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研究生:林秋妏
研究生(外文):Chio-Wen Lin
論文名稱:胃幽門螺旋桿菌之嗜中性白血球激活蛋白的表達、純化與定性
論文名稱(外文):Overexpression, Purification and Characterization of Helicobacter pylori Neutrophil-Activating Protein
指導教授:傅化文傅化文引用關係
指導教授(外文):Hua-Wen Fu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子與細胞生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:英文
論文頁數:57
中文關鍵詞:胃幽門螺旋桿菌嗜中性白血球激活蛋白
外文關鍵詞:H. pyloriHelicobacter pyloriNAPneutrophil-activating protein
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胃幽門螺旋桿菌被認為是最常見的感染源,幾乎有一半以上的人口都感染有胃幽門螺旋桿菌。近來發現,有一種胃幽門螺旋桿菌的蛋白會高度引起人體的免疫反應,此蛋白被稱為嗜中性白血球激活蛋白。然而,直接從胃幽門螺旋桿菌中純化出嗜中性白血球激活蛋白是非常困難的,因為胃幽門螺旋桿菌一定要生長在微厭氧的環境下,而且產量也不多。迄今,沒有報導指出可以從大腸桿菌的表達系統中得到具有功能的重組嗜中性白血球激活蛋白。為了確定是否可以從大腸桿菌的表達系統中得到具有功能且由十二個單體所組成的嗜中性白血球激活蛋白,從大腸桿菌表達系統中被純化出的重組自然嗜中性白血球激活蛋白和帶有六個組胺酸的嗜中性白血球激活蛋白進一步地被定性。經由超高速離心與凝膠過濾之分析,重組自然嗜中性白血球激活蛋白和帶有六個組胺酸的嗜中性白血球激活蛋白呈現多體聚合的情況。利用旋光儀分析,確定了純化出來的重組蛋白都具有a-螺旋狀的二級結構。同時,經由化學冷光與西方墨點法,證明純化出來的重組蛋白能使細胞產生過氧化物並增加ERK的磷酸化。從此一大腸桿菌的表達系統中,可以純化得到大量的重組嗜中性白血球激活蛋白。而重組的嗜中性白血球激活蛋白之多聚體與活性都與從胃幽門螺旋桿菌來的嗜中性白血球激活蛋白相似。此結果證明著從大腸桿菌純化來的重組嗜中性白血球激活蛋白具有功能上的活性。更重要的是,大腸桿菌的表達系統能夠產生大量的重組嗜中性白血球激活蛋白以提供基礎研究、疫苗的開發、或是藥物的設計。
Helicobacter pylori is considered the most common infectious agent among humans worldwide. Recently, the neutrophil-activating protein (NAP), a H. pylori protein that is highly immunogenic in human, has been identified. However, purification of NAP from H. pylori is difficult due to its fastidious growth in microaerophilic condition and poor yield. To date, it has never been reported that the functional recombinant NAP were purified from E. coli expression system. To determine whether the functional dodecameric NAP could be obtained from E. coli expression system, both recombinant native and His-tag fusion NAPs expressed in E. coli were purified and characterized. By size exclusion and analytical ultracentrifuge analyses, both recombinant native and His-tag fusion NAPs are assembled into multimeric forms. By circular dichroism analysis, the secondary structures of both purified recombinant NAPs were identified as the typical a-helix. By luminol-enhanced chemiluminescent assay and westen blotting, both the recombinant native and His-tag fusion NAPs were able to cause the O2.- release from cells and increase phosphorylation of extracellular regulated kinase (ERK). From my E. coli expression system, a large amount of recombinant NAPs were obtained. Multimeric assembly and activity of both recombinant native and His-tag fusion NAPs are similar to those of native NAP from H. pylori. The results indicate that the recombinant NAPs obtained from our E. coli expression system is functionally active. Importantly, this E. coli expression system can provide a lot of recombinant NAPs for basic studies, vaccinal development, or drug design.
References
Chang,C.C., Tsai,C.T., and Chang,C.Y. (2002). Structural restoration of inactive recombinant fish growth hormones by chemical chaperonin and solvent restraint approaches. Protein Eng, 15, 437-441.

Cooksley,C., Jenks,P.J., Green,A., Cockayne,A., Logan,R.P., and Hardie,K.R. (2003). NapA protects Helicobacter pylori from oxidative stress damage, and its production is influenced by the ferric uptake regulator. J. Med. Microbiol., 52, 461-469.

Del Giudice,G., Covacci,A., Telford,J.L., Montecucco,C., and Rappuoli,R. (2001). The design of vaccines against Helicobacter pylori and their development. Annu. Rev. Immunol., 19, 523-563.

Dundon,W.G., Nishioka,H., Polenghi,A., Papinutto,E., Zanotti,G., Montemurro,P., Del,G.G., Rappuoli,R., and Montecucco,C. (2002). The neutrophil-activating protein of Helicobacter pylori. Int. J. Med. Microbiol., 291, 545-550.

Dundon,W.G., Polenghi,A., Del Guidice,G., Rappuoli,R., and Montecucco,C. (2001). Neutrophil-activating protein (HP-NAP) versus ferritin (Pfr): comparison of synthesis in Helicobacter pylori. FEMS Microbiol. Lett., 199, 143-149.

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. Infect. Immun., 63, 2213-2220.

Garnier, J., Osguthorpe, D.J., Robson, B. (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol., 120, 97-120.

Ilari,A., Stefanini,S., Chiancone,E., and Tsernoglou,D. (2000). The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site. Nat. Struct. Biol., 7, 38-43.

Leakey,A., Hirst,R., and La Brooy,J. (2001). A low molecular weight factor is a significant mediator of non-opsonic neutrophil activation by Helicobacter pylori. J. Med. Microbiol., 50, 787-794.

Leakey,A., La Brooy,J., and Hirst,R. (2000). The ability of Helicobacter pylori to activate neutrophils is determined by factors other than H. pylori neutrophil-activating protein. J. Infect. Dis., 182, 1749-1755.

Montecucco,C. and de Bernard,M. (2003). Molecular and cellular mechanisms of action of the vacuolating cytotoxin (VacA) and neutrophil-activating protein (HP-NAP) virulence factors of Helicobacter pylori. Microbes. Infect., 5, 715-721.

Montecucco,C. and Rappuoli,R. (2001). Living dangerously: how Helicobacter pylori survives in the human stomach. Nat. Rev. Mol. Cell Biol., 2, 457-466.
Montemurro,P., Barbuti,G., Dundon,W.G., Del Giudice,G., Rappuoli,R., Colucci,M., De Rinaldis,P., Montecucco,C., Semeraro,N., and Papini,E. (2001). Helicobacter pylori neutrophil-activating protein stimulates tissue factor and plasminogen activator inhibitor-2 production by human blood mononuclear cells. J. Infect. Dis., 183, 1055-1062.

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. Eur. J. Immunol., 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. Infect. Immun., 66, 444-447.

Niccolai,A., Fontani,S., Kapat,A., and Olivieri,R. (2003). Maximization of recombinant Helicobacter pylori neutrophil activating protein production in Escherichia coli: improvement of a chemically defined medium using response surface methodology. FEMS Microbiol. Lett., 221, 257-262.

Nishioka,H., Baesso,I., Semenzato,G., Trentin,L., Rappuoli,R., Del Giudice,G., and Montecucco,C. (2003). The neutrophil-activating protein of Helicobacter pylori (HP-NAP) activates the MAPK pathway in human neutrophils. Eur. J. Immunol., 33, 840-849.

Papinutto,E., Dundon,W.G., Pitulis,N., Battistutta,R., Montecucco,C., and Zanotti,G. (2002). Structure of two iron-binding proteins from Bacillus anthracis. J. Biol. Chem., 277, 15093-15098.

Roche,N., Angstrom,J., Hurtig,M., Larsson,T., Boren,T., and Teneberg,S. (2004). Helicobacter pylori and complex gangliosides. Infect. Immun., 72, 1519-1529.

Satin,B., Del Giudice,G., Della,B., 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. J. Exp. Med., 191, 1467-1476.

Shiraishi R., Itou T., Sugisawa H., Shoji Y., Endo T., and Sakai T. (2002) The respiratory burst activity of bottlenose dolphin neutrophils elicited by several stimulants. J. Vet. Med. Sci., 64, 711-714

Teneberg,S., Jurstrand,M., Karlsson,K.A., and Danielsson,D. (2000). Inhibition of nonopsonic Helicobacter pylori-induced activation of human neutrophils by sialylated oligosaccharides. Glycobiology, 10, 1171-1181.

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. J. Biol. Chem., 272, 19067-19071.

Tomb,J.F., White,O., Kerlavage,A.R., Clayton,R.A., Sutton,G.G., Fleischmann,R.D., Ketchum,K.A., Klenk,H.P., Gill,S., Dougherty,B.A., Nelson,K., Quackenbush,J., Zhou,L., Kirkness,E.F., Peterson,S., Loftus,B., Richardson,D., Dodson,R., Khalak,H.G., Glodek,A., McKenney,K., Fitzegerald,L.M., Lee,N., Adams,M.D., Venter,J.C., and . (1997). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature, 388, 539-547.

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. Mol. Microbiol., 34, 238-246.

Vajdy,M., Singh,M., Ugozzoli,M., Briones,M., Soenawan,E., Cuadra,L., Kazzaz,J., Ruggiero,P., Peppoloni,S., Norelli,F., del Giudice,G., and O'Hagan,D. (2003). Enhanced mucosal and systemic immune responses to Helicobacter pylori antigens through mucosal priming followed by systemic boosting immunizations. Immunology, 110, 86-94.

Yoshida,N., Granger,D.N., Evans,D.J., Jr., Evans,D.G., Graham,D.Y., Anderson,D.C., Wolf,R.E., and Kvietys,P.R. (1993). Mechanisms involved in Helicobacter pylori-induced inflammation. Gastroenterology, 105, 1431-1440.

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. J. Mol. Biol., 323, 125-130.
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