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標題: | 單鏈抗體預防白蝦經由攝食感染白點病毒效果之研究 Single chain antibody fragment against the horizontal transmission of white spot syndrome virus in Penaeus vannamei |
作者: | Lung-Yi Chen 陳龍怡 |
指導教授: | 韓玉山(Yu-San Han) |
關鍵字: | 白點病毒,巨大芽孢桿菌表現系統,大腸桿菌表現系統,中和抗體,單鏈抗體片段, white spot syndrome virus,B. megaterium expression system,E. coli expression system,neutralizing antibody,single-chain variable fragment, |
出版年 : | 2015 |
學位: | 碩士 |
摘要: | 養殖蝦是現今水產養殖產業中重要的一環,根據聯合國糧食及農業組織的統計,全球養殖蝦佔水產養殖總產值的15%。但各種疾病的爆發對養殖蝦產業造成了極大的影響及經濟損失,白點症即是其中之一。白點症是由白點病毒感染所引起,病蝦蝦殼上會有白點的病徵,且會在出現病徵後10天內造成養殖池內的蝦大量死亡 (可達100%)。然而目前為止仍缺乏有效抵抗、治療白點病的養殖策略及療法被提出。
抗體是一種能有效對抗病毒的免疫蛋白,但在產業大規模的應用上有其限制。冠群科技利用抗白點病毒的單株抗體 (AP-1),取其Fab區的變異片段,設計出一單鏈抗體片段重組蛋白:sAP-1。在先前的研究中發現sAP-1有取代AP-1、保護蝦子對抗白點病毒的潛力,本實驗則進一步的去測試投餵方式的可行性及抵抗病毒的效果。此外為了使sAP-1的生產更有經濟效益且降低食安疑慮,本實驗中也嘗試使用巨大芽孢桿菌 (Bacillus megaterium) 來生產sAP-1。 實驗結果顯示巨大芽孢桿菌蛋白質表現系統沒有成功的表現sAP-1,但在大腸桿菌系統中則有成功的優化而增加其產量。在口服試驗中,白點病毒能有效的經由攝食來傳播,而有添加sAP-1的組別其存活率皆有大幅提升,且蝦體內病毒數量也有大幅度的下降。本實驗也利用酵素連結免疫吸附分析及超氧化物產量分析來尋找sAP-1抵抗病毒的機制,結果發現sAP-1能與VP28蛋白接合,可能藉此干擾病毒與細胞的接觸進而影響了病毒的感染力。 Shrimp farming is very important in aquaculture today. According to Food and Agriculture Organization of the United Nations (FAO), the production of global shrimp aquaculture nowadays accounts for about 15 percent of global aquaculture output value. However, numerous disease outbreaks have caused huge economic losses, among them, one of the most threatening pathogens is the white spot syndrome virus (WSSV). WSSV can cause up to 100% mortality within 10 days in shrimp farms, and to date, no therapeutics has been published to effectively inhibit WSSV infection in the field. A single chain antibody fragment against WSSV, sAP-1 (derived from AP-1, a monoclonal antibody against WSSV), was constructed by Transcending Biotechnologies Inc. (U.S.A.). sAP-1 could protect shrimps from the infection of WSSV in virus challenge experiments through injection, indicating the possibility of substituting the use of AP-1. In order to produce secreted sAP-1 more economic and meet the requirements for food safety, a probiotic, B. megaterium, was used for sAP-1 expression. Our goal in this study is to test the efficiency of sAP-1 against WSSV by oral administration. The results showed that sAP-1 can’t be expressed by B. megaterium, but the production of sAP-1 in E. coli expression system can be optimized and increased. In the oral challenge experiments, WSSV could infect shrimps via ingestion more efficient than via immersion, the mortality and the viral loads of shrimps decreased when fed with additional sAP-1. According to the results of ELISA (sAP-1 can bind to VP28 in competition assay with AP-1 as competitor) and superoxide anion production (sAP-1 doesn’t stimulate shrimp’s immune system), sAP-1 may interfere the attachment between WSSV and cells by binding to VP28. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53966 |
全文授權: | 有償授權 |
顯示於系所單位: | 漁業科學研究所 |
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