進階搜尋


下載電子全文  
系統識別號 U0026-2308201216010000
論文名稱(中文) Cathepsin L在點帶石斑魚上的功能性分析
論文名稱(英文) Functional Analysis of Cathepsin L (CTSL) in Orange-Spotted Grouper (Epinephelus coioides)
校院名稱 成功大學
系所名稱(中) 生物科技研究所碩博士班
系所名稱(英) Institute of Biotechnology
學年度 100
學期 2
出版年 101
研究生(中文) 蔡侑翰
研究生(英文) You-Han Cai
學號 l66991099
學位類別 碩士
語文別 中文
論文頁數 98頁
口試委員 指導教授-林翰佑
口試委員-楊惠郎
口試委員-邱品文
中文關鍵字 點帶石斑魚  半胱胺酸蛋白酶  細胞自溶酵素L 
英文關鍵字 Epinephelus coioides  cysteine protease  cathepsin L 
學科別分類
中文摘要 細胞自溶酵素L在動物的生理機制上,扮演著相當重要的作用機制,在過去研究中指出,這類蛋白水解酶和細胞的廢料降解、代謝有著很重要的關聯,而在免疫相關的研究中,位於細胞中溶小體內的細胞自溶酵素L,和對抗外來之抗原、抗原之降解、抗原的呈現有著很密不可分的關係。
因此本研究主要透過大腸桿菌重組蛋白表現系統,來了解點帶石斑魚細胞自溶酵素L的基本生化特性,並對其是否具備對抗細菌能力進行測試;並輔以即時聚合酶鏈鎖反應進行組織分布以及免疫刺激下的表現情形,結果顯示,細胞自溶酵素L必須在pH 3~6間才具有生物活性,其酵素可再4~65 ℃環境下作用,在40 ℃時其活性最大。作用溫度在25℃時 仍然保有最大活性之80%,而在最佳作用環境下其作用之半衰期約為2.07小時。在抗菌測試方面,將活菌與酵素共同培養過後,發現其具有水解細菌蛋白的情形,而在抗菌圖盤結果發現,細菌在處理酵素過後,的確會有菌數降低的情況,因此初步推測點帶石斑魚細胞自溶酵素可能具備有殺死及分解細菌之能力。而在即時聚合酶鏈鎖反應去偵測細胞自溶酵素L之表現,發現在所有採取的臟器中均會表現,而在以Vibrio anguillarum進行免疫刺激過後,在肝臟的表現量有顯著增加,我們推斷在石斑魚免疫機制上可能扮演著特定角色。
英文摘要 Cathepsin L plays a very important role in animal physiological defend mechanism. In previous studies, this proteolysis enzyme was present in the lysosome, and degradated the cell waste. But in the immune cell, the same mechanism of cathepsin L in the lysosome was help in the foreign antigens digest, and antigen presentation.
Since there were little informations of the teleost cathepsin L, the cathepsin L gene of orange spotted grouper (Epinephelus coioides) was cloned and the recombinant was expressed by E. coli expression system, the recombinant teleost cathepsin L was used to ananlysis the biochemical properties, and whether cathepsin L has ability to against pathogens in this study. Meanwhile, the cathepsin L mRNA expression pattern in various organs at different time was detected by real-time polymerase chain reaction after immune stimulation. The results showed the grouper cathepsin L was strictly work between pH 3 to 6, between 4 65℃and has maximal activity at 40℃; and it retain 80% maximal activity at 25℃. The half-life of this enzyme is about 2.07 hrs under the working condition. In the bactericidal test, the enzyme degradated bacterial protein while culture together under working condition, and that will kill the bacterial in this situation. In the tissues tropism, cathepsin L can be detected at all the organs, the mRNA expression raised significanly in the liver after Vibrio anguillarum stimulation. Since the relative mRNA expression will highly expression in the infectious situation and the bactericidal bioactive we observed. We speculate that orange-spotted grouper cathepsin L may play a specific role in immune mechanisms and against pathogen.
論文目次 中文摘要……………………………………………………………… I
英文摘要…………………………………………………………III
誌謝………………………………………………………………V
目錄………………………………………………………………VIII
圖表目錄…………………………………………………………..X
第壹章、前言…………………………………………………………1
1.1 研究背景………………………………………………..1
1.2 免疫系統………………………………………………..1
1.2.1 先天性免疫 (Innate immunity) …………..……..2
1.2.2 適應性免疫 (Adaptor immunity) ……………..…..2
1.3 蛋白分解酶 (protease) 與免疫系統……………..…..3
1.4 細胞自溶酵素L (cathepsin L) …………………..…..4
1.4.1 細胞自溶酵素L在哺乳動物上之免疫相關研究……..6
1.4.2 細胞自溶酵素L (cathepsin L)在魚類之研究……..8
第貳章、研究目的………………………………..……..…….9
第參章、材料方法及步驟…….. …….. …….. …….. …….. ..…….10
3-1實驗材料…….. …….. …….. …….. ……………….. ..…….10
3-2實驗方法…….. …….. …….. …….. ……………….. ..…….15
第肆章、研究成果…….. …….. …….. …….. ………………….43
第伍章、討論…….. ………….. …….. .. ……..……………….51
第陸章、結論…….. ………….. …….. .. ……..………………...62
第柒章、參考文獻…….. ………….. …….. ………………….63
第捌章、圖表…….. ………….. …….. ……....…………………….68
第玖章、附錄…….. ………….. …….. ……....…………………….97
參考文獻 Aranishi, F. (1999). Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel. Fish physiology and biochemistry 20, 37-41.
Aranishi, F., and Nakane, M. (1997). Epidermal proteinases in the European eel. Physiological zoology 70, 563-570.

Aranishi, F., Ogata, H., Hara, K., Osatomi, K., and Ishihara, T. (1997). Purification and characterization of cathepsin L from hepatopancreas of carp Cyprinus carpio. Comparative biochemistry and physiology Part B, Biochemistry & molecular biology 118, 531-537.

Arunachalam, B., Phan, U.T., Geuze, H.J., and Cresswell, P. (2000). Enzymatic reduction of disulfide bonds in lysosomes: characterization of a gamma-interferon-inducible lysosomal thiol reductase (GILT). Proceedings of the National Academy of Sciences of the United States of America 97, 745-750.

Bainton, D.F. (1981). The discovery of lysosomes. The Journal of cell biology 91, 66s-76s.

Barrett, A.J., Kembhavi, A.A., Brown, M.A., Kirschke, H., Knight, C.G., Tamai, M., and Hanada, K. (1982). L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. The Biochemical journal 201, 189-198.

Chertov, O., Ueda, H., Xu, L.L., Tani, K., Murphy, W.J., Wang, J.M., Howard, O.M., Sayers, T.J., and Oppenheim, J.J. (1997). Identification of human neutrophil-derived cathepsin G and azurocidin/CAP37 as chemoattractants for mononuclear cells and neutrophils. The Journal of experimental medicine 186, 739-747.

Cho, J.H., Park, I.Y., Kim, H.S., Lee, W.T., Kim, M.S., and Kim, S.C. (2002). Cathepsin D produces antimicrobial peptide parasin I from histone H2A in the skin mucosa of fish. The FASEB journal : official publication of the Federation of American Societies for Experimental Biology 16, 429-431.

Colbert, J.D., Matthews, S.P., Miller, G., and Watts, C. (2009). Diverse regulatory roles for lysosomal proteases in the immune response. European journal of immunology 39, 2955-2965.

Ishidoh, K., and Kominami, E. (1994). Multi-step processing of procathepsin L in vitro. FEBS letters 352, 281-284.

Kawai, T., and Akira, S. (2010). The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11, 373-384.

Kirschke, H., Kembhavi, A.A., Bohley, P., and Barrett, A.J. (1982). Action of rat liver cathepsin L on collagen and other substrates. The Biochemical journal 201, 367-372.

Kirschke, H., and Wiederanders, B. (1987). Lysosomal proteinases. Acta histochemica 82, 2-4.

Lennon-Duménil, A.-M., Bakker, A.H., Wolf-Bryant, P., Ploegh, H.L., and Lagaudrière-Gesbert, C. (2002). A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Current Opinion in Immunology 14, 15-21.

Liu, H., Yin, L., Zhang, N., Li, S., and Ma, C. (2006). Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix). Journal of agricultural and food chemistry 54, 9584-9591.

Magnadottir, B. (2006). Innate immunity of fish (overview). Fish & shellfish immunology 20, 137-151.

Mindell, J.A. (2012). Lysosomal acidification mechanisms. Annual review of physiology 74, 69-86.

Nomura, T., Fujishima, A., and Fujisawa, Y. (1996). Characterization and crystallization of recombinant human cathepsin L. Biochemical and biophysical research communications 228, 792-796.

Pandey, K.C., Barkan, D.T., Sali, A., and Rosenthal, P.J. (2009). Regulatory Elements within the Prodomain of Falcipain-2, a Cysteine Protease of the Malaria Parasite Plasmodium falciparum. PloS one 4, e5694.

Raptis, S.Z., Shapiro, S.D., Simmons, P.M., Cheng, A.M., and Pham, C.T. (2005). Serine protease cathepsin G regulates adhesion-dependent neutrophil effector functions by modulating integrin clustering. Immunity 22, 679-691.

Repnik, U., Stoka, V., Turk, V., and Turk, B. (2012). Lysosomes and lysosomal cathepsins in cell death. Biochimica et biophysica acta 1824, 22-33.

Schulze, H., Kolter, T., and Sandhoff, K. (2009). Principles of lysosomal membrane degradation: Cellular topology and biochemistry of lysosomal lipid degradation. Biochimica et biophysica acta 1793, 674-683.

Sina, C., Lipinski, S., Gavrilova, O., Aden, K., Rehman, A., Till, A., Rittger, A., Podschun, R., Meyer-Hoffert, U., Haesler, R., et al. (2012). Extracellular cathepsin K exerts antimicrobial activity and is protective against chronic intestinal inflammation in mice. Gut.

Smith, S.M., and Gottesman, M.M. (1989). Activity and deletion analysis of recombinant human cathepsin L expressed in Escherichia coli. The Journal of biological chemistry 264, 20487-20495.

Sol-Church, K., Picerno, G.N., Stabley, D.L., Frenck, J., Xing, S., Bertenshaw, G.P., and Mason, R.W. (2002). Evolution of placentally expressed cathepsins. Biochemical and biophysical research communications 293, 23-29.

Spitznagel, J.K. (1990). Antibiotic proteins of human neutrophils. The Journal of clinical investigation 86, 1381-1386.

Sriraman, V., and Richards, J.S. (2004). Cathepsin L gene expression and promoter activation in rodent granulosa cells. Endocrinology 145, 582-591.

Thorne, K.J., Oliver, R.C., and Barrett, A.J. (1976). Lysis and killing of bacteria by lysosomal proteinases. Infection and immunity 14, 555-563.

Uinuk-Ool, T.S., Takezaki, N., Kuroda, N., Figueroa, F., Sato, A., Samonte, I.E., Mayer, W.E., and Klein, J. (2003). Phylogeny of antigen-processing enzymes: cathepsins of a cephalochordate, an agnathan and a bony fish. Scandinavian journal of immunology 58, 436-448.

Whang, I., De Zoysa, M., Nikapitiya, C., Lee, Y., Kim, Y., Lee, S., Oh, C., Jung, S.J., Oh, M.J., Choi, C.Y., et al. (2011). Molecular characterization and expression analysis of Cathepsin B and L cysteine proteases from rock bream (Oplegnathus fasciatus). Fish & shellfish immunology 30, 763-772.

Yamaguchi, T., Naruishi, K., Arai, H., Nishimura, F., and Takashiba, S. (2008). IL-6/sIL-6R enhances cathepsin B and L production via caveolin-1-mediated JNK-AP-1 pathway in human gingival fibroblasts. Journal of cellular physiology 217, 423-432.

Zhu, X.L., Ohta, Y., Jordan, F., and Inouye, M. (1989). Pro-sequence of subtilisin can guide the refolding of denatured subtilisin in an intermolecular process. Nature 339, 483-484.

Randelli, E., Buonocore, F., and Scapigliati, G. (2008). Cell markers and determinants in fish immunology. Fish & shellfish immunology 25, 326-340.

Hsu, K.F., Wu, C.L., Huang, S.C., Wu, C.M., Hsiao, J.R., Yo, Y.T., Chen, Y.H., Shiau, A.L., and Chou, C.Y. (2009). Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells. Autophagy 5, 451-460.

Ratovitski, T., Chighladze, E., Waldron, E., Hirschhorn, R.R., and Ross, C.A. (2011). Cysteine proteases bleomycin hydrolase and cathepsin Z mediate N-terminal proteolysis and toxicity of mutant huntingtin. The Journal of biological chemistry 286, 12578-12589.
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2017-09-04起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2017-09-04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