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研究生:黃文杰
研究生(外文):Wen-chie Huang
論文名稱:幽門桿菌ftsX基因突變株之特性分析
論文名稱(外文):Characterization of ftsX gene of Helicobacter pylori
指導教授:林念璁林念璁引用關係
指導教授(外文):Nien-tsung Lin
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微免暨分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
畢業學年度:95
語文別:中文
論文頁數:69
中文關鍵詞:離子運輸細胞分裂幽門桿菌型態
外文關鍵詞:transporterftsXHelicobacter pyloricell divisionftsE
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FtsE和FtsX是屬於ABC(ATP-binding cassette)運輸系統的同源家族蛋白,並很廣泛地保留在各菌種中。ftsE和ftsX基因所扮演的角色目前仍是有爭議的,它們最初被報導參與了大腸桿菌(Escherichia coli)的細胞分裂,隨後也被認為和細胞隔膜的形成、細胞分裂構成要素的傳遞皆有相關。在此研究中,我們希望能對幽門桿菌(Helicobacter pylori)的ftsE和ftsX基因突變株進行特性分析。我們藉由一段不完整的同源基因,並以chloramphenicol acetyl transferase(cat)基因當作選擇標記,經同源基因重組,將野生株NCTC 11637的染色體ftsE和ftsX破壞而得到突變株。結果ftsE和ftsX突變株的生長曲線和野生株11637有明顯的不同,且破壞ftsE和ftsX基因後的確使細胞分裂受到影響而出現絲狀化菌體;此外,ftsX的突變株也產生染色體分離不正常,藉由螢光染色觀察到部分沒有DNA的菌體。另一方面ftsX的突變株呈現較野生株佳的高鹽耐受程度。最後我們建構了ftsX的互補株,希望藉由flaA promoter重新表達ftsX,但菌體型態並沒恢復為短桿狀。
FtsE and FtsX belong to the ABC(ATP-binding cassette) transporter superfamily of and appear to be widely conserved among bacteria. So far, the role of the ftsE and ftsX genes are controversial. They were initially reported to be involved in cell division in Escherichia coli, and were subsequently proposed to be components of the septalsome, translocating periplasmic components of the cell division apparatus. In this study, we would like to characterize the ftsE and ftsX function in Helicobacter pylori. We constructed ftsE and ftsX mutant of H. pylori NCTC 11637 by homologous recombination with chloramphenicol-resistant marker. The growth curve of ftsE and ftsX mutant showed the distinct from wild type, and disrupted ftsE and ftsX gene would affect the cell division then resulted in the filamentation phenotype. Also, the chromosome distribution exhibited unseparated DNA of ftsX mutant, caused part of bacterium absence of DNA. On the other hand, the tolerance of high salt medium in ftsX mutant was superior to wild type. In addition, we constructed ftsX complementary strain using flaA promoter to express ftsX, but didn’t restore the function.
致 謝 1
英文摘要 2
中文摘要 3
前言 7
一、幽門桿菌的發現 7
二、幽門桿菌特性 7
三、幽門桿菌流行病學 8
四、幽門桿菌致病機轉 9
五、fts與細菌細胞分裂 11
六、FtsE和FtsX所扮演的運輸角色 12
七、研究目的 12
材料與方法 14
一、材料 14
1. 引子及菌種、質體 14
2. 試藥 14
二、實驗方法 14
1. 製備細菌培養基 14
2. 聚合酶鏈鎖反應 (Polymerase Chain Reaction,PCR) 15
3. DNA補齊(Fill-in) 15
4. 限制酶切割載體(Vector cut with Restriction enzymes) 15
5. 電泳凝膠中的DNA回收(Gel extraction) 16
6. 黏接作用(Ligation) 16
7. 製備勝任細胞 (Competent cell) 16
8. 大腸桿菌轉型作用(Transformation) 17
9. 質體DNA快速篩選(Rapid screening) 17
10. 質體DNA萃取(Plasmid extraction) 17
11. 自然轉型作用(Natural transformation) 18
12. 細菌染色體DNA萃取 18
13. 南方墨漬雜交法(Southern blotting hybridization) 18
14. 大腸桿菌表現蛋白的誘導 20
17. 西方墨點法(Western blotting) 21
18. 電泳 21
19. 以顯微鏡觀察螢光染色 22
結果 24
一、ftsX基因突變株的成功構築 24
二、完成ftsX基因上下游之選殖與定序 24
三、ftsE基因突變株的成功構築 26
四、野生株與突變株的型態觀察與生長曲線(Growth curve)之差異 27
五、幽門桿菌之ftsX基因與細胞分裂及染色體分離有關 28
六、幽門桿菌ftsX基因突變株對於高鹽的耐受性優於野生株 29
七、ftsX基因互補株的成功構築並未恢復桿狀 29
八、表達大量FtsX和FtsE蛋白質 31
討論 32
參考文獻 35


圖表目錄
表一、引子 41
表二、菌種 42
表三、質體 43
圖一、幽門桿菌ftsX突變株的構築和確認。 45
圖二、經由自我黏合獲得NCTC 11637 ftsX基因序列。 46
圖三、由11637-△0747染色體獲得完整ftsX基因。 47
圖四、完成包含ftsX在內上下游九個基因的選殖及定序。 48
圖五、FtsX氨基酸比對。 50
圖六、FtsE氨基酸比對。 52
圖七、幽門桿菌ftsE突變株的構築和確認。 54
圖八、突變株與野生株生長曲線。 55
圖九、以革蘭氏染色法觀察NCTC 11637、11637-△ftsX及11637-△ftsE三菌株在各時間點的外觀型態 57
圖十、NCTC 11637各時間點在SYTO-9和PI螢光染劑下之死活情形。 58
圖十一、11637-△ftsX各時間點在SYTO-9和PI螢光染劑下之死活情形。 59
圖十二、NCTC 11637在FM 4-64和DAPI螢光染劑下細胞膜(紅)與染色體(藍)之分佈情形。 60
圖十三、11637-△ftsX在FM 4-64和DAPI螢光染劑下細胞膜與染色體之分佈情形。 61
圖十四、以革蘭氏染色法觀察NCTC 11637、11637-△ftsX及11637-△ftsE三菌株在高鹽濃度下生長各時間點的外觀型態 63
圖十五、NCTC 11637和11637-△ftsX對於高鹽培養液的耐受性。 64
圖十六、培養在高鹽培養液中的野生株和ftsX突變株,經過二十四小時後分別再重新接種到一般培養液中,重新生長二十四小時後的型態觀察。 65
圖十七、構築基因互補株所需的質體。 66
圖十八、利用南方墨漬雜交法確定互補株之正確性。 67
圖十九、將FtsX 共268個完整氨基酸序列以網路軟體進行預測。 68
圖二十、用SDS-PAGE及西方氏點墨法證明大量表達的蛋白。 69
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