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研究生:周妍
研究生(外文):Yen Chou
論文名稱:選殖表現重組人類第一型間質金屬蛋白酶於大腸桿菌表現系統
論文名稱(外文):Cloning and Expression of Recombinant Human Matrix Metalloproteinase-1 from Escherichia coli 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:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:120
中文關鍵詞:選殖大腸桿菌表現第一型人類間質金屬蛋白酶組合式聚合酶連鎖反應
外文關鍵詞:ExpressionCloningEscherichia coliHuman skin fibroblast collagenaseAssembly PCR
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第一型間質金屬蛋白酶 (Matrix metalloproteinase-1, MMP-1)可分解細胞外間質而引起皮膚皺縮老化及癌症之轉移。本研究以Escherichia coli表現系統來進行人類MMP-1之選殖與表現。根據人類MMP-1之胺基酸序列,以Assembly PCR原理設計出48條引子,以引子為模板之方式重新合成出重組基因,再將其構築於pET-23a(+)表現載體中,以E. coli BL21(DE3) pLysS作為表現系統。在Lac promoter調控下,經由IPTG誘導後可表現出高量重組人類第一型間質金屬蛋白酶(recombinant human MMP-1, rhMMP-1, 386 amino acids)之不溶體。經蛋白質復性及純化後,根據SDS–PAGE、活性染色(gelatin zymogram gels)及N端胺基酸定序後,確定可獲得高量具活性之rhMMP-1蛋白 (44-kDa)。上述結果顯示rhMMP1-pET 23a(+) -Escherichia coli BL21(DE3)pLysS表現系統可提供一個方便及有效之生產重組人類第一型間質金屬蛋白酶平台。未來將大量表現具有活性之重組MMP-1蛋白作為探針,以搜尋可抑制rhMMP-1活性之天然抑制劑,應用於抗皺產品或抑制腫瘤細胞轉移之研究。
Human skin fibroblast collagenase belongs to the family 1 of metalloproteinases which is considered to participate in the degradation of extracellular matrix components and is one of the critical enzymes causing skin wrinkle and malignant tumor metastasis. In this study, the recombinant DNA encoding mature human MMP-1 (1158 bp) was synthesized by assembly PCR, integrated into pET 23a(+) expression vector and transformed into Escherichia coli BL21(DE3)pLysS expression host. Under control of Lac promoter, high level of insoluble form of recombinant human MMP-1 (rhMMP1, 386 amino acids) was expressed through IPTG induction. After renaturation and purification processes, high level of active form of rhMMP1 (44-kDa) was obtained according to the SDS–PAGE, activity staining gelatin zymogram gels, and N-terminal amino acid sequence. These data suggest that rhMMP1- pET 23a(+)- Escherichia coli BL21 (DE3) pLysS expression system offers a convenient and efficient mean to produce recombinant human MMP-1. The recombinant rhMMP-1 will be used as probe to screening the natural MMP-1 inhibitors as anti-wrinkle ingredients using in cosmetic industries and anti-cancer research.
中文摘要 I
英文摘要 II
誌謝 III
名詞縮寫 IX
第一章、前言 1
第二章、文獻回顧 3
第一節 皮膚的構造與老化的相關機制 3
第二節 膠原蛋白 9
第三節 間質金屬蛋白酶 (Matrix metalloproteinases, MMPs) 15
第四節 第一型間質金屬蛋白酶 (Matrixmetalloproteinases-1, MMP-1 EC 3.4.24.7) 25
第五節 Escherichia coli表現系統 36
第六節 人工合成基因 40
第三章、 材料與方法 43
第一節 實驗設計 43
第二節 材料與方法 44
第一部分 實驗材料: 44
第二部分 人類間質金屬蛋白酶第一型(MMP-1)組合式PCR 48
引子之設計 48
第三部分 Escherichia coli表現系統 58
第四部分 重組人類第一型間質金屬蛋白酶之純化與活性分析 64
第四章、結果與討論 70
A. 重組人類第一型間質金屬蛋白酶基因之建立 70
B. 重組人類第一型間質蛋白酶選殖構築於大腸桿菌表現系統 72
C. 重組人類第一型間質金屬蛋白酶基因之表現 72
D. SDS-PAGE與活性染色之電泳圖分析 73
E. N端定序 74
F. rhMMP-1 之inclusion body 復性 (renature) 75
G. rhMMP-1 蛋白之純化 77
第五章、結論 79
第六章、圖表 80
第七章、參考文獻 104
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