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系統識別號 U0026-0302201517164400
論文名稱(中文) 重組人類表皮調節素的純化與分析
論文名稱(英文) Purification and characterization of recombinant human epiregulin
校院名稱 成功大學
系所名稱(中) 生物科技研究所
系所名稱(英) Institute of Biotechnology
學年度 103
學期 1
出版年 104
研究生(中文) 楊偉宏
研究生(英文) Wei-Hung Yang
電子信箱 jackplm1234@gmail.com
學號 L66011085
學位類別 碩士
語文別 中文
論文頁數 94頁
口試委員 指導教授-蕭世裕
口試委員-陳宗嶽
口試委員-陳俊榮
中文關鍵字 大腸癌  表皮調節素  雙硫鍵分析  重組蛋白 
英文關鍵字 colon cancer  epiregulin  disulfide bond identification  recombinant protien 
學科別分類
中文摘要 人類表皮調節素(epiregulin)為表皮生長因子家族成員之一,過去其他
的研究者發現其具有抑制A431 等癌症細胞株生長,以及促進人類表皮角
質細胞等正常細胞生長的生物活性,而實驗室先前的研究中發現,大腸
癌患者中有較高表現量的人類表皮調節素,則其存活率較高,為探討人
類表皮調節素是否具有抑制或促進大腸癌細胞之生長,本研究以基因工
程之方式將人類表皮調節素以大腸桿菌進行生產。表現之重組蛋白主要
具有組胺酸標籤、凝血酶切割位、人類表皮調節素編碼區等三個部分,
並以鎳親和力管柱與逆向層析管柱進行純化或分析,所純化分離之六產
物分子量均與預期之重組人類表皮調節素相同,且均形成三對雙硫鍵,
且六產物的α 螺旋與β 摺版之比例均與原生人類表皮調節素相似。在經過氧化再摺疊的過程後,對其主要產物進行雙硫鍵分析,發現其三對雙硫鍵結合之情形與原生人類表皮調節素之雙硫鍵形成均相同,最後以受體結合測試證明該產物具有與其受體EGFR 結合之能力,且其結合能力
在移除其組胺酸標籤後有增強的現象。本研究成功以大腸桿菌生產出重
組人類表皮調節素,其二級結構之比例與原生人類表皮調節素相似,而
雙硫鍵形成位置則與之完全相同,並具有與其受體EGFR 結合之活性。
英文摘要 In previous study, we know that patients suffer from colon cancer have higher survival rate when they have higher epiregulin mRNA expression level. To study the potential of epiregulin to be a novel protein drug, recombinant human epireguiln is produced by E.coli expression system. There are at least six conformational isomer found in HPLC analysis. Oxidative refolding method was introduced to get more stable conformational isomer. According the result of LC-MS-MS, the disulfide bond linkages of the stable conformational isomer are the same as native epiregulin. Finally, receptor binding assay
was also used to judge the binding ability of recombinant human epireguiln. In conclusion, purified recombinant human epireguiln has correct disulfide bond linkages and the ability to bind its receptor.
論文目次 目錄
中文摘要.................................................... I
英文摘要................................................... II
誌謝....................................................... VI
目錄...................................................... VII
圖目錄...................................................... X
縮寫表.................................................... XII
一、前言.................................................... 1
1-1 癌症與EGFR (Epidermal growth factor receptor)........... 1
1-2 EGFR 在癌細胞中的角色................................... 2
1-3 表皮調節素(Epiregulin).................................. 4
1-4 蛋白質藥物.............................................. 5
1-5 蛋白質的表現............................................ 6
二、研究目的................................................ 8
三、材料與方法.............................................. 9
3-1 實驗材料................................................ 9
3-2 實驗方法............................................... 12
3-2-1 重組人類epiregulin (rhEREG) 製備..................... 12
3-2-2 以高效液相層析儀(HPLC)分析重組人類Epiregulin......... 15
3-2-3 重組人類EREG 的再折疊(refolding) .................... 15
3-2-4 重組人類EREG 的雙硫鍵分析............................ 15
3-2-5 受體結合試驗(receptor binding assay) ................ 16
3-2-6 以圓二色光譜分析rhEREG 之二級結構.................... 18
四、結果................................................... 19
4-1 建構pET28a-EREG 質體................................... 19
4-2 以親和性色層分析法純化rhEREG........................... 19
4-3 以高效液相層析儀(HPLC)分析rhEREG....................... 20
4-4 以液相層析質譜儀(LC-MS)分析rhEREG...................... 20
4-5 以圓二色光譜分析rhEREG 之二級結構...................... 21
4-6 rhEREG 的再折疊(refolding) ............................ 23
4-7 以液相層析串聯式質譜儀分析rhEREG 雙硫鍵................ 23
4-8 以受體結合試驗(receptor binding assay)測試活性......... 25
五、討論................................................... 28
5-1 rhEREG 之表現.......................................... 28
5-2 rhEREG 之純化.......................................... 29
5-3 以液相層析質譜儀(LC-MS)分析rhEREG...................... 31
5-4 以圓二色光譜分析rhEREG 之二級結構...................... 32
5-5 rhEREG 的再折疊........................................ 34
5-6 以液相層析串聯式質譜儀分析rhEREG 雙硫鍵................ 36
5-7 以受體結合試驗測試rhEREG 活性.......................... 38
六、參考文獻............................................... 42
七、附錄................................................... 91
參考文獻 施慶彬,基因重組骨粘連蛋白對臍帶間質幹細胞、牙隨幹細胞與脂肪幹
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