簡易檢索 / 詳目顯示

研究生: 李國緯
Kuo-Wei Lee
論文名稱: 發展雞源抗人類乳癌細胞乙醯膽鹼受器之抗體
Development of chicken anti-human breast cancer nAchR antibody
指導教授: 楊沂淵
Yang, Yi-Yuan
方剛
Fang, Kang
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2013
畢業學年度: 101
語文別: 中文
論文頁數: 68
中文關鍵詞: 噬菌體展示乳癌乙醯膽鹼受器單鏈多樣性片段
英文關鍵詞: phage display, breast cancer, AchR, scFv
論文種類: 學術論文
相關次數: 點閱:363下載:1
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 尼古丁型乙醯膽鹼受器(nAchR)是一種具有神經傳導物質受器和離子通道功能的膜蛋白。過去的研究指出,在尼古丁的長期暴露下,nAchR會大量表現並造成癌症如肺癌、大腸癌、膀胱癌等。其中的α9 nAchR的大量表現則跟乳癌形成相關。故此蛋白未來在乳癌篩檢或治療上是一個相當有潛力的標的。而為了偵測α9 nAchR的表現,能夠專一性辨識此蛋白的抗體就變得十分重要。
      本計畫以重組之人類α9 nAchR膜外片段作為抗原,對雞隻進行免疫注射後將雞隻犧牲取脾臟以噬菌體展示(phage display)技術建立重組單鏈抗體片段(single chain variable fragment,scFv)基因庫。之後將此基因庫進行四次篩選(panning)後把篩選好的基因群轉殖到top10F’大腸桿菌表現抗α9 nAchR scFv。之後再用重組之人類α9 nAchR以酵素結合免疫分析法 (ELISA) 和西方墨點法 (Western blot) 篩選出具專一性辨識能力之scFv。最後再以α9 nAchR高表現量的MDA-MB-231人類乳腺癌細胞株以西方墨點法、流式細胞儀分析 (Flow cytometry) 以及MTT測試scFv是否能辨識細胞所表現的α9 nAchR以及是否能抑制癌細胞生長。最後我們得到一株具有明顯專一性結合細胞原生α9 nAchR能力之scFv。希望這株抗體在未來可以應用在乳癌臨床篩檢甚至是標靶治療上。

    Nicotine acetylcholine receptor (nAchR) is a kind of membrane proteins with neurotransmitter receptor and ion channel function. Previous studies have pointed out that long-term exposure to nicotine, nAchR will be up-regulated expression and cause cancers such as lung cancer, colon cancer, and bladder cancer. Overexpression of α9 nAchR is associated with the formation of breast cancer. Therefore, α9 nAchR is a considerable potential molecule target in screening or treatment breast cancer in the future. In order to detect the overexpression of α9 nAchR, specific α9 nAchR antibody is very important.
    We produce truncated recombinant human α9 nAchR outer membrane fragments as antigen for chicken immunization. Chicken will be sacrificed after immunization, and be established recombinant antibody fragments (single chain variable fragment, scFv) library from spleen cell by phage display technology. After four panning, enriched anti-α9 nAchR scFv phage library will be transformed to Top10F' E. coli to express anti-α9 nAchR scFv. α9 nAchR specific scFv will be selected with recombinant α9 nAchR by ELISA and Western blot. Then, we test native α9 nAchR binding ability of scFv with high α9 nAchR expression MDA-MB-231 breast cancer cell by Western blot, Flow cytometry, and immunofluorescence staining. Further, scFv will be test if can inhibit MDA-MB-231 cell proliferation in MTT test. We get a clone scFv with obvious specific binding ability to native α9 nAchR of MDA-MB-231 cell. Hope this scFv can be applied in clinical screening and treatment for breast cancer in the future.

    中文摘要 6 Abstract 8 表目錄 10 圖目錄 11 一. 緒論 12 1. 論文評述 12 (1) 尼古丁型乙醯膽鹼受器 (nicotinic acetylcholine receptor, nAchR) 簡介 12 (2) α9 nAchR誘發乳癌之機制 13 (3) 乳癌簡介 14 (4) 乳癌之篩檢與治療 15 (5) 多株抗體與單株抗體 15 (6) 融合瘤單株抗體生產 16 (7) 噬菌體展示技術 17 二. 研究目標 19 三. 實驗材料 20 1. 藥品類 20 2. 抗體類 22 3. 其它 22 4. 各式溶液 23 四. 實驗方法 27 1. 篩選噬菌體基因庫 (panning) 27 2. 細菌質體萃取 29 3. 瓊脂糖凝膠 (agarose gel) 電泳 29 4. Top 10F’ 勝任細胞製備 30 5. 轉型作用 (transformation) 30 6. 表現 scFv蛋白 31 7. scFv純化與透析 32 8. scFv 質體DNA序列分析 33 9. 聚丙烯膠體 (SDS-PAGE) 電泳法 33 10. 西方墨點法 (Western blot) 34 11. 酵素結合免疫分析法 (Enzyme-linked immunosorbent assay, ELISA) 34 12. 細胞培養 35 13. 細胞蛋白質萃取 35 14. 蛋白質定量 36 15. 流式細胞儀分析 (Flow cytometry) 36 16. 癌細胞生長測試分析 37 五. 實驗結果 38 1. 篩選抗人類α9 nAchR scFv噬菌體基因庫 (panning) 38 2. 抗人類α9 nAchR scFv表現 39 (1) 轉型作用 39 (2)以Isopropyl-beta-D-thiogalactopyranoside (IPTG) 誘導Top10F’大腸桿菌表現抗人類α9 nAchR scFv 39 3. 以重組α9 nAchR片段測試抗人類α9 nAchR scFv之結合能力 40 4. 抗人類α9 nAchR scFv之序列分析 41 5. 抗人類α9 nAchR scFv純化 41 6. 以α9 nAchR高表現量之人類乳腺癌細胞MDA-MB-231測試抗人類α9 nAchR scFv之結合能力 42 (1) 西方墨點法 42 (2) 流式細胞儀分析 42 7. 抗人類α9 nAchR scFv之透析與過濾 43 8. 抗人類α9 nAchR scFv S1抑制人類乳腺癌細胞MDA-MB -231生長測試 43 六. 討論 45 七. 參考文獻 49 八. 圖表 53

    Ahmad, Z.A., et al. (2012) scFv antibody: principles and clinical application, Clinical & developmental immunology, 2012, 980250.
    Akita, E.M. and Li-Chan, E.C. (1998) Isolation of bovine immunoglobulin G subclasses from milk, colostrum, and whey using immobilized egg yolk antibodies, Journal of dairy science, 81, 54-63.
    Burioni, R., et al. (1997) An improved phage display vector for antibody repertoire cloning by construction of combinatorial libraries, Research in virology, 148, 161-164.
    Burritt, J.B., et al. (1996) Filamentous phage display of oligopeptide libraries, Analytical biochemistry, 238, 1-13.
    Chen, R.J., et al. (2010) Long-term nicotine exposure-induced chemoresistance is mediated by activation of Stat3 and downregulation of ERK1/2 via nAChR and beta-adrenoceptors in human bladder cancer cells, Toxicological sciences : an official journal of the Society of Toxicology, 115, 118-130.
    Coughlin, S.S. and Ekwueme, D.U. (2009) Breast cancer as a global health concern, Cancer epidemiology, 33, 315-318.
    Dasgupta, P. and Chellappan, S.P. (2006) Nicotine-mediated cell proliferation and angiogenesis: new twists to an old story, Cell Cycle, 5, 2324-2328.
    Elgoyhen, A.B., et al. (1994) Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells, Cell, 79, 705-715.
    Glidewell-Kenney, C., et al. (2005) ERE-independent ERalpha target genes differentially expressed in human breast tumors, Mol Cell Endocrinol, 245, 53-59.
    Holliger, P. and Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains, Nature biotechnology, 23, 1126-1136.
    Jin, Z., et al. (2004) Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis, The Journal of biological chemistry, 279, 23837-23844.
    Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256, 495-497.
    Lee, C.H., et al. (2011) Crosstalk between nicotine and estrogen-induced estrogen receptor activation induces alpha9-nicotinic acetylcholine receptor expression in human breast cancer cells, Breast cancer research and treatment, 129, 331-345.
    Lee, C.H., et al. (2010) Overexpression and activation of the alpha9-nicotinic receptor during tumorigenesis in human breast epithelial cells, Journal of the National Cancer Institute, 102, 1322-1335.
    Lindstrom, J. (1997) Nicotinic acetylcholine receptors in health and disease, Molecular neurobiology, 15, 193-222.
    Luebke, A.E., et al. (2005) Alpha-9 nicotinic acetylcholine receptor immunoreactivity in the rodent vestibular labyrinth, The Journal of comparative neurology, 492, 323-333.
    Lustig, L.R. and Peng, H. (2002) Chromosome location and characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) gene, Cytogenetic and genome research, 98, 154-159.
    McGehee, D.S., et al. (1995) Nicotine enhancement of fast excitatory synaptic transmission in CNS by presynaptic receptors, Science, 269, 1692-1696.
    Minna, J.D. (2003) Nicotine exposure and bronchial epithelial cell nicotinic acetylcholine receptor expression in the pathogenesis of lung cancer, J Clin Invest, 111, 31-33.
    Nguyen, V.T., Ndoye, A. and Grando, S.A. (2000) Novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by Pemphigus vulgaris autoimmunity, Am J Pathol, 157, 1377-1391.
    Parmley, S.F. and Smith, G.P. (1988) Antibody-selectable filamentous fd phage vectors: affinity purification of target genes, Gene, 73, 305-318.
    Poola, I., et al. (2005) Identification of MMP-1 as a putative breast cancer predictive marker by global gene expression analysis, Nature medicine, 11, 481-483.
    Smith, G.P. (1985) Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface, Science, 228, 1315-1317.
    Vincler, M., et al. (2006) Molecular mechanism for analgesia involving specific antagonism of alpha9alpha10 nicotinic acetylcholine receptors, Proceedings of the National Academy of Sciences of the United States of America, 103, 17880-17884.
    Wei, P.L., et al. (2009) Tobacco-specific carcinogen enhances colon cancer cell migration through alpha7-nicotinic acetylcholine receptor, Ann Surg, 249, 978-985.
    West, K.A., et al. (2003) Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells, J Clin Invest, 111, 81-90.
    Winter, G. and Milstein, C. (1991) Man-made antibodies, Nature, 349, 293-299.
    Wolff, A.C., et al. (2007) American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer, Journal of clinical oncology : official journal of the American Society of Clinical Oncology, 25, 118-145.

    下載圖示
    QR CODE