半滑舌鳎（Cynoglossus semilaevis）非特异性免疫相关基因SAP和PTX1的克隆、表达和功能研究

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	半滑舌鳎（Cynoglossus semilaevis）非特异性免疫相关基因SAP和PTX1的克隆、表达和功能研究
	王婷
学位类型	硕士
导师	孙黎
	2016-05-19
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	生物工程
关键词	半滑舌鳎非特异性免疫血清淀粉样蛋白p成分正五聚蛋白-1 细菌凝集作用抗菌作用抗病毒作用
其他摘要	非特异性免疫是机体抵抗外源病原菌入侵的第一道防线，同时在清除病原菌、修复损伤组织、激活和调节特异性免疫应答反应等方面发挥着重要作用。非特异性免疫系统由细胞免疫和体液免疫组成，体液免疫系统中最重要的组成成分是能识别病原体相关分子模式的体液模式识别分子。在哺乳动物中，血清淀粉样P成分（serum amyloid P component, SAP）和正五聚蛋白-1（pentraxin-1, PTX1）作为经典的体液模式识别分子，拥有进化上保守的功能，如激活和调控补体级联反应，发挥凝集、中和和调理作用，调节炎症反应等。然而，目前有关鱼类SAP和PTX1的功能研究十分有限，特别是免疫功能研究。在本论文中，我们克隆了半滑舌鳎SAP（CsSAP）和PTX1（CsPTX1）基因，分析了它们在mRNA水平上的表达变化情况，并检测了CsSAP和CsPTX1重组蛋白（rCsSAP和rCsPTX1）的抗细菌和抗病毒免疫作用。 CsSAP的开放性阅读框有672个碱基，共编码224个氨基酸。CsSAP与已知鱼类的SAP样蛋白有46％-58％的序列相似性，且在细菌和病毒感染的情况下表达量显著上调。功能研究结果表明，rCsSAP能结合多种革兰氏阳性和革兰氏阴性细菌，且这种结合活性有蛋白浓度依赖性和菌种特异性。体外实验发现rCsSAP结合细菌后，能显著增强宿主吞噬细胞的吞噬活性。体内组织侵染实验发现，rCsSAP能有效帮助宿主抵抗细菌和病毒感染。与此相反，特异性抗体封闭宿主体内天然的CsSAP后，半滑舌鳎清除入侵细菌的能力显著降低。这些结果首次证明了鱼类的SAP蛋白在宿主抗细菌和抗病毒免疫反应中发挥着重要作用。 CsPTX1蛋白包含223个氨基酸，与其它已知鱼类的正五聚蛋白有38.8％- 49.3％的序列相似性。正常生理条件下，CsPTX1在多个组织中均有表达，且在细菌和病毒感染的情况下表达量显著上调。由于CsPTX1包含一个能结合胞外抗原的正五聚蛋白结构域，因此rCsPTX1能与多种革兰氏阳性和革兰氏阴性细菌相互作用。同时，rCsPTX1也能以钙依赖的方式使细菌发生凝集。体外实验发现，rCsPTX1对细菌包膜的渗透性有一定的影响，进而抑制荧光假单胞菌的生长和增殖。体内组织侵染实验发现，rCsPTX1能有效帮助宿主抵抗细菌感染。上述结果表明，CsPTX1作为经典的模式识别分子，能促进半滑舌鳎抗细菌感染，在宿主非特异性免疫反应中发挥着重要作用。; Innate immunity is the first line of defense against pathogens and plays an important role in pathogen clearance in vertebrates. It also affects the activation and orientation of adaptive immunity and the maintenance of tissue integrity and repair. The innate immune system comprises a cellular and a humoral arm, components of the latter including humoral pattern recognition molecules that recognize pathogen associated molecular patterns. As classical humoral pattern recognition molecules, in mammals, Serum amyloid P component (SAP) and pentraxin-1 (PTX1), share basic, evolutionarily conserved functions such as opsonization, complement activation, agglutination, neutralization, and regulation of inflammation. However, research on the function of fish SAP and PTX1 is very limited, in particular that concerning immunology. In this study, we cloned the tongue sole SAP (CsSAP) and PTX1 (CsPTX1), analyzed their expression and examined the potential of recombinant CsSAP (rCsSAP) and CsPTX1 (rCsPTX1) in antibacterial and antiviral immunity. The open reading fream of CsSAP contains 672 bp which encoded a polypeptide of 224 amino acids. CsSAP shares 46%-58% overall sequence identities with known fish SAP and was upregulated in expression by bacterial and viral infection. rCsSAP exhibited differential binding capacities to a wide range of Gram-positive and Gram-negative bacteria and promoted uptake of the bound bacteria by host phagocytes. When introduced in vivo, rCsSAP enhanced host resistance not only to bacterial infection but also to viral infection. Consistently, antibody blockage of CsSAP significantly weakened the ability of tongue sole to clear invading bacteria. These results provide the first evidence that fish SAP contributes significantly to both antibacterial and antiviral immunities. CsPTX1 contains 223 amino acids and shares 38.8% - 49.3% overall sequence identity with other known fish pentraxins. CsPTX1 is expressed in multiple tissues and is upregulated by bacterial and viral infection. CsPTX1 contains a pentraxin domain, which is known to bind extracellular antigens, and recombinant CsPTX1 bound a wide range of Gram-positive and Gram-negative bacteria. rCsPTX1 also agglutinated all the bacteria tested in a Ca²⁺-dependent manner and the agglutinating capacity of rCsPTX1 was abolished in the absence of calcium. As well as its ability to agglutinate bacterial cells, rCsPTX1 displayed apparent bacteriostatic activity against Pseudomonas fluorescens in vitro by influencing the permeability of the microbial envelope. When introduced in vivo, rCsPTX1 enhanced the host’s resistance to bacterial infection. These results indicate that CsPTX1 is a classic pattern recognition molecule that defends Cynoglossus semilaevis against bacterial infection.
学科领域	地球科学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/112544
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
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	王婷. 半滑舌鳎（Cynoglossus semilaevis）非特异性免疫相关基因SAP和PTX1的克隆、表达和功能研究[D]. 北京. 中国科学院大学,2016.

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