神經胺酸酶(neuraminidase [NA] )是A型流感病毒膜上的醣蛋白，可切割宿主細胞醣蛋白受器上與病毒血清凝集素 (hemagglutinin [HA] ) 連接的唾液酸 (sialic acid)，協助新複製出的病毒脫離宿主細胞，始之散播感染新細胞，先前文獻指出，流感疫苗誘發的NA抑制抗體能抑制病毒傳播並減緩症狀。在我們先前研究指出，pH1N1重組神經胺酸酶突變蛋白I365T/S366N疫苗誘發之抗體能抑制多種NA亞型，說明誘發廣效型抗體之抗原位點可能在活性位點周圍，因此本研究以此概念搭配pandemic H1N1 (pH1N1)(A/Texas/05/2009)、H3N2 (A/Udorn/307/1972)、H5N1 (A/Vietnam/1203/2004)和H7N9 (A/Shanghai/02/2013) NA亞型序列比對結果，在H5N1重組神經胺酸酶蛋白活性位點344、365、366和369胺基酸位置設計突變，利用點突變產生H5N1重組神經胺酸酶突變蛋白，其突變蛋白為Y344N、Y344H、T365I/N366S、T365E/N366D、N366A以及S369N，進一步探討H5N1重組神經胺酸酶突變蛋白是否能誘發廣效型神經胺酸酶抑制性抗體。結果顯示，H5N1重組神經胺酸酶突變蛋白無法誘發神經胺酸酶廣效型抑制性抗體抑制H5N1病毒株，我們希望這個發現能夠對於未來之廣效性神經胺酸酶疫苗發展提供有用的資訊。

Neuraminidase (NA) is an influenza virus envelope glycoprotein that cleaves sialic acids from cellular receptors and facilitates the virus release and spread from infected cells. NA-inhibiting (NI) antibodies are known to limit virus spreading and to mitigate clinical symptoms caused by influenza A virus infection. Previous report showed that cross reactive NI antibodies can be elicited by immunizations with pH1N1 NA proteins with I365T/S366N mutations (Liu et al., 2015). In this study, mutant H5N1 NA with mutations around the active site (344、365、366 and 369 NA residues) were examined according to sequence analyses. Cross-reactive NI antibodies elicited by the immunizations of mutant H5N1 NA proteins (Y344N、Y344H、T365I/N366S、T365E/N366D、N366A以及S369N). Our result shows that these six mutant H5N1 NA proteins did not elicit more cross-reactive NI antibodies against different NA subtypes (H1N1 NA, H5N1 NA, H3N2 NA and H7N9 NA). It is our hope that these findings provide useful information for the development of more cross-reactive NA-based influenza vaccines.

中文摘要 I
Abstract II
致謝 III
1. 簡介 1
1.1. 流感病毒 1
1.2. A型流感病毒 2
1.3. 人類與禽類A型流感病毒 3
1.4. A型流感神經胺酸酶 4
1.5. 神經胺酸酶抑制性抗體(Neuraminidase-inhibiting (NI) antibodies) 6
1.6. 研究目標 7
2. 材料與方法 9
2.1. 細胞株 9
2.2. Bac-to-Bac® Baculovirus 表現系統 9
2.2.1. 構築與表現流感病毒重組神經胺酸酶 (rNA) 9
2.2.2. 點突變聚合酶連鎖反應 (site-direct mutagenesis PCR)........................10
2.2.3. 構築以及生產Bacmids 12
2.2.4. 生產重組Baculovirus 14
2.3. 生產與純化重組神經胺酸酶 14
2.3.1. 可溶性神經胺酸酶之生產 14
2.3.2. 重組神經胺酸酶之純化...........................................................................15
2.4. 重組神經胺酸酶之特性測定…...………………………………………………………………...16
2.4.1. 十二烷基硫酸鈉聚丙烯醯胺凝膠(Sodium dodecyl sulfate polyacrylamide(SDS-PAGE))之準備及電泳 16
2.4.2. 西方墨點法(Western blotting) 17
2.5. 老鼠之免疫時程設計 17
2.6. 利用Fetuin-based酵素結合免疫吸附分析法(ELISA)測定神經胺酸酶抑制抗體(Neuraminidase-inhibiting (NI) antibodies)分析 18
2.7. 酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay (ELISA)...............................................................................................................19
2.8. 螢光素酶定量細胞融合測定………………………………………..……….......................19
2.9. 統計學分析 21
3. 結果 22
3.1. 表現和純化野生型pH1N1, H5N1, H3N2和H7N9病毒重組神經胺酸酶蛋白 22
3.2. 比對分析pH1N1, H5N1, H3N2和H7N9病毒神經胺酸酶序列......................23
3.3. H5N1 NA,pH1N1 NA, H3N2 NA和H7N9 NA蛋白結構 24
3.4. 生產以及純化H5N1重組神經胺酸酶突變蛋白(Y344N, Y344H, T365I/N366S, T365E/N366D, N366A,S369N)…..…………………………................................….....24
3.5. 分析各H5N1-rNA突變蛋白誘發的anti-NA IgG抗體效價 25
3.6. 分析各H5N1-rNA突變蛋白誘發的NI抗體效價.............................................25
3.7. 分析各H5N1-rNA突變蛋白誘發的抗體是否有anti-HA fusion的能力…...26
4. 討論…………………………………………..………………………………………………………..…..….…27
5. 圖表 30
Fig. 1表現重組水溶性神經胺酸酶蛋白（rNA） 30
Fig. 2 pH1N1, H5N1, H3N2和H7N9神經胺酸酶蛋白胺基酸序列比對….……..31
Fig. 3 pH1N1, H5N1, H3N2和H7N9神經胺酸酶的3D蛋白結構. 32
Fig. 4表現H5N1突變重組神經胺酸酶蛋白(Y344N, Y344H, T365I/N366S, T365E/N366D, N366A,S369N). 33
Fig. 5 H5N1突變重組神經胺酸酶蛋白疫苗誘發之IgG抗體效價(Y344N, Y344H, T365I/N366S, T365E/N366D, N366A,S369N)…………………………………..……..…34
Fig. 6由野生型或是突變型重組神經胺酸酶蛋白所誘導的N1神經胺酸酶抑制性抗體抑制病毒神經胺酸酶活性(Y344N, Y344H, T365I/N366S, T365E/N366D, N366A,S369N) 35
Fig. 7 測試H5N1重組神經胺酸酶蛋白誘發抗體之Anti-HA fusion活性(Y344N,Y344H, T365I/N366S, T365E/N366D, N366A,S369N)………………..… 36
Table. 1比對H5N1 rNA、pH1N1 rNA、H3N2 rNA和H7N9 rNA序列上344、365、366以及369胺基酸位點………………………………………………………………...…..37
Fig. S1描述H5N1突變重組神經胺酸酶蛋白(T365I/N366S/369A, T365I/N366S/S369K, T365I/N366S/S369N) 38
Fig. S2 H5N1 NA突變蛋白疫苗誘發之IgG抗體效價( T365I/N366S/369A, T365I/N366S/S369K, T365I/N366S/S369N) 39
Fig. S3由野生型或是突變型重組神經胺酸酶蛋白所誘導的N1神經胺酸酶抑制性抗體抑制病毒神經胺酸酶活性( T365I/N366S/369A, T365I/N366S/S369K, T365I/N366S/S369N) 40
6. 參考文獻 41

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