跳到主要內容

臺灣博碩士論文加值系統

(3.236.110.106) 您好!臺灣時間:2021/07/24 07:00
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

: 
twitterline
研究生:施毓桓
研究生(外文):Yu-Huan Shih
論文名稱:利用抗體噬菌體呈現系統篩選抗麩胱甘肽-硫-轉移酶和嚴重急性呼吸道症候群冠狀病毒殼包核酸蛋白質的抗體片段
論文名稱(外文):Generation of Antibodies Fragment against Glutathione-S-transferase and SARS-CoV Nucleocapsid protein by Antibody Phage Display System
指導教授:許萬枝許萬枝引用關係
指導教授(外文):Wan-Jr Syu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:80
中文關鍵詞:噬菌體呈現系統
外文關鍵詞:phage display system
相關次數:
  • 被引用被引用:0
  • 點閱點閱:151
  • 評分評分:
  • 下載下載:22
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要

單鏈抗體是將抗體中的重鏈變異區與輕鏈變異區利用連接子(linker)連接的蛋白質。因為單鏈抗體的分子量小、操作容易、具專一性,能夠提供單株抗體以外的應用在臨床上和實驗研究。透過抗體噬菌體呈現系統篩選到能夠和特定抗原結合的單鏈抗體。我們目標是產生抗日本血吸蟲的麩胱甘肽-硫-轉移酶和嚴重急性呼吸道症候群冠狀病毒的殼包核酸蛋白質單鏈抗體。先用麩胱甘肽-硫-轉移酶和殼包核酸蛋白質分別免疫小鼠,然後摘取小鼠的脾臟分離其mRNA並且進行反轉錄聚合酵素連鎖反應產生輕鏈和重鏈的cDNA。將輕鏈和重鏈的cDNA利用重疊聚合酵素連鎖反應產生單鏈抗體基因庫。然後利用抗體噬菌體呈現系統篩選出有結合能力的單鏈抗體。透過鎳離子親合管柱純化單鏈抗體並且確定對於抗體的專一性。我們的結果顯示純化的單鏈抗體可以結合麩胱甘肽-硫-轉移酶和殼包核酸蛋白質在酵素聯合免疫吸附測定。此外,利用單鏈抗體製成的抗原抓取酵素聯合免疫吸附測定可以測得重組的殼包核酸蛋白質,證明了篩選過的單鏈抗體可以專一地認得抗原。本論文所產生的單鏈抗體可應用在麩胱甘肽-硫-轉移酶和嚴重急性呼吸道症候群冠狀病毒殼包核酸蛋白質的偵測使用。
Abstract

A single-chain variable fragment (ScFv) is the combination of heavy chain variable region (VH) and the light chain variable region (VL) of an antibody, connected by a polypeptide linker. Due to the small size and the simplicity in engineering, yet with specificity to antigen, ScFv is a promising alternative to monoclonal antibody in both clinic and research applications. Conventionally, specific ScFv is isolated from a ScFv library by screening against an interested antigen using phage-display technology. The aim of the study is to generate ScFv against either recombinant Schistosoma japonicum glutathione-S-transferase (GST) or recombinant SARS-CoV nucleocapsid protein. Mice were immunized with either GST or nucleocapsid protein. The VH and VL cDNA fragments were generated by RT-PCR using mRNA isolated from spleens of immunized mice. The ScFv libraries were constructed by overlapping PCR, with VH and VL cDNA as templates. Potential ScFv clones were isolated from the libraries by phage-display screening. To further confirm the specificity of ScFv to their antigens, the ScFv candidates were purified by immobilized metal affinity chromatography and then were assayed. We showed that the purified ScFv was able to bind either GST or nucleocapsid protein, respectively by ELISA. In addition, recombinant nucleocapsid protein could be detected by ScFv using nucleocapsid protein-capture ELISA. These data indicated that the ScFv could specifically recognize its antigens. The ScFv might be applied to the detection of GST and nucleocapsid protein.
參考文獻
Azzazy, H. M., and Highsmith, W. E., Jr. (2002). Phage display technology: clinical applications and recent innovations. Clin Biochem 35, 425-445.
Barbas, C. F., 3rd, Kang, A. S., Lerner, R. A., and Benkovic, S. J. (1991). Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88, 7978-7982.
Che, X. Y., Qiu, L. W., Pan, Y. X., Wen, K., Hao, W., Zhang, L. Y., Wang, Y. D., Liao, Z. Y., Hua, X., Cheng, V. C., and Yuen, K. Y. (2004). Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome. J Clin Microbiol 42, 2629-2635.
Chowdhury, S., Chester, K. A., Bridgewater, J., Collins, M. K., and Martin, F. (2004). Efficient retroviral vector targeting of carcinoembryonic antigen-positive tumors. Mol Ther 9, 85-92.
Clackson, T., Hoogenboom, H. R., Griffiths, A. D., and Winter, G. (1991). Making antibody fragments using phage display libraries. Nature 352, 624-628.
Danese, P. N., and Silhavy, T. J. (1998). Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli. Annu Rev Genet 32, 59-94.
Dauvillier, S., Merida, P., Visintin, M., Cattaneo, A., Bonnerot, C., and Dariavach, P. (2002). Intracellular single-chain variable fragments directed to the Src homology 2 domains of Syk partially inhibit Fc epsilon RI signaling in the RBL-2H3 cell line. J Immunol 169, 2274-2283.
Falsey, A. R., and Walsh, E. E. (2003). Novel coronavirus and severe acute respiratory syndrome. Lancet 361, 1312-1313.
Gao, C., Mao, S., Ronca, F., Zhuang, S., Quaranta, V., Wirsching, P., and Janda, K. D. (2003). De novo identification of tumor-specific internalizing human antibody-receptor pairs by phage-display methods. J Immunol Methods 274, 185-197.
Gu, Z., Weidenhaupt, M., Ivanova, N., Pavlov, M., Xu, B., Su, Z. G., and Janson, J. C. (2002). Chromatographic methods for the isolation of, and refolding of proteins from, Escherichia coli inclusion bodies. Protein Expr Purif 25, 174-179.
Holliger, P., and Hoogenboom, H. (1998). Antibodies come back from the brink. Nat Biotechnol 16, 1015-1016.
Holt, L. J., Enever, C., de Wildt, R. M., and Tomlinson, I. M. (2000). The use of recombinant antibodies in proteomics. Curr Opin Biotechnol 11, 445-449.
Hon, K. L., Leung, C. W., Cheng, W. T., Chan, P. K., Chu, W. C., Kwan, Y. W., Li, A. M., Fong, N. C., Ng, P. C., Chiu, M. C., et al. (2003). Clinical presentations and outcome of severe acute respiratory syndrome in children. Lancet 361, 1701-1703.
Hoogenboom, H. R., and Chames, P. (2000). Natural and designer binding sites made by phage display technology. Immunol Today 21, 371-378.
Hudson, P. J. (1998). Recombinant antibody fragments. Curr Opin Biotechnol 9, 395-402.
Hudson, P. J. (1999). Recombinant antibody constructs in cancer therapy. Curr Opin Immunol 11, 548-557.
Hudson, P. J., and Kortt, A. A. (1999). High avidity scFv multimers; diabodies and triabodies. J Immunol Methods 231, 177-189.
Lobato, M. N., and Rabbitts, T. H. (2003). Intracellular antibodies and challenges facing their use as therapeutic agents. Trends Mol Med 9, 390-396.
Marasco, W. A., LaVecchio, J., and Winkler, A. (1999). Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. J Immunol Methods 231, 223-238.
Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A., Butterfield, Y. S., Khattra, J., Asano, J. K., Barber, S. A., Chan, S. Y., et al. (2003). The Genome sequence of the SARS-associated coronavirus. Science 300, 1399-1404.
McCafferty, J., Griffiths, A. D., Winter, G., and Chiswell, D. J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552-554.
Neri, D., Petrul, H., and Roncucci, G. (1995). Engineering recombinant antibodies for immunotherapy. Cell Biophys 27, 47-61.
Nielsen, U. B., and Marks, J. D. (2000). Internalizing antibodies and targeted cancer therapy: direct selection from phage display libraries. 3, 282-291.
Peiris, J. S., Chu, C. M., Cheng, V. C., Chan, K. S., Hung, I. F., Poon, L. L., Law, K. I., Tang, B. S., Hon, T. Y., Chan, C. S., et al. (2003a). Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet 361, 1767-1772.
Peiris, J. S., Lai, S. T., Poon, L. L., Guan, Y., Yam, L. Y., Lim, W., Nicholls, J., Yee, W. K., Yan, W. W., Cheung, M. T., et al. (2003b). Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319-1325.
Penichet, M. L., and Morrison, S. L. (2001). Antibody-cytokine fusion proteins for the therapy of cancer. J Immunol Methods 248, 91-101.
Poon, L. L., Chan, K. H., Wong, O. K., Yam, W. C., Yuen, K. Y., Guan, Y., Lo, Y. M., and Peiris, J. S. (2003). Early diagnosis of SARS coronavirus infection by real time RT-PCR. J Clin Virol 28, 233-238.
Rader, C. (2001). Antibody libraries in drug and target discovery. Drug Discov Today 6, 36-43.
Rietsch, A., and Beckwith, J. (1998). The genetics of disulfide bond metabolism. Annu Rev Genet 32, 163-184.
Rodi, D. J., and Makowski, L. (1999). Phage-display technology--finding a needle in a vast molecular haystack. Curr Opin Biotechnol 10, 87-93.
Rondot, S., Koch, J., Breitling, F., and Dubel, S. (2001). A helper phage to improve single-chain antibody presentation in phage display. Nat Biotechnol 19, 75-78.
Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli, R., Icenogle, J. P., Penaranda, S., Bankamp, B., Maher, K., Chen, M. H., et al. (2003). Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394-1399.
Schickli, J. H., Zelus, B. D., Wentworth, D. E., Sawicki, S. G., and Holmes, K. V. (1997). The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range. J Virol 71, 9499-9507.
Smith, G. P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315-1317.
Stadler, K., Masignani, V., Eickmann, M., Becker, S., Abrignani, S., Klenk, H. D., and Rappuoli, R. (2003). SARS--beginning to understand a new virus. Nat Rev Microbiol 1, 209-218.
Verma, R., Boleti, E., and George, A. J. (1998). Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems. J Immunol Methods 216, 165-181.
Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T., and Winter, G. (1989). Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544-546.
Wege, H., Schliephake, A., Korner, H., and Flory, E. (1993). An immunodominant CD4+ T cell site on the nucleocapsid protein of murine coronavirus contributes to protection against encephalomyelitis. J Gen Virol 74 ( Pt 7), 1287-1294.
Williams, A. F., and Barclay, A. N. (1988). The immunoglobulin superfamily--domains for cell surface recognition. Annu Rev Immunol 6, 381-405.
Zhong, N. S., Zheng, B. J., Li, Y. M., Poon, Xie, Z. H., Chan, K. H., Li, P. H., Tan, S. Y., Chang, Q., Xie, J. P., et al. (2003). Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People''s Republic of China, in February, 2003. Lancet 362, 1353-1358.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top