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| 研究生： |
何倫 Haroon Afzal |
|---|---|
| 論文名稱： |
鞭毛蛋白N末端與傳染性華氏囊病病毒抗原融合時之佐劑效應評估 Evaluation of the Adjuvancy of Flagellin N Terminal When Fused to An Infectious Bursal Disease Virus Antigen |
| 指導教授： |
鄭力廷
Li-Ting Cheng 吳幸潔組長 Hsing-Chieh Wu |
| 學位類別： |
碩士 Master |
| 系所名稱： |
國際學院 - 動物用疫苗國際學位專班 International Program in Animal Vaccine Technology |
| 畢業學年度： | 109 |
| 語文別： | 英文 |
| 論文頁數： | 64 |
| 中文關鍵詞： | 鞭毛蛋白N末端與傳染性華氏囊病病毒抗原融合時之佐劑 、高變域。 、表位 、二聚化 |
| 外文關鍵詞： | Evaluation of the adjuvancy of flagellin N terminal when fused to an infectious bursal disease virus antigen. |
| DOI URL： | http://doi.org/10.6346/NPUST202100222 |
| 相關次數： | 點閱：51 下載：28 |
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鞭毛蛋白N末端與傳染性華氏囊病病毒抗原融合時之佐劑效應評估
先天免疫系統的 TLR 5 和NAIP5-NLRC4 可偵測細菌鞭毛蛋白。鞭毛蛋白有兩個保守域 D0 和 D1，而 D2、D3 被認為是可變異。與 C 端結構域相比， N 端保守結構域在引發 TLR5 免疫活性和對初級和次級二聚化的實質性支持方面具有更多作用。結構研究發現，N 端結構域為主要（ 70%）支持 TLR5 結合和信號傳導，而 C 端結構域僅扶左與 TLR5 的結合。一項研究表明 NAIP5-NLRC4 複合物被 C 端的 35 個殘基激活。儘管這些鞭毛蛋白結構域就其結構和功能進行了廣泛的研究，但這些結構域並未作為嵌合產物單獨應用於抗原。這項研究揭示了 N 末端在免疫激活和保護免受致命病毒攻擊中的作用。在這項研究中，我們結合來自傳染性法氏囊病病毒 VP2 的 N 末端 (1-176) 的保守域和高變域（包含中和抗體表位）。進行PCR克隆以構建嵌合DNA，然後通過大腸桿菌表達系統表達蛋白質。我們評估了單獨由嵌合體和抗原提供的免疫功效和保護。通過細胞因子分析驗證免疫功效。通過ELISA分析總IgG力價。收集PMC並用抗原刺激用於細胞增殖測定。測量嵌合構建體的刺激指數並與抗原和對照組進行比較。體外中和試驗有助於了解中和抗體力價以應對病毒。關鍵詞：嵌合、表位、二聚化、高變域。
Receptors of the innate immune system including Toll-like receptor 5, and neuronal apoptosis inhibitory protein 5 signal in response to bacterial flagellins. Flagellin has two conserve domains D0 and D1 while D2, D3 are considered as the variable domain. N terminal conserved domains were found to have more role in eliciting TLR5 immune activity and substantial support to primary and secondary dimerization compared to C-terminal domains. Structural studies explain that N -terminal domain almost 70% support TLR5 binding and signaling, while the C-terminal domain only support primary binding with TLR5. Although these flagellin domains were extensively studied regarding their structure and function, yet these domains are not applied individually to antigen as the chimeric product. This study put light on the flagellin N-terminus(1-176aa) in immune activation as adjuvant. In this study, our focus was fusion of the conserved domains from the N terminus of flagellin (1-176 residues) to hypervariable domain (contain neutralizing antibody epitopes) from VP2 of infectious bursal disease virus. PCR cloning was performed to construct a chimeric DNA, followed by chimeric and tVP2 expression of the protein by the E. coli expression system. We evaluated the immune efficacy and protection offered by chimeric and antigen alone. Immune efficacy was verified by the cytokine analysis. Total IgG titers were analyzed by the ELISA. Peripheral blood mononuclear cell (PBMC) were collected and stimulated with antigen for cell proliferation assay. The stimulation index of the chimeric construct was measured and compared with the antigen and control group. In vitro neutralization assay helps to understand the neutralizing antibody titers to cope with virus.
Keywords: Chimeric, epitopes, dimerization, hypervariable domain.
Table of Contents
ABSTRACT 2
ACKNOWLEDGEMENTS 6
LIST OF TABLES 9
LIST OF FIGURES 10
INTRODUCTION 12
1. LITERATURE REVIEW 14
2.1 Mineral Salts 15
2.2 Emulsions 15
2.3 Toll-like receptors agonists 15
2.4 Toll-like receptors recognition and signaling 16
2.5 Flagellin as agonists of TLR5 16
2.6 Flagellin binding with TLR5 and truncated flagellin designs 17
2.7 Infectious bursal disease model to analyze the effectiveness of flagellin as an adjuvant 22
2.8 Considering the lethality of IBDV, the only way to limit control is vaccination. Two way of vaccination is in practice 24
2.9 The crystal structure of nFlic and VP2 projection domains that we cloned and expressed for our vaccine design 25
3. Materials and Methods 26
3.1 Bacterial strains 26
3.2 Cloning of desired flagellar and IBDV antigenic segments and production of chimeric vaccine. 26
3.2.1 Production of N and C terminal flagellar clones 26
3.2.2 Production of infectious bursal disease VP2 clone 26
3.3 Primer used in PCR amplification of nFlic, cFlic, and Chimeric Clone 27
3.4 Designing of chimeric flagellin-VP2 clone 28
3.5 Expression of recombinant proteins and analysis by SDS and western blot 29
3.5.1 Protein expression and release 29
3.5.2 Protein purification 29
3.5.3 Quantification assay 29
3.5.4 Western blot 30
3.6 Vaccine Preparation and Immunization 30
3.7 Immune analysis of birds was supported by the following experiments 30
3.7.1 The enzyme-linked immunosorbent assay (ELISA) 30
3.7.2 ELISA Protocol 31
3.8 Lymphocyte proliferation assay 31
3.9 Cytokine Analysis 32
3.10 Total RNA extraction 32
3.11 Analysis of cytokine fold change by qPCR 33
3.12 Primers for cytokine genes 34
3.13 Neutralization Assay Protocol 36
4. RESULTS AND DISCUSSION 37
4.1 Cloning of flagellin N-terminus 1-176aa(nFlic) 37
4.2 Sequence result of flagellin N-terminus 37
4.3 Cloning of flagellin C-terminus (406-496aa) 38
4.4 Construction of Chimeric nFlic-VP2 that lead to the generation of a subunit vaccine 39
4.5 Subunit vaccine was designed by making antigen-adjuvant chimeric protein 42
4.6 SDS analysis of protein size and quantification 43
4.7 SDS-PAGE analysis of antigen VP2 44
4.8 SDS, Western blot comparison of purified chimeric, unpurified and pET32a 45
4.9 Animal experiment results 46
4.10 Lymphocyte proliferation assay 47
4.11 Th1 and Th2 type cytokines analysis 48
4.12 Neutralizing assay 49
5. DISCUSSIONS 51
6. CONCLUSION 54
7. REFERENCES 55
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