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Alternative TitleMolecular cloning and characterization of NUB1 and subcellular localization of IRF3 and IRF7 in Crucian Carp (Carassius auratus L.)
Thesis Advisor张义兵
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 干扰素 干扰素刺激基因 诱导表达 亚细胞定位
Abstract干扰素(Interferon, IFN)系统在鱼类非特异性免疫中发挥重要作用。本实验室已成功建立研究鱼类IFN系统基因的细胞模型系统,并分离和鉴定了一系列病毒诱导基因。本研究在此基础上,克隆鉴定了IFN系统基因NEDD8 Ultimate Buster-1 (NUB1)并对其诱导表达特性进行了分析,同时对另外两个IFN系统基因Interferon regulator factor3 (IRF3)和Interferon regulator factor7 (IRF7)的亚细胞定位进行了研究。 NUB1是近年来发现的一种干扰素诱导表达基因,哺乳类的研究表明,过量表达该基因能抑制细胞生长。本实验通过RACE-PCR方法从产生干扰素的鲫囊胚培养细胞(Carassius auratus blastulae embryonic cells, CAB)中克隆出鲫NUB1基因。鲫NUB1全长cDNA为2298bp,编码一个由589个氨基酸残基组成的蛋白。推导的鲫NUB1蛋白具有哺乳类同源蛋白保守的结构域,包括N端的UBL结构域(Ubiquitin like domain)和C端两个UBA结构域(Ubiquitin associated domain),与已知的哺乳类包括人和小鼠、鸟类和两栖类的同源蛋白具有41-45%的相似性。诱导表达分析显示:Poly I:C和LPS均能诱导CAB细胞NUB1 mRNA的上调,表明NUB1基因在鱼类的抗病免疫反应中发挥某种重要作用。 IRF3和IRF7是哺乳类干扰素抗病毒信号通路中两个关键的调节因子,能够调控type I IFN和IFN刺激基因(Interferon stimulated genes, ISGs)的表达。本实验首先利用同源克隆法克隆了鲫IRF3基因, 其全长cDNA为1839bp, 编码一个由458个氨基酸组成的蛋白。推导的鲫IRF3蛋白具有哺乳类同源蛋白保守的结构域,N端DBD结构域(DNA binding domain)包含5个保守的色氨酸(Trp)重复序列。C端与其它物种的同源性较低,但也具有IAD结构域(IRF association domain)的核心序列。C端富含丝氨酸(Ser)残基,推测是磷酸化位点。其中DBD结构域的70-92氨基酸序列是一段富含K/R的序列(70NGDHSVWKRNFRSALRAKGFKM92),推测是一个核定位信号(nuclear localization signal, NLS)。软件推测I327可能与核输出信号(nuclear export signal, NES)相关。将IRF3与IRF7的ORF与GFP相连构建表达载体,转染COS7细胞,发现IRF3和IRF7主要定位在细胞质,转染5h加入100-300g/ml polyI:C诱导,19h后观察, IRF3和IRF7蛋白主要定位在细胞质,但均有少量定位在细胞核内。
Other AbstractMore attention has been paid to fish interferon system since it plays a key role in fish innate immune system. A cDNA library of CAB cells has been established for isolation and identification of cellular genes involved in IFN antiviral response. In this study, we cloned and identified the gene NEDD8 Ultimate Buster-1 (NUB1) and its expression was subsequently characterized. The other two genes involved in fish interferon system, interferon regulator factor 3 (IRF3) and interferon regulator factor 7 (IRF7), were characterized on subcellular localization. NUB1(NEDD8 Ultimate Buster-1)is identified recently as an interferon inducible gene, and overexpression of human NUB1 results in inhibition of cell growth. In the present study, Carassius auratus NUB1 (CaNUB1) gene was cloned from UV-inactivated grass carp haemorragic virus (GCHV)-infected and interferon-producing Carassius auratus blastulae embryonic cells (CAB) by RACE-PCR. The full-length cDNA of CaNUB1 is 2298 bp, which encodes a protein of 589 amino acids. Structurally, the putative CaNUB1 protein contains a ubiquitin like (UBL) motif in N terminus and two ubiquitin associated domains (UBA) in C terminus, showing 41-45% homology to known counterparts from mammals, birds and amphibian. Expression analysis showed that Poly I:C and LPS were able to upregulate the expression of CaNUB1, indicating that CaNUB1 play some roles in fish antiviral immune response. IRF3 and IRF7 are two transcription factors important for regulation of expression of type I interferon genes and interferon stimulated genes (ISGs). In the present study, Carassius auratus IRF3 (CaIRF3) gene was cloned by homology cloning method. The full-length cDNA of CaIRF3 is 1839bp encoding a 458-amino-acid protein. CaIRF3 protein shows 28-32% homology to known counterparts from mammals and amphibian. Stucturally, CaIRF3 protein possesses a putative DNA-binding domain (DBD) containing five conserved tryptophan repeats, and an IRF association domain (IAD) in the C terminus. Similar to mammalian IRF3 proteins, CaIRF3 has a serine-rich stretch in C terminus, which might be the phosphorylation site. The sequence (70NGDHSVWKRNFRSALRAKGFKM92) is rich in K / R, which may represent a nuclear localization signal (NLS). Subcellular localization analysis revealed that CaIRF3 and CaIRF7 are located in cytoplasm when IRF3 and IRF7 constructs are transfected into COS7 cells, respectively. Transfection of IRF3 and IRF7 constructs followed Poly I:C treatment for 19h showed that a small amount of IRF3 and IRF7 was present in nucleus.
Document Type学位论文
Recommended Citation
GB/T 7714
甘力. 鲫NUB1基因的克隆表达及IRF3和IRF7的亚细胞定位研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2009.
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