【作者】 陈利军；

【导师】 苏建国；

【作者基本信息】 西北农林科技大学 ， 水生生物学， 2013， 硕士

【摘要】 草鱼（Ctenopharyngodon idella）是我国重要的经济鱼类，养殖规模居“四大家鱼”之首，然而草鱼易受许多疾病的困扰。其中，由草鱼呼肠孤病毒（grass carp reovirus，GCRV）所引起的草鱼出血病死亡率较高且严重困扰着草鱼养殖业的顺利发展。作为低等的脊椎动物，草鱼的获得性免疫系统欠发达，因此主要依赖于先天性免疫来抵抗病原微生物的入侵。目前，还没有特别有效的药物和方法针对该病的治疗。维甲酸诱导基因I（RIG-I，retinoic acid-inducible gene-I）是细胞质内关键的模式识别受体（PRRs，pattern recognition receptors）之一，能感知各种各样的病原分子相关模式（PAMPs，pathogen-associated molecular patterns），进而诱导I型干扰素（IFN-I，TypeI interferon）的产生和效应分子（例如Mx蛋白、干扰素刺激基因15（IFN-stimulated gene15）和RNA的腺苷酸脱胺酶（ADAR，adenosine deaminases acting on RNA））的表达。RIG-I包含三个重要的结构域：N端两个串联的半胱天冬酶激活募集结构域（CARDs，caspase activation and recruitment domains）、位于中间的解旋酶结构域（DExD/H helicasedomain）和C末端的调节结构域（RD，regulatory domain）。在哺乳动物中，RIG-I的CARDs结构域主要与IPS-1（interferon-β promoter stimulator1）的CARD结构域结合，传递下游的信号；DExD/H helicase结构域主要负责双链RNA的识别和ATP酶的结合，引起RIG-I的二聚化和结构的改变；RD结构域主要参与负调控，也有研究表明其能与RNA结合。与哺乳动物类似，鱼类的RIG-I也有同样的三个结构域。尽管RIG-I的免疫识别机制在哺乳类已经研究的比较全面，但是在鱼类上，具体的RIG-I功能的研究还处于起步阶段。因此，对鱼类RIG-I基因的各个结构域的研究将有助于进一步了解鱼类的先天性免疫信号转导途径和具体的效应结构区域。本研究利用之前已经克隆得到的RIG-I基因的全长cDNA序列，成功构建了草鱼RIG-I基因（简称为CiRIG-I，Ctenopharyngodon idella RIG-I）6个结构域的过表达载体，并将其转染到CIK细胞（Ctenopharyngodon idella kidney cells）中，得到稳定的CiRIG-I各个结构域的过表达细胞。接着用病毒GCRV感染和人工双链RNA类似物poly（I:C）（polyinosine-polycytidylic acid）、革兰氏阴性菌的类似物LPS（lipopolysaccharide）以及革兰氏阳性菌的类似物PGN（peptidoglycan）细胞，利用实时荧光定量PCR（qRT-PCR，quantitative real-time RT-PCR）技术，检测其信号通路上的三个基因（CiIPS-1、CiIFN-I和CiMx2）在各个转染细胞中的表达。病毒感染或者PAMPs刺激后，单独的RD结构域，对于信号通路上的基因表达起到了抑制作用。病毒GCRV感染和poly（I:C）刺激后，CiRIG-I的CARDs结构域起到了关键的作用。LPS和PGN刺激后，CiRIG-I的DExD/H helicase结构域发挥了非常重要的调节功能。在稳定表达的各个结构域细胞中，96孔板染色和病毒滴度检测表明各个构建体均具有或多或少的抗病毒活性。有趣的是，病毒感染后，在pΔCARDs（pCMV-EGFP-CMV-SV40-CiRIG-I-ΔCARDs）转染的细胞中，定量检测信号通路上的基因表达上调。同时，通过直接的病毒定量结果表明，草鱼RIG-I基因中CARDs结构域的缺失（相对应于对照），也能适当的抵抗病毒复制。本研究表明，全长的CiRIG-I能够引起广泛的基因表达，而且CiRIG-I能够介导RLRs（RIG-I-like receptors）信号通路的激活，具有广泛的抗病毒和抗菌效应。通过一系列的研究，我们初步证实了草鱼RIG-I基因的不同结构域发挥了直接的抗病毒和抗菌效应，为今后进一步探索RNA病毒识别和抗病毒天然免疫奠定了良好的理论基础，并最终为草鱼出血病的防治提供新的研究思路。更多还原

【Abstract】 Grass carp (Ctenopharyngodon idella) is considered one of the economically importantaquaculture species in China with the largest production among the “Great Four CulturedFish”. However, grass carp is susceptible to a wide variety of diseases, and grass carphemorrhage caused by grass carp reovirus (GCRV)(a dsRNA virus) was one of the mostserious epidemics in grass carp. As lower vertebrates, grass carp rely mainly on innateimmunity against pathogens infection for the defective adaptive immune system. To date, noeffective method is provided for the prevention and treatment of this disease.RIG-I (retinoic acid inducible gene-I) is one of the key cytosolic pattern recognitionreceptors (PRRs) for detecting pathogen associated molecular patterns (PAMPs) andmediating the induction of type I interferon and expreeeions of effector molecules (such asMx, ISG15(IFN-stimulated gene15) and ADAR (adenosine deaminases acting on RNA)).RIG-I consists of three distinct domains: N-terminal two tandem caspase activation andrecruitment domains (CARDs), central DExD/H box RNA helicase domain and C-terminalregulatory/repressor domain (RD). In mammals, CARDs of RIG-I mediate the interactionwith the CARD of interferon-β promoter stimulator1(IPS-1) and transmit downstreamsignaling molecules. The central DExD/H helicase domain is responsible for dsRNArecognition and ATPase binding, which leads to the dimerization and structural alterations ofRIG-I. The C-terminal RD inhibits RIG-I signaling cascade, however, recent report indicatesthat RD is responsible for RNA binding.Like mammals, RIG-I also possess three classical domains in teleosts. The mechanism iswell analysed in mammals, however,the study of the accurate function of RIG-I is still in itsinfancy in teleosts. Therefore, the research for RIG-I domains will contribute to the ofunderstanding signaling pathways and the detail effector regions in innate immunty inteleosts.Based on the full-length cDNA sequence of CiRIG-I (Ctenopharyngodon idella RIG-I)of the previous study, six representative overexpression plasmids were constructed for stablyexpressing recombinant proteins in CIK cells, respectively. The quantitative real-time RT-PCR (qRT-PCR) detected mRNA expressions of CiIPS-1, CiIFN-I and CiMx2post virus(GCRV) infection and dsRNA viral PAMP (poly(I:C), polyinosine-polycytidylic acid),gram-negative bacterial PAMP (LPS, lipopolysaccharide) or gram-positive bacterial PAMP(PGN, peptidoglycan) stimulation in the steadily transfected cells.After viral infection and PAMPs stimulation, repressor domain (RD) exerted inhibitoryfunction of signaling channels. After GCRV infection and poly(I:C) stimulation, CARDs ofCiRIG-I played positive and pivotal roles. The DExD/H helicase motif was crucial forsignaling pathway upon LPS and PGN stimulation. Virus titer test and96-well plate stainingassay showed that all constructs exhibited the antiviral activity more or less. Interestingly,pΔCARDs (pCMV-EGFP-CMV-SV40-CiRIG-I-ΔCARDs) transfected cells showed a postivemodulation in RIG-I signal transduction post GCRV infection. According to viral quantities, itdemonstrated that ΔCARDs of CiRIG-I (relative to the control) were moderately resistant toGCRV replication.The results show that the full-length RIG-I played a key role in RLRs (RIG-I-likereceptors) pathway mediating strikingly broad expressions in grass carp, responding to notonly dsRNA virus or synthetic dsRNA but also bacterial PAMPs. According to a series ofresearch, we preliminary evidence that the distinct domians of CiRIG-I exert differentantiviral and antibacterial functions, which provids a theoretical foundation for furtherrecognition of RNA viruses and the antiviral innate immunity. Ultimately, it will supply anovel sight for the defense of grass carp hemorrhage.更多还原