半滑舌鳎（Cynoglossus semilaevis）先天性免疫基因TLR2、TLR7、CD94和Bf的功能研究

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	半滑舌鳎（Cynoglossus semilaevis）先天性免疫基因TLR2、TLR7、CD94和Bf的功能研究
其他题名	海洋生物学
	李学鹏
学位类型	博士
导师	孙黎研究员
	2017-05-22
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	半滑舌鳎 Tlr2 Tlr7 Cd94 补体因子b Myd88 先天性免疫
摘要	半滑舌鳎（Cynoglossus semilaevis）是我国重要养殖鱼类。半滑舌鳎养殖面临着病害频发的问题，目前尚没有有效的控制方法，由此限制了养殖业的发展。在本文中，我们开展了半滑舌鳎先天性免疫分子的基础性研究，目的是揭示鱼体自身抵抗病原感染的机制，从而为安全高效的生物免疫防控疾病奠定基础。我们研究了半滑舌鳎先天性免疫基因Toll-Like Receptor TLR2（命名为CsTLR2）、TLR7（命名为CsTLR7）、Cluster of Differentiation 94（命名为CsCD94）和complement factor B（命名为CsBf），分析了这些基因在不同条件下的表达情况以及在抵抗病原侵染过程中的作用。 TLR2是TLR家族中重要的成员之一，在先天免疫系统中发挥着重要的作用。在哺乳动物中，TLR2能够识别微生物特异性的胞壁成分，进而引发MyD88依赖型信号途径的激活，导致多种细胞因子的产生。在本论文中，我们研究了CsTLR2的表达模式和抗菌功能特点。CsTLR2包含898个氨基酸残基，和其它的硬骨鱼的同源性在25.6% 到27.3% 之间。CsTLR2作为一个跨细胞膜蛋白含有一个典型的TIR胞内结构域和一个富含LRR的胞外结构域。CsTLR2在多种组织中组成型表达。细菌刺激后，CsTLR2表达量显著上调。CsTLR2信号途径的激活能够增强MyD88依赖型途径相关信号分子的上调表达。重组的CsTLR2胞外蛋白 (rCsTLR2) 能够结合多种细菌。无论在体内还是体外条件下，rCsTLR2都能显著降低细菌的侵染。这些结果为硬骨鱼TLR2的信号途径及功能的研究提供了一个新的视角。在哺乳动物和硬骨鱼中，TLR7主要是通过识别病毒的RNA而参与抗病毒免疫。然而鱼类的TLR7的抗病原免疫功能并不清楚。在本研究中，我们分析了CsTLR7在抗细菌免疫中的作用。CsTLR7蛋白由1052个氨基酸组成，和其它硬骨鱼TLR7的同源性在64.0%到75.9%之间。CsTLR7包含一个胞内TIR结构域和和由六个亮氨酸重复（LRR）序列组成的胞外结构域。CsTLR7是组成型表达的，高表达于肾脏、脾脏和肝脏中。细菌刺激能够上调CsTLR7的表达，然而病毒刺激却下调CsTLR7的表达。利用RNA干扰技术干扰CsTLR7表达显著削弱了宿主抵抗细菌在组织中侵染的能力。进一步研究发现，咪唑喹啉复合物R848（TLR7激活剂）能够增强半滑舌鳎抗细菌的能力，氯喹CQ（TLR7抑制剂）能够减弱半滑舌鳎的抗细菌能力。这些结果表明，CsTLR7在半滑舌鳎的抗细菌免疫中起到非常重要的作用。 CD94属于C型凝集素的V家族，含有一个细胞膜外C型凝集素样结构域（CTLD）。我们对CsCD94的表达模式和功能进行了研究。CsCD94包含209个氨基酸残基，和其它硬骨鱼CD94的同源性在43.0％到50.2%之间。CsCD94含有一个CTLD。CsCD94在多种组织中表达，在细菌的刺激下会呈显著性的上调表达。重组的CsCD94 (rCsCD94)能够以钙离子的依赖型的方式结合多种细菌，同时能够对细菌产生凝集作用。rCsCD94能够促进外周血白细胞（PBL）对细菌的吞噬作用，而且rCsCD94能够杀死多种细菌。这些结果说明rCsCD94在半滑舌鳎吞噬和杀菌过程中起到重要作用。补体B因子（Bf）是补体系统中重要的成分之一。在辅助激活补体旁路途径的同时，B因子也被切割成Ba和Bb两个片段。在鱼类当中，我们只知道虹鳟鱼的B因子能够作为C3转化酶的组分之一，而鱼类的Ba片段的功能尚未知。在本论文中，我们研究了CsBf的表达模式以及CsBa片段的抗菌活性。CsBf拥有Bf保守的结构域，和其它硬骨鱼的Bf的同源性在39.9%到56.4%之间。CsBf在肝脏、肌肉和心脏中的表达量较高，在肠道、血液和肾脏中的表达量较低。细菌感染能够以一种时间依赖性的方式显著诱导肾脏、脾脏和肝脏中的Bf上调表达。重组的CsBa蛋白 (rCsBa) 能够显著结合细菌和PBL，rCsBa在结合细菌之后显著性地抑制细菌生长。当CsBa在半滑舌鳎中过量表达时，能够显著性地抑制细菌在鱼体组织中的扩散。这些结果第一次指出鱼类的Ba因子拥有抗细菌的活性和细胞结合活性，说明CsBa在宿主抵抗细菌的侵染过程中起到重要的作用。
其他摘要	Tongue sole Cynoglossus semilaevis is an important fish cultured in China. Microbial disease is a severe problem of tongue sole aquaculture, and currently there is no effective means of disease control. In this study, in order to understand the immune defense mechanism against pathogens and provide a basis for the development of safe and effective methods of disease control, we examined the functions of four innate immune genes, i.e., Toll-Like Receptor (TLR) 2 (named CsTLR2)、TLR7 (named CsTLR7)、Cluster of Differentiation 94 (named CsCD94), and complement factor B (named CsBf). Toll-like receptor (TLR) 2 is a member of the TLR family that plays a pivotal role in innate immunity. In mammals, TLR2 is known to recognize specific microbial structures and trigger MyD88-dependent signaling to induce various cytokine responses. In this study, we examined the expression and function of the tongue sole Cynoglossus semilaevis TLR2, CsTLR2. CsTLR2 is composed of 898 amino acid residues and shares 25.6%–27.3% overall sequence identities with known teleost TLR2. CsTLR2 is a transmembrane protein with a toll/interleukin-1 receptor domain and eight leucine-rich repeats. Expression of CsTLR2 occurred in multiple tissues and was upregulated during bacterial infection. Stimulation of the CsTLR2 pathway led to enhanced expression of MyD88-dependent signaling molecules. Recombinant CsTLR2 (rCsTLR2) corresponding to the extracellular region was able to bind to a wide range of bacteria. Under both in vitro and in vivo conditions, rCsTLR2 significantly reduced bacterial infection. These observations add new insights into the signaling and function of teleost TLR2s. In mammals as well as in teleost, toll-like receptor 7 (TLR7) is known to be involved in antiviral immunity by recognizing viral RNA. However, the antibacterial potential of fish TLR7 is unclear. In this study, we analyzed the TLR7 of tongue sole (Cynoglossus semilaevis), CsTLR7, and examined its potential involvement in antibacterial immunity. CsTLR7 is composed of 1052 amino acid residues and shares 64.0%-75.9% overall sequence identities with known teleost TLR7. CsTLR7 possesses a toll/interleukin-1 receptor domain and six leucine-rich repeats. Constitutive expression of CsTLR7 occurred in relatively high levels in kidney, spleen and liver. Bacterial infection upregulated CsTLR7 expression, whereas viral infection downregulated CsTLR7 expression. Knockdown of CsTLR7 significantly enhanced bacterial dissemination in the tissues of tongue sole. Treatment of tongue sole with the imidazoquinoline compound R848 (TLR7 activator) and the endosomal acidification inhibitor chloroquine (TLR7 inhibitor) caused enhanced and reduced resistance against bacterial infection respectively. These results indicate that CsTLR7 plays an essential role in the antibacterial immunity of tongue sole. In this study, we examined the expression patterns and the functions of the tongue sole Cynoglossus semilaevis CD94, CsCD94. CsCD94 is composed of 209 amino acid residues and shares 43.0–50.2% overall identities with known teleost CD94 sequence. CsCD94 has a C-type lectin-like domain. Expression of CsCD94 occurred in multiple tissues and was upregulated during bacterial infection. Recombinant CsCD94 (rCsCD94) exhibited apparent binding and agglutinating activities against both Gram-positive and Gram-negative bacteria in a Ca²⁺-dependent manner. Treatment of bacteria with rCsCD94 enhanced phagocytosis of the bacteria by peripheral blood leukocytes. Furthermore, incubation of rCsCD94 with bacteria reduced the survival of the bacteria in vitro. Taken together, these results indicate that rCsCD94 is a key factor in the bactericidal and phagocytic effects of tongue sole, and reveal for the first time an essential role of fish CD94 in antibacterial immunity, thereby adding insight into the function of CD94. Complement factor B (Bf) is a component of the complement system. Following activation of the alternative pathway of the complement system, factor B is cleaved into Ba and Bb fragments. In fish, the functions of Bf and Ba are essentially scarce. In this study, we examined the expression patterns of tongue sole Cynoglossus semilaevis Bf (named CsBf) and the biological activity of the Ba fragment of CsBf (named CsBa). CsBf possesses the conserved domains of Bf and shares 40.3%-56.5% sequence identities with other fish Bf. CsBf expression was high in liver, muscle, and heart, and low in intestine, blood, and kidney. Bacterial infection significantly induced CsBf expression in kidney, spleen, and liver in a time-dependent manner. Recombinant CsBa (rCsBa) exhibited apparent binding capacities to bacteria and tongue sole peripheral blood leukocytes, and binding of rCsBa to bacteria inhibited bacterial growth. When overexpressed in tongue sole, CsBa significantly reduced bacterial dissemination in fish tissues. Together these results indicate for the first time that a fish Ba possesses antibacterial effect as well as immune cell-binding capacity, and thus probably plays a role in host immune defense against bacterial infection.
学科领域	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/337002/136547
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	李学鹏. 半滑舌鳎（Cynoglossus semilaevis）先天性免疫基因TLR2、TLR7、CD94和Bf的功能研究[D]. 北京. 中国科学院大学,2017.

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