半滑舌鳎（Cynoglossus semilaevis）免疫相关基因HMGB2和IFIT1功能研究

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	半滑舌鳎（Cynoglossus semilaevis）免疫相关基因HMGB2和IFIT1功能研究
	龙昊
学位类型	博士
导师	孙黎
	2015-05-24
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	海洋生物学
关键词	半滑舌鳎 Hmgb2 Ifit1 抗细菌感染抗病毒感染
摘要	本研究从半滑舌鳎（Cynoglossus semilaevis）中克隆了两个免疫相关基因HMGB2（high-mobility group protein B2）和IFIT1（i nterferon-induced protein with tetratricopeptide repeats 1），命名为CsHMGB2和CsIFIT1，并研究了它们的表达模式以及在宿主抵抗病原感染过程中的作用。在哺乳动物中，高速泳动族蛋白（HMG）已知有多种不同的功能，而在鱼类中HMG相关的研究较少。本研究中，我们从半滑舌鳎cDNA文库中克隆到了HMGB家族的一个基因，CsHMGB2，并研究其生物学功能。CsHMGB2蛋白共有245个氨基酸残基，两个HMG结构域和一个由23个天冬氨酸/谷氨酸残基组成的酸性羧基尾部。荧光定量RT-PCR（qRT-PCR）实验结果表明CsHMGB2在多个组织表达，并且其表达在病毒或细菌后显著上调。体外细胞实验发现，重组蛋白rCsHMGB2和缺失了C端酸性氨基酸区域的突变型重组蛋白rCsHMGB2M都能显著提高半滑舌鳎外周血淋巴细胞（PBL）抵抗细菌侵染的能力。qRT-PCR分析发现rCsHMGB2显著增强PBL中多种细胞因子和趋化因子的表达。体内实验表明，rCsHMGB2显著抑制病原在半滑舌鳎脾脏和肾脏中的侵染。上述实验结果表明，CsHMGB2具有免疫调节功能和抗病原感染作用，并且这种能力与其酸性羧基端无关。干扰素诱导基因IFIT在哺乳动物免疫系统中具有抗病毒功能，而在鱼类中的功能仍然未知。在本研究中，我们从半滑舌鳎cDNA文库中克隆到了IFIT家族的一个基因，CsIFIT1，并研究了它的抗病毒感染的活性。CsIFIT1由436个氨基酸残基组成，与其它鱼类IFIT蛋白的序列相似度介于30.9%和58.7%之间。软件分析鉴定出两个TPR（tetratricopeptide repeats）结构域。qRT-PCR实验表明，CsIFIT1的表达在免疫相关的器官组织中最高，并且在鱼体感染肿大细胞病毒后明显上调。在半滑舌鳎中过量表达CsIFIT1可以显著抑制肿大细胞病毒在鱼体中的复制。同样，使用RNA干扰技术降低CsIFIT1表达后，病毒在组织中的复制量显著增高。这些结果表明，CsIFIT1在鱼类抗病毒免疫中起关键作用。
其他摘要	In this study, we have identified two immune related genes HMGB2（high-mobility group protein B2） and IFIT1（Interferon-induced protein with tetratricopeptide repeats1） from half smooth tongue sole （Cynoglossus semilaevis）, and analyzed their functions in host defense pathogen. We also determinated the best internal references for qRT-PCR analysis of gene expression while the fish was infected with pathogen. High mobility group box （HMGB） proteins are known to be involved in diverse functions in mammalian cells. In teleost, very limited studies on HMGB proteins have been documented. In this study, we reported identification of a HMGB homologue （named CsHMGB2） from tongue sole and examined its biological property. CsHMGB2 is 245 residues in length and contains two basic HMG boxes and an acidic C-terminal tail composed of 23 Asp/Glu residues. Quantitative real time RT-PCR （qRT-PCR） analysis showed that CsHMGB2 expression occurred in multiple tissues and was upregulated by bacterial and viral infection in a time-dependent manner. In vitro studies showed that when tongue sole peripheral blood leukocytes were treated with recombinant CsHMGB2 （rCsHMGB2） and the mutant rCsHMGB2M, which bears a deletion of the C-terminal acidic region, significant and comparable increases in cellular resistance against bacterial infection were observed. qRT-PCR detected enhanced expression of proinflammatory cytokines and chemokines in rCsHMGB2-treated cells. In vivo studies showed that when tongues sole were administered with rCsHMGB2 or rCsHMGB2M before being subjected to bacterial and viral infection, the pathogen loads in the spleen and kidney of the fish were significantly reduced. Taken together, these results suggest that CsHMGB2 possesses immunoregulatory properties that promote resistance against bacterial and viral infection in a manner that is largely independent on the highly conserved C-terminal acidic domain. Interferon-induced protein with tetratricopeptide repeats （IFIT） is involved in antiviral immunity in mammalian cells. In fish, the function of this group of proteins is unknown. In this study, we report the identification of an IFIT homologue （named CsIFIT1） from tongue sole （Cynoglossus semilaevis） and examined its antiviral potential. CsIFIT1 is composed of 436 residues and shares 30.9%-58.7% overall sequence identities with the known IFITs of a number of teleost species. In silico analysis identified two tetratricopeptide repeats in CsIFIT1. Quantitative real time RT-PCR analysis showed that CsIFIT1 expression was most abundant in immune relevant organs and upregulated, in a time-dependent manner, by experimental infection with the fish viral pathogen megalocytivirus. When CsIFIT1 was overexpressed in tongue sole before megalocytivirus infection, viral replication in fish tissues was blocked to significant extents. Consistently, when CsIFIT1 expression in tongue sole was knocked down by siRNA, viral replication in fish tissues was significantly enhanced. Taken together, these results indicate that CsIFIT1 is likely a key factor of antiviral immunity and is required for optimal defense against viral infection
学科领域	海洋生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/22738
专题	实验海洋生物学重点实验室研究生部
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	龙昊. 半滑舌鳎（Cynoglossus semilaevis）免疫相关基因HMGB2和IFIT1功能研究[D]. 北京. 中国科学院研究生院,2015.

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