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  1. NTU Theses and Dissertations Repository
  2. 醫學院
  3. 醫學檢驗暨生物技術學系
請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89214
完整後設資料紀錄
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dc.contributor.advisor林靜宜zh_TW
dc.contributor.advisorJing-Yi Linen
dc.contributor.author白念平zh_TW
dc.contributor.authorNain-Ping Baien
dc.date.accessioned2023-09-05T16:08:31Z-
dc.date.available2023-11-09-
dc.date.copyright2023-09-05-
dc.date.issued2023-
dc.date.submitted2023-08-07-
dc.identifier.citationUncategorized References
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/89214-
dc.description.abstract嚴重急性呼吸道冠狀病毒2型 ( Severe acute respiratory syndrome coronavirus 2,SARS-CoV-2 )造成嚴重特殊傳染性肺炎( Coronavirus disease 2019,COVID-19 )並造成全球大流行。因此了解病毒與宿主的相互作用有利於抗病毒藥物的研發。其中病毒的3CL蛋白酶是由病毒基因組中的開放閱讀框( ORF1a )轉譯出來的蛋白質,其分子量為33 kDa,具有半胱胺酸( Cysteine )活性。其結構中包含組氨酸( Histidine 41,His 41 )和半胱胺酸( Cysteine 145,Cys145 )形成的催化二元組( catalytic dyad )。在不同冠狀病毒間的3CL序列具有高度保留性因此常作為藥物靶點之一。目前已知3CL蛋白酶除了可藉由切割病毒多蛋白質產生出多個非結構蛋白質來幫助病毒複製。此外3CL蛋白酶也可切割宿主蛋白質影響宿主免疫反應及病毒複製。但目前對於3CL蛋白酶和宿主蛋白質間如何影響病毒複製的詳細機制還有很多是未知的,因此本研究想探討有那些宿主蛋白質可被SARS-CoV-2的3CL蛋白酶切割以及探討其對病毒複製及宿主的影響。論文主軸分成三個部分來建立3CL蛋白酶的切割系統。第一部分是建立體外切割系統:首先建構具有蛋白酶活性的3CL蛋白酶質體和不具蛋白酶活性的3CL-C145A蛋白酶質體且利用大腸桿菌系統純化出3CL蛋白質,並使用酵素活性測試3CL蛋白質活性及已知可被SARS-CoV-2 3CL切割的宿主蛋白質p62、SNX6和DCP1A進行不同體外切割溶液系統的測試。結果顯示以250 ug的Calu-3細胞萃取液和體外切割溶液A和10 ug的SARS-CoV-2-3CL-WT跟SARS-CoV-2-3CL-C145A蛋白質一起於37度下反應12小時為我們最佳體外切割系統條件,這部分也將此反應條件的樣品進行質譜分析以鑑定出可被3CL蛋白酶切割的宿主蛋白質。第二部分是建立在293TACE2細胞中大量表達SARS-CoV-2-3CL和SARS-CoV-2-3CL-C145A質體的細胞模式觀察宿主蛋白質p62、SNX6和DCP1A是否有蛋白質降解現象。結果顯示當大量表達3CL時,DCP1A出現預期的切割產物,而p62和SNX6則無影響。第三部分是建立病毒感染之細胞模式是否有切割現象。結果顯示以0.1 MOI SARS-CoV-2感染CaLu-3細胞24和48小時後,p62、SNX6和DCP1A的蛋白質量減少。同時在0.1 MOI的HCoV-229E病毒感染A549細胞24和48小時後,p62和SNX6的蛋白質量減少,且DCP1A出現預期的切割產物。整合此研究結果我們建立3CL蛋白質在體外及體內的切割系統,未來有利於探討病毒與宿主相互作用機轉。zh_TW
dc.description.abstractSevere acute respiratory syndrome coronavirus 2 ( SARS-CoV-2 ) causes Coronavirus disease 2019 ( COVID-19 ) and global pandemic. Therefore, understanding the interaction between viruses and their hosts is beneficial for the development of antiviral drugs. The SARS-CoV-2 3CL protease is a protein translated from the open reading frame (ORF1a) in the viral genome. It has a molecular weight of 33 kDa and possesses Cysteine activity. Its structure contains a catalytic dyad formed by Histidine 41 ( His 41 ) and Cysteine 145 ( Cys145 ). Other research demonstrated that 3CL protease can cleavage viral polyprotein to help viral replication and it is highly conserved in β-coronaviruses. Therefore, it has been recognized as a key target for preventing and treating COVID-19. Increasing evidence demonstrated that 3CL protease can cleavage host proteins and further affect host immune response and viral replication. However, the detailed mechanism of how the SARS-CoV-2 3CL protease and host proteins affect viral replication is still unknown. Therefore, this study wants to identify which host proteins can be cleaved by the SARS-CoV-2 3CL protease and investigate the mechanism of viral replication and host. The main focus of the paper is divided into three parts to establish the cleavage system of the 3CL protease.. The first part involves the establishment of an in vitro cleavage system. Firstly, we constructed a 3CL protease expression plasmid with proteinase activity and a 3CL-C145A protease expression plasmid without proteinase activity. Subsequently, we purified the 3CL protein using the Escherichia coli system. Enzymatic activity assays are performed to evaluate the activity of the 3CL protein, as well as to test different in vitro cleavage buffer systems using known host proteins p62, SNX6 and DCP1A, which can be cleaved by SARS-CoV-2 3CL protease. The results indicate that the optimal in vitro cleavage system for us was to incubate 250 ug of Calu-3 cell lysate with in vitro cleavage buffer A and 10 μg of SARS-CoV-2-3CL-WT and SARS-CoV-2-3CL-C145A proteins together at 37°C for 12 hours. Subsequently, samples from this reaction condition were sent for Mass spectrometry analysis to identify host proteins that can be cleavage by the 3CL protease. The second part involves the establishment of a cell model in 293TACE2 cells to overexpress the SARS-CoV-2-3CL and SARS-CoV-2-3CL-C145A plasmids. This allows the observation whether there is protein degradation of host proteins p62, SNX6, and DCP1A.. The results show that when 3CL is overexpressed, DCP1A exhibits the expected cleavage products, while p62 and SNX6 remain unaffected. The third part investigates the occurrence of cleavage in cell models infected with the virus. It is found that when CaLu-3 cells are infected with 0.1 MOI SARS-CoV-2 for 24 and 48 hours, the protein levels of p62, SNX6 and DCP1A decreased. Similarly, when A549 cells are infected with 0.1 MOI HCoV-229E for 24 and 48 hours also results in decreased protein levels of p62 and SNX6, accompanied by the appearance of cleavage products in DCP1A. Based on the results of this study, we have established an in vitro and in vivo cleavage system for the 3CL protein. This system will be beneficial for investigating the mechanisms underlying the interaction between the virus and the host.en
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dc.description.tableofcontents口試委員審定書 i
致謝 ii
中文摘要 iii
Abstract v
目錄 viii
第一章 前言 1
1-1 嚴重急性呼吸道冠狀病毒2型與嚴重特殊傳染性肺炎 1
1-1-1 流行病學 1
1-1-2 病毒變異株 1
1-1-3 COVID-19臨床症狀 2
1-1-4 COVID-19預防及治療方式 3
1-2 嚴重急性呼吸道冠狀病毒2型( SARS-CoV-2 )特性 4
1-2-1 SARS-CoV-2簡介 4
1-2-2 病毒的分類及結構 4
1-2-3 病毒生命週期 5
1-2-3-1 病毒的進入( virus entry ) 5
1-2-3-2 病毒的內化( internalization )及融合( fusion ) 5
1-2-3-3 病毒的複製( replication ) 6
1-2-3-4 病毒的組裝( assemble )及出芽( budding ) 6
1-2-4 3C樣蛋白酶( 3C-like protease,3CL )特性 7
1-2-5 3CL蛋白酶切割宿主蛋白質後對SARS-CoV-2複製及宿主本身的影響 8
1-2-6 SARS-CoV-2-3CL蛋白酶之應用 9
第二章 研究目的 11
第三章 實驗方法與材料 12
3-1實驗材料 12
3-1-1細胞 12
3-2實驗方法 14
3-2-1 細胞培養 14
3-2-2 人類冠狀病毒229E培養與放大 15
3-2-3 病毒效價測定( plaque assay ) 15
3-2-4 收取229E感染A549細胞之萃取液( cell lysate ) 16
3-2-5 西方墨點法 16
3-3 建構pET-21a-SARS-CoV-2-3CL、pET-21a-SARS-CoV-2-3CL-C145A質體 17
3-3-1 大量繁殖pET-21a-SARS-CoV-2-3CL以及pET-21a-SARS-CoV-2-3CL-C145A質體的菌種 17
3-3-2 抽取質體 17
3-3-3 聚合酶連鎖反應( Polymerase chain reaction ) 18
3-3-4 定點突變聚合酶連鎖反應( Site Directed Mutagenesis PCR) 18
3-3-5 膠體純化( Gel extraction ) 19
3-3-6 酶解作用( Enzyme digestion ) 19
3-3-7 接合( Ligation ) 20
3-3-8 形質轉型作用(Transformation) 20
3-4 在pET-21a系統表達之SARS-CoV-2-3CL-WT、SARS-CoV-2-3CL-C145A蛋白質純化及表現 20
3-4-1 IPTG蛋白質誘導 20
3-4-2 在pET-21a系統表達之SARS-CoV-2-3CL-WT和SARS-CoV-2-3CL-C145A蛋白質純化 21
3-4-3 將純化出的蛋白質進行透析( dialysis ) 21
3-5 以快速蛋白質液相色譜法( Fast protein liquid chromatography,FPLC )純化在pET-29a系統之SARS-CoV-2-3CL-WT跟SARS-CoV-2-3CL-C145A蛋白質 21
3-6 體外切割試驗 22
3-6-1 CaLu-3細胞萃取液製備 22
3-6-2 濃度測定 23
3-6-3體外切割實驗結合西方墨點法偵測 23
3-7在293TACE2中過表達SARS-CoV-2-3CL及SARS-CoV-2-3CL-C145A觀察宿主蛋白質切割之細胞模式建立 23
3-7-1 建構pRH-EV-A71-5’ UTR-SARS-CoV-2-3CL、pRH-EV-A71-5’ UTR -SARS-CoV-2-3CL-C145A質體 23
3-7-2 轉染( Transfection ) pRH-EV-A71-5’ UTR-SARS-CoV-2-3CL、pRH-EV-A71-5’ UTR -SARS-CoV-2-3CL-C145A質體 24
3-8 SARS-CoV-2 3CL蛋白酶切割活性測試 25
第四章 結果 26
4-1 在原核E.Coli系統中建構pET-21a-SARS-CoV-2-3CL及pET-21a- SARS-CoV-2-3CL-C145A質體 26
4-2 以SDS-PAGE和西方墨點法確認SARS-CoV-2-3CL-WT及SARS-CoV-2-3CL-C145A的表現 26
4-3 SARS-CoV-2 3CL蛋白酶活性測試 27
4-4 高濃度的SARS-CoV-2-3CL-WT蛋白質具有切割活性但仍切割效率不佳 28
4-5 以SDS-PAGE確認Fast protein liquid chromatography ( FPLC )方法純化出的SARS-CoV-2-3CL-WT及 SARS-CoV-2-3CL-C145A蛋白質表現 28
4-6 FPLC方法純化的在pET-21a系統表達之SARS-CoV-2-3CL-WT蛋白質活性極低而SARS-CoV-2-3CL-C145A蛋白質則無活性 29
4-7 以SDS-PAGE確認在pET-29a系統的SARS-CoV-2-3CL-WT及 SARS-CoV-2-3CL-C145A蛋白質表現並進行蛋白酶切割活性測試 29
4-8 SARS-CoV-2-3CL-WT在體外切割溶液A和B中可切割DCP1A 30
4-9 SARS-CoV-2-3CL-WT在體外切割溶液A中可切割DCP1A及減少p62和SNX6蛋白質量 31
4-10 在293TACE2細胞中建構出pRH-EV-A71-5’UTR -SARS-CoV-2-3CL及pRH-EV-A71-5’UTR - SARS-CoV-2-3CL-C145A質體並確認其表現 33
4-11 在293TACE2細胞中大量表達SARS-CoV-2-3CL時觀察到DCP1A 有切割產物的出現而表達SARS-CoV-2-3CL-C145A時皆沒有變化 34
4-12 DCP1A、p62及SNX6蛋白質在SARS-CoV-2感染下隨時間延長有減少現象 34
4-13 在HCoV-229E感染下會造成SNX6及p62蛋白質表現量降低及DCP1A有切割產物 35
第五章 討論 37
5-1 比較找尋SARS-CoV-2-3CL蛋白酶之宿主蛋白質方法學優缺點 37
5-2 分析實驗結果和已知文獻結果的差異處 38
5-3不同大腸桿菌系統pET-21a跟pET-29a的比較 39
第六章 圖表 41
表一、本研究使用的抗體 41
表二、本研究使用的引子 42
表三、可被SARS-CoV-2 3CL蛋白酶切割的宿主蛋白質 43
圖一.在原核E.coli系統中建構出pET-21a-SARS-CoV-2-3CL及pET-21a- SARS-CoV-2-3CL-C145A質體 44
圖二. 以SDS-PAGE和西方墨點法確認SARS-CoV-2-3CL-WT及 SARS-CoV-2-3CL-C145A的表現 45
圖三. SARS-CoV-2-3CL蛋白酶切割活性測試 47
圖四.以切割溶液A和B透析後的SARS-CoV-2-3CL-WT蛋白質在50 μg量具有活性 48
圖五. 以SDS-PAGE確認FPLC方法純化出的SARS-CoV-2-3CL-WT及 SARS-CoV-2-3CL-C145A蛋白質表現 49
圖六. FPLC方法純化出的SARS-CoV-2-3CL-WT蛋白質活性極低而SARS-CoV-2-3CL-C145A蛋白質則無活性 50
圖七. 以SDS-PAGE確認在pET-29a系統的SARS-CoV-2-3CL-WT及 SARS-CoV-2-3CL-C145A蛋白質表現 51
圖八. 在pET-29a系統的SARS-CoV-2-3CL-WT蛋白質具有活性而SARS-CoV-2-3CL-C145A蛋白質則沒有 52
圖九. SARS-CoV-2-3CL-WT在體外切割溶液A和B中可切割DCP1A 53
圖十. SARS-CoV-2-3CL在體外切割溶液A中可切割DCP1A和減少p62及SNX6蛋白質量 55
圖十一.在293TACE2細胞中建構出pRH-EV-A71-5’UTR -SARS-CoV-2-3CL及pRH-EV-A71-5’UTR - SARS-CoV-2-3CL-C145A質體並確認其表現 57
圖十二. 在293TACE2細胞中大量表達SARS-CoV-2-3CL時觀察到DCP1A 有切割產物出現而表達SARS-CoV-2-3CL-C145A時皆沒有變化 59
圖十三. DCP1A、 p62及SNX6蛋白質在SARS-CoV-2感染下隨時間延長有減少現象 60
圖十四. 在HCoV-229E感染下會造成SNX6及p62蛋白質表現量量降低及DCP1A有切割產物 62
第七章 參考文獻 63
-
dc.language.isozh_TW-
dc.title建立嚴重急性呼吸道冠狀病毒第2型3CL蛋白酶體外及體內切割系統zh_TW
dc.titleDevelopment of in vitro and in vivo SARS-CoV-2 3CL protease cleavage systemsen
dc.typeThesis-
dc.date.schoolyear111-2-
dc.description.degree碩士-
dc.contributor.oralexamcommittee張淑媛;顏莉蓁;黃幸宜zh_TW
dc.contributor.oralexamcommitteeSui-Yuan Chang;Li-Chen Yen;Hsing-I Huangen
dc.subject.keyword嚴重特殊傳染性肺炎,宿主蛋白質,SARS-CoV-2-3CL蛋白酶,體外切割試驗,蛋白質體學,zh_TW
dc.subject.keywordCOVID-19,host proteins,SARS-CoV-2-3CL proease,In vitro cleavage assay,protemics,en
dc.relation.page72-
dc.identifier.doi10.6342/NTU202302837-
dc.rights.note未授權-
dc.date.accepted2023-08-07-
dc.contributor.author-college醫學院-
dc.contributor.author-dept醫學檢驗暨生物技術學系-
顯示於系所單位:醫學檢驗暨生物技術學系

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