(3.238.130.97) 您好!臺灣時間:2021/05/10 13:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

: 
twitterline
研究生:黃琬茹
研究生(外文):Wan-Ju Huang
論文名稱:白蝦熱休克蛋白60之功能性分析
論文名稱(外文):The functional analysis of heat shock protein 60 in white shrimp (Litopenaeus vannamei)
指導教授:陳歷歷
指導教授(外文):Li-Li Chen
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:海洋生物研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:69
中文關鍵詞:
相關次數:
  • 被引用被引用:0
  • 點閱點閱:161
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:13
  • 收藏至我的研究室書目清單書目收藏:0
前人研究指出,熱休克蛋白60以伴護子的功能以及對哺乳動物先天免疫系統的重要性而聲名大噪。然而,熱休克蛋白60對於海洋無脊椎動物生理作用的影響,我們卻所知甚少。因此,本實驗分為三個部分來調查南美白蝦中熱休克蛋白60所扮演的角色。首先,分析熱休克蛋白60的序列,得知其具有熱休克蛋白60家族的保守性序列及代表性的重複性二級結構單位,是為典型的粒線體-熱休克蛋白60。其次,調查白蝦在不同環境壓力的變化下,熱休克蛋白60基因的表現情形,結果發現不論是養殖環境因素的變動、熱休克作用及病原感染時,熱休克蛋白60都會有所變化。尤其是熱休克作用及病原感染,熱休克蛋白60有被誘導上升的現象,說明了熱休克蛋白60在蝦類免疫系統中具有一定重要的指標性意義,視為一指標分子。第三,以熱休克蛋白60做為免疫刺激物,雖然效益不佳,但卻證實熱休克蛋白60是以胞內刺激免疫為導向。此外,熱聚集實驗也證明了白蝦的熱休克蛋白60具有伴護子的功能。綜合以上結論,熱休克蛋白60在蝦子處於逆境及免疫反應之間是為一關鍵性的因子。
謝辭 i
摘要 ii
Abstract iii
一、 文獻回顧 1
1. 台灣養蝦歷史之簡述 1
2. 蝦類的防禦機制 2
3. 熱休克蛋白 (Heat shock proteins, HSPs) 之介紹 3
4. 熱休克蛋白60 (Heat shock protein60, HSP60) 對先天免疫系統 (innate immunity) 之研究 4
二、 研究動機 6
三、 材料與方法 7
1. LvHSP60之序列分析 7
2. 親緣關係 (phylogenetic analysis) 7
3. 組織趨性 (tissue tropism) 8
3.1 樣本製備 8
3.2 組織全RNA之萃取 8
3.3 互補DNA (complementary DNA, cDNA) 之合成 8
3.4 反轉錄-聚合酶連鎖反應 (reverse transcription-polymerase chain reaction, RT-PCR) 9
4. 大腸桿菌 (Escherichia coli) 基因表現 9
4.1 全長序列選殖 9
4.2 全長蛋白表現 10
4.3 部分序列選殖 11
4.4 部分蛋白表現 11
5. 抗體 (antibody) 製備 12
6. 西方轉印法 (Western blot) 12
7. 環境逆境實驗 (environmental stresses experiment) 12
7.1 熱休克反應 (heat shock) 13
7.2 高溫變化 (hyperthermic treatment) 13
7.3 滲透壓處理 (osmotic exposure) 13
7.4 缺氧 (hypoxia) 13
7.5 白點症病毒 (white spot syndrome virus, WSSV) 感染 14
7.6 溶藻弧菌 (Vibrio alginolyticus) 感染 14
8. 定量即時PCR (quantitative real-time PCR) 15
9. 數據統計 15
10. 蛋白表現定量 16
11. 熱聚集實驗 (thermal aggregation assay) 16
12. 免疫刺激實驗 16
12.1 劑量測試 16
12.2 免疫指標 (immunity indices) 之分析 17
12.2.1 血球細胞數目 (total hemocyte count, THC) 之計數 17
12.2.2 酚氧化酶 (phenoloxidase, PO) 活性之檢測 17
四、 結果 18
1. LvHSP60之序列分析 18
2. 親緣關係 (phylogenetic analysis) 18
3. 組織趨性 (tissue tropism) 18
4. LvHSP60重組蛋白表現 19
5. 抗體專一性 19
6. 熱休克反應前後LvHSP60的表現量變化 19
7. 熱休克反應前後LvHSP60蛋白的表現量變化 20
8. 高溫變化前後LvHSP60的表現量變化 20
9. 滲透壓處理前後LvHSP60的表現量變化 20
10. 缺氧前後LvHSP60的表現量變化 21
11. WSSV感染前後LvHSP60的表現量變化 21
12. 溶藻弧菌感染前後LvHSP60的表現量變化 21
13. 伴護子功能分析 (chaperone assay) 22
14. 以LvHSP60做為免疫刺激物之分析 22
五、 討論 24
六、 參考文獻 28
七、 表與圖 33

宋延齡 (1997). 水產養殖動物疾病的防治策略。漁業推廣月刊,131, 14-21.
宋延齡,余俊億 (1997). 養殖蝦的白點病毒、水產養殖永續經營的轉捩點。漁業推廣月刊,135, 13-18.
張碧芳 (2004). 溫度對作物的影響及熱休克蛋白質的功能。植物重要防疫檢疫病害診斷鑑定技術研習會專刊,(3),119-156.
廖一久 (1969). 蝦類養殖試驗。中國水產學會五十八年年會專題演講。
Beal, M.F. 1995. Aging, energy, and oxidative stress in neurodegenerative diseases. Ann Neurol. 38:357-366.
Brocchieri, L., and Karlin, S. 2000. Conservation among HSP60 sequences in relation to structure, function, and evolution. Protein Sci. 9:476-486.
Brudzynski, K., Martinez, V., and Gupta, R. S. 1992 . Immunocytochemical localization of heat-shock protein 60-related protein in beta-cell secretory granules and its altered distribution in non-obese diabetic mice. Diabetologia 35:316.
Brudzynski, K. 1993. Insulitis-caused redistribution of heat-shock protein HSP60 inside beta-cells correlates with induction of HSP60 autoantibodies. Diabetes 42:908.
Chen, K.Y., Hsu, T.C., Huang, P.Y., Kang, S.T., Lo, C.F., Huang, W.P., and Chen, L.L. 2009. Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection. Fish & Shellfish Immunology . 27:460-465.
Chen, W., Syldath, U., Bellmann, K., Burkart, V., and Kolb, H. 1999 . Human 60-kDa heat-shock protein: a danger signal to the innate immune system. J. Immunol. 162:3212.
Cheng, M. Y., Hartl, U. and Kirschning, C. J. 1990. The mitochondrial chaperonin hsp60 is required for its own assembly. Nature 348:455.
Choresh, O., Loya, Y., Muller, W.E.G., Wiedenmann, J., and Azem, A. 2004. The mitochondrial 60-kDa heat shock protein in marine invertebrates: biochemical purification and molecular characterization. Cell Stress Chaperon. 9:38-47.
Clavero-Salas, A., Sotelo-Mundo, R.R., Gollas-Galvan, N., Hernandez-Lopez, J., Peregrino-Uriarte, A.B., Muhlia-Almazan, A., and Yepiz-Plascencia, G. 2007. Transcriptome analysis of gills from the white shrimp Litopenaeus vannamei infected with white spot syndrome virus. Fish & Shellfish Immunology. 23:459-472.
De la Vega, E., Hall, M.R., Degnan, B.M., and Wilson, K.J. 2006. Short-term hyperthermic treatment of Penaeus monodon increases expression of heat shock protein 70 (HSP70) and reduces replication of gill associated virus (GAV). Aquaculture. 253:82-90.
Feder, M.E., and Hofmann, G.E. 1999. Heat-shock proteins, molecular chaperones, and the stress response:evolutionary and ecological physiology. Annu Rev Physiol. 61:243-282.
Gething, M.J., and Sambrook, J. 1992. Protein folding in the cell. Nature. 355:33-45.
Habich, C., Baungart, K., Kolb, H., and Burkhart, V. 2002. The receptor for hsp60 on macrophages is saturable, specific and distinct from the receptors for other heat shock proteins. J Immunol. 168:569-576.
Haas, I.G. 1991. BiP — a heat shock protein involved in immunoglobulin chain assembly. Curr. Top. Microbiol Immunol. 167:71-82.
Hatefi, Y. 1985. The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem. 54:1015-1069.
Hauton, C., and Smith, V.J. 2007. Adaptive immunity in invertebrates:a straw house without a mechanistic foundation. Bioessays. 29:1138-1146.
Hoffmann, J.A., Kafatos, F.C., Janeway, C.A., and Ezekowitz, R.A. 1999. Phylogenetic perspectives in innate immunity. Science. 284:1313-1318.
Huang, P.Y., Kang, S.T., Chen, W.Y., Hsu, T.C., Lo, C.F., Liu, K.F., and Chen, L.L. 2008. Identification of the small heat shock protein, HSP21, of shrimp Penaeus monodon and the gene expression of HSP21 is inactivated after white spot syndrome virus (WSSV) infection. Fish & Shellfish Immunology. 25:250-257.
Iwanaga, S., Kawabata, S., Miura,Y., Seki, N., Shigenaga, T., and Muta, T. 1994. Clotting cascade in the immune response of horseshoe crab.p. 79-96. In Hoffmann, J.A., Janeway, C.A.J., and Natori, S.【edi.】, Phylogenetic perspective in immunity:the insect host defense.
Kampmueller, K.M., and Miller, D.J. 2005. The Cellular Chaperone Heat Shock Protein 90 facilitates Flock House virus RNA replicationin Drosophilan. CellsJ Virol. 79:6827-6837.
Kol, A., Bourcier, T., Lichtman, A. H., and Libby, P. 1999. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth muscle cells, and macrophages. J. Clin. Invest. 103:571.
Kurtz, J., and Armitage, S.A. 2006. Alternative adaptive immunity in invertebrates. Trends Immunol. 27:493-6.
Lackie, A.M. 1988. Hemocyte behavior. Adv. Insect Physiol. 21:85-178.
Liao, I.C. 1989a. Penaeus monodon culture in Taiwan:Through two decades of growth. Int. J. Aquacult. Fish. Technol. 1:16-24.
Liao, I.C. 1989b. Taiwanese shrimp culture:A molting industry, p55-83. In:K. Chauvun, P. Menesses, W. Chauvin and A. Cuccia. 【edi.】, Proc. Shrimp World IV (The Fourth Shrimp World Marketing Conference), Shrimp World Incorporated, New Orleans, Louisiana, U.S.A.
Lindquist, S. 1986. The heat-shock response. Annu Rev Biochem. 55:1151-1191.
Liu, C.H., Chemg, W., Hsu, J.P., and Chen, J.C. 2004. Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencecing. Dis Aquat Org. 61:169-174.
McMullen, T.W., and Hallberg, R.L. 1987. A normal mitochondrial protein is selective synthesized and accumulated during heat shock in Tetrahymena thermophila. Molec. Cell. Biol. 7:4414-4423.
Morimoto, R.I. 1998. Regulation of the heat shock transcriptional response:cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev. 12:3788-3796.
Parsell, D.A., and Lindquist, S. 1993. The function of heat-shock proteins in stress tolerance:degradation and reactivation of damaged proteins. Annu Rev Genet. 27:437-496.
Ritossa, F.A. 1962. New puffing pattern induced by temperature shock and DNP in Drosophila. Experientia. 18:571-573.
Sanchez, G.I., Carucci, D.J., Sacci, J.J., Resau, J.H., Rogers, W.O., Kumar, N., and Hoffman, S.L. 1999. Plasmodium yoelli:cloning and characterization of the gene encoding for the mitochondrial heat shock protein 60. Exp Parasitol. 93:181-190.
Scheffler, I.E. 1999. Structure and morphology:integration into the cell. In:Scheffler IE, ed. Mitochondria. New York:Wiley-Liss, 15-47.
Soderhall, K., and Cerenius, L. 1998. Role of prophenoloxidase-activating system in invertebrate immunity. Curr Opin Immunol. 10:23-28.
Sritunyalucksana, K., and Soderhall, K. 2000. The proPO and clotting system in crustaceans. Aquaculture. 191:53-59.
Srivastava, P. 2002. Roles of heat-shock proteins in innate and adaptive immunity. Nature Immunology. 2:185-194.
Sung, H.H., Chang, H.J., Her, C.H., Chang, J.C., and Song, Y.L. 1998. Phenoloxidase activity of hemocytes derived from Penaeus monodon and Macrobrachium rosenbergii. J Invertebr Pathol. 71:26-33.
Sung, H.H., and Song, Y.L. 1996. Tissue location of Vibrio antigen delivered by immersion to tiger shrimp (Penaeus monodon). Aquaculture. 145:41-54.
Tissieres, A., Mitchell, H.K., and Tracy, U.M. 1974. Protein synthesis in salivary glands of Drosophila melanogaster relation to chromosome puffs. J Mol Biol. 84:389-398.
Tsing, A., Arcier, J.M., and Brehelin, M. 1989. Hemocytes of penaeid and palaemonid shrimps:morphology, cytochemistry, and hemograms. J Invertebr Pathol. 53:64-77.
Vargas-Albores, F., Jimknez-Vega, F., and Söderhäll, K. 1996. A plasma protein isolated from brown shrimp (penaeus californiensis) which enhances the activation of prophenoloxidase system by beta-1,3-glucan. Developmenatl & Comparative Imunology. 20:299-306.
Wang, H.C.,Wang, H.C., Leu, J.H., Kou, G.H.,Wang, A.H., and Lo, C.F. 2007. Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection. Dev Comp Immunol. 31:672-686.
Wu, W.L., Zong, R.R., Xu, J.Y., and Zhang, X.B. 2008. Antiviral phagocytosis is regulated by a novel Rab-dependent complex in shrimp Penaeus japonicus. J Proteome Res. 7:424-431.
連結至畢業學校之論文網頁點我開啟連結
註: 此連結為研究生畢業學校所提供,不一定有電子全文可供下載,若連結有誤,請點選上方之〝勘誤回報〞功能,我們會盡快修正,謝謝!
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔