金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試

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涂文峰(Wen-Feng Tu) 查詢紙本館藏

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生命科學系

論文名稱

金屬硫蛋白在大腸桿菌的表達與金屬累積能力測試
(Metallothionein expression in Escherichia coli and metal accumulation ability test)

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摘要(中)

在於自然環境中，具有強烈的毒性，易造成人體危害。當生物暴露在重金屬環境時，為處理體內的重金屬會表現金屬硫蛋白，並與重金屬結合降低重金屬的毒性。根據前人文獻，建構並且比較3種形式的持續表現金屬硫蛋白(metallothionein, MT)載體在大腸桿菌中的表現情形，在大腸桿菌中只有單獨表現金屬硫蛋白的情況，因金屬硫蛋白是不穩定的小分子蛋白，因此在大腸桿菌中的表達並不是很穩定；然而當金屬硫蛋白接上綠螢光蛋白(EGFP)或是谷胱甘肽S-轉移酶(GST)時，金屬硫蛋白能在大腸桿菌中穩定表達。而在測試重金屬抗性，確定大腸桿菌能生長於150 ppm Ni2+ 和 Cd2+之培養基。因為大腸桿菌的鎳離子外排系統，使鎳離子不能長時間存在於大腸桿菌內，無法應用在鎳的生物復育，然而在大腸桿菌表達金屬硫蛋白之融合蛋白的情況，在培養24小時後能穩定結合著鎳離子，避免鎳離子在大腸桿菌體內的大量流失，有效吸收鎳離子1.5 mg/g，顯示金屬硫蛋白對於增加鎳的吸收量是有效的。

摘要(英)

Heavy metals exist in the natural environment and have strong toxicity, which is easy to cause harm to the human body. When organisms are exposed to heavy metals, metallothioneins combine with heavy metals to reduce the toxicity of heavy metals. The three types of sustained expression of metallothionein (MT) plasmid in Escherichia coli were constructed and compared. In Escherichia coli, only metallothionein was expressed alone, because metallothionein is a small molecule protein not stable, however, metallothionein is attached to green fluorescent protein (EGFP) or glutathione S-transferase (GST), metallothionein can be stably expressed in Escherichia coli. While testing for heavy metal resistance, it was confirmed that Escherichia coli can grow in medium of 150 ppm Ni2+ or Cd2+. Because of the nickel ion efflux system of Escherichia coli, nickel ions cannot be present in Escherichia coli for a long time, and cannot be used for bioremediation of nickel. However, the expression of metallothionein fusion protein in Escherichia coli, after 24 hours, to avoid the massive loss of nickel ions from Escherichia coli, and effectively absorb nickel ions 1.5 mg/g, indicating that metallothionein is effective for increasing the absorption of nickel.

關鍵字(中)

★ 重金屬
★ 生物復育
★ 金屬硫蛋白
★ 基因工程

關鍵字(英)

★ heavy metals
★ biological remediation
★ metallothionein
★ genetically engineered bacteria

論文目次

中文摘要 I
Abstract II
目錄 III
表目錄 VI
圖目錄 VI
一、緒論 1
1.1 研究緣起 1
1.2 重金屬特性 2
1.2.1 「鎘」 2
1.2.2 「鎳」 2
1.3 重金屬生物復育 3
1.4 基因工程與生物整治 5
1.4.1 金屬硫蛋白(Metallothionein) 5
1.4.2 基因工程菌與生物復育 5
二、研究動機與目的 7
三、實驗材料與方法 8
3.1 實驗材料 8
3.1.1 使用儀器與廠牌 8
3.1.2 常用藥品與試劑 9
3.2 實驗方法 13
3.2.1 核酸聚合反應(PCR) 13
3.2.2 DAN 純化 14
3.2.3 DNA電泳 14
3.2.4 限制酵素處裡(Digestion) 15
3.2.5 DNA膠體純化 15
3.2.6 DNA接合反應 (Ligation) 16
3.2.7 大腸桿菌電穿孔勝任細胞製備 16
3.2.8 大腸桿菌熱休克勝任細胞製備 17
3.2.9 大腸桿菌電穿孔轉型法 18
3.2.10 大腸桿菌熱休克轉型法 19
3.2.11 表現金屬硫蛋白之質體建構 20
3.2.12 質體萃取傳統法 20
3.2.13 質體萃取試劑組 21
3.2.14 蛋白質萃取(超音波震盪法) 22
3.2.15 蛋白質萃取(法式細胞破碎儀) 23
3.2.16 蛋白質濃度測定(Bradford protein assay) 24
3.2.17 蛋白質電泳 (SDS Poly-acrylamide gel electrophoresis) 24
3.2.18 蛋白質膠體染色 (Coomassie Brilliant Blue R染色法) 25
3.2.19 蛋白質純化 (IMAC-Ni column純化) 26
3.2.20 生長曲線 28
3.2.21 重金屬生長曲線分析 28
3.2.22 重金屬生物累積實驗 29
3.2.23 菌種保存與繼代 29
3.2.24 重金屬分析 30
四、實驗結果 32
4.1 持續表現MT蛋白載體pJBM之建構 32
4.2 持續表現MT-EGFP融合蛋白載體pJBME2之建構 32
4.3 持續表現MT-EGFP融合蛋白載體pJBME2之建構 32
4.4 Escherichia coli BL21大腸桿菌持續表現MT分析 33
4.5 Escherichia coli BL21大腸桿菌持續表現MT-EGFP融合蛋白分析 33
4.6 Escherichia coli BL21大腸桿菌持續表現GST-MT融合蛋白分析 33
4.7 轉殖基因Escherichia coli BL21大腸桿菌之生長曲線 34
4.8 轉殖基因Escherichia coli BL21大腸桿菌在75 ppm Ni2+之生長曲線 34
4.9 轉殖基因Escherichia coli BL21大腸桿菌在150 ppm Ni2+之生長曲線 34
4.10 轉殖基因Escherichia coli BL21大腸桿菌在200 ppm Ni2+之生長曲線 35
4.11 轉殖基因Escherichia coli BL21大腸桿菌在75 ppm Cd2+之生長曲線 35
4.12 轉殖基因Escherichia coli BL21大腸桿菌在150 ppm Cd2+之生長曲線 36
4.13 轉殖基因Escherichia coli BL21大腸桿菌在200 ppm Cd2+之生長曲線 36
4.14 轉殖基因Escherichia coli BL21大腸桿菌在250 ppm Cd2+之生長曲線 36
4.15 Escherichia coli BL21與基因工程菌重金屬生物固定分析 37
4.16 MT2-EGFP蛋白質純化分析 37
五、討論 38
5.1 MT以及融合蛋白之表現 38
5.2 表現MT以及融合蛋白影響Escherichia coli BL21生長速率分析 38
5.3 重金屬對於轉殖基因工程菌生長影響分析分析 39
5.4 Escherichia coli BL21與基因工程菌重金屬生物固定分析 40
結論 42
圖表 43
參考文獻 54
附表 60
附圖 61

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指導教授

陳師慶(Ssu-Ching Chen)

審核日期

2019-7-11

推文