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

作　　者：张贞[1] 柯涛[2] 周玉玲[1] 马向东[1] 马立新[1] 

机构地区：[1]湖北大学生命科学学院,武汉430062 [2]南阳师范学院生命科学与技术学院,南阳473061

出　　处：《生物工程学报》2009年第8期1247-1253,共7页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30760009);湖北省教育厅重点项目(No.D20081004)资助~~

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。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.

关 键 词：抗菌肽 融合蛋白 高效表达 

分 类 号：Q78[生物学—分子生物学] S511.03[农业科学—作物学]