博士 / 國立臺灣大學 / 動物學研究所 / 92 / 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.
| Identifer | oai:union.ndltd.org:TW/092NTU00312023 |
| Date | January 2004 |
| Creators | Shih-Hu Ho, 何詩琥 |
| Contributors | Yen-Ling Song, 宋延齡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 87 |
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