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Molecular Pathogenesis of Extracellular Proteases of Pathogenic Vibrio Species from Marine Fish and Shellfish Cultured in Taiwan (II)
|Contributors: ||NTOU:Department of Aquaculture|
Amino acid sequence,Gene sequence,Marine fish and shellfish,Protease,molecular pathogenesis,Vibrio alginolyticus;V. carchariae;V. harveyi;Vibrioalginolyticus;V. carchariae;V. harveyi
|Issue Date: ||2011-06-28T07:45:18Z
|Abstract: ||摘要:本研究探討台灣養殖海水魚蝦貝類主要病原弧菌各菌株間之細胞外蛋白分解酵素之相關性及致病機制。實驗所使用之菌株包括Vibrio alginolyticus、V. carchariae 及V. harveyi共7 株係分離自罹病石斑魚、海鱺、草蝦、斑節蝦及九孔且皆對各該生物具病原性，經再確認後保存備用。以石斑魚、海鱺、草蝦及九孔為對象，進行各菌株細胞外產物的毒性試驗。之後以各菌株間之細胞外產物於快速蛋白液體層析儀利用各種管柱進行蛋白分解酵素之純化，進行氨基酸序列分析試驗，以了解對各不同菌株間此酵素之相關性。利用已得知之氨基酸序列推定其基因組成，進行酵素基因相關性之分析比較，供作疫苗開發及防治之參考。以各菌株活菌107 colony forming units(CFU)╱g fish劑量攻擊石斑魚(每組六尾)。結果顯示，分離自石斑魚及九孔的菌株具較強之毒性，死亡率皆高於50﹪。各菌株之細胞外產物皆具蛋白分解酵素活性且對石斑魚具毒性(10 μg protein╱g fish)，但其蛋白分解酵素在不同菌種間不具相關性。V. carchariae蛋白分解酵素(33 kDa)經存化後經氨基酸序列後，於NCBI BLAST search比對僅與V.vulnificus CMCP6 Signal transduction histidinekinase有18％ 相似，顯示此33 kDa 蛋白分解酵素為一新的酵素。|
Abstract:The present study investigated the molecular pathogenesis of extracellular proteases of major pathogenic Vibrio species from marine fish and shellfish cultured in Taiwan. Seven strains of pathogenic V. alginolyticus, V. carchariae and V. harveyi originally isolated from diseased grouper, cobia, tiger prawn, kuruma prawn or small abalone were used in the study. All the strains were re-identified and stocked prior to the experiment. Virulence tests were conducted in batches of grouper, cobia, tiger prawn and small abalone using each bacterial extracellular products (ECP). Protease activity of each ECP were also conducted to understand the presence of possible variations. Extracellular protease were purified from each ECP using various columns on FPLC (Fast Protein Liquid Chromatography) System. The amino acid composition of purified protease was sequenced for further comparison. Nucleotide composition of the protease were deduced through the amino acid sequence. The results may be useful for vaccine development and disease control. In the virulence tests, groupers were challenged with a dose of 107colony forming units (CFU)╱g fish of each strain. The results revealed that strains isolated from grouper and small abalone were more virulent (mortality higher than 50%) than other strains indicating the presence of variations. Extracellular products from each strain all exhibited protease activities and were lethal to the grouper at a dose of 10 .mu.g protein╱g fish. The 33 kDa serine protease of V. carchariae was purified on FPLC System, then characterized on Native-PAGE and 2-D PAGE. The amino acid sequences of the 33 kDa protease were compared with other bacterial protease amino acid sequencesusing NCBI BLAST search. The result revealed that it possessed 18% similarity with Signal transduction histidine kinase of V. vulnificus CMCP6. The protease was therefore suggested to be a novel protease since there were no other similar Vibrio protease existed in the NCBI BLAST search.
|Appears in Collections:||[水產養殖學系] 研究計畫|
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