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系統識別號 U0026-0812200910450169
論文名稱(中文) 創傷弧菌其質體之特性分析
論文名稱(英文) Characterization of a plasmid in Vibrio vulnificus YJ016
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 91
學期 2
出版年 92
研究生(中文) 徐立芳
研究生(英文) Li-Fang Hsu
電子信箱 coma2914@yahoo.com.tw
學號 s4690403
學位類別 碩士
語文別 中文
論文頁數 65頁
口試委員 口試委員-陳建華
召集委員-吳俊忠
指導教授-何漣漪
中文關鍵字 質體  創傷弧菌 
英文關鍵字 plasmid  Vibrio vulnificus 
學科別分類
中文摘要 創傷弧菌為一棲居於海水環境的嗜鹽性革蘭氏陰性菌,對一些有潛在性疾病或免疫缺陷的人會造成嚴重的傷口感染和致死性敗血症。創傷弧菌依其在生化反應及生物特性上的差異,可大略區分成三種生物型。會引起人類感染疾病的菌株主要為生物1型。在將一創傷弧菌臨床株,YJ016的全基因體定序後,發現有一48.5 kb的質體存在,我們將它稱為pYJ016。分析不同創傷弧菌臨床株或環境株質體存在的情形發現,並沒有和pYJ016相同的質體存在,顯示pYJ016之獨特性。為了解pYJ016在細菌的生長及毒力中所扮演的角色,我們藉由acridine orange將pYJ016從YJ016中移除。實驗結果顯示,將pYJ016從YJ016中移除後,其於基礎或營養培養基中的生長曲線及對小鼠的毒力與野生株YJ016並沒有明顯差異。由此推論,pYJ016與細菌在培養基中的生長及其對小鼠的毒力無關。另一方面,pYJ016上有多個與質體轉移功能相關的基因存在,且其DNA序列與排列順序和F質體上所帶有的tra基因類似,只是缺少部分調控mating signal及合成sex pilus的基因,例如: traM,traJ,traP,traV,traR,traE,traQ,traS及traT等。雖然如此,我們的實驗結果顯示pYJ016仍能以接合作用的方式轉移到其他生物1型的創傷弧菌中,其頻率約為10-3。然而,pYJ016並無法以接合作用或轉形作用轉移到大腸桿菌DH5α中,由此推測pYJ016並無法在大腸桿菌中複製。

英文摘要 Vibrio vulnificus is a gram-negative, marine bacterium that causes septicemia and serious wound infection in persons who are immunocompromised or have underlying diseases such as liver cirrhosis. V. vulnificus is divided into three distinct biotypes based on the phenotypic and host-range differences. Typically, the biotype 1 strains are associated with human illness. A plasmid (designated pYJ016) of 48.5 kb has been identified in the completed genome sequence of a biotype 1 clinical isolate, YJ016. pYJ016 was uniquely present in strain YJ016 among the various clinical or environmental V. vulnificus strains tested. To examine the role of the plasmid in bacterial growth and virulence, this plasmid was cured from the organism by acridine orange treatment. The growth rate of the cured strain was not distinctly different from the plasmid-harboring strain, and the virulence of the cured strain in mice is comparable to the wild-type strain YJ016. These results indicate that pYJ016 is not required for the growth and virulence of YJ016. On the other hand, the organization and nucleotide sequences of some of the open reading frames (ORFs) in pYJ016 are strikingly like those of the F plasmid, except that pYJ016 lacks some genes for the formation of the sex pilus, like traM, traJ, traP, traV, traR, traE, traQ, traS, and traT. Nevertheless, pYJ016 could still be transferred to other biotype 1 strains by conjugation at a frequency of approximately 10-3. However, pYJ016 could not be transferred to Escherichia coli DH5α by either conjugation or transformation, suggesting that this plasmid may not replicate in E. coli.

論文目次 中文摘要i
英文摘要iii
致謝iv
目錄v
表目錄viii
圖目錄ix
符號及縮寫x
緒論1
材料與方法
一、實驗菌株、質體與實驗動物
1.實驗菌株與質體7
2.實驗動物7
二、菌種的培養與保存
1.細菌培養液的配置7
2.細菌培養方法8
3.細菌保存方法8
三、創傷弧菌基因之選殖
1.質體的純化與其DNA處理方法9
(1)小量純化質體的方法9
(2)商業化抽取質體DNA套件法10
(3)限制酶切割質體DNA12
(4)DNA電泳分析12
(5)聚合酶連鎖反應13
(6)PCR放大片段之選殖14
(7)DNA片段之分離與回收15
(8)DNA片段之去磷酸化反應16
(9)DNA接合反應 (Ligation)16
2.質體轉移方法
(1)轉形作用 (Transformation)17
(2)細菌接合作用 (Conjugation)17
3.菌落雜交法 (Colony hybridization)20
四、創傷弧菌質體pYJ016之特性分析
1.pYJ016所含基因之序列比對與分析22
2.將抗藥性基因插入創傷弧菌質體22
(1)質體構築22
(2)具抗氯黴素基因質體之構築23
3.創傷弧菌質體之移除(Plasmid curing)24
4.細菌生長曲線 (Growth curve) 之測定25
5.創傷弧菌對小鼠的毒力試驗25
6.創傷弧菌突變株之製作25
(1)對streptomycin具抗性突變株之分離25
(2)lacZ基因突變株分離26
7.創傷弧菌接合作用頻率 (conjugation frequency )之測定27
結果28
一、創傷弧菌YJ016 所含質體 pYJ016 於創傷弧菌生長及毒力中所扮演之角色
1.創傷弧菌YJ016之質體在其他創傷弧菌中的分佈情形28
2.含氯黴素(Chloramphenicol) 抗藥性之pYJ016衍生質體pYJ016Cmr之構築29
3.質體pYJ016之移除 29
4.野生菌株與移除質體菌株在培養液中生長速率之比較30
5.野生菌株與移除質體菌株對小鼠毒力之比較30
二、分析質體pYJ016 之轉移特性
1.分離創傷弧菌lacZ基因突變株30
2.質體pYJ016 藉接合作用 (conjugation) 於不同創傷弧菌宿主中轉移之頻率31
3.質體pYJ016Cmr由創傷弧菌轉移至大腸桿菌之試驗32
討論33
參考文獻38
圖表46
自述65
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