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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蕭超隆(Chiaolong Hsiao) | |
| dc.contributor.author | Lu-Hao Wang | en |
| dc.contributor.author | 王麓豪 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:40:03Z | - |
| dc.date.available | 2022-02-21 | |
| dc.date.copyright | 2017-02-21 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-01-20 | |
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Finn, Cell targeting with hybrid Qbeta virus-like particles displaying epidermal growth factor. Chembiochem, 2011. 12(16): p. 2441-7. 52. Schagger, H., Tricine-SDS-PAGE. Nat Protoc, 2006. 1(1): p. 16-22. 53. Tomisawa, S., et al., A new approach to detect small peptides clearly and sensitively by Western blotting using a vacuum-assisted detection method. Biophysics (Nagoya-shi), 2013. 9: p. 79-83. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78027 | - |
| dc.description.abstract | 從粒線體12S核糖體核糖核酸之編碼中發現一段粒線體衍生性多肽：MOTS-c (一段位於粒線體12S核糖體核糖核酸之開放讀序框架)。在細胞層級中，MOTS-c同時被發現可調節胰島素敏感性及代謝恆定性。有賴生物科技與醫學的發展，對於以奈米尺度在生物體內運輸小分子之快捷運輸平臺研究日趨重要。我們實驗室業已發展一套以類病毒顆粒為轉染媒介的核糖核酸運輸系統。類病毒顆粒是一種可將蛋白質或核糖核酸包裹在內的生物性腔室。在本研究中，我使用一種從Qβ核糖核酸噬菌體衍生而來的重組型20面體奈米微粒：Qβ類病毒顆粒直接包裹MOTS-c之信使核糖核酸(QβMOTS-c 類病毒顆粒)。接著以QβMOTS-c 類病毒顆粒處理HeLa細胞使其產生MOTS-c多肽。我們發展數種生化分析方法以鑑定產物，像是西方墨點法以及競爭型酶聯免疫分析法。我們的目標是生產在生物體層級中能有效調節代謝恆定性的MOTS-c多肽。 | zh_TW |
| dc.description.abstract | MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) is a mitochondrial-derived peptide that is found to be encoded within the mitochondrial 12S rRNA. The MOTS-c is also found to regulate insulin sensitivity and metabolic homeostasis at the cellular level. Convenient platforms for delivering small molecules in vivo in nanoscale of interest are becoming more desirable as the development of biotechnology and medicine. An RNA-delivery system utilizes VLPs (virus-like particles) as a transfection agent is developed prior in our lab. The VLP is a biological compartment that allows one to pack with proteins or RNAs. Here I use the Qβ VLP, a recombinant icosahedral nanoparticle derived from the RNA bacteriophage Qβ, to directed pack the MOTS-c mRNA (QβMOTS-c VLP). The QβMOTS-c VLP is then used to produce the MOTS-c peptide in vivo in the HeLa cells. Several biochemistry assays such as western blot and the competitive enzyme-linked immunosorbent assay (ELISA) are developed to identify the product. Our goal is to produce an effective MOTS-c peptide to functionally regulate metabolic homeostasis at the organismal level. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:40:03Z (GMT). No. of bitstreams: 1 ntu-106-R03b46003-1.pdf: 1981480 bytes, checksum: aa16d4a7508c6391394f623132fe6c4c (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 國立臺灣大學碩士學位論文口試委員會審定書 I
誌謝 II 摘要 III Abstract IV 目錄 V 圖目錄 IX 表目錄 X Chapter 1: Introduction 1 1.1 Qβ virus-like particles (Qβ VLPs) 1 1.2 Mitochondrial open reading frame of the 12S rRNA-c 2 1.3 Motivation and objective 3 Chapter 2: Materials and Methods 4 2.1 Materials 4 2.2 Methods 12 2.2.1 Cloning 12 2.2.2 Digestion of pUC57-Simple and pT7CFE1-NHA 13 2.2.3 The hairpin RNA gene construction 14 2.2.4 DNA agarose gel electrophoresis 14 2.2.5 DNA purification 15 2.2.6 Further DNA purification and concentration 16 2.2.7 DNA ligation 16 2.2.8 Engineer the ΔHA-pT7CFE1-NHA vector 17 2.2.9 Heat-shock transformation 18 2.2.10 Colony polymerase chain reaction 19 2.2.11 Streaking and isolating bacteria on an LB agar plate 20 2.2.12 Liquid bacteria culture and storage 20 2.2.13 Plasmid DNA extraction 21 2.2.14 Competent cells production 21 2.2.15 Electroporation transformation 22 2.2.16 Virus-like particles (VLPs) production and purification 24 2.2.17 SDS-PAGE electrophoresis 26 2.2.18 Coomassie blue staining 28 2.2.19 RNA extraction from virus-like particles (VLPs) 28 2.2.20 In vitro transcription 29 2.2.21 RNA urea gel electrophoresis 30 2.2.22 SYBR green staining 31 2.2.23 Cell culture 32 2.2.24 Native PAGE electrophoresis 32 2.2.25 Competitive enzyme linked immunosorbent assays (ELISA) 33 2.2.26 MOTS-c purification by ammonium sulfate precipitation 34 2.2.27 MOTS-c purification by C18 column 35 2.2.28 Cell lysate extraction 37 2.2.29 Western blot 38 2.2.30 Direct enzyme linked immunosorbent assays (ELISA) 39 Chapter 3: Results 41 3.1 Construction of the [MOTS-c plus Qβ hairpin] plasmid 41 3.2 Production of the MOTS-c mRNA in vivo and in vitro 41 3.3 Development of the MOTS-c competition ELISA assay 43 3.3.1 rAb (anti-MOTS-c) titer between BSA and BSA-MOTS-c 44 3.4 Identification of the ex vivo production of the MOTS-c from the HeLa cells 44 Chapter 4: Discussions 47 4.1 Combination of the process for QβMOTS-c VLP 47 4.2 Detection of the cell produced MOTS-c peptides 48 Chapter 5: References 51 Chapter 6: Appendix 58 6.1 MOTS-c sequence (51 bp) 58 6.2 MOTS-c amino acid sequence (16 AA) 58 6.3 Sequence of MOTS-c with BamHI and SacI (63 bp) 58 6.4 Qβ hairpin 59 6.4.1 Qβ hairpin: Forward primer (43 bp) 59 6.4.2 Qβ hairpin: Reverse primer (43 bp) 59 6.5 pT7CFE1-(MOTS-c)-(Qβ hairpin) plasmid sequence 59 6.6 T7 sequences for PCR 63 6.6.1 T7 promoter: Forward primer (20 bp) 63 6.6.2 T7 terminator: Reverse primer (19 bp) 63 | |
| dc.language.iso | en | |
| dc.subject | 轉譯作用 | zh_TW |
| dc.subject | 類病毒顆粒 | zh_TW |
| dc.subject | 轉錄作用 | zh_TW |
| dc.subject | MOTS-c | zh_TW |
| dc.subject | 信使核糖核酸 | zh_TW |
| dc.subject | virus-like particle | en |
| dc.subject | MOTS-c | en |
| dc.subject | translation | en |
| dc.subject | mRNA | en |
| dc.subject | VLP | en |
| dc.subject | transcription | en |
| dc.title | 在哺乳類細胞中藉由Qβ類病毒顆粒快捷運輸平臺過度表現粒線體衍生性多肽(MOTS-c) | zh_TW |
| dc.title | Qβ Virus-Like Particles: An Accessible Delivery Platform for Overexpressing Mitochondrial-Derived Peptide MOTS-c in Mammalian Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張震東(Geen-Dong Chang),冀宏源(Hung-Yuan Chi) | |
| dc.subject.keyword | MOTS-c,類病毒顆粒,信使核糖核酸,轉譯作用,轉錄作用, | zh_TW |
| dc.subject.keyword | MOTS-c,virus-like particle,VLP,mRNA,transcription,translation, | en |
| dc.relation.page | 77 | |
| dc.identifier.doi | 10.6342/NTU201700127 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-01-23 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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