臺灣博碩士論文加值系統

本實驗是研究一個從Lactobacillus plantarum ATCC 14917分離出來的新質體pLF1，並將其全序列完成定序，經由核酸序列的分析結果可知，在質體上有16個可合成出分子量大於50個氨基酸的開放性閱讀框架，再經過與基因資料庫比對結果顯示，有3個開放性閱讀框架可被定義：orf-66是冷休克蛋白；orf-195相似於其它乳酸桿菌質體的重組酵素或插入酵素；轉位子，ISLp1，而其餘的開放性閱讀框架則無比對到相似性。 利用放射性標定的pLF1及 ISLp1當探針，經過南方雜配結果顯示，有些乳酸桿菌表現出限制切位片段長度的多樣性，且在將Lb. plantarum ATCC 14917破菌後的沈澱物與放射性標定的pLF1做雜配的實驗中，可知質體pLF1是利用θ的模式來進行複製。
將來自pE194的抗紅黴素基因與pBluescriptSK做連接反應，以建構一個複製區篩選載體，pBluSKEⅡ。此載體包含來自pBluescriptSK的多重選殖區，抗紅黴素的特徵，及完整的lacZ’基因，可直接在大腸桿菌中篩選嵌入核酸的重組載體。 一旦此重組載體包含了來自乳酸菌質體的複製區，則送入乳酸菌中後可利用其抗紅黴素的特徵來做篩選。 本實驗中，質體pLF1的複製區尚未被找到。

The complete nucleotide sequence of cryptic plasmid pLF1 isolated from Lactobacillus plantarum ATCC 14917 has been determined. Sequence analysis revealed 16 ORFs encoding polypeptides larger than 50 amino acids. Based on sequence similarity, three predicated products have been identified in pLF1: orf-66 codes for a cold-shock protein; orf-195 corresponds to a putative integrase/recombinase that shows similarities to integrase/recombinase from plasmids of other Lactobacillus; IS element, ISLp1. No function could be assigned to the other putative ORFs found. As determined by Southern hybridization with pLF1 and ISLp1-radioactively labeled probes, some Lactobacillus species show restriction fragment length polymorphism(RFLP). According to the result of hybridization of lysate of Lb. plantarum ATCC 14917 with pLF1-radioactively labeled probe, plasmid pLF1 could use a θ-mode of replication.
The Emr gene from pE194 was ligated into pBluescriptSK to construct replication origin(RO)-screening vector, pBluSKEⅡ. This plasmid contains pBluescriptSK-derived multiple cloning site, Em-resistance trait, and the lacZ’ gene, which enable direct screening for recombinants in E. coli. Once the recombinant contains a RO from plasmids of Lactobacillus, Emr trait is used as a selection marker as it is transformed into Lactobacillus. In this work, the RO of plasmid pLF1 has not been characterized.

英文摘要-------------------------------------------------------1
中文摘要-------------------------------------------------------2
前言-----------------------------------------------------------3
材料與方法-----------------------------------------------------9
結果----------------------------------------------------------25
一.尋找乳酸菌中的質體-----------------------------------------25
二.Plasmid pLF1的限制切位分析---------------------------------26
三.pLF1定序後的序列比較---------------------------------------26
四.確認pLF1的複製方式-----------------------------------------28
五.建構含抗藥基因Erythromycin的載體---------------------------29
六.Lactobacillus rhamnosus TCELL-1的生長情況------------------31
七.尋找pLF1中複製源之策略-------------------------------------31
表三：過去研究顯示在Lb. plantarum曾發現的質體-----------------33
圖一：pLF1經過各種限制酵素作用後經瓊脂凝膠電泳分析的結果------34
圖二：質體pLF1中限制酵素切位及三個主要ORF的分佈位置-----------35
圖三：Orf-66與其他乳酸菌cold-shock protein氨基酸序列的相似度比較------------------------------------------------------------36
圖四：Orf-195(pLF1)，pRC18，pLH1中integrase/recombinase氨基酸序列的比較------------------------------------------------------37
圖五：ISLp1與Pediococcus pentosaceus中ISPp1的氨基酸序列比較氨基酸序列的比較--------------------------------------------------38
圖六： ISLp1的DNA序列及轉位的氨基酸轉譯序列-----------------39
圖七(a)：乳酸菌染色體DNA經HindⅢ作用的電泳結果----------------41
圖七(b)：以南方雜配的方法分析ISLp1在乳酸菌的分佈情況----------42
圖八(a)：乳酸菌染色體DNA經EcoRⅠ作用的電泳結果----------------43
圖八(b)：以南方雜配的方法分析ISLp1在乳酸菌的分佈情況----------44
圖九(a)：乳酸菌染色體DNA經EcoRⅠ作用的電泳結果----------------45
圖九(b)：以南方雜配的方法分析質體pLF1在乳酸菌的分佈情況-------46
圖十 ：以膠體電泳與南方雜配的方法分析pLF1的複製方式----------47
圖十一：建構pBluSKEⅡ的流程-----------------------------------48
圖十二：pBluSKEⅡ經過限制酵素作用及PCR確認，經膠體電泳分析的結果------------------------------------------------------------49
圖十三：以PCR確認pBluSKEⅡ上插入的Emr基因方向性---------------50
表四 ：Lb. rhamnosus TCELL-1之培養時間與OD值及菌數之間的整理表51
圖十四：Lb. rhamnosus TCELL-1初期log phase的生長曲線圖--------51
圖十五：尋找pLF1中replication origin之策略--------------------52
圖十六：利用限制酵素切位確認利用pBluSKEⅡ接上H1與H2-----------53
討論----------------------------------------------------------54
參考文獻------------------------------------------------------60
附錄----------------------------------------------------------64

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