实验海洋生物学重点实验室知识库

       牙鲆（Paralichthys olivaceus）和大菱鲆（Scophthalmus maximus）是我国主 要的海水养殖鱼类，具有重要的经济价值。补体系统是免疫系统抵抗微生物病原 的第一道防线，在抗感染免疫中发挥重要作用。开展牙鲆和大菱鲆补体相关分子 的研究，有利于开发绿色安全的病害防治手段，促进水产养殖业的健康发展。 补体因子 C3 是补体系统的中心成分，在补体激活、免疫防御和免疫调节过 程中发挥着不可缺少的作用。补体系统可以通过 3 条途径激活，进而发挥清除病 原体的作用。C3 的活化是所有补体激活途径中最关键的一步，C3 经 C3 转化酶 切割形成 C3a 和 C3b，进一步激活补体后续通路。补体系统的激活受到系列调节 因子的调控。C3b 发挥功能后可以在补体调控因子的作用下降解为 iC3b，iC3b 进 一步降解为 C3c 和 C3d，从而将补体从激活状态恢复至静默状态。CD55 分子是 一种补体调节蛋白，通过加速 C3 转化酶的衰变解离来阻止补体系统的异常激活。 本论文研究了牙鲆 C3（PoC3）以及其激活片段 C3a（PoC3a）与 C3dg（PoC3dg） 和大菱鲆 CD55（SmCD55）的生物学功能。 PoC3 由 1655 个氨基酸残基构成，序列中包含 C3 家族高度保守的 GCGEQ 序列。PoC3 在牙鲆 9 种不同的组织中均有表达，并且病原体的感染会显著上调 PoC3 的表达水平。血清中的 PoC3 可以结合多种细菌，纯化的天然 PoC3 可以直 接杀伤哈维氏弧菌和海豚链球菌。敲降了 PoC3 的牙鲆的血清的杀菌活性显著降 低，并且加重迟缓爱德华氏菌在牙鲆中的感染。重组表达的 PoC3a（rPoC3a）可 以结合多种细菌和牙鲆外周血淋巴细胞（PBL）并可以诱导 PBL 发生趋化反应。 rPoC3a 可以提高牙鲆抗感染的能力。重组的 PoC3dg（rPoC3dg）可以与多种细 菌和 PBL 结合。 SmCD55 在大菱鲆 9 种不同的组织中均有表达。利用重组的 SmCD55 （rSmCD55）处理大菱鲆血清，可以显著抑制血清的溶血活性和杀菌活性，说明 rSmCD55 能够抑制补体激活。另外，rSmCD55 还可以结合多种细菌。综上所述， 这些结果表明 PoC3 是牙鲆补体系统激活的关键成分，其激活片段 PoC3a 和 PoC3dg 在抗感染免疫过程中发挥着重要作用，而 SmCD55 是大菱鲆重要的补体调节蛋白。

Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) are the main fish cultured in China and have important economic values. The complement system is the first line of the immune system against microbial pathogens and plays an important role in antibacterial immunity. The research on the complement system of flounder and turbot will be helpful to develop green and safe means of disease control and to promote the healthy development of aquaculture industry. Complement component 3 (C3) is the central component of complement system and plays an indispensable role in complement activation, immune defense and immune regulation. Complement system can be activated through three ways, and then play a role in the elimination of pathogens. The most critical step in all complement activation pathways is that C3 is cleaved by C3 convertase to form C3a and C3b, and then initiate the complement activation pathway. The complement system has a complex and strict regulation mechanism, complement regulators cleaves C3b to iC3b, and further degrades iC3b to C3c and C3d, to prevent excessive complement activation and tissue damage. CD55 is a complement regulatory protein that exists on the surface of cells and can prevent abnormal activation of the complement system by accelerating the disintegration and dissociation of C3 convertase. In this study, we investigated the biological functions of C3 (PoC3), its activated fragments C3a (PoC3a) and C3dg (PoC3dg) from Japanese flounder, and the complement regulatory protein CD55 (SmCD55) from Turbot. PoC3 consists of 1655 amino acid residues and contains the highly conserved GCGEQ sequence of C3 family. PoC3 was expressed in 9 different tissues of Japanese flounder, and the expression level of PoC3 was significantly up-regulated during pathogen infection. Serum PoC3 coul bind to a variety of bacteria, and the purified natural PoC3 protein directly killed Vibrio harveyi and Streptococcus iniae. Knockdown of PoC3 in flounder significantly reduced the serum bactericidal activity and led to elevated Edwardsiella tarda proliferation in fish. Recombinant PoC3a (rPoC3a) could bind to a variety of bacteria. rPoC3a could also bind to the peripheral leukocytes (PBL) of Japanese flounder and induce chemotaxis of PBL. Japanese flounder injected with rPoC3a protein showed better resistance to infection. Recombinant PoC3dg (rPoC3dg) was able to bind to a variety of bacteria and PBL. SmCD55 was expressed in 9 different tissues of turbot. Recombinant SmCD55 (rSmCD55) could significantly inhibit the hemolytic and bactericidal activity of turbot serum, indicating that rSmCD55 repressed complement activation. In addition, rSmCD55 could bind to a variety of bacteria. In conclusion, these results indicate that PoC3 is a key component in the activation of complement system in flounder, PoC3a and PoC3dg play an important role in anti-infection immunity, and SmCD55 is an important complement regulatory protein in turbot.

第 1 章 绪论... 1

1.1 免疫系统... 1

1.2 补体系统... 2

1.2.1 补体系统的组成... 2

1.2.2 补体激活途径... 3

1.3 补体系统中心分子C3. 4

1.4 C3分子在发挥作用过程中生成的活性片段... 6

1.4.1 C3a. 6

1.4.2 C3dg. 7

1.5 补体调节蛋白CD55. 8

1.6 本论文研究的目的与意义... 9

第2章 牙鲆重要补体因子C3在补体系统中的功能探究... 9

2.1 实验材料与方法... 10

2.1.1 实验所用动物及菌株... 10

2.1.2 实验试剂与实验仪器... 11

2.1.3 实验中常用的分析工具... 13

2.1.4 序列比对与分析... 13

2.1.5 牙鲆各组织在正常生理条件下PoC3的表达... 14

2.1.6 在病原菌感染条件下牙鲆各组织中PoC3的表达... 16

2.1.7 通过天然纯化方式获取PoC3蛋白与其特异性抗体的制备... 17

2.1.8 检测血清中PoC3蛋白是否可以结合细菌... 18

2.1.9 检测PoC3蛋白的杀菌作用... 19

2.1.10 利用RNA干扰（RNAi）的方式探究PoC3的功能... 19

2.2 实验结果... 22

2.2.1 PoC3序列特征分析... 22

2.2.2 PoC3的组织特异性表达谱... 25

2.2.3 在病原菌感染条件下牙鲆各组织中PoC3的表达... 26

2.2.4 PoC3蛋白的纯化... 27

2.2.5 血清中PoC3结合多种细菌... 27

2.2.6 天然PoC3蛋白的杀菌作用... 28

2.2.7 PoC3敲降对血清中补体激活的影响... 29

2.2.8 PoC3敲降对细菌感染的影响... 30

2.3 讨论... 31

第3章 牙鲆C3激活片段C3a和C3dg的功能研究... 35

3.1 实验材料与方法... 35

3.1.1 实验所用菌株与培养条件... 35

3.1.2 实验试剂与实验仪器... 35

3.1.3 PoC3a重组蛋白（rPoC3a）原核表达载体的构建... 35

3.1.4 rPoC3a蛋白的原核表达纯化... 38

3.1.5 检测rPoC3a与细菌的结合作用... 39

3.1.6 rPoC3a与PBL细胞之间的相互作用... 40

3.1.7 检测rPoC3a对细菌感染的影响... 42

3.1.8 PoC3dg重组蛋白（rPoC3dg）原核表达载体的构建... 43

3.1.9 rPoC3dg蛋白的原核表达纯化... 44

3.1.10 重组蛋白rPoC3dg与细菌的相互作用... 44

3.1.11 免疫荧光法检测rPoC3dg与PBL细胞的结合... 45

3.2 实验结果... 45

3.2.1 rPoC3a蛋白的表达纯化... 45

3.2.2 rPoC3a蛋白结合多种细菌... 46

3.2.3 rPoC3a结合PBL细胞... 47

3.2.4 rPoC3a诱导PBL细胞产生趋化效应... 48

3.2.5 rPoC3a对细菌感染的影响... 49

3.2.6 rPoC3dg蛋白的表达纯化... 50

3.2.7 rPoC3dg蛋白结合多种细菌... 51

3.2.8 rPoC3dg结合枯草芽孢杆菌... 52

3.2.9 rPoC3dg结合PBL细胞... 53

3.3 讨论... 54