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半滑舌鳎 (Cynoglossus semilaevis) 中性粒细胞胞外陷阱抗 菌组分研究
温丽联
Subtype硕士
Thesis Advisor孙黎
2018-05
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name农学硕士
Degree Discipline水产养殖
Keyword中性粒细胞胞外陷阱 组蛋白 弹性蛋白酶 半滑舌鳎 抗菌作用
Abstract

中文:中性粒细胞胞外陷阱(neutrophil extracellular traps, NETs)是中性粒细胞产生的一种胞外网状结构,在防御病原入侵中起重要作用。NETs在高等脊椎动物中通过捕获和局部高浓度抗菌蛋白(组蛋白、弹性蛋白酶等)杀伤病原菌。在低等脊椎动物如硬骨鱼类中都能观察到NETs现象,但是鱼类NETs的抑菌机制尚不清楚。在本文中,我们研究了组蛋白和弹性蛋白酶在半滑舌鳎(Cynoglossus semilaevisNETs抗菌过程中的作用。为此,我们首先从半滑舌鳎中克隆了两种组蛋白(CsH2BCsH4)和两种弹性蛋白酶(CsEla1CsEla2)的基因。序列分析表明,CsH2BCsH4分别具有组蛋白H2B H4的保守结构域,CsEla1CsEla2则具有胰蛋白酶样丝氨酸蛋白酶的保守结构域。我们在大肠杆菌中表达、纯化了上述四个半滑舌鳎蛋白,获得了重组蛋白rCsH2BrCsH4rCsEla1rCsEla2。细菌结合实验表明,这四个重组蛋白对革兰氏阴性细菌和革兰氏阳性细菌均具有广泛的结合能力,且在结合后对其中一些细菌的生长具有显著的抑制作用。免疫荧光显微镜研究发现,CsH2BCsH4CsEla1CsEla2均定位于细胞所分泌的NETs中。抗体封闭实验发现,当用抗CsH2BCsH4CsEla1CsEla2特异性抗体封闭NETs后,NETs的抗微生物作用被显著降低。以上研究结果表明,组蛋白(CsH2BCsH4)和弹性蛋白酶(CsEla1CsEla2)是硬骨鱼类NETs的重要组成成分,它们在NETs参与的抗微生物免疫过程中发挥着重要的作用。

英文:Neutrophil extracellular traps (NETs) are a form of extracellular bacterial killing structure of neutrophils observed in higher and lower vertebrates, the latter including the teleost fish tongue sole Cynoglossus semilaevis. Histone and elastase are main components of NETs presence in high local concentrations which enables NETs to kill microorganisms in higher vertebrates. However, the bactericidal mechanism of fish NETs is unknown. In the present study, we examined the potential contribution of histones and elastases to the antibacterial effect of tongue sole NETs. For this purpose, two histones (CsH2B and CsH4) and two elastases (CsEla1 and CsEla2) were cloned from tongue sole and investigated. The histones and elastases possess the conserved domain structures characteristic of that of histones H2B/H4 and trypsin-like serine protease, respectively. Recombinant CsH2B, CsH4, CsEla1, and CsEla2 bound a wide range of Gram-negative and Gram-positive bacteria, and some of the bound bacteria were inhibited in growth by the bound proteins. CsH2B, CsH4, CsEla1, and CsEla2 were all localized in NETs induced by various stimuli including bacterial pathogen. Treatment of NETs with antibodies targeting CsH2B, CsH4, CsEla1, and CsEla2 significantly reduced the antimicrobial effect of NETs. These results indicate that histones and chymotrypsin-like elastases are components of teleost NETs that contribute to the antimicrobial activity of NETs.

Subject Area水产学
MOST Discipline Catalogue农学 ; 农学::水产
Table of Contents

... I

Abstract II

第一章 绪论... 1

1.1 鱼类免疫系统概论... 1

1.1.1 鱼类免疫细胞... 1

1.2 中性粒细胞... 4

1.2.1 颗粒和脱颗粒... 5

1.2.2 中性粒细胞中的抗菌蛋白... 6

1.2.3 呼吸爆发... 9

1.2.4 吞噬... 9

1.3 中性粒细胞胞外陷阱(NETs... 10

1.3.1 NETs的形成机制... 10

1.3.2 NETs的抗微生物作用及机制... 13

1.3.3 组蛋白... 14

1.3.4 弹性蛋白酶... 15

1.4 鱼类NETs的研究现状... 17

1.5 本论文的研究目的与意义... 17

第二章 实验材料... 21

2.1 实验动物与实验菌株... 21

2.1.1 实验动物... 21

2.1.2 实验菌株... 21

2.2 实验试剂... 22

2.3 实验仪器和设备... 23

第三章 实验方法... 25

3.1 序列分析... 25

3.2 半滑舌鳎总RNA提取及cDNA合成... 25

3.3 重组蛋白原核表达载体的构建... 27

3.4 重组蛋白的表达和纯化... 33

3.5 重组蛋白多克隆抗体的制备及效价检测... 35

3.6 重组蛋白对细菌的结合作用... 37

3.7 免疫荧光检测重组蛋白对细菌的结合作用... 37

3.8 重组蛋白对细菌增殖的影响... 38

3.9 半滑舌鳎中性粒细胞的提取及中性粒细胞胞外陷阱(NETs)的产生... 38

3.10 显微镜观察组蛋白和弹性蛋白酶与NETs共定位... 40

3.11 抗体封闭NETs对细菌活性的影响... 40

第四章 实验结果... 43

4.1 半滑舌鳎两个组蛋白和两个弹性蛋白序列分析... 43

4.2 rCsH2BrCsH4对革兰氏阳性和革兰氏阴性细菌的结合能力... 45

4.3 rCsH2BrCsH4对细菌生长的影响... 48

4.4 rCsEla1rCsEla2对革兰氏阳性和革兰氏阴性细菌的结合... 50

4.5 rCsEla1 rCsEla2对细菌生长的影响... 52

4.6组蛋白和弹性蛋白与NETs的共定位... 53

4.7 抗体封闭组蛋白和弹性蛋白酶后NETs对细菌生长的影响... 55

第五章 讨论... 57

第六章 结论... 59

参考文献... 61

... 75

... 77

作者简历及攻读硕士期间发表的学术论文与研究成果... 78

Pages78
DOI10.1016/j.fsi.2017.11.004
URL查看原文
Funding ProjectNational Natural Science Foundation of China[31530079]
Language中文
Citation statistics
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/154515
Collection实验海洋生物学重点实验室
First Author Affilication中国科学院海洋研究所;  实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
温丽联. 半滑舌鳎 (Cynoglossus semilaevis) 中性粒细胞胞外陷阱抗 菌组分研究[D]. 中国科学院海洋研究所. 中国科学院大学,2018.
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