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研究生:陳昱文
研究生(外文):Yu-Wen Chen
論文名稱:表現自Pichiapastoris表現系統之羊乳鐵蛋白素胜肽生化特性分析
論文名稱(外文):Characterization of Biochemical Properties of Lactoferricin-Related Peptides from Pichia pastoris Expression System
指導教授:陳俊宏陳俊宏引用關係江善宗
指導教授(外文):Gen-Hung ChenShann-Tzong Jiang
學位類別:碩士
校院名稱:靜宜大學
系所名稱:化粧品科學系碩士班
學門:民生學門
學類:美容學類
論文種類:學術論文
論文出版年:2010/07/
畢業學年度:98
語文別:中文
論文頁數:101
中文關鍵詞:抗菌乳鐵蛋白素抑菌胜肽
外文關鍵詞:antimicrobial peptidelactoferricinantimicrobial activity
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近年來由於抗生素的濫用,使微生物開始具有抗藥性,而將乳鐵蛋白用胃液中的胃蛋白酶處理,純化之後即可得到同樣具有抗菌效果的 lactoferricin,經研究證實它的抗菌效果比乳鐵蛋白好,同時它的抗菌效果不受鐵離子的影響。乳鐵蛋白素的抗菌機制與其高度正電性相關聯,帶正電的蛋白質片段與帶負電的細菌細胞壁結合,進而破壞細菌的細胞壁,因而殺死或抑制細菌生長。
本實驗於酵母菌系統表現羊乳鐵蛋白素GLFcin、GLFcin-(His)6、GLFcin II、GLFcin II-(His)6,經由甲醇誘導後,可於胞外大量表現重組的羊乳鐵蛋白素。經由兩次Sephadex G-25膠體過濾管柱層析純化後,分別進行最小抑菌濃度試驗 (Minimum inhibitory concentration,MIC)、血管收縮素轉換酶(Angiotensin converting enzyme, ACE)抑制活性測定、細胞毒性試驗 (MTT assay),結果顯示這四條重組的羊乳鐵蛋白素都能對Escherichia coli BCRC 11549、Pseudomonas aeruginosa BCRC 12450、Bacillus cereus BCRC 10603、Staphylococcus aureus BCRC 25923、Propioni bacterium acnes BCRC 10723、 Listera monocytogenes
BCRC 14845有明顯的抑菌活性,其最小抑菌濃度為4.07~16.00 mg/mL。
抑制ACE活性測定部分,GLFcin II、GLFcin II-(His)6經由chymotrypsin 和trypsin 水解後其IC50值為115.01μg/mL 、 41.07μg/mL、74.69μg/mL 和 55.23μg/mL結果顯示水解後其抑制血管收縮素轉換酶效果更佳。
細胞毒性試驗針對最小抑菌濃度去對人類纖維母細胞 (Detroit 551) 做毒性試驗,由結果顯示最小抑菌濃度對於人類纖維母細胞有明顯的毒性,因而將濃度往下降低為一半後,其無顯著的毒性。因此,往後要添加重組羊乳鐵蛋白素時要考慮到毒性的部分,可搭配其它的抗生素使用。
因此,利用基因選殖方式可大量表現重組羊乳鐵蛋白素,並有助於重組羊乳鐵蛋白素的資料建立。
Goat lactoferricin (GLfcin) is released from N-terminal of goat lactoferricin by pepsin digestion. Four recombinant goat Lactoferricin-related peptide cDNAs (GLFcin and GLFcin II) with/without (His)6-Tag were cloned into pPICZαC and transformed into Pichia pastoris SMD1168H.After methanol induction, these peptides were expressed and secreted into broth. They were purified by sephadex G-25 chromatographies. The yield of purified rGLFcin was 0.15 mg/mL of broth. In this study, the optimal culture condition for over-expression of the peptide and their biochemical properties including MIC, MTT and ACEI were determined.These results suggest that rGLFcin might be good materials for nutritional supplements, functional foods and cosmetics.
中文摘要 I
Abstract III
謝誌 IV
目錄 VI
圖表目錄 VIII
附錄目錄 IX
Abbreviation 1
第一章 前言 2
第二章 文獻回顧 4
第一節 乳鐵蛋白(Lactoferrin) 4
第二節 乳鐵蛋白素(Lactoferricin) 10
第三節 高血壓與血管收縮素轉換酶抑制劑 17
第四節 抗菌胜肽 25
第三章 材料與方法 30
第一節 實驗設計 30
第二節 材料與方法 31
第一部份 重組Goat LFcin 於P. pastoris系統之表現與純化 34
第二部份 抑菌分析 36
第三部份 ACE 抑制活性測定 40
第四部份 最適培養條件探討 41
第五部份 細胞毒性試驗 44
第四章 結果與討論 48
第一節 重組羊乳鐵蛋白素之表現與純化 48
第二節 抑菌分析 49
第三節 重組羊乳鐵蛋白素抑制ACE活性 50
第四節 最適培養成份探討 50
第五節 細胞毒性試驗 53
第五章、結論 55
第六章、參考文獻 57
第七章、附錄 89
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