and therapeutic strategies.

石斑鱼是我国南方重要的海水名贵养殖鱼类之一，经济价值极高。病害频繁爆发是其可持续健康发展的主要瓶颈，新加坡石斑鱼虹彩病毒（Singapore grouper iridovirus, SGIV）是其中最严重的传染性病毒病原之一，目前，有关石斑鱼对SGIV感染的免疫应答机理仍不十分清楚。我们前期研究表明，石斑鱼半胱氨酸组织蛋白酶B（Cathepsin B）参与了SGIV感染过程，而半胱氨酸蛋白酶抑制剂B（Cystatin B）是Cathepsin B的天然胞内抑制剂。本项目拟利用石斑鱼脾脏转录组文库中筛选得到的EST克隆石斑鱼Cystatin B基因，并进一步通过体外过表达、RNA干扰、流式细胞、免疫共沉淀及共聚焦显微镜等技术研究Cystatin B在SGIV感染中的功能与作用机制。研究结果不仅有助于我们进一步了解鱼类在病毒感染中的免疫应答机制，还为虹彩病毒病的防治提供新的理论依据和潜在靶点。

半胱氨酸蛋白酶抑制剂B（Cystatin B）是半胱氨酸组织蛋白酶B（Cathepsin B）的天然胞内抑制剂, 在免疫防御中发挥重要的作用。但鱼类Cystatin B在病毒感染中的作用及其分子机制仍不清楚。本研究从石斑鱼中克隆鉴定了Cystatin B基因（Ec-CysB）。Ec-CysB的cDNA全长为656bp, 具有303bp的开放阅读框, 编码101个氨基酸。Real-time PCR结果表明Ec-CysB在12个组织中均有分布, 且主要在肠道、鳃和头肾中表达量相对较高。经新加坡石斑鱼虹彩病毒（SGIV）刺激后, Ec-CysB在脾脏中的表达量在24h有明显上调。亚细胞定位结果显示Ec-CysB定位在细胞质和细胞核中。Ec-CysB过表达显著促进了病毒基因的表达, 同时, Ec-CysB过表达在SGIV感染过程中抑制了炎性因子TNF-α、IL-1β和IL8的mRNA表达。此外, Ec-CysB过表达促进了SGIV诱导的细胞凋亡和Caspase-3活性。以上研究结果表明Ec-CysB在病毒感染诱导的炎症及细胞凋亡中发挥重要的作用。