臺灣博碩士論文加值系統

本實驗室從 Lactobacillus casei ATCC 393中誘導出一株潛溶性噬菌體 ΦA3， 其已定序之部分染色體 DNA序列和NCBI database比對， 發現一個插入序列（insertion sequence ， IS）， 可能為一個新的轉位子， 將之命名為 ISLC3。
轉位子 ISLC3（NCBI accession No. AF445084）， 其全長為 1,351 bp， 兩端含 37 bp 的反向重複序列（inverted repeat，IR）， ISLC3 胺基酸編碼區（coding region）部分重疊的 orfA 及 orfB 及特定具有功能的胺基酸區域（DD35E motif）均與現公認的轉位子 IS3 family 之轉位子類似，故在分類上歸屬於 IS3 family。
以此 ISLC3 轉位子內部引子對經 PCR 反應後， 使用放射線標識 [α-P32] —dATP 標誌核酸探針以進行南方雜配實驗， 分析 ISLC3 在乳酸桿菌的分佈（distribution）情形及估算其套數， 發現在六株乳酸桿菌中具有南方雜配訊號，可作為分類學上的輔助工具。
以 ISLC3 內一對反向的引子對偵側其direct repeat， 結果並未發現direct repeat。 以同一組反向引子對以有 ISLC3 存在之六株乳酸桿菌進行PCR反應， 其中兩株得到342 bp的PCR產物， 經定序分析之後， 推測 ISLC3 會經環化作用形成 circular form， 其IRR 和 IRL銜接， IRL出現25 bp的缺失的情形。 推測此 junction circular form 形成一處新的-35區， 和-10區相距18 bp， 此組promoter有較強的驅動力。
本研究在於探討轉位子ISLC3之特性及其應用。 構築一自殺性載體（suicide vector）， 在IS 內置入一erythromycin 抗藥性基因， 將此載體轉形到 L. casei ATCC 393中，進行轉位實驗，篩選產生轉位之菌株。 若此方法可行， 可將EryR基因置換成其他的基因，如病毒的表面抗原，即可用以發展口服性的乳酸菌疫苗。

Bacteriophage ΦA3 is isolated from Lactobacillus casei ATCC 393 by our lab. After sequence its partial chromosome DNA, we found a 1,351 bp insertion sequence ISLC3 (NCBI accession No. AF445084).
ISLC3 contains 37 bp inverted repeats and two overlapping open reading frames, named orfA and orfB, with an A7G motif in the overlapping region. ISLC3 orfB also contains a DD(35)E motif of transposase, so it is a member of IS3 family.
According to Southern hybridization, ISLC3 is present 1-6 copies in the Lactobacilli genome. Southern hybridization analyses the distribution of the new insertion sequence ISLC3.
Sequence analysis of 4 independent genomic copies of inverse PCR products indicated that the direct repeats of ISLC3 is non-detected. Formation of circle junction is shown here to occur a 25 bp deletion of IRL and create a new stronger promoter.
After analyzing ISLC3 characteristics and distribution, the further research is trying to construct a suicide integration vector, which carries an erythromycin resistance gene within ISLC3. By transforming the vector into L. casei ATCC 393, the erythromycin resistance gene may be transposed into the Lactobacillus chromosome. If the experiment is successful, it is possible to choosing another gene, such as virus epitop gene, to develop Lactobacillus oral vaccine.

縮寫字對照表…………………………………………………………………………1.
Abstract…………………………………………………………………………………3
摘要………………………………………………….………………………………....4
前言…………………………………………….…………………………….………...6
材料與方法……………………………………….………………………………..…..11
一、菌株與質體……………………………….………………………………..……..11
二、藥品、酵素及放射性同位素……………………………………………………11
三、噬菌體DNA的抽取………………………………………………………….…...11
四、小量抽取Lactobacilli 染色體DNA……….……………………………..……..13
五、小量抽取E. coli質體DNA………………………………………………..………14
六、DNA片段的回收及純化………………………………………………..……….15
七、聚合酵素連鎖反應（Polymerase Chain Reaction，PCR）…………….………16
八、DNA 黏接反應……………………………………………….…………...……..17
九、勝任細胞（competent cell）之製備…………………………………...………..18
十、轉形作用（transformation）…….……………………………………………....19
1. 熱休克法（heat shock），適用於E. coli………………………………………..…19
2. 電擊法（electroporation），適用於革蘭氏陽性菌………………………….…….19
十一、內鑑識酵素圖譜分析………………………………………………..…….…..20
十二、南方雜配分析………………………………………………………….………21
1. 探針的製備………………………………………………………………..….…….21
2. 南方氏轉漬………………………………………………………………..…....…..21
3. 雜配反應………………………………………………………………..….……….23.
結果…………………………………………………………………………….……….24
一、噬菌體ΦA3來源…………………………………………………………...…….24
二、噬菌體ΦA3內鑑識圖譜…………………………………………………...…….24
三、轉位子ISLC 3序列之特性分析…………………………………………...……25
四、反向重複序列（inverted repeats）的分析………………………………...……25
五、質體p2EN的構築…………………………………………………...…………..26
六、ISLC3在乳酸桿菌的分佈（distribution）情況…………………...……………26
七、反向聚合酵素連鎖反應（Inverse PCR）………………………………...…….28
八、質體p2EN-Ery之構築與轉位（transposition）之應用………………………..29
討論………………………………………………………………………….……..…..30
參考文獻 References…...…………………………………………..……….…………35
Introduction………………………………………………………….…………………39
Material and methods. ……………………………………………….…………….…..43
1. Bacterial strains and plasmids……………………………………………….………43
2. Chemicals, enzymes, and isotope…………………………………………….……..43
3. Preparation of bacteriophage DNA…………………………………………….……43
4. Small scale preparation of Lactobacilli chromosome DNA…………………….…...45
5. Small scare preparation of E. coli plasmid DNA……………………………….……47
6. DNA fragments extraction and purification……………………………….…………48
7. Polymerase Chain Reaction (PCR) …………………………………………….……49
8. DNA ligation………………………………………………………….………….….51
9. Small scale preparation of efficient competent cells………………….……………..51
10. Transformation………………………………………………………….………….52
A. Heat shock for E. coli……………………………………………….………………52
B. Electroporation for Gram positive bacteria…..………………….………………….53.
11. Restriction map analysis……………………………………………….…………..54
12. Southern hybridization……………………………………………….…………….54
A. Preparation of probe……………………………………………….…………….….54
B. Southern blot………………………………………………………….……………..55
C. Hybridization………………………………………………………….……………56
Results………………………………………………………….……………………….58
1. Source of bacteriophage ΦA3……………………………….……………………….58
2. Restriction map of bacteriophage ΦA3 DNA……………….……………………….58
3. Characteristics of ISLC3 sequence……………………….………………………….58
4. Analysis of inverted repeats……………………….…………………………………59
5. Construction of plasmid p2EN. ……………………….…………………………….59
6. Distribution of ISLC3 in Lactobacillus spp. ……………………….………………..60
7.Inverse PCR.………………………………………………………….………………61
8. Construction of plasmid p2EN-Ery and application of transposition.……………….62
Discussion………………………………………………………….……………………64
圖一、噬菌體ΦA3電子顯微鏡下的形態…………………………………………..…..69
圖二、噬菌體ΦA3之內鑑識圖譜及轉位子 ISLC3所在之位置…………………....70
圖三、ISLC3 之核酸序列……………………………………………………….……71
圖四、ISLC3與其他IS3家族之轉位子Orf B胺基酸序列的比對………..……….72
圖五之一、ISLC3 之IRL與其他IS3家族之轉位子核酸序列的比對…………….73
圖五之二、ISLC3 之IRR與其他IS3家族之轉位子核酸序列的比對…………...… .73
圖五之三、ISLC3 之IRL、IRR與其他IS3家族之轉位子核酸序列的比對..…... 73
圖六、質體p2EN之構築及ISLC3之內鑑識圖譜……………………………..….. 74
圖七之一、以內鑑識酵素EcoRI酵解Lactobacillui各菌株之全DNA…………………………………………………………………………………… 75
圖七之二、以ISLC3 探針對Lactobacillui各菌株之全DNA經內鑑識酵素EcoRI酵解後進行南方氏雜配分析……….…………….…………………….…………….. 76
圖八之一、以內鑑識酵素EcoRV酵解Lactobacillui各菌株之全DNA..………… 77
圖八之二、以ISLC3 探針對Lactobacillui各菌株之全DNA經內鑑識酵素EcoRV酵解後進行南方氏雜配分析……….…………….…………………………..…….… 78
圖九、以引子對 149out / 999out 對質體XL1-Blue（p2EN）轉形株及L. casei ATCC393、L. sanfrancisco ATCC 27651 進行PCR反應…………...……….…………...………...…………...……….……………………… 79
圖十、環化作用產生一處新的強 promoter 區…………………………………….. 80
圖十一、質體p2EN-Ery之構築………………………………………………….…… 81
圖十二、mRNA形成之stem-loop二級結構…………………………………….…… 82
圖十三、-1 frameshift 與 OrfAB 的形成……………………………………….… 83
表一、本論文所使用之菌株…………………………………………..……………….84
表二、本論文所使用之質體…………………………………………..……………… 85
表三、本論文所使用之引子對……………………………….……………………..... 86
表四、乳酸桿菌之ISLC3套數…………………………………………………..…… 87

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