臺灣博碩士論文加值系統

在中東呼吸道症候群冠狀病毒 (MERS-CoV)的成熟複製過程中，需要兩種半胱胺酸蛋白水解酶進行多肽鏈剪切，其中之一為主要蛋白水解酶 (Mpro)，此蛋白水解酶對於病毒的複製成熟具有關鍵性，因此常作為抗病毒的理想研究目標。相較於嚴重急性呼吸道症候群冠狀病毒 (SARS-CoV)之主要水解酶，MERS-CoV主要水解酶在結構與功能的相關性仍尚未明瞭，即使兩者之間的胺基酸序列有50%的一致性，而根據先前SARS-CoV主要水解酶的研究，已知需要形成同二聚體才具有催化活性，且有受質誘發二聚體化現象 (substrate-induced dimerization)。在本論文研究中，首先用大腸桿菌表現並純化MERS-CoV主要水解酶，再分析其活性及利用分析型超高速離心機分析其四級結構與解離常數，有趣的是，有別於SARS-CoV主要蛋白水解酶，MERS-CoV主要蛋白水解酶以單體單元的方式存在於溶液中，這令我們想進一步了解其結構與功能相關性。由近期解出的MERS-CoV主要蛋白水解酶結構及序列比對發現，其結構雖與其他冠狀病毒之主要水解酶相似，但一些與二聚體化相關的胺基酸並不相同，因此突變這些胺基酸，並分析突變株的活性與四級結構，結果顯示若將Met-298突變成與SARS-CoV相對位置一樣的Arg，將可使蛋白水解酶形成穩定的同二聚體，而另一個胺基酸Glu-169，相當於SARS-CoV主要蛋白水解酶Glu-166，也同樣在MERS-CoV主要水解酶扮演受質誘導二聚體化的關鍵角色。

The Middle East respiratory syndrome coronavirus (MERS-CoV) main protease (Mpro) is one of two cysteine proteases involved in the proteolytic processing of the viron polyproteins which is required for virus maturation. Different to SARS-CoV, until now, its structure and function is still not fully characterized; albeit their sequence identity is 50%. Previous studies on SARS-CoV have suggested the functional form of Mpro is a dimer. Here, we expressed and purified MERS-CoV Mpro and then analyzed its quaternary structure and dissociation constant by analytical ultracentrifugation (AUC). Interestingly, different to SARS-CoV Mpro, MERS-CoV Mpro is a monomer. It attracts us to clarify the correlation between the structure and function of MERS-CoV Mpro. Besides, recent studies suggested that the crystal structure of MERS-CoV Mpro shares a similar scaffold to other coronaviral Mpro, although some residues in dimer interface are different. Therefore, we generated several mutants related to dimerization and studied their enzyme activity and dimerization. The results suggest that mutation of residue Met-298 to Arg can result in Mpro dimerization. Another residue Glu-169, the equivalent residue of Glu-166 in SARS-CoV Mpro, also plays an important role in substrate induced dimerization of MERS-CoV Mpro.

致謝、感謝 I
目錄 II
表目錄 IV
圖目錄 IV
中英文對照表 VII
縮寫表 VIII
摘要 IX
Abstract X
第一章、緒論 1
一、中東呼吸症候群 1
二、中東呼吸症候群冠狀病毒 1
三、中東呼吸症候群冠狀病毒之生命週期 3
四、冠狀病毒主要蛋白水解酶 4
五、研究目的與設計 5
第二章、實驗材料 7
一、藥品試劑 7
二、儀器設備 8
三、培養液與溶液配製 8
四、菌種 10
第三章、實驗方法 11
一、製備勝任細胞 (Competent cell) 11
二、大腸桿菌表現株之轉型 (Transform) 11
三、質體製備與純化 11
四、定點突變 (Site-directed mutagenesis, SDM) 12
五、重組蛋白質表現與純化 13
六、蛋白質定量 15
七、SDS-聚丙烯胺 (Sodium dodecyl sulfate-polyacrylamide)凝膠電泳法 15
八、MERS-CoV主要蛋白水解酶的酵素活性分析 16
九、恆溫滴定熱卡計 (Isothermal titration calorimetry, ITC) 17
十、分析型超高速離心機 (Analytical ultracentrifugation, AUC) 17
第四章、結果與討論 19
一、MERS-CoV主要蛋白水解酶與突變株的製備與純化 19
二、MERS-CoV主要蛋白水解酶之活性測定 20
三、MERS-CoV的受質誘導二聚體化 20
四、Glu-169在MERS-CoV的受質誘導二聚體化扮演重要角色 20
五、E169A單點突變株與野生型MERS-CoV Mpro對受質結合具有相近的親和力 21
六、V4R、T126S、M298R突變點的選擇 21
七、V4R、T126S、M298R突變株之活性測定 22
八、MERS-CoV主要蛋白水解酶與V4R、T126S、E169A、M298R突變株之解離常數 (Kd)之分析 22
九、活性與不含受質狀態下二聚體解離常數 (Kd)之比較 22
十、V4R/M298R、T126S/M298R雙突變株的活性與二聚體解離常數 (Kd)與單點突變株V4R、T126S之比較 23
第五章、圖表 24
表目錄
Table 1, Primer sequence list. 24
Table 2, The total protein quantity of MERS-CoV Mpro and mutants in 800 ml LB. 25
Table 3, Kinetic parameters and dissociation of MERS-CoV Mpro and mutants with and without substrates. 26
圖目錄
附圖 一、新型冠狀病毒之分類 (phylogenetic tree) 2
附圖 二、中東呼吸症候群冠狀病毒之基因組 (genome) 3
附圖 三、中東呼吸道症候群冠狀病毒之生活史 3

Figure 1.中東呼吸症候群冠狀病毒刺突蛋白 (Spike)與宿主細胞膜表面的第四型雙基胜肽酶 (DPP-4)結合 4
Figure 2.主要蛋白水解酶之三級結構 5
Figure 3. MERS-CoV與SARS-CoV主要蛋白水解酶之結構比較 6
Figure 4. Sequence alignment of various group 2a coronaviral Mpros. 27
Figure 5. Purification of MERS-CoV recombinant Mpro by size-exclusion chromatography after Ni-NTA agarose elution. 28
Figure 6. Purification of MERS-CoV recombinant Mpro by size-exclusion chromatography after Ni-NTA agarose elution. 29
Figure 7. Purification of MERS-CoV recombinant Mpro by size-exclusion chromatography after Ni-NTA agarose elution. 30
Figure 8. The pET 28a expression plasmid consists a histidine-tagged recombinant DNA. 31
Figure 9. The SDS-PAGE analysis of MERS-CoV recombinant Mpro 32

Figure 10. The SDS-PAGE analysis of MERS-CoV recombinant V4R Mpro mutant and T126S Mpro mutant. 33
Figure 11. The SDS-PAGE analysis of MERS-CoV recombinant M298R Mpro mutant. 34
Figure 12. The SDS-PAGE analysis of MERS-CoV recombinant V4R/M298R Mpro mutant and T126S/M298R Mpro mutant. 35
Figure 13. Initial velocity pattern of MERS-CoV Mpro and its mutants. 36
Figure 14. Initial velocity pattern of Mpro mutants. 37
Figure 15. Sedimentation Velocity patterns of MERS-CoV Mpro by AUC. 38
Figure 16. Isothermal calorimetric titration for the substrate TQ6-pNA binding to MERS-CoV Mpro (A) and E169A mutant (B). 39
Figure 17. The dimer interface of SARS-CoV Mpro. 40
Figure 18. Global analysis of the sedimentation velocity data of MERS-CoV Mpro without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 41
Figure 19. Global analysis of the sedimentation velocity data of MERS-CoV Mpro V4R mutant without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 42
Figure 20. Global analysis of the sedimentation velocity data of MERS-CoV Mpro T126S mutant without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 43
Figure 21. Global analysis of the sedimentation velocity data of MERS-CoV Mpro E169A mutant without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 44

Figure 22. Global analysis of the sedimentation velocity data of MERS-CoV Mpro M298R mutant without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 45
Figure 23. Global analysis of the sedimentation velocity data of MERS-CoV Mpro V4R/M298R mutant without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 46

Figure 24. Global analysis of the sedimentation velocity data of MERS-CoV Mpro T126S/M298R mutant without (A-C) and with 600 μM TQ6-pNA (D-F) at three protein concentrations. 47

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