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系統識別號 U0026-2306201117342300
論文名稱(中文) 利用噬菌體人類單鏈抗體庫進行新抗體之研發
論文名稱(英文) Discovery of antibodies by phage display human scFv antibody library
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 99
學期 2
出版年 100
研究生(中文) 張憶文
研究生(英文) Yi-Wen Chang
學號 T36984085
學位類別 碩士
語文別 英文
論文頁數 88頁
口試委員 指導教授-張權發
口試委員-吳漢忠
口試委員-吳華林
口試委員-施桂月
中文關鍵字 噬菌體人類單鏈抗體庫 
英文關鍵字 phage display 
學科別分類
中文摘要 抗體除了在生物體內扮演防禦的角色外,其也參與了基礎研究、臨床診斷和臨床治療。而製造單株抗體除了廣為人知的細胞融合瘤技術外,噬菌體人類單鏈抗體庫也是製造單株抗體很有潛力的技術。在此研究中,我們利用反轉錄聚合酶連鎖反應技術獲得人類抗體的重鏈和輕鏈的cDNA,接上連接子(linker)後以PCR與質體pHEN2接合,於大腸桿菌TG1中建構出大於1×108的噬菌體人類單鏈抗體庫,其多樣性也透過序列鑑定確認。Biopanning是一種利用噬菌體人類單鏈抗體庫針對特殊抗原篩選出抗體的一種方法,其包含且重複著連結(binding)、洗提(elution)以及放大(amplification)等步驟。我們利用兩種不同來源的噬菌體人類單鏈抗體庫(2.5×1010 和1.15×108),針對重組蛋白thrombomodulin (rTM)和腸病毒71型病毒顆粒進行抗體的篩選。針對rTM進行五次Biopanning流程,其中菌落rTM 3-9的單鏈抗體的重鏈和輕鏈序列已確認來自人類的抗體。故我們針對進行rTM 3-9的水溶性單鏈抗體(scFv)的表現與純化,並其偵測對rTM的親和力,然而實驗結果發現其親和力是不佳的。針對腸病毒71型的部分也進行了五次的Biopanning流程,其中EV3-1A, EV3-1E, EV3-3H和EV4-11E單鏈抗體序列確定來自人類的抗體。此外,EV3-3H單鏈抗體也具有明顯的劑量依賴性(dose dependent manner),故進行EV3-3H的水溶性單鏈抗體(scFv)的表現與純化,並發現無法測得其對EV71的親和力。本論文成功建構了一個>1×108的噬菌體人類單鏈抗體庫,並測試兩種不同來源的抗體庫,嘗試找出蛋白以及病毒的新抗體,然而我們所建構的抗體庫並無法獲得具有明顯結合力的單鏈抗體。
英文摘要 Antibodies not only participate in immune protection from pathogens, but also involve in clinical diagnosis, basic research and treatments. Instead of producing monoclonal antibody from hybridoma technology, phage display antibody library is another powerful tool for generating specific antibodies. Here, we constructed a human single chain variable fragment (scFv) antibody library for exploring new antibodies. Lymphocytes isolated from pooled human blood (from 38 volunteers) were subjected to RNA extraction. After reverse transcription, the cDNA of heavy chain and light chain were amplified and connected with linker to form scFv. The scFv repertoires were ligated into pHEN2 phagemid and electroporated into Escherichia coli TG1 competent cells. Colony formation on ampicillin plate indicated that a >1×108 phage scFv library was constructed. In addition, DNA sequencing of colonies were distinct from each other. Biopanning, that included repeating binding, elution and amplification procedure, were used to explore high affinity scFv for specific antigens. Two libraries (2.5×1010 and 1.15×108) were used in our biopanning to explore new scFvs for recombinant thrombomodulin (rTM) and enterovirus 71 (EV-71). After 5 rounds of biopanning, the colony rTM 3-9F specific to rTM was confirmed to be a human antibody on database. The soluble form rTM 3-9F scFv was purified, but the binding affinity was low. For EV-71 viral particles, 5 rounds of biopanning were performed and colonies EV3-1A, EV3-1E, EV3-3H and EV4-11E were confirmed to be human antibodies. In addition, the binding of EV3-3H phage to EV-71 was in a dose-dependent manner. In conclusion, we had constructed a >1×108 human scFv phage library, although we could not obtain good results from our phage library. And we had explore new scFvs for rTM and EV-71 by biopanning.
論文目次 Contents

摘要 I
Abstract II
誌謝 III
Contents IV
Figure contents VI
Table contents VIII
Abbreviations IX
Introduction 1
Antibodies 1
Phage 2
Phage display technology 3
Application of phage display 5
Biopanning 6
Antibody phage-displayed library 6
Thrombomodulin 7
Enterovirus 71 8
Study Design 10
Materials and Methods 11
Materials 11
Bacteria Strain 11
Helper phage 11
RNA isolation from Peripheral Blood lymphocytes 11
Reverse Transcription Reaction (RT) 12
Amplification of VH and VL repertoires 13
Overlapping PCR for producing the scFv repertoires 14
Electroporation 14
Rescue of phage library 15
Biopanning for recombinant thrombomodulin (rTM) 16
Biopanning for Enterovirus 71 (EV-71) 18
Colonies selected for binding screening 19
ELISA assay for binding screening 20
Purification of soluble rTM 3-9F scFv and EV 3-3H scFv 21
Western Blot 22
Results 24
Construction of scFv gene repertoires 24
Construction of phage scFv library 24
Biopanning of recombinant thrombomodulin (rTM) 26
Biopanning of Enterovirus 71 viral particles (EV-71) 27
Purification of soluble rTM 3-9F scFV 28
Purification of soluble EV3-3H scFv 28
Characterization of scFv-phage and scFvs for rTM and EV-71 29
Discussion 30
Conclusion 36
Reference 37
Figure 45
Table 70
Appendix 84
作者簡歷 88

參考文獻 Reference
1 Brekke, O. H. & Sandlie, I. Therapeutic antibodies for human diseases at the dawn of the twenty-first century. Nat Rev Drug Discov 2, 52-62, doi:10.1038/nrd984 nrd984 [pii] (2003).
2 Hudson, P. J. & Souriau, C. Engineered antibodies. Nat Med 9, 129-134, doi:10.1038/nm0103-129 nm0103-129 [pii] (2003).
3 Holliger, P. & Hudson, P. J. Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23, 1126-1136, doi:nbt1142 [pii] 10.1038/nbt1142 (2005).
4 Kohler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-497 (1975).
5 Nelson, P. N. et al. Monoclonal antibodies. Mol Pathol 53, 111-117 (2000).
6 Majidi, J., Barar, J., Baradaran, B., Abdolalizadeh, J. & Omidi, Y. Target therapy of cancer: implementation of monoclonal antibodies and nanobodies. Hum Antibodies 18, 81-100, doi:L52261175568N6R8 [pii] 10.3233/HAB-2009-0204 (2009).
7 Specthrie, L. et al. Construction of a microphage variant of filamentous bacteriophage. J Mol Biol 228, 720-724, doi:0022-2836(92)90858-H [pii] (1992).
8 Liu, T. J., You, B. Y., Lin, N. T., Yang, M. T. & Tseng, Y. H. Purification and expression of the gene III protein from filamentous phage phi Lf. Biochem Biophys Res Commun 242, 113-117, doi:S0006-291X(97)97932-8 [pii] 10.1006/bbrc. 1997.7932 (1998).
9 Marvin, D. A. Filamentous phage structure, infection and assembly. Curr Opin Struct Biol 8, 150-158 (1998).
10 Hoess, R. H. Protein design and phage display. Chem Rev 101, 3205-3218, doi:cr000056b [pii] (2001).
11 Barbas, C. F., 3rd, Kang, A. S., Lerner, R. A. & Benkovic, S. J. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88, 7978-7982 (1991).
12 Smith, G. P. & Petrenko, V. A. Phage Display. Chem Rev 97, 391-410, doi:cr960065d [pii] (1997).
13 Smith, G. P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315-1317 (1985).
14 Scott, J. K. & Smith, G. P. Searching for peptide ligands with an epitope library. Science 249, 386-390 (1990).
15 Hoogenboom, H. R. et al. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res 19, 4133-4137 (1991).
16 Greenwood, J., Hunter, G. J. & Perham, R. N. Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA. J Mol Biol 217, 223-227, doi:0022-2836(91)90534-D [pii] (1991).
17 Pande, J., Szewczyk, M. M. & Grover, A. K. Phage display: concept, innovations, applications and future. Biotechnol Adv 28, 849-858, doi:S0734-9750(10)00097-2 [pii] 10.1016/j.biotechadv.2010.07.004 (2010).
18 Rodi, D. J. & Makowski, L. Phage-display technology--finding a needle in a vast molecular haystack. Curr Opin Biotechnol 10, 87-93, doi:S0958-1669(99)80016-0 [pii] (1999).
19 Lowman, H. B. Bacteriophage display and discovery of peptide leads for drug development. Annu Rev Biophys Biomol Struct 26, 401-424, doi:10.1146/annurev.biophys.26.1.401 (1997).
20 Choo, Y. & Klug, A. Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc Natl Acad Sci U S A 91, 11168-11172 (1994).
21 Parmley, S. F. & Smith, G. P. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73, 305-318 (1988).
22 Cull, M. G., Miller, J. F. & Schatz, P. J. Screening for receptor ligands using large libraries of peptides linked to the C terminus of the lac repressor. Proc Natl Acad Sci U S A 89, 1865-1869 (1992).
23 Oh, M. Y., Joo, H. Y., Hur, B. U., Jeong, Y. H. & Cha, S. H. Enhancing phage display of antibody fragments using gIII-amber suppression. Gene 386, 81-89, doi:S0378-1119(06)00533-6 [pii] 10.1016/j.gene.2006.08.009 (2007).
24 Zhang, J. L. et al. Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen. Hepatobiliary Pancreat Dis Int 5, 237-241, doi:824 [pii] (2006).
25 Lee, T. Y., Lin, C. T., Kuo, S. Y., Chang, D. K. & Wu, H. C. Peptide-mediated targeting to tumor blood vessels of lung cancer for drug delivery. Cancer Res 67, 10958-10965, doi:67/22/10958 [pii]10.1158/0008-5472.CAN-07-2233 (2007).
26 Su, J. L. et al. A novel peptide specifically binding to interleukin-6 receptor (gp80) inhibits angiogenesis and tumor growth. Cancer Res 65, 4827-4835, doi:65/11/4827 [pii] 10.1158/0008-5472.CAN-05-0188 (2005).
27 Marasco, W. A., LaVecchio, J. & Winkler, A. Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. J Immunol Methods 231, 223-238, doi:S0022-1759(99)00159-3 [pii] (1999).
28 Chang, D. K., Lin, C. T., Wu, C. H. & Wu, H. C. A novel peptide enhances therapeutic efficacy of liposomal anti-cancer drugs in mice models of human lung cancer. PLoS One 4, e4171, doi: 10.1371/journal.pone.0004171 (2009).
29 Chang, D. K. et al. Antiangiogenic targeting liposomes increase therapeutic efficacy for solid tumors. J Biol Chem 284, 12905-12916, doi:M900280200 [pii]10.1074/jbc.M900280200 (2009).
30 Kirsch, M. I. et al. Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV). BMC Biotechnol 8, 66, doi:1472-6750-8-66 [pii]10.1186/1472-6750-8-66 (2008).
31 Hust, M. et al. Improved microtitre plate production of single chain Fv fragments in Escherichia coli. N Biotechnol 25, 424-428, doi:S1871-6784(09)00052-1 [pii]10.1016/j.nbt.2009.03.004 (2009).
32 Smothers, J. F., Henikoff, S. & Carter, P. Tech.Sight. Phage display. Affinity selection from biological libraries. Science 298, 621-622, doi:10.1126/science.298.5593.621298/5593/621 [pii] (2002).
33 Kolonin, M. G., Saha, P. K., Chan, L., Pasqualini, R. & Arap, W. Reversal of obesity by targeted ablation of adipose tissue. Nat Med 10, 625-632, doi:10.1038/nm1048 nm1048 [pii] (2004).
34 Arap, W. et al. Steps toward mapping the human vasculature by phage display. Nat Med 8, 121-127, doi:10.1038/nm0202-121 nm0202-121 [pii] (2002).
35 Wen, D. Z. et al. Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene. Biochemistry 26, 4350-4357 (1987).
36 Dzionek, A. et al. BDCA-2, BDCA-3, and BDCA-4: three markers for distinct subsets of dendritic cells in human peripheral blood. J Immunol 165, 6037-6046 (2000).
37 Fuentes-Prior, P. et al. Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex. Nature 404, 518-525, doi:10.1038/35006683 (2000).
38 Suzuki, K. et al. Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 6, 1891-1897 (1987).
39 Esmon, C. T., Esmon, N. L. & Harris, K. W. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 257, 7944-7947 (1982).
40 Esmon, C. T. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB J 9, 946-955 (1995).
41 Solomon, T. et al. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10, 778-790, doi:S1473-3099(10)70194-8 [pii] 10.1016/S1473-3099(10)70194-8 (2010).
42 McMinn, P. C. An overview of the evolution of enterovirus 71 and its clinical and public health significance. FEMS Microbiol Rev 26, 91-107, doi:S0168644502000700 [pii] (2002).
43 Schmidt, N. J., Lennette, E. H. & Ho, H. H. An apparently new enterovirus isolated from patients with disease of the central nervous system. J Infect Dis 129, 304-309 (1974).
44 Ho, M. et al. An epidemic of enterovirus 71 infection in Taiwan. Taiwan Enterovirus Epidemic Working Group. N Engl J Med 341, 929-935, doi:10.1056/NEJM199909233411301 (1999).
45 Lee, T. C. et al. Diseases caused by enterovirus 71 infection. Pediatr Infect Dis J 28, 904-910, doi:10.1097/INF.0b013e3181a41d6300006454- 200910000-00012 [pii] (2009).
46 Yamayoshi, S. et al. Scavenger receptor B2 is a cellular receptor for enterovirus 71. Nat Med 15, 798-801, doi:nm.1992 [pii] 10.1038/nm.1992 (2009).
47 Nishimura, Y. et al. Human P-selectin glycoprotein ligand-1 is a functional receptor for enterovirus 71. Nat Med 15, 794-797, doi:nm.1961 [pii] 10.1038/nm.1961 (2009).
48 Marks, J. D. et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 222, 581-597, doi:0022-2836(91)90498-U [pii] (1991).
49 Keiko Sakai, Y. S., Tomoki Chiba, Ayano Matsumoto-Takasaki, Yu Kusada, Wei Zhang,| Munehiro Nakata,§,| Naoya Kojima,| Kazunori Toma, Atsushi Takayanagi, Nobuyoshi Shimizu, and Yoko Fujita-Yamaguchi. Isolation and Characterization of Phage-Displayed Single Chain Antibodies Recognizing Nonreducing Terminal Mannose Residues. 1. A New Strategy for Generation of Anti-Carbohydrate Antibodies. Biochemistry 46, 253-262 (2007).
50 ANGRAY S. KANG, TERESA M. JONES & BURTON, D. R. Antibody redesign by chain shuffling from random combinatorial immunoglobulin libraries. Proc. Nati. Acad. Sci. USA 88, 11120-11123 (1991).
51 Cadwell, R. C. & Joyce, G. F. Randomization of genes by PCR mutagenesis. Genome Research 2, 28-33, doi:10.1101/gr.2.1.28 (1992).
52 SHENLAN MAO, C. G., CHIH-HUNG L. LO, PETER WIRSCHING, CHI-HUEY WONG, AND KIM D. JANDA*. Phage-display library selection of high-affinity human single-chainantibodies to tumor-associated carbohydrate antigens sialyl Lewisx and Lewisx. Proc. Natl. Acad. Sci. USA 96, 6953–6958 (1999).
53 Goffinet, M. et al. Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection. BMC Biotechnol 8, 34, doi:1472-6750-8-34 [pii]10.1186/1472-6750-8-34 (2008).


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