臺灣博碩士論文加值系統

澱粉磷解酶（starch phosphorylase, SP；EC 2.4.1.1）為高等植物澱粉代謝酵素之一。先前已獲得綠豆 (Vigna radiata L.) SP之原生型全長cDNA (Vrsp-wt, 2961bp, GenBank GQ465004)；本研究目的是將Vrsp-wt選殖到以大腸桿菌為宿主的表現載體並表現出重組蛋白。實驗方法為將兩端含 EcoRI 與 SalI 限制酶切位的Vrsp-wt，接入 pMAL-c2X 載體，再轉型至 JM 109 選殖宿主；結果發現只接入 1500 bp 長度的原生型重組質體，推測是受 Vrsp-wt 本身序列影響，使其重組質體在宿主複製時產生斷裂或重組現象；因此設計修飾型的Vrsp (Vrsp-modify)，兩端為含有 SalI 與 BamHI 的切位接入；所獲正株系之重組質體大小約為10 kb，利用Vrsp-modify 序列的引子以PCR、限制酶切割與內部序列確認，已成功建立含有重組載體 pMAL-c2X-Vrsp-modify 的株系。在重組蛋白質的誘導方面，以BL21(DE3) 為表現宿主，當誘導溫度37℃、6小時、IPTG 濃度為0.6 mM，觀察到誘導出之蛋白質分子大小為 54 kDa 之可溶性未知蛋白質，但比預估為157 kDa之 rVrSP 重組蛋白質小；此 54 kDa 蛋白質待進一步的身份鑑定，西方墨點等實驗來確定是否為一部分的rVrSP 重組蛋白質，以及再試誘導條件，或需更換表現宿主來得到預期分子量蛋白質。

總　目 錄 I
圖 目 錄 V
表 目 錄 VIII
中文摘要 1
Abstract 2
第一章　前言 3
第二章 文獻回顧 5
第一節 澱粉生合成作用 5
1.1 澱粉的結構與組成 5
1.2 參與澱粉生合成之酵素 9
1.3 澱粉生合成之路徑 11
1.4 酵素性修飾澱粉的發展 13
第二節 澱粉磷解酶研究歷史 15
2.1 澱粉磷解酶背景 15
2.2 催化特性 18
2.3 綠豆中澱粉磷解酶 19
2.4 研究目的 22
第三章 材料與方法 23
第一節 材料 23
1. 1 樣品 23
1. 2 載體 23
1.3 勝任細胞 23
第二節　藥品 24
第三節 儀器設備 26
第四節 實驗流程設計 28
第五節　DNA 片段的擴增與純化 29
5.1 聚合酶鏈鎖反應 ( polymerase chain reaction, PCR) 29
5.2 DNA 洋菜膠體電泳分析法 30
5. 3 DNA片段純化方法 31
第六節 重組質體之建構與轉形 32
6.1 電轉勝任細胞製備 32
6.2 重組質體之構築 33
6.3 重組載體轉形 (transformation) 至勝任細胞 37
第七節　重組質體分析 38
7. 1 小量質體DNA抽取 38
7. 2 表現質體以PCR 增幅目標片段確認 39
7. 3　表現質體以限制酶切割確認 40
7. 4 DNA 序列定序 41
第八節 重組蛋白質的誘導與分析 41
8.1 重組蛋白質誘導表現 41
8.2 重組蛋白質之抽取 42
8.2.1 SDS 煮沸破菌法 42
8.2.2 冷凍解凍破菌法 43
8.3 以電泳確定目標蛋白表現 44
第四章 結果與討論 47
第一節 pMAL-c2X-Vrsp-wt重組質體的構築與分析 47
1.1 Vrsp-wt 內部序列限制酶切位分析 47
1.2 表現載體與宿主的挑選 48
1.3 Vrsp-wt 量化與片段純化 48
1.4 pMAL-c2X-Vrsp-wt重組質體的構築 50
1.5 pMAL-c2X-Vrsp-wt重組質體的分析 50
第二節 pMAL-c2X-Vrsp-modify重組載體的構築與分析 52
2.1 pMAL-c2X-Vrsp-modify重組載體的構築 52
2.2 pMAL-c2X-Vrsp-modify重組載體的分析 53
第三節 重組蛋白質的表現與分析 54
3.1不同IPTG濃度誘導重組蛋白質的表現 54
3.2 不同誘導時間重組蛋白質的表現 55
3.3　誘導 rVrSP重組蛋白質的探討 56
第五章　結論 57
結果圖與表 58
參考文獻 80
附錄...... 89

















圖 目 錄

圖一、直鏈澱粉與支鏈澱粉的鍵結形式圖 6
圖二、支鏈澱粉的分支結構圖 7
圖三、澱粉顆粒的結構與組成 8
圖四、澱粉生合成由光合作用產物開始的路徑圖 12
圖五、Vrsp 編碼出的內部胺基酸序列與其他物種之SP經平行並列後之比較 21
圖六、建構重組質體pMAL-c2X-Vrsp之實驗流程 28
圖七、pMAL-c2X載體結構圖 (New England BioLabs ) 40
圖八、帶有Vrsp-modify的pMK-RQ載體結構圖 (GENEART) 53
圖九、Vrsp-wt DNA限制酶切位圖譜 58
圖十、以 Vrsp-wt為模板以SP-EcoRI-F3 及SP-SalI-R3引子對使用58~62℃引子黏合溫度進行PCR擴增產物之瓊酯膠體電泳圖 60
圖十一、Vrsp-wt全長片段經EcoRI及SalI限制酶切割作用產物切膠純化後之瓊脂醣膠體電泳圖 61
圖十二、載體　pMAL-c2X 經限制酶EcoRI及SalI處理後之瓊脂醣膠體電泳圖 62
圖十三、挑選六個單一菌落抽取質體之瓊脂膠體電泳圖 63
圖十四、隨意挑選七個pMAL-c2X-Vrsp-wt positive colony之劃線次培養 64
圖十五、轉形至E. coli JM-109選殖宿主之重組質體pMAL-c2X- Vrsp-wt之瓊脂醣膠體電泳圖 65
圖十六、以SP-EcoRI-F3和SP-SalI-R3引子對進行PCR確認pMAL-c2X-Vrsp 之插入序列之瓊酯膠體電泳圖 66
圖十七、pMK-RQ載體經BamHI及SalI限制酶切割後 67
之瓊脂醣膠體電泳圖 67
圖十八、莫耳數比1：3進行接合反應並利用電穿孔轉形至BL21 (DE3)宿主細胞所獲得之positive colonies. 68
圖十九、莫耳數比1：1進行接合反應並利用電穿孔轉形至BL21 (DE3)宿主細胞所獲得之positive colonies. 69
圖二十、挑選5個positive colonies抽取質體進行瓊脂醣膠體電泳圖 70
圖二十一、轉型進入BL21(DE3)宿主的重組質體DNA經由PCR反應量化插入片段之瓊脂膠體電泳圖 71
圖二十二、轉型進入BL21(DE3)宿主的重組質體DNA經由限制酶　EcoRI 與SalI進行雙限制酶剪切作用之瓊脂膠體電泳圖 72

圖二十三、pMAL-c2X-Vrsp-modify DNA 999 bp 片段定序結果與 Vrsp-modify DNA進行並列比對 74
圖二十四、pMAL-c2X-Vrsp-modify DNA 904 bp 片段定序結果與 Vrsp-modify DNA進行並列比對 76
圖二十五、不同IPTG濃度誘導 rVrSP重組蛋白質蛋白質在大腸桿菌 BL21 (DE3)細胞表現之粗萃可溶性蛋白質SDS-PAGE 分析 77
圖二十六、不同誘導時間rVrSP重組蛋白質在大腸桿菌BL21 (DE3)宿主表現之粗萃可溶性蛋白質SDS-PAGE分析 79





















表 目 錄

表一、根據分子量、對醣類親合性及來源的不同，可將 SP 分為兩種型態 18　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
表二、本研究所使用的引子序列表. 59
表三、不同誘導時間之 BL21 (DE3) 宿主細胞之　pellet 濕重與　　　　　　　　　　　可溶性蛋白質濃度 78

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