抗微生物胜肽，天蠶素及對蝦素，在草蝦免疫上之研究

Study on Antimicrobial Peptides, Cecropin and Penaeidin, in the Immunity of Tiger Shrimp Penaeus monodon

何詩琥(Shih-Hu Ho)

指導教授 ： 宋延齡

國立臺灣大學/生命科學院/動物學研究研究所/博士(2004年)

https://doi.org/10.6342/NTU.2004.01421

摘要

本論文從兩方面進行抗微生物胜肽(antimicrobial peptides, AMPs)在草蝦Penaeus monodon免疫上的研究，一為測試外源AMP，天蠶素(cecropin)，在草蝦抗病應用上可行性之評估；另一為研究草蝦本身的AMP，對蝦素(penaeidin)，包括基因選殖、定性、重組表現及弁鄐尷R。
Cecropin應用研究方面，選用性質已被確定的抗菌雙極性(amphipathic)胜肽cecropin A和P1，測試其對抗水產動物病原菌的活性。結果顯示cecropin可抑制15株細菌，包括Aeromonas hydrophila, A. salmonicida, Photobacterium damselae subsp. damselae, Pseudomonas anguilliseptica, Vibrio alginolyticus, V. anguillarum, V. carchariae, V. harveyi, V. ordalii, V. parahemolyticus, V. tubiashii and V. vulnificus biogroup I等革蘭氏陰性菌。Cecropins也可抑制淡水長臂大蝦的兩株病原性酵母菌Debaryomyces hanseni菌株。Cecropin A對抗造成養殖蝦很大威脅的弧菌，其最低抑制濃度(minimal inhibitory concentration, MIC)為0.98到7.81 μM；最低殺菌濃度(minimal bactericidal concentration, MBC)從3.90到15.59 μM。但由NaCl對cecropin殺菌能力的影響測試，得知在高鹽度環境下，cecropin對抗病原菌的效果會減弱。在鉻51釋出試驗中，當cecropin A在最低殺菌濃度時，對草蝦血球沒有測出顯著影響，但是在50 μM (僅最低殺菌濃度的3.2倍)或更高濃度時，發現對蝦血球有細胞破壞作用。這些結果暗示在應用cecropin在海水養殖蝦活體的感染防治上，可能要審慎處理。
草蝦對蝦素(penaeidin)方面，我們選殖出對蝦素完整的open reading frame序列225 bp，推譯出的蛋白質為74個胺基酸，在第19和20胺基酸間有一secretary signal peptide的切位，估計其成熟胜肽分子量為6.1 kDa，pI值為9.1。北方墨點分析表示對蝦素主要在血球中合成。在基因調控上，用β-glucan浸泡草蝦，以即時定量RT-PCR (real-time quantitive reverse transcription polymerase chain reaction)偵測血球中對蝦素mRNA的表現，未發現有誘發的效果。免疫組織染色顯示在lymphoid organ、心臟和鰓部等器官中有明顯的訊號。在血清學上，抗白蝦Litopenaeus vannamei對蝦素N端之20個胺基酸之胜肽的抗血清對草蝦對蝦素沒有交叉反應(cross reaction)；反之抗草蝦P. monodon對蝦素N端之20個胺基酸之胜肽的抗血清對白蝦對蝦素亦無交叉反應。從序列比對目前已發現的各種對蝦素，結果顯示草蝦對蝦素不同於Litopenaeus屬和Marsupenaeus japonicus的penaeidin-1到penaeidin-4族群；而和Fenneropenaeus chinensis的penaeidin-5較相似，可見對蝦素在蝦不同屬間有相當差異。將此cDNA利用昆蟲細胞-桿狀病毒表現系統進行生物合成重組對蝦素，其對Aerococcus viridans可形成抑菌圈，但對Vibrio屬的細菌和酵母菌Debaryomyces hansenii則無。此外，重組對蝦素可延遲絲狀真菌Neurospora crassa孢子的萌發及生長。在其他免疫弁鄐W，亦發現重組對蝦素能增加蝦血球的附著(attachment)。

關鍵字

草蝦 ； 抗菌 ； 天蠶素 ； 昆蟲細胞-桿狀病毒表現系統 ； 抗微生物胜肽對蝦素

並列摘要

In this thesis, two aspects were studied on antimicrobial peptides (AMPs) in the immunity of tiger shrimp Penaeus monodon. One is a testing of a foreign AMP, cecropin, for applying on the shrimp against diseases. The other is a study of a shrimp’s AMP, penaeidin, including gene cloning, characterization, protein overexpression and functional analyses.
The effects of two well-characterized antimicrobial amphipathic peptides (cecropins A and P1) on pathogens of aquatic organisms were assessed. Results showed that cecropins inhibited growth of 15 strains of bacteria, including Gram-negative bacteria: Aeromonas hydrophila, A. salmonicida, Photobacterium damselae subsp. damselae, Pseudomonas anguilliseptica, Vibrio alginolyticus, V. anguillarum, V. carchariae, V. harveyi, V. ordalii, V. parahemolyticus, V. tubiashii and V. vulnificus biogroup I. Cecropins also inhibited growth of two yeast strains, Debaryomyces hanseni, pathogenic to freshwater prawn. Minimal inhibitory concentration (MIC) of cecropin A ranged from 0.98 to 7.81 μM and minimal bactericidal concentration (MBC) ranged from 3.90 to 15.59 μM against the most prevalent vibrios pathogenic to cultured shrimp. A higher level of cecropin was required in a high salt environment to exhibit the effect against the pathogens. Influence of NaCl on bactericidal activity of cecropin A was also assayed. 51Cr-release assay showed a non-cytotoxic effect of cecropin A on tiger shrimp hemocytes at MBC, but cytotoxic effect at 50 μM (3.2-fold MBC) or higher concentration. This suggests that cecropin peptides may be difficult to be applied in vivo to treat infected marine shrimp.
A penaeidin cDNA sequence of tiger shrimp was obtained. In the sequence, an open reading frame that coded for a peptide composed of 74 amino acids was found. A cleavage site of secretory signal peptide was predicted between amino acids 19 and 20. The calculated molecular mass of mature penaeidin was about 6.1 kDa and the estimated pI of this peptide was 9.1. Northern blot analysis indicated that the penaeidin was mainly synthesized in the hemocytes. Concerning the regulation of penaeidin gene, the effect of β-glucan immersion was not found. Immunohistochemical staining positive signals were observed in lymphoid organ, hemocytes in heart and gill. In serological study, no cross reaction was observed between penaeidins of P. monodon and Litopenaeus vannamei using serum against L. v. and P. m. penaeidin N-20 mers respectively. The comparison and clustering analysis of penaeidin sequences from P. monodon, Litopenaeus vannamei, L. setiferus, Marsupenaeus japonicus and Fenneropenaeus chinensis was discussed. The recombinant penaeidin has been expressed using insect cell-baculovirus expression system. The recombinant penaeidin could form an inhibition zone against a bacterium Aerococcus viridans, but not Vibrio species or a yeast Debaryomyces hansenii. In addition, it could delay spore germination and growth of a filamentous fungus, Neurospora crassa. Regarding the other immuno-related function of penaeidin, the enhancement of hemocyte attachment was determined.

並列關鍵字

tiger shrimp ； penaeidin ； Penaeus monodon ； insect cell-baculovirus expression system ； antibacterial ； cecropin ； antimicrobial peptide

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