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研究生:吳世怡
研究生(外文):SHIH-YI WU
論文名稱:創傷弧菌核酸分解基因之DNA定序與該酵素之特性分析
論文名稱(外文):Nucleotide sequencing of the nuclease gene of vibrio vulnificus and characterization of its product
指導教授:何漣漪
指導教授(外文):LIEN-I HOR
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:1998
畢業學年度:86
語文別:中文
論文頁數:75
中文關鍵詞:創傷弧菌核酸分解
相關次數:
  • 被引用被引用:4
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中文摘要
創傷弧菌為生存於海水環境中之嗜鹽性革蘭氏陰性細菌,在臨床上常造成
嚴重的敗血症及組織壞死,特別常見於具有肝硬化或血色素沉著症的病人。
在感染後病程發展相當快速且死亡率高。此菌具有DNA分解的活性;為
了研究此活性對細菌本身之生理意義,以及對創傷弧菌存活於宿主體內的
重要性,我們對此酵素進行基因選殖與其DNA定序。從創傷弧菌基因庫中
共篩選出8株具有DNA分解活性的選殖株,它們的重組質體均具有相同的
核酸分解基因。針對其中一株SL006進行次選殖後,得到一仍具有DNA分
解活性的DNA插入片段,長度為1.7 kb。將此1.7 kb片段插入pUC19載體
中進行DNA定序,得到二個開放閱讀區域,其中之一長度為696 bp,可轉
譯出232個胺基酸,且其胺基酸序列與霍亂弧菌之成熟型DNA分解有78%
的相似性。該基因產物具有分解環狀與線狀DNA及RNA之活性,故將之命名
為創傷弧菌核酸分解(Vvn)。該酵素在100℃煮沸30分鐘後仍具有分解
DNA的活性,為一對熱安定的酵素。依據蛋白質電泳的結果及由其DNA序列
推衍出的胺基酸序列計算,其分子量約24 kDa,而其等電點推算為8.61。
此核酸分解有一由18個胺基酸組成的訊息序列,而訊息之切割位
置在Ala-Ala之間。此核酸分解活性存在於創傷弧菌與大腸桿菌選殖株
之細胞間隙中,且其還原型不具有酵素活性。表現此酵素之大腸桿菌選殖
株進行轉形、電擊轉形及接合作用之頻率都比不表現此酵素之對照菌株要
低,尤其是轉形作用,表示此核酸分解會阻礙遺傳物質的轉移。因此若
能將此創傷弧菌核酸分解基因破壞,使更容易進行基因之移轉,將有助
於未來對創傷弧菌的遺傳學研究。這樣的突變株也可以用來探討此酵素在
創傷弧菌的致病機轉中的重要性。
英文摘要
Vibrio vulnificus is a halophilic gram-negative marine
bacterium that when infected could cause fulminant septicemia
with a high mortality rate and serious wound infection in
persons who have underlying conditions, especially those with
liver cirrhosis or hemochromatosis. This organism exhibits
DNase activity on the DNase test agar. In order to study the
role of the DNase activity in the pathogenesis and physiology
of V. vulnificus, we cloned and determined the nucleotide
sequence of the gene encoding this enzyme. From the gene
library of V. vulnificus YJ016, 8 nuclease-producing clones
were screened and were proved to contain the same nuclease gene.
From these clones, SL006 was selected for extensive studies.
The nuclease gene was mapped to a 1.7-kb MluI-BamHI fragment by
deletion analysis. This fragment was then subcloned to pUC19
to facilitate nucleotide sequence determination. Two open
reading frames were identified in this sequence. One was 696 bp
long encoding a protein of 232 amino acids(aa), and the
deduced amino acid sequence of it shared 78 % homology with
that of the mature Vibrio cholerae DNase. Its gene product
(designated Vvn) was able to digest both DNA, either circular
or linear form, and RNA, and remained active for digesting DNA
after a heat treatment at 100℃for 30 min. The molecular weight
of Vvn estimated either by protein gel electrophoresis or from
the deduced aa sequence was approximately 24 kDa, and the pI
value estimated from the deduced aa sequence was 8.61. Vvn
contains a signal sequence of 18 aa with a signal peptidase
cleavage site between two alanine residues. The nuclease
acativity was detected in the periplasmic fraction of either
V. vulnificus or the recombinant E. coli strain, and was lost
when Vvn was reduced by treatment with 2-mercaptoethanol.
E. coli K12 strain DH5a expressing Vvn from a high copy number
plasmid showed a lower frequency, when served as the recipient,
in conjugation, electroporation, and especially, transformation
, compared with that was not expressing the nuclease. This
suggests that Vvn might hamper the gene transfer processes,
perhaps would not allow the recipient to retain the transferred
gene. Therefore, disruption of the nuclease gene in
V. vulnificus should render gene transfer to this organism
easier and facilitate further genetic studies of this organism.
Such mutants should also be useful for determining the role of
V. vulnificus nuclease in the pathogenesis.
目錄
頁數
中文摘要 i
英文摘要 ii
誌謝 iv
目錄 v
表目錄 vii
圖目錄 viii
符號及縮寫 x
緒論 1
材料與方法 5
(1)菌株與質體 5
(2)細菌之培養與保存 5
(3)產DNA分解細菌之測試 6
(4)快速純化質體DNA之方法 7
(5)小量純化質體DNA之方法 8
(6)大量純化質體DNA之方法 8
(7)商業化抽取質體DNA套件法 10
(8)限制切割質體DNA 11
(9)DNA電泳分析 12
(10)核酸回收法 13
(11)DNA片段之黏端補齊反應 13
(12)DNA片段之去磷酸化反應 14
(13)DNA接合反應 15
(14)細胞轉形作用 16
(15)細胞電擊轉形作用 17
(16)細胞接合作用 18
(17)細菌之間隙蛋白的製備 19
(18)蛋白質濃度測定 19
(19)放射狀酵素擴散作用 20
(20)硫酸十二酯鈉聚丙烯醯氨膠電泳法 21
(21)核酸分解活性染色分析 24
(22)DNA定序 24
(23)DNA序列之電腦分析 27
結果 29
(一)核酸分解基因之選殖 29
(二)核酸分解基因之DNA序列分析 30
(三)核酸分解N端胺基酸序列之分析 32
(四)核酸分解基因產物之活性分析 33
(五)核酸分解對轉形作用、電擊轉形
作用及接合作用之影響 34
討論 36
參考文獻 40
圖表 50
附錄 73
自述 74
授權書 75
參考文獻
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