实验海洋生物学重点实验室知识库

具有clip 结构域的丝氨酸蛋白酶（clip domain serine protein，cSP）在甲壳动
物天然免疫体系中发挥着重要作用，然而该蛋白酶在同一物种中不同cSP 功能的
比较研究较少，更是缺乏对免疫信号调节作用的研究。可变淋巴受体基因
（variable lymphocyte receptors，VLRs）是脊索动物七鳃鳗（Petromyzon marinus）
获得性免疫的分子基础，近年来已有报道VLRs 在无脊椎动物的适应性免疫防御
中发挥独特作用。本研究基于基因克隆、原核系统蛋白表达、RNA 干扰、实时
定量PCR 等分子生物学实验技术对3 个三疣梭子蟹（Portunus trituberculatus）
clip 结构域丝氨酸蛋白酶（PtcSP1、PtcSP2、PtcSP3）进行了免疫功能比较分析，
探讨了PtcSPs 与其他免疫基因间的调控关系，并对中华绒螯蟹（Eriocheir
sinensis）可变淋巴受体（EsVLRA）进行了结构分析及免疫致敏效应的研究，从
先天免疫和免疫致敏两个维度分别研究了PtcSPs 基因在蛋白水平的功能多样性
以及EsVLRA 的免疫记忆性。本文主要研究内容及结果如下：
PtcSPs-C 的序列比对结果显示，PtcSP1、PtcSP3 具有胰蛋白酶所特有的
Asp189、Gly216 和Gly226 位点，而PtcSP2 在底物结合口袋位点序列则更为多
样，进化树分析结果表明，PtcSP2 与胰凝乳蛋白酶聚为一枝。经原核重组蛋白
表达系统，分别重组表达了三疣梭子蟹PtcSPs 的SP 结构域（PtcSPs-C）和clip
结构域（PtcSPs-N）。胰蛋白酶和胰凝乳蛋白酶活性验证实验进一步证明，重组
表达复性的PtcSP1、PtcSP3 具有胰蛋白酶活性，而复性的重组蛋白PtcSP2 具有
胰凝乳蛋白酶活性。PtcSP1、PtcSP3 的clip 结构域（PtcSPs-N）对测试的革兰氏
阴性（铜绿假单胞菌Pseudomonas aeruginosa 和溶藻弧菌Vibrio alginolyticus）和
革兰氏阳性菌（金黄色葡萄球菌Staphylococcus aureus 和藤黄微球菌Micrococcus
luteus）菌具有抑制作用。值得注意的是，PtcSP1、PtcSP3 抗菌特性有所不同，
rPtcSP1-N 对革兰氏阳性菌藤黄微球菌的抗菌活性最高（MIC 值小于0.99 μM），
rPtcSP3-N 则对革兰氏阴性菌溶藻弧菌的生长（MIC 值小于0.58 μM）表现出最
强抗菌活性。然而，并没有观察到重组蛋白针对酵母的明显的抗菌活性，也未检
测到明显的PtcSP1-C 和PtcSP3-C 的抗菌活性。此外，菌结合试验中发现，
rPtcSP1-N 和rPtcSP3-N 均对检测的革兰氏阴性菌（ P. aeruginosa 和V.alginolyticus）革兰氏阳性菌（S. aureus 和M. luteus）以及真菌（毕赤酵母菌Pichia
pastoris）表现出菌结合活性。菌清除试验结果表明，PtcSP1 具有溶藻弧菌的菌
清除活性并呈现时间依赖性响应模式，暗示PtcSP1 可能作为调理素参与免疫反
应。
三疣梭子蟹PtcSP1、PtcSP2 基因沉默后，除抗脂多糖因子5（PtALF5）之
外，其他PtALFs 表达均受到抑制，而PtcSP3 沉默后所有检测的PtALFs 表达均
受到抑制，提示PtcSPs 调控PtALFs 合成的多样性；PtcSP1 和PtcSP3 的沉默可
上调补体样相关分子的表达，暗示丝氨酸蛋白酶基因与补体样基因之间的拮抗关
系。此外，PtcSP2 基因沉默后机体酚氧化酶（Phenoloxidase，PO）活性并未受
到影响与PO 激活相关的丝氨酸蛋白酶抑制剂相关基因表达也未受到明显干扰，
而PtcSP1 和PtcSP3 的低表达则使机体内PO 活性明显降低。
中华绒螯蟹EsVLRA 的ORF 长度为2400 bp，编码一个799 个氨基酸长的
蛋白。包括一个LRR_NT 结构域，十三个LRRs 结构域和一个LRR_CT 结构域，
同时，在其C 末端发现了一个明显的凸出环结构。氨基酸序列的多序列比对分
析表明，EsVLRA 具有与其他物种VLRA 一致保守的氨基酸序列组成；系统发
育分析表明，EsVLRA 被归为VLRA 一类，说明中华绒螯蟹中的VLR 是VLRAs
家族的新成员。在所有检测的血细胞、心脏、肌肉、肝胰腺、腮和性腺组织中都
检测到EsVLRA 的mRNA 表达，其中，在肝胰腺、性腺和鳃中具有高表达水平；
值得注意的是，EsVLRA 在雄性蟹的肝胰腺、性腺、鳃和脑中的表达明显高于雌
性。
重组表达的rEsVLRA 可以与革兰氏阴性菌副溶血性弧菌（ Vibrio
parahaemolyticus）和铜绿假单胞菌，革兰氏阳性菌藤黄微球菌和金黄色葡萄球
菌以及毕赤酵母强烈结合，但并没有显示出任何抗菌活性，说明EsVLRA 主要
作为一种病原微生物的受体参与免疫识别。免疫致敏试验中，发现健康蟹经过甲
醛灭活的副溶血弧菌刺激时首先在第3 小时表现出显著升高（P < 0.05）；在第
二次注射活菌后，EsVLRA 的表达在注射后的第1 小时便显著上调，并且比第一
次注射时表达量高得多（约为第一次峰值的50 倍，P < 0.01）；最高的表达水平
出现在第二次攻击后的第6 小时（约为第一次峰值的240 倍，P < 0.01），这种
显著的高表达水平还一直持续到第48 小时（P < 0.01）。表明EsVLRA 可能对
于病原刺激具有记忆性。

对三疣梭子蟹3 丝氨酸蛋白酶的免疫功能研究表明，它们具有丰富多样的免
疫活性。研究认为PtcSPs 可分别通过抗菌活性，病原微生物识别，调理素介导
的细胞免疫，酚氧化酶原激活反应介导的体液免疫以及调控其他先天免疫基因等
多个方面在甲壳动物先天免疫中执行免疫抵御功能。对中华绒螯蟹免疫致敏相关
基因可变淋巴受体的研究表明，EsVLRA 在结构上与七鳃鳗中的VLRA 具有较
高相似性，在功能上作为模式识别受体参与到甲壳动物的免疫致敏过程中。综上，
本研究丰富了丝氨酸蛋白酶的多样性免疫功能的理论基础，同时为甲壳动物的免
疫致敏现象研究提供了新的研究线索。

Clip domain serine proteins (cSPs) play an important role in the innate immune
response of crustacean. However, few comparative studies have been reported on the
function of this protein in different cSPs of the same species, and there is a lack of
research on the regulation of immune signals. The variable lymphocyte receptor (VLR)
genes are the molecular basis of the acquired immunity in Petromyzon marinus, and it
has been reported that VLR plays a special role in the adaptive immune defense of
invertebrates in the recent years. This study based on molecular cloning, prokaryotic
system protein expression, RNA interference, real-time quantitative PCR etc.,
compared the immune functions of three clip domain serine proteases (PtcSP1,
PtcSP2 and PtcSP3) of Portunus trituberculatus and studied the regulatory
relationships among other immune genes of PtcSPs. Also, the structure of the variable
lymphocyte receptor (EsVLRA) of Eriocheir sinensis was analyzed and the effect of
immune priming was investigated. This research compared the immune functional
diversity based on its recombinant proteins and explained the immune memory of
EsVLRA. The research essays and results are as following:
Sequence alignment of PtcSPs-C showed that PtcSP1 and PtcSP3 had
trypsin-specific Asp189, Gly216, and Gly226 binding site, but PtcSP2 had more
diverse sequences in the binding pocket sites, and phylogenetic tree analysis showed
that PtcSP2 from P. trituberculatus and the chymotrypsins were clustered together.
SP domain (PtcSPs-C) and the clip domain (PtcSPs-N) of PtcSPs of P. trituberculatus
were expressed by the prokaryotic protein expression system separately. Proteinase
activity verification-experiments further proved that the purified PtcSP1 or PtcSP3
had trypsin-like activity, while PtcSP2 recombinant protein had chymotrypsin-like
activity. Moreover, rPtcSP1-N and rPtcSP3-N protein could inhibit all the tested
Gram-negative bacteria (Pseudomonas aeruginosa and Vibrio alginolyticus) and
Gram-positive bacteria (Staphylococcus aureus and Micrococcus luteus). Remarkably,
PtcSP1 and PtcSP3 showed different antibacterial MIC: RPtcSP1-N had the highest
antibacterial activity against gram-positive bacteria M. luteus (MIC value less than
0.99 μM), while rPtcSP3-N strongly inhibited growth of the Gram-negative bacteria V.
alginolyticus (MIC value of less than 0.58 μM). However, no obvious antimicrobialactivity was observed against yeast, and no significant antibacterial activity of
PtcSP1-C or PtcSP3-C was detected. In addition, in the bacterial-binding assay,
rPtcSP1-N and rPtcSP3-N were detected against Gram-negative bacteria (P.
aeruginosa and V. alginolyticus), Gram-positive bacteria (S. aureus and M. luteus),
and the fungus (Pichia pastoris) showed bacterial-binding activity. The results of the
bacteria clearance test showed that PtcSP1 has the bacteria clearance activity of V.
alginolyticus and presents a time-dependent pattern, suggesting that PtcSP1 may
participate in the immune response as an opsonin.
Under PtcSP1 or PtcSP2 gene RNAi, all PtALF genes were inhibited except
anti-lipopolysaccharide factor 5 (PtALF5), and the expression of all detected PtALFs
was suppressed after PtcSP3 was silenced, indicated that PtcSPs could regulate the
diverse PtALFs synthesis. Silencing of PtcSP1 or PtcSP3 could up-regulate the
expression of complement-like genes, suggesting an antagonistic relationship between
serine protease genes and complement-like genes. In addition, the PO activity of the
crab was not affected after PtcSP2 gene silencing, and the expression level of serine
protease inhibitor-related genes, which are related to PO activation did not change,
while in the low expression of PtcSP1 or PtcSP3 crab the PO activity was
significantly reduced.
EsVLRA ORF contains 2400 bp, and encodes 799 amino acids. Including one
LRR_NT domain, thirteen LRRs domain and one LRR_CT domain, meanwhile a
large protruding-structure at the C-terminal. Multi-sequence alignment analysis of
amino acid sequences showed that EsVLRA has a conserved amino acid sequence,
which is consistent with VLRA from other species; phylogenetic analysis showed that
EsVLRA and VLRAs were clustered together, indicating the VLR from E. sinensis is
a new member of VLRAs. The mRNA transcript of EsVLRA was detected in all
examined tissues, including hemocytes, heart, muscle, hepatopancreas, gill, intestine
and gonads. EsVLRA highly expressed in hepatopancreas, gonads and gill. Besides,
the expression level of EsVLRA in hepatopancreas, gonads, gill and brain of male
crabs was significantly higher than that in female crabs.
rEsVLRA could strongly bind to Gram-negative bacteria Vibrio.
parahaemolyticus and P. aeruginosa, Gram-positive bacteria M. luteus and S. aureus
and yeast P. pastoris. However, no significant antimicrobial activity of rEsVLRA was
observed, indicating that EsVLRA is mainly involved in immune recognition as a
receptor. In the immune priming assay, it was found that when healthy crabs werestimulated by formaldehyde-inactivated V. parahaemolyticus firstly, EsVLRA
showed a significant increase at third hour (P < 0.05). After injected with live V.
parahaemolyticus, EsVLRA was significantly up-regulated at the first hour and was
much higher than that in the first injection (approximately 50 times to the first
injection, P < 0.01). The highest expression level appeared at the sixth hour after the
second challenge (approximately 240 times to the first injection, P < 0.01), this high
expression level is lasting to the 48th hour (P < 0.01). It showed that EsVLRA might
be memorable for the pathogen-stimulation.
Studies on the immunological function of the 3 kinds of serine protease of P.
trituberculatus showed that they have diverse immune activities. PtcSPs functioned as
an immune factor in crustacean innate immunity through various functions including
antibacterial activity, pathogen recognation protein, opsonin-mediated cellular
immunity, proPO activation-mediated humoral immunity, and the regulation of other
innate immune genes. Investigations of the variable lymphocyte receptors of mitten
crab immune priming-related genes had shown that EsVLRA is structurally similar to
VLRA in lamprey, and functions as a pattern recognition receptor to participate in the
immunity of crustaceans. In summary, this study enriched the theoretical basis of
serine proteases diverse immune functions, and provided new clues for the study of
crustacean immune priming.

第1 章 绪论 .................................................................................................................. 1
第1 节 无脊椎动物免疫防御机制 ...................................................................... 1
1 前言 ............................................................................................................ 1
2 物理防御 .................................................................................................... 3
3 细胞免疫 .................................................................................................... 4
4 体液免疫 .................................................................................................... 4
第2 节 丝氨酸蛋白酶免疫功能研究 .................................................................. 6
1 丝氨酸蛋白酶的结构 ................................................................................ 7
2 丝氨酸蛋白酶参与的免疫反应 ................................................................ 8
2.1 凝血过程中的丝氨酸蛋白酶 ......................................................... 8
2.2 酚氧化酶原激活系统中的丝氨酸蛋白酶 ..................................... 8
2.3 补体系统中的丝氨酸蛋白酶 ......................................................... 9
3 甲壳动物丝氨酸蛋白酶研究进展 .......................................................... 10
第3 节 无脊椎动物免疫致敏 ............................................................................ 11
1 无脊椎动物免疫记忆的发现 .................................................................. 11
2 免疫致敏的概念 ...................................................................................... 12
2.1 免疫致敏的基本特性 ................................................................... 12
3 免疫致敏机制研究 .................................................................................. 14
3.1 抗菌活性 ....................................................................................... 14
3.2 吞噬活性 ....................................................................................... 14
3.3 模式识别分子 ............................................................................... 14
3.4 协同交互作用与剂量效应 ........................................................... 16
4 甲壳动物中免疫致敏研究现状 .............................................................. 17
第4 节 VLR 的免疫特异性 ............................................................................... 18
1 LRR 基序 .................................................................................................. 18
2 VLR 基因研究进展 .................................................................................. 19
第5 节 蟹类SP 基因与VLR 基因研究目的和意义 ....................................... 20第2 章 实验材料与方法 ............................................................................................ 23
第1 节 三疣梭子蟹3 种PtcSP 基因免疫功能研究 ........................................ 23
1 实验材料 .................................................................................................. 23
1.1 动物和菌株 ................................................................................... 23
1.2 主要试剂 ........................................................................................ 23
1.3 仪器设备 ....................................................................................... 25
2 实验方法 .................................................................................................. 26
2.1 质粒提取 ....................................................................................... 26
2.2 目的片段的扩增 ............................................................................ 26
2.3 序列分析与系统进化树构建 ....................................................... 29
2.4 重组蛋白表达载体的构建 ........................................................... 29
2.5 重组蛋白诱导表达 ....................................................................... 29
2.6 重组蛋白收集、纯化与复性 ....................................................... 30
2.7 复性蛋白浓缩与浓度测定 ........................................................... 30
2.8 重组蛋白的蛋白酶活性分析 ....................................................... 31
2.9 抗菌活性试验 ............................................................................... 31
2.10 菌结合试验 ................................................................................. 31
2.11 溶藻弧菌清除活性的鉴定 ......................................................... 32
2.12 siRNA 合成与PtcSP1-RNAi 实验 ............................................. 32
2.13 dsRNA 合成及PtcSP2-RNAi、PtcSP3-RNAi 实验 .................. 34
2.14 血细胞RNA 提取及cDNA 合成 .............................................. 34
2.15 干扰效率检测 ............................................................................. 36
2.16 基因沉默后相关免疫基因表达 ................................................. 36
2.17 干扰后酚氧化酶（PO）活力测定 ........................................... 37
2.18 统计分析 ..................................................................................... 37
第2 节 中华绒螯蟹VLR 基因克隆、表达及免疫功能研究 .......................... 37
1 实验材料 .................................................................................................. 37
2 实验方法 .................................................................................................. 38
2.1 VLR 的组织分布实验样品制取 ................................................... 38
2.2 EsVLRA 的RNA 分离、cDNA 合成和基因克隆 ...................... 382.3 序列和系统发育分析 ................................................................... 39
2.4 EsVLRA 的实时定量PCR 分析 .................................................. 40
2.5 重组质粒的构建 ........................................................................... 40
2.6 重组EsVLRA 的表达和纯化 ....................................................... 41
2.7 抗菌活性测定 ............................................................................... 41
2.8 菌结合活性的测定 ....................................................................... 42
2.9 “疫苗”接种与二次刺激实验 ........................................................ 42
2.10 统计分析 ..................................................................................... 43
第3 章 实验结果 ........................................................................................................ 44
第1 节 三疣梭子蟹PtcSP1 重组蛋白功能及免疫基因互作分析 .................. 44
1 重组蛋白PtcSP1-C 和PtcSP1-N 表达 .................................................. 44
2 重组蛋白底物结合位点分析及酶活性验证 .......................................... 44
3 rPtcSP1-N 的最小抗菌浓度 .................................................................... 47
4 重组蛋白的菌结合活力 .......................................................................... 47
5 菌清除实验 .............................................................................................. 48
6 最佳干扰时间检测 .................................................................................. 49
7 PtcSP1 干扰后相关免疫基因表达情况 .................................................. 50
8 干扰后的PO 活性 .................................................................................. 51
第2 节 三疣梭子蟹PtcSP2 蛋白酶活性及免疫基因互作分析 ...................... 52
1 PtcSP2 的同源和系统发育分析 .............................................................. 52
2 蛋白酶活性 .............................................................................................. 55
3 PtcSP2 的干扰效率 .................................................................................. 55
4 PtcSP2 干扰对AMPs 和SPIs 表达的影响 ............................................ 56
5 PtcSP2 沉默蟹的总PO 活性 ................................................................... 57
第3 节 三疣梭子蟹PtcSP3 重组蛋白功能及免疫基因互作分析 .................. 58
1 重组蛋白PtcSP3-C 和PtcSP3N 的表达 ................................................ 58
2 重组蛋白底物结合位点分析及酶活性验证 .......................................... 59
3 rPtcSP3-N 最小抗菌浓度 ........................................................................ 61
4 重组蛋白的菌结合活力 .......................................................................... 62
5 菌清除实验 .............................................................................................. 636 PtcSP3 最佳干扰时间检测 ...................................................................... 64
7 SP 干扰后相关免疫基因表达情况 ......................................................... 65
8 干扰后的PO 活性 .................................................................................. 66
第4 节 中华绒螯蟹VLR 基因克隆、表达及免疫功能研究 .......................... 67
1 EsVLRA 的序列分析 ............................................................................... 67
2 多序列比对、系统发育分析和三维结构模型 ...................................... 69
3 EsVLRA 表达模式分析 ........................................................................... 72
4 EsVLRA 的重组表达与纯化 ................................................................... 73
5 抗菌活性和菌结合活性 .......................................................................... 73
6 疫苗接种后蟹的存活率 .......................................................................... 75
7 疫苗接种和活菌二次刺激 ...................................................................... 75
第4 章 讨论 ................................................................................................................ 77
第1 节 三疣梭子蟹3 种PtcSP 基因免疫功能 ................................................ 77
1 PtcSP1、PtcSP2、PtcSP3 蛋白酶活性多样 ........................................... 77
2 PtcSP1、PtcSP2、PtcSP3 抗菌活性各不相同 ....................................... 78
3 PtcSP1、PtcSP2、PtcSP3 均为PRP ....................................................... 80
4 PtcSP1 具有调理活性 .............................................................................. 82
5 PtcSP1、PtcSP2、PtcSP3 对AMP 合成具有调节作用 ........................ 83
6 PtcSP2 不参与proPO 激活 ...................................................................... 84
7 PtcSP1 与PtcSP3 具有补体样分子的调节作用 .................................... 85
第2 节 中华绒螯蟹VLR 的免疫功能 .............................................................. 86
1 EsVLRA 的序列特征 ............................................................................... 86
2 EsVLRA 广泛的组织分布 ....................................................................... 88
3 EsVLRA 是一种PRP ............................................................................... 89
4 EsVLR 参与免疫致敏 .............................................................................. 90
第5 章 总结与展望 .................................................................................................... 92
第1 节 结论与创新点 ........................................................................................ 92
1 三疣梭子蟹PtcSPs 具有多样的生物学功能 ........................................ 92
2 三疣梭子蟹PtcSP1、PtcSP2、PtcSP3 参与多种免疫基因的调控 ..... 92
3 三疣梭子蟹PtcSP2 不参与proPO 激活 ................................................ 934 中华绒螯蟹中存在七鳃鳗中的免疫适应性基因VLR ......................... 93
5 中华绒螯蟹EsVLRA 可能是一种PRP ................................................. 93
6 中华绒螯蟹EsVLRA 参与免疫致敏 ..................................................... 93
第2 节 存在问题 ................................................................................................ 93
1 三疣梭子蟹中的VLR 基因 .................................................................... 93
2 中华绒螯蟹EsVLRA 的记忆时效 ......................................................... 94
第3 节 展望 ........................................................................................................ 94
参考文献 ...................................................................................................................... 96
致 谢 .......................................................................................................................... 119
作者简历及攻读学位期间发表的学术论文与研究成果 ........................................ 121