請用此 Handle URI 來引用此文件：
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97340完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林翰佑 | zh_TW |
| dc.contributor.advisor | Han-You Lin | en |
| dc.contributor.author | 陳盈芳 | zh_TW |
| dc.contributor.author | Ying-Fang Chen | en |
| dc.date.accessioned | 2025-05-07T16:05:42Z | - |
| dc.date.available | 2025-05-08 | - |
| dc.date.copyright | 2025-05-07 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-04-22 | - |
| dc.identifier.citation | Reference
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/97340 | - |
| dc.description.abstract | 神經壞死病毒（Nervous Necrosis Virus, NNV）是一種高致病性的病毒，會導致病毒性神經壞死症（Viral Nervous Necrosis, VNN），對水產養殖業，特別是魚類幼苗造成嚴重的經濟損失。NNV的殼體為T=3二十面體結構，由RNA2所編碼的180個次單位殼蛋白組成，在病毒的穩定性與感染力中扮演關鍵角色。利用其自我組裝成T=3二十面體結構之特性，我們開發了NNV病毒樣顆粒（Virus-Like Particles, VLPs）作為攜帶重組石斑魚介白素-6（Recombinant Grouper Interleukine-6; rgIL-6）的奈米載體，rgIL-6對於對石斑魚幼苗免疫調節來說，是一個相當重要的細胞激素。奈米包覆的目的，希望能夠降低細胞激素傳遞時受到環境的影響，以及組織障壁的阻隔。首先必須將rgIL-6成功報復在NNV殼蛋白之中。我們先使用EGTA解聚緩衝液將NNV殼體蛋白解離為單體，接著再利用含二價金屬離子（如鈣離子）的重組緩衝液，在rgIL-6的存在下重新組裝回二十面體結構。這一個策略成功地實現了rgIL-6的包覆，並可以計算出重組蛋白的被包覆率。在適當的條件下，rgIL-6的被包覆效率可達55.67%（w/w），對應於約5:4的分子質量比例（NNV殼蛋白對rgIL-6）。此外，包覆於具蛋白酶耐受性的NNV VLPs內，rgIL-6可能免受酶解，進一步提升其穩定性與免疫刺激功能。與目前廣泛應用於人類醫學但成本高昂的脂質奈米粒子（Lipid Nanoparticles, LNPs）相比，NNV VLP是一種可規模化生產且具生物相容性的替代方案。先前的研究已證實，NNV VLPs可作為DNA疫苗質體的遞送載體，其具有進一步的應用的潛力。我們的研究結果顯示，與其他常見的二十面體結構VLPs相較，NNV VLPs作為細胞激素奈米載體展現出不錯的包覆效率。本研究針對NNV殼蛋白的單體解離與重組條件進行了優化，確保成功封裝rgIL-6，這個結果有望應用於石斑魚苗水產養殖產業，為功能性重組蛋白，特別是免疫促進劑的投遞策略提供一項新選擇。 | zh_TW |
| dc.description.abstract | Nervous Necrosis Virus (NNV) is a highly pathogenic virus responsible for viral nervous necrosis (VNN), causing significant economic losses in aquaculture, particularly among fish larvae. The NNV capsid, an icosahedral T=3 structure composed of 180 capsid protein subunits encoded by RNA2, plays a crucial role in viral stability and infectivity. Leveraging its self-assembling properties, we developed NNV Virus-Like Particles (VLPs) as nanocarriers for recombinant grouper interleukin-6 (rgIL-6), a cytokine critical for immune modulation in grouper larvae. To optimize encapsulation, we employed an EGTA-based disassembly buffer to break down the capsid into monomers, followed by a calcium-rich assembly buffer to restore its icosahedral structure in the presence of rgIL-6. This strategy successfully encapsulated rgIL-6 with an efficiency of 55.67% by mass, corresponding to a molecular ratio of approximately 5:4 (capsid protein to rgIL-6). Encapsulation within protease-resistant NNV VLPs protects rgIL-6 from enzymatic degradation, potentially enhancing its stability and immunostimulatory function.
Unlike lipid nanoparticles (LNPs), which are widely used in human medicine but cost-prohibitive for aquaculture applications, NNV VLPs offer a scalable and biocompatible alternative. Previous studies with NNV VLPs have demonstrated the efficacy of VLPs in delivering DNA vaccine plasmid for cellular delivery, further reinforcing the versatility of this approach. Our findings highlight the potential of NNV VLPs as a cytokine-based nanocarrier platform, addressing key challenges in antigen stability and immune stimulation in aquaculture. Nervous Necrosis Virus (NNV), a highly pathogenic virus causing viral nervous necrosis (VNN), poses significant economic threats to aquaculture due to its devastating impact on fish species, particularly larvae. The capsid of NNV has been reported to adopt a T=3 icosahedral architecture, consisting of 180 capsid protein subunits encoded by RNA2, which contributes to viral stability and infectivity. Building upon this structural framework, we engineered virus-like particles (VLPs) from NNV capsid proteins as nanocarriers for recombinant grouper interleukin-6 (rgIL-6), a cytokine essential for immune modulation in grouper larvae. The study addressed key challenges in capsid protein disassembly and reassembly, employing EGTA-based disassembly buffers to break down the capsid into monomers and calcium-rich assembly buffers to restore its icosahedral architecture. Optimization of conditions ensured successful reassembly and encapsulation of rgIL-6. The encapsulation efficiency was optimized by determining the ideal ratio of capsid proteins to rgIL-6, resulting in VLPs that encapsulated rgIL-6 with an efficiency of 55.67% by mass, corresponding to a molecular ratio of approximately 5:4 (capsid protein to rgIL-6). Encapsulation within protease-resistant NNV VLPs may theoretically protect rgIL-6 from enzymatic degradation and enhance its delivery potential in clinical use. Leveraging this approach could pave the way for developing cytokine-based nanocarrier delivery methods for grouper aquaculture. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-05-07T16:05:42Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-05-07T16:05:42Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Table of Contents
口試委員會審定書 II Acknowledgement III 中文摘要 Chinese Abstract IV English Abstract V Table of Contents VI List of Tables IX List of Figures X Chapter 1: Introduction 1 1.1 Overview of Nervous Necrosis Virus (NNV) 1 1.2 VLP Assembly and NNV Capsids: Nature's Blueprint for Nanocarrier Design 1 1.3 Structure of NNV and Its Capsid Proteins 3 1.4 Principles of Icosahedral Symmetry and Packaging Efficiency in Viral Capsids 4 1.5 Application of NNV VLPs: Mechanisms and Biocompatibility 5 1.6 VLPs of Nodaviruses as Nanocarriers in Aquaculture 6 1.7 Rationale for Encapsulating Recombinant Grouper Interleukin-6 in NNV VLPs 7 1.8 Research Hypothesis 9 Chapter 2: Materials and Methods 10 2.1 Research Methodology Overview 10 2.2 Expression and Inclusion Body Purification of Nodavirus Capsid Proteins 10 2.3 SDS-PAGE and Western Blotting 11 2.4 Transmission Electron Microscopy (TEM) 12 2.5 Expression and Purification of Recombinant Grouper IL-6 (rgIL-6) 12 2.6 SDS-PAGE and Western Blotting for Confirmation of Recombinant Grouper IL-6 12 2.7 Expression and Purification of Recombinant Grouper IL-6 Receptor (rgIL-6R) 13 2.8 The Encapsulation of Recombinant Grouper IL-6 in NNV VLPs 13 2.9 Purification of rgIL-6-Encapsulated NNV VLPs 14 2.10 Densitometry Analysis of SDS-PAGE Bands with Standard for Encapsulation Ratios 14 2.10.1 BSA Standard Preparation and Dilution 14 2.10.2 Protein Sample Preparation and SDS-PAGE Electrophoresis 14 2.10.3 Gel Imaging and ImageJ Analysis 15 2.10.4 Quantification of rgIL-6 Using BSA Standard Curve and Densitometric Analysis 15 2.11 Optimization of the Encapsulation Efficiency and Statistical Analysis 15 2.12 Investigating the Protein Interaction Between NNV Capsid and rgIL-6 17 2.13 Detection of Protein Interaction Using Dot Blotting 18 Chapter 3: Results 19 3.1 Expression and Purification of NNV Capsid Proteins 19 3.2 Refolding and Self-Assembly Conditions of NNV Capsid Proteins 19 3.3 Transmission Electron Microscopy (TEM) 19 3.4 Expression and Purification of Recombinant Grouper IL-6 (rgIL-6) 20 3.5 Expression and Purification of Recombinant Grouper IL-6 Receptor (rgIL-6R) 20 3.6 Encapsulation of rgIL-6 in Nodavirus Capsid Proteins 20 3.7 Densitometry Analysis of SDS-PAGE Bands with Standard for Encapsulation Ratios 20 3.8 Encapsulation Efficiency of rgIL-6 in NNV VLPs at Different Ratios 21 3.9 Time-Dependent Optimization of Encapsulation Duration 22 3.10 Results of Dot Blot Experiment 22 Chapter 4: Discussion 23 4.1 Optimization of VLP Refolding and Assembly Process 23 4.2 Formulation and Composition of NNV VLPs Encapsulating rgIL-6 24 4.3 The Statistical Encapsulation and Comparison with Other Statistical VLP Encapsulation 24 4.4 Theoretical Benefits and Limitations of the Nodavirus VLP Delivery System 26 4.5 Implications of the Nodavirus VLP Encapsulation System Based on Proof of Concept 27 Reference 28 Table 1. Recombinant Grouper IL-6 (rgIL-6) Concentrations Derived from BSA Densitometric Standard Curve 36 Table 2. Comparison of Encapsulation Efficiency and Protein Interaction Between Cargo and Virus Capsids in Different VLP Types 37 Figure 1. DXXDXD Motif of the NNV Capsid Protein 38 Figure 2. Schematic of the Self-Assembly and Encapsulation of NNV-VLPs with rgIL-6 39 Figure 3. Expression and Purification of NNV Capsid Protein 40 Figure 4. TEM Image of NNV VLPs 41 Figure 5. Expression and Purification of rgIL-6 42 Figure 6. Expression and Purification of Recombinant Grouper IL-6 Receptor (rgIL-6R) Protein 43 Figure 7. SDS-PAGE and Western Blotting Analysis of Nodavirus Capsids and rgIL-6 VLPs 44 Figure 8. SDS-PAGE Quantification of rgIL-6 Protein Concentration Using a BSA Standard Curve 45 Figure 9. SDS-PAGE Analysis of rgIL-6 Encapsulation Efficiency in NNV VLPs 46 Figure 10. Statistical Encapsulation Ratio for rgIL-6 Loading in NNV VLPs 47 Figure 11. SDS-PAGE Analysis of Protein Encapsulation Efficiency at Different Time Durations 48 Figure 12. Optimized Statistical Encapsulation Duration for rgIL-6 in NNV VLPs 49 Figure 13. Dot Blot Analysis of rgIL-6 Interaction with rgIL-6R, NNV Capsid Proteins, and BSA 50 | - |
| dc.language.iso | en | - |
| dc.subject | 病毒性神經壞死症（VNN) | zh_TW |
| dc.subject | 鈣離子結合基序 | zh_TW |
| dc.subject | 殼蛋白包埋 | zh_TW |
| dc.subject | 奈米載體 | zh_TW |
| dc.subject | 蛋白質包覆效率 | zh_TW |
| dc.subject | 病毒樣顆粒（VLPs） | zh_TW |
| dc.subject | 石斑魚介白素-6（rgIL-6） | zh_TW |
| dc.subject | 神經壞死病毒（NNV） | zh_TW |
| dc.subject | Capsid Protein | en |
| dc.subject | Nanocarrier | en |
| dc.subject | Interleukin-6 (IL-6) | en |
| dc.subject | Virus-Like Particles (VLPs) | en |
| dc.subject | Viral Nervous Necrosis (VNN) | en |
| dc.subject | Nervous Necrosis Virus (NNV) | en |
| dc.subject | Encapsulation | en |
| dc.subject | Calcium-Binding Motif | en |
| dc.title | 以神經壞死病毒類病毒顆粒作為石斑魚IL-6重組蛋白之奈米載體 | zh_TW |
| dc.title | Nervous Necrosis Virus Virus-Like Particles (NNV VLPs) as Nanocarriers for Recombinant Grouper Interleukin-6 (IL-6) | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 林翰佐;蘇豐傑 | zh_TW |
| dc.contributor.oralexamcommittee | Han-Tso Lin;Feng-Jie Su | en |
| dc.subject.keyword | 神經壞死病毒（NNV）,病毒性神經壞死症（VNN),石斑魚介白素-6（rgIL-6）,病毒樣顆粒（VLPs）,蛋白質包覆效率,奈米載體,殼蛋白包埋,鈣離子結合基序, | zh_TW |
| dc.subject.keyword | Nervous Necrosis Virus (NNV),Viral Nervous Necrosis (VNN),Virus-Like Particles (VLPs),Interleukin-6 (IL-6),Nanocarrier,Capsid Protein,Calcium-Binding Motif,Encapsulation, | en |
| dc.relation.page | 50 | - |
| dc.identifier.doi | 10.6342/NTU202500831 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-04-22 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 獸醫學系 | - |
| dc.date.embargo-lift | 2030-04-16 | - |
| 顯示於系所單位： | 獸醫學系 | |
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