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Alternative TitleMolecular cloning and functional characterization of interferon system genes TLR3 and IRF1 in Crucian Carp (Carassius auratus L.)
Thesis Advisor桂建芳
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鲫鱼囊胚细胞 草鱼出血病病毒 Ifn系统 Tlr3基因 Irf1基因 诱导表达 细胞定位 核定位信号 功能分析 信号通路 抗病毒机制
Abstract本论文对IFN系统两个重要基因TLR3和IRF1进行了克隆鉴定、诱导表达和初步功能分析。TLR3是识别dsRNA的重要免疫识别受体。CaTLR3基因全长cDNA 3312bp,编码904aa。CaTLR3具有同源TLR3蛋白的基本结构,与斑马鱼的TLR3同源性最高。利用病毒及polyI:C处理CAB细胞,CaTLR3基因的表达均有所增强;但表达模式不同。CaTLR3基因广泛分布于健康鲫鱼的各种组织,而在病毒感染的鲫鱼头肾组织中有明显的表达上调。CaTLR3基因可以激活NF-κB,从而启动与NF-κB相连的荧光素酶报告基因的表达。IRF1是与type I IFN的表达调控相关的转录因子之一。CaIRF1基因全长cDNA 1450bp,编码289aa。基因组结构包括8个外显子和7个内含子,全长1.57kb。CaIRF1基因具有广泛的组成型表达,在病毒和polyI:C等诱导下,体外和体内均呈现表达上调。CaIRF1是一个典型的核定位蛋白,存在两个核定位信号(NLS)。其中一个与哺乳类IRF1的NLS一致,位于CaIRF1序列117-146aa;另一个新的NLS在DBD结构域内73-115aa。过量表达CaIRF1的细胞具有一定的抗病毒作用和细胞凋亡相关功能。在筛选的稳定表达CaIRF1的细胞中,IFN基因的组成型表达有所增强,诱导后IFN系统相关基因STAT1和IFI58的表达也显著增强。本论文的研究结果表明鱼类存在通过TLR3识别dsRNA激活NF-κB,诱导IFN表达的信号通路。同时,鱼类IRF1可以通过调节IFN基因和ISG基因的表达,调控细胞抗病毒状态的形成,但调控机制可能比哺乳类更复杂。
Other AbstractIn this study, two important genes of fish interferon system, toll-like receptor 3 (TLR3) and interferon regulator factor (IRF1), were characterized on inductive expression and preliminary function analysis. Toll-like receptor 3 (TLR3) is an important innate immune-recognition receptor that recognize double-stranded RNA (dsRNA). The CaTLR3 cDNA is 3312bp in length that encodes 904 amino acid residues. CaTLR3 is homologous to mammalian TLR3 in the primary structure and has the highest homology with zebrafish TLR3 in amino acid level. Real-time PCR analysis reveals a wide tissue distribution of CaTLR3 constitutive expression in healthy crucian carp and increased expression in virus-infected head kidney. In addition, expression pattern of CaTLR3 is different in CAB cells by different stimulus, such as virus and poly I:C. CaTLR3 can induce luciferase expression of NF-κB-luc vector. IRF1 is a member of transcription factors involved in regulating type I IFN gene transcription. The CaIRF1 cDNA contains 1450 nucleotides that translate into a 289 amino-acid putative peptide. The CaIRF1 gene spans the open reading frame (ORF) is 1.57 kb long consisting of 8 exons and 7 introns. Similar to the known IRF1 genes, CaIRF1 is ubiquitously expressed, and is upregulated in vitro and in vivo in response to virus and poly I:C. Subcellular localization analysis confirms the nuclear distribution of CaIRF1 protein, and reveals two nuclear localization signals (NLS). One NLS locates to amino acids 117-146, and appears to be the equivalent of the NLS in mammalian IRF1. The second NLS (amino acids 73-115) is found within the DNA binding domain (DBD) of CaIRF1. In addition, Overexpression of CaIRF1 not only enhanced cell viability under virus infection, but also regulates cell apoptosis. Real-time PCR also confirm that expression levels of STAT1 and IFI58 gene are obviously increased in CaIRF1-overexpressed cells in response to polyI:C stimulation. These results suggest fish may be similar to mammals in the recognition mechanism of dsRNA and inductive expression of IFN gene. At the same time,CaIRF1 can mediate the antiviral state in cells by regulating the expression of IFN and ISG genes. However, it is a different and complex mechanism of regulation in fish.
Document Type学位论文
Recommended Citation
GB/T 7714
史燕. 鲫鱼IFN系统基因TLR3和IRF1的克隆鉴定及功能分析[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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