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系統識別號 U0026-2308201011124500
論文名稱(中文) 創傷弧菌生物二型之毒力質體中與該菌於鰻魚血清存活相關的基因之鑑識
論文名稱(英文) Identification of genes in biotype 2 Vibrio vulnificus virulence plasmid associated with survival of this organism 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 98
學期 2
出版年 99
研究生(中文) 謝政原
研究生(英文) Cheng-Yuan Hsieh
學號 s4696106
學位類別 碩士
語文別 中文
論文頁數 56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-陳建華
中文關鍵字 創傷弧菌  鰻魚血清 
英文關鍵字 vibrio vulnificus  eel serum 
學科別分類
中文摘要 創傷弧菌是一種對於人類和鰻魚具有致病力的海洋性細菌。目前創傷弧菌分成三種生物型,其中會對於鰻魚有毒力的菌株被分類為生物二型。實驗室先前的研究已證實創傷弧菌生物二型具有一共有的質體,而此質體與此型細菌在鰻魚血清中的存活對鰻魚的毒力密切相關。一個創傷弧菌生物二型菌株的毒力質體pR99,其所攜帶的基因vep07已被發現為該菌在鰻魚血清中的存活所必需。然而,當此基因單獨存在於無質體的菌株中時,並不能使該菌對鰻魚血清具有抗性,暗示著此毒力質體中可能尚有其他的基因參與其中。本研究中我們以篩選鰻魚血清抵抗力減弱的突變株來鑑別此毒力質體中其他參與抵抗鰻魚血清殺菌作用的基因。首先,將帶有mini-Tn10的質體pBSL181送入創傷弧菌進行轉位突變以建構突變株庫,接著,在這些突變菌株中送入一個接合質體以協助帶有突變的質體轉移到無質體的菌株。經過鰻魚血清的篩選,我們挑選出3株鰻魚血清抗性減弱的突變株並鑑定出受轉位子影響的基因。其中之一的vep71已確認其與菌體對鰻魚血清的抗性相關,不過,其功能需要進一步研究。我們也將鰻魚血清生存不受影響的突變株的突變位置在pR99圖譜中標定出來。
英文摘要 Vibrio vulnificus is a marine bacterial species pathogenic for humans and eels. There are three biotypes (BTs) of V. vulnificus, and the strains virulent for eels are classified as BT2. We have previously demonstrated that the BT2 strains possess a common plasmid that is associated with their virulence in eels. A gene, vep07, carried by the virulence plasmid, pR99, of a BT2 strain was found to be essential for bacterial virulence in eels. However, this gene alone in a plasmid-cured strain did not confer the virulence for eels, indicating that other genes in the virulence plasmid might be involved. Both the virulence plasmid and vep07 are required for the bacterial resistance to killing by eel serum. In the study we identified other virulence genes in the plasmid by screening for mutants with reduced resistance to eel serum. A mutant bank has been constructed by transposon mutagenesis with a mini-Tn10-carrying plasmid, pBSL181. The virulence plasmids with mutations have been transferred to a strain cured of the virulence plasmid with the help of a conjugative plasmid. Three mutants with reduced resistance to eel serum have been thus isolated and the mutated genes have been mapped. The association of vep71 one of these genes and is annotated as a hypothetical protein, with resistance to eel serum has been confirmed. The function of vep71 needs to be further studied. We have also mapped those mutations in those with normal survival in eel serum in pR99.
論文目次 中文摘要 I
Abstract II
致謝 III
目錄 IV
表目錄 VI
圖目錄 VII
符號及縮寫 VIII
緒論 1
創傷弧菌的感染 1
創傷弧菌的分類 2
創傷弧菌的毒力因子 3
實驗動機與目的 4
材料與方法 6
I. 實驗菌株與質體: 6
II. 實驗菌種的培養與保存方法: 6
III. 核酸技術: 6
1. 小量純化質體DNA (TENS法) 6
2. 以商業化套組純化質體DNA 7
4. 聚合酶連鎖反應 (PCR) 8
5. DNA電泳分析 8
6. PCR放大片段選殖 (TA cloning) 8
7. 限制酶切割DNA 9
8. DNA片段之分離與回收 9
9. DNA片段之去磷酸化反應 10
10. DNA接合反應 10
IV. 轉位子突變技術 ( transposon mutagenesis) 10
V. 隨意引子聚合鏈反應 (AP-PCR) 11
VI. 創傷弧菌∆vep71突變株之建構: 11
VII. 創傷弧菌∆vep71互補株之建構 12
VIII. 雜交實驗: 12
1. DNA探針製備 12
2. 南方雜交法 (southern hybridization) 13
IX. 質體轉移方法: 13
1. 熱休克轉形作用 (heat shock transformation) 13
2. 接合生殖作用 (conjugation) 14
XI. 細菌對鰻魚血清殺菌作用的抗性之測定: 14
XII. 細菌生長曲線之測定: 15
XIII. 資料分析: 15
1. DNA序列決定 15
2. 胺基酸資料庫搜索分析 15
3. 胺基酸序列之分析 16
結果 17
創傷弧菌生物二型CECT4999菌株之轉位子突變株庫的建立: 17
鰻魚血清抗性降低之突變株挑選: 18
轉位子插入之質體DNA序列分析: 19
創傷弧菌CECT4999∆vep71突變株對鰻魚血清之抗性分析: 19
Vep71蛋白之生物資訊分析: 20
pR99毒力質體中與鰻魚血清抗性無關之基因: 20
討論 22
參考文獻 26
自述 56
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