石斑魚腸道菌抗神經壞死症病毒物質之分析與作用機制探討

Analysis of Antiviral Substance Against Nervous Necrosis Virus (NNV) and Antiviral Mechanism of The Intestinal Bacteria Isolated From Grouper

邱莉璇(Li-Shiuan Chu)

指導教授 ： 齊肖琪

國立臺灣大學/生命科學院/動物學研究研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02881

摘要

神經壞死症病毒 (Nervous necrosis virus,NNV) 會感染世界各地多種養殖的海水魚及某些淡水魚，造成魚苗及幼魚的高死亡率。現有的防治方法，除了以物理化學方式消毒及疫苗免疫外，益生菌的使用也逐漸受到重視。本研究室自石斑魚腸道中分離出益生菌，經由細胞及活體實驗證實有抗NNV活性 (Chang, 2004)。本研究目的，在分析一株抗NNV菌株0409所分泌的抗病毒物質，利用物理化學方法進行特性分析，進而採用高效能液相層析技術及快速蛋白液相色譜純化分離抗病毒物質，並利用石斑魚鰭細胞GF-1研究抗病毒物質對NNV的作用機制。結果發現，抗病毒物質具熱穩定性、耐酸鹼值的變化、不易受proteinase K等蛋白酶影響、具有高極性，且分子量小於或等於 5 kDa。菌液經由凝膠過濾管柱層析分離後，在第40管之分管液具有抗病毒活性，比對分管樣品的總醣與蛋白質吸收光圖譜，發現該物質可能為帶有芳香基團的小胜肽。細胞實驗結果發現，抗病毒物質不直接作用於病毒顆粒上，亦不會阻斷細胞表面的病毒受器，但根據real-time PCR及 Western Blot 的時序檢測結果，在有菌液和受NNV感染細胞共培養的情況下，病毒核酸及外鞘蛋白質合成量會受到抑制，並且是藉由抑制NNV核酸的複製而達到抑制病毒增殖的結果。

關鍵字

神經壞死症病毒, 抗病毒物質

並列摘要

Nervous necrosis virus (VNN) has caused mass mortality of many cultured fish species at larval stage. Probiotics become more important in the control of virus infection other than physical or chemical methods and immunization by vaccine. A few strains of bacteria were isolated from grouper intestine, including strain 0409, and were found to exhibit anti-NNV activity in vitro and in vivo (Chang, 2004). The aim of the present study is to analyze the biological and physiochemical properties of antimicrobial metabolites derived from strain 0409, use HPLC and FPLC to pure antiviral substrate, and to characterize its anti-NNV mechanism in GF-1 cell line. The anti-NNV components in the metabolites of strain 0409 were found to be heat-stable, wide-pH tolerant, proteinase K-resistant, high polar and smaller than 5 kD. The anti-NNV components were separated by Gel filtration, and the collected fractions were analyzed by absorbance of protein, total glycan staining and antiviral activity. It is suggested that the anti-NNV substance secreted by strain 0409 is highly to be a small peptide with aromatic group. During in vitro experiment, the culture supernatent of strain 0409 did not neutralize virions directly or block the viral receptor on cell membrane. According to the results of real-time PCR and Western blot, it was found that the amplification of NNV nucleic acids and capsid protein were inhibited only in NNV-infected cells co-cultured with the supernatants of strain 0409, therefore, it is suggested the mechanism of anti-NNV substances is through the depression of viral nucleic acids.

並列關鍵字

Nervous Necrosis Virus, anti viral substance

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