鋸緣青蟹，學名Scylla serrata，與其他三種Scylla 屬分佈於西太平洋及印度洋，此蟹 於幼苗期分佈於河口鹹水地區，等到成熟時，遷徙到海洋產卵。此蟹已廣泛養殖於此地區 超過30 年。關於此蟹的幼苗飼育，適當生存鹽度，以及在不到鹽度下之耗氧、排泄、滲 透壓及離子調節已有一些研究，但有關此蟹的免疫系統幾乎沒有相關研究報告。 已知環境惡化，衰弱宿主及病原聚集造成宿主疾病爆發。已知蟹曾受到幾種病菌及幾 種弧菌包括Vibrio parahaemolyticus 的感染。無脊椎動物缺乏適應性反應，而完全依賴先 天性免疫。因此關於先天性免疫防禦的研究，提供必要的疾病感染及防止疾病感染必要管 理是當務之急。一旦細菌或黴菌等異物入侵宿主的體腔內時，宿主啟動一連串複雜的先天 性免疫反應系統。有幾種參與先天性免疫系統的蛋白質，他們分別為 1)認知蛋白包括 LBP、βGBP、LGBP、PGRP、lectin 及hemolin 等。 2)原酚氧化酵素活化系統相關蛋白質 包括proPO 、peroxinectin 、serine proteinase 、proteinase inhibitors ( 如pacifastin 及 α2-macroglobulin) 等。 3)參與吞噬作用及形成反應氧族群的相關蛋白質如SOD、peroxidase 及catalase 等。 4)凝集系統相關蛋白質包括clottable protein 及transglutaminase 等。 5)抗 菌勝.如penaeidin 及chelonianin 等。 6)抗菌蛋白lysozyme。關於十足目甲殼類參與原酚 氧化酵素活化系統的相關蛋白質，已知已分別自螯蝦Pacifastacucus leniusculus、草蝦 Penaeus monodon、白蝦Litopenaeus vannamei、斑節蝦Marsupenaeus japonicus 選殖LGBP、 βGBP、proPO、peroxinectin、serine proteinase、及α2-macroglobulin。但在鋸緣蟹青蟹的免 疫相關蛋白的選殖、特性及相關功能的研究幾乎空白。 因此，本計畫擬在第一年研究此等參與proPO 活化系統相關免疫蛋白質(proPO、 peroxinectin、serine proteinase 及α2-macroglobulin)基因選殖，第二年研究此等蛋白質基因 在組織的分佈，不同脫殼期，以及當蟹在受到外來刺激物如LPS 及zymosan 和環境因子 變化如水溫、鹽度及pH 的此等蛋白質基因的表現情形。第三年擬用酵母Pichia pastoris 重組表現系統來生產表現此等免疫相關基因之蛋白質，進而研究此等重組免疫相關蛋白質 之功能及活性。 Mud crab belonging to the genus Scylla are distributed throughout the west Pacific and Indian Oceans. Among the four species of mud crab that have been described to date, S. serrata is the most common. S. serrata spends its juvenile phase in brackish water, then moves to the sea during the adult period to spawn and subsequently releases zoeae into seawater. This specie has been cultured throughout the Indo-Pacific regions for more than 30 year. Concerning the larval rearing, optimal salinity level, and the oxygen consumption, and ammonia excretion have been reported. Osmotic and ionic regulations of crab under salinity stress have also been reported. However, none is known on the immune system of crab. It is known that deteriorated environment, weakening host and aggregation of pathogen cause an occurrence of acute diseases. Several viral diseases and vibriosis caused by Vibrio parahaemolyticus have been reported to infect crab. It is known that invertebrate lack a true adaptive immune system, and rely instead on innate responses against invading pathogens. Research on the innate immune system will provide insights into the management and control of infectious diseases. Once pathogen enter the hemocoel of the host, they may encounter a complex system of innate immune mechanisms. There are several proteins involved in the innate immune system. They are 1) pattern recognition proteins like LPS binding protein (LBP), β-1,3-glucan binding protein (βGBP), LPS and β-1,3-glucan binding protein (LGBP), peptidoglcan recognition protein (PGRP), lectin and hemolin, 2) prophenoloxidase activating system proteins like prophenoloxidase, peroxinectin, serine proteinase (porphenoloxidase activating factor, PPAF), proteinase inhibitors like pacifastin and α2-macroglobulin, 3) phagocytosis and reactive oxygen intermediates related proteins like superoxide dismutase (SOD), peroxidase and catalase, 4) clotting system related proteins like clottable protein and transglutaminase, 5) anti-microbial peptides (AMP) like penaeidin and chelonianin, and 6) anti-bacterial protein, lysozyme. Concerning the immune related proteins in the prophenoloxidase activating system of decapod crustaceans, several proteins including LGBP, βGBP, proPO, peroxinectin, serine proteinase and α2-macroglobulin have been cloned and characterized from crayfish Pacifastacucus leniusculus, tiger shrimp Penaeus monodon, white shrimp Litopenaeus vannamei and kuruma shrimp Marsupenaeus japonicus. However, none is kown on the cloning of these proteins and their related functions in the mud crab. This proposal is to study on cDNA cloning and its characterization of prophenoloxidase activating system related proteins of mud carb during the first year. This proposal is to study tissue gene and protein expression of mud crab in different tissues, molting stages, when the crab receive LPS, zymosan, and under environmental stress like water temperature, salinity and pH during the second year. This proposal is to study the protein expression level of immune related proteins using yeast Pichia pastoris expression system to produce the recombinant protein (immune related proteins of prophenoloxidase activating system) and to study their functions and activities of the mud crab during the third year. The prophenoloxidase activating system related proteins that we plan to study are proPO, peroxinectin, serine proteinase and α2-macroglobulin.
Prophenoloxidase activating system