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研究生:何國銓
論文名稱:檸檬蛋白質雙硫鍵異構酶與樟芝麩胱甘肽轉移酶之基因選殖、蛋白質表現及酵素特性分析
論文名稱(外文):cloning, expression and characterization of Citrus limonum protein disulfide isomerase and Taiwanofungus camphoratus glutathione S-transferase
指導教授:林棋財
指導教授(外文):Chi-Tsai Lin
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:生物科技研究所
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:2011
畢業學年度:99
語文別:中文
論文頁數:92
中文關鍵詞:檸檬樟芝蛋白質雙硫鍵異構酶麩胱甘肽轉移酶
外文關鍵詞:Citrus limonumTaiwanofungus camphoratusprotein disulfide isomeraseglutathione S-transferase
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本論文分為兩部份,第一部分為檸檬蛋白質雙硫鍵異構酶之基因選殖、蛋白質表現及酵素特性分析。第二部份為樟芝麩胱甘肽轉移酶之蛋白質表現及酵素特性分析。
蛋白質雙硫鍵異構酶對雙硫鍵具有氧化、還原之功能,因而可幫助蛋白質形成新的雙硫鍵及促進蛋白質重新摺疊。利用聚合酶鏈鎖反應,從檸檬選殖出蛋白質雙硫鍵異構酶之全長 cDNA,轉譯區包含1503 bp、500個胺基酸,分子量為61.5 kD。發現具有兩個活性區位,分別位於胺基酸60-63、405-408,具有氧化還原的功能。以 pET-20b(+) 作為表現載體,轉形到 E. coli Rosetta ( DE3 ) pLysS 作表現,再利用含鎳螯合物凝膠之親和性管柱來純化,最後利用 scrambled RNase A 測試蛋白質雙硫鍵異構酶之活性,其 KM、Vmax 和 Kcat 的值分別為 0.015 nM、0.35 nM h -1 和 1.06 x 10-4 h-1。
麩胱甘肽轉移酶屬於第二型代謝解毒酶,高度表現於人類肝臟和肺臟組織中,其主要催化還原態麩胱甘肽與環氧化合物結合,以達到保護個體對抗細胞毒素和致癌物質之傷害。樟芝麩胱甘肽轉移酶之全長 cDNA 包含651 bp 轉譯區、216 個胺基酸,分子量為25.0 kD。以 pYEX-S1 作為表現載體,轉形到 S.cerevisiae CK3,再利用含鎳螯合物凝膠之親和性管柱來純化,最後利用1-氯-2,4-二硝基苯來測試樟芝麩胱甘肽轉移酶之活性,其 KM、Vmax 和 Kcat 的值分別為8.49 mM、0.39 nMh-1和13.03 h-1。

目錄

謝辭 I
摘要 II
Abstract IV
縮寫表 IX
壹、前言 1
Part I 檸檬蛋白質雙硫鍵異構酶 1
一、檸檬簡介 1
二、 蛋白質雙硫鍵異構酶 ( protein disulfide isomerase,PDI ) 2
三、蛋白質雙硫鍵異構酶體系結構 ( domain architecture ) 5
四、人類蛋白質雙硫鍵異構酶家族 ( human PDI family ) 6
Part II 樟芝麩胱甘肽轉移酶 7
一、樟芝簡介 7
二、麩胱甘肽 ( glutathione, GSH ) 8
三、麩胱甘肽轉移酶 ( glutathione S-transferase, GST ) 10
貳、實驗材料 12
一、材料 12
(一) 材料來源 12
(二) 載體 ( vector ) 12
(三) 宿主 ( host ) 12
二、培養基 ( medium ) 13
(一) Escherichia coli 13
(二) yeast 13
三、儀器設備 14
四、實驗套組 15
五、實驗藥品試劑 15
(一) 酵素 ( enzyme ) 15
(二) 標準品 ( marker ) 15
(三) 染劑 15
(四) 緩衝液 16
(五)培養基添加物 16
(六)蛋白質電泳相關藥品 16
(七)純化相關藥品 16
(八)活性測試相關物品 16
(九)其他 17
參、實驗方法 19
一、檸檬mRNA之萃取 19
二、cDNA之合成 21
三、檸檬之基因選殖 23
(一) 檸檬蛋白質雙硫鍵異構酶 ( ClPDI ) 之基因比對 23
(二) 選殖檸檬蛋白質雙硫鍵異構酶基因 23
(三) 瓊脂膠體電泳 ( agarose gel electrophoresis ) 24
(四) 瓊脂膠體回收與目標 DNA 純化 ( gel elution ) 25
(五) 選殖載體之建構與 cDNA 片段序列確認 26
(六) 檸檬蛋白質雙硫鍵異構酶之全長分析 27
四、檸檬蛋白質雙硫鍵異構酶與樟芝麩胱甘肽轉移酶基因之建構( construction ) 28
(一) 基因全長之選殖 28
(二) 抽取質體 DNA ( plasmid extraction ) 28
(三) 限制酶切割 29
(四) 勝任細胞之製備 30
(五) 重組質體與轉形作用 30
五、檸檬蛋白質雙硫鍵異構酶與樟芝麩胱甘肽轉移酶重組蛋白質之表現與純化 31
(一) 誘導蛋白質之表現 31
(二) 蛋白質之純化 32
(三) SDS-聚丙烯醯胺凝膠電泳( sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE ) 33
(四) 西方墨點轉漬 ( western blotting ) 36
(五) 蛋白質濃縮透析與濃度測定 38
(六) 蛋白質濃度測定 38
六、活性測定 ( activity assay ) 39
(一) 蛋白質雙硫鍵異構酶活性測定之反應式及原理 39
(二) 麩胱甘肽轉移酶活性測定之反應式及原理 41
七、酵素特性分析 ( enzyme characterization ) 42
(一) 熱穩定性 42
(二) pH 值 42
(三) imidazole 43
(四) DTT 43
肆、結果與討論 44
Part I 檸檬蛋白質雙硫鍵異構酶 44
一、檸檬蛋白質雙硫鍵異構酶之基因選殖 44
二、檸檬蛋白質雙硫鍵異構酶重組蛋白質之表現與純化 47
三、檸檬蛋白質雙硫鍵異構酶家族歸類 49
四、檸檬蛋白質雙硫鍵異構酶之活性測試 49
五、檸檬蛋白質雙硫鍵異構酶之特性分析 51
Part II 樟芝麩胱甘肽轉移酶 53
一、樟芝麩胱甘肽轉移酶重組蛋白質之表現與純化 53
二、樟芝麩胱甘肽轉移酶之酵素動力 55
三、樟芝麩胱甘肽轉移酶之特性分析 56
伍、附圖 57
Figure 1. Agarose gel analysis. 58
Figure 2. DNA and amino acid sequence of ClPDI. 59
Figure 3. Alignment of the amino acid sequence of PDI with other organisms and 3-D homology structure. 60
Figure 4. Purification of the recombinant ClPDI. 61
Figure 5. Western blot demonstration of the presence of ClPDI in Rosetta ( DE3 ) pLysS. 61
Figure 6. Western blots of TcGST and AnPDIA. 62
Figure 7. The activity of PDI was assayed. 63
Figure 8. The standard curve using different concentrations of RNase A to RNA in 5 min. 63
Figure 9. The initial rate of the enzymatic reaction was measured with the varied sRNase A concentration. 64
Figure 10. Double-reciprocal plot of ClPDI activity at varying concentrations of sRNase A. 64
Figure 11. Effect of temperature, pH, imidazole, DTT on the purified ClPDI. 65
Figure 12. Alignment of the amino acid sequence of GST with other organisms and 3-D homology structure. 66
Figure 13. Purification of the recombinant TcGST. 67
Figure 14. Molecular mass of TcGST was determined via ESI-Q-TOF mass spectrometry(Micromass, Manchester, England). The molecular mass obtained was 14304 Da. 68
Figure 15. The initial rate of the enzymatic reaction was measured with the varied CDNB concentration. 69
Figure 16. Double-reciprocal plot of TcGST activity at varying concentrations of CDNB. 69
Figure 17. Effect of temperature, pH, imidazole on the purified TcGST. 70
陸、References 71
柒、Appendix 76
Appendix 1. Citrus limonum. 77
Appendix 2. Protein disulfide isomerase and thioredoxin expression in response to hypersaline stress. 78
Appendix 3. ER lumen and calreticulin(CRT)/calnexin (CNX) cycle. 79
Appendix 4. Molecular interactions in antigen presentation and host responses to virus infections. 80
Appendix 5. PDI contains an active-site with two reduced cysteine sulfhydryl (–SH) groups. 81
Appendix 7. The human PDI family. 83
Appendix 8. Taiwanofungus camphoratus. 84
Appendix 9. pCR2.1-TOPO vector. 85
Appendix 10. pCR4-TOPO vector. 86
Appendix 11. pET-20b(+) vector. 87
Appendix 12. Map and cloning site of pYEX-S1. 88
Appendix 13. Amino Acid Sequence of Bovine Ribonuclease. 89
Appendix 14. Reestablishing correct disulfide pairing. 90
Appendix 15. GST assay. 91
Appendix 16. DTNB only assay. 91
Figure 17. GST active sites. 92
Figure 18. Mammalian GST structures. 92


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