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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 韓玉山(Yu-San Han) | |
| dc.contributor.author | Lung-Yi Chen | en |
| dc.contributor.author | 陳龍怡 | zh_TW |
| dc.date.accessioned | 2021-06-16T02:34:56Z | - |
| dc.date.available | 2020-07-30 | |
| dc.date.copyright | 2015-07-30 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-07-28 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/53966 | - |
| dc.description.abstract | 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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T02:34:56Z (GMT). No. of bitstreams: 1 ntu-104-R02b45003-1.pdf: 4743762 bytes, checksum: 13f3dce58ef4406b188db0b46b33ec98 (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 誌謝 i
中文摘要 ii ABSTRACT iii CONTENTS v LIST OF FIGURES viii LIST OF TABLES x Chapter 1 Introduction 1 1.1 Shrimp farming 1 1.2 White Spot Syndrome Virus 2 1.3 Neutralization Antibody & Single chain Antibody Fragments 4 1.4 Bacillus megaterium Protein Production System 6 Chapter 2 Materials and methods 8 2.1 Experimental animals and WSSV 8 2.1.1 Penaeus vannamei 8 2.1.2 White Spot Syndrome Virus 8 2.2 Expression and purification of sAP-1 8 2.2.1 Plasmid construction 8 2.2.2 Protein production 9 2.2.3 Protein extraction and purification 9 2.2.4 Analysis of expressed sAP-1 10 2.3 Oral administration and challenge assay 11 2.3.1 Preparation of cannibalism-like meal and sAP-1 coated feed pellets 11 2.3.2 Oral challenge model 11 2.3.3 Oral administration of sAP-1 12 2.3.4 Oral administration of sAP-1 coated feeds 12 2.4 Analysis of neutralizing activity 13 2.4.1 Competitive enzyme-linked immunoassays 13 2.4.2 Quantitative real time polymerase chain reaction 13 2.4.3 Analysis of superoxide anion 14 Chapter 3 Results 16 3.1 Analysis of expressed sAP-1 in B. megaterium Protein Production System 16 3.1.1 Cloning of sAP-1 gene and construction of plasmid 16 3.1.2 Expression of sAP-1 16 3.1.3 Purification of sAP-1 16 3.2 Analysis of expressed sAP-1 in E. coli Protein Production System 16 3.2.1 Expression of sAP-1 in different condition 17 3.2.2 Purification of sAP-1 17 3.3 Oral challenge model 17 3.3.1 Oral challenge assay 17 3.3.2 Quantification of WSSV 18 3.4 Analysis of sAP-1 activity 18 3.4.1 Competition ELISA 18 3.4.2 Oral administration of sAP-1 and challenge assay 18 3.4.3 Oral administration of sAP-1 coated feeds and challenge assay 19 3.4.4 Analysis of superoxide anion 19 Chapter 4 Discussion 20 4.1 The expression of sAP-1 in B. megaterium Protein Production System 20 4.2 The expression of sAP-1 in E. coli Protein Production System 20 4.3 Oral challenge model 22 4.4 Neutralizing activity of sAP-1 23 Chapter 5 Conclusion 25 References 26 Figures 39 Tables 58 | |
| dc.language.iso | en | |
| dc.subject | 單鏈抗體片段 | zh_TW |
| dc.subject | 白點病毒 | zh_TW |
| dc.subject | 巨大芽孢桿菌表現系統 | zh_TW |
| dc.subject | 大腸桿菌表現系統 | zh_TW |
| dc.subject | 中和抗體 | zh_TW |
| dc.subject | B. megaterium expression system | en |
| dc.subject | single-chain variable fragment | en |
| dc.subject | neutralizing antibody | en |
| dc.subject | E. coli expression system | en |
| dc.subject | white spot syndrome virus | en |
| dc.title | 單鏈抗體預防白蝦經由攝食感染白點病毒效果之研究 | zh_TW |
| dc.title | Single chain antibody fragment against the horizontal transmission of white spot syndrome virus in Penaeus vannamei | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 廖一久(I-Chiu Liao),彭正(Cheng Peng),廖文亮(Wen-Lian Liao),王涵青(Han-Ching Wang) | |
| dc.subject.keyword | 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, | zh_TW |
| dc.subject.keyword | white spot syndrome virus,B. megaterium expression system,E. coli expression system,neutralizing antibody,single-chain variable fragment, | en |
| dc.relation.page | 61 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-07-28 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
| Appears in Collections: | 漁業科學研究所 | |
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| ntu-104-1.pdf Restricted Access | 4.63 MB | Adobe PDF |
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