臺灣博碩士論文加值系統

胃幽門螺旋桿菌是一種會引發人類胃炎、消化性潰瘍，以及胃癌的主要的致病菌。胃幽門螺旋桿菌嗜中性白血球活化蛋白是胃幽門螺旋桿菌重要的毒性因子。胃幽門螺旋桿菌嗜中性白血球活化蛋白可能藉由活化人類白血球細胞使其釋放細胞激素和活性氧分子來引發因胃幽門螺旋桿菌感染而導致的胃黏膜發炎反應。阻斷胃幽門螺旋桿菌嗜中性白血球活化蛋白的活性很有可能可以減緩胃黏膜的發炎反應。在這篇論文中，我發現到抗體X可以偵測到重組的胃幽門螺旋桿菌嗜中性白血球活化蛋白。藉由西方墨點法、酵素酶連免疫法以及原態西方墨點法，發現到抗體X可以偵測變性態以及原態的重組胃幽門螺旋桿菌26695菌株的胃幽門螺旋桿菌嗜中性白血球活化蛋白。為了尋找抗體X辨識胃幽門螺旋桿菌嗜中性白血球活化蛋白的表位，利用改良且以聚合酶鍊鎖反應為基礎的定點突變技術產生出帶有突變的胃幽門螺旋桿菌嗜中性白血球活化蛋白基因的質體。並利用二乙氨基乙基陰離子交換樹脂純化法純化出這些帶有突變的胃幽門螺旋桿菌嗜中性白血球活化蛋白。藉由抗體X偵測這些帶有突變的胃幽門螺旋桿菌嗜中性白血球活化蛋白，我發現到抗體X偵測胃幽門螺旋桿菌嗜中性白血球活化蛋白是藉由一個與原抗體X抗原序列不同的表位，並且該表位序列於所有幽門螺桿菌菌株是保守的。於是，針對在所有的胃幽門螺旋桿菌株中鄰近胃幽門螺旋桿菌嗜中性白血球活化蛋白表位的不同胺基酸位點進行定點突變，發現抗體X能夠偵測到這些突變的胃幽門螺旋桿菌嗜中性白血球活化蛋白，代表抗體X可能可以偵測到各種胃幽門螺旋桿菌菌株中的胃幽門螺旋桿菌嗜中性白血球活化蛋白。另外，抗體X是可以用來抑制胃幽門螺旋桿菌嗜中性白血球活化蛋白刺激人類嗜中性白血球而產生的活性氧分子。因此，抗體X是可以偵測胃幽門螺旋桿菌嗜中性白血球活化蛋白以及抑制它的活性。

Helicobacter pylori (H. pylori) is a major pathogen involved in gastritis, peptic ulcer disease, and gastric cancer. Helicobacter pylori neutrophil-activating protein (HP-NAP) is an important virulence factor of H. pylori. The inflammation of the gastric mucosa caused by H. pylori infection might be resulted from the cytokines and reactive oxygen species (ROS) produced by HP-NAP-stimulated human leukocytes. Thus, H. pylori-induced inflammation of the gastric mucosa could be attenuated by blocking the activity of HP-NAP. Here, I found that antibody X not only detected their target protein but also detected recombinant HP-NAP. By western-blot, enzyme linked immunosorbent assay (ELISA) and native western-blot analyses, the antibody X detects denatured and native form recombinant HP-NAP of H. pylori 26695 strain. To determine the epitope sequence of the antibody X on HP-NAP, HP-NAP mutants were generated by using the modified PCR-based site-directed mutagenesis method and then purified by one-step DEAE anion-exchange chromatography. The antibody X is able to recognize HP-NAP through a new set of epitope sequence which is different from the original epitope of antibody X. The epitope sequence is conserved in all H. pylori strains. The non-identical amino acid residues which nearby the epitope sequence of HP-NAP in various H. pylori strains were then subjected to site-directed mutagenesis. I found that the antibody X could detect these mutated HP-NAP, indicating that antibody X is able to detect HP-NAP of various H. pylori strains. Furthermore, antibody X is able to inhibit HP-NAP-stimulated ROS production by human neutrophils. Thus, antibody X is able to detect HP-NAP and block its activity through the new epitope sequence of HP-NAP.

Table of Contents
Abstract i
中文摘要 iii
致謝 v
Abbreviation vi
Table of Contents viii
List of Figures x
List of Tables xii
Chapter 1 Introduction 11
General introduction 11
Objective of this thesis 17
Chapter 2 Production of Helicobacter pylori neutrophil-activating protein mutants by a modified PCR-based site-directed mutagenesis method with introduction of silent restriction sites in the primer 19
Abstract 19
Introduction 21
Materials and Methods 24
Results and Discussion 27
Conclusion 33
Chapter 3 Purification of Helicobacter pylori neutrophil-activating protein mutants and maltose binding protein-tagged protein containing residues Arg77 to Glu116 of Helicobacter pylori neutrophil activating protein 43
Abstract 43
Introduction 44
Materials and Methods 46
Results and Discussion 55
Conclusion 61
Chapter 4 A commercial monoclonal antibody X is a new tool for detecting Helicobacter pylori neutrophil-activating protein and blocking its activity 67
Abstract 67
Introduction 69
Materials and Methods 71
Results 81
Discussion 93
Chapter 5 Conclusion 114
Chapter 6 References 115
Chapter 7 Biography 123

References

Amedei, A., Cappon, A., Codolo, G., Cabrelle, A., Polenghi, A., Benagiano, M., Tasca, E., Azzurri, A., D'Elios, M.M., Del Prete, G., et al. (2006). The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses. Journal of Clinical Investigation 116, 1092-1101.

Arentzen, R., and Ripka, W.C. (1984). Introduction of restriction enzyme sites in protein-coding DNA sequences by site-specific mutagenesis not affecting the amino acid sequence: a computer program. Nucleic Acids Research 12, 777-787.

Blaser, M.J. (1993). Helicobacter pylori: microbiology of a 'slow' bacterial infection. Trends in Microbiology 1, 255-260.

Brisslert, M., Enarsson, K., Lundin, S., Karlsson, A., Kusters, J.G., Svennerholm, A.M., Backert, S., and Quiding-Jarbrink, M. (2005). Helicobacter pylori induce neutrophil transendothelial migration: role of the bacterial HP-NAP. FEMS Microbiology Letters 249, 95-103.

Brizzard, B.L., Chubet, R.G., and Vizard, D.L. (1994). Immunoaffinity purification of FLAG epitope-tagged bacterial alkaline phosphatase using a novel monoclonal antibody and peptide elution. BioTechniques 16, 730-735.

Burne, R.A., and Chen, Y.Y. (2000). Bacterial ureases in infectious diseases. Microbes and infection / Institut Pasteur 2, 533-542.

Censini, S., Lange, C., Xiang, Z., Crabtree, J.E., Ghiara, P., Borodovsky, M., Rappuoli, R., and Covacci, A. (1996). cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proceedings of the National Academy of Sciences of the United States of America 93, 14648-14653.

Ching, C.K., Wong, B.C., Kwok, E., Ong, L., Covacci, A., and Lam, S.K. (1996). Prevalence of CagA-bearing Helicobacter pylori strains detected by the anti-CagA assay in patients with peptic ulcer disease and in controls. The American Journal of Gastroenterology 91, 949-953.

Codolo, G., Fassan, M., Munari, F., Volpe, A., Bassi, P., Rugge, M., Pagano, F., D'Elios, M.M., and de Bernard, M. (2012). HP-NAP inhibits the growth of bladder cancer in mice by activating a cytotoxic Th1 response. Cancer Immunology, Immunotherapy 61, 31-40.

Couturier, M.R., Tasca, E., Montecucco, C., and Stein, M. (2006). Interaction with CagF is required for translocation of CagA into the host via the Helicobacter pylori type IV secretion system. Infection and Immunity 74, 273-281.

Cover, T.L., Glupczynski, Y., Lage, A.P., Burette, A., Tummuru, M.K., Perez-Perez, G.I., and Blaser, M.J. (1995). Serologic detection of infection with cagA+ Helicobacter pylori strains. Journal of Clinical Microbiology 33, 1496-1500.

Evans, D.J., Jr., Evans, D.G., Lampert, H.C., and Nakano, H. (1995a). Identification of four new prokaryotic bacterioferritins, from Helicobacter pylori, Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by analysis of gene sequences. Gene 153, 123-127.

Evans, D.J., Jr., Evans, D.G., Takemura, T., Nakano, H., Lampert, H.C., Graham, D.Y., Granger, D.N., and Kvietys, P.R. (1995b). Characterization of a Helicobacter pylori neutrophil-activating protein. Infection and Immunity 63, 2213-2220.

Evans, P.M., and Liu, C. (2005). SiteFind: a software tool for introducing a restriction site as a marker for successful site-directed mutagenesis. BMC Molecular Biology 6, 22.

Fox, J.G., and Wang, T.C. (2007). Inflammation, atrophy, and gastric cancer. The Journal of Clinical Investigation 117, 60-69.

Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., and Pease, L.R. (1989). Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.

Howden, C.W., and Hunt, R.H. (1998). Guidelines for the management of Helicobacter pylori infection. Ad Hoc Committee on Practice Parameters of the American College of Gastroenterology. The American Journal of Gastroenterology 93, 2330-2338.

Iankov, I.D., Allen, C., Federspiel, M.J., Myers, R.M., Peng, K.W., Ingle, J.N., Russell, S.J., and Galanis, E. (2012a). Expression of immunomodulatory neutrophil-activating protein of Helicobacter pylori enhances the antitumor activity of oncolytic measles virus. Molecular Therapy : The Journal of the American Society of Gene Therapy 20, 1139-1147.

Iankov, I.D., Federspiel, M.J., and Galanis, E. (2013). Measles virus expressed Helicobacter pylori neutrophil-activating protein significantly enhances the immunogenicity of poor immunogens. Vaccine 31, 4795-4801.

Iankov, I.D., Haralambieva, I.H., and Galanis, E. (2011). Immunogenicity of attenuated measles virus engineered to express Helicobacter pylori neutrophil-activating protein. Vaccine 29, 1710-1720.

Iankov, I.D., Penheiter, A.R., Carlson, S.K., and Galanis, E. (2012b). Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein. Journal of Immunological Methods 384, 1-9.

Kang, Q.Z., Duan, G.C., Fan, Q.T., and Xi, Y.L. (2005). Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli. World Journal of Gastroenterology 11, 454-456.

Karnik, A., Karnik, R., and Grefen, C. (2013). SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites. BMC Bioinformatics 14, 105.

Kottakis, F., Befani, C., Asiminas, A., Kontou, M., Koliakos, G., and Choli-Papadopoulou, T. (2009). The C-terminal region of HP-NAP activates neutrophils and promotes their adhesion to endothelial cells. Helicobacter 14, 177-179.

Kottakis, F., Papadopoulos, G., Pappa, E.V., Cordopatis, P., Pentas, S., and Choli-Papadopoulou, T. (2008). Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region even without dodecamer formation, which is a prerequisite for DNA protection--novel approaches against Helicobacter pylori inflammation. Federation of European Biochemical Societies 275, 302-317.

Laurence, J.S., Blanpain, C., De Leener, A., Parmentier, M., and LiWang, P.J. (2001). Importance of basic residues and quaternary structure in the function of MIP-1 beta: CCR5 binding and cell surface sugar interactions. Biochemistry 40, 4990-4999.

Liao, J.H., Sun, Y.H., Hsu, C.H., Lin, Y.C., Wu, S.H., Kuo, C.J., Huang, C.H., and Chiou, S.H. (2013). Up-regulation of neutrophil activating protein in Helicobacter pylori under high-salt stress: structural and phylogenetic comparison with bacterial iron-binding ferritins. Biochimie 95, 1136-1145.

Little, J.W., and Mount, D.W. (1984). Creating new restriction sites by silent changes in coding sequences. Gene 32, 67-73.

Liu, H., and Naismith, J.H. (2008). An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnology 8, 91.

Liu, J., Liu, H., Zhang, T., Ren, X., Nadolny, C., Dong, X., Huang, L., Yuan, K., Tian, W., and Jia, Y. (2014). Serum Helicobacter pylori NapA antibody as a potential biomarker for gastric cancer. Scientific Reports 4, 4143.

Long, M., Luo, J., Li, Y., Zeng, F.Y., and Li, M. (2009). Detection and evaluation of antibodies against neutrophil-activating protein of Helicobacter pylori in patients with gastric cancer. World Journal of Gastroenterology 15, 2381-2388.

Marshall, B.J., and Warren, J.R. (1984). Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1, 1311-1315.
Meng, G., Grabiec, A., Vallon, M., Ebe, B., Hampel, S., Bessler, W., Wagner, H,, Kirschning, C.J. (2003) Cellular recognition of tri-/di-palmitoylated peptides is independent from a domain encompassing the N-terminal seven leucine-rich repeat (LRR)/LRR-like motifs of TLR2. The Journal of Biological Chemistry 10, 39822-39829

Montecucco, C., and Rappuoli, R. (2001). Living dangerously: how Helicobacter pylori survives in the human stomach. Nature Reviews Molecular Cell Biology 2, 457-466.

Montemurro, P., Nishioka, H., Dundon, W.G., de Bernard, M., Del Giudice, G., Rappuoli, R., and Montecucco, C. (2002). The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a potent stimulant of mast cells. European Journal of Immunology 32, 671-676.

Namavar, F., Sparrius, M., Veerman, E.C., Appelmelk, B.J., and Vandenbroucke-Grauls, C.M. (1998). Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin. Infection and Immunity 66, 444-447.

Nelson, M., and McClelland, M. (1992). Use of DNA methyltransferase/endonuclease enzyme combinations for megabase mapping of chromosomes. Methods in Enzymology 216, 279-303.

Nomura, A., Stemmermann, G.N., Chyou, P.H., Kato, I., Perez-Perez, G.I., and Blaser, M.J. (1991). Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. The New England Journal of Medicine 325, 1132-1136.

Parsonnet, J., Friedman, G.D., Vandersteen, D.P., Chang, Y., Vogelman, J.H., Orentreich, N., and Sibley, R.K. (1991). Helicobacter pylori infection and the risk of gastric carcinoma. The New England Journal of Medicine 325, 1127-1131.

Paziak-Domanska, B., Chmiela, M., Jarosinska, A., and Rudnicka, W. (2000). Potential role of CagA in the inhibition of T cell reactivity in Helicobacter pylori infections. Cellular Immunology 202, 136-139.

Polenghi, A., Bossi, F., Fischetti, F., Durigutto, P., Cabrelle, A., Tamassia, N., Cassatella, M.A., Montecucco, C., Tedesco, F., and de Bernard, M. (2007). The neutrophil-activating protein of Helicobacter pylori crosses endothelia to promote neutrophil adhesion in vivo. Journal of Immunology 178, 1312-1320.

Prickett, K.S., Amberg, D.C., and Hopp, T.P. (1989). A calcium-dependent antibody for identification and purification of recombinant proteins. BioTechniques 7, 580-589.

Ramachandran, M., Jin, C., Yu, D., Eriksson, F., and Essand, M. (2014). Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration. Journal of Immunology 193, 2287-2296.

Ramachandran, M., Yu, D., Wanders, A., Essand, M., and Eriksson, F. (2013). An infection-enhanced oncolytic adenovirus secreting H. pylori neutrophil-activating protein with therapeutic effects on neuroendocrine tumors. Molecular Therapy : The Journal of the American Society of Gene Therapy 21, 2008-2018.

Roosild, T.P., Castronovo, S., and Choe, S. (2006). Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins. Acta Crystallographica Section F, Structural Biology and Crystallization Communications 62, 835-839.

Rossi, G., Ruggiero, P., Peppoloni, S., Pancotto, L., Fortuna, D., Lauretti, L., Volpini, G., Mancianti, S., Corazza, M., Taccini, E., et al. (2004). Therapeutic vaccination against Helicobacter pylori in the beagle dog experimental model: safety, immunogenicity, and efficacy. Infection and Immunity 72, 3252-3259.

Satin, B., Del Giudice, G., Della Bianca, V., Dusi, S., Laudanna, C., Tonello, F., Kelleher, D., Rappuoli, R., Montecucco, C., and Rossi, F. (2000). The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor. Journal of Experimental Medicine 191, 1467-1476.

Shih, K.S., Lin, C.C., Hung, H.F., Yang, Y.C., Wang, C.A., Jeng, K.C., and Fu, H.W. (2013). One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis. PloS One 8, e60786.

Slootstra, J.W., Kuperus, D., Pluckthun, A., and Meloen, R.H. (1997). Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5. Molecular Diversity 2, 156-164.

Stockman, B.J., Bannow, C.A., Miceli, R.M., Degraaf, M.E., Fischer, H.D., and Smith, C.W. (1995). Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition. International Journal of Peptide and Protein Research 45, 11-16.

Suggs, S.V., Wallace, R.B., Hirose, T., Kawashima, E.H., and Itakura, K. (1981). Use of synthetic oligonucleotides as hybridization probes: isolation of cloned cDNA sequences for human beta 2-microglobulin. Proceedings of the National Academy of Sciences 78, 6613-6617.

Teneberg, S., Miller-Podraza, H., Lampert, H.C., Evans, D.J., Jr., Evans, D.G., Danielsson, D., and Karlsson, K.A. (1997). Carbohydrate binding specificity of the neutrophil-activating protein of Helicobacter pylori. The Journal of Biological Chemistry 272, 19067-19071.

Tonello, F., Dundon, W.G., Satin, B., Molinari, M., Tognon, G., Grandi, G., Del Giudice, G., Rappuoli, R., and Montecucco, C. (1999). The Helicobacter pylori neutrophil-activating protein is an iron-binding protein with dodecameric structure. Molecular Microbiology 34, 238-246.

Tsai, C.C., Kuo, T.Y., Hong, Z.W., Yeh, Y.C., Shih, K.S., Du, S.Y., and Fu, H.W. (2015). Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells. Virulence 17, 755-765.

Wang, C.A., Liu, Y.C., Du, S.Y., Lin, C.W., and Fu, H.W. (2008). Helicobacter pylori neutrophil-activating protein promotes myeloperoxidase release from human neutrophils. Biochemical and Biophysical Research Communications 377, 52-56.

Wang, T., Liu, X., Ji, Z., Men, Y., Du, M., Ding, C., Wu, Y., and Kang, Q. (2015). Antitumor and immunomodulatory effects of recombinant fusion protein rMBP-NAP through TLR-2 dependent mechanism in tumor bearing mice. International immunopharmacology 29, 876-883

Yang, Y., Mayo, K.H., Daly, T.J., Barry, J.K., and La Rosa, G.J. (1994). Subunit association and structural analysis of platelet basic protein and related proteins investigated by 1H NMR spectroscopy and circular dichroism. The Journal of Biological Chemistry 269, 20110-20118.

Yang, Y.C., Kuo, T.Y., Hong, Z.W., Chang, H.W., Chen, C.C., Tsai, T.L., and Fu, H.W. (2015). High yield purification of Helicobacter pylori neutrophil-activating protein overexpressed in Escherichia coli. BMC biotechnology 15, 23.

Yokota, S., Toita, N., Yamamoto, S., Fujii, N., and Konno, M. (2013). Positive relationship between a polymorphism in Helicobacter pylori neutrophil-activating protein a gene and iron-deficiency anemia. Helicobacter 18, 112-116.

Zanotti, G., Papinutto, E., Dundon, W., Battistutta, R., Seveso, M., Giudice, G., Rappuoli, R., and Montecucco, C. (2002). Structure of the neutrophil-activating protein from Helicobacter pylori. Journal of Molecular Biology 323, 125-130.

Zheng, L., Baumann, U., and Reymond, J.L. (2004). An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Research 32, e115.