实验海洋生物学重点实验室知识库

Caspase（cysteinyl aspartate specific proteinase, CASP）是一类含半胱氨酸的天冬氨酸蛋白水解酶，其介导细胞凋亡等多种形式的细胞死亡，在生物体生长、发育和免疫过程中发挥重要作用。哺乳动物中, 至少有18种caspase被研究和报道。仿刺参（Apostichopus japonicus）是我国重要海洋经济物种。基因组测序表明，仿刺参拥有多个caspase基因。然而，目前对仿刺参caspase的研究大多是检测其在不同条件下的转录表达变化，这些caspase的具体免疫功能和作用机制有待深入探索。

在本研究中，我们从仿刺参中鉴定出两种未见报道的caspase，分别命名为AjCASPX1和AjCASPX2。AjCASPX1和AjCASPX2分别由426和427个氨基酸组成，分子量分别为47.52 kDa和49.17 kDa，理论等电点分别为6.58和5.91。二者均由一个N端CARD结构域和一个C端caspase 结构域（CASc）构成。系统发育分析和关键位点分析结果表明AjCASPX1和AjCASPX2分别与哺乳动物CASP9和CASP2家族有较近的进化关系。然而，AjCASPX1和AjCASPX2具有一些独特的结构特征。不同于人Homo sapiens CASP2/9，AjCASPX1和AjCASPX2在p20和p10亚基连接区存在一个α-螺旋。细胞实验结果显示，AjCASPX1能够诱导HEK293T细胞发生凋亡，在此过程中伴随CASP3/7激活。在AjCASPX1诱发的凋亡晚期，lactate dehydrogenase (LDH) 释放以及Annexin V⁺/PI^- 细胞显著增加。不同于AjCASPX1，AjCASPX2只能诱导非常微弱的细胞凋亡。AjCASPX2以及AjCASPX2的CARD结构域（命名为CARD_AjCASPX2）能够结合多种革兰氏阳性/阴性细菌以及细菌表面组分脂多糖（LPS）与肽聚糖（PGN）。AjCASPX2以及CARD_AjCASPX2与病原菌哈维氏弧菌（Vibrio harveyi）结合后能够显著抑制细菌生长。AjCASPX1在大肠杆菌中表达后能杀死宿主细菌。qRT-PCR结果表明，正常生理条件下，AjCASPX1和AjCASPX2在呼吸树、肌肉、体腔细胞以及肠道中均有分布，分别在肌肉和体腔细胞中呈现最高量表达。V. harveyi感染仿刺参后，AjCASPX1和AjCASPX2在各组织中的表达均出现显著上调。与此类似，V. harveyi体外感染仿刺参体腔细胞后，也能诱导AjCASPX1和AjCASPX2的表达变化。

上述结果表明，AjCASPX1和AjCASPX2参与细胞凋亡和抗感染免疫。本研究加深了对仿刺参caspase免疫功能的理解，并为海参养殖病害防控提供了理论指导。

Caspase (CASP) is a protein family of cysteine-dependent aspartate-specific proteases that mediate apoptosis and other forms of cell death. CASPs play a vital role in growth, development, and immune response. At least 18 CASPs have been reported and studied in mammals. Sea cucumber Apostichopus japonicus is an important marine economic species in our country. Genome sequencing indicated that A. japonicus possessed a number of caspase genes. To date, most of the studies on A. japonicus caspases focused on the expression of caspases under different conditions. The immune functions and mechanisms of A. japonicus caspases remain largely to be explored.

In this study, we reported the identification and characterization of two CASPs, named AjCASPX1 and AjCASPX2, respectively, from A. japonicus. AjCASPX1 contains 426 amino acids, with a molecular weight of 47.52 kDa and a theoretical pI of 6.58. AjCASPX2 contains 427 amino acids, with a molecular weight of 49.17 kDa and a theoretical pI of 5.91. AjCASPX1 and AjCASPX2 both contain an N-terminal CARD domain and a C-terminal CASP domain (CASc). Phylogenetic and key functional motif analysis showed that AjCASPX1 and AjCASPX2 were close to the mammalian CASP9 and CASP2 lineages, respectively. However, AjCASPX1 and AjCASPX2 possessed distinct structural features different from that of human (Homo sapiens) CASP2/9. Compared to human CASP2/9, AjCASPX1 and AjCASPX2 exhibited an extra α-helix at the linker region between the p20 and p10 subunits. Cellular study showed that AjCASPX1 was able to trigger apoptosis of HEK293T cells with significant increase of lactate dehydrogenase (LDH) release and Annexin V⁺/PI^- cells at the late stage of apoptosis, and the process was accompanied by significant activation of CASP3/7. Unlike AjCASPX1, AjCASPX2 could only induce slight apoptosis. The recombinant proteins of AjCASPX2 and the CARD domain of AjCASPX2(CARD_AjCASPX2) were able to bind to a wide range of bacteria (including G^- and G⁺ bacteria), as well as bacterial cell wall components LPS and PGN. The binding of AjCASPX2 and CARD_AjCASPX2 to the pathogenic bacteria Vibrio harveyi inhibited bacterial growth. When expressed in Escherichia coli, AjCASPX1 was able to kill the host bacteria. qRT-PCR indicated that under normal conditions, AjCASPX1 and AjCASPX2 were expressed in the respiratory tree, muscle, coelomocyte and intestine of A. japonicus, with the highest expression levels occurring in muscle and coelomocytes, respectively. When infected with V. harveyi, AjCASPX1 and AjCASPX2 expressions were significantly upregulated in A. japonicus tissues. Similar results were observed in V. harveyi-infected coelomocytes.

Together, these results indicated that AjCASPX1 and AjCASPX2 played a role in apoptosis and antibacterial infection. These findings deepened our understanding of the immune function of A. japonicus caspases, and provided a theoretical guidance to the development of disease control approaches for sea cucumber in aquaculture.

第1章 绪论 1

1.1 免疫系统概述 1

1.1.1 免疫的基本概念与功能 1

1.1.2 海参的免疫系统 2

1.2 Caspase概述 6

1.2.1 Caspase的结构与分类 6

1.2.2 Caspase与细胞死亡 8

1.2.3 海参caspase的研究进展 11

1.3 研究目的及意义 11

第2章 实验材料与方法 13

2.1 实验材料 13

2.1.1 实验动物 13

2.1.2 实验细胞系及菌株 13

2.1.3 实验仪器 13

2.1.4 实验试剂 15

2.1.5 常用实验溶液的配制方法 16

2.1.6 序列分析及数据处理软件 18

2.2 实验方法 19

2.2.1 基因克隆 19

2.2.2 AjCASPX1和AjCASPX2的生物信息学分析 22

2.2.3 构建AjCASPX1/X2的真核与原核表达载体 23

2.2.4 HEK293T细胞转染与显微镜观察 27

2.2.5 LDH释放检测 29

2.2.6 流式细胞术检测细胞凋亡 30

2.2.7 转染细胞酶活检测 30

2.2.8 Western Blot检测转染细胞AjCASPX1/X2的表达和激活 31

2.2.9 重组蛋白原核表达纯化 32

2.2.10 AjCASPX2/CARDAjCASPX2 与细菌体外结合能力检测 34

2.2.11 AjCASPX2/CARDAjCASPX2 与病原相关分子模式体外结合能力检测 35

2.2.12 AjCASPX2/CARDAjCASPX2对细菌生长的影响 35

2.2.13 AjCASPX1对细菌存活的影响 36

2.2.14 正常生理条件下AjCASPX1/X2的组织分布情况 36

2.2.15 细菌感染后AjCASPX1/X2的表达情况变化 38

第3章 实验结果 40

3.1 AjCASPX1和AjCASPX2的生物信息学分析 40

3.2 AjCASPX1/X2能够引起HEK293T细胞发生凋亡 44

3.3 AjCASPX2/CARDAjCASPX2与细菌的结合特征 47

3.4 AjCASPX2/CARDAjCASPX2与病原相关分子模式的结合特征 48

3.5 AjCASPX2/CARDAjCASPX2能够抑制细菌生长 49

3.6 AjCASPX1具有杀菌效应 50

3.7 正常生理状态下AjCASPX1/X2的组织分布 50

3.8 感染状态下AjCASPX1/X2的表达变化 51

第4章 讨论 54

第5章 结论与展望 57

参考文献 58

致  谢 64

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 66