斑马鱼新的I型干扰素分子的抗病毒机制研究

Type I interferon (IFN) is a kind of innate antiviral cytokine, and plays an important role in host immunity. Fish type I IFN, like its counterpart in mammals, can mediate JAK-STAT signaling to participate in immune response against virus infection. . In addition to the report of four type I IFNs in zebrafish, we have found a new type I IFN gene in zebrafish, named temporarily as IFN5. This is the first IFNf subtype gene of type I IFN in zebrafish. Our study also showed that, compared to the known zebrafish type I IFN genes, ifn5 had a different expression pattern in adult fish tissues, suggesting that it may possess some new functional traits. During the development of zebrafish embryos, ifn5 showed a maternal expression pattern and its specific signal was detected in brain, esophageal wall and otic vesicle 24 hours post fertilization. Following poly I:C stimulation, ifn5 expression level increased obviously in different tissues. These results indicate that IFN5 may have some unknown effect in the early development stage of zebrafish, and may indeed have antiviral function. To answer these questions, we plan to 1) study the expression and regulation mechanism of ifn5 by the approach of WISH, promoter analysis and dual luciferase reporter assay; 2) identify the components of IFN5 receptor by overexpression, knockdown and CoIP; 3) analyze the mediated signaling pathway and antiviral function of IFN5 by overexpression, immunoblotting and CoIP. This work will clarify the mechanism of IFN5, contributing to our understanding of fish IFN system.

I型干扰素是先天性抗病毒细胞因子，在宿主免疫中有重要作用。鱼类I型干扰素类似于其哺乳动物同源基因，受病毒刺激后，能介导JAK-STAT信号通路发挥抗病毒作用，但鱼类I型干扰素信号传递网络复杂，尚未明了。我们发现了斑马鱼一个新的I型干扰素分子（暂命名IFN5），是斑马鱼I型干扰素IFNf亚型首个成员，它在成鱼组织表达分布不同于已知的四个斑马鱼I型干扰素，且polyI:C刺激后ifn5表达明显上调，这些都提示它可能具有抗病毒作用以及新的特性。因此，我们以斑马鱼为研究对象，拟1）通过原位杂交、启动子分析、双荧光素酶报告实验等研究ifn5的表达调控机制；2）以过表达、敲降、免疫共沉淀等鉴定IFN5的受体亚基组成；3）采取过表达、免疫印迹和免疫共沉淀等方法系统分析IFN5介导的信号通路及其抗病毒功能。基于此，阐明IFN5调控与作用机制，完善对鱼类免疫网络的认识。

干扰素是一类重要的细胞因子，在抗病毒免疫反应中起重要作用。而鱼类I型干扰素系统比其他脊椎动物的信号传递网络更高的复杂度，使其成为了研究热点。本项目以斑马鱼新的I型干扰素ifn5为关注点，克隆ifn5序列信息，分析了其序列与已知的斑马鱼4种I型干扰素的异同点，发现ifn5是斑马鱼首个ifn f亚型成员；获得了ifn5在斑马鱼中的表达模式，显示它与已知的4种I型干扰素的组织表达存在差异；通过双荧光素酶报告系统实验，表明了病毒可通过启动子激活ifn5表达；通过过表达和敲降的方法，鉴定了IFN5的受体亚基组成为crfb1与crfb5，并通过stat1a与irf9进行信号传递调控下游基因表达；用感染实验，证明了IFN5具有抗病毒功能，对细胞有保护作用。此外还完成了对斑马鱼I型干扰素受体表达模式的研究，并获得了crfb2/ crfb5的敲除品系。通过上述结果，我们探讨了斑马鱼I型干扰素IFN5的表达调控机制，以此有助于增加对鱼类病毒防御免疫的理解与治疗手段。目前在该项目资助下撰写论文2篇，正在审稿中。