臺灣博碩士論文加值系統

肝癌為台灣十大癌症之一，其中患有肝癌的台灣人中，有高達 80% 為 B 型肝炎或 C 型肝炎病毒所致。而傳統檢測 B 型肝炎方法需要專業人員以及昂貴設備才能進行。故此，本研究欲開發攜帶型 B 型肝炎表面抗原快篩試片並且可直接以肉眼觀察檢測結果，以協助偏鄉或醫療資源不足地區達到早期篩檢、防疫。而研究中利用抗原與單鍊抗體之間的專一性結合力配合酵素免疫分析法，並以氧化鎳箔為 B 型肝炎表面抗原檢測之平台。
本研究所應用 B 型肝炎表面抗原a決定位之單鍊抗體 (scFv anti-HBsAg_a determinant) 於大腸桿菌中表現後，目標蛋白多聚集於包涵體當中，此問題不利蛋白進行後續純化生產。為增加此蛋白水溶性，研究中運用基因工程技術將目標基因與七種可能增加蛋白水溶性的標籤蛋白融合。其中以 Tsf (E. coli elongation factor) 或 Bla (beta-lactamase) 融合後，所產出的 B 型肝炎表面抗原 a 決定位之單鍊抗體水溶性確實增加，表現量也大幅提高。
隨後，因產出的 B 型肝炎表面抗原a決定位之單鍊抗體中擁有 6×His tags，其與氧化鎳具有親和力。所以選用氧化鎳箔開發 B 型肝炎表面抗原檢測平台。
研究中將鎳箔分別以硝酸及氫氧化鋰浸泡，運用高溫氧化金屬表面製備氧化鎳箔，並鑑定其材料表面形貌與組成。接著將經過不同程序處理所製備的氧化鎳箔，修飾上 B 型肝炎表面抗原 a 決定位之單鍊抗體進行酵素免疫分析，以驗證此平台對 B 型肝炎表面抗原辨識能力。當中以 1% 氫氧化鋰浸泡 72 小時並退火 800℃ 1 小時之氧化鎳箔為 B 型肝炎表面抗原檢測平台其靈敏度最佳，偵測極限為34 ng/mL。

Hepatocellular carcinoma is one of the most serious cancers in Taiwan. People who infect hepatitis B or hepatitis C virus are high risk in developing hepatocellular carcinoma. Since the traditional way of hepatitis B virus examination requires professional technicians and expensive equipment, we aim to develop a feasible and simple method for HBsAg diagnosis.
In this study, an ELISA assay is employed for quantitative analysis detection of HBsAg a-determinant. An scFv, obtained from our previous study through a phage display screening, was planed to immobilize on a lab-made NiO chip to function as the capture layer for HBsAg a determinant recognition. However, most of scFvs against a-determinant of HBsAg aggregated to form an inclusion body when they were expressed in E.coli. To improve the solubility of target scFv, the corresponding gene was fused with seven carrier proteins, separately. Among the various tags, Tsf and Bla enhance the expression and solubility of the target scFv.
Since the surface of material containing NiO has been shown to possess strong affinity towards protein containing polyhistidine tags, we select NiO foil as the substrate of chip for scFv immobilization and subsequently for HBsAg detection. The NiO foil was prepared through thermal oxidation, which facilitates the formation of metal-oxide nanostructures from metallic materials. By using NiO chip, we successfully demonstrated the sensitivity of HBsAg detection was significantly improved and the detection limit was 34 ng/mL.

摘要 Ⅰ
Abstract Ⅱ
謝誌 Ⅲ
目錄 Ⅳ
圖目錄 Ⅵ
表目錄 Ⅶ
第一章 緒論 1
1-1 B型肝炎病毒介紹 1
1-1-1 肝炎 1
1-1-2 B型肝炎病毒流行病學 2
1-1-3 B型肝炎病毒分類學 3
1-1-4 B型肝炎病毒結構及基因結構 5
1-1-5 B型肝炎表面抗原 7
1-1-6 B型肝炎病毒感染之檢測 9
1-1-7 B型肝炎病毒血清學 10
1-2 免疫分析方法 11
1-2-1 放射免疫分析法 12
1-2-2 酵素免疫分析法 12
1-2-3 螢光免疫分析法 14
1-3 以融合標籤蛋白提高目標蛋白溶解度 15
1-4 鎳離子與多重組胺酸親和力之應用 17
1-5 研究動機 19
第二章 實驗方法 20
2-1 實驗藥品 20
2-2 實驗儀器 20
2-3 培養基、電泳、緩衝液及反應配方法 21
2-4 scFv anti-HBsAg_a determinant融合標籤蛋白 24
2-4-1 質體構築 24
2-4-2 熱休克轉型 25
2-4-3 scFv anti-HBsAg_a determinant融合標籤蛋白之表現 25
2-4-4 scFv anti-HBsAg_a determinant融合標籤蛋白之純化 25
2-4-5 SDS-PAGE分析 26
2-4-6 scFv anti-HBsAg_a determinant融合標籤蛋白之定量 26
2-4-7 scFv anti-HBsAg_a determinant融合標籤蛋白之保存 27
2-5 B型肝炎表面抗原a決定位 27
2-5-1 B型肝炎表面抗原重組蛋白質之表現 27
2-5-2 B型肝炎表面抗原重組蛋白質之純化 28
2-5-3 西方墨點法 28
2-6 酵素免疫分析法 29
2-7 氧化鎳箔製備 30
2-8 氧化鎳箔應用於酵素免疫分析法 31
第三章 結果與討論 34
3-1 scFv anti-HBsAg_a determinant之重組蛋白 34
3-1-1 scFv anti-HBsAg_a determinant與七個SET融合 34
3-1-2 Tsf-scFv anti-HBsAg_a determinant誘導條件 37
3-1-3 Bla-scFv anti-HBsAg_a determinant誘導條件 39
3-1-4 Tsf-scFv anti-HBsAg_a determinant純化 41
3-1-5 Bla-scFv anti-HBsAg_a determinant純化 42
3-2 B型肝炎表面抗原表現與純化 43
3-3 以96孔盤為檢測平台之酵素免疫分析結果與討論 44
3-4 以氧化鎳箔為B型肝炎表面抗原檢測平台 47
3-4-1 氧化鎳箔之鑑定 47
3-4-2 以氧化鎳箔為檢測平台之酵素免疫分析結果與討論 52
第四章 結論與未來展望 57
4-1 結論 57
4-2 未來展望 58
參考資料 59
附錄 63

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