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Sequence analysis of endogenous plasmids isolated from Lactobacillus pentosus F03

By 龔琳舒 and Lin-Shu Kung

Abstract

乳酸菌 (Lactic acid bacteria) 作為益生菌 (probiotic) 使用已有長久的歷史,不但能增進食物風味、改變營養成分、提高保存時間,還能抑制特定病原菌、改善宿主腸胃道健康與提高免疫力等。 例如乳酸桿菌屬 (Lactobacillus) 就是一般認可為安全 (generally recognized as safe, GRAS) 的益生菌種。 質體 (plasmids) 是一種能自主複製的染色體外遺傳物質,廣泛存在於細菌中。有些質體具有特殊功能或表現特徵,例如能表現抵抗抗生素的蛋白質;但是這些具有抗生素抵抗性質體,可能會在宿主腸胃道中轉移到其他病原菌中,不利於宿主的健康。因此,益生菌菌種的篩選條件應包括其內源性質體的分析。另外,質體具有方便進行基因改造的特性,常用來建構蛋白質表現載體,生產具經濟效益的蛋白質。 本實驗室從豬的腸道篩選出一Lactobacillus pentosus F03 (戊糖乳酸桿菌F03) 菌株,含有5個以上的內源性質體。將其中的5個質體進行定序與分析,命名為pF03-1 (3282 bp)、pF03-2 (3293 bp)、pF03-3 (1787 bp)、pF03-4 (2138 bp)、pF03-5 (1949 bp)。經資料庫比對結果顯示,除了pF03-5屬於θ複製型質體以外,pF03-1~4皆屬於滾輪複製型 (rolling-circle replication, RCR) 質體;依複製蛋白質序列的不同,可將pF03-1歸類於pMV158 family,pF03-2 ~ pF03-4則歸類於pC194 family。生物資訊學分析結果顯示,質體pF03-1 ~ pF03-5都未比對到任何已知的抗生素抗性基因。而質體pF03-5的DNA序列中具有一段特殊複製起始區為9 bp序列直接重複四次,以及22 bp串聯重複四次半的序列,符合θ複製型質體pUCL287 family的典型特徵。此外,pF03-5與已知的抗噬菌體質體pLKS序列有高達89%的一致性,且為罕見的小於2 kb之θ複製型質體,具有進一步研究及應用的價值。It has a long history for lactic acid bacteria (LAB) being recognized as probiotic. LAB has been suggested to be able to enhance food flavor, change nutrient composition, prolong the storage time, inhibit some specific pathogen, and improve gastrointestinal health and immunity of the host. Lactobacillus, a lactic acid bacteria, is generally recognized as safe (GRAS) probiotic strain of lactic acid bacteria. Plasmids are considered as autonomously replicating extrachromosomal genetic elements, widely present in bacteria. Some plasmids have particular function or features, for example, can produce antibiotic-resistance proteins. These antibiotic-resistance plasmids may be transferred to other gastrointestinal pathogens of the host and increase health risks. Thus, it is necessary to investigate the endogenous plasmids present in the bacterial strains used for probiotics purposes. In addition, plasmids can provide cost-effective production of proteins, since they can be easily modified as expression vectors. Lactobacillus pentosus F03, a strain isolated from pig intestine in our laboratory, which contains more than five endogenous plasmids. In this study, we report the cloning and sequence analysis of the five plasmids present in this strain, named pF03-1 (3282 bp), pF03-2 (3293 bp), pF03-3 (1787 bp), pF03-4 (2138 bp) and pF03-5 (1949 bp), respectively. Results of BLAST analyses indicate that these plasmids, with the exception of pF03-5, belong to rolling-circle replication (RCR) plasmid families. Plasmid pF03-5 belongs to θ-replicating type. By comparing the sequences of their replication proteins, plasmids pF03-1 and pF03-2~4 belong to pMV158 family and pC194 family, respectively. Bioinformatic analyses revealed that plasmids pF03-1~pF03-5 do not contain any known antibiotic resistant genes. The nucleotide sequence of plasmid pF03-5 shows a special replication origin containing 9 bp direct repeats (four times) and 22 bp iterons, tandemly repeated four and one-half times. These features meet the typical configuration of the pUCL287 family of θ-replicating plasmids. In addition, plasmid pF03-5 shows 89% identity with that of pLKS, a phage resistant plasmid. Plasmid pF03-5 is a rare small plasmid (< 2 kb) of θ-replication type, and has potential for further research and application.中文摘要 …………………………………………………………………i Abstract …………………………………………………………………ii 目次………………………………………………………………………iii 圖表目次…………………………………………………………………v 縮寫字對照表……………………………………………………………vi 前言………………………………………………………………………1 一、益生菌簡介…………………………………………………………1 (一)定義 …………………………………………………………………1 (二)功效 …………………………………………………………………1 (三)菌種選擇與風險 ……………………………………………………2 二、細菌內源性質體介紹………………………………………………4 (一)內源性質體的特性與功能 …………………………………………4 (二)質體DNA複製 (plasmid DNA replication) 形式 …………………5 三、戊糖乳酸桿菌 (Lactobacillus pentosus)之簡介…………………6 (一)分類 …………………………………………………………………6 (二)內源性質體研究 ……………………………………………………6 (三)其他應用 ……………………………………………………………6 四、乳酸菌內源性質體之研究動機與應用……………………………7 材料與方法………………………………………………………………9 一、材料…………………………………………………………………9 (一)菌株與質體 …………………………………………………………9 (二)培養基與緩衝溶液 …………………………………………………9 (三)試劑藥品 ……………………………………………………………9 (四)實驗套組……………………………………………………………10 (五)酵素…………………………………………………………………10 (六)引子…………………………………………………………………10 二、實驗方法……………………………………………………………11 (一)染色體與質體DNA純化方法 ………………………………………11 1.乳酸桿菌染色體DNA萃取……………………………………………11 2.乳酸桿菌質體DNA萃取………………………………………………11 3.大腸桿菌質體DNA萃取………………………………………………12 (二)乳酸桿菌內源性質體電泳分析與切膠純化 ………………………12 1.以洋菜膠電泳分析質體DNA…………………………………………12 2.自洋菜膠純化質體DNA………………………………………………13 (三)酵素作用和產物純化 ………………………………………………13 1.限制酶 (Restriction enzyme) 作 用…………………………………13 2.去磷酸酶 (Calf intestinal alkaline phosphatase) 作用 ……………14 3.純化經酵素作用之DNA片段 …………………………………………14 4.以洋菜膠電泳分離純化經限制酶切割之DNA ………………………14 (四)DNA黏合作用 (DNA ligation)………………………………………14 (五)大腸桿菌電勝任細胞 (Electro-competent cells)製備……………15 (六)聚合酶鏈鎖反應 (polymerase chain reaction, PCR) ……………15 1.利用PCR進行DNA增幅與PCR產物之純化…………………………15 2.即時定量PCR (Real-Time PCR) ……………………………………16 3.PCR菌落篩選 (Colony PCR screening)……………………………16 (七)PCR選殖 (PCR cloning)……………………………………………17 (八)DNA轉形作用(DNA Transformation)………………………………17 1.熱休克法 (Heat-shock) ………………………………………………17 2.電穿孔法 (Electroporation)……………………………………………17 結果 ………………………………………………………………………18 一、戊糖乳酸桿菌F03質體萃取和限制酶作用圖譜……………………18 二、戊糖乳酸桿菌F03質體片段選殖與定序……………………………18 三、F03各質體序列與分析………………………………………………19 (一)圖譜建構………………………………………………………………19 (二)開放閱讀框(ORF)分析 ………………………………………………19 1.pF03-1……………………………………………………………………20 2.pF03-2……………………………………………………………………20 3.pF03-3……………………………………………………………………21 4.pF03-4……………………………………………………………………21 5.pF03-5……………………………………………………………………21 (三)複製形式分析 …………………………………………………………22 四、複製套數 (Copy number) 檢測結果…………………………………22 討論 …………………………………………………………………………23 一、乳酸桿菌質體萃取方法探討 …………………………………………23 二、乳酸桿菌內源性質體定序方式探討 …………………………………23 三、戊糖乳酸桿菌F03內源性質體比較 …………………………………24 四、建構表現載體之可行性 ………………………………………………24 五、質體pF03-5之應用價值 ………………………………………………25 參考文獻 ……………………………………………………………………27 圖表 …………………………………………………………………………35 附錄一、培養基與緩衝液 …………………………………………………50 附錄二、Lactobacillus pentosus F03各質體之序列 ……………………5

Topics: 戊糖乳酸桿菌, 益生菌, 內源性質體, Lactobacillus pentosus, probiotic, endogenous plasmid
Publisher: 生命科學院碩士在職專班
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81145
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