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研究生:施孚奕
研究生(外文):Fu-Yi Shih
論文名稱:雞丙型干擾素重組蛋白與抗體製備
論文名稱(外文):Recombinant protein expression of chicken interferon-gamma and its antibodies preparation
指導教授:邱繡河
口試委員:周寬基潘慧錦
口試日期:2018-01-26
學位類別:碩士
校院名稱:國立中興大學
系所名稱:微生物暨公共衛生學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:43
中文關鍵詞:雞丙型干擾素重組蛋白一氧化氮抗體
外文關鍵詞:recombinant chIFN-γnitric oxideantibodies
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本研究主要目的是表現雞丙型干擾素(chicken Interferon-gamma, chIFN-γ) 的重組蛋白並且製備其抗體。丙型干擾素(interferon-gamma, IFN-γ)是一種細胞素,主要是由輔助型T細胞第一型(T helper type 1 cells, Th1)與自然殺手(natural killer, NK)細胞分泌,可以活化巨噬細胞產生一氧化氮(nitric oxide, NO),並且具有促進Th1細胞分化與抑制Th2細胞分化的功能,因此丙型干擾素在免疫反應調節中扮演了重要的角色。本研究將研究室先前選殖的雞丙型干擾素cDNA基因置入原核系統的蛋白表現載體pET32-a中,進而利用大腸桿菌BL-21(DE3)菌株表現雞丙型干擾素重組蛋白(recombinant chIFN-γ, rchIFN-γ),分子量約為33.7 kDa,再進一步利用基質輔助雷射脫附游離飛行時間質譜儀分析法(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF-MS)確認表現的蛋白質為雞的丙型干擾素。我們利用金屬螯合層析法純化重組的雞丙型干擾素,並且儲存於適合的保存液中,再進行生物活性測試,結果雞丙型干擾素重組蛋白可隨濃度增加而成正比地活化巨噬細胞產生一氧化氮(nitric oxide, NO),顯示具有生物的活性。此外,我們將雞丙型干擾素重組蛋白與佛氏佐劑混合施打進入小鼠腹腔,再進行多次的補強免疫後,利用西方墨漬法分析小鼠血清確定得到抗重組雞丙型干擾素的多株抗體,隨後將小鼠的B細胞(B cell)與小鼠的骨髓瘤(myeloma) NS-1細胞株進行細胞融合,篩選會分泌辨識重組雞丙型干擾素抗體的融合瘤細胞珠(hybridoma),利用ELISA技術與西方墨漬法測得抗體存在,未來可以將融合瘤細胞單株化得到單株抗體。總而言之,我們期待所表現的雞丙型干擾素重組蛋白與其抗體可以運用於獸醫學術研究或應用。
The specific aims of this study are to express recombinant protein of chicken interferon-gamma (chIFN-γ) and to prepare its antibodies. Interferon-gamma (IFN-γ) is mainly secreted by type 1 T helper (Th1) cells and natural killer (NK) cells. It can promote Th1 cell differentiation while inhibit Th2 subtype. In addition, IFN-γ can activate macrophages to produce nitric oxide (NO). Therefore, IFN-γ plays important roles in the regulation of immune responses. In a previous study, our laboratory obtained a chIFN-γ cDNA clone. In this study, we introduced the chIFN-γ cDNA into pET32-a expression vector. Subsequently, recombinant chIFN-γ (rchIFN-γ) protein was expressed in Escherichia coli (BL21-DE3), with a molecular mass of about 33.7 kDa. The identity of rchIFN-γ protein was further confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We purified the rchIFN-γ protein by metal chelation affinity chromatography and stored it in an adequate storage solution. Macrophages could be activated by rchIFN-γ to produce nitric oxide (NO) in a dose-dependent manner, indicating that the rchIFN-γ protein expressed in this study is bioactive. Furthermore, we injected the mixture of rchIFN-γ and Freund''s adjuvant into the abdominal cavity of mice. After boosting several times, we detected elevated anti-rchIFN-γ antibodies in mouse sera based on Western blot analysis. Mouse B cells and mouse myeloma cells(NS-1) were fused to obtain hybridoma cells producing anti-rchIFN-γ antibodies. We detected anti-rchIFN-γ antibodies in culture medium of hybridoma cells based on Western blot analysis and enzyme-linked immunosorbent assay (ELISA). We will proceed with the development of anti-rchIFN-γ monoclonal antibodies. In conclusion, we expect that rchIFN-γ protein and anti-rchIFN-γ antibodies generated in this study can be useful for veterinary studies or applications.
摘要 i
Abstract ii
目次 iii
圖次 v
中英文對照表 vi
第一章 序論 1
第二章 文獻探討 2
第一節 細胞素(cytokines) 2
1-1 細胞素簡介 2
第二節 干擾素(Interferon) 2
2-1 干擾素簡介 2
第三節 丙型干擾素(Interferon-gamma) 3
3-1 丙型干擾素簡介 3
第四節 雞的丙型干擾素(chicken Interferon-gamma) 4
4-1 雞的丙型干擾素(chicken Interferon-gamma, chIFN-γ)發現 4
4-2 chIFN-γ的生物來源與活性 4
4-3 chIFN-γ的蛋白質結構與受器 4
4-4 目前chIFN-γ的應用 5
第五節 單源抗體(monoclonal antibody)的起源、原理與應用 5
5-1 單源抗體(monoclonal antibody)之起源 5
5-2 細胞融合之技術原理 5
5-3 單源抗體之融合瘤細胞篩選技術 6
5-4 單源抗體之應用 6
第三章 材料與方法 7
第一節 構築雞的干擾素載體 7
1-1 增幅雞丙型干擾素基因序列與原核表現載體 7
1-2 利用TA cloning技術獲得擁有成熟的雞丙型干擾素片段之載
體 8
1-3 限制酶處理 8
1-4 構築重組雞丙型干擾素之載體與載體保存 9
第二節 重組雞丙型干擾素蛋白和載體蛋白大量表現和純化 9
2-1 誘導表現重組雞丙型干擾素蛋白(rchIFN-γ)和載體蛋白及所
需時間 9
2-2 重組雞丙型干擾素蛋白和載體蛋白大量表現 10
2-3 重組雞丙型干擾素蛋白和載體蛋白純化蛋白 10
2-4 重組雞丙型干擾素蛋白和載體蛋白純化蛋白去除內毒素 10
第三節 生物活性測試 11
3-1 雞周邊血單核細胞分離與誘導獲得巨噬細胞 11
3-2 雞丙型干擾素誘導巨噬細胞生成一氧化氮 11
3-3 一氧化氮濃度測試 12
第四節 抗體製備 12
4-1 誘導小鼠抗體生成 12
4-2 形成融合瘤細胞(hybridoma) 13
4-3 融合瘤細胞(hybridoma)分泌抗重組雞丙型干擾素抗體
(anti-rchIFN-γ antibody)偵測 14
4-4 融合瘤細胞極限稀釋後抗體偵測 14
第四章 結果 15
第一節 構築雞丙型干擾素(chIFN-γ)之原核表現載體 15
第二節 蛋白質表現和純化 15
2-1 誘導重組雞丙型干擾素蛋白和載體蛋白的表現 15
2-2 重組雞丙型干擾素蛋白和載體蛋白的純化 16
2-3 純化蛋白去除內毒素 16
第三節 生物活性測試 17
3-1 重組雞的干擾素誘導雞巨噬細胞產生一氧化氮 17
第四節 重組雞的丙型干擾素單株抗體製備 17
4-1 誘導小鼠生成抗rchIFN-γ抗體 17
4-2 融合瘤細胞製備 18
4-3 多株抗體篩選 18
4-4 極限稀釋後抗體篩選 19
第五章 討論 20
圖表 24
參考文獻 39
附錄 43
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