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研究生:鍾雅薇
研究生(外文):Ya-Wei Zhong
論文名稱:使用具有INT和TEV裂解反應的表面表達系統同時生產EGFP和CecropinXJ
論文名稱(外文):Simultaneous Production of EGFP and Cecropin XJ using surface expression system with both INT and TEV cleavage reactions.
指導教授:劉永銓
指導教授(外文):Yung-Chuan Liu
口試委員:胡念仁郭家宏陳柏庭
口試日期:2023-07-06
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學工程學系所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:中文
論文頁數:83
中文關鍵詞:大腸桿菌冰核蛋白蛋白質內含肽綠螢光蛋白酵素酶切抗菌胜肽天蠶素
外文關鍵詞:E. coliIce nucleation proteinInteinEGFPTEV ProteaseAntibacterial peptidesCecropin XJ
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由於抗生素的過量生產和濫用,許多細菌開始表現出對這些抗生素的抗藥性,進而產生了超級細菌。而抗菌胜肽被認為是一種極具潛力的替代藥物,是生物界最早演化形成的先天免疫系統。此外,抗菌胜肽的殺菌機制具備多項優點,如殺菌譜廣泛、對人畜無害以及無抗藥性等問題。並且在醫學上也有抗菌胜肽成功抑制超級細菌的先例。
本研究中所要生產的 Cecropin XJ 是屬於 Cecropin-B 家族,是一種源自家蠶幼蟲的陽離子抗菌胜肽。其具有殺死癌細胞的能力,並且對人體紅血球細胞不具有溶血活性,對某些正常細胞也沒有毒性。
利用冰核蛋白表面表達系統 (Ice nucleation protein, INP) 加上蛋白質內含肽 (Intein, INT) 表達重組蛋白,該重組蛋白由增強型綠螢光蛋白 (Enhanced green fluorescent protein, EGFP)、TEV 蛋白酶和抗菌胜肽天蠶素 Cecropin XJ (CecXJ) 組成。構建形成了 pET28a-inp-int-his-egfp-tev-cecXJ 並將其轉形至不同的大腸桿菌宿主中,篩選出最適表達重組蛋白的宿主,並研究其最佳培養條件。
將表面表達的重組蛋白進行 INT 自斷裂反應,得到重組蛋白EGFP-TEV-CecXJ 後,再使用 TEV 蛋白酶進行酶切反應以獲得 EGFP 和 CecXJ。透過 Ni-NTA column 的 6xHis tag 純化獲得 CecXJ,並研究了 CecXJ 對革蘭氏陽性菌和革蘭氏陰性菌的抗菌活性。
實驗結果證明,利用細胞表面表達系統融合 INT 與 TEV 蛋白,可以同時生產 EGFP 與 CecXJ,並且 CecXJ 對革蘭氏陰性菌株與革蘭氏陽性菌株同時具有抑菌效果。
Due to the overproduction and misuse of antibiotics, many bacteria have begun to show resistance to these antibiotics, thus producing superbugs. Antimicrobial peptides are considered to be a potential alternative drug, and are the earliest evolutionary innate immune system in the biological world. In addition, the bactericidal mechanism of antimicrobial peptides has many advantages, such as a broad bactericidal spectrum, harmless to humans and animals, and no drug resistance. And there are also medical precedents of antibacterial peptides successfully inhibiting superbugs.
The Cecropin XJ to be produced in this study belongs to the Cecropin-B family and is a cationic antibacterial peptide derived from silkworm larvae. It has the ability to kill cancer cells, has no hemolytic activity on human red blood cells, and is not toxic to some normal cells.
Using the surface expression system (Ice nucleation protein, INP) plus protein intein (Intein, INT) to express the recombinant protein, the recombinant protein is composed of enhanced green fluorescent protein (Enhanced green fluorescent protein, EGFP), TEV protease and antibacterial win Peptide Cecropin XJ (CecXJ) composition. The pET28a-inp-int-his-egfp-tev-cecXJ was constructed and transformed into different Escherichia coli hosts, the most suitable host for expressing the recombinant protein was screened, and the optimal culture conditions were studied.
The surface-expressed recombinant protein was subjected to INT self-cleavage reaction to obtain the recombinant protein EGFP-TEV-CecXJ, and then the enzyme cleavage reaction was performed with TEV protease to obtain EGFP and CecXJ. CecXJ was obtained through 6xHis tag purification of Ni-NTA column, and the antibacterial activity of CecXJ against Gram-positive and Gram-negative bacteria was studied.
The experimental results proved that EGFP and CecXJ can be produced simultaneously by fusing INT and TEV proteins using the cell surface expression system, and CecXJ has antibacterial effects on both Gram-negative and Gram-positive strains.
摘要 i
Abstract ii
目錄 iii
圖目錄 vi
表目錄 viii
一、緒論 1
二、文獻回顧 2
2.1 抗生素與抗菌胜肽 2
2.1.1 抗生素之由來及問題 2
2.1.2 抗菌胜肽 (Antimicrobial peptides, AMPs) 2
2.1.3 天蠶素XJ (Cecropin XJ, CecXJ) 3
2.2 增強型綠螢光蛋白 (Enhanced green fluorescent protein, EGFP) 3
2.3 菸草蝕刻病毒蛋白酶 (Tobacco etch virus protease, TEV protease) 4
2.4 表現系統 5
2.4.1 表現系統之宿主 5
2.4.2 基因重組 6
2.4.3 pET 載體系統 6
2.4.4 蛋白質表達 6
2.4.5 冰核蛋白 (Ice nucleation protein, INP) 7
2.4.6 蛋白質內含肽 (Intein, INT) 9
2.5 重組蛋白純化技術 (Ni-NTA) 10
三、實驗材料與方法 11
3.1 實驗儀器與設備 11
3.2 實驗材料與藥品 13
3.2.1 實驗所用之配方 15
3.3 實驗菌株與質體來源 18
3.4 重組基因之構築 20
3.4.1 大腸桿菌質體 DNA 抽取 21
3.4.2 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 22
3.4.3 DNA 洋菜膠電泳分析 (Agarose gel electorphorsis) 22
3.4.4 DNA 純化回收 23
3.4.5 勝任細胞 (Competent cell) 之製備 24
3.4.6 轉形作用 (Transformation by heat shock) 25
3.4.7 基因酵素剪切 (Digestion) 25
3.5 重組蛋白質之最適表達與純化 26
3.5.1 大腸桿菌之醱酵培養 26
3.5.2 INT 斷裂反應 26
3.5.3 TEV 酶切反應 26
3.5.4 重組蛋白之純化 27
3.6 目標蛋白分析方法 28
3.6.1 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 分析 28
3.7 抑菌活性測試 29
3.7.1 底菌之製備 29
3.7.2 Soft agar製作 29
四、結果與討論 30
4.1 pET28a-inp-int-his-egfp-tev-cecXJ 之基因構築 30
4.1.1 PCR 擴增 Vector 30
4.1.2 PCR 擴增 Insert 31
4.1.3 In-fusion cloning 之確認與定序 32
4.2 在大腸桿菌宿主中之穩定性 34
4.2.1 轉形至不同大腸桿菌宿主中的生長狀況 34
4.2.2 以 PCR 擴增方式確認轉形之結果 42
4.2.3 以限制酶酵切方式確認轉形之結果 47
4.3 重組蛋白表達 48
4.3.1 在不同大腸桿菌宿主中的重組蛋白表達之比較 48
4.3.2 不同細胞 OD 下添加誘導劑對重組蛋白表達之影響 53
4.3.3 誘導時間對重組蛋白表達之影響 55
4.3.4 誘導溫度對重組蛋白表達之影響 57
4.3.5 誘導劑濃度對重組蛋白表達之影響 59
4.3.6 重組蛋白表達之最適化條件結果 61
4.4 重組蛋白之 INT 斷裂反應 61
4.4.1 反應細胞 OD 對 INT 斷裂之影響 62
4.4.2 反應時間對 INT 斷裂之影響 63
4.4.3 反應溫度對 INT 斷裂之影響 64
4.4.4 INT 斷裂之最適化反應條件結果 65
4.5 TEV 酶切反應 65
4.6 抑菌活性測試 66
4.6.1 對 E.coli pUC19 之抑菌活性 67
4.6.2 對 Staphylococcus aureus 之抑菌活性 68
4.6.3 高濃度 Imidazole 抑菌活性測試 69
4.6.4 抑菌測試總結 70
五、結論與未來展望 71
5.1 結論 71
5.2 未來展望 72
參考文獻 73
附錄一、定序報告結果 78
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