臺灣博碩士論文加值系統

許多種類的微生物具有集約反應 (Quorum sensing)的基因調控機制，藉著可自由在細胞間質中擴散的autoinducer分子 (AI)─ acyl-L-homoserine lactone (AHL)濃度的改變，來作為調控相關基因活化的信號。此種機制也與許多致病菌的感染過程有關，因此信號分子AI在病原菌表現毒性相關基因上扮演著重要的角色。AI除了直接影響病菌之致病能力以外，也會對免疫系統活化造成某種程度的抑制作用，故如何中和AI的生理活性，在細菌感染治療上是一相當重要的課題。
由於AHL存在於不同菌種間結構上的變異性，在實驗中我們將succiny-BSA接上homoserine lactone (HSL)作為抗原來免疫小鼠，以期能產生專一性辨識其共通官能基HSL的抗體。並且以目前已知的抗體基因序列，使用一系列的degenerate primers以RT-PCR將免疫後小鼠脾臟細胞VH、VL 基因放大，並構築抗體噬菌體展示基因庫 (Antibody phage display library)。在構築過程中發現由於抗體基因庫因較為複雜且變異度高，在實驗操作上有諸多困難。但在重複嘗試後，已獲得構築抗體基因庫的最佳條件。
構築完成之單鏈抗體基因庫經BstNI限制酶圖譜分析 (BstNI Restriction fingerprinting)後確認基因庫的多樣性後，即進行篩選。經重複三次的親合力篩選 (Affinity selection)後，初步單離出三株對AHL具有微弱親合力之噬菌體株。分析後證實三株噬菌體皆帶有全長之單鏈抗體片段，並可表現全長蛋白分子 (~27kDa)。經轉殖至適合大量表現之菌株後便可進一步評估此類抗體在抑制集約反應上的效果及應用的潛力。

Many kinds of microorganisms are capable of regulating genes by Quorum sensing (QS) mechanism. When the cell density reaches at a certain level high, the quorum sensing regulated genes will be activated. In fact, quorum sensing genes are trigerred by organic signal molecules that are able to freely diffuse though the cell membrane. The signal molecules, also called autoinducer (AI), are derivatives of acyl homoserine lactone (AHL). Virulence genes found in many pathogenic bacteria were recently proved to be associated with AHL regulated QS system. Since AHLs regulate most virulence activity of pathogenic mechanisms, it might be significant to neutralize AHL in the course of prevention bacteria infection.
Since AIs have common structure, homoserine lactone ring, we have designed and synthesized homoserine lactone ring conjugated with succinyl-BSA as an immunogen in order to produce antibodies which can bind to variety of AHL. After immunization, we use a series of degenerate primers covering recently known murine Ig gene family and performing RT-PCR to amplify splenic VH/ VL genes. Then, the amplified VH/ VL were assembled to construct an antibody phage display library. In the preliminary work we found the complexity and variation of germline VH/ VL DNA increase the difficulty of DNA manipulation, sometimes even hinder the reaction. After repetitive attempts, we have gained the best reaction parameters.
The constructed antibody phage display library was being selected against immunogen after comfirming the diversity by BstNI restriction fingerprinting analysis. After three rounds of selection, we have isolated three phage clones with weak affinity against AHL. Further analysis showed these clones bear full-length of scFv DNA fragment and are able to express scFv. After transforming to the other E. coli suitable of protein expression, we will assess their ability of inhibiting QS system and evaluate the potential of medical use.

中文摘要…………………………………………………………………… 1
英文摘要…………………………………………………………………… 2
研究背景
Origin of quorum sensing……………………………………………… 4
Mechanism of AHL induced gene activation………………………… 4
Functional activity of AHL…………………………………………… 5
Structure of acyl-homoserine lactone……………………………… 7
實驗方向與策略
Antibody against N-Acyl homoserine lactones……………………… 9
Single chain fragment of variable region, scFv………………… 13
Expression of soluble scFv…………………………………………… 17
實驗材料與方法
1. Primer………………………………………………………… 19
2. Vector………………………………………………………… 22
3. Bacterial strain…………………………………………… 23
4. Baceriophage strain……………………………………… 23
5. Media………………………………………………………… 23
6. Synthesis of immunogen…………………………………… 24
7. Immunization………………………………………………… 24
8. Tail bleeding of mice…………………………………… 25
9. Indirect ELISA…………………………………………… 26
10. Competitive ELISA………………………………………… 27
11. SDS-PAGE…………………………………………………… 28
12. Western blotting………………………………………… 29
13. Agarose DNA electrophoresis…………………………… 30
14. Total RNA extraction from spleen cell……………… 31
15. Reverse transcription…………………………………… 32
16. PCR amplification of VH/VL and assembly of scFv…… 33
17. Restriction digestion of DNA…………………………… 35
18. Ligation of scFv DNA……………………………………… 36
19. Preparation of competent cell, heat shock method… 37
20. Preparation of electro-competent cell……………… 38
21. Heat shock Transformation……………………………… 39
22. Electroporation…………………………………………… 39
23. Generation of phage antibody from E.coli…………… 40
24. Selection of phage antibody library by panning…… 41
25. BstNI fingerprinting analysis of scFv library……… 42
26. Reinfection of E. coli HB2151 by the selected phage… 43
27. Expression of scFv from E.coli HB2151……………… 44
結果與討論
1. 抗原合成…………………………………………………… 45
2. 抗體可變區域基因庫構築………………………………… 48
3. 單鏈抗體基因庫的分析…………………………………… 53
4. Affinity Panning………………………………………… 54
縮寫字對照表…………………………………………………………… 58
參考文獻………………………………………………………………… 60

1. Amersdorfer P., Wong, C., Chen, S., Smith, T., Deshpande, S., Sheridan R., Finnern, R. & Marks, J. D. (1997). Infection and Immunity 65, 3743-752.
2. Barbas III, C. F., Burton, D. R., Scott, J. K. & Silverman, G. J. (2001). Phage Display: A laboratory manual, CSHL Press.
3. Bassler, B.L. (1999). Curr. Opin. Microbiol. 2, 582-587.
4. Bauminger, S. & Wilchek, M. (1980). Methods in Enzymology, Vol. 70, pp. 151-159. Academic Press.
5. Borrebaeck, C. A. K. (1992). Antibody Engineering, W.H.Freeman.
6. Burmester, J. & Pluckthun, A. (2001). In Antibody Engineering, pp. 19-40. Springer-Verlag.
7. Chhabra, S. R., Stead. P., Bainton, N. J., Salmmond, G. P. C., Stewart, G. S. A. B., Williams, P. & Bycroft, B. W. (1992). Journal of Antibiotics 46, 441-454.
8. Chowdhury, P. S., Viner, J. L., Beers, R., and Pastan, I. (1998). Proc. Natl. Acad. Sci. USA 95, 669-674.
9. Clackson, T., Hoogenboom, H. R., Griffiths, A. D. & Winter, G. (1991). Nature 352, 624-628.
10. De Kievit, T. R. & Iglewski, B. H. (2000). Infection and Immunity 68, 4839-4849.
11. Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L., Nealson, K. H. & Oppenheimer, N. J. (1981). Biochemistry 20, 2444-2449.
12. Finch, R. G., Pritchard, D. I., Bycroft, B. W., Williams, P. & Stewart,G. S. A. B.. (1998). Journal of Antimicrobial Chemotherapy 42, 569-571.
13. Gambello, M. J. & Iglewski, B.H. (1991). J. Bacteriol. 173, 3000-3009.
14. Gram, L., Christensen, A. B., Ravn, L., Molin, S., and Givskov, M. (1999). Appilied and Environmental Microbiology 65, 3458-3463.
15. Griffiths, A. D., Williams, S. C., Harltey, O., Tomlinson, I. M., Waterhouse, P., Crosby, W. L., Kontermann, R. E., Jones, P. T., Low, N. M., Allison, T. J., Prospero, T. D., Hoogenboom, H. R., Nissim, A., Cox, J. P. L., Harrison, J. L., Zaccolo, M., Gherardi, E. & Winter, G. (1994). EMBO J. 13, 3245-3260.
16. Hanock and Benz. (1986). BBA 860, 699-707.
17. Harrison, J. L., Williams, S. C., Winter, G. & Nissim, A. (1996). Method in Enzymology, Vol. 267, pp. 83-109. Academic Press.
18. Hawkins, R. E., Russell, S. J. & Winter, G. (1992). J. Mol. Biol. 226, 889-896.
19. Hentzer, M., Riedel, K., Rasmussen, T. B., Heydorn, A., Andersen, J. B., Parsek, M. R., Rice, S. A., Eberl, L., Molin, S., Hoiby, N., Kjelleberg, S. & Givskov, M. (2002). Microbiology 148, 87?02.
20. Hoogenboom, H. R. & Winter, G. (1992). J. Mol. Biol. 227, 381-388.
21. Horton, R. M., Cai, Z., Ho, S. N. & Pease, L. R. (1990). Biotechniques 8, 528-535.
22. Ig BLAST, http://www.ncbi.nlm.nih.gov/igblast/
23. Knappik, A., Ge, L., Honegger, A., Fischer, P. P., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A. & Virneka, B. (2000). J. Mol. Biol. 296, 57-86.
24. Krebber, A., Bornhauser, S., Burmerter, J., Honegger, A., Willuda, J., Bosshard, H. R. & Pluckthun, A. (1997). Journal of Immunological Methods 201, 35-55.
25. Leger, O. J. P., Saldanha, J. W. (2002). In Monoclonal Antibody: A practial approach, pp. 25-57. Oxford Press.
26. Lindner, P. & Pluckthun, A. (2001). Antibody Engineering, pp. 657-647. Springer-Verlag.
27. Low, N. M., Holliger, P. & Winter, G. (1996). J. Mol. Biol. 260, 359-368.
28. Marks, J. D., Griffiths, A. D., Malmqvist, M., Clackson, T. P., Bye, J. M. & Winter, G. (1992). Biotechnology 10, 779-783.
29. Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., Mccafferty, J., Griffiths, A. D. & Winter, G. (1991). J. Mol. Biol. 222, 581-597.
30. McCafferty, J. & Johnson, K. S. (1996). Phage Display and Peptides and Proteins, 79-111. Academic Press.
31. McCafferty, J., Griffiths, A. D., Winter, G. & Chiswell, D. J. (1990). Nature 348, 552-554.
32. Nealson, K. H., Platt, T. & Hastings, J. W. (1970). J. Bacteriol. 104, 313-322.
33. Nissim, A., Hoogenboom, H. R., Tomlinson, I. M., Flynn, G., Midgley, C., Lane, D. & Winter, G. (1994). EMBO J. 13, 692-698.
34. Oikawa, Y., Sugano, K. & Yonemitsu, O. (1978). J. Org. Chem. 43, 2087-2088.
35. Osbourn, J. K. (2002). 67-89. Oxford Press.
36. Parsek, M. R. & Greenberg, E. P. (2000). Proc. Natl. Acad. Sci. USA 97, 8789-793.
37. Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard, A., Iglewski, B. H. & Greenberg, E. P. (1994). Proc. Natl. Acad. Sci. USA 91, 197-201.
38. Pesci, E. C., Milbank, J. B. J., Pearson, J. P., McKnight, S., Kende, A. S., Greenberg, E. P. & Iglewski, B. H. (1999). Proc. Natl. Acad. Sci. USA 96, 11229-11234.
39. Quorum sensing site, http://www.nottingham.ac.uk/quorum/
40. Rodelas, B., Lithgow, J. K., Wisniewski-Dye, F., Hardman, A., Wilkinson, A., Economou, A., Williams, P., and Downie, J. A. (1999). Journal of Bacteriology 181, 3816-823.
41. Sapre, A. G., Ramjilal, Sivaprasad, N. & Vavia, P. R. (1996). J. Radioanal. Nucl. Chem. Letters 214, 291-301.
42. Shaw, P. D., Ping, G., Daly, S. L., Cha, C., Cronan, J. E., Rinehart, K. L. & Farrand, S. K.. (1997). Proc. Natl. Acad. Sci. USA 94, 6036-6041.
43. Sheets, M. D., Amersdorfer, P., Finnern, R., Sargent, P., Lindqvist, E., Schier, R., Hemingsen, G., Wong, C., Gerhart, J. C., and Marks, J. D. (1998). Proc. Natl. Acad. Sci. 95, 6157-6162.
44. Shih, P. C. & Huang, C. Y. (2002). J. Antimicrobial Chemotherapy 49, 309-314.
45. Smith, G. P. & Petrenko, V. A. (1997). Phage Display. Chem. Rev. 97, 391-410.
46. Warrens, A. N., Jones, M. D., Lechler, R. I.. (1997). Gene 186, 29-35.
47. Winson, M. K., Camara, M., Latifi, A., Foglino, M., Chhabra, S. R., Daykin, M., Bally, M., Chapon, V., Salmond, G. P., Bycroft, B. W., Lazdunski, A., Stewart, G. S. A. B., and Williams, P. (1995). Proc. Natl. Acad. Sci. USA 92, 9427-9431.
48. Winter, G. (1998). FEBS Letter 430, 92-94.
49. Winter, G., Griffiths, A. D., Hawkins, R. E. & Hoogenboom, H. R. (1994). Annu. Rev. Immunol. 12, 433-455.
50. Worn, A. & Pluckthun, A. (2001). J. Mol. Biol. 305, 989-1010.
51. Zhang, L., Murphy, P. J., Kerr, A. & Tate, M. E. (1993). Nature 362, 446-448.
52. Zhang, R. G., Pappas, T., Brace, J. L., Miller, P. C., Oulmassov, T., Molyneaux, J. M., Anderson, J. C., Bashkin, J. K., Winans, S. C. & Joachimiak, A. (2002). Nature 417, 971-974.