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Title:
菲律宾蛤仔免疫效应因子的多样性和免疫功能研究
Author: 杨顶珑
Degree Level: 博士
Issued Date: 2017-06-18
Degree Grantor: 中国科学院大学
Place of Degree Grantor: 北京
Supervisor: 赵建民
Keyword: 菲律宾蛤仔 ; 先天性免疫 ; 防御素 ; 溶菌酶 ; 免疫应答
Subject: 生物学
Major: 海洋生物学
Abstract:     菲律宾蛤仔广泛分布于我国南北沿海,是我国四大养殖贝类之一。作为单产较高的经济软体动物,2009年产量达到300多万吨,约占我国海水养殖贝类产量的30%,接近我国海水养殖总产量的20%。菲律宾蛤仔通常生活在富含微生物的水环境中,依靠滤食为生,经常面临着环境中多种病原微生物的侵袭。由于菲律宾蛤仔缺乏类似于脊椎动物的获得性免疫系统,因此,先天性免疫系统中的免疫效应分子在免疫防御过程中的作用尤为重要。
    作为机体先天性免疫的第一道屏障,免疫效应分子在宿主免疫防御过程中发挥着重要作用,其分子特征、功能分化及其与微生物的互作方式很大程度上决定了机体对外界环境中病原体的抵御能力。目前,针对菲律宾蛤仔抗菌效应因子的分子特征及相关功能的研究仍然缺乏系统深入的了解。本研究以菲律宾蛤仔为实验对象,较为系统地研究了防御素和溶菌酶的抗菌活性,并对防御素的杀菌机制和调理作用进行了探讨,研究结果可为深入了解软体动物的免疫防御机制提供借鉴,也可为新型水产免疫制剂的研发提供基础。本论文主要获得以下研究结果:
    (一)大防御素的分子克隆与免疫功能分析
采用巢式PCR技术,扩增获得了菲律宾蛤仔两种大防御素(分别命名为VpBDef-1和VpBDef-2)的全长cDNA序列。通过序列分析、同源性分析及系统发育树分析,证明VpBDef-1和VpBDef-2为大防御素家族的重要成员。通过TaqMan PCR技术,发现VpBDef-1和VpBDef-2转录本在所检测组织中均有表达;结合免疫组化和原位杂交技术,发现VpBDef-1主要存在于肝胰腺腺泡腔上皮细胞或邻近腺泡腔的上皮细胞中,而VpBDef-2主要存在于鳃组织的瓣鳃腔和鳃丝中。受到鳗弧菌刺激后,VpBDef-1和VpBDef-2 mRNA的表达量均显著上调,提示其参与了机体的免疫应答反应。重组蛋白rVpBDef-1和rVpBDef-2对多种供试菌株均具有杀菌作用,且可抑制微生物被膜的形成;其中,rVpBDef-1对哈氏弧菌、副溶血弧菌和藤黄微球菌具有较强的杀菌活性,而rVpBDef-2对哈氏弧菌、灿烂弧菌、大肠杆菌和藤黄微球菌表现出较强的杀菌作用。此外,VpBDef-1和VpBDef-2还具有免疫调理功能和自由基清除活性,在机体的免疫应答和免疫调节过程中发挥着重要作用。
    (二)防御素的分子克隆与免疫功能分析
菲律宾蛤仔防御素(VpDef)基因编码70个氨基酸,预测分子量为5.3 kDa,蛋白等电点为7.8。多序列比对和系统发育树分析表明,VpDef是无脊椎动物防御素家族成员之一。qRT-PCR分析发现,VpDef转录本在所检测组织中均有表达,且在肝胰腺组织中的表达量最高。受到鳗弧菌刺激后,血细胞中VpDef mRNA表达量在6 h、12 h和72 h显著上调,提示VpDef参与了机体的免疫应答反应。此外,重组VpDef蛋白(rVpDef)对藤黄微球菌和灿烂弧菌均表现出较强的杀菌活性。细菌膜完整性分析结果表明,rVpDef处理能够增加大肠杆菌的通透性。以上结果表明,VpDef在菲律宾蛤仔清除细菌过程中发挥着重要作用,对细胞膜的破坏作用可能是VpDef发挥作用的潜在途径之一。
    (三)C型溶菌酶的分子克隆与免疫功能分析
菲律宾蛤仔C型溶菌酶(VpCLYZ)基因编码153个氨基酸,预测分子量为15.2 kDa,蛋白等电点为4.78。多序列比对和系统发育树分析表明,VpCLYZ属于C型溶菌酶基因家族的一员。VpCLYZ mRNA在所检测的组织中均有表达,且在肝胰腺和鳃组织中表达量较高。鳗弧菌刺激可导致VpCLYZ mRNA表达量(12 h)显著上调,提示其参与了机体的免疫应答反应。重组蛋白rVpCLYZ对所检测细菌均有溶菌作用,其对金黄色葡萄球菌、藤黄微球菌和鳗弧菌等均具有较强的杀菌作用,最小抑菌浓度为1.7 μM,且这种溶菌作用主要是由于其对细胞壁的分解作用所造成。综上所述,VpCLYZ是无脊椎动物C型溶菌酶家族成员,在菲律宾蛤仔免疫防御中发挥着重要作用。
    (四)P型溶菌酶的分子克隆与免疫功能分析
在菲律宾蛤仔中克隆获得了一种分泌型P型溶菌酶(VpPLYZ)的全长cDNA序列,编码177个氨基酸,蛋白分子量为19.6 kDa,蛋白等电点为9.05。多序列比对和系统发育树分析表明,VpPLYZ隶属于P型溶菌酶家族,是动物界中发现的少数几种P型溶菌酶之一。VpPLYZ转录本在所检测组织中均有表达,且在血细胞和鳃组织中表达量较高。在鳗弧菌刺激6 h、24 h、48 h和72 h后,血细胞中VpPLYZ的mRNA表达量显著上调。重组蛋白rVpPLYZ对阴沟肠杆菌和金黄色葡萄球菌表现出较高活性,而对产气肠杆菌和鳗弧菌溶菌作用较弱。以溶壁微球菌为供试菌株,研究rVpPLYZ的最适杀菌条件,发现在pH、温度和金属离子浓度分别为5.5、50℃和5 mM(离子强度)时,rVpPLYZ的杀菌活性最强。以上研究结果表明,VpPLYZ作为研究较少的一类溶菌酶,在保护菲律宾蛤仔免受病原微生物入侵方面发挥了重要作用。
    综上所述,本论文研究了菲律宾蛤仔中防御素和溶菌酶的分子特征、分布规律及抗菌作用,较全面的揭示了大防御素的杀菌作用,可为深入了解菲律宾蛤仔的免疫防御机制提供科学依据。
English Abstract:     Manila clam Venerupis philippinarum, as one of the four mariculture species, is widely distributed in coastal areas of China. In 2009, the output was more than 3 million tons, accounting for about 20% of the entire mariculture production. At present, the frequent outbreak of severe diseases hampered the health development of clam culture. Mollusks lack the acquaried immune systems, and rely mainly on their innate immunity to defend against the invasion of various pathogens.
    As the first line of immune defense, antibacterial effectors play an important role in the innate immune responses. The resistibility of host against pathogens is potentially determined by the molecular diversities and functional differentiation of their antibacterial effectors. However, the molecular characteristics, functional differentiation and the interaction between antibacterial effectors and microbe still need to be studied. In the present study, the antimicrobial activities of defensins and lysozymes from V. philippinarum, their action mode and the biological significances of functional differentiation were investigate to better understand the innate immune responses of manila clam, which will hopfully facilitate the development of clam aquaculture. This study mainly includes the following contents:
    (1)Molecular cloning and immune functions of big defensins: Nested PCR assay was used to clone the full-length cDNA of VpBDef-1 and VpBDef-2, respectively. According to multiple alignments and phylogenic analysis, VpBDef-1 and VpBDef-2 were found to be new members of the big defensin family. VpBDef-1 and VpBDef-2 were found in all detected tissues. For VpBDef-1, it was mainly determined in acinus cavity epithelial cells of hepatopancreas, while VpBDef-2 existed dominantly in gill cavities and filaments. After stimulated by Vibrio anguillarum, the expression of VpBDef-1 and VpBDef-2 transcripts was significantly up-regulated. Furthermore, recombinant VpBDef-1 and VpBDef-2 (rVpBDef-1 and rVpBDef-2) showed antibacterial activities against all tested bacteria and fungi. For example, rVpBDef-1 showed high antibacterial activity against Vibrio harveyi, Vibrio parahaemolyticus and Micrococcus luteus, while rVpBDef-2 showed high activities against V. harveyi, Vibrio splendidus, Escherichia coli and M. luteus. What's more, both VpBDef-1 and VpBDef-2 possessed opsonic activity and radical scavenging activities.
    (2)Molecular cloning and immune functions of a defensin: The full-length cDNA of VpDef was of 333 bp, encoding a polypepide of 70 amino acids. Multiple alignments and phylogenetic analysis strongly suggested that VpDef was a member of the defensin family. In non-stimulated clams, VpDef transcript was constitutively expressed in all tissues detected, especially in hepatopancreas. After V. anguillarum infection, the mRNA expression of VpDef was significantly up-regulated in hemocytes at 6 h, 12 h and 72 h. The recombinant VpDef (rVpDef) showed high antibacterial activities against M. luteus and V. splendidus. Furthermore, rVpDef could increase the membrane permeability of M. luteus and then resulted in cell death. Overall, these results suggested that VpDef played an important role in the elimination of invading bacteria by membrane-disruptive activity.
    (3)Molecular cloning and immune functions of a c-type lysozyme: The full-length cDNA of VpCLYZ was of 736 bp, encoding a polypepide of 153 amino acids. The deduced amino acid sequences of VpCLYZ showed high similarity to other known invertebrate c-type lysozymes. Multiple alignments and phylogenetic relationship strongly suggested that VpCLYZ belonged to the c-type lysozyme family. VpCLYZ transcript was constitutively expressed in a wide range of tissues, especially in the tissues of hepatopancreas and gills. The mRNA expression of VpCLYZ was significantly up-regulated at 12 h post V. anguillarum challenge. The recombinant VpCLYZ (designed as rVpCLYZ) exhibited lytic activities against all tested bacteria, especially against M. luteus and V. anguillarum. Overall, our results suggested that VpCLYZ belonged to the c-type lysozyme family, and played important roles in the immune responses of manila clam, especially in the elimination of pathogens.
    (4)Molecular cloning and immune functions of a phage-type lysozyme: The full-length cDNA of VpPLYZ was of 699 bp, encoding a polypepide of 177 amino acids. Multiple alignments and phylogenetic analysis strongly suggested that VpPLYZ was a new member of the phage-type lysozyme family. The mRNA transcript of VpPLYZ was found mainly in hemocytes and mantles. The relative expression of VpPLYZ mRNA in hemocytes was significantly up-regulated at 6 h, 24 h, 48 h and 72 h after V. anguillarum challenge. The recombinant VpPLYZ (rVpPLYZ) showed high activity against Entherobacter cloacae and Staphyloccocus aureus, and less effective activity towards Entherobacter aerogenes and V. anguillarum. Moreover, the optimal pH, temperature and ionic strength for rVpPLYZ activities were determined to be 5.5, 50 ℃ and 5 mM, respectively. These results suggested that VpPLYZ was a member of the phage-type lysozyme family, and played important roles in the immune responses of manila clam, especially in the elimination of bacteria. 
    In summary, the present study investigated the molecular characteristics, tissue distribution and antimicrobial functions of immune effectors in manila clam, hopfuly sheding new light on the elucidation of the immune responses of V. philippinarum.
Language: 英语
Content Type: 学位论文
URI: http://ir.yic.ac.cn/handle/133337/22448
Appears in Collections:中科院烟台海岸带研究所知识产出_学位论文

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Recommended Citation:
杨顶珑. 菲律宾蛤仔免疫效应因子的多样性和免疫功能研究[D]. 北京. 中国科学院大学. 2017.
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