臺灣博碩士論文加值系統

在生物系統中，Reactive oxygen species(ROS) (活性氧分子)藉由粒線體的電子傳遞鏈和NADP氧化酶的活化作用一直持續被製造產生。然而這些氧自由基在細胞體內卻會去破壞DNA、改變生化反應，損害細胞膜並且直接殺死細胞；以致這些氧自由基分子在癌症、心血管等疾病的形成上亦扮演著非常重要的角色。另一方面，Superoxide Dismutase(SOD) 、glutathione peroxidase (GPx) 、glutathione(GSH)和ascorbic acid這些抗氧化劑能夠藉由不同機轉去清除自由基，減低ROS所造成的傷害。Metallothionein(MT)是一個低分子量且富有cysteine的金屬結合蛋白，它能夠鏊合多種類的重金屬,而其中cysteine在則在清除自由基中扮演了最重要的角色。在水生環境裡，吳郭魚常被視為受污染水域之指標魚種而且其對重金屬耐受性比其他的魚類都好。正因如此，我們進行兩種不同自由基的體外試驗,比較吳郭魚和人類的MT清除自由基能力。在這項研究中，被純化的吳郭魚和人類的MT能夠有效地清除︰2,2-azinobis3- ethylbenzothiazoline- 6-sulfonic acid (ABTS•+) 和 1,1-diphenyl- picrylhydrazyl radical (DPPH• )等自由基，且其清除能力和MT濃度是呈現正相關的。雖然人與魚的MT皆遠高於glutathione的清除能力，但兩者的能力並無明顯的差別。另一方面，由重組大腸菌中純化出的陽性菌汞離子結合蛋白 (MerP)並且以化學合成革蘭氏陽性菌(G+ peptide：GMDCCPP)和革蘭氏陰性菌(G- peptide：GMTCAACP) MerP的金屬離子結合區域之胜肽，以DPPH•和ABTS•+進行自由基清除能力測試，同時以GSH作為此實驗的抗氧化劑對照組，在DPPH•自由基抑制百分率((OD control-OD sample)/OD control x 100%))試驗中，MerP和GSH幾乎是相同的；對於ABTS•+自由基的部分，MerP的清除率則約多出對照組GSH多達20%。此實驗證明了汞離子結合蛋白MerP也能扮演一種抗氧化劑的角色。ABTS•+自由基清除試驗中，合成的胜肽和GSH具有同等的清除能力；而在DPPH•自由基的部分，合成的胜肽抑制率則約多出對照組GSH超過30%。同時，當他們的半胱胺酸(cysteine)被丙氨酸(alanine)替換掉時即喪失清除的能力. 這些結果可以反應出細菌的重金屬結合蛋白質亦具有清除自由基的功能。

Reactive oxygen species (ROS) are continuously produced in biological system by the action of mitochondrial electron transport system and nicotinamide adenine dinucleotide phosphate (NADP) oxidase.These oxygen free radicals are cellular renegades and wreak havoc in biological system by damaging DNA,altering biochemical compounds,corroding cell membranes and killing cells outrightly.Such molecular mayhem plays a major role in the development of ailments like cancer,heart and lung diseases and cataracts.There are various key antioxidants such as superoxide dismutase (SOD),glutathione peroxidase (GPx),glutathione reductase (GR),catalase (CAT),glutathione (GSH) and ascorbic acid (AA) which mitigate the impact of ROS by different mechanisms.Antioxidant role for Metallothionein(MT) is well documented.MT is a low molecular weight and cysteine-rich metal-binding protein which could chelate many kinds of heavy metal and cysteine residues of MT are proposed to be the main contributors for its radical scavenging ability.In the water environment,tilapia shows higher tolerance than other kinds of fishes and was known as a biological marker for heavily polluted water areas.For these purposes,we compare the free radical scavenging activity using two different in vitro assays between Tilapia and Human metallothionein.In this study,the purified Tilapia and Huaman MT effectively scavenged the following free radicals：2,2-azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS•+) and 1,1-diphenyl-picrylhydrazyl radical (DPPH• ).The radical scavenging effectwas found to be concentration-dependent.However,there is no differences on the inhibition ability between Tilapia and Human MT proteins.On the other hand, Bacillus mercuric ion binding protein (MerP) was purified from recombinant Escherichia coli host and oligopeptides that according to the metal binding motif of MerP occurred from either Gram-positive bacteria (G+ peptide：GMDCCPP) or Gram-negative bacteria (G- peptide：GMTCAACP) were synthesized.The DPPH• and ABTS•+ were used as free radicals while GSH was used as the control antioxidant.Though the free radicals scavenging ratio ((OD control-OD sample)/OD control x 100%)) in DPPH• assay was almost the same between MerP and GSH while MerP was 20% higher than GSH in ABTS•+ free radicals assay.The result suggest that MerP can play as an antioxidant.Furthermore,both of the synthesized oligopeptides performed scavenging ability as good as GSH in ABTS•+ assay and over 30% higher than GSH in DPPH• assay.Meanwhile,the scavenging abilities were lost while their cysteine residues were replaced by alanine residues.These results may reflect the duel functions of those heavy metals binding proteins.

摘要…………………………………………………………………………………….I
abstract…………………………………………………………………………….…..II
目錄…………………………………………………………………………………..IV
第一章 研究背景與目的……………………………………………………………..1
第二章 文獻探討……………………………………………………………………..3
2-1自由基之特性簡介……………………………………………………….….3
2-1-1自由基之定義……………………………………………………...…..3
2-1-2自由基在生物體內的生理作用……………………………………….3
2-1-3自由基的來源………………………………………………..…..4
2-1-4過多自由基造成的傷害…………………………………………….…5
2-1-5抗氧化與自由基…………………………………………………….....5
2-2吳郭魚的介紹…………………………………………………………6
2-3金屬硫蛋白質…………………………………………………………6
2-3-1金屬硫蛋白質的簡介…………………………………………..…….6
2-3-2 金屬硫蛋白質的生理功能………………………………………..…7
2-3-3 金屬硫蛋白質的分類………………………………………………..7
2-3-4 金屬硫蛋白質的合成…………………………………………….….9
2-4 MerP的介紹……………………………………………………..…..10
第三章 研究材料與方法……………………………………………………………11
3-1菌種及質體…………………………………………………………..……..11
3-2藥品及酵素………………………………………………………..………..11
3-3試劑與緩衝溶液…………………………………………………...……….11
3-4培養基………………………………………………………………..……..12
3-5 細菌的培養與保存………………………………………………...………12
3-6質體DNA的少量製備…………………………………………………..…13
3-7 聚合酶鏈反應 (Polymerase chain reaction, PCR)......................................13
3-7-1 操作方法………...........................................................................…13
3-7-2 反應條件……………..................................................................….13
3-8 核酸洋菜膠體電泳 (agarose gel electrophoresis) 的分析與記錄….....…14
3-9 PCR產物片段回收與純化………...........................................................….14
3-10 DNA的黏接反應（Ligation）.......................................................................15
3-11 細胞轉形作用（Transformation）................................................................15
3-11-1 勝任細胞（competent cells）之製備…………............................…15
3-11-2轉形作用（Transformation）..............................................................16
3-12 藍白篩(Blue – white screen ).....................................................................16
3-13快速質體篩選法……………………………………………………….….16
3-14 pET30aHMT及pET30aTMT質體的構築……….............................……16
3-14-1 pET30aHMT質體的構築………....................................................16
3-14-2 pET30aTMT質體的構築……………...................................……..17
3-15重組菌金屬硫蛋白的大量表現……………..........................................…18
3-16蛋白的粗萃取…………………………………............…………………..18
3-17 Liquid chromatography(LC)純化人類和吳郭魚的金屬硫蛋白…............18
3-18 Sodium dodecyl sulphate polyacrylamide gel electro phoresis (SDS-PAGE)...............................................................................................18
3-19重組菌MerP蛋白的大量表現……………………….......................…….19
3-20 Liquid chromatography(LC)純化MerP蛋白………………..............……19
3-21 蛋白質定量…………………………............................................……….20
3-22 化學合成MerP汞離子結合區域胜肽……………….…………………..20
3-23 自由基的能力清除分析…………………………………………...……..21
3-23-1 DPPH radical assay..........................................................................21
3-23-2 ABTS radical assay..........................................................................21
第四章 實驗結果………………........................................................................……22
4-1 大量表現吳郭魚和人類的金屬硫蛋白並分析其自由基清除能力…...…22
4-1-1 pET30aHMT和pET30aTMT質體構築……………………………22
4-1-2金屬硫蛋白的純化……………………………………………...…..22
4-1-2-1人類金屬硫蛋白在DEAE 管柱層析的結果………………22
4-1-2-2人類金屬硫蛋白在Gel filtration管柱層析的結果………...22
4-1-2-3吳郭魚金屬硫蛋白在DEAE 管柱層析的結果………...….23
4-1-2-4吳郭魚金屬硫蛋白在Gel filtration管柱層析的結果....23
4-1-3 吳郭魚和人類的金屬硫蛋白在自由基清除能力分析………...…23
4-1-3-1 DPPH自由基之分析……………………………………..…23
4-1-3-2 ABTS自由基之分析……………………………………..…23
4-2 MerP的自由基清除能力分析………………………………………….….24
4-2-1 MerP的純化……………………………………………………...…24
4-2-1-1 MerP蛋白在DEAE 管柱層析的結果…………………..…24
4-2-1-2 MerP蛋白在phenyl sepharose 管柱層析的結果……...…..24
4-2-1-3 MerP蛋白在Gel filtration 管柱層析的結果…………...….24
4-2-2 MerP在清除DPPH和ABTS自由基之分析…………………...….24
4-2-2-1 DPPH自由基之分析………………………………………..24
4-2-2-2 ABTS自由基之分析………………………………………..25
4-3化學合成MerP汞離子結合區域胜肽之清除自由基能力………..………25
4-3-1 G(+) peptide和G(-) peptide在DPPH和ABTS自由基之分析…....25
4-3-2 G(+)m peptide和G(-)m peptide在DPPH和ABTS自由基之分析..25
4-3-3 G(+)m peptide、G(+)m2 peptide和G(+)m3 peptide在DPPH和ABTS自由基之分析…………………………………………………………..…25
第五章 討論………………………………………………………………..……27
5-1 關於吳郭魚和人類的金屬硫蛋白在自由基清除能力的探討……...……27
5-2 MerP的自由基清除能力的探討…………………………………….…….28
5-3 探討化學合成MerP汞離子結合區域胜肽之清除自由基能力…...28
5-4 金屬硫蛋白質的作用功能與應用…………………………………….…..29
5-5 Future work………………………………………………………..………..30
參考文獻………………………………………………………………………..……32
圖表…………………………………………………………………………………..37
附錄………………………………………………………………………………..…56

謝儒樑.2002.應用汞離子結合蛋白MerP於重金屬生物修復上之研究.國立中興大學生命科學研究所碩士論文.
彭雨菁.2005.吳郭魚之金屬硫蛋白質在重金屬之生物檢測及其生物復育之應用.國立中興大學生命科學研究所碩士論文.
林天送.自由基與健康.科學月刊.25, 515-21, 1994.
林天送.人體自由基知多少？,健康世界雜誌, 228,20-24, 1994.
曾文陽.1976.本省吳郭魚的種類.漁牧科學叢書3本省吳郭魚的種類及養殖方法,田渙玉編,台北,台灣.pp. 9-28.
謝嘉珍、鐘文松、李宗翰.1997.莫三比克吳郭魚(Oreochromis mossambicus)生殖及育幼行為之描述.中國水產月刊第540期,台北,台灣.pp. 3-13.
A.P. Roberts, J.T. Oris, G.A. Burton Jr., W.H. Clements. 2005.Gene expression in caged fish as a first-tier indicator of contaminant exposure in streams, Environ. Toxicol. Chem. 24.3092–3098.
A. Viarengo, B. Burlando, N. Ceratto, I. Panfoli. 2000.Antioxidant role
of metallothioneins: a comparative overview, Cell. Mol. Biol. 46.407–417.
Barry Halliwell,John M.C. Gutteridge.2007.Free Radicals In Biology And Medicine. Fourth edition. New York.
Belliveau, B. H. and J. T. Tevirs. 1989. Mercury resistance and detoxification in bacteria. Appl. Organometal. Chem. 3:283-294.
Bogdanova ES, Bass IA, Minakhin LS, Petrova MA, MindlinSZ, Volodin AA, et al. 1998.Horizontal spread of mer operons among Gram-positive bacteria in natural environments. Microbiology;144:609–20.
C. Capasso, V. Carginale, O. Crescenzi, D. Di Maro, E. Parisi,
R. Spadaccini, P.A. Temussi. 2003.Solution structure of MT nc, a novel metallothionein from the Antarctic fish Notothenia coriiceps,Structure 11.435–443.
Chen, W., J. A. C. John, C. H. Lin, H. F. Lin, S. U. Wu, C. H. Lin, and C. Y. Chang. 2004. Expression of metallothionein gene during embryonic and early larval development in zebrafish. Aquatic Toxicology. 69.215-217.
Chen X., A. Agarwal, and D. P. Giedroc. 1998. Structural and functional heterogeneity among the zinc fingers of human MRE-binding transcription factor-1. Biochemistry. 37:11152-11161.
Cherian, M.G., Jayasurya, A. and Bay, B.H. 2003.Metallothioneins in human tumors and potential roles in carcinogenesis. Mutat Res, 533, 201-209.
Chieh-Chen Huang, Chi-Chia Su, Ju-Liang Hsieh, Chiao-Ping Tseng,Ping-Jei Lin, Jo-Shu Chang.2003. Polypeptides for heavy-metal biosorption: capacity and specificity of two heterogeneous MerP proteins. Enzyme and Microbial Technology 33. 379–385.
Cui, Y., Wang, J., Zhang, X., Lang, R., Bi, M., Guo, L. and Lu, S.H. 2003.ECRG2, anovel candidate of tumor suppressor gene in the esophageal carcinoma, interacts directly with metallothionein 2A and links to apoptosis. Biochem Biophys Res Commun, 302, 904-915.
Edwards, S. W. 1995. Cell signaling by integrins and immunoglobulin receptors in primed neutrophils. TIBS. 20, 362-7.
Fahim Atif, Manpreet Kaur, Seema Yousuf, Sheikh Raisuddin.2006. In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch. Chemico-Biological Interactions 162 .172–180
Fang Yang, Min Zhou, Zhimin He, , Xiaorong Liu, Lin Sun, Yu Sun, Zhuchu Chen.2007. High-yield expression in Escherichia coli of soluble human MT2A with native functions. Protein Expression and PuriWcation 53.186–194
Florianczyk, B. 2003.Copper and metallothioneins in cancer cells. Ann Univ Mariae Curie Sklodowska, 58, 390-393.
Freedman, J.H. and Peisach, J. 1989. Intracellular copper transport in cultured hepatoma cells. Biochem Biophys Res Commun, 164, 134-140.
G. Miliauskas, P.R. Venskutonis, T.A. van Beek.2003. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chemistry 85. 231–237.
Gregory, M., A. Leach, P. Kling, P. Olsson, and C. Hogstrand. 2003. Activation of the rainbow trout metallothionein-A promoter by silver and zinc. CBP J. 134.181- 188.
Hamer, D.H. 1986.Metallothionein. Annu Rev Biochem, 55, 913-951.
Hijova, E. 2004.Metallothioneins and zinc: their functions and interactions. Bratisl Lek Listy, 105, 230-234.
Ibolya B., J. Ahlqvist, A. Mulchandani, W. Chen, W. Bae, R. K. Mehra, A. Mortari, and E. Csoregi. 2003. Novel synthetic phytochelatin-based capacitive biosensor for heavymetal ion detection. Biosensors and Bioelectronics. 18:547-553
Irina V., D. C. Bittel, R. Ravindra, H. Jiang, and G. K. Andrews. 2000. Zinc and cadmium can promote rapid nuclear translocation of metal response element-binding tranxcription factor-1. J. Biological Chem. 275:9377-9384.
Jeremias, H. R. 1991. Overview of metallothionein. Methods in enzymology. 205:613-626.
Jeremias, H. R., and A. Schaffer. 1988. Biochemistry of metallothionein. Biochemistry. 27:8509-8515.
Ju-Liang HSIEH, Ching-Yi CHEN, Jo-Shu CHANG, Ginro ENDO, and Chieh-Chen HUANG.2007. Overexpression of a Single Membrane Component from the Bacillus mer Operon Enhanced Mercury Resistance in an Escherichia coli Host. Biosci. Biotechnol. Biochem., 71 (6), 1494–1499.
Ju-Ling Hsieh.2007.Molecular Characteristics and Appilcation of Mercury-Ion-Binding Protein,MerP from Bacillus megaterium MB1.Department of Life Sciences National Chung Hsing University.
Kagi, J.H. 1991. Overview of metallothionein. Methods Enzymol, 205, 613-626.
Kimura, T., N. Itoh, M. Takehara, I. Oguro, J. Ishizaki, T. Nakanishi, and K. Tanaka. 2002. MRE-binding transcription factor-1 is activated during endotoxemia: a central role for metallothionein. Toxicol. Lett. 129:77-84.
Kiyono M, Pan-Hou H. 1999.DNA sequence and expression of a defective
mer operon from Pseudomonas K-62 plasmid pMR26. Biol Pharm Bull;22:910–4.
Linchtlen, P., Y. Wang, T. Belser, O. Georgiev, U. Certa, R. Sack, and W. Schaffner. 2001.Target gene search for the metal-responsive transcription factor MTF-1. Nucleic Acids Research. 29:1514-1523.
Liu, J., Kershaw, W.C. and Klaassen, C.D. 1991.The protective effect of
metallothionein on the toxicity of various metals in rat primary hepatocyte culture.Toxicol Appl Pharmacol, 107, 27-34.
Margoshes, M. and B. L. Vallee. 1957. A Cadmium Protein from Equine Kidney Cortex. J. Am. Chem.Soc. 79:4813-4814.
Miles, A.T., Hawksworth, G.M., Beattie, J.H. and Rodilla, V. 2000.Induction,regulation, degradation, and biological significance of mammalian metallothioneins.Crit Rev Biochem Mol Biol, 35, 35-70.
Mindlin S, Kholodii G, Gorlenko Z, Minakhina S, Minakhin L,Kalyaeva E, et al. 2001 .Mercury resistance transposons of Gram-negative
environmental bacteria and their classification. Res Microbiol;152:811–22.
Moffatt, P. and Denizeau, F. 1997. Metallothionein in physiological and physiopathological processes. Drug Metab Rev, 29, 261-307.
Moyle, P. B. and J. J. Cech. 1996. Fishes, an Introduction to Ichthyology, 3rd ed., Prentice Hall ,NJ, USA, pp.137-138;170-171.
M. Satoh, A. Shimada, B. Zhang, C. Tohyama. 2000.Renal toxicity caused by cisplatinum in glutathione-depleted metallothioneinnull mice, Biochem. Pharm. 60.1729–1734.
M.V. Kumari, M. Hiramatsu, M. Ebadi. 1998.Free radical scavenging action of metallothionein isoforms I and II, Free Rad. Res. 29.93–101.
M. Zafarullah, K. Bonham, L. Gedamu. 1988.Structure of the rainbow trout metallothionein B gene and characterization of its metalresponsive region, Mol. Cell. Biol. 8.4469–4476.
Piersma, A.H., Roelen, B., Roest, P., Haakmat-Hoesenie, A.S., van Achterberg, T.A. and Mummery, C.L. (1993) Cadmium-induced inhibition of proliferation and differentiation of embryonal carcinoma cells and mechanistic aspects of protection by zinc. Teratology, 48, 335-341.
P.J. Thornalley, M. Vasak. 1985.Possible role for metallothionein in protection against radiation induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals, Biochim. Biophys. Acta 827.36–44.
Roberta Re,Nicoletta Pellegrini,Anna Proteggente,Ananth Pannala,Min Yang,and Catherine Rice-Evans.1999.Antioxidant activity applying an improved ABTS radical cation decolorization assay.Free Radical Biology & Medical,vol.26,Nos.9/10,pp.1231-1237
Rochelle LG. Fischer BM. Adler KB.1998, Concurrent production of reactive
oxygen and nitrogen species by airway epithelial cells in vitro. Free Radic.
Bio.Med., 24, 863-8.
Rodenas, J., Mitjavila, M. T. 1995. Stimultaneous generation of nitric oxide and superoxide by inflammatory cells in rats., Free Radic. Bio. Med., 18, 869-
875.
R. Scudiero, V. Carginale, M. Riggio, C. Capasso, A. Capasso,P. Kille, G. di Prisco, E. Parisi. 1997.Difference in hepatic metallothionein content in Antarctic red-blooded and haemoglobinless fish: undetectable metallothionein levels in haemoglobinless fish is accompanied by accumulation of untranslated metallothionein mRNA, Biochem. J. 332.207–211.
Sambrook, J., E. F. Fritsch and T. Maniatis. 2000. Molecular cloning.: A laboratory manual, 3nd edition. Cold Spring Laboratory. Cold Spring Harbor, New York.
Shosuke Kawanishi, Yusuke Hiraku, Shinji Oikawa. 2001, Mechanism of guanine-specific DNA damage by oxidative stress and its role in carcinogenesis and aging. Mutation Research 488,65-76 .
Thornalley, P.J. and Vasak, M. 1985.Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim Biophys Acta, 827, 36-44.
T. Kimura, I. Fujita, N. Itoh, N. Muto, T. Nakanishi, K. Takahashi,J. Azuma, K. Tanaka. 2000.Metallothionein acts as a cytoprotectantagainst doxorubucin toxicity, J. Pharm. Exp. Ther. 292.299–302.
Toren Finkel& Nikki J. Holbrook. 2000, Oxidants, oxidative stress and the biology of ageing, NATURE.,VOL 408,9 NOVEMBER.
Wang, J. F., Komarov, P. and Groot, H.1993, Luminol-chemiluminescence in rat macrophages and granulocytes : the role of NO, O2-•/H2O2 and HOCl.,Arch. Biochem. Biophys. 304, 189-196.
Http：blog.udn.com/jakwang4664/864801