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研究生:翁敬堯
研究生(外文):Jing-Yao Wong
論文名稱:單株抗體 5B3G、6G9C、6G10F 免疫化學技術上之應用及 5B3G 目標抗原之鑑定
論文名稱(外文):Applications of monoclonal antibody 5B3G, 6G9C and 6G10F in immunological techniques and identification of the target antigen for monoclonal antibody 5B3G
指導教授:楊秋英
指導教授(外文):Chiou-Ying Yang
口試委員:楊明德林念璁
口試委員(外文):Ming-Te YangNien-Tsung Lin
口試日期:2017-07-28
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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:2017
畢業學年度:105
語文別:中文
論文頁數:51
中文關鍵詞:單株抗體豬霍亂沙門氏桿菌克雷伯氏肺炎桿菌
外文關鍵詞:Monoclonal antibodiesSalmonella choleraesuisKlebsiella pneumonia
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5B3G (IgG1)、6G9C (IgG2b) 和 6G10F (IgG2a) 為實驗室以加熱去活性之豬霍亂沙門氏桿菌 (Salmonella choleraesuis, SC) 之全菌免疫小鼠製備的單株抗體。在西方墨點法分析中,三株單株抗體之目標抗原不僅能在沙門氏屬中亦能在E. coli中被辨認。本研究中,我先以西方墨點法分析此三株單株抗體對克雷伯氏肺炎桿菌 (Klebsiella pneumonia, KP)、腦膜炎雙球菌、卡他莫拉菌及鮑氏不動桿菌的辨識情形,發現只有單株抗體 5B3G 能辨認 KP,顯示 5B3G 之目標抗原為腸內菌科 (Enterobacteriaceae) 之共通抗原。本研究透過 ELISA 及免疫螢光檢測法分析三株單株抗體對於 KP、SC及E. coli的辨認情形,實驗結果顯示在全菌 ELISA 及免疫螢光檢測法中 5B3G 皆無法辨識 KP,但能夠辨識 SC 及 E. coli; 6G10F 在 ELISA 及免疫螢光檢測法中皆能有效率地辨認 SC 及 E. coli; 而 6G9C 只能在 ELISA 中辨識 E. coli。在 Sandwich ELISA 實驗中更加證實 6G10F 能作為偵測 SC 及 E. coli 之捕捉抗體使用。上述實驗結果的重要性,在於提供不同免疫檢測方式所需選擇相對應之抗體資訊。我進一步針對 5B3G 之目標抗原進行身份鑑定,鑑定結果為外膜蛋白 OmpA,並分析出 5B3G 之 epitope 位在 OmpA 胺基酸序列 219~336 之間。最後,我將單株抗體 6G10F 輕鏈及重鏈變異區基因構築至哺乳類表現質體,透過 Sp2/0-Ag14 哺乳類細胞生產出 6G10F 之重組抗體 (IgG2b)。由此實驗得知單株抗體 6G10F 輕鏈及重鏈變異區專一性配對之基因序列,透過此序列可生產出與原始單株抗體具有相同特性之重組抗體,以預防因意外而失去單株抗體之細胞株。
Monoclonal antibodies (mAbs) 5B3G (IgG1)、6G9C (IgG2b) and 6G10F (IgG2a) were previously prepared from mice immunized with the heat-killed Salmonella choleraesuis (SC) in this laboratory. Target antigens are detected not only in Salmonella but also E. coli lysates in Western blot analysis. In this study, I first performed Western blot analysis to test the cross-reactivity of these mAbs to Klebsiella pneumonia (KP), Neisseria meningitidis, Moraxella catarrhalis and Acinetobacter baumannii. Immunoreactive band was only observed in the KP lysates detected with the mAb 5B3G indicating that the target antigen of 5B3G may be an enterobacterial common antigen. Next, the reactivity of these mAbs against SC, E. coli, and KP were examined by ELISA and immunofluorescence microscopy. The results showed that 5B3G did not recognize KP but could bind to SC and E. coli in whole cell-ELISA and immunofluorescence microscopy; 6G10F efficiently recognized SC and E. coli in both methods; 6G9C detected E. coli only in ELISA. Sandwich ELISA further demonstrated that 6G10F can be used as a capture antibody for SC and E. coli. These data ate important for selecting the right antibody for different immunoassays. In addition, I also identified that target antigen of 5B3G to be the outer membrane protein OmpA and mapped its minimal binding region to amino acid residues 219 to 336. Finally, I have generated a recombinant 6G10F (IgG2b) by cloning the variable genes into a mammalian expression plasmid and expressing in the Sp2/0-Ag14 cells. This work verified the specific paired heavy and light chain variable regions and allows regenerating the antibody with the same specificity if an unexpected event occurs in the corresponding hybridoma.
摘要 i
Abstract ii
目次 iii
圖表目次 v
壹、前言 1
貳、材料與方法 5
I. 實驗材料 5
一、 菌株及培養條件 5
二、 細胞株及培養條件 5
三、 實驗動物 5
四、 質體與核酸引子 5
II、實驗方法 6
一、 構築表現載體 6
二、 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 6
三、 DNA 洋菜膠電泳分析 (DNA agarose electrophoresis) 6
四、 DNA 連結反應 (Ligation) 7
五、 製備 E. coli 勝任細胞 (Competent cell) 7
六、 熱休克轉形作用 (Heat shock transformation) 7
七、 小量質體 DNA 之萃取 7
八、 重組蛋白質之表現及純化 8
九、 蛋白質膠體電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 9
十、 西方墨點法 (Western blot) 9
十一、 Coomassie blue staining 10
十二、 蛋白質濃度之測定 10
十三、 酵素結合免疫吸附法 (Enzyme-linked immunosorbent assay, ELISA)….. 11
十四、 硫酸銨 [(NH4)2SO4] 沉澱純化菌體蛋白 11
十五、 免疫螢光染色 (Immunofluorescence) 12
十六、 純化單株抗體 12
參、結果 13
一、 分析三株單株抗體在免疫化學技術上之應用 13
二、 分析 5B3G 之目標抗原 14
(一) SDS-PAGE 及西方墨點法分析之蛋白質鑑定 14
(二) 分析單株抗體 5B3G 之 epitope 於 KpOmpA 蛋白上的位置 15
三、分析三株單株抗體之基因序列 15
(一) 將單株抗體輕鏈及重鏈變異區基因構築至哺乳類雙向表現載體 15
(二) 將單株抗體之哺乳類雙向表現質體轉染至 SP2/0-Ag14 哺乳類細胞 16
肆、討論 17
伍、參考文獻 19
陸、圖表 22
柒、附錄 44
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