利用融合蛋白EDDIE在大肠杆菌中高效表达抗菌肽Cecropin AD

High expression of antimicrobial peptide Cecropin AD in Escherichia coli by fussion with EDDIE

摘要:

本研究采用猪瘟病毒(Classical swine fever virus, CSFV)定点突变外壳蛋白(EDDIE)为融合蛋白, 对抗菌肽Cecropin AD (CAD)基因进行了高效融合表达, 获得了有抗菌活性的抗菌肽CAD。首先采用重叠PCR基因合成技术将编码抗菌肽的CAD基因与猪瘟病毒定点突变外壳蛋白EDDIE编码基因合成为e-cad融合基因, 接着将融合基因e-cad采用定点同源重组的方法连接到载体pET30a上, 构建成pETED表达载体, 然后转化大肠杆菌BL21(DE3)表达, 表达的融合蛋白在大肠杆菌中主要以包涵体形式存在, 表达量占菌体总蛋白的40%以上。蛋白质在体外复性, 融合蛋白中EDDIE自我剪切, 产生抗菌肽CAD。抑菌试验表明抗菌肽CAD能有效地抑制大肠杆菌和藤黄八叠球菌的生长, 并且对酵母菌的生长也有微弱地抑制作用。以EDDIE为融合蛋白是在大肠杆菌中高效表达抗菌肽的一种好方法。

Abstract:

In this study, we efficiently expressed the active antimicrobial peptide (CAD), which fused with the site-mutated coat protein (EDDIE) of the classical swine fever virus, in Escherichia coli. First, we obtained the e-cad fusion gene from the CAD gene and the EDDIE gene using overlapping PCR. Then to get the recombinant expression vector (pETED), the e-cad fusion gene was cloned into the pET30a vector by a site-directed homologous recombination technique. The EDDIE-CAD fusion protein expressed in E. coli as inclusion bodies, and its yield was more than 40% of total bacterial proteins. After renaturated in vitro and self-cleavage of the fusion protein, we obtained the antimicrobial peptide Cecropin AD. Antimicrobial experiments showed that the Cecropin AD efficiently inhibited the growth of G+ and G- bacteria, but it weakly inhibited the growth of Saccharomyces. This method provides an excellent way for high expression of antimicrobial peptides when fused with EDDIE.