O1型霍乱弧菌Fosmid文库的构建及分析

Construction and Characterization of a Fosmid Library of Vibrio cholera Serogroup O1

为构建O1型霍乱弧菌Fosmid文库并对文库进行分析,采用低熔点琼脂糖包埋法提取O1群霍乱弧菌N16961完整基因组,用随机剪切法将基因组进行片段化处理,脉冲电泳分离片段化DNA并切胶回收33～48kb之间的DNA片段,然后对DNA片段进行末端平滑化和磷酸化处理,与pCC1FOS载体连接,经包装、转染后构建霍乱弧菌Fosmid文库。平均插入片段为37kb,共保存9 600个克隆,空载率小于2%。本文库符合文库的构建要求,文库的建立为O1霍乱弧菌重要基因及其功能的研究奠定了基础。

A fosmid library was constructed and characterized for Vibrio cholera serogroup O1 in this study. Intact genomic DNA of Vibrio cholera N16961 was extracted with low melting point agarose embedment and p-agarase I digestion methods, fragmented with pipette and separated through pulse field gel eleetrophoresis. The fragments with desirable lengths ranging from 33 to 48kb were recovered from agarose gel with the ends blunted and phosphorylated. These fragments were ligated with pCC1FOS vector, packaged and and transfected into hosting bacteria, yielding a fosmid library containing 9600 clones. The inserts were 37kb in length in average. Vector containing clones were less than 2%. The constructed reached the standard of a fosmid library, which will certainly facilitate cloning the genes of Vibrio cholera N16961 and deciphering their functions.