出版年:

2000

研究生:

羅仕淦

研究生(英文姓名):

Lo Shi-Kan

論文名稱:

創傷弧菌不產核酸分解突變株之分離和特性分析

英文論文名稱:

Isolation and characterization of Vibrio vulnificus mutant deficient in nuclease

指導教授:

何漣漪

指導教授(英文姓名):

Hor L. I.

學位類別:

碩士

校院名稱:

國立成功大學　

系所名稱:

微生物暨免疫學研究所

學號:

s46851097

學年度:

88

語文別:

中文

論文頁數:

66

關鍵詞:

創傷弧菌 ; 核酸分解

英文關鍵詞:

Vibrio vulnificus ; nuclease

被引用次數:

0

[ 摘要 ]

創傷弧菌為嗜鹽性革蘭氏陰性細菌，會在免疫機能不全或是有肝硬化或血色素沉著症等潛在性疾病的病人造成嚴重的敗血症及皮膚病變。我們曾經從此菌選殖出一種能分解DNA和RNA，並且對熱穩定的核酸分解(Vvn)。當大腸桿菌中大量表現Vvn時，會降低轉形作用的效率，因此，此核酸分解可能會在對創傷弧菌進行傳統方式的轉形作用時干擾外來DNA的進入。此外，細菌的核酸分解可以提供細菌碳和氮的來源，並且在有些細菌感染宿主過程中，幫助其在小腸黏膜繁殖。本論文藉分離並分析創傷弧菌不產核酸分解的突變株，以了解Vvn在細菌毒力方面和在防止外來DNA進入細菌所扮演的角色。我們利用DNA同源性重組的方式，將一段刪除335 bp片段的vvn基因和創傷弧菌臨床株YJ016染色體中的完整vvn基因互換，而得到不產核酸分解突變株SK005。此突變株在營養培養基中的生長速率和對老鼠的毒力都和野生株YJ016相似，顯示Vvn並非是一個重要的毒力因子。此外，突變株的接合作用及轉形作用效率也只比野生株稍微高一些。我們發現突變株在核酸分解測試培養盤或是突變株的細胞間質蛋白質之核酸分解活性分析試驗中，仍然可以測得核酸分解活性。我們推測創傷弧菌細胞間質中可能含有其他具有核酸分解活性的蛋白質，也參與阻擾外來核酸進入菌體的過程。

[ 英文摘要 ]

Vibrio vulnificus is a halophilic gram-negative bacterium that causes septicemia and severe wound infection in persons who are immunocompromised or having underlying diseases, such as liver cirrhosis or hemochromatosis. This organism produces a cell-associated nuclease the gene of which has been cloned in our laboratory, shown to be a thermostable enzyme capable of digesting both DNA and RNA. Expression of V. vulnificus nuclease (designated Vvn) in Escherichia coli resulted in a dramatic decrease of transformation efficiency indicating that this nuclease may be responsible for failure of introducing foreign DNA into V. vulnificus by conventional transformation or electroporation. Bacterial nucleases have also been proposed to be involved in providing carbon and nitrogen sources for the microorganisms, and promoting infection of enteric pathogens by facilitating their colonization in the mucosa of the small intestinal. To determine the role of Vvn in bacterial virulence and in preventing the bacterial cell from uptake of foreign DNA, a V. vulnificus mutant with a deletion in the nuclease gene (vvn) was isolated and characterized in this study. This V. vulnificus mutant, SK005, was generated by allelic exchange in which a deletion of 335 bp that resulted in apparent abolishment of the nuclease activity of Vvn was introduced into the vvn gene in the chromosome of a clinical V. vulnificus isolate, YJ016. SK005 exhibited a wild-type growth rate in rich medium and virulence in mice, indicating that Vvn is probably not an important virulence factor. Furthermore, it showed only a slight increase in the frequency of transformation and virtually no difference in conjugation, compared with its parental strain. When tested with DNase test agar plate or by the nuclease assay with various DNA substrates, substantial nuclease activity could still be detected in the periplasmic preparation of SK005. These data suggested that nuclease(s) other than Vvn might be present in the periplasm of V. vulnificus and take part in preventing uptake of foreign nucleic acids by th