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研究生:紀承宇
研究生(外文):Cheng-yu Chi
論文名稱:聚乙二醇化藍藻蛋白之特性分析
論文名稱(外文):The characterization of PEGylated cyanophycin
指導教授:曾文祺曾文祺引用關係
指導教授(外文):Wen-Chi Tseng
口試委員:曾文祺
口試日期:2011-06-24
學位類別:碩士
校院名稱:國立臺灣科技大學
系所名稱:化學工程系
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:2011
畢業學年度:99
語文別:中文
論文頁數:82
中文關鍵詞:聚乙二醇化藍藻蛋白
外文關鍵詞:PEGylated cyanophycin
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藍藻蛋白(cyanophycin)為一種非核醣體合成的聚合物,結構上由aspartic acid、arginine與lysine等三種胺基酸所組成,而且目前在市面上還沒有廣泛的應用;而藍藻蛋白在其組成上包含了胺基,容易利用化學修飾的方式進行接枝反應;本研究即是對藍藻蛋白進行聚乙二醇化,期望能拓展藍藻蛋白的應用。
  因為聚乙二醇末端為羥基,不易與胺基反應,故先將聚乙二醇末端的羥基改質為醛基,使其更容易與藍藻蛋白進行接枝反應;且藉由改變藍藻蛋白與聚乙二醇的反應莫耳比,去觀察其特性的變化,且嘗試去對DNA進行包覆,觀察其有無對DNA包覆的能力。
  經過聚乙二醇化的藍藻蛋白,利用TLC、液態超導核磁共振儀與傅立葉轉換紅外線光譜儀確定其結構,並經由熱重/熱示差分析儀、熱示差掃瞄卡量計以及廣角X-ray繞射測試觀察其物性的變化。聚乙二醇對藍藻蛋白的結構影響很大,結晶度會因為聚乙二醇而變高,熱穩定性與熔點也會因為聚乙二醇而升高;至於在對DNA包覆能力測試上,雖然由DNA電泳上可以看出有影響,但無法完整包覆住DNA,所以在細胞轉染上看不出有轉染效果。
  聚乙二醇化藍藻蛋白如果再進一步進行改質,除了有機會當成一種藥物載體,也期望能當成一種新型的生醫材料去應用。
Cyanophycin is a non-ribosomal protein. The constituents consist of aspartic acid, arginine and lysine. Cyanophycin includes amino group which allows chemical modification to improves the shortcomings of cyanophycin. In this study, polyethylene glycol was used to modify cyanophycin.
 The hydroxyl functional group at the end of PEG was first modified to aldehyde group. The modified PEG then reacted with cyanophycin. Different molar ratios of cyanophycin and PEG were employed to investigate the effect of PEGylation on cyanophycin.
 The physical properties of PEGylated cyanophycin were characterized by TLC, NMR, FTIR, TGA, DSC and XRD. The results showed that the crystallinity, thermal stability and melting point increased as the degree of PEGylation increased in comparision to unmodified cyanophycin.
 The PEGylated cyanophycin could serve as a new biomaterial in the future.
目錄
中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VI
第一章 緒論 1
第二章 文獻回顧 3
2.1. 藍藻蛋白介紹 3
2.2. 藍藻蛋白的應用 7
2.3. 聚乙二醇簡介 8
2.4. 聚乙二醇化(PEGylation) 9
2.5. 聚乙二醇化對高分子的影響 10
2.6. 聚乙二醇的使用限制 13
第三章 實驗部分 15
3.1. 實驗材料 15
3.1.1. 菌株 15
3.1.2. 細胞株 15
3.1.3. 細胞培養基 15
3.1.4. 抗生素 15
3.1.5. 其他 15
3.2. 實驗藥品 16
3.3. 實驗儀器 18
3.4. 溶液配製 20
3.5. 實驗步驟 22
3.5.1. 藍藻蛋白的生產 22
3.5.1.1. 培養微生物於瓊脂板上 22
3.5.1.2. 培養微生物於2 mL LB培養基 22
3.5.1.3. 培養微生物於70 mL LB培養基 23
3.5.1.4. 培養微生物於3 L 醱酵槽 23
3.5.1.5. 自E. coli BL21(DE3) CodonPlus菌體純化出藍藻蛋白 24
3.5.2. 聚乙二醇化藍藻蛋白之製備 30
3.5.2.1. 聚乙二醇單甲基醚(mPEG)的改質(-OH →-CHO) 30
3.5.2.2. 聚乙二醇的改質(-OH →-CHO) 31
3.5.2.3. Cyanophycin-mPEG的製備 32
3.5.2.4. Cyanophycin-PEG-cyanophycin的製備 33
3.5.3. 聚乙二醇化之藍藻蛋白對DNA包覆測試 34
3.5.3.1. DNA電泳膠配製 34
3.5.3.2. DNA-Polymer製備 34
3.5.4. 細胞培養與轉染效率測定 35
3.5.4.1. 動物細胞的繼代培養(Cellular subculture) 35
3.5.4.2. 細胞轉染(Transfection) 36
3.5.5. 物性測試 36
3.5.5.1. 液態超導核磁共振儀分析 36
委託國立台灣師範大學貴重儀器中心操作分析。 36
3.5.5.2. 熱重/熱示差分析儀(TGA)測試 36
3.5.5.3. 熱示差掃瞄卡量計(DSC)測試 36
3.5.5.4. 傅立葉轉換紅外線光譜儀( FTIR )鑑定 37
3.5.5.5. 廣角X-ray繞射測試 37
第四章 結果與討論 38
4.1. 聚乙二醇改質分析 38
4.1.1. 聚乙二醇單甲基醚(mPEG)的改質結果 38
4.1.2. 聚乙二醇的改質結果 39
4.2. 聚乙二醇化藍藻蛋白(CGP-mPEG)結果分析 40
4.3. 聚乙二醇化藍藻蛋白(CGP-PEG-CGP)結果分析 41
4.4. 傅立葉轉換紅外線光譜儀( FTIR )鑑定結果 42
4.4.1. CGP-mPEG FTIR鑑定 42
4.4.2. CGP-PEG-CGP FTIR鑑定 43
4.5. XRD結果分析 43
4.6. 熱重/熱示差分析儀結果分析 44
4.6.1. CGP-mPEG TGA結果分析 44
4.6.2. CGP-PEG-CGP TGA結果分析 45
4.7. 熱示差掃瞄卡量計結果分析 46
4.7.1. CGP-mPEG DSC結果分析 46
4.7.2. CGP-PEG-CGP DSC結果分析 46
4.8. DNA電泳結果分析 47
4.9. 溶解度測試 47
第五章 結論 49
參考文獻 50


圖目錄
圖1藍藻蛋白結構示意圖 5
圖2藍藻蛋白之化學結構 6
圖3由基因重組菌株培養後之藍藻蛋白結構示意圖 6
圖4聚乙二醇之化學結構。 9
圖5聚乙二醇單甲基醚之化學結構 9
圖6 Synechocystis sp. PCC6803(cphA)E. coli BL21(DE3) CodonPlus-RIL培養流程圖 26
圖7藍藻蛋白純化流程圖A 27
圖8藍藻蛋白純化流程圖B 28
圖9藍藻蛋白純化流程圖C 29
圖10 mPEG2000改質反應式 31
圖11 PEG2000改質的反應式 32
圖12合成mPEG-CHO TLC測試(碘染) 53
圖13 mPEG2000於D2O,500MHz 1H-NMR圖譜 54
圖14 mPEG2000-CHO於D2O,500MHz 1H-NMR圖譜 55
圖15合成CHO-PEG-CHO TLC測試(碘染) 56
圖16 PEG2000於D2O,500MHz 1H-NMR圖譜 57
圖17 CHO-PEG-CHO於D2O,500MHz 1H-NMR圖譜 58
圖18純化CGP-mPEG之TLC測試(碘染) 59
圖19水溶性藍藻蛋白於D2O,500MHz 1H-NMR圖譜 60
圖20 CGP-mPEG(1:0.15)於D2O,500MHz 1H-NMR圖譜 61
圖21 CGP-mPEG(1:0.6)於D2O,500MHz 1H-NMR圖譜 62
圖22 CGP-mPEG(1:2)於D2O,500MHz 1H-NMR圖譜 63
圖23純化CGP-PEG-CGP之TLC測試(碘染) 64
圖24 CGP-PEG-CGP(1:0.5)於D2O,500MHz 1H-NMR圖譜 65
圖25 CGP-PEG-CGP(1:1)於D2O,500MHz 1H-NMR圖譜 66
圖26 CGP-PEG-CGP(1:2)於D2O,500MHz 1H-NMR圖譜 67
圖28聚乙二醇化藍藻蛋白(CGP-PEG-CGP)FTIR比較 69
圖31 TGA圖譜-不同比例之藍藻蛋白與聚乙二醇單甲基醚反應比較 71
圖32 TGA圖譜-不同比例之藍藻蛋白與聚乙二醇反應比較 71
圖33 DSC圖譜-不同比例之藍藻蛋白與聚乙二醇單甲基醚反應比較 72
圖34 DSC圖譜-不同比例之藍藻蛋白與聚乙二醇反應比較 72
圖35 CGP-mPEG(1:0.15)以不同DNA-polymer比例對DNA進行包覆 73
圖36 CGP-mPEG(1:0.6)以不同DNA-polymer比例對DNA進行包覆 73
圖37 CGP-mPEG (1:2)以不同DNA-polymer比例對DNA進行包覆 74
圖38 CGP-PEG-CGP(1:0.5)以不同DNA-polymer比例對DNA進行包覆 74
圖39 CGP-PEG-CGP (1:1)以不同DNA-polymer比例對DNA進行包覆 75
圖40 CGP-PEG-CGP(1:2)以不同DNA-polymer比例對DNA進行包覆 75
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