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乌鳢免疫相关基因的克隆鉴定与特征分析
Alternative TitleMolecular cloning, identification and characterization of immune-relevant genes in snakehead Channa argus
贾伟章
Subtype博士
Thesis Advisor徐旭东 ; 郭琼林
2007-06-16
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword差减cdna文库 干扰素系统 干扰素调控因子 干扰素刺激基因 信号通路 免疫相关基因
Abstract鱼类具备两种基本的免疫应答类型:先天性免疫应答和适应性免疫应答。头肾主要由网状内皮系统支持下的淋巴造血组织构成,包括淋巴细胞、单核细胞、浆细胞、两种或三种的粒细胞以及巨噬细胞等,担负着机体特异性和非特异性免疫反应,在个体发育过程中可能为免疫系统的生发中心。为了解乌鳢在病原刺激后的免疫反应,以及为鱼类分子免疫学研究提供一定的科学依据,本研究以poly I:C体内诱导乌鳢为测试组,对照鱼为驱动组,通过抑制差减杂交构建富含诱导后差异表达基因的乌鳢头肾差减cDNA文库。利用PCR和斑点杂交筛选文库,揭示了一批与哺乳类IFN系统基因同源,参与干扰素信号转导和转录调控的相关基因,如信号转导和转录激活因子(STAT)、干扰素调控因子(IRF),IFN刺激基因(IFN stimulated gene, ISG)或抗病毒基因,如Viperin、ISG15和ISG12,以及其他的免疫相关基因包括鼠李糖凝集素(RBL)、组织相容性抗原复合物(MHC I)、免疫球蛋白重链和轻链(IgM和IgL)、热休克蛋白70(HSP70),受体激活蛋白C(RACK)等。 通过RACE扩增的方法获得乌鳢STAT1、IRF1、Viperin、ISG15 cDNA全长。在此基础上,根据乌鳢IRF1以及已知IRF2和IRF7同源分子,设计位于开放阅读框保守位置的IRF2和IRF7兼并引物,扩增IRF2和IRF7片段。进而完成了三个干扰素调控因子cDNA和基因组克隆。分析三种鱼类干扰素调控因子的基因结构,并与人类IRF1、IRF2和IRF7基因进行了比较基因组学研究,发现IRF1和IRF2基因在进化上具有高度的保守性,外显子和内含子的组织模式在乌鳢和人基本一致,并且IRF1和IRF2基因之间也存在进化的保守性,尤其DNA结合区的外显子区段;乌鳢IRF7 5′ 端非编码区未出现内含子,这使该基因的组织模式与人IRF7显著不同。同时检测了三种IRF的组织分布,并且证实这些基因在脾脏和肝脏中能够被poly I:C诱导表达。为了解干扰素调控因子表达的分子基础,通过基因步移的方法,获得乌鳢三个干扰素调控因子的启动子序列。分析乌鳢干扰素调控因子启动子区的目的是寻找可能的转录因子结合位点,了解IRFs的表达调控,以及干扰素调控因子能够被病毒和双链RNA诱导表达的分子机理。此外,通过基因步移的方法,我们还获得了STAT1、Viperin和ISG15的启动子序列,鉴定了两种干扰素刺激基因,并比较了这两种干扰素刺激基因的启动子区特征。我们发现在Viperin和ISG15启动子区存在保守的干扰素刺激反应元件(ISRE)和γ-干扰素激活序列(GAS)。此外,Viperin启动子区包含保守的NF-κB结合位点,这是保守的NF-κB结合位点以一致的序列模式(GGGRNNYYCC)出现在鱼类干扰素刺激基因启动子区的首次报道。通过分析乌鳢STAT1和IRF7启动子序列特征,表明鱼类存在类似哺乳动物信号反馈的传导通路调控细胞对干扰素信号的反应。这为研究干扰素系统基因的调控奠定了基础。在此基础上,我们讨论了干扰素调控因子在调控免疫相关基因和病毒或干扰素介导的信号传导通路中潜在的角色。从低等脊椎动物到高等脊椎动物,鱼类和哺乳类干扰素和抗病毒蛋白呈现不同水平的差异,这从一定程度上反映了它们在不同生境中遭遇不同病原的选择压力。然而,根据我们的结果来看,不仅干扰素信号通路成员是保守的,而且干扰素调控因子和干扰素刺激基因的调控方式均与哺乳动物类似。此外,用RACE-PCR扩增的方法克隆到MHC I类分子,免疫球蛋白重链和轻链,RBL等全长cDNA序列。通过基因步移的方法获得RBL基因全长及其启动子序列。检测了STAT1、Viperin、ISG15、MHC I和RBL基因在各组织中的表达情况,同时还检测了poly I:C诱导后Viperin和ISG15在肝脏中的诱导表达。 在差减杂交和同源PCR的基础上,通过RACE和基因步移,我们克隆并鉴定了STAT1、IRF1、IRF2、IRF7、Viperin、ISG15,以及其他免疫相关基因。着重对干扰素系统几个基因的结构特征、表达特性、诱导机制、分子进化等进行了比较研究,初步揭示出鱼类存在复杂的调控网络介导IFN系统基因的表达。乌鳢免疫相关基因的研究工作对了解鱼类免疫机制以及鱼类抗病药物的开发具有重要的理论指导意义。
Other AbstractFish immune system contains innate immune response and acquired immune response. The parenchyma of head kidney is supported by the stroma consisting of reticular cells and fibroblasts. The cells are responsible for the nonspecific and specific immune defenses including lymphocytes, monocytes, plasma cells, granulocytes (two or three types) and macrophages. The immune cells in head kidney are abundant, and may be an important germinal center. In an effort to develop reagents to clarify the immune mechanism involved in snakehead responsive to pathogen infection, and importantly to make a breakthrough in molecular study on fish immune system. A model system for studying fish immune-relevant genes has been established through the mRNA of the stimulated group with poly I:C used as tester and the control group used as driver. A subtractive cDNA library accumulating an enhanced mRNA level of fish immune-relevant genes has been constructed by suppression subtractive technology (SSH). Differential ESTs have been identified through randomly screening colonies from the subtractive cDNA library by both PCR and dot blot. Sequencing analysis revealed that these genes have homology to that in other species. These genes are implicated in signal transduction and modulation, STAT and IRF, antivirus protein, Viperin, ISG15 and ISG12, other important immune genes including Rhamnose binding lectin (RBL), MHC class I, IgH, IgL, HSP70, receptor activated for C protein kinase (RACK), et al. The cDNA sequences of snakehead STAT1, IRF1, Viperin, ISG15 were obtained by RACE-PCR. Together with snakehead IRF1 and known IRF2 and IRF7 molecules, degenerate primers of IRF2 and IRF7 were designed from a conserved region located at the start of the open reading frame (ORF) to specifically amplify IRF2 and IRF7 gene fragments. We performed the cloning cDNA sequences and genomic organization of three IRF genes from the snakehead. The gene organization of IRF-1 and IRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes revealed a common evolutionary structure, notably within the exons encoding the N-terminal portions of the two factors. However, the gene organization of IRF-7 consisting of ten exons and nine introns is different to human IRF-7a gene which has an intron in 5′ UTR. Moreover, we detected their distribution in ten tissues, and further demonstrated that these genes are rapidly and transiently induced by poly I:C in spleen and liver. The regulatory sequences of the IRF1, IRF2 and IRF7 5′ flanking region have been cloned and sequenced through gene walking, the analysis of snakehead IRF promoters was aim to determine the critical factors that regulate the expression of IRF genes as well as the possible mechanism by which expression of IRF genes are capable of induction by virus and dsRNA. Moreover, the 5′ flanking region of STAT1, Viperin and ISG15 were obtained using a genome walking approach. The present study was performed to identify two IFN stimulated genes and to compare the characterization of Viperin and ISG15 promoters in the snakehead.The important characteristic features in the Viperin and ISG15 promoter regions were the presence of interferon stimulating response elements (ISRE) and γ-IFN activation sites (GAS). One conserved site for NF-κB was found in the promoter region of Viperin. This is first reported conservative binding motif for NF-κB in accordance with the consensus sequence (GGGRNNYYCC) among teleost ISG promoters. The characteristics of snakehead STAT1 and IRF7 promoter suggested that mammal intracellular amplifier circuit also exist controlling cellular responsiveness to the IFNs in fish. Subsequently, we discuss the potential roles of these IRFs in regulating immune-related genes and virus- or IFN-mediated signaling pathways. From the lower fish to higher mammals, though IFN and antiviral proteins present a different level of divergence, reflecting to some extent that selection pressures from different pathogens encountered in fish and other organisms because they stay at different environments and have very different life-styles. However, according to our results, not only IFN signal pathway is substantially conserved but also the regulation patterns of IFN regulatory factors and the expression patterns of IFN stimulated gene similar to mammals. In addation, the cDNA sequences of MHC class I, Ig, RBL were obtained by RACE-PCR. Genomic sequences of RBL and its promoter were obtained by genome walking. Expression of STAT1, Viperin, ISG15, MHC class I and RBL genes in tissues were analyzed by RT-PCR. The induced effect of Viperin and ISG15 were detected by the stimulated of poly I:C in liver. In this paper, through SSH and homology cloning, we have cloned, identified and characterized IFN system genes, including STAT1, IRF1, IRF2, IRF7, Viperin, ISG15, and other immune genes. A complex regulatory network implicated in modulating the expressions of fish IFN system genes during virus infection has been preliminary revealed by further studying the structure, expression, induction and evolution of some important genes. The researches of snakehead immune system are essential for understanding immune mechanism and antiviral drug discovery in fish.
Pages190
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12276
Collection学位论文
Recommended Citation
GB/T 7714
贾伟章. 乌鳢免疫相关基因的克隆鉴定与特征分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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