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系統識別號 U0026-2208201314351800
論文名稱(中文) 生物二型創傷弧菌毒力質體中為該菌在鰻魚血清中存活所需基因之探討
論文名稱(英文) Determination of genes in biotype 2 Vibrio vulnificus virulence plasmid required for bacterial survival in eel serum
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 101
學期 2
出版年 102
研究生(中文) 武冠志
研究生(英文) Kuan-Chih Wu
電子信箱 s46001024@mail.ncku.edu.tw
學號 S46001024
學位類別 碩士
語文別 中文
論文頁數 51頁
口試委員 指導教授-何漣漪
口試委員-吳俊忠
口試委員-鄧景浩
口試委員-黃一修
口試委員-許鴻猷
中文關鍵字 創傷弧菌  鰻魚 
英文關鍵字 Vibrio vulnificus  eel 
學科別分類
中文摘要 創傷弧菌為一種革蘭氏陰性的弧狀桿菌,可以造成人類及鰻魚的嚴重感染症,並導致宿主的死亡。創傷弧菌菌株中,只有一部分能夠感染鰻魚,這一群菌株被分類為生物二型。我們實驗室之前的研究顯示,生物二型菌株感染鰻魚的能力跟其內專有的毒力質體pR99有關,而進一步使用大規模剔除質體上未知區域進行的分析則指出,此毒力質體所含的基因中,能夠幫助菌株在鰻魚血清中存活的基因可能有三個:vep07、vep20及vep71。然而,當把這三個基因以兩個質體送入生物二型質體剔除株進行回補時,卻無法恢復該菌在鰻魚血清中存活的能力。本研究的主要目標為確認創傷弧菌在鰻魚血清中生存的能力是否只需要毒力質體中這三個基因就能達成。首先,我確認了這三個基因中的vep07及vep20對於生物二型菌株生存在鰻魚血清中是很重要的,而vep71對於處於stationary phase的細菌很重要,而對於處在exponential phase的細菌而言不重要。之後,我藉由插入一組毒素-抗毒素系統到pBBR1MCS4此一和我們實驗室常用的pIT009相容,但是在創傷弧菌中穩定度不高的廣宿主性的載體,以提高其穩定度後,和pIT009搭配,建立了一個雙載體系統,讓我能夠藉以把這三個基因轉殖到創傷弧菌各菌株中,以進一步確認這三個基因對於創傷弧菌在鰻魚血清中生存的重要性。實驗結果顯示,當質體剔除株得到這三個基因之後,其在鰻魚血清中生存的能力就大幅的回升了;然而,當我將這三個基因送入生物一型菌株YJ016之後發現,同時得到三個基因的轉殖株在鰻魚血清中的生存能力大約只有生物二型的1/3左右,且和只得到vep71的轉殖株的生存能力相當,以即時偵測反轉錄PCR偵測這些基因的mRNA表現量及使用西方墨點法偵測Vep07的蛋白質表現,結果顯示在YJ016轉殖株中vep07及vep20的mRNA及Vep07蛋白皆可以被正常表現。因此,在這三個基因當,vep71對於YJ016在鰻魚血清中是最重要的,然而只有vep71仍然是不夠的,故我推論除了在毒力質體上的基因之外,可能有些位在生物二型染色體上的基因,也參與在細菌對鰻魚血清的抵抗能力中,而造成生物一型及生物二型菌株在鰻魚血清中生存能力的差異。
英文摘要 Vibrio vulnificus, a Gram-negative, curved-bacillus, may cause severe infectious diseases in both the human and eels. Only a fraction of V. vulnificus can infect the eels, and they are classified as biotype (BT) 2. We have previously demonstrated that the ability of BT2 V. vulnificus strains to infect eels is associated with a BT2 specific virulence plasmid. Further, by characterization of mutants with large sectional deletion in the virulence plasmid, pR99, of BT2 strain CECT4999, it was suggested that in this plasmid only vep07, vep20 and vep71, are required for bacterial survival in eel serum. However, the ability of pR99-cured strain complemented with these three genes to survive in eel serum was not observed. The aim of this study is to determine whether vep07, vep20 and vep71 together are sufficient to convey resistance to eel serum. First, I determined the importance of vep07, vep20 and vep71 for survival in eel serum by deleting each gene individually from pKT391, and confirmed that vep07 and vep20 are essential while vep71 is important only when the bacteria were in stationary, but not exponential phase. Then, I inserted a toxin-antitoxin module into a broad-host range vector, pBBR1MCS4, which is compatible with pIT009, to increase its stability, and used the resulting plasmid in combination with pIT009 to establish a double vector system. With this system, I transferred vep07, vep20 and vep71 to V. vulnificus strains to determine the importance of these genes for bacterial survival in eel serum. The results showed that these three genes together are sufficient to restore the survival of pR99-cured BT2 strain. However, these three genes together or vep71 alone in the BT1 strain YJ016 conferred only 1/3 of the level in BT2 for survival in eel serum. By real-time reverse transcription PCR and Western blotting, it was shown that both vep07 and vep20 were transcribed and Vep07 was expressed in YJ016. These data suggest that, among these three genes, only vep71 conveyed partial survival of YJ016 in eel serum. Therefore, it is likely that some other BT2-specific chromosomal genes are also involved in resistance to eel serum, thus leading to a better survival of BT2 than BT1 strains in eel serum.
論文目次 目錄 I
表目錄 III
圖目錄 IV
中文摘要 V
Abstract VI
誌謝 VII
符號及縮寫 VIII
緒論 1
創傷弧菌( Vibrio vulnificus) 1
創傷弧菌的生物型( biotypes) 分類及其感染症 1
創傷弧菌對小鼠之致病因子與毒力因子 3
創傷弧菌對於鰻魚之致病因子 5
研究動機與目的 7
材料與方法 9
I. 實驗菌株、質體與核酸引子 9
1. 實驗菌株 9
2. 實驗菌種的培養與保存方法 9
3. 質體 9
II. 基礎核酸及分子生物學技術 9
1. 以商業化套組萃取小量質體DNA 9
2. 以商業化套組萃取大量質體DNA 10
3. 以傳統方法萃取小量質體DNA 11
4. 聚合酶連鎖反應( polymerase chain reaction, PCR) 11
5. DNA電泳分析 12
6. DNA片段之分離與回收 12
7. 限制酶切割DNA 13
8. DNA 片段之去磷酸化反應 13
9. DNA黏合作用 13
10. 熱休克勝任細胞製備 13
11. 熱休克轉型作用 (heat shock transformation) 14
12. 接合作用 14
13. 質體之穩定及相容性測試 15
14. 細菌RNA之萃取 15
15. 反轉錄作用 (reverse transcription) 16
16. 即時偵測聚合酶連鎖反應 (real-time PCR) 17
III. 創傷弧菌突變株之構築 17
IV. 創傷弧菌互補株之構築 18
V. 細菌生長曲線之測定 18
VI. 蛋白質分析方法 19
1. 誘導Vep07蛋白質表現 19
2. 以商業化套件萃取全細胞蛋白質 19
3. 以商業化套組定量蛋白質樣品 19
4. 蛋白質變性處理 20
5. 蛋白質電泳分析 20
6. 蛋白質膠體染色 (coomasie blue stain) 20
7. 西方墨點轉漬反應 (Western blotting analysis) 21
VII. 鰻魚血液抽取與血清分離 21
VIII. 創傷弧菌抵抗鰻魚血清殺菌作用能力之分析 22
IX. 統計分析方法 22
結果 23
I. 毒力質體pR99之衍生質體pKT391上所含之vep07, vep20及vep71基因為生物二型創傷弧菌生存於鰻魚血清所必需 23
II. 生物二型質體剔除株之回補系統之建立及回補株之特性分析 24
II-1回補系統之建立 24
II-2 生物二型質體剔除回補株於鰻魚血清中生存能力之測試 25
III. 生物一型vep07, vep20及vep71轉殖株之分析 25
III-1回補系統於生物一型菌株之穩定度及相容性分析 25
III-2生物一型轉殖株於鰻魚血清中之生存能力測試 26
III-3生物一型轉殖株中vep07, vep20及vep71之表現分析 26
討論 27
參考文獻 31
圖表集 35

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