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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 莊榮輝(Rong-Huay Juang) | |
dc.contributor.author | Ho-Chen Tsai | en |
dc.contributor.author | 蔡和成 | zh_TW |
dc.date.accessioned | 2021-06-13T15:33:52Z | - |
dc.date.available | 2013-07-21 | |
dc.date.copyright | 2008-07-21 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-11 | |
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Mazor, Y., Van Blarcom, T., Mabry, R., Iverson, B.L. and Georgiou, G. (2007) Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat Biotechnol 25, 563-5. McCafferty, J., Griffiths, A.D., Winter, G. and Chiswell, D.J. (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552-4. Ning, Y., Wang, Y., Li, Y., Hong, Y., Peng, D., Liu, Y., Wang, J., Hao, W., Tian, X., Wu, F., Dong, W., Wang, L., Wu, Q., Liu, X., Gao, J., He, F., Qian, X., Sun, Q.H. and Li, M. (2006) An alternative strategy for high throughput generation and characterization of monoclonal antibodies against human plasma proteins using fractionated native proteins as immunogens. Proteomics 6, 438-48. O'Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250, 4007-21. Pieper, R., Su, Q., Gatlin, C.L., Huang, S.T., Anderson, N.L. and Steiner, S. 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(1989) Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544-6. Wasinger, V., Cordwell, S. J., Cerpa-Poljak, A., Gooley, A. A., Wilkins M. R., Duncan, M., Williams, K. L., and Humphery-Smith, I. (1995) Progress with gene-product mapping of the mollicutes: Mycoplasma genitaliu. Electrophoresis 16, 1090-1094. Wu, Y.J., Chen, H.M., Wu, T.T., Wu, J.S., Chu, R.M. and Juang, R.H. (2006) Preparation of monoclonal antibody bank against whole water-soluble proteins from rapid-growing bamboo shoots. Proteomics 6, 5898-902. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37587 | - |
dc.description.abstract | 融合瘤技術是常用的單株抗體生產技術,藉由B細胞與骨髓瘤細胞的融合,得到分泌專一性抗體的融合瘤細胞株,此技術於發明以來三十年沒有太大的改變。本實驗室於2006年提出抗體庫概念,並以綠竹筍水溶性蛋白質體免疫小鼠,由兩階段細胞融合得到192株單株抗體。本論文承襲抗體庫的概念,進一步提出差異性抗體庫的想法,希望製備出能夠區別綠竹筍生長過程中,各成長階段差異性蛋白質之對應抗體。第一階段實驗,以差異性吸附管柱製備差異性蛋白質之抗體庫,結果因為吸附後之蛋白質差異性不大而不甚理想。於是改變製備策略,第二階段實驗則改以流式細胞儀進行差異性分選。利用實驗室已建立的融合瘤細胞 (H7c、C7a、D5b),先進行小規模預備實驗,以抗小鼠IgG抗體的FITC結合探針,確定融合瘤細胞會保留膜上抗體。然後以抗原標定的方式,成功將融合瘤細胞篩選出來。正式操作時,以綠竹筍之兩個生長階段的全體蛋白質免疫小鼠,分別進行細胞融合,於T80-flask培養3週,然後以流式細胞儀進行負篩選 (去除相同的抗體) 及正篩選 (挑出差異性抗體),限數稀釋後再以免疫染色做確認。最後結果,得到數株單株抗體,但無法有效得到未出土或出土60公分綠竹筍專一性抗體,因此,此實驗技術及流程仍然做進一步改進。 | zh_TW |
dc.description.abstract | Hybridoma technology is the most frequently used method for preparing monoclonal antibody. It is produced by fusing antibody secreting splenic B cells with myeloma cells. This technology was basically unchanged since its first introduction in 1975 by Kohler and Milstein. The concept of monoclonal antibody bank was proposed by our lab in 2006, and totally 192 specific monoclonal antibodies was collected by immunizing mice with the total water-soluble proteins from bamboo shoot, and then following an improved fusion and screening procedure. In this project, we further proposed the concept of differential antibody bank, in which the antibodies recognizing those proteins that were specific to variuos growing stages of bamboo shoots were screened out. First attempt to construct this bank by immunoaffinity column was not successful, since we cannot produce the protein banks with significant difference. Flow cytometer which could differentially sort out the cells labeled with specific proteins was utilized. In preliminary tests, we confirmed that hybridma cells could produce membrane-bound antibodies as detected by anti-mouse IgG-FITC antibody; and several established hybridoma lines (H7c, C7a, and D5b) could be sorted successfully by antigen labeling. Then we immunize mice with total proteins from bamboo shoots (BS0TP or BS60TP), and fuse the B cells with myeloma cells. After 3 wk cultured in T80-flask, the specific antibody producing lines were sorted out by negative selection and subsequently positive selection. The antibody produced was then checked by Western blotting after limiting dilution. Finally, we get some monoclonal antibody, but it can’t recognize BS0TP or BS60TP specifically. Therefore, we have to improve this technique and the procedure. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T15:33:52Z (GMT). No. of bitstreams: 1 ntu-97-R95b47202-1.pdf: 4690385 bytes, checksum: 807fbcfcdf29a8dbce8000088ef75946 (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 中文摘要 i
英文摘要 ii 第一章 緒論 1 1.1 竹 1 1.1.1 分布 1 1.1.2 型態 1 1.1.3 獨特的生理現象 2 1.1.4 應用與經濟價值 3 1.2 蛋白質體學 4 1.2.1 蛋白質體學技術 4 1.3 單株抗體及抗體庫 5 1.3.1 B細胞的成熟 5 1.3.2 單株抗體技術 7 1.3.3 抗體製備流程及其它製備技術 8 1.4 研究動機及目的 10 第二章 差異性蛋白質體抗體庫 13 2.1 以差異性吸附親和性管柱建立抗體庫 13 2.1.1 建立竹筍未出土及出土40公分水溶性蛋白質體多株抗體 14 2.1.2 差異性吸附親和性管柱 14 2.1.3 差異性抗體庫建立 15 2.2 以流式細胞儀建立差異性抗體庫 15 2.2.1 確定融合瘤細胞是否有membrane-bound抗體 16 2.2.2 利用抗原標定分開不同的融合瘤細胞 17 2.2.3 建立未出土及出土60公分綠竹筍水溶性蛋白質體單株抗體庫 17 2.2.4 未出土及出土60公分綠竹筍水溶性蛋白質體進行Biotinylation 17 2.2.5 以抗原標定配合流式細胞儀進行差異性分選 18 2.2.6 差異性抗體庫細胞株單株化 19 2.3 差異性抗體庫概念的創新 19 2.3.1 抗體庫概念 (高產能) 19 2.3.2 針對未知蛋白質的研究 (差異性概念) 20 2.3.3 加速抗體篩選 20 第三章 材料與方法 21 3.1 一般分析法 21 3.1.1 蛋白質定量法 21 3.2 一般電泳檢定法 22 3.2.1 原態膠體電泳 23 3.2.2 SDS膠體電泳 27 3.2.3 膠體染色法:Coomassie Brilliant Blue R-250 (CBR) 蛋白質染色法 29 3.2.4 膠片乾燥法及護貝 30 3.2.5 蛋白質電泳轉印法 31 3.3 免疫學檢定法 33 3.3.1 酵素免疫分析法 33 3.3.2 酵素免疫染色法 35 3.4 二維電泳相關實驗 38 3.4.1 二維電泳樣本蛋白質萃取法 38 3.4.2 蛋白質樣本溶解方法 39 3.4.3 二維電泳之蛋白質定量方法 40 3.4.4 二維電泳 41 3.5 以差異性吸附親和性管柱建立抗體庫 43 3.5.1 多株抗體製備 43 3.5.2 親和性管柱製備及差異性吸附 49 3.5.3 差異性抗體庫製備 51 3.6 以流式細胞儀建立差異性抗體庫 53 3.6.1 小白鼠免疫 53 3.6.2 細胞融合 54 3.6.3 未出土及出土60公分綠竹筍水溶性蛋白質體進行Biotinylation 58 3.6.4 流式細胞儀差異性分選 59 3.6.5 細胞保存法 60 3.6.6 單株抗體的生產 62 第四章 結果 63 4.1 以差異性吸附親和性管柱建立抗體庫 63 4.1.1 建立竹筍未出土及出土40公分水溶性蛋白質體多株抗體 63 4.1.2 抗體純化 63 4.1.3 差異性吸附親和性管柱 64 4.1.4 差異性抗體庫建立 64 4.2 以流式細胞儀建立差異性抗體庫 64 4.2.1 確定融合瘤細胞是否有membrane-bound抗體 64 4.2.2 利用抗原標定分開不同的融合瘤細胞 65 4.2.3 建立未出土及出土60公分綠竹筍水溶性蛋白質體單株抗體庫 66 4.2.4 未出土及出土 60 公分綠竹筍水溶性蛋白質體進行 Biotinylation 66 4.2.5 以抗原標定配合流式細胞儀進行差異性分選 66 4.2.6 差異性抗體庫細胞株單株化及免疫染色測定 67 第五章 討論 81 5.1 差異性抗體庫技術 81 5.2 以差異性吸附親和性管柱建立抗體庫 81 5.3 以流式細胞儀建立差異性抗體庫 82 5.3 未來方向 83 參考文獻 85 答問錄 89 | |
dc.language.iso | zh-TW | |
dc.title | 綠竹筍生長過程差異性蛋白質體及其抗體庫之建立 | zh_TW |
dc.title | The Differential Proteome of the Fast Growing Bamboo Shoots and Construction of Its Antibody Bank | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 吳建興(Jiann-Shing Wu),陳翰民(Han-Min Chen),張世宗(Shih-Chung Chang) | |
dc.subject.keyword | 融合瘤細胞,抗原標定, | zh_TW |
dc.subject.keyword | hybridoma,antigen labeling, | en |
dc.relation.page | 91 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2008-07-14 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 微生物與生化學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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