Study the nuclease of Vibrio vulnificus by DNA shuffling

• Main Authors: Ying-Chou Chen, 陳盈州
• Other Authors: Ching-Mei Hsu
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/94680979718682604432

碩士 === 國立中山大學 === 生物科學系研究所 === 89 === 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.