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系統識別號 U0026-2308201116105400
論文名稱(中文) 製造B型肝炎病毒大型表面蛋白特異性抗體用於肝癌診斷
論文名稱(英文) Production of HBV large surface protein specific antibodies for the diagnosis of hepatocellular carcinoma
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 99
學期 2
出版年 100
研究生(中文) 張博硯
研究生(英文) Po-Yen Chang
學號 T36981045
學位類別 碩士
語文別 中文
論文頁數 99頁
口試委員 指導教授-葉才明
口試委員-張權發
口試委員-黃國珍
中文關鍵字 B型肝炎病毒  肝癌  單鏈抗體 
英文關鍵字 HBV  hepatocellular carcinoma  scFv 
學科別分類
中文摘要 在臺灣B型肝炎病毒 (HBV) 的感染是造成肝細胞癌 (hepatocellular carcinoma, HCC) 的一個主要的因素。慢性B型肝炎的帶原者轉變至肝細胞癌一般需要十幾年的時間,如何去早期診斷出帶原者轉變為肝細胞癌的風險是對於病人的治療方面是一個重要的因素。在過去的研究中指出了突變型的HBV大型表面蛋白 (Large HBV Surface protein, LHBS) 會出現在B型肝炎病患的毛玻璃狀肝細胞 (ground glass hepatocytes , GGH) 中,之後也證實了這些突變的大型表面蛋白,尤其是在Pre-S2區域有缺失的突變型,跟肝癌的發生有很大的關係。因此此類的突變型大型表面蛋白是可以作為一個HBV帶原者轉化為肝癌的指標。在本篇的研究之中,我們將嘗試著去利用融合瘤或是噬菌體表現之單鏈抗體去製造Pre-S2突變型B型肝炎病毒大型表面蛋白的特異性單株抗體以及一個大型表面蛋白的特異性抗體。在使用野生型、Pre-S2突變型B型肝炎病毒大型表面蛋白、LHBS的Pre-S1區域以及一個大型表面蛋白N端高度保留區域的勝肽去免疫小鼠或兔子之後,收集到的動物血清中大部分都是同時辨識到野生型或是突變型蛋白的抗體。為了增加抗體之特異性,我們利用了免疫小鼠的脾臟去製造出融合瘤或是噬菌體表現之單鏈抗體,並且經過篩選以及確認之後順利得到兩種特異性單株抗體,分別是會產生大型表面蛋白特異性抗體的融合瘤細胞株,以及是對於野生型大型表面蛋白有不錯特異性的噬菌體表現之單鏈抗體。即使並沒有一個Pre-S2突變型B型肝炎病毒大型表面蛋白特異性抗體,但是現有的兩種抗體仍然是可以應用在發展一個利用偵測突變型HBV大型表面蛋白作為慢性B型肝炎患者轉化為肝細胞癌的診斷試劑組。
英文摘要 Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Taiwan. Because HBV may persist in carriers for decades before HCC develops, it will be very important for HBV carriers to be able to estimate their risk of HCC at early stage. In previous studies, mutant large HBV surface proteins (LHBS) were found in HCC patients with ground glass hepatocytes (GGH). These mutant type LHBS, especially the Pre-S2 deletion type, is highly associated with HCC.Taken together, these mutant proteins have potential to be a risk factor of HCC. In this study, we tried to select Pre-S2 mutant and LHBS specific antibodies using phage display technique to construct single chain variable fragment (scFv) library and hybridoma library. Mice were immunized with Pre-S2 mutant, wild type recombinant protein, LHBS Pre-S1 region or LHBS N-terminal conserved region peptide. Antisera in all these mice recognized both wild type and mutant Pre-S2 proteins. To select specific antibodies, we used spleens from immunized mice to construct scFv and hybridoma libraries. After several times of confirmation, we successfully found a scFv preferentially bind to wild type LHBS and several hybridomas which could secrete antibodies against both wild type and Pre-S2 deletion type LHBS. In the future, these two reagents will be used in developing diagnostic kit for the presence Pre-S2 mutant protein from patients’ specimens as a risk factor for HCC.
論文目次 總目錄 i
圖目錄 iv
縮寫指引 vi
1. 緒論 1
1.1 B型肝炎病毒 1
1.1.1 B型肝炎病毒概論 1
1.1.2 B型肝炎病毒生活史 2
1.1.3 B型肝炎病毒致病病程 4
1.1.4 B型肝炎病毒表面蛋白 6
1.1.5 B型肝炎病毒大型表面蛋白突變型與其致癌機轉 8
1.2 實驗動機與目標 10
1.3 以融合瘤 (hybridoma) 技術製造之單株抗體 11
1.3.1 單株抗體 11
1.3.2 融合瘤細胞技術 11
1.4 以噬菌體(phage)表現技術之單煉抗體 (single chain fragment of variable region, scFv) 12
1.4.1 噬菌體 12
1.4.2 噬菌體表現之單鏈抗體 16
2. 實驗目標與設計 21
3. 實驗方法與材料 22
3.1 小鼠免疫 22
3.1.1 小鼠品系與來源 22
3.1.2 重組蛋白免疫小鼠之前處理 22
3.1.3 合成胜肽免疫小鼠之前處理 22
3.1.4 免疫小鼠之技術和流程 23
3.1.5 免疫小鼠血清中抗體之測定 25
3.2 融合瘤技術製造之單株抗體 27
3.2.1 骨髓瘤細胞(myeloma cell)種類 27
3.2.2 免疫小鼠脾臟細胞之取得 27
3.2.3 骨髓瘤細胞與免疫小鼠脾臟細胞之融合 28
3.2.4 融合瘤細胞之培養 28
3.2.5 篩選製造特異性抗體之融合瘤細胞 29
3.2.6 融合瘤細胞細胞之單株化 29
3.2.7 單株抗體之製造與純化 30
3.3 噬菌體表現技術製造之單煉抗體 30
3.3.1 免疫小鼠脾臟cDNA的取得 30
3.3.2 抗體變異區基因之純化 32
3.3.3 單鏈抗體基因庫之製造 33
3.3.4 單鏈抗體基因庫與噬菌體質體(phagemid)之接合 35
3.3.5 幫助噬菌體(helper phage)之製備 36
3.3.6 單煉抗體表現之噬菌體庫之製造 37
3.3.7 特異性單煉抗體之生物篩選(bio-panning) 39
3.3.8 特異性單煉抗體表現之單一噬菌體之篩選 40
3.4 特異性抗體結合能力之確認 42
3.4.1 西方墨點法(western blot) 42
3.4.2 免疫螢光染色法(immunofluorescence assay, IFA) 45
4. 結果 46
4.1 由Pre-S1 高保留區域合成胜肽製造大型表面蛋白特異性單株抗體 47
4.1.1 免疫小鼠之血清效價分析 47
4.1.2 免疫小鼠之血清特異性抗體 47
4.1.3 篩選由免疫小鼠所製備融合瘤 47
4.1.4 篩選出之單株抗體分析 48
4.2 Pre-S2區域缺失突變型大型表面蛋白特異性單株抗體 48
4.2.1 各種重組蛋白免疫小鼠之血清效價分析 48
4.2.2 免疫動物之血清特異性抗體 49
4.2.3 Pre-S2區域缺失突變型大型表面重組蛋白免疫小鼠所製造之單株抗體 52
4.2.4 Pre-S2區域缺失突變型大型表面重組蛋白免疫小鼠所製造之單鏈抗體 53
4.2.5 Pre-S1 區域重組蛋白免疫小鼠所製造之單株抗體 56
5. 討論 57
5.1 B型肝炎病毒大型表面蛋白特異性抗體 57
5.2 Pre-S2區域缺失突變型B型肝炎病毒大型表面蛋白特異性抗體 57
5.3 特異性抗體之抗原決定位探討 59
6. 結論 61
7. 參考文獻 62
附錄一、 試劑配方 90
附錄二、試劑及實驗耗材和抗體 95
附錄三、儀器 99
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