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研究生:林明義
研究生(外文):Ming-Yi Lin
論文名稱:靈芝免疫調節蛋白質及其衍生功能性胜肽之設計、合成並於大腸桿菌及枯草桿菌中表現純化
論文名稱(外文):Design, synthesis, expression and purification of recombinant Ganoderma lucidum immunomdulatory protein Ling ZHi-8 derivative functional peptides in Bacillus subtilis and Escherichia coli
指導教授:葉娟美
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系
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:2008
畢業學年度:96
語文別:中文
論文頁數:77
中文關鍵詞:生物活性胜肽降膽固醇胜肽融合蛋白質雙體結構高效能液相層析
外文關鍵詞:bioactive peptideshypocholesterolemic peptidefusion proteindimmer structureHPLC
相關次數:
  • 被引用被引用:1
  • 點閱點閱:255
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
近幾年來,許多具有生物活性功能的胜肽(Bioactive peptides)已陸續從各種不同的蛋白質食品中被分離出來,包括;抗氧化胜肽(antioxidant peptide) 、降膽固醇胜肽(hypocholesterolemic peptide) 等等,從牛奶中所取得之抗氧化胜肽不僅可以防止酵素性及非酵素性氧化反應、與抗氧化劑如BHA、BHT 混合後更具有協同的作用;從大豆蛋白中所取得之降膽固醇胜肽 I-I-A-E-K 更被證實具有活化cholesterol 7α-hydroxylase gene 的表現。傳統上欲獲得生物活性胜肽,是將含此胜肽之蛋白質進行酵素水解後純化所得,隨著科技發展與生物活性胜肽價值的提高,人工合成胜肽的技術也逐漸成熟。目前胜肽合成方式可分為三種:化學合成法,酵素合成法及基因重組法。本論文則嘗試利用基因重組方式來生產生物活性胜肽。
本實驗室先前成功於 GRAS (generally recognized as safe)級之菌種中表現重組靈芝免疫調節蛋白質 rLZ-8,且經體外證實具有免疫調節之活性;為了成功表現生物活性胜肽,因此本實驗利用遺傳工程技術將欲表現之三重複降膽固醇胜肽及抗氧化胜肽基因序列以OE-PCR的方式接續於SLZ-8基因之3’端,並於rLZ-8及短胜肽之間插入 trypsin 、pepsin 及chymotrypsin之切位,而後利用 B. subtilis WB800 及 E. coli BL21為表現宿主進行融合蛋白之表現生產。
實驗中成功構築出五個表現質體 pOASLZFC 、 pOASLZFCO、 pETLZFC 、 pETLZFCO 及 pETSLZFCO 以 B. subtilis WB800 為宿主可分泌融合蛋白質 rLZFC 約達8.9mg/L 之產量;所表現之融合蛋白質 rLZFCO 則可能遭受枯草桿菌蛋白酶分解,以西方墨點法配合His tag 抗體無法觀察到表現。在以 E. coli BL21(DE3)為宿主的蛋白表現中, rLZFC 與 rLZFCO 皆可利用 IPTG 進行誘導表現。融合蛋白質特性分析方面,由大腸桿菌所表現之融合蛋白質 rLZFC,經 trypsin 水解之後即可藉由交聯試劑觀察到雙體結構,且水解之產物,由高效能液相層析 (HPLC) 分析證實與化學合成法所得之標準品為同ㄧ物質。由此可知,融合蛋白質具有 rLZ-8 與生物活性胜肽兩部份,因此,本研究所設計之融合蛋白質具有開發成預防保健食品之潛力。
內容目錄

中文摘要---------------------------------------------------------------------------------------------i
英文摘要---------------------------------------------------------------------------------------------ii
壹、 前言--------------------------------------------------------------------------------------------1
一、 預防醫學的觀點-----------------------------------------------------------------------1
(一) 預防醫學的起源-----------------------------------------------------------------1
(二) 健康促進的概念-----------------------------------------------------------------2
(三) 我國健康促進政策之發展-----------------------------------------------------2
二、生技產業的發展與現況--------------------------------------------------------------2
(一)台灣生物技術產業發展歷程--------------------------------------------------2
(二)台灣地區生物技術產業發展現況---------------------------------------------3
(三)台灣生物技術產業發展優劣勢分析-----------------------------------------3
三、大腸桿菌表現系統簡介--------------------------------------------------------------4
四、枯草桿菌表現系統--------------------------------------------------------------------4
(一)枯草桿菌之特性與應用---------------------------------------------------------4
(二)枯草桿菌作為遺傳工程宿主之優缺點--------------------------------------5
(三)枯草桿菌表現相關元件--------------------------------------------------------5
五、靈芝免疫調節蛋白質簡介-----------------------------------------------------------6
(一) 靈芝免疫調節蛋白質發現與起源--------------------------------------------6
(二) 結構與功能-----------------------------------------------------------------------6
六、生物活性胜肽簡介--------------------------------------------------------------------7
(一) 生物活性胜肽來源及定義-----------------------------------------------------7
(二) 食品中生物活性胜肽種類與生理功能--------------------------------------7
(三) 生物活性胜肽製備方式--------------------------------------------------------8
七、降膽固醇胜肽簡介--------------------------------------------------------------------8
(一) 膽固醇恆定-----------------------------------------------------------------------8
(二) 高膽固醇血症--------------------------------------------------------------------9
(三) 降膽固醇胜肽作用機制--------------------------------------------------------9
八、抗氧化胜肽簡介-----------------------------------------------------------------------9
(一) 自由基與抗氧化胜肽-----------------------------------------------------------9
(二) 抗氧化胜肽結構與特性--------------------------------------------------------10
貳、實驗緣起與目的-----------------------------------------------------------------------------11
參、實驗策略--------------------------------------------------------------------------------------12
肆、材料與方法-----------------------------------------------------------------------------------13
一、菌種與質體-----------------------------------------------------------------------------13
二、實驗使用儀器、藥品與試劑--------------------------------------------------------14

三、質體 DNA之抽取及確定-----------------------------------------------------------14
(一) 大腸桿菌質體DNA之抽取---------------------------------------------------14
(二) 枯草桿菌質體DNA之抽取--------------------------------------------------15
(三) 質體確認-------------------------------------------------------------------------16
四、聚合酶鏈鎖反應(PCR)及其增幅條件--------------------------------------------16
(一) 使用引子及其序列-------------------------------------------------------------16
(二) 反應溶液之配置----------------------------------------------------------------17
(三) 重組靈芝免疫調節蛋白質 rLZ-8 基因之增幅---------------------------18
(四) 利用 over-lap PCR 增幅胜肽片段基因 FC、FO------------------------18
(五) 融合蛋白質 LZFC 基因之增幅---------------------------------------------18
(六) 聚合酶鏈鎖反應產物之回收-------------------------------------------------18
五、DNA電泳、剪切、回收、黏合---------------------------------------------------18
(一) DNA分子之電泳----------------------------------------------------------------19
(二) DNA分子之限制酶剪切-------------------------------------------------------19
(三) DNA分子之膠體回收----------------------------------------------------------19
(四) DNA分子之黏合反應----------------------------------------------------------19
六、電勝任細胞之製備及電轉形條件--------------------------------------------------20
(一)大腸桿菌-------------------------------------------------------------------------20
(二)枯草桿菌-------------------------------------------------------------------------20
七、表現質體之構築----------------------------------------------------------------------20
(一) pOASLZFC 之構築------------------------------------------------------------21
(二) pOASLZFCO之構築-----------------------------------------------------------21
(三) pETLZFC之構築---------------------------------------------------------------21
(四) pETLZFCO之構築-------------------------------------------------------------21
(五) pETSLZFCO之構築------------------------------------------------------------21
八、Colony PCR篩選轉形株-------------------------------------------------------------22
九、重組融合蛋白質之表現與偵測-----------------------------------------------------22
(一) 枯草桿菌-------------------------------------------------------------------------22
(二) 大腸桿菌-------------------------------------------------------------------------22
十、利用西方轉漬法偵測重組融合蛋白質之表現------------------------------------23
(一) 西方轉漬法----------------------------------------------------------------------23
(二) 呈色反應-------------------------------------------------------------------------23
十一、大腸桿菌內融合蛋白質之純化、濃縮與定量---------------------------------23
十二、重組融合蛋白質雙體結構之觀察------------------------------------------------24
十三、大腸桿菌融合蛋白質之 trypsin 水解作用-------------------------------------24
十四、生物活性胜肽之高效能液相層析-----------------------------------------------25
(一)HPCL 分析條件-----------------------------------------------------------------25
(二)樣品中目標物之測定-----------------------------------------------------------25
十五、DNA 與蛋白質分析軟體---------------------------------------------------------26
伍、結果與討論----------------------------------------------------------------------------------27
一、生物活性胜肽片段之選定設計及基因之增幅----------------------------------27
二、枯草桿菌分泌表現載體之構築----------------------------------------------------27
(一) pOASLZFC 之構築------------------------------------------------------------27
(二) pOASLZFCO 之構築----------------------------------------------------------28
三、大腸桿菌誘導表現載體之構築----------------------------------------------------28
四、重組融合蛋白質於枯草桿菌之表現與偵測------------------------------------28
(一) rLZFC 於Bacillus subtilis WB800 之表現情形--------------------------28
(二) rLZFCO 於Bacillus subtilis WB800 之表現情形------------------------29
五、重組融合蛋白質於大腸桿菌之誘導表現情形----------------------------------29
六、不同來源之重組融合蛋白質之純化----------------------------------------------30
七、融合蛋白質之 trypsin 水解作用-------------------------------------------------30
八、不同來源之融合蛋白質之雙體結構觀察----------------------------------------31
九、生物活性胜肽之 HPLC 分析-----------------------------------------------------32
十、生產融合蛋白質之問題與改進----------------------------------------------------32
陸、結論-------------------------------------------------------------------------------------------33
柒、參考文獻-------------------------------------------------------------------------------------68
捌、附錄------------------------------------------------------------------------------------------78
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行政院衛生署國民健康局網站 http://www.bhp.doh.gov.tw/BHPnet/Portal/Default.aspx

經濟部生物技術與醫藥工業發展推動小組全球資訊網http://www.biopharm.org.tw/

生技產業白皮書 2007經濟部生物技術與醫藥工業發展推動小組

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