利用乳酸菌表達大腸桿菌之酸性磷酸

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曾季清(Chih-Ching Tseng) 查詢紙本館藏

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生命科學系

論文名稱

利用乳酸菌表達大腸桿菌之酸性磷酸

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摘要(中)

本研究首先利用聚合?鏈鎖反應（PCR）的方式成功的篩選出大腸桿菌（Escherichia coli B strain CCRC 12567）之酸性磷酸?基因，其長度為1237 bp；接著將該基因送入大腸桿菌之蛋白質表現系統（pET system），並證明所篩選出之基因的確能轉譯成具有能夠分解抗營養因子植酸之酵素。
然後將酸性磷酸?基因與穿梭載體pGIT032重新構築後，以電穿孔（electroporation）將此重組載體送入Lactobacillus plantarum CCRC 10069、Lactobacillus casei subsp. rhamnosus GG、Lactobacillus casei Shirota等三種不同乳酸菌。結果顯示轉殖後之菌種胞內酵素活性表現以Lactobacillus casei subsp. rhamnosus GG轉殖株之植酸?表現最高，達未轉殖單一宿主菌株之62.4倍，而Lactobacillus casei Shirota之轉殖株之植酸?表現亦分別為2倍，但Lactobacillus plantarum的活性幾乎與對照菌株相同。
最後將此三種轉殖菌株進行模擬體內消化道環境酸度、膽鹽耐受性試驗。在酸度耐受性試驗方面，三種轉殖株經pH 2.0之Rogosa and Sharpe（MRS）處理、培養後均不生長；但經pH 3.0處理後，任一轉殖菌均發現當酸度處理時間愈長，其菌液於培養皿內培養後之生成菌落數愈少。此外，三種菌轉殖株均不具0.3和0.5％Oxygall膽鹽之耐受性。

摘要(英)

The acid phosphatase gene of Escherichia coli B strain was cloned using polymerase chain reaction (PCR) technique. Subsequently, the gene was transferred into pET expression system using BL21 cells as host. The recombinant Escherichia coli successfully expressed fused protein affirmatively carrying phytase activity.
The same gene was set to transform the Lactobacillus using shuttle vector pGIT032. After the recombinant vector was constructed, it was transferred to Lactobacillus plantarum, Lactobacillus casei subsp. rhamnosus GG and Lactobacillus casei Shirita using electroporation technique. The results showed the intracellular phytase activity of the transformants of Lactobacillus casei subsp. rhamnosus GG increased 62.4 times and Lactobacillus casei Shirita increased 2 times while Lactobacillus plantarum remained unchanged.
All three different transformed Lactobacillus were subjected to simulation tests for gastrointestinal tract in animals including acid tolerance and bile-salt tolerance. In pH 2.0, all three showed no tolerance. In pH 3.0, the tolerance decreased as the incubation time increased. Furthermore, no tolerance was observed in the treatments by bile salt of 0.3 and 0.5% of Oxygall.

關鍵字(中)

★ 大腸桿菌
★ 酸性磷酸
★ 電穿孔
★ 植酸酵素
★ 乳酸菌

關鍵字(英)

★ Escherichia coli
★ acid phosphatase
★ electroporation
★ phytase
★ Lactobacillus

論文目次

中文摘要…………………………………………………………...I
英文摘要…………………………………………………………...III
目錄…………………………………………………………...IV
圖目錄…………………………………………………………...VI
表目錄…………………………………………………………...VIII
第一章前言……………………………………………………...1
1.1研究緣起………………………………………………...1
1.2研究目的與內容………………………………………...3
第二章文獻回顧………………………………………………...4
2.1基因工程在乳酸菌之應用……………………………...4
2.2植酸與植酸分解?……………………………………...7
2.3大腸桿菌植酸分解?…………………………………...12
第三章實驗材料與方法………………………………………...15
3.1研究材料與儀器設備…………………………………...17
3.1.1菌種與宿主細胞………………………………………...17
3.1.2引子(primer)……………………………………………..17
3.1.3載體(vecter)……………………………………………...21
3.1.4蛋白質表現系統（expression system）………………..23
3.1.5培養基…………………………………………………...23
3.1.6化學藥品與耗材………………………………………...24
3.1.7儀器設備………………………………………………...24
3.2研究方法………………………………………………...26
3.2.1酵素基因之篩選與活性確認…………………………...26
3.2.2乳酸菌的蛋白質表現…………………………………...38
3.2.3模擬動物體內環境耐受試驗…………………………...40
第四章結果與討論……………………………………………...43
4.1植酸分解酵素酸性磷酸?基因之篩選與活性之確認...43
4.2乳酸菌的轉型作用與酵素活性表現測定……………...53
4.3模擬動物體內環境耐受試驗…………………………...60
第五章結論與建議……………………………………………...63
5.1結論……………………………………………………...63
5.2建議……………………………………………………...65
參考文獻……………………………………….…………………..66

參考文獻

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指導教授

董啟功(Chi-Gong Tong)

審核日期

2000-7-18

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