臺灣博碩士論文加值系統

家禽流行性感冒(Avian Influenza, AI)是由A型流行性感冒病毒所引起之全身性或呼吸性感染的疾病。依其表面醣蛋白抗原血球凝集素(Hemagglutinin, HA)可分為15種血清亞型(H1~H15)、而依神經胺酸酶(Neuraminidase, NA)亦可分為9種血清亞型(N1~N9)。其中H5及H7可能為強毒型AI病毒，感染後可能會造成嚴重的全身症狀及高死亡率。傳統上區分AI病毒血清亞型的方法為血球凝集抑制試驗(hemagglutination inhibition test, HI)，但此方法缺乏敏感性且耗時。本研究利用H5血清亞型之HA重組蛋白，經E. coli BL21(DE3)表現後純化蛋白免疫BALA/c小鼠，製備出H5血清亞型之HA單源抗體，單源抗體經純化後進行競爭性ELISA (competitive enzyme-linked immunosorbent assay, CELISA)之試驗，以期能建立強毒型AI病毒H5血清亞型之快速診斷技術。實驗結果顯示H5血清亞型之單源抗體僅能被H5血清亞型之高免血清所競爭。且有極高的特異性。H5高免血清在512HI抗體力價與單源抗體10號競爭結果有91.5的競爭百分比。而與單源抗體11號的試驗結果，有86.1的競爭百分比。因此利用原核系統表現之H5血清亞型HA重組蛋白，配合以H5血清亞型重組蛋白製備之單源抗體所建立之競爭性ELISA套組，可用於偵測H5強毒型AI之抗血清，此一診斷技術的建立將可協助高病原性H5血清亞型AI病毒之診斷。

Avian Influenza (AI) is caused by type A influenza virus, which might cause a systemic or respiratory infection and disease. According to their surface glycoprotein, fifteen HA subtypes (HA1-HA15) and nine NA subtypes (NA1-NA9) have been identified. Avian influenza viruses of subtypes H5 and H7 might be highly pathogenic, which cause a severe systemic disease and a high mortality after infection. The conventional method for differentiating HA subtypes of AIV is hemagglutination inhibition test (HI test), which is both time-consuming and insensitive. In this study, we used E. coli BL21 (DE3) as the host to express recombinant HA protein of H5 subtype, and immunized BALB/c mice with the recombinant HA protein to prepare monoclonal antibody against the HA protein. After purification, the recombinant HA protein and the monoclonal antibody were used to develop a competitive-ELISA that could differentiate the antisera of H5 subtype from antisera other subtypes. Our results show that, for this competitive-ELISA, the binding of H5 monoclonal antibodies could be specifically competed by H5-specific serum. The H5-specific serum (512 HI), showed 91.5% and 86.1% of competition when competed with monoclonal antibody number 10 and number 11. Therefore, the competitive-ELISA developed by using recombinant HA proteins and monoclonal antibody, might serve as a tool for rapid identification of highly pathogenic influenza virus of the H5 subtype.

中文摘要…………………………………………………………………. Ⅰ
英文摘要…………………………………………………………………. Ⅱ
目次………………………………………………………………………. Ⅲ
表次………………………………………………………………………. Ⅴ
圖次………………………………………………………………………. Ⅵ
第一章 緒言……………………………………………………………. 1
第二章 文獻探討………………………………………………………. 2
2-1 歷史背景…………………………………………………... 2
2-2 病毒特徵…………………………………………………... 6
2-3 結構與功能………………………………………………... 7
2-3.1 血球凝集素蛋白…………………………………… 8
2-3.2 神經胺酸酶………………………………………… 9
2-3.3 核蛋白……………………………………………… 9
2-3.4 聚合酶蛋白………………………………………… 9
2-3.5 基質蛋白…………………………………………… 10
2-3.6 非結構蛋白………………………………………… 10
2-4 病毒複製…………………………………………………... 10
2-5 診斷方法…………………………………………………... 11
2-5.1 偵測抗原………………………………………….... 11
2-5.2 偵測抗體…………………………………………… 13
2-6 單源抗體…………………………………………………... 14
2-6.1單源抗體之歷史背景………………………………... 14
2-6.2融合瘤細胞篩選之原理……………………………... 14
2-6.3 單株抗體之特性…………………………………….. 15
第三章 材料與方法……………………………………………………. 16
3-1 病毒的增殖與純化………………………………………... 16
3-1.1 病毒來源………………………………………........ 16
3.1.2 病毒增殖………………………………………........ 16
3.1.3 病毒純化………………………………………........ 16
3.1.4 病毒抗原力價測定………………………………… 17
3-1.5 應用RT-PCR確認純化之病毒…………………… 17
3-2 重組蛋白之表現與確認…………………………………... 19
3-2.1 重組蛋白之表現…………………………………… 20
3-2.2 重組蛋白之純化…………………………………… 20
3-2.3 重組蛋白之電泳分析與確認……………………… 21
3-2.4 重組蛋白特異性分析與確認……………………… 21
3-3 單源抗體之製備………………………………………....... 22
3-3.1 小鼠免疫計畫……………………………………… 22
3-3.2 骨髓瘤細胞培養…………………………………… 22
3-3.3 細胞融合………………………………………........ 23
3-3.4 融合瘤細胞篩選…………………………………… 23
3-3.5 融合瘤細胞單株化………………………………… 24
3-3.6 融合瘤細胞之確認………………………………… 24
3-3.7 腹水之生產……………………………………….... 25
3-3.8 單源抗體之純化…………………………………… 25
3-3.9 純化之單源抗體特異性分析……………………… 26
3-4 競爭性酵素連結免疫吸附分析法之架構………………... 27
3-4.1 以棋盤式力價測定法決定單源抗體與重組HA蛋白之最佳稀釋倍數………………………………... 27
3-4.2 家禽流行性感冒病毒H5血清亞型之高免血清與單原抗體之CELISA試驗………………………... 27
3-4.3 單原抗體與15種血清亞型之家禽流行性感冒高免血清CELISA試驗……………………………... 28
3-4.4 應用於偵測野外陰性血清建立CLISA陰性競爭百分比(competition percentage)…………………… 28
第四章 結果………………………………………................................. 29
4-1 病毒純化之結果……………………………………………. 29
4-2 重組蛋白之表現與確認……………………………………. 29
4-3融合瘤細胞製備之結果…………………………………….. 29
4-4 單原抗體之生產與分析……………………………………. 30
4-5 競爭性ELISA之架構與分析……………………………… 31
4-5.1 單原抗體與重組HA蛋白之最佳稀釋倍數……… 31
4-5.2 AI病毒H5血清亞型之雞高免血清與單原抗體CELISA試驗………………………………………. 31
4-5.3 單原抗體與15種血清亞型之家禽流行性感冒高免血清CELISA試驗……………………………… 31
4.5.4 應用於偵測野外陰性血清建立CLISA陰性競爭百分比……………………………………………… 32
第五章 討論……………………………………………………………. 54
參考文獻…………………………………………………………………. 59

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