实验海洋生物学重点实验室知识库

NLRs是一类细胞内模式识别受体，可识别病原相关分子模式（pathogen-associated molecular patterns, PAMPs）或损伤相关分子模式（damage-associated molecular patterns, DAMPs），在动物的先天免疫中发挥着重要作用。在低等无脊椎动物中，NLR家族基因发生明显扩张，其编码蛋白的结构域组成呈现多样化。目前对于无脊椎动物NLR家族基因免疫功能的了解非常有限。本论文通过分析凡纳滨对虾（Litopenaeus vannamei）组学数据鉴定了16条NLR家族基因，选取其中3条结构域组成及时空表达特征具有代表性的基因，开展了免疫学功能及作用机制研究。论文的主要进展如下：

1. 凡纳滨对虾NLR家族基因（LvNLRs）的鉴定及表达特征分析。从凡纳滨对虾组学数据中获得了16条NLR家族基因，序列特征分析显示，它们均可编码NLR蛋白中保守的NACHT结构域。其中一条基因编码氨基酸序列的C端含有LRRs结构域，而其他基因编码氨基酸序列的C端和N端均不含已知功能的结构域。对不同动物中NLR家族蛋白的NACHT结构域进行系统发育分析，结果表明，编码C端LRRs结构域的NLR基因属于NLRC亚家族，其与低等动物的NLRs聚在一起；而另外15条基因则属于NLRP亚家族，它们单独聚为一支。组织表达分析显示，对虾NLR家族基因大多在血细胞和淋巴器官（Oka）等免疫相关组织中呈现高表达。病原感染后，大多数基因的表达呈现相同的表达趋势。在Oka中，多数LvNLRs基因在WSSV或副溶血弧菌感染后的病原快速增殖期显著上调；而在血细胞中，多个LvNLRs基因在WSSV感染后期显著上调。基于序列特征和表达分析结果，选取3条有代表性的基因LvNLRPL1、LvNLRPL2和LvNLRC进行了免疫学功能和作用机制研究。

2. LvNLRPL1与LvNLRPL2的免疫学功能及作用机制研究。LvNLRPL1与LvNLRPL2为非典型的NLR，仅具有NACHT结构域。LvNLRPL1和LvNLRPL2均在血细胞中呈现高表达；在副溶血弧菌感染后3~24 h的Oka或WSSV感染晚期阶段的血细胞中，其表达量均发生了明显的变化。将LvNLRPL1或LvNLRPL2进行敲降后用副溶血弧菌感染对虾，与对照组相比，对虾体内弧菌的繁殖速度和对虾的死亡率均显著增加，然而当用WSSV感染对虾时，其体内的病毒增殖明显受到抑制，表明LvNLRPL1和LvNLRPL2在对虾应对弧菌和WSSV感染中的功能可能存在差异。当LvNLRPL1或LvNLRPL2被敲降后，对虾血细胞中凋亡相关基因LvCaspase2、3、5的转录表达水平显著上调，且对虾中凋亡血细胞的比例明显增加，表明它们可调控对虾血细胞的凋亡过程。免疫共沉淀分析发现，LvNLRPL1的NACHT结构域可形成同型二聚体，而LvNLRPL2的NACHT结构域则不能形成同型二聚体，但可以与LvNLRPL1通过其NACHT结构域形成异型二聚体。这与已报道的有关哺乳动物中NLRs通过形成同型或异型寡聚体实现对配体的识别从而激活下游信号通路的功能作用方式类似。以上结果表明，对虾中这两种NLRs可通过异型或同型聚合来调控血细胞的凋亡过程，进而在细菌或WSSV感染对虾的过程中发挥功能。

3. LvNLRC基因的免疫学功能及作用机制研究。LvNLRC是对虾中已鉴定NLR家族中唯一在C端含有已知结构域LRRs的蛋白。LvNLRC在组织中呈泛表达的特征，且明显响应副溶血弧菌和WSSV的感染。与LvNLRPL1和LvNLRPL2相似，LvNLRC的敲降可引起对虾体内弧菌载量的明显升高或使WSSV在对虾体内的增殖速度明显减慢。LvNLRC的C端LRRs结构域体外重组蛋白对革兰氏阴性菌的PAMP脂多糖（LPS）和DNA病毒核酸模拟物poly(dA:dT)均具有结合活性。免疫共沉淀分析发现LvNLRC既可与自身形成同型二聚体，也可与LvNLRPL1形成异型二聚体，但不能与LvNLRPL2发生异型聚合。但与LvNLRPL1和LvNLRPL2不同的是，LvNLRC的敲降不会引起血细胞的凋亡。由此推测，LvNLRC不直接调控抗血细胞凋亡过程，而是发挥传感器作用，在识别PAMPs后通过异型聚合激活LvNLRPL1-LvNLRPL2复合物及其介导的血细胞凋亡过程，在细菌或病毒感染对虾的过程中参与重要的免疫功能。此外，通过酵母双杂交筛选及免疫共沉淀分析，发现LvNLRC可通过其NACHT结构域与亲环素A（cyclophilin A，CypA）结合。LvCypA敲降后的对虾被WSSV感染后，病毒在对虾体内的增殖也明显受到抑制。进一步的作用机制分析发现，LvNLRC或LvCypA的敲降均可上调对虾的类干扰素基因LvVago5的表达。同时，LvNLRC的NACHT结构域还与对虾类干扰素转录激活通路中的LvSTING具有直接相互作用，表明其对类干扰素途径的调控是直接通过LvSTING实现的。以上结果表明，LvNLRC作为对虾中一个新的病毒核酸模式识别受体，不仅能够通过激活非典型NLR介导的信号通路促进病毒感染，也可以通过与LvCypA和LvSTING互作参与类干扰素基因的表达调控，从而在WSSV感染过程中发挥免疫调控作用。

本研究首次揭示了甲壳动物NLRs家族基因在细菌或病毒感染过程中的免疫学功能及其作用机制，即LvNLRPL1和LvNLRPL2复合物通过协同调控血细胞的凋亡过程参与细菌或病毒的感染过程；而具有LRRs结构域的LvNLRC既可以识别细菌或病毒的PAMPs，也可与LvNLRPL1聚合参与其调控的细胞凋亡过程或通过与CypA/STING互作直接调控抗病毒类干扰素途径。本研究不但明确了无脊椎动物非典型NLRs抗病原感染作用机制，还首次发现了CypA与NLR直接互作，调控STING-IRF抗病毒干扰素途径。研究结果丰富了无脊椎动物先天免疫的基础理论，为了解NLRs的起源与进化提供了新的线索和分子证据，也为对虾的病害防治提供了新思路。

NLRs are a class of cytoplasmic pattern recognition receptors. They can recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and play important roles in innate immunity of animals. In lower invertebrates, NLR family genes are significantly expanded and the domain-composition of their encoding proteins is diversified. However, the current knowledge about the immune function of NLRs in invertebrates is very limited. In this study, sixteen NLR family genes were identified based on the omics data analysis of Litopenaeus vannamei. Three genes with representative domain-composition and spatio-temporal expression profiles were selected for the immunological functional and mechanistic study. The main research progress of this study is as follows:

1. Identification and expression characterization of NLR family genes in L. vannamei (LvNLRs). Sixteen NLR family genes were obtained from the omics data of L. vannamei. Sequence characteristic analysis showed that they all encoded the conserved NACHT domain of NLRs. One of these genes encoded the C-terminal LRRs domain, while the C-terminal and N-terminal amino acid sequences of the other 15 proteins did not contain domains with known functions. The phylogenetic analysis of the NACHT domains in NLR family proteins from different animals showed that the protein containing the C-terminal LRRs domain belonged to the NLRC subfamily and was clustered with NLRs of lower species, while the other 15 proteins belonged to the NLRP subfamily and were clustered into a single branch. Tissue distribution analysis revealed that most of the LvNLRs were highly expressed in the immune-related tissues, such as hemocytes and lymphoid organ (Oka). Most LvNLRs showed a similar expression profile after pathogens infection. In Oka, most of the LvNLRs were significantly up-regulated during the rapid proliferation stage of WSSV or Vibrio parahaemolyticus infection. In hemocytes, several LvNLRs were significantly up-regulated during the late stage of WSSV infection. Based on their sequence and expression characteristics, three representative genes, including LvNLRPL1, LvNLRPL2 and LvNLRC, were selected for the study on their immunological functions and functioning mechanisms.

2. Study on the immunological functions and functioning mechanisms of LvNLRPL1 and LvNLRPL2. LvNLRPL1 and LvNLRPL2 are atypical NLRs only containing NACHT domains. Both LvNLRPL1 and LvNLRPL2 were highly expressed in shrimp hemocytes. The expression levels of LvNLRPL1 and LvNLRPL2 were both significantly changed in Oka during 3~24 h post V. parahaemolyticus infection or in hemocytes during the late stage of WSSV infection. Compared to the control group, knockdown of LvNLRPL1 or LvNLRPL2 could markedly increase the in vivo Vibrio proliferation and the mortality of shrimp infected with V. parahaemolyticus, whereas inhibit in vivo virus propagation of shrimp infected with WSSV, indicating their different functions in shrimp responding to different pathogens. After LvNLRPL1 or LvNLRPL2 knockdown, the expression levels of apoptosis-related genes, including LvCaspase2, 3 and 5, in shrimp hemocytes were significantly up-regulated, and the apoptosis rate of shrimp hemocytes was significantly increased, suggesting that LvNLRPL1 or LvNLRPL1 could regulate the apoptosis process of shrimp hemocytes. The co-immunoprecipitation assay revealed that the NACHT domain of LvNLRPL1 could form homo-dimers, while the NACHT domain of LvNLRPL2 could not form homo-dimers, instead of forming hetero-dimers with the NACHT domain of LvNLRPL1. This interaction mechanism is similar to the reported way that mammalian NLRs recognize ligands and activate downstream signaling pathways through forming homo- or hetero-oligomers. The results suggest that shrimp NLRs could regulate the apoptosis process of hemocytes through heterotypic or homotypic aggregation, thus exerting immune functions in shrimp during Vibro or WSSV infection.

3. Study on the immunological function and functioning mechanism of LvNLRC. LvNLRC is the only member containing the C-terminal LRRs domain in all of the identified LvNLRs. LvNLRC was ubiquitously expressed in shrimp tissues and was significantly responsive to V. parahaemolyticus and WSSV infection. Similar to LvNLRPL1 and LvNLRPL2, knockdown of LvNLRC could markedly increase the in vivo Vibrio proliferation in shrimp infected with V. parahaemolyticus or significantly inhibit the in vivo virus proliferation rate in shrimp infected with WSSV. The in vitro recombinant protein of C-terminal LRRs domain in LvNLRC had binding activity to lipopolysaccharide (LPS) or poly(dA:dT), which were the PAMP of Gram-negative bacteria or the nucleic acid mimic of DNA virus, respectively. Co-immunoprecipitation assay found that LvNLRC could form homo-dimers with itself or hetero-dimers with LvNLRPL1, but could not aggregate with LvNLRPL2. However, unlike LvNLRPL1 and LvNLRPL2, knockdown of LvNLRC did not induce apoptosis of shrimp hemocytes. Therefore, it is speculated that LvNLRC did not directly regulate the anti-apoptotic process of hemocytes, but acted as a sensor and activate the LvNLRPL1-LvNLRPL2 complex and their mediated hemocytes apoptotic process through hetero-polymerizing with LvNLRPL1 after PAMPs recognition, thus participating in important immune functions during the bacterial or WSSV infection process of shrimp. In addition, yeast two-hybrid screening and co-immunoprecipitation assay showed that LvNLRC could interact with shrimp cyclophilin A (CypA) through its NACHT domain. Knockdown of LvCypA also inhibited in vivo WSSV propagation in shrimp. Further mechanism analysis found that knockdown of LvNLRC or LvCypA could up-regulate the expression level of the shrimp interferon-like gene LvVago5. Meanwhile, the NACHT domain of LvNLRC could also interact directly with LvSTING, a regulatory molecule in the shrimp interferon-like pathway, indicating that LvNLRC regulated the interferon-like pathway directly through LvSTING. These results suggest that LvNLRC, act as a novel pattern recognition receptor in shrimp for viral nucleic acid, could not only promote viral infection by activating the pathway mediated by atypical NLR, but also participate in the regulation of interferon-like gene expression by interacting with LvCypA and LvSTING, thus playing an immune regulatory role in WSSV infection.

This study revealed for the first time the immunological functions and functioning mechanisms of crustacean NLRs family genes during bacterial or viral infection. The complexes formed by LvNLRPL1 and LvNLRPL2 participate in bacterial or viral infection by synergistically regulating the apoptosis process of hemocytes, while LvNLRC with LRRs domain can not only recognize bacterial or viral PAMPs, but also participate in the apoptosis process regulated by LvNLRPL1 by polymerizing with LvNLRPL1 or directly regulate the antiviral interferon-like pathway by interacting with CypA/STING. This study not only clarified the mechanism of resistance to pathogenic infection of atypical invertebrate NLRs, but also discovered for the first time that CypA directly interacted with NLR to regulate the STING-IRF antiviral interferon pathway. The results of this research have enriched the fundamental theory of invertebrate innate immunity, and provided new clues and molecular evidence for understanding the origin and evolution of NLRs, as well as new ideas for disease control of shrimp.

第一章  绪论... 1

1.1  先天免疫系统... 1

1.1.1  先天免疫系统概述... 1

1.1.2  体液免疫... 1

1.1.3  细胞免疫... 2

1.2  模式识别受体... 3

1.2.1  模式识别受体的类型... 4

1.2.2  无脊椎动物的核酸识别受体... 5

1.3  脊椎动物NLRs的分类与功能... 7

1.3.1  NLRs的结构与分类... 7

1.3.2  NLRs的免疫功能... 9

1.4  无脊椎动物NLRs的研究进展... 14

1.4.1  无脊椎动物NLRs的结构与分类... 14

1.4.2  无脊椎动物NLRs的免疫功能研究现状... 18

1.5  本研究的目的和意义... 20

第二章  凡纳滨对虾NLR家族基因的鉴定与序列分析... 21

2.1  引言... 21

2.2  材料与方法... 21

2.2.1  实验动物... 21

2.2.2  序列鉴定及分析... 21

2.2.3  组织分布分析... 24

2.2.4  病原感染及组织取样... 24

2.2.5  总RNA提取... 26

2.2.6  cDNA合成... 26

2.2.7  实时荧光定量PCR.. 27

2.2.8  统计分析... 28

2.2.9  引物列表... 28

2.3  结果... 30

2.3.1  LvNLRs的序列鉴定和系统发育分析... 30

2.3.2  LvNLRs的组织表达分析... 34

2.3.3  LvNLRs对不同病原感染的响应... 35

2.4  讨论... 37

第三章  凡纳滨对虾LvNLRPL1基因的免疫学功能研究... 41

3.1  引言... 41

3.2  材料与方法... 41

3.2.1  实验动物... 41

3.2.2  取样与组织分布检测... 41

3.2.3  序列特征分析... 41

3.2.4  总RNA提取与cDNA合成... 42

3.2.5  序列获取与验证... 43

3.2.6  双链RNA合成及最优干扰剂量筛选... 45

3.2.7  LvNLRPL1敲降与细菌感染实验... 49

3.2.8  LvNLRPL1敲降对虾血细胞凋亡相关实验... 51

3.2.9  统计分析... 53

3.2.10  引物列表... 53

3.3  结果... 54

3.3.1  LvNLRPL1基因的序列特征分析... 54

3.3.2  LvNLRPL1基因的组织表达分析... 57

3.3.3  LvNLRPL1基因对副溶血弧菌感染的响应特征分析... 57

3.3.4  LvNLRPL1基因沉默对副溶血弧菌感染对虾的影响... 58

3.3.5  LvNLRPL1基因对血细胞凋亡的抑制作用... 60

3.4  讨论... 63

第四章  凡纳滨对虾LvNLRPL2基因的免疫学功能研究... 67

4.1  引言... 67

4.2  材料与方法... 67

4.2.1  实验动物... 67

4.2.2  组织分布... 68

4.2.3  序列特征分析... 68

4.4.4  总RNA提取与cDNA合成... 68

4.4.5  序列获取与验证... 68

4.2.6  基因敲降与病原感染... 68

4.2.7  对虾基因组DNA提取... 69

4.2.8  WSSV拷贝数检测... 70

4.2.9  LvNLRPL2敲降对虾血细胞凋亡相关实验... 71

4.2.10  免疫共沉淀分析... 71

4.2.11  统计分析... 79

4.2.12  引物列表... 79

4.3  结果... 81

4.3.1  LvNLRPL2基因的序列特征分析... 81

4.3.2  LvNLRPL2基因的组织表达分析... 83

4.3.3  LvNLRPL2与LvNLRPL1基因的病原感染响应特征分析... 84

4.3.4  LvNLRPL2基因沉默对副溶血弧菌或WSSV感染对虾的影响... 85

4.3.5  LvNLRPL2基因对血细胞凋亡的抑制作用... 88

4.3.6  LvNLRPL2与LvNLRPL1的同型或异型二聚化作用... 90

4.4  讨论... 91

第五章  凡纳滨对虾LvNLRC基因的免疫学功能及作用机制研究... 95

5.1  引言... 95

5.2  材料与方法... 95

5.2.1  实验动物... 95

5.2.2  组织分布... 95

5.2.3  序列特征分析... 96

5.2.4  dsRNA合成及最优干扰剂量选择... 96

5.2.5  基因敲降与病原感染实验... 96

5.2.6  凡纳滨对虾组织的cDNA文库构建... 96

5.2.7  酵母双杂交筛选互作蛋白... 100

5.2.8  酵母双杂交点对点验证LvNLRC与LvCypA的互作... 102

5.2.9  免疫共沉淀验证与LvNLRC互作的蛋白... 103

5.2.10  Pull-down与质谱联用筛选LvNLRC的N端互作蛋白... 104

5.2.11  ELISA检测LvNLRC的LRRs结构域对PAMPs的识别作用... 108

5.2.12  引物列表... 111

5.3  结果... 113

5.3.1  LvNLRC基因的序列特征分析... 113

5.3.2  LvNLRC基因的组织表达分析... 115

5.3.3  LvNLRC基因在WSSV感染过程中的免疫作用... 116

5.3.4  LvNLRC基因在副溶血弧菌感染过程中的免疫作用... 118

5.3.5  LvNLRC-LRRs结构域的识别作用探究... 119

5.3.6  LvNLRC的同型和异型寡聚化作用探究... 120

5.3.7  酵母双杂交文库筛选与LvNLRC互作的蛋白... 122

5.3.8  LvCypA与LvNLRC的互作验证... 124

5.3.9  LvCypA在WSSV感染过程中的调控作用... 126

5.3.10  LvNLRC对STING抗病毒通路的调控作用探究... 128

5.3.11  LvNLRC的N端互作蛋白探究... 131

5.4  讨论... 133

结论与展望... 139

参考文献... 141

致谢... 157

作者简历及攻读学位期间发表的学术论文与研究成果    159