創傷弧菌核酸分解酵素基因（vvn）序列長度為696 bp，可轉譯出含 232 個胺基酸的蛋白質（Vvn），Vvn必須存在於細胞周質而且呈氧化態才能表現活性。DNA shuffling是活體外製造基因突變的好方法，可集合同源重組和點突變兩種突變方式。DNA shuffling的方法共有四個步驟：（1）大量製備欲進行突變的基因，（2）以DNase I 隨機切割基因成為小片段DNA，（3）利用未加入引子的PCR反應進行小片段DNA重組，（4）以加入引子的PCR反應擴大重組後的基因。DNA shuffling具有的優點為不需事先研究特定蛋白質構造而可以快速地改造特定蛋白質。本研究的目的為希望利用DNA shuffling的方法，在短時間內製造多樣突變，經由活體內菌株分解DNA和周質蛋白分解DNA的活性分析結果，將突變後的Vvn活性變化歸類為三個族群，酵素分解核酸的活性升高、降低或沒有差異，從這三個族群中隨機挑選出14株轉形菌定序其vvn基因，定序後與原始vvn基因序列作比對，結果僅得到一個鹼基突變，突變位置為蛋白質N端第22個胺基酸Ser突變成Ile。突變Vvn與野生型Vvn活性相比，其DNase相對活性為82 %，RNase相對活性為59 %。此單一胺基酸的變化對Vvn兩種活性的影響不同，符合假說中Vvn可能具有兩個但有部分重疊的活性中心。

The nuclease gene of Vibrio vulnificus, vvn, is 696 bp long encoding a protein（Vvn）of 232 amino acids. Vvn is a periplasmic protein and is active in the oxidized form. DNA shuffling is a powerful method for in vitro mutational mechanism by homologous recombination with a low level of point mutation . DNA shuffling consists of four steps：（1）preparation of genes to be shuffled, （2）random fragmentation with DNase I, （3）fragment reassembly by primerless polymerase chain reaction（PCR）, and（4）amplification of reassembled products by a conventional PCR. The advantage of this process is that it can be used to rapidly evolve any protein, without any knowledge of its structure. The goal of this work was using DNA shuffling to generate a diversity of mutation in vvn within a short time. Followed by analyzing the DNase activity of periplasmic protein or in vivo, the mutants were divided into three groups for increase, decrease or no change in DNase activity. Randomly DNA sequencing vvn gene of fourteen transformed clones from the three groups showed only one clone has one base change with comparison to wild-type sequence. The mutation is at amino acid 22 of the N-terminus of Vvn, the change is from serine to isoleucine. The relative activity of mutant Vvn was 82 % in DNase and 59 % in RNase. The effect of a single amino acid change on the DNase and RNase activity of Vvn is different. It supports the postulation that there are two distinct but overlapping active sites exist in Vvn.

目 錄
中文摘要 ……………………………………………………………… I
英文摘要 ……………………………………………………………… II
目錄 …………………………………………………………………… III
圖目錄 ………………………………………………………………… V
表目錄 ………………………………………………………………… VI
緒論 …………………………………………………………………… 1
材料與方法 …………………………………………………………… 7
1. 菌株和質體 …………………………………………………… 7
2. 菌種之培養與保存 …………………………………………… 7
3. 質體的小量製備 ……………………………………………… 7
4. DNA shuffling ………………………………………………… 8
（1）vvn的製備 …………………………………………………… 8
（2）DNA電泳分析 ……………………………………………… 9
（3）以DNase I水解vvn ………………………………………… 9
（4）核酸回收 …………………………………………………… 10
（5）不加入引子（primer）的PCR反應 ……………………… 10
（6）加入引子的PCR反應 ……………………………………… 11
5. 轉形作用 …………………………………………………… 11
（1）限制酵素切割 ……………………………………………… 11
（2）接合作用（ligation） ……………………………………… 12
（3）勝任細胞（competent cell）之製備 ……………………… 12
（4）轉形作用（transformation） ……………………………… 13
6. PCR檢驗菌株是否攜帶vvn基因 …………………………… 13
7. 活體內菌株分解DNA的活性測定 ………………………… 13
8. 細菌之周質蛋白（periplasmic protein）的製備 …………… 14
9. 周質蛋白分解DNA的活性測定 …………………………… 15
10. 蛋白質膠體電泳法（SDS-PAGE） ………………………… 15
11. 西方墨點法（Western blot） ………………………………… 16
12. 定序 ………………………………………………………… 17
13. 純化Vvn ……………………………………………………… 17
14. 蛋白質濃度測定 …………………………………………… 17
15. RNase活性分析 ……………………………………………… 18
結果…………………………………………………………………… 19
1. 利用DNA shuffling的方法重組vvn基因 ………………… 19
2. 轉形作用 …………………………………………………… 19
3. 活體內菌株分解DNA的活性測定 ………………………… 20
4. 周質蛋白分解DNA的活性測定 …………………………… 21
5. 西方墨點法（Western blot） ………………………………… 21
6. 定序 ………………………………………………………… 22
7. 純化轉形菌株51之Vvn ………………………………… 22
8. mutant Vvn分解DNA及RNA的活性測定 …………… 23
討論 …………………………………………………………………… 24
參考文獻 ……………………………………………………………… 29
圖 ……………………………………………………………………… 34
表 ……………………………………………………………………… 50

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