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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 朱雪萍 | zh_TW |
| dc.contributor.advisor | Hsueh-Ping Chu | en |
| dc.contributor.author | 徐榮澤 | zh_TW |
| dc.contributor.author | JUNG-TSE HSU | en |
| dc.date.accessioned | 2024-09-15T16:44:20Z | - |
| dc.date.available | 2024-09-16 | - |
| dc.date.copyright | 2024-09-14 | - |
| dc.date.issued | 2024 | - |
| dc.date.submitted | 2024-08-14 | - |
| dc.identifier.citation | 1. Al-Qahtani, A. A. (2022). Mutations in the genome of severe acute respiratory syndrome coronavirus 2: implications for COVID-19 severity and progression. J Int Med Res, 50(3), 3000605221086433. https://doi.org/10.1177/03000605221086433
2. An, W., Dai, L., Niewiadomska, A. M., Yetil, A., O'Donnell, K. A., Han, J. S., & Boeke, J. D. (2011). Characterization of a synthetic human LINE-1 retrotransposon ORFeus-Hs. Mob DNA, 2(1), 2. https://doi.org/10.1186/1759-8753-2-2 3. Baldwin, E. T., van Eeuwen, T., Hoyos, D., Zalevsky, A., Tchesnokov, E. P., Sanchez, R., Miller, B. D., Di Stefano, L. H., Ruiz, F. X., Hancock, M., Isik, E., Mendez-Dorantes, C., Walpole, T., Nichols, C., Wan, P., Riento, K., Halls-Kass, R., Augustin, M., Lammens, A., . . . Taylor, M. S. (2024). Structures, functions and adaptations of the human LINE-1 ORF2 protein. Nature, 626(7997), 194-206. https://doi.org/10.1038/s41586-023-06947-z 4. Briggs, E. M., McKerrow, W., Mita, P., Boeke, J. D., Logan, S. K., & Fenyo, D. (2021). RIP-seq reveals LINE-1 ORF1p association with p-body enriched mRNAs. Mob DNA, 12(1), 5. https://doi.org/10.1186/s13100-021-00233-3 5. Cost, G. J., Feng, Q., Jacquier, A., & Boeke, J. D. (2002). Human L1 element target-primed reverse transcription in vitro. EMBO J, 21(21), 5899-5910. https://doi.org/10.1093/emboj/cdf592 6. Kazachenka, A., & Kassiotis, G. (2021). SARS-CoV-2-Host Chimeric RNA-Sequencing Reads Do Not Necessarily Arise From Virus Integration Into the Host DNA. Front Microbiol, 12, 676693. https://doi.org/10.3389/fmicb.2021.676693 7. Khalid, K., & Poh, C. L. (2023). The development of DNA vaccines against SARS-CoV-2. Adv Med Sci, 68(2), 213-226. https://doi.org/10.1016/j.advms.2023.05.003 8. Kines, K. J., Sokolowski, M., deHaro, D. L., Christian, C. M., Baddoo, M., Smither, M. E., & Belancio, V. P. (2016). The endonuclease domain of the LINE-1 ORF2 protein can tolerate multiple mutations. Mob DNA, 7, 8. https://doi.org/10.1186/s13100-016-0064-x 9. Lee, S., Lee, Y. S., Choi, Y., Son, A., Park, Y., Lee, K. M., Kim, J., Kim, J. S., & Kim, V. N. (2021). The SARS-CoV-2 RNA interactome. Mol Cell, 81(13), 2838-2850 e2836. https://doi.org/10.1016/j.molcel.2021.04.022 10. Loubalova, Z., Konstantinidou, P., & Haase, A. D. (2023). Themes and variations on piRNA-guided transposon control. Mob DNA, 14(1), 10. https://doi.org/10.1186/s13100-023-00298-2 11. Machhi, J., Herskovitz, J., Senan, A. M., Dutta, D., Nath, B., Oleynikov, M. D., Blomberg, W. R., Meigs, D. D., Hasan, M., Patel, M., Kline, P., Chang, R. C., Chang, L., Gendelman, H. E., & Kevadiya, B. D. (2020). The Natural History, Pathobiology, and Clinical Manifestations of SARS-CoV-2 Infections. J Neuroimmune Pharmacol, 15(3), 359-386. https://doi.org/10.1007/s11481-020-09944-5 12. Reece-Hoyes, J. S., & Walhout, A. J. M. (2018). Gateway Recombinational Cloning. Cold Spring Harb Protoc, 2018(1), pdb top094912. https://doi.org/10.1101/pdb.top094912 13. Smits, N., Rasmussen, J., Bodea, G. O., Amarilla, A. A., Gerdes, P., Sanchez-Luque, F. J., Ajjikuttira, P., Modhiran, N., Liang, B., Faivre, J., Deveson, I. W., Khromykh, A. A., Watterson, D., Ewing, A. D., & Faulkner, G. J. (2021). No evidence of human genome integration of SARS-CoV-2 found by long-read DNA sequencing. Cell Rep, 36(7), 109530. https://doi.org/10.1016/j.celrep.2021.109530 14. Zhang, L., Richards, A., Barrasa, M. I., Hughes, S. H., Young, R. A., & Jaenisch, R. (2021). Reverse-transcribed SARS-CoV-2 RNA can integrate into the genome of cultured human cells and can be expressed in patient-derived tissues. Proc Natl Acad Sci U S A, 118(21). https://doi.org/10.1073/pnas.2105968118 15. Zhang, X., Zhang, R., & Yu, J. (2020). New Understanding of the Relevant Role of LINE-1 Retrotransposition in Human Disease and Immune Modulation. Front Cell Dev Biol, 8, 657. https://doi.org/10.3389/fcell.2020.00657 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/95674 | - |
| dc.description.abstract | 嚴重急性呼吸道症候群冠狀病毒2型 (SARS-CoV-2) 在2020年大量爆發並導致世界各地頻發死亡病例，主要是透過上呼吸道接觸細胞表面的血管收縮素轉化酶2(ACE2)受體感染細胞，導致肺部功能障礙甚至可能導致死亡。然而，在Rudolf Jaenisch, April 19, 2021 的論文中他們用奈米孔定序發現病患身上有人類與冠狀病毒2型的序列嵌合體。這個驚人的發現意味著有某個特殊分子的存在可以幫助冠狀病毒2型的RNA反轉錄成DNA，並嵌入進病患的基因組之中，不過嵌入的DNA都只有部分序列並非全長，也尚未發現是否會對患者造成影響。先前我們的研究發現被冠狀病毒2型感染的細胞會顯著提升長散在核元件 (Long interspersed nuclear element 1, LINE-1)RNA的表現量，藉由直接RNA互作蛋白質鑑定質譜法(iDRiP-MS)發現冠狀病毒2型會與長散在核元件(Long interspersed nuclear element 1, LINE-1)內所編碼的蛋白質(LINE1-ORF2p) 有交互作用。
因此本篇主要想更深入探討LINE-1所編碼的蛋白質（LINE1-ORF2p）是否是幫助冠狀病毒2型基因嵌入的主要因子，我以人胚腎293T細胞(HEK293T)過來表現LINE-1所編碼的蛋白質LINE1-ORF2p並進行純化，過程中我嘗試改變可能的影響因素，包括：實驗的細節步驟、不同來源的抗體、高通量基因克隆(Gateway Cloning)產生的新質體，但是卻始終無法有效產出蛋白。考慮到LINE1-ORF2p在自然界哺乳類動物的細胞中大多是被抑制的狀態，想更換整體的蛋白表現系統，藉由使用桿狀病毒表現系統(Bac-to-Bac expression system)來表現LINE1-ORF2p蛋白於昆蟲細胞(hi five)免於被細胞自身的防禦系統辨識。如果可以順利產出蛋白，我們就可以在體外測試其是否可幫助冠狀病毒2型的RNA進行嵌入作用。 | zh_TW |
| dc.description.abstract | Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a massive outbreak in 2020, leading to frequent fatalities worldwide. The virus primarily infects cells through the upper respiratory tract by binding to the angiotensin-converting enzyme 2 (ACE2) receptor on the cell surface, causing lung dysfunction and potentially leading to death. It was reported by Rudolf Jaenisch ‘s group that chimeric sequences of human and SARS-CoV-2 in patients was observed from long-read nanopore sequencing. This surprising finding suggests the presence of a particular molecule that facilitates the reverse transcription of SARS-CoV-2 RNA into DNA and its integration into the host genome. The integrated DNA consists only of partial sequences, not the full length, and it is not yet known if this integration affects the patients. In our previous research, the expression of Long Interspersed Nuclear Element-1 (LINE-1) was upregulated significantly in Calu3 cells infected with SARS-CoV-2. According to the results from Chia-Yu Guh, using Identification of Direct RNA-Interacting Proteins-Mass Spectrometry (iDRiP-MS), it was shown that LINE-1 open reading frame 2 protein (LINE1-ORF2p) interacts with SARS-CoV-2. To investigate whether LINE1-ORF2p is a key factor in assisting the integration of SARS-CoV-2 genes, I tried to overexpress and purify the ORF2 protein in human embryonic kidney 293T (HEK293T) cells. However, ORF2 protein was unable to be expressed in HEK293 cells. Given that LINE elements are usually repressed but derepressed upon inflammation and infection, I will change the protein expression system by using the Bac-to-Bac expression system to overexpress L1 ORF2p in insect cells (HiFive) after virus infection, and use the synthetic ORFeus-Hs L1 with modified sequences for expressing ORF2 protein. If ORF2 protein is successfully produced, I can test whether it directly facilitates the integration of SARS-CoV-2 RNA in vitro. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2024-09-15T16:44:20Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2024-09-15T16:44:20Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 中文摘要 iii Abstract v Content vii Chapter 1 Introduction 1 1-1 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) 1 1-2 Long Interspersed Nuclear Element-1 2 1-3 Previous research on LINE1's role in the genetic integration of SARS-CoV-2 4 Chapter 2 Materials and Methods 5 2-1 SARS-CoV2 cDNA synthesis. 5 2-2 Amplifcations of SARS-CoV-2 1000bp, 351bp, LINE1 351bp, ORF2p sequences for cloning 6 2-3 TA cloning 7 2-4 Transformation 8 2-5 Gateway cloning 8 2-6 Cell culture and transfection 10 2-7 Cell lysis and protein purification 10 2-8 Western blot 11 2-9 RNA transcription in vitro and RNA electrophoresis 14 2-10 Target-Primed Reverse Transcription (TPRT) 15 2-11 Bac-to-Bac Baculovirus Expression System 15 Chapter 3 Results 16 3-1 Constructions of LINE1.3 and SARS-CoV-2 into expression vectors 16 3-2 RNA transcription in vitro 17 3-3 L1 ORF2 protein purification 18 3-4 Overexpressing ORF2p using plasmids generated by Gateway cloning 19 3-5 Producing ORF2p using the Bac-to-Bac Baculovirus Expression System 20 Chapter 4 Discussion 21 Chapter 5 Supplementary data 29 Chapter 6 Reference 34 | - |
| dc.language.iso | en | - |
| dc.subject | 嚴重急性呼吸道症候群冠狀病毒2型 | zh_TW |
| dc.subject | SARS-CoV-2 | en |
| dc.title | 探討SARS-CoV2病毒是否經由ORF2蛋白將病毒嵌入宿主DNA中 | zh_TW |
| dc.title | Investigation of SARS-CoV-2 integration into host genome via ORF2 | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 112-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 冀宏源;吳青錫 | zh_TW |
| dc.contributor.oralexamcommittee | Hung-Yuan Chi;Ching-Shyi Wu | en |
| dc.subject.keyword | 嚴重急性呼吸道症候群冠狀病毒2型, | zh_TW |
| dc.subject.keyword | SARS-CoV-2, | en |
| dc.relation.page | 35 | - |
| dc.identifier.doi | 10.6342/NTU202403995 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2024-08-14 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 分子與細胞生物學研究所 | - |
| 顯示於系所單位： | 分子與細胞生物學研究所 | |
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