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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 翁啟惠(Chi-Huey Wong) | |
| dc.contributor.author | Jia-Yan Chen | en |
| dc.contributor.author | 陳佳妍 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:26:31Z | - |
| dc.date.copyright | 2022-09-29 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-09-26 | |
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Chen, J.R., et al., Vaccination of monoglycosylated hemagglutinin induces cross-strain protection against influenza virus infections. Proc Natl Acad Sci U S A, 2014. 111(7): p. 2476-81. 66. Wu, C.Y., et al., Glycosite-deleted mRNA of SARS-CoV-2 spike protein as a broad-spectrum vaccine. Proc Natl Acad Sci U S A, 2022. 119(9). 67. Gagne, M., et al., mRNA-1273 or mRNA-Omicron boost in vaccinated macaques elicits comparable B cell expansion, neutralizing antibodies and protection against Omicron. 2022. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85854 | - |
| dc.description.abstract | 自從嚴重特殊傳染性肺炎 (COVID 19)大流行以來，已有數種疫苗在全球被使用。然而嚴重急性呼吸道症候群冠狀病毒2型 (SARS-CoV-2)快速地突變，並且可能逃脫現有疫苗帶來的保護力。因此對所有冠狀病毒都有保護作用的廣泛保護性疫苗需求日益增高。棘蛋白的醣基化在逃脫免疫反應中扮演重要的角色。根據先前的研究，單醣棘蛋白疫苗和醣位點缺失mRNA疫苗可以對高關注變異株(VOCs)產生更好的免疫反應。棘蛋白中有12個保守抗原表位：7個在受體結合區(RBD)，五個在CD和HR2。然而在Omicron變異株中，三個在受體結合區的保守抗原表位發生突變。因此CD和HR2是更不易突變的區域。此外，CD和HR2的保守抗原表位被大量的聚醣覆蓋。所以我想研究CD和HR2的醣基化對mRNA疫苗免疫反應的影響，及使用哪種VOCs棘蛋白序列做為mRNA疫苗對不同變異株有最廣的保護。細胞實驗中發現去除CD和HR2的醣基化影響棘蛋白正確摺疊。另外也測試了mRNA的運送工具：in vivo-jetRNA®、 mRNA-LNP及實驗室合成的ZG 奈米載體。小鼠實驗發現施打兩劑的mRNA-ZG 奈米載體後，CD和HR2去醣基化的棘蛋白mRNA可以引起針對Delta及Omicron變異棘蛋白更強的抗體反應，並且也可以引起IFNγ及IL-4的T細胞反應。和全醣基化棘蛋白mRNA相比，CD和HR2去醣基化的棘蛋白mRNA疫苗和可以針對變種病毒有更好的保護力，具有開發廣效COVID-19疫苗的潛力。 | zh_TW |
| dc.description.abstract | Since the pandemic of Coronavirus disease 2019 (COVID-19), there are several vaccines have been used on the global scale. However, SARS-CoV-2 mutates quickly and the mutants that can escape protection from existing vaccines have emerged. So broadly protective vaccine is urgently needed. Glycosylation on the spike protein plays a role in immune response evasion. Monoglycosylated spike protein (SMG) and glycosite-deleted mRNA vaccine induced better immune responses and protection against SARS-CoV-2 variant of concerns (VOCs). Variants of SARS-CoV-2 impact the vaccine efficiencies, because of large number of mutations on the spike protein. According to a previous study, connection domain (CD) and heptad repeat 2 (HR2) domains are more conserved region in the spike protein and largely shielded by glycans [1]. So, I want to investigate the impacts of glycosylation on CD and HR2 domain and which variant of SARS-CoV-2 with CD and HR2 deglycosylation can provide broader protection against variant virus. The in vitro expression experiments showed that deglycosylation on the CD and HR2 domains impacted the spike protein folding. Several mRNA delivery tools were tested: in vivo-jetRNA®, mRNA-LNP (syringe injection and microfluidic device), and mRNA-ZG nanocarrier. After two doses of mRNA-ZG nanocarrier, WT dG (CD+HR2) mRNA vaccine induced stronger antibody response against Delta and Omicron spike protein compared to WT mRNA vaccine. IFNγ and IL-4 T cell responses were also induced. My results showed that CD and HR2 glycosites-deleted spike mRNA vaccine provides better protection against SARS-CoV-2 variants and has the potential to develop the broadly protective COVID-19 vaccine. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T23:26:31Z (GMT). No. of bitstreams: 1 U0001-2209202215550900.pdf: 9650629 bytes, checksum: 93db645c91b18d0bac876acd41cb4dd0 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | Table of contents Acknowledgement……………………………………………………………….……………….... i Chinese abstract….……………………………………………………………….......................... ii English abstract…..……………………………………………………………………...……...... iii Table of contents….………………………………………………………………………..…….. iv Figures……………………………………………………………………….…...………………. vi List of abbreviations…………………………………………………………………..…………. vii 1. INTRODUCTION……………………………………………………………………..……… 1 Transmission routes for SARS-CoV-2 infection…...……………………………………………… 1 Structure of SARS-CoV-2………………………………………………………………………… 2 Structure of spike protein………………………………………………………………..………… 3 Viral entry mechanisms of SARS-CoV-2……………………………………………….………… 3 Feature and function of N-glycans on the spike protein……………………………….………… 4 Development of the SARS-CoV-2 vaccine………………………………………..…….………… 6 The goal of this study……..……………………………………………………………..…..…… 10 2. MATERIALS AND METHODS…………………………………………………..………… 11 2.1 Materials………………………………………………………………...…………………… 11 2.2 Construction of mRNA synthesis plasmid……………………………………………..…….. 12 2.3 Colony PCR……………………………………………………………………..……..…….. 12 2.4 Site-directed mutagenesis……………………………………………………….……..…….. 13 2.5 Production of mRNA……………………………………………………...…….……..…….. 13 2.6 RNA Agarose gel electrophoresis……………………………………………….……..…….. 14 2.7 Encapsulation of mRNA by in vivo-jetRNA®………………………………...………..…….. 14 2.8 Encapsulation of mRNA by lipid nanoparticle…………………………………..……..…….. 15 2.9 Formulation mRNA-ZG nanocarrier complex…………………………...…….……..…….. 15 2.10 Characterization of particle size of mRNA lipid nanoparticles……...………………..…….. 16 2.11 Quantification of encapsulated mRNA……………………………..………….……..…….. 16 2.12 Cell culture……………………………………………………………….…….……..…….. 16 2.13 Transfection of DNA or mRNA to HEK293T cell……………………….…….……..…….. 17 2.14 Cell lysis…...……………………………………………………………..…….……..…….. 17 2.15 Immunoblotting analysis………………….…………………………………………..…….. 18 2.16 LC-MS/MS analysis (protein identification) ………………………….……….……..…….. 18 2.17 Size-exclusion chromatography………………………………….…………….……..…….. 19 2.18 Mice immunization………………….………………………………………………..…….. 20 2.19 ELISA determination…………………………………………………………..……..…….. 20 2.20 enzyme-linked immune absorbent spot (ELISpot) …………………………..……..…...….. 21 3. RESULTS…………………………..…………….…………………………..….……..…….. 22 3.1 Design of mRNA vaccine candidate……………………………………………..……..…….. 22 3.2 In vitro expression of spike protein and identity confirmation………..…………..…….….. 23 3.3 Characterization of the spike protein size……………………………….……….……..…….. 24 3.4 Test of mRNA delivery tools…………………………………………………….……..…….. 25 3.5 Immunogenicity study………………………………………………………..….……..…….. 27 4. DISCUSSIONS……..……………………………………….…………..……….……..…….. 30 5. REFFERENCES………………….………………………………………………..…..…….. 32 Figures Fig. 1 The flowchart of the experiment design...……………………..….……………..…….. 38 Fig. 2 Schematic of spike mRNA vaccine candidate design……..….……………...…..…….. 39 Fig. 3 mRNA production and expression of spike protein in HEK293T cell. .…..………….. 40 Fig. 4 Spike protein analsis by LC-MS/MS. ……………………………………………..….. 41 Fig.5 Immunoblotting analysis of size fractions of the spike protein in cell lysates. .…..….. 42 Fig. 6 Encapsulation efficiency of mRNA delivery tools. .………………………………….. 43 Fig. 7 Particle size and polydispersity index (PDI) of mRNA delivery tools. …………..….. 44 Fig. 8 Transfection efficiencies of mRNA using different delivery tools. ………….…….….. 45 Fig. 9 Mice immunization with in vivo-jetRNA® and immunogenicity. ………………...….. 46 Fig. 10 Spike protein expression using in vivo-jetRNA® transfection. ………………..….….. 47 Fig. 11 IgG titer of mRNA-LNP, mRNA-ZG nanocarrier, and DNA vaccine……………….... 48 Fig. 12 T cell responses of mice immunized with mRNA-ZG-nanocarrier or DNA vaccine...... 50 | |
| dc.language.iso | en | |
| dc.subject | 嚴重特殊傳染性肺炎 | zh_TW |
| dc.subject | 免疫反應 | zh_TW |
| dc.subject | 棘蛋白 | zh_TW |
| dc.subject | 醣基化 | zh_TW |
| dc.subject | 嚴重急性呼吸道症候群冠狀病毒2型 | zh_TW |
| dc.subject | COVID-19 | en |
| dc.subject | immunogenicity | en |
| dc.subject | glycosylation | en |
| dc.subject | spike protein | en |
| dc.subject | SARS-CoV-2 | en |
| dc.title | 醣化對棘突蛋白mRNA疫苗設計之影響 | zh_TW |
| dc.title | Effects of glycosylation on spike mRNA vaccine design | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 馬徹(Che Ma),林國儀(Kuo-I Lin) | |
| dc.subject.keyword | 嚴重特殊傳染性肺炎,嚴重急性呼吸道症候群冠狀病毒2型,棘蛋白,醣基化,免疫反應, | zh_TW |
| dc.subject.keyword | COVID-19,SARS-CoV-2,spike protein,glycosylation,immunogenicity, | en |
| dc.relation.page | 50 | |
| dc.identifier.doi | 10.6342/NTU202203829 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-09-26 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-09-29 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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