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研究生:李金蓉
研究生(外文):Chin- Jung Li
論文名稱:利用LAE技術製造抗IFN-γ抗體作為診斷之用
論文名稱(外文):Generation of anti-IFN-γantibodies for IFN-γdiagnosis by LAE technique
指導教授:黃昭蓮曾銘仁
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
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:2005
畢業學年度:93
語文別:中文
論文頁數:65
中文關鍵詞:干擾素線性陣列抗原決定
外文關鍵詞:IFN-γlinear array epitope ( LAE )
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干擾素( Interferon gamma;IFN-γ),為一種相當重要的細胞激素,由活化的T淋巴球( T lymphocytes )以及NK細胞( natural killer cells) 所分泌。IFN-γ最早發現其具抗病毒感染弁遄A但是它在一些免疫反應中也參與調節,包括刺激吞噬細胞對抗病原菌、增加MHC (major histocompatibility complex)Ⅰ、Ⅱ分子呈獻抗原,誘導IgG2a轉變,抑制TH2的分化,並調節延遲型過敏反應以及影響細胞增生和凋亡。由於IFN-γ的生理弁鄐w知在與發炎、發燒、抗病毒、過敏及自體免疫疾病中扮演重要角色,所以血液中的IFN-γ含量具有臨床診斷指標之價值,本研究旨在製造抗IFN-γ之抗體,以作為開發快速檢測細胞激素之抗體晶片的材料。選擇位於IFN-γ分子表面之區域作為抗原辨認區(epitopes),利用線性陣列重複抗原技術,製造含線性陣列抗原決定區(linear array epitope)之多胜肽,並與綠膿桿菌外毒素A接受器結合區(PEDIa)或麩胱甘肽轉移酵素(GST)結合成融合蛋白,作為抗原, 純化後注射實驗兔及鼠後激發抗IFN-γ抗體。以酵素免疫分析來檢驗此獲得之抗體的效價。結果我們成弗o到四段抗原決定區的多株抗體,但經由西方點墨法( Western blot )以及點漬法( Dot blot )初步分析只有一組抗血清在十倍稀釋下可以辨認原態IFN-γ。同時為了製造具高度效價及穩定性之抗體,也利用融合瘤技術試圖製造抗IFN-γ單株抗體。
Interferon gamma ( IFN-γ) is an important cytokine secreted by activated T lymphocyte and natural killer ( NK ) cells. Although originally defined as an agent with direct antiviral activity, various roles of IFN-γwere reported ,including regulation of several aspects of the immune response, stimulation of bactericidal activity of phagocytes, increment of the antigen presentation through class I and class II major histocompatibility complex ( MHC ) molecules, induction of class switch to IgG2a, inhibition of TH2 proliferation, mediation of various effects important in delayed–type hypersensitivity, and influence of cell proliferation and apoptosis. All these physiological functions suggested that IFN-γplayed important roles in inflammation, fever, antiviral, hypersensitivity, and autoimmune diseases, therefore, it’s concentration could be as a good indicator for clinical diagnosis. In this study, we first selected different regions of IFN-γexposed at the molecular surface as antigenic targets. The linear array epitope ( LAE ) technique was applied to generate LAE immunogen, which fused with Pseudomonas exotoxin domain Ia ( PEDIa ) or glutathione S-transfera for production of fusion protein. These purified LAE immunogens were used to immunized rabbits or mice. The harvested antibodies were collected and assayed by enzyme–linked immunosorbent assay ( ELISA ). We were succeeded in getting polyclonal antibodies induced by selected regions as analyzed by both Western blot and Dolt blot analysis,However ,Only one of the antisera can recognize the native IFN-γprotein. In order to generate antibodies with high titer and stability, currently we are planning to generate monoclonal antibodies against IFN-γwith hybridoma technique.
頁次
中文摘要……………………………………………………………………………………….3
英文摘要………………………………………………………………………………….……4
壹、緒論………………………………………………………………………………………...7
一、Gamma Interferon (IFN-γ) …………………………………………………………….7
二、抗體產生的機轉………………………………………………………………………...9
三、 線性陣列抗原決定區(linear array epitope;LAE)…………………………..............10
四、單株抗體產生原理…………………………………………………………………….11
五、結合外源性載體蛋白(carrier protein)與IFN-γ的…………………………………...12
線性陣列抗原決定區的IFN-γ來產生疫苗的設計
六、研究目的與實驗策略……………………………………………………….................14
貳、材料與方法……………………………………………………………………………….16
一、材料與試劑…………………………………………………………………………….16
二、實驗方法……………………………………………………………………………….20
參、結果……………………………………………………………………………………….29
一、免疫原的製造…………………………………………………………………………...29
二、動物免疫與抗體分析…………………………………………………………………...32
肆、討論……………………………………………………………………………………….35
參考文獻……………………………………………………………………………………...39
圖表…………………………………………………………………………………………...45
附圖…………………………………………………………………………………………...64
圖表、附錄目錄 頁次

表一、實驗中使用到的引子序列…………………………………………………………….45
圖一、實驗策略流程圖……………………………………………………………………….46
圖二、IFN-γ三維結構與選定抗原辨認區示意圖………………………………………….47
圖三、模板重複聚合酶鏈鎖反應的模板設計圖…………………………………………….48
圖四、模板重複聚合酵素連鎖反應的示意圖……………………………………………….49
圖五、模板重複聚合酵素連鎖反應結果…………………………………………………….50
圖六、轉接端聚合酵素連鎖反應示意圖與結果…………………………………………….51
圖七、本論文用以表現免疫原蛋白的質體………………………………………………….52
圖八、誘導表現PEDIa-IFN-γ-017-His6與純化情形……………………………………….53
圖九、本研究所製造重組蛋白純化後的情形……………………………………………….54
圖十、兔子免疫PEDIa-IFN-γ-017-His6四次後的抗體產生情形………………………….55
圖十一、兔子免疫PEDIa-IFN-γ-02P8-His6四次後的抗體產生情形……………………..56
圖十二、兔子免疫GST -IFN-γ-0310-His6四次後的抗體產生情形………………………..57
圖十三、兔子免疫GST -IFN-γ-049-His6四次後的抗體產生情形………………...............58
圖十四、小鼠免疫PEDIa-IFN-γ-02P8-His6四次後的抗體產生情形……………………..59
圖十五、兔子血清專一性分析……………………………………………………………….60
圖十六、兔子抗IFN-γ多株抗體(Chemicon)對於完整蛋白及融合蛋白辨識的
程度…………………………………………………………………………………...61
圖十七、兔子血清對於recombinant Human interferon-γ所辨認的程度…………………...62
圖十八、兔子抗IFN-γ多株抗體對於完整蛋白及融合蛋白辨識的程度………………....63
附錄一、人類IFN-γ與小鼠IFN-γ之胺基酸序列比對………………………....................64
附錄二、人類IFN-γ與兔子IFN-γ之胺基酸序列比對…………………………………..65
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