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研究生:謝銘峰
研究生(外文):Mmig-Fong Hsieh
論文名稱:以LactobacillusrhamnosusTCELL-1建構乳酸菌食品級載體
論文名稱(外文):Construct a food-grade cloning vector for Lactobacillus rhamnosus TCELL-1
指導教授:林志侯
指導教授(外文):Thy-Hou Lin
學位類別:碩士
校院名稱:國立清華大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:96
中文關鍵詞:乳酸桿菌乳酸鏈球菌素食品級載體穿梭載體
外文關鍵詞:LactobacillusnisinnisIfood-grade vectorshuttle vector
相關次數:
  • 被引用被引用:1
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本實驗室從國內健康成人腸道分離出一全新的乳酸菌種,Lactobacillus rhamnosus TCELL-1,未發現有內源性質體存在。本實驗是爲了構築一個能在此菌中表現的食品級選殖載體。
我們利用乳酸鏈球菌素免疫蛋白,nisI,作為篩選指標,並利用此菌的乳糖調控子的啟動子,pLac,作為nisI基因的啟動子,再利用乳酸菌廣域宿主複製源pAMβ1及pUC origin構築成一可在乳酸菌及大腸桿菌中複製的穿梭載體pNI。在此一穿梭載體中還接有一段nucT報導基因,作為篩選指標。
食品級選殖載體pNI經由電轉型方式送入Lb. rhamnosus TCELL-1內後,我們利用南方轉漬雜交法證實質體pNI確實存在於此菌內。再利用乳糖誘導表現ni I基因,測定此菌抵抗乳酸鏈球菌素的生長曲線,發現含有質體pNI的Lb. rhamnosus TCELL-1可以抵抗乳酸鏈球菌素達60 IU/ml,並利用plate diffusion assay測定此菌株與wild-type Lb. rhamnosus TCELL-1抗乳酸鏈球菌素之差別。
One new strain of Lactobacillus, identified as Lactobacillus rhamnosus TCELL-1, was isolated from the healthy adult rectum biopsies in our laboratory. A new food-grade shuttle cloning vector for Lb. rhamnosus TCELL-1 and E. coli was constructed using the nisin immunity gene nisI as a selection marker. The food-grade shuttle cloning vector, pNI, was constructed using the pAMβ1 replicon, the pUC origin, the nisI gene, the promoter Lac for nisI expression, and the nucT reporter gene. Electroporation into Lb. rhamnosus TCELL-1 was selected with the nucT reporter gene. Plasmid pNI was confirmed in Lb. rhamnosus TCELL-1 with southern hybridization. Lb. rhamnosus TCELL-1 carrying pNI was shown to be able to grow in medium containing a maximum of 60 IU nisin/ml. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in Lb. rhamnosus TCELL-1 in order to construct improved starter bacteria for food applications.
致謝辭
中文摘要---------------------------------------------------1
英文摘要---------------------------------------------------2
序論-------------------------------------------------------3
研究動機--------------------------------------------------13
材料與方法------------------------------------------------14
一.材料
二.培養條件
三.方法
1.質體DNA的抽取
1-1. E.coli質體DNA的抽取(微量)
1-2. E. coli質體DNA的抽取(中量)
1-3. Lactobacillus質體DNA的抽取
2.瓊脂膠電泳分析
3.DNA片段回收
4.DNA純度鑑定及定量分析
5.聚合酶連鎖反應
6.連接反應
7.質體的轉形作用
7-1.勝任細胞的製備
(1)E. coli.勝任細胞的製備
(2)Lactobacillus勝任細胞之製備
7-2.轉型作用
(1)化學方式
(2)電穿孔法
8.Lactic acid bacteria全DNA的抽取
9.南方雜配
(1)放射性標定探針的製備
(2)南方轉漬及雜配反應
10.蛋白質電泳分析
10-1.膜蛋白之收集
(1).E.coli膜蛋白之收集
(2).Lactobacillus膜蛋白之收集
10-2.SDS-PAGE
11.生長曲線測定
12.Agar diffusion assay
13.質體穩定度分析
表一:本實驗中所用到的質體及其來源
表二:本實驗中所用到的菌株及其來源
結果------------------------------------------------------33
一.抗乳酸鏈球菌素基因之選殖
二.pGN載體之構築
三.可在Lb. rhamnosus TCELL-1複製之複製源的尋找
四.乳酸菌表現載體pNuc10上NucT報導基因的表現測試
五.建構食品級載體pNI
六.測試穿梭載體pNI是否可在Lb. rhamnosus TCELL-1中複製
七.蛋白質電泳分析
(一).nisI gene 在E.coli之表現
(二).nisI gene 在Lb. rhamnosus TCELL-1之表現
八.食品及載體pNI抗乳酸鏈球菌測試
(一). Lb. rhamnosus TCELL-1抗乳酸鏈球菌的生長曲線
(二)含有pNI質體的Lb. rhamnosus TCELL-1抗乳酸鏈球菌的生長曲線
(三)Agar Diffusion Assay
九.質體pNI在Lb. rhamnosus TCELL-1中的穩定性測試
討論------------------------------------------------------44
圖一:ColE1之複製起始機制
圖二:pSC101的複製起始機制
圖三:RCR質體之複製機制
圖四:Organization of the nisin gene cluster
圖五:nisin各基因之調控
圖六:利用overlapping extension方式連接pLac及nisI之策略
圖七:overlapping extension之產物確認
圖八:plasmid pGN 之map
圖九:質體pGN之限制酵素分析
圖十:pLac-nisI之序列
圖十一:NucT reporter gene 測試
圖十二:建構食品級載體pNI之策略
圖十三:plasmid pNI之map
圖十四:以nucT reporter gene篩選TG1(pNI) colony
圖十五:質體pNI之限制酵素分析
圖十六:以nucT reporter gene篩選Lb. rhamnosus
TCELL-1(pNI) colony
圖十七:以southern hybridization證明plasmid pNI存在於Lb.
rhamnosus TCELL-1(pNI)內
圖十八:nisI基因在E. coli之表現
圖十九:Lb.rhamnosus TCELL-1抗乳酸鏈球菌素的生長情況
圖二十:含質體pNI的Lb.rhamnosus TCELL-1抗乳酸鏈球菌素的生長
情況
圖二十一:Agar diffusion assay
圖二十二:食品級載體pNI之穩定度分析
參考文獻--------------------------------------------------71
附錄------------------------------------------------------77
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