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半滑舌鳎半乳糖凝集素-8的免疫功能研究
张腾飞
Subtype硕士
Thesis Advisor孙黎
2021-05-17
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name工程硕士
Degree Discipline生物工程
Keyword半滑舌鳎 半乳糖凝集素-8 细菌结合 杀菌 免疫防御
Abstract

半乳糖凝集素(galectin)是一种对β-半乳糖苷(β-galactosides)具有高亲和力的凝集素。在哺乳动物中已发现半乳糖凝集素具有多种生物学功能。在鱼类中发现半乳糖凝集素是重要的模式识别受体,能够识别多种病原体。然而对鱼类半乳糖凝集素的其他免疫功能的研究还不多。我们从半滑舌鳎(Cynoglossus semilaevis)基因组中鉴定了galectin-8(CsGal-8),对其免疫功能进行了研究。

CsGal-8的开放阅读框含有939 bp核苷酸,编码312个氨基酸。CsGal-8属于串联重复型半乳糖凝集素,含有两个糖识别结构域(carbohydrate recognition domains,CRDs),由48个氨基酸残基的多肽链相连接。CsGal-8与其他鱼类galectin-8序列相似性在~70%-80%之间。CsGal-8在多种组织中表达分布,在肾和脾等免疫相关器官中表达水平很高。迟缓爱德华氏菌(Edwardsiella tarda)刺激后,CsGal-8在半滑舌鳎肾、脾和血液中表达水平显著上调。亚细胞定位分析发现CsGal-8主要分布在细胞质中,并能够以囊泡的形式被分泌到细胞外。CsGal-8的重组蛋白(rCsGal-8)对乳糖、半乳糖、果糖和海藻糖具有非常高的亲和性,并且rCsGal-8与糖的亲和性与rCsGal-8的剂量呈正相关。rCsGal-8能够广泛的结合革兰氏阳性菌和革兰氏阴性菌,尤其是对鳗弧菌(Vibrio anguillarum)和哈维氏弧菌(Vibrio harveyi)有很强的结合能力。用鳗弧菌侵染过表达CsGal-8的RAW264.7细胞发现CsGal-8能够与侵入细胞内的鳗弧菌共定位。rCsGal-8与细菌的结合能够被糖所抑制。rCsGal-8能够对三种革兰氏阴性菌(鳗弧菌、哈维氏弧菌和大肠杆菌Escherichia coli)造成直接的杀伤作用。rCsGal-8能够对鳗弧菌的细胞膜造成严重破坏,并使细菌的形态发生改变。此外,rCsGal-8能够抑制外周血白细胞产生活性氧(ROS)。

本论文的研究结果表明CsGal-8在半滑舌鳎的免疫识别、抗细菌感染以及免疫调控过程中发挥重要作用。在鱼类中首次发现半乳糖凝集素具有直接的杀菌作用。研究结果为利用凝集素防控鱼类病害提供了新的思路。

Other Abstract

Galectins are a family of lectins with a high affinity for β-galactosides. In mammals, galectins have been found to have multiple biological functions. In fish, galectins have been found to be important pattern recognition receptors and recognize a wide range of pathogens. However, other immune functions of fish galectins have rarely been reported. In the present study, we identified a galectin-8 gene from genome of Cynoglossus semilaevis (designated CsGal-8), and investigated the immune functions of CsGal-8.

The open reading frame of CsGal-8 contains 939 bp nucleotides, which encodes 312 amino acids. CsGal-8 belongs to the tandem repeat type of galectins which has two carbohydrate recognition domains (CRDs) connected by a linker peptide of 48 amino acids residues. The amino acids sequence similarities between CsGal-8 and the galectin-8 of other teleost species range from ~70% to ~80%. CsGal-8 mRNA was constitutively expressed in many tissues and highly expressed in kidney and spleen, which are immune associated organs. Expressions of CsGal-8 mRNA in kidney, spleen and blood were up-regulated after Edwardsiella tarda challenge. Subcellular distribution analysis showed that CsGal-8 was mainly distributed in the cytoplasm, and CsGal-8 could be secreted into the extracellular milieu in the form of microvesicles. Recombinant CsGal-8 (rCsGal-8) showed high affinity for lactose, galactose, glucose and trehalose. The affinity of rCsGal-8 to carbohydrates had a positive relation with the dose of rCsGal-8. rCsGal-8 bound to a wide range of gram-positive and gram-negative bacteria. The binding ability of rCsGal-8 toward Vibrio harveyi and Vibrio anguillarum was especially strong. Colocalization of CsGal-8 with V. anguillarum was observed in RAW264.7 cells expressing CsGal-8 and infected with V. anguillarum. Binding of rCsGal-8 to bacteria could be inhibited by carbohydrates. rCsGal-8 exhibited potent bactericidal ability against three gram-negative bacteria (V. harveyi, V. anguillarum and Escherichia coli). rCsGal-8 disrupted the membrane integrity of V. anguillarum, and the morphology of V. anguillarum treated with rCsGal-8 changed greatly. In addition, rCsGal-8 inhibited reactive oxygen species (ROS) production in peripheral blood lymphocytes.

These results indicated that CsGal-8 plays an important role in immune recognition, antibacterial infection and immune regulation processes. Galectins have been found to have direct bactericidal activities in fish for the first time. The results provide new insights for controlling fish diseases with lectins.

Language中文
Table of Contents

第 1 章 绪论..............................................................................................................1

1.1 半乳糖凝集素.....................................................................................................1

1.1.1 半乳糖凝集素定义与分类..........................................................................1

1.1.2 半乳糖凝集素的结构..................................................................................2

1.1.3 半乳糖凝集素与糖的结合机制..................................................................3

1.1.4 半乳糖凝集素的组织分布..........................................................................4

1.1.5 半乳糖凝集素的分泌..................................................................................4

1.1.6 半乳糖凝集素的功能..................................................................................5

1.2 半滑舌鳎简介.....................................................................................................8

1.3 半乳糖凝集素在鱼类中的研究现状.................................................................8

1.4 研究目的及意义.................................................................................................9

第 2 章 半滑舌鳎半乳糖凝集素-8 的序列分析..........................................11

2.1 实验材料与方法...............................................................................................11

2.2 实验结果...........................................................................................................12

2.2.1 半滑舌鳎半乳糖凝集素-8 序列分析 .......................................................12

2.2.2 序列比对....................................................................................................13

2.2.3 三维结构预测............................................................................................14

2.3 讨论...................................................................................................................15

第 3 章 半滑舌鳎半乳糖凝集素-8 的组织表达与亚细胞定位..............17

3.1 实验材料与方法...............................................................................................17

3.1.1 实验动物....................................................................................................17

3.1.2 实验细胞....................................................................................................17

3.1.3 实验菌株....................................................................................................17

3.1.4 实时荧光定量 PCR...................................................................................18

3.1.5 真核细胞过表达载体构建........................................................................22

3.1.6 CsGal-8 在细胞中的定位分析 .................................................................27

3.2 实验结果...........................................................................................................29

3.2.1 CsGal-8 的组织表达分布 ..........................................................................29

3.2.2 CsGal-8 的亚细胞定位 ..............................................................................31

3.3 讨论...................................................................................................................32

第 4 章 半滑舌鳎半乳糖凝集素-8 的免疫功能研究.................................35

4.1 实验材料与方法...............................................................................................35

4.1.1 实验细胞....................................................................................................35

4.1.2 实验菌株....................................................................................................35

4.1.3 重组蛋白的表达与纯化............................................................................35

4.1.4 酶联免疫吸附试验....................................................................................37

4.1.5 CsGal-8 与侵入细胞内细菌的共定位分析 ..............................................39

4.1.6 流式细胞术检测细胞对细菌的吞噬........................................................39

4.1.7 重组蛋白对细菌的杀伤能力检测............................................................40

4.1.8 碘化丙啶染色分析....................................................................................41

4.1.9 电子显微镜分析........................................................................................41

4.1.10 ROS 测定..................................................................................................41

4.2 实验结果...........................................................................................................42

4.2.1 rCsGal-8 的 SDS-PAGE 分析....................................................................42

4.2.2 rCsGal-8 与糖的结合.................................................................................43

4.2.3 rCsGal-8 与细菌的结合.............................................................................44

4.2.4 CsGal-8 与鳗弧菌在细胞中共定位 ..........................................................46

4.2.5 rCsGal-8 对细菌的杀伤作用.....................................................................47

4.2.6 rCsGal-8 对革兰氏阳性菌调理作用的检测.............................................51

4.2.7 rCsGal-8 对外周血白细胞产生活性氧的影响.........................................52

4.3 讨论:...............................................................................................................54

结论............................................................................................................................59

参考文献...................................................................................................................61

附录 本研究所用溶液配制方法.......................................................................71

致谢............................................................................................................................75

作者简历及攻读学位期间发表的学术论文与研究成果..........................77

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/170642
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
张腾飞. 半滑舌鳎半乳糖凝集素-8的免疫功能研究[D]. 中国科学院海洋研究所. 中国科学院大学,2021.
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