霍乱弧菌中弧菌素合成酶VibF的A结构域基因的克隆、表达与纯化研究

摘要

弧菌素合成酶的A结构域在弧菌素的合成过程中发挥着重要作用.使用DNAStar软件对A结构域的蛋白质二级结构进行预测,截取了多组不同氨基酸长度的A结构域片段.使用PCR方法从霍乱弧菌N16961的基因组中克隆了A结构域基因,通过酶切、连接等方法将其构建到表达载体pET- 21b(+)上,并在大肠杆菌BL21 (DE3)中得到高效表达.在30个不同氨基酸长度的A结构域蛋白片段中获得5个可溶性表达的片段；并且通过三步分离纯化的方法获得了一性状良好、均一和高纯度的A结构域蛋白(N861-1410),从而为A结构域蛋白质晶体生长提供了基础.%The A domain of vibriobactin synthetas VibF plays a key role in the biosynthesis of vibriobactin in the pathogenic bacteria Vibrio cholerae. DNAStar software was used to predict the secondary structure of the A domain for chosing several fragments with various amino acid lengths. The A domain gene was cloned from the genome of Vibrio cholerae strain N16961 and constructed into the expression vector pET-21b( + ) ,and then was overexpressed in E. Coli BL21 (DE3). Five soluble A domain fragments from 30 different fragments with various molecular weight were obtained. One A domain protein,N861 -1410,with homogeneous state and high purity, was purified via three steps of chromatography, providing fundamental step for its crystallization.