鳜伽玛-干扰素的剪接异构体及其受体的转录调控与功能研究

Interferons (IFNs) are important molecules mediating protection against virus infection. They can be characterized into three types, i.e. types I, II and III. In fish, two types, that is, types I and II IFNs have been reported in literature. In vertebrates, with the exception of fish, the type II IFN, i.e. IFN-gamma is characteristic of a single coding gene, which plays important role in innate as well as in adaptive immune responses. However, one IFN-gamma gene and an IFN-gamma related gene have been identified in several species of fish, with another IFN-gamma gene reported in some species of fish. In our research on mandarin fish (Siniperca chuatsi) immune system, six functional spliced isoforms of IFN-gamma have been identified. However, spliced isoforms of IFN-gamma have never been reported in any species of vertebrates. This proposed research is thus designed to illustrate the gene organization of all possible isoforms of IFN-gamma and their receptors. Then, the splicing pattern and the mechanisms involved in the splicing are to be further investigated in order to understand the formation of these spliced isoforms. To reveal the role of these isoforms, it is necessary to understand the interaction between each splicing isoform and its corresponding receptor. The effect of different isoforms on innate and adaptive immunity will be evaluated eventually by examining their direct antiviral and possible anti-bacterial roles, and by examining the expression of immune factors, such as antimicrobial peptides, interferon inducible proteins, and those molecules involved in antigen presenting, such as MHC I and II, as well as immunoglobulin heavy chain genes. It is believed that this proposed research will for the first time provide novel information in relation to the structural and possibly functional diversity of IFN-gamma in vertebrates, being thus significantly important in understanding the role of type II IFN in fish, and the evolutionary implication of IFN system in fish as well as in vertebrates.

干扰素是机体抗病毒感染的重要分子，包括三种类型，即Ⅰ型、Ⅱ型和Ⅲ型干扰素；鱼类只有Ⅰ型和Ⅱ型干扰素。除鱼类外，其它脊椎动物的Ⅱ型干扰素，即伽玛-干扰素，由一个基因编码，在先天性和适应性免疫中具有重要作用。一些鱼类中报道了一个伽玛-干扰素基因和一个伽玛-干扰素相关基因，有些鱼类还有另外一个伽玛-干扰素基因。我们在鳜的伽玛-干扰素研究中发现6种有功能活性的剪接异构体，这种剪接形式还没有报道于其他任何一种脊椎动物的伽玛-干扰素。本项目将在此基础上，研究我国重要的水产养殖对象鳜的伽玛-干扰素及其受体的基因结构，阐明其剪接异构体的剪接形式及转录调控机理，解析不同剪接异构体与受体的相互作用，并进一步阐明不同剪接异构体在先天免疫和适应性免疫中的作用，揭示Ⅱ型干扰素结构与功能的多样性及其产生机制，丰富对Ⅱ型干扰素功能与作用机理的认识，从而在干扰素系统的研究方面具有重要意义。

干扰素是重要的细胞因子，可分为三种类型，即I型、II型和III型。硬骨鱼类只有I型和II型干扰素，且其II型干扰素有别于其他脊椎动物类群，具有两个成员，即伽玛干扰素（IFN-γ）和伽玛干扰素相关因子（IFN-γrel）。本项目以我国重要经济鱼类鳜为对象，对其基因组进行了survey测序，克隆了鳜II型干扰素的两个成员，IFN-γ和IFN-γrel，以及IFN-γ的剪接异构体，分析了它们的表达规律，揭示IFN-γ和IFN-γrel分别以同源二聚体形式存在，发现IFN-γ利用细胞因子受体家族基因B 6（chemokine receptor family member B 6，CRFB6）和CRFB13发挥其生活学活性，而IFN-γrel则需要CRFB17。发现CRFB13胞内结构域酪氨酸残基Y386在IFN-γ信号转导中起着必不可少的作用，而CRFB17胞内结构域中酪氨酸残基Y324和Y370对于IFN-γrel的功能是必需的。发现STAT1分子参与IFN-γ和IFN-γrel的信号通路，IRF9与STAT1和STAT2分子之间存在相互作用，IL-10和ISG15可以调控II型干扰素表达，阐明了鳜II型干扰素的两个成员在启动干扰素诱导基因（IFN-stimulated genes, ISGs）表达及其抗病毒活性方面的机理。本项目还克隆了参与并影响鳜干扰素信号通路的关键分子，包括鳜的3个I型干扰素基因（IFNh、IFNc、IFNd）及其受体基因（CRFB1、CRFB2、CRFB3、CRFB5）、干扰素调节因子家族基因1-11（IRF1-IRF11）、抗病毒基因MX、信号通路分子STAT1、STAT2和STAT3、以及干扰素诱导基因ISG15，揭示了3个I型干扰素分子的受体利用、以及在STAT1和STAT2分子磷酸化和诱导ISGs表达方面的作用。发现ISG15能抑制鳜传染性脾肾坏死病毒繁殖，且能诱导I型干扰素、IRF3和VIPERIN的表达，具有直接的抗病毒作用并能调节免疫应答。.另一方面，对中华鳖的II型干扰素、绿色安乐蜥II型和III型干扰素以及非洲爪蟾I型进行了系统研究，解析了低等脊椎动物干扰素系统的组成、功能和演化。本项目对鳜的I型和II型干扰素系统的研究，增加了对硬骨鱼类干扰素系统的认识，鉴定的干扰素分子及其诱导的抗病毒分子对鳜鱼的健康养殖具有重要实践意义。