Exploration of the antibacterial proteins in pearl oyster Pinctada fucata induced by bacterial inoculation

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  • 細菌の接種によって誘導されるアコヤガイ中の抗菌性タンパク質の探索

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The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 1012 colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.本研究で対象とするアコヤガイは，日本において重要な養殖真珠の母貝として用いられる二枚貝である。腸炎ビブリオをアコヤガイ閉殻筋に直接接種したところ，鰓から得られた酸抽出物に，非接種の対照よりも強い抗菌活性を示す成分が存在することを見出した。酸抽出物はグラム陽性菌および陰性菌に抗菌活性を示し，とくにビブリオ属に強く作用した。鰓より2種の抗菌タンパク質APg-1（分子量約210 kDa）およびAPg-2（分子量約30 kDa）を分離し，これらはMALDI-TOF MS分析により，新規の抗菌タンパク質である可能性が推察された。

source:https://doi.org/10.1007/s12562-017-1084-2

source:https://jsfs.jp/

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