淡水長臂大蝦血漿內抗菌素的分析

Abstract

以常見之水產動物的病原菌為測試菌，利用抑菌圈分析法檢測 cecropin A、 cecropin P1 及溶菌酵素（lysozyme) 對不同菌種的抗菌能力，結果發現 cecropin 可以抑制淡水菌 Aeromonas hydrophila 及海水菌 A . salmonicida 之生長；但對淡水菌 Edwardsiella tarda沒有抗菌活性；除了從病鰻分離出的 Vibrio vulnificus 菌株外，cecropin 對其餘常見 10 種 Vibrio 菌都有抗菌活性；cecropin 對 2 株淡水酵母菌 Debaryomyces 有抗菌活性：對 3 株 E . coli D21 , D31 及JM107 亦有抗菌效果。溶菌酵素則僅對 2 株酵母菌 Debaryomyces 有抗菌活性。 以大腸桿菌 E . coli JM 107 為免疫刺激物，注射到淡水長臂蝦圍心腔內，經過 1 、 2 、 3 、 4 、 5 、 6 、 12 、 18 、 24 、 48 、 72 小時後，從圍心腔抽取血淋巴液，將抗凝處理過的血淋巴液離心取血漿，再利用超過濾膜略純化並縮減體積約三倍後利用抑菌圈分析法檢測其是否含抗菌物質。以大腸桿菌 JM107 及 D21 為測試菌，結果發現血漿中確實有抗菌物質被誘發出來。注射後 6 小時， 31％的測試蝦血漿開始出現抗菌活性，迄 24 小時，79％的測試蝦出現抗菌活性，至 72 小時所有蝦子的抗菌活性皆消失，顯示一旦被激發後多數測試蝦在血漿中會合成抗菌素，估算其濃度最高不會超過 0.21 μM，且為短效性。將含抗菌物質的蝦血漿跑SDS-PAGE，轉印至 PVDF 膜上，再以兔抗 cecropin A 血清作免疫染色，發現在 35kd 附近有一色帶，因此推測淡水長臂大蝦注射大腸桿菌後血漿中誘發的抗菌物質可能類似野蠶的 cecropin。

Cecropin A, cecropin P1 and lysozyme susceptibilities of the common aquatic pathogens were tested with an inhibition zone assay. Results showed that cecropins could inhibit growth of the 16 stains of bacteria, including Aeromonas hydrophila, A. salmonicida, Escherichia coli, Vibrio alginolyticus, V. anguillarum, V. carchariae, V. damsela, V. fluvialis, V. harveyi, V. ordalii ，V, parahemolytics ，V. tubiashii and V. vulnificus. Cecropins also inhibited growth of 2 pathogenic yeast stains, Debaryomyces hansenii. However, antibacterial activities of cecropins could not reach to Edwardsiella tarda, as well as, V. vulnificus strains isolated from a diseased eel. Microbicidal activity provided by the lysozyme only acted on D. hansenii. We used the inhibition zone assay to detecte the presence of antimicrobial substances produced by giant freshwater prawn (Macrobrachium rosenbergii). When prawn were injected into the pericardial cavity with E. coli cells, hemolymph were collected at 1,2,3,4,5,6,1,18,24,48, 72 hours post - injection. Assay of the plate showed an induced bacterial substance (s). The antibacterial activity appeared in 31 % of tested prawn plasma 6 hrs post- injection. Same activity was shown in 79 % of tested plasma 24 hrs post-injection. However, it was not detected in all tested plasma synthesize antibacterial substance (s) once stimulated. However ，this activity was short - term and concentration was estimated no more than 0.2luM in prawn plasma. Western blot analysis of plasma with an antibacterial acti-rabbit-anti-cecropin A serum. These results indicated that the antibacterial substance induced in giant freshwater prawn may be a cecropin-like.