臺灣博碩士論文加值系統

日本腦炎病毒在東亞地區廣泛流行,西起印度,東至日本都曾經發現日本腦炎病毒的感染病例. 在日本腦炎病毒中,其封套蛋白扮演著舉足輕重的角色,舉凡與寄主細胞的受器結合,免疫系統的反應,病毒本身的再製,以及處於低pH值的融合反應都與封套蛋白有關係. 其中,封套蛋白的第三區塊為一111個殘基,分子量約為12千道耳吞的蛋白質. 利用核磁共振儀,吾人得到此蛋白質之骨架序列光譜標定關係 (使用HNCA, HNCO, HN(CO)CA, HN(CA)CO, CBCANH, and CBCA(CO)NH 光譜). 每個殘基側鏈之氫光譜則是經由TOCSY-HSQC, HCCH-TCOSY,HCCH-COSY 以及H(CC)(CO)NH光譜實驗所觀測. 3JHNHA 耦合常數值是使用HNHA光譜標定. 除此之外,吾人亦完成了氫氘交換以及骨架動力學的光譜實驗. NOE光譜的關連性,氨基之氫氘,以及 3JHNHA 耦合常數以及化學位移索引之統合分析,此第三區塊蛋白質之二級結構可清楚地判斷出來. 日本腦炎病毒封套蛋白第三區塊之二級結構是由由七個beta-strands所組成,並且與免疫球蛋白G的二級結構相似.

Japanese encephalitis virus (JEV) is found throughout Asia from India in the west to Japan in the east. JEV envelope protein plays an important role in cell receptor binding, immunological response, virion assembly and fusion activity at low pH state. The domain III of JEV envelope protein is a 111 amino acid protein with a molecular weight of 12 kDa. The backbone sequential assignments were determined from six triple resonance experiments (HNCA, HNCO, HN(CO)CA, HN(CA)CO, CBCANH, and CBCA(CO)NH). Proton side chain assignments were obtained from TOCSY-HSQC, H(CC)(CO)NH, HCCH-TOCSY and HCCH-COSY. 3JHNHA coupling constants were accomplished based on HNHA experiment. Moreover, hydrogen-deuterium exchange experiments and backbone dynamics experiments were achieved. Secondary structures were identified via NOE correlations, amide exchange, 3JHNHA coupling constants, and CSI analysis. This protein is comprised of seven beta strands and shared as an immunoglobulin-G like domain.

Content
List of Figures III
List of Tables IV
Abbreviations V
Abstract (English Version) VI
Abstract (中文) VII
Chapter 1 Introduction: 1
1.1. Flaviviridae Family 1
1.2 Japanese Encephalitis Virus 1
1.3. Envelope protein of JEV 3
1.4. Summary. 4
Chapter 2 Materials and Methods 15
2.1. Cell culture 15
2.1.1 Non-isotope Labeled Condition. 15
2.1.2 Isotope Labeled Condition. 15
2.2. Sample Protein Preparations 17
2.2.1 pET32a clone 17
2.2.2 pET22b clone 18
2.3. Buffer Compositions 18
2.3.1 PBS (phosphate-buffered saline) 18
2.3.2 Wash buffer for S-tagged column 19
2.3.3 Elute buffer for S-tagged column 19
2.4. NMR Spectroscopy. 19
2.4.1 General information 19
2.4.2 NMR experiments 20
2.4.3 Dynamics experiments 21
2.4.4 Data processing and analysis 21
Chapter 3 Results 24
3.1. Backbone assignment 24
3.2. Side chain assignment 25
3.3. Secondary structure determination 26
3.4. NMR relaxation data and backbone dynamics 27
3.5. Hydrogen-deuterium exchange 28
Chapter 4
Discussions74
Reference 80

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