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研究生:蕭睿
研究生(外文):Jui Hsiao
論文名稱:開發診斷流感病毒H7N9亞型血球凝集素單株抗體
論文名稱(外文):Development of monoclonal antibodies against the hemagglutinin of influenza virus subtype H7N9 for diagnosis application
指導教授:莊榮輝莊榮輝引用關係
口試委員:陳翰民吳裕仁張世宗陳慧文
口試日期:2015-07-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:56
中文關鍵詞:H7N9病毒亞型血球凝集素單株抗體酵素連結免疫吸附分析法
外文關鍵詞:H7N9hemagglutininmonoclonal antibodyELISA
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中國於2013年2月出現人類感染禽流感首例,經分析為低致病率之H7N3、H7N9和H9N2重組而來之新型流感H7N9。此新型流感病毒隨後於中國沿海地區快速傳播,不僅有多項確診案例,疫情也分布廣泛。經序列分析,此新型流感為其他3型低致病性禽流感H7N3、H7N9和 H9N2在中間宿主體內重組而產生。此新型流感轉移到人類體內,會成為發病期短、重症率高且致死率高的流感。但H7N9潛伏在禽類體內,並不會有明顯的病徵,故篩檢環境中的H7N9,則成為疫情控管的重要議題。病毒顆粒表面之血球凝集素 (hemagglutinin, HA) 是病毒歸類時,不同亞型間主要的差異點,也是表面數量最多之蛋白質,此外還有高抗原性的特點,適合作為篩選H7N9之目標。本研究針對病毒表面蛋白HA進行單株抗體製備,待得到專一性及效價皆理想之單株抗體之後,將開發H7N9快篩檢驗技術平台。首先於HA中挑出抗原性和序列特異性均高的片段,同時避開可能帶有轉譯後修飾之區域,最後再標示至HA結晶構型,確認所選的片段位於結構表面、容易被抗體所辨識。得出之3段epitopes用以免疫小鼠、生產專一性高之單株抗體。接著以表現之重組蛋白H7N9 HA1及H6N1 HA1,分別作為正負向篩選用蛋白,篩出只專一性辨識H7N9 HA1之細胞株,即為符合實驗需求之單株抗體。未來將結合blocking ELISA和antigen-capture ELISA兩種實驗方法,確認單株抗體確實有抓病毒顆粒能力。符合實驗需求之單株抗體,將應用於病毒快篩系統,可有助於田間禽流感疫情之控管。

In February 2013, a human-infecting avian influenza virus subtype H7N9 was first reported in China. It was reasserted from 3 low-pathogenicity avian influenza (LPAI) H7N3, H7N9 and H9N2. While the novel H7N9 causes mild illness in birds, most human cases showed rapidly progressive pneumonia, severe respiratory illness and even high mortality. Thus, research for profound understanding of the highly pathogenic avian influenza (HPAI) virus H7N9 as well as an accurate detection method for this virus is important for disease control in Taiwan. Hemagglutinin (HA) is a surface glycoprotein with high specificity and high antigenicity, which is suitable for the development an early diagnosis platform with monoclonal antibody (mAb) against the novel H7N9 viral proteins. In this study, 3 peptides which showed high immunogenicity and high specificity were synthesized for mice immunization. After 5 boosts, mice with efficient immune response were sacrificed to produces monoclonal antibodies (mAb). The hybridoma strains were first screened by enzyme-linked immunosorbent assay (ELISA) and then tested by western blot using H7N9 HA1 and H6N1 HA1 as antigens, respectively. Two mAb that are highly specific to 2 different epitopes of H7N9 HA1 were isolated. They will be used in developing the blocking ELISA and antigen-capture ELISA, as well as in clinical diagnosis and quick viral detection in the field.

內容
中文摘要 1
英文摘要 1
縮寫表 1
第一章 緒論 1
1.1 H7N9的爆發以及病例 1
1.2 流行性感冒病毒 2
1.2.1 流感病毒之分類 2
1.2.2 A型流感病毒 3
1.2.3 流感病毒感染宿主之機制 5
1.2.4 病毒 HA 和宿主表面受器之結合 6
1.3 新型禽流感病毒H7N9 8
1.3.1 H7N9介紹 8
1.3.2 新型流感病毒H7N9序列特性 8
1.3.3 新型H7N9和人類受器有高度親和力 10
1.4 單株抗體製備 12
1.4.1 單株抗體技術 12
1.4.2 抗原的製備 12
1.5 研究動機 13
第二章 材料與方法 15
2.1 抗原決定基之預測 15
2.2 單株抗體之製備 16
2.2.1 小白鼠免疫 16
2.2.2 小鼠抗體效價測定 (酵素連結免疫分析法,ELISA) 16
2.2.3 細胞融合 17
2.2.4 抗體單株化篩選 18
2.2.5 細胞冷凍法 19
2.2.6 細胞解凍法 19
2.2.7 單株抗體的量產 20
2.2.8 腹水之純化 20
2.3 製備H7N9 HA1及H6N1 HA1重組蛋白 20
2.3.1 H7N9 HA1重組蛋白之構築 21
2.3.2 H7N9 HA1及 H6N1 HA1重組蛋白之表現 21
2.3.3 H7N9 HA1及H6N1 HA1重組蛋白之純化 21
第三章 結果與討論 23
3.1 抗原片段之挑選 23
3.1.1 流感亞型HA之序列校準 23
3.1.2 抗原序列特性之預測 24
3.1.3 抗原於HA立體結構上之位置 25
3.2 抗原製備 32
3.2.1 小鼠免疫及效價測試 32
3.2.2 細胞融合及單株抗體篩選 33
3.2.3 以重組蛋白篩作為篩選之抗原 34
3.3 H6和H7兩種亞型之HA重組蛋白製備 42
3.3.1 H6N1 HA1重組蛋白之製備 42
3.3.2 正向篩選H7N9 HA1重組蛋白之製備 42
3.4 單株抗體專一性測試 47
第四章 未來研究方向 50
參考文獻 51
問答錄 54


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