進階搜尋


 
系統識別號 U0026-0812200915190733
論文名稱(中文) 薄膜式醣晶片的發展及應用
論文名稱(英文) Development and Application of Carbohydrate Membrane Array
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 97
學期 2
出版年 98
研究生(中文) 劉詠儀
研究生(英文) Weng-I Lao
學號 t3694113
學位類別 碩士
語文別 中文
論文頁數 106頁
口試委員 指導教授-張權發
口試委員-張長泉
口試委員-梁碧惠
中文關鍵字 薄膜晶片  醣類  高通量篩選 
英文關鍵字 high throughput screening  glycan  membrane array 
學科別分類
中文摘要 生物體內細胞表面醣類分子與蛋白質間相互作用具有很多重要的功能,包括調控細胞分化、細胞附著、免疫反應、蛋白質運輸、腫瘤細胞轉移及微生物感染等重要的生理功能。這些功能皆透過特定的凝集素、抗體或蛋白質與細胞表面上醣蛋白或醣脂質末端的醣類分子發生交互作用所引發的。過去發展許多技術用於蛋白質與醣類間交互作用的研究上,例如酵素結合免疫吸附分析、平衡透析、表面等離子共振技術、等溫滴定微量熱法、親和性層析及醣類晶片。其中最為成功的技術則是醣類晶片,它的原理是透過疏水作用力、biotin與streptavidin作用或共價鍵等將醣類分子固定在玻璃、金屬或矽等材質的表面。本研究目的是建立薄膜式醣類晶片平台來偵測凝集素、蛋白質和病毒與醣類間作用的關係。本論文使用了88種biotin-PAA(polyacrylamide)-sugar,分別測試透過streptavidin、抗biotin抗體或直接固定在薄膜上,研究中使用了三種不同的薄膜:包括西方墨點法所用的轉漬膜PVDF、DNA晶片常用的帶正電尼龍膜及適用於蛋白質晶片的UltraBind薄膜;結果的觀察則是透過標記有鹼性磷酸酶的凝集素或抗體來進行。本研究結果顯示biotin-PAA-sugar能成功地透過streptavidin固定在UltraBind薄膜上,而最適用於本薄膜的填塞緩衝液則是含有酪蛋白的TBST裡。透過18個已知凝集素及4個抗醣類抗體測試,結果分析符合過去研究的結果。再來是應用這平台分析人類和禽類的感冒病毒在晶片上的結合圖譜,我們測試了臨床分離的流感病毒16株B型、1株A型及2株禽類的H6N1,結果發現禽類流感病毒主要與alpha 2-3鏈結的唾液酸結合,而人類流感則是與alpha 2-6鏈結的唾液酸結合為主,此外我們也發現了毒性較高的禽類流感病毒與醣類結合能力較強,因此推測禽類感冒病毒其致病能力與結合醣類能力有關聯。
英文摘要 Protein-carbohydrate interactions involved in many important biological processes including cell differentiation, adhesion, immune response, protein trafficking and tumor metastasis. All of these functions occur through the interaction between glycans on glycoprotein or glycolipid displayed on cell surfaces with specific lectins, antibodies, or proteins. Many techniques have been developed to study protein-carbohydrate interactions including enzyme-linked lectin sorbent assay (ELLSA), equilibrium dialysis, surface plasmon resonance, isothermal titration calorimetry, frontal affinity chromatography (FAC) and carbohydrate microarray. Carbohydrate microarray, which glycans are immobilized on the surface of glass or silicon through hydrophobic interactions, biotin-streptavidin interactions or covalent bonding, is one of the most successful approaches. Here, we established a carbohydrate membrane array platform to explore protein-carbohydrate interaction of lectins, proteins and microorganisms. Eighty-eight biotin conjugated polyacrylamide (PAA) based glycan epitopes were immobilized directly or through streptavidin/anti-biotin antibody on three membranes (PVDF, nylon membrane or UltraBind membrane). The binding signal was observed by alkaline phosphatase conjugated on lectins or antibodies. It showed that PAA based glycan epitopes could be immobilized on UltraBind membranes through streptacidin and casein in Tris buffer saline (TBS) with 0.05% Tween 20 was the best buffer for blocking. The binding profiles of eighteen known lectins and four anti-Lewis sugar antibodies were analyzed and the results were comparable to literature. In addition, this platform was applied to investigate the carbohydrate binding profiles of influenza virus. The profiles of twenty clinical isolates of influenza B viruse and avian influenza viruse (H6N1) were examined and the results showed that avian viruse selectively bound with alpha 2-3 linked sialosides (sialylated glycans) and human viruse selectively bound with alpha 2-6 linked sialosides, respectively. Furthermore, the binding strength of high virulence avian influenza virus was obviously stronger than low one. The finding has opened a new direction for inspecting the relationship between the carbohydrate epitopes and the avian flu.
論文目次 目錄
摘要 I
Abstract II
誌謝 III
目錄 IV
表目錄 VI
圖目錄 VII
緒論 1
醣類分子與蛋白交互作用 1
醣類晶片的種類及製作 2
醣類與病毒細菌的交互作用 3
流行性感冒病毒的介紹 4
研究目的 7
研究架構 7
材料與方法 8
材料 8
測試醣受質不同濃度及streptavidin不同濃度的晶片製作 9
醣晶片與不同濃度帶鹼性磷酸酶標記的凝集素WGA測試結合反應 10
測試醣的固定方式及不同材質的薄膜晶片製作 10
三種不同薄膜的醣晶片與不同濃度帶鹼性磷酸酶標記的凝集素WGA結合反應測試 11
醣晶片製備(54種) 11
各類緩衝液的結合反應 12
醣晶片製備(88種) 13
醣晶片與帶鹼性磷酸酶標記的凝集素進行結合反應 13
醣晶片與凝集素及相應抗體進行結合反應 14
醣晶片與帶有FITC標記的凝集素進行結合反應 14
醣晶片與醣類抗體進行結合反應 15
流感醣晶片製備 16
醣晶片與流行性感冒病毒進行結合反應 16
晶片結合反應結果判讀 17
結果 18
利用不同濃度帶鹼性磷酸酶標記的凝集素WGA,測試晶片上醣受質的不同濃度及streptavidin不同濃度的結果 18
利用不同濃度帶鹼性磷酸酶標記的凝集素WGA,測試固定醣的方式及不同材質薄膜晶片的結果 18
測試不同緩衝液結合反應的結果 21
測試本研究所使用的相關抗體與醣類晶片有否發生非專一性結合的情況 22
88種醣類晶片與帶鹼性磷酸酶標記的凝集素結合反應的結果 22
凝集素及相應抗體結合反應的結果 23
帶有FITC標記的凝集素結合反應的結果 25
88種醣類晶片與醣類抗體結合反應的結果 26
感冒病毒醣類晶片與感冒病毒結合反應的結果 26
討論 28
製備醣類晶片討論 28
相關抗體與醣類晶片發生非專一性結合的討論 28
凝集素在54種醣類晶片與88種醣類晶片結合得到結果的差異 28
凝集素以不同方式與88種醣類晶片結合得到結果的差異 29
凝集素在88種醣類晶片偵測得到的結果與其特性相異 30
流感病毒偵測得到的結果討論 31
結論 32
參考文獻 33
自述 106
參考文獻 1. Bertozzi, C. R., and Kiessling, L. L. (2001) Science 291, 2357-2364
2. Crocker, P. R., Paulson, J. C., and Varki, A. (2007) Nat Rev Immunol 7, 255-266
3. Lis, H., and Sharon, N. (1998) Chem Rev 98, 637-674
4. Robinson, M. J., Sancho, D., Slack, E. C., LeibundGut-Landmann, S., and Reis e Sousa, C. (2006) Nat Immunol 7, 1258-1265
5. Smith, E. A., Thomas, W. D., Kiessling, L. L., and Corn, R. M. (2003) J Am Chem Soc 125, 6140-6148
6. van Vliet, S. J., den Dunnen, J., Gringhuis, S. I., Geijtenbeek, T. B., and van Kooyk, Y. (2007) Curr Opin Immunol 19, 435-440
7. Feizi, T., and Loveless, R. W. (1996) Am J Respir Crit Care Med 154, S133-136
8. van Kooyk, Y., and Rabinovich, G. A. (2008) Nat Immunol 9, 593-601
9. Huang, C. Y., Thayer, D. A., Chang, A. Y., Best, M. D., Hoffmann, J., Head, S., and Wong, C. H. (2006) Proc Natl Acad Sci U S A 103, 15-20
10. Dube, D. H., and Bertozzi, C. R. (2005) Nat Rev Drug Discov 4, 477-488
11. Sharon, N., and Goldstein, I. J. (1998) Science 282, 1049
12. Sharon, N., and Lis, H. (2004) Glycobiology 14, 53R-62R
13. Hirabayashi, J. (2004) Glycoconj J 21, 35-40
14. Wu, A. M., Lisowska, E., Duk, M., and Yang, Z. (2008) Glycoconj J
15. Angeloni, S., Ridet, J. L., Kusy, N., Gao, H., Crevoisier, F., Guinchard, S., Kochhar, S., Sigrist, H., and Sprenger, N. (2005) Glycobiology 15, 31-41
16. Hsu, K. L., and Mahal, L. K. (2006) Nat Protoc 1, 543-549
17. Hsu, K. L., Pilobello, K. T., and Mahal, L. K. (2006) Nat Chem Biol 2, 153-157
18. Kuno, A., Uchiyama, N., Koseki-Kuno, S., Ebe, Y., Takashima, S., Yamada, M., and Hirabayashi, J. (2005) Nat Methods 2, 851-856
19. Pilobello, K. T., Krishnamoorthy, L., Slawek, D., and Mahal, L. K. (2005) Chembiochem 6, 985-989
20. Zheng, T., Peelen, D., and Smith, L. M. (2005) J Am Chem Soc 127, 9982-9983
21. Blixt, O., Collins, B. E., van den Nieuwenhof, I. M., Crocker, P. R., and Paulson, J. C. (2003) J Biol Chem 278, 31007-31019
22. Mega, T., and Hase, S. (1991) J Biochem 109, 600-603
23. Shinohara, Y., Kim, F., Shimizu, M., Goto, M., Tosu, M., and Hasegawa, Y. (1994) Eur J Biochem 223, 189-194
24. Dam, T. K., and Brewer, C. F. (2004) Methods Enzymol 379, 107-128
25. Tateno, H., Nakamura-Tsuruta, S., and Hirabayashi, J. (2007) Nat Protoc 2, 2529-2537
26. Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M. E., Alvarez, R., Bryan, M. C., Fazio, F., Calarese, D., Stevens, J., Razi, N., Stevens, D. J., Skehel, J. J., van Die, I., Burton, D. R., Wilson, I. A., Cummings, R., Bovin, N., Wong, C. H., and Paulson, J. C. (2004) Proc Natl Acad Sci U S A 101, 17033-17038
27. Feizi, T., Fazio, F., Chai, W., and Wong, C. H. (2003) Curr Opin Struct Biol 13, 637-645
28. Fukui, S., Feizi, T., Galustian, C., Lawson, A. M., and Chai, W. (2002) Nat Biotechnol 20, 1011-1017
29. Manimala, J. C., Roach, T. A., Li, Z., and Gildersleeve, J. C. (2006) Angew Chem Int Ed Engl 45, 3607-3610
30. Stevens, J., Blixt, O., Glaser, L., Taubenberger, J. K., Palese, P., Paulson, J. C., and Wilson, I. A. (2006) J Mol Biol 355, 1143-1155
31. Stevens, J., Blixt, O., Paulson, J. C., and Wilson, I. A. (2006) Nat Rev Microbiol 4, 857-864
32. Galanina, O. E., Mecklenburg, M., Nifantiev, N. E., Pazynina, G. V., and Bovin, N. V. (2003) Lab Chip 3, 260-265
33. Dyukova, V. I., Dementieva, E. I., Zubtsov, D. A., Galanina, O. E., Bovin, N. V., and Rubina, A. Y. (2005) Anal Biochem 347, 94-105
34. Lee, J. C., Wu, C. Y., Apon, J. V., Siuzdak, G., and Wong, C. H. (2006) Angew Chem Int Ed Engl 45, 2753-2757
35. Wang, D., Liu, S., Trummer, B. J., Deng, C., and Wang, A. (2002) Nat Biotechnol 20, 275-281
36. Houseman, B. T., and Mrksich, M. (2002) Chem Biol 9, 443-454
37. Fazio, F., Bryan, M. C., Blixt, O., Paulson, J. C., and Wong, C. H. (2002) J Am Chem Soc 124, 14397-14402
38. Manimala, J. C., Roach, T. A., Li, Z., and Gildersleeve, J. C. (2007) Glycobiology 17, 17C-23C
39. Tateno, H., Mori, A., Uchiyama, N., Yabe, R., Iwaki, J., Shikanai, T., Angata, T., Narimatsu, H., and Hirabayashi, J. (2008) Glycobiology 18, 789-798
40. Rogers, G. N., and Paulson, J. C. (1983) Virology 127, 361-373
41. Rogers, G. N., Paulson, J. C., Daniels, R. S., Skehel, J. J., Wilson, I. A., and Wiley, D. C. (1983) Nature 304, 76-78
42. Tumpey, T. M., Maines, T. R., Van Hoeven, N., Glaser, L., Solorzano, A., Pappas, C., Cox, N. J., Swayne, D. E., Palese, P., Katz, J. M., and Garcia-Sastre, A. (2007) Science 315, 655-659
43. Yamada, S., Suzuki, Y., Suzuki, T., Le, M. Q., Nidom, C. A., Sakai-Tagawa, Y., Muramoto, Y., Ito, M., Kiso, M., Horimoto, T., Shinya, K., Sawada, T., Usui, T., Murata, T., Lin, Y., Hay, A., Haire, L. F., Stevens, D. J., Russell, R. J., Gamblin, S. J., Skehel, J. J., and Kawaoka, Y. (2006) Nature 444, 378-382
44. Chandrasekaran, A., Srinivasan, A., Raman, R., Viswanathan, K., Raguram, S., Tumpey, T. M., Sasisekharan, V., and Sasisekharan, R. (2008) Nat Biotechnol 26, 107-113
45. Disney, M. D., and Seeberger, P. H. (2004) Chem Biol 11, 1701-1707
46. Walz, A., Odenbreit, S., Mahdavi, J., Boren, T., and Ruhl, S. (2005) Glycobiology 15, 700-708
47. Chessa, D., Dorsey, C. W., Winter, M., and Baumler, A. J. (2008) J Biol Chem 283, 8118-8124
48. Neumann, G., and Kawaoka, Y. (2006) Emerg Infect Dis 12, 881-886
49. Taubenberger, J. K., Reid, A. H., Lourens, R. M., Wang, R., Jin, G., and Fanning, T. G. (2005) Nature 437, 889-893
50. Scholtissek, C., Burger, H., Kistner, O., and Shortridge, K. F. (1985) Virology 147, 287-294
51. Ito, T., Couceiro, J. N., Kelm, S., Baum, L. G., Krauss, S., Castrucci, M. R., Donatelli, I., Kida, H., Paulson, J. C., Webster, R. G., and Kawaoka, Y. (1998) J Virol 72, 7367-7373
52. Hay, A. J., Gregory, V., Douglas, A. R., and Lin, Y. P. (2001) Philos Trans R Soc Lond B Biol Sci 356, 1861-1870
53. Nobusawa, E., and Sato, K. (2006) J Virol 80, 3675-3678
54. Kanegae, Y., Sugita, S., Endo, A., Ishida, M., Senya, S., Osako, K., Nerome, K., and Oya, A. (1990) J Virol 64, 2860-2865
55. McCullers, J. A., Wang, G. C., He, S., and Webster, R. G. (1999) J Virol 73, 7343-7348
56. Nakagawa, N., Kubota, R., Maeda, A., Nakagawa, T., and Okuno, Y. (2000) J Clin Microbiol 38, 3467-3469
57. Nakagawa, N., Kubota, R., Morikawa, S., Nakagawa, T., Baba, K., and Okuno, Y. (2001) J Med Virol 65, 745-750
58. Nakagawa, N., Nukuzuma, S., Haratome, S., Go, S., Nakagawa, T., and Hayashi, K. (2002) J Clin Microbiol 40, 3068-3070
59. Nakagawa, N., Kubota, R., Maeda, A., and Okuno, Y. (2004) J Clin Microbiol 42, 3295-3297
60. Nakagawa, N., Kubota, R., and Okuno, Y. (2005) J Clin Microbiol 43, 4212-4214
61. Nakagawa, N., Suzuoki, J., Kubota, R., Kobatake, S., and Okuno, Y. (2006) J Clin Microbiol 44, 1564-1566
62. Matsuzaki, Y., Sugawara, K., Mizuta, K., Tsuchiya, E., Muraki, Y., Hongo, S., Suzuki, H., and Nakamura, K. (2002) J Clin Microbiol 40, 422-429
63. Taubenberger, J. K., and Morens, D. M. (2008) Annu Rev Pathol 3, 499-522
64. Matsuzaki, Y., Katsushima, N., Nagai, Y., Shoji, M., Itagaki, T., Sakamoto, M., Kitaoka, S., Mizuta, K., and Nishimura, H. (2006) J Infect Dis 193, 1229-1235
65. Katagiri, S., Ohizumi, A., and Homma, M. (1983) J Infect Dis 148, 51-56
66. Matrosovich, M. N., Matrosovich, T. Y., Gray, T., Roberts, N. A., and Klenk, H. D. (2004) Proc Natl Acad Sci U S A 101, 4620-4624
67. Shinya, K., Ebina, M., Yamada, S., Ono, M., Kasai, N., and Kawaoka, Y. (2006) Nature 440, 435-436
68. von Itzstein, M. (2007) Nat Rev Drug Discov 6, 967-974
69. Schnell, J. R., and Chou, J. J. (2008) Nature 451, 591-595
70. Zambon, M. C. (1999) J Antimicrob Chemother 44 Suppl B, 3-9
71. Matrosovich, M. N., Gambaryan, A. S., Teneberg, S., Piskarev, V. E., Yamnikova, S. S., Lvov, D. K., Robertson, J. S., and Karlsson, K. A. (1997) Virology 233, 224-234
72. Kogure, T., Suzuki, T., Takahashi, T., Miyamoto, D., Hidari, K. I., Guo, C. T., Ito, T., Kawaoka, Y., and Suzuki, Y. (2006) Glycoconj J 23, 101-106
73. Kumlin, U., Olofsson, S., Dimock, K., and Arnberg, N. (2008) Influenza and Other Respiratory Viruses 2, 147-154
74. Wu, W., and Air, G. M. (2004) Virology 325, 340-350
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2014-07-21起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2019-07-21起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw