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研究生:王介民
研究生(外文):Chieh-Min Wang
論文名稱:LactoferricinII立體結構之研究
論文名稱(外文):Studies on the solution structures of lactoferricin II
指導教授:鄭梅芬
指導教授(外文):Mei-Fen Jeng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:79
中文關鍵詞:Lactoferricin II立體結構
外文關鍵詞:solution structures of lactoferricin II
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乳鐵蛋白 (lactoferrin)主要是從動物體中的乳液、唾液及淚液等分泌物中所分離純化出,在黏膜表面以及嗜中性白血球中也含有乳鐵蛋白,是一種能和鐵離子鍵結的醣蛋白,屬於運鐵蛋白 (transferrin)族群之一。在功能上它對於許多的革蘭氏陽性、革蘭氏陰性細菌以及真菌等都具有優異的抗菌活性。
將乳鐵蛋白用胃液中的pepsin水解,會得到氨基酸N端序列具抗菌活性的一段,稱之為lactoferricin (Lfcin),而lactoferricin II (Lfcin II)則是此片段中含有11個氨基酸的胜,它是乳鐵蛋白的N端區域中結構上屬於易彎折部分的一段,來自不同物種的Lfcin II在氨基酸序列上有相當高的同源性,且使用化學合成方式所合成的Lfcin II在對抗革蘭氏陽性、陰性菌時,也同樣具有優異的抗菌活性。
由於來自不同脊椎動物的乳鐵蛋白及Lfcin與牛的乳鐵蛋白和Lfcin在氨基酸序列上有很高的同源性。相對應於牛的BLFcin II (於牛的乳鐵蛋白氨基酸序列中N端39-49 ),利用化學合成法(solid-phase peptide synthesis)合成人類 (HLFcin II)、老鼠 (MLFcin II)以及豬 (PLFcin II)。在抗菌活性的研究上,這四種不同來源的Lfcin II有不同的抗菌效果,其中以BLFcin II對E. coli 及 S. aureus的抗菌效果最佳。從目前已發表的文獻得知,BLFcin II的抗菌活性和其帶有高正電性及許多疏水性氨基酸有關。
為了更進一步瞭解lactoferricin抗菌作用的機制,我們使用核磁共振NMR光譜及結構計算法來獲得各種不同動物來源的Lfcin II在與擬細胞膜作用狀態下的立體結構,並經由比較這四種Lfcin II立體結構上之差異,進一步了解結構與抗菌活性間的相關性,以及Lfcin II抗菌作用在分子層級的作用機制。
Lactoferrin is an avidly iron-binding glycoprotein of the transferring family. It was found at mucosal surfaces, within the specific granules of neutrophils, and in biological fluids, such as milk, from which it was first isolated. Lactoferrin has been shown to have antimicrobial activity against a broad range of gram-positive, gram-negative bacteria, and fungi.
Pepsin digestion of lactoferrin releases antibacterial peptides from N -terminal named lactoferricin (Lfcin). Lactoferricin II (Lfcin II) includes an 11 amino acids of amphipathic region, which is exhibited on the outer surface of the amino-terminal lobe of lactoferrin. Synthetic peptides homologous to this region exhibited potent antibacterial activity against a selected range of both gram-positive and gram-negative bacteria.
Based on the high amino acid sequence homology between bovine lactoferrin and lactoferrins isolated from other mammals, we have located the regions corresponding to residue 39-49 of bovine lactoferrin (BLFcin II) and synthesized human (HLFcin II), murine (mouse; MLFcin II) and porcine (pig; PLFcin II) lactoferricin derivatives using standard solid-phase peptide synthesis. Studies on the antibacterial activity of BLFcin II, HLFcin II, MLFcin II and PLFcin II have shown different efficiency. BLFcin II was found to be the most active agent against both E. coli and S. aureus. Previous reports have pointed out that BLFcin possess the highest net charge and the largest amount of hydrophobic residues among all the lactoferricin derivatives might be the reason for its high efficiency.
In order to better understand the mechanism of the antimicrobial action of lactoferricin, we propose to use NMR spectroscopy and structure calculation to obtain solution structures of lactoferricin II from different animals. Comparing the comformational differences of these lactoferricin IIs will help us a better understanding the structure and function relationship. Further, the knowledges put together will give a foundation to understand the antimicrobial mechanism at molecular level.
目 錄
中文摘要-------------------------------------------------------------------------------- I
英文摘要------------------------------------------------------------------------------ III
致謝------------------------------------------------------------------------------------- V
目錄------------------------------------------------------------------------------------ VI
表目錄------------------------------------------------------------------------------- VIII
圖目錄--------------------------------------------------------------------------------- IX
縮寫檢索表-------------------------------------------------------------------------- XII
第一章 序論
第一節 抗微生物胜之簡介------------------------------------------------ 2
第二節 乳鐵蛋白與Lactoferricin之簡介---------------------------------- 3
第三節 乳鐵蛋白與Lactoferricin的研究---------------------------------- 5
第二章 圓形偏極光(Circular Dichroism, CD)與核磁共振(NMR)光譜
第一節 Circular Dichroism (CD)光譜------------------------------------- 10
第二節 核磁共振(NMR)光譜---------------------------------------------- 11
第三章 使用材料與方法
第一節 樣品來源------------------------------------------------------------- 21
第二節 Circular Dichroism (CD)光譜-------------------------------------- 22
第三節 核磁共振光譜與結構計算----------------------------------------- 23
第四章 實驗結果
第一節 CD實驗結果--------------------------------------------------------- 30
第二節 NMR實驗結果------------------------------------------------------ 30
第三節 Lfcin II的立體結構------------------------------------------------- 31
第五章 討論與展望---------------------------------------------------------------- 55
參考文獻------------------------------------------------------------------------------ 61
附錄
附錄一 牛、人類、老鼠及豬的完整乳鐵蛋白氨基酸序列------------- 65
自述
表 目 錄
表1-1 抗菌性胜的分類----------------------------------------------------------- 7
表1-2 牛、人類、老鼠及豬的Lfcin II之
氨基酸序列、分子量及等電點--------------------------------------------- 8
表4-1(a) BLFcin II 2.5mM於250mM SDS / 10mM KH2PO4緩衝
溶液pH 4.8,300K下的1H化學位移表------------------------------ 43
表4-1(b) HLFcin II 2.5mM於250mM SDS / 10mM KH2PO4緩衝
溶液pH 4.8,300K下的1H化學位移表------------------------------ 43
表4-1(c) MLFcin II 2.5mM於250mM SDS / 10mM KH2PO4緩衝
溶液pH 4.8,300K下的1H化學位移表------------------------------ 44
表4-1(d) PLFcin II 2.5mM於250mM SDS / 10mM KH2PO4緩衝
溶液pH 4.8,300K下的1H化學位移表------------------------------ 44
圖 目 錄
圖1-1 LfcinB在水溶液下的立體結構圖----------------------------------------- 8
圖2-1 一般紫外光的吸收光譜與CD的吸收光譜---------------------------- 15
圖2-2 左右旋前進紫外光所形成的圓形偏極光------------------------------ 15
圖2-3 典型的蛋白質或胜二級結構CD光譜-------------------------------- 16
圖2-4 各種電磁波波長範圍------------------------------------------------------ 16
圖2-5 2D FT-NMR實驗進行的過程--------------------------------------------- 17
圖2-6 2D COSY實驗的脈衝序列------------------------------------------------ 17
圖2-7 2D TOCSY實驗的脈衝序列---------------------------------------------- 17
圖2-8 2D NOESY實驗的脈衝序列---------------------------------------------- 18
圖2-9 一般蛋白質或胜的核磁共振氫原子二維光譜分佈圖------------ 19
圖3-1 固態化學合成法(solid phase peptide synthesis)合成的流程圖----- 25
圖3-2 各種氨基酸的在核磁共振光譜中的自旋系統------------------------ 26
圖3-3 由COSY光譜判斷TOCSY光譜中各個氨基酸的
α、β、γ等氫原子的位置---------------------------------------------------- 27
圖3-4 以TOCSY及NOESY光譜做循序判定--------------------------------- 28
圖4-1(a) BLFcin II在30、40及50℃下的CD光譜---------------------------- 32
圖4-1(b) HLFcin II在30、40及50℃下的CD光譜---------------------------- 32
圖4-1(c) MLFcin II在30、40及50℃下的CD光譜---------------------------- 33
圖4-1(d) PLFcin II在30、40及50℃下的CD光譜---------------------------- 33
圖4-2 BLFcin II、HLFcin II及PLFcin II在30℃下的CD光譜比較-------- 34
圖4-3(a) BLFcin II在10mM KH2PO4緩衝溶液中的NOESY光譜--------- 35
圖4-3(b) HLFcin II在10mM KH2PO4緩衝溶液中的NOESY光譜--------- 36
圖4-3(c) MLFcin II在10mM KH2PO4緩衝溶液中的NOESY光譜-------- 37
圖4-3(d) PLFcin II在10mM KH2PO4緩衝溶液中的NOESY光譜--------- 38
圖4-4(a) BLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的TOCSY NH-side chain區域光譜--------------- 39
圖4-4(b) HLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的TOCSY NH-side chain區域光譜--------------- 40
圖4-4(c) MLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的TOCSY NH-side chain區域光譜--------------- 41
圖4-4(d) PLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的TOCSY NH-side chain區域-------------------- 42
圖4-5(a) BLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜-------------------------------------- 45
圖4-5(b) HLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜--------------------------------------- 46
圖4-5(c) MLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜--------------------------------------- 47
圖4-5(d) PLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜--------------------------------------- 48
圖4-6(a) BLFcin II 在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜循序判定圖------------------------ 49
圖4-6(b) HLFcin II 在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜循序判定--------------------------- 49
圖4-6(c) MLFcin II 在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜循序判定圖------------------------ 50
圖4-6(d) PLFcin II 在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的NOESY光譜循序判定圖------------------------ 50
圖4-7(a) BLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的15組結構重疊顯示圖---------------------------- 51
圖4-7(b) HLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的15組結構重疊顯示圖---------------------------- 52
圖4-7(c) MLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的15組結構重疊顯示圖---------------------------- 53
圖4-7(d) PLFcin II在250mM SDS / 10mM KH2PO4緩衝溶液
pH 4.8,300K下的15組結構重疊顯示圖---------------------------- 54
圖5-1(a) BLFcin II的單一分子立體結構圖------------------------------------ 58
圖5-1(b) HLFcin II的單一分子立體結構圖------------------------------------ 58
圖5-1(c) MLFcin II的單一分子立體結構圖------------------------------------ 59
圖5-1(d) PLFcin II的單一分子立體結構圖------------------------------------ 59
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