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研究生:黃鉉文
研究生(外文):Shiuan-Wen Huang
論文名稱:鮑氏不動桿菌噬菌體尾纖維基因的特性分析
論文名稱(外文):Characterization of the tail fiber gene of Acinetobacter baumannii phages
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:微生物學免疫學暨生物化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:44
中文關鍵詞:鮑氏不動桿菌噬菌體尾纖維蛋白
外文關鍵詞:Acinetobacter baumanniiphagetail fiber
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鮑氏不動桿菌會造成嚴重的院內感染,且世界上有越來越多的多重抗藥性菌株被報導,已造成治療上的嚴重問題。噬菌體療法是治療多重抗藥性細菌感染有潛力的替代療法。此療法的優點在於噬菌體對宿主的專一性很高,即使是同一種細菌的不同分離株也可能不會被同一種噬菌體感染。目前本實驗室由環境中分離到七株僅能感染鮑氏不動桿菌之噬菌體,以電子顯微鏡觀察此些噬菌體之型態係屬於Podoviridae科,但感染宿主範圍卻不相同。目前phiAB1、phiAB2和phiAB6的基因體已定序完成,此三者序列相似度達90.8%,唯獨phiAB1 gp41,phiAB2 gp41及phiAB6 gp40皆被註解尾纖維基因,但胺基酸序列之相似度卻僅有34%,顯示此基因可能扮演辨識宿主的角色。我們利用免疫膠體金標記phiAB1 Gp41在噬菌體上的位置,結果顯示此蛋白位在噬菌體的尾部。接著為了證實gp41是辨識宿主功能的基因,將phiAB1 gp41置換為phiAB6 gp40 ,並將此重組噬菌體命名為phiAB1tf6。經由酵素切割、PCR、結構和蛋白分析確認phiAB1tf6確實係帶有phiAB6 gp40之phiAB1重組噬菌體。而且phiAB1tf6的宿主範圍轉變為和phiAB6一樣,無法感染?淲B1的宿主。此外也發現phiAB6 Gp40具有exo-beta-1,3-glucanase,這表示phiAB6 Gp40可能具有分解beta-1,3-glucan的能力。
Acinetobacter baumannii is one of the bacteria causing serious nosocomial infections and multidrug-resistant strains of A. baumannii (MDRAB) are increasingly being reported worldwide. Bacteriophage therapy is potentially an alternative treatment of MDR bacterial infections. The advantages of phage therapy include the specificity of phage to host, rendering the infection restricted to specific host certain strains or even to specific strain of the same bacterium. Our laboratory has previously isolated seven A. baumannii-specific lytic phages from the environments. Electron microscopy revealed that they belong to Podoviridae family but their host range was difference. Three of them, phiAB1, phiAB2 and phiAB6, were completely genome sequenced. The phiAB1 gp41, phiAB2 gp41 and phiAB6 gp40 were annotated the tail fiber gene of the phages respectively. The DNA sequence of phiAB1 and phiAB6 genome were 90.8% identical but the amino acid sequences of phiAB1 Gp41 and phiAB6 Gp40 share only 34% identity, suggesting the gene may play a role for host recognition. Using immunogold labeling, phiAB1 Gp41was localized at the phage tail. To verify that phiAB1 gp41 was indeed the gene responsible for host recognition, phiAB1 gp41 gene was replaced with the phiAB6 gp40. Restriction digestion, PCR amplification, structure and protein analysis were performed to verify that the desired replacement was successful, and named phiAB1tf6. The phiAB1tf6 was found to infect the host of phiAB6 instead of the host of phiAB1, indicating that these tail fiber proteins recognized the host receptor. Additionally, the tail fiber protein of phiAB6 had exo-beta-1,3-glucanase that may allow it to degrade the beta-1,3-glucan.
目錄
致謝 I
Abstract II
摘要 III
目錄 IV
圖表目錄 VI
前言 1
一、 鮑氏不動桿菌 (Acinetobacter baumannii) 1
二、 鮑氏不動桿菌的抗藥性問題 1
三、 噬菌體 (bacteriophage;phage)的發現及命名 2
四、 噬菌體之分佈及構造 2
五、 噬菌體之分類 2
六、 噬菌體的生命週期 (life cycle) 3
七、 噬菌體的感染過程 3
八、 噬菌體療法 (bacteriophage therapy) 4
九、 噬菌體尾纖維之研究 4
十、 研究動機 5
材料與方法 6
實驗材料 6
實驗方法 6
1. 培養基之配製 6
2. 噬菌體的來源 6
3. 噬菌體的大量繁殖 (propagation) 7
4. 噬菌體濃度 (titer)之測定 7
5. 點測試法 (Spot test) 7
6. 吸附試驗 (Adsorption assay) 7
7. 電穿孔 (Electroporation) 7
8. 質體DNA之抽取 (Plasmid extraction) 8
9. 限制酶切割載體與質體 8
10. 電泳膠體回收 (Gel extraction) 8
11. 黏合作用 (ligation) 9
12. 勝任細胞 (Commpetent cell)的備製 9
13. 大腸桿菌轉型作用 (Transformation) 9
14. 質體 DNA快速篩選 (Rapid screening) 9
15. 聚合酵素連鎖反應 (Polymerase Chain Reaction,PCR) 10
16. 西方墨點法 (Western blot) 10
17. pET30a::?淲B1TF質體之構築 11
18. pET30a::?淲B6TF質體之構築 11
19. 噬菌體之純化 11
20. 以anti-Gp41?淲B1抗體妨礙 (block) ?淲B1和?淲B6感染宿主 12
21. Infected center試驗 12
22. 噬菌體之繁殖試驗 12
23. 蛋白質表達與純化 12
24. 免疫膠體金標記 (immunogold labeling) 13
25. pCH64質體之構築 13
26. ?淲B1與?淲B6的尾纖維基因置換 13
27. 尾纖維蛋白質之功能試驗 14
結果 15
1. phiAB1、phiAB2和phiAB6噬菌體尾纖維蛋白序列比對 15
2. ?淲B1尾纖維蛋白能大量表達 15
3. 以anti-Gp41抗體偵測phiAB1和phiAB6蛋白 16
4. anti-Gp41抗體能妨礙 (block) phiAB1和phiAB6感染宿主 16
5. 以免疫膠體金標記 (immunogold labeling)證實phiAB1 Gp41位在phiAB1尾部位置 16
6. phiAB1與phiAB6不能吸附於非自身宿主的A. baumannii分離株 17
7. phiAB1與phiAB6能在彼此的宿主中繁殖 17
8. phiAB1與phiAB6的尾纖維基因置換 17
9. Gp40?淲B6能分解細菌的細胞壁或胞外物質,但沒有殺菌的效果 18
討論 20
參考文獻 22
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