摘要
根据从实验室克隆的牙鲆抗菌肽hepcidin基因（GenBank登陆号DQ129693）的序列,设计了特异性引物,PCR扩增其成熟肽片段.重组至融合表达载体pGEX-4T-1中,构建成牙鲆抗菌肽hepcidin基因融合表达载体pGEX-fhep,转化至大肠杆菌BL21（DE3）plyS中,筛选得到的阳性克隆用IPTG进行诱导.SDS-PAGE电泳显示在29kD处有特异性的蛋白条带出现,Western-blotting 检测表明已经成功表达了融合蛋白,表达的融合蛋白最高占总蛋白的21%.纯化得到的GST-fhep用凝血酶切割后,用亲合层析得到牙鲆的抗菌肽hepcidin.抑菌实验结果表明抗菌肽对大肠杆菌（ATCC25922）有一定程度的抑制作用.
Based on the sequence of the hepcidin gene from Paralichthys olivaceus, gene-specific primers were designed and synthesized to amplify the hepcidin mature peptide sequence. The amplified fragment was inserted into pGEX-4T-1 and the fusion expression plasmid pGEX-fhep was constructed. The plasmid was transferred into E. col BL21（DE3）plyS and induced by IPTG. SDS-PAGE and Western-blotting analysis showed that the recombinant had expressed a 29kD specific protein. By Glutathione Sepharase 4B affinity chromatography, the fusion protein GST-fhep was obtained and purified. After cleaved by thrombin, hepcidin was obtained. The recombinant GST-fhep showed antibacterial activities to E. coli by liquid growth inhibition method.
出处
《高技术通讯》
CAS
CSCD
北大核心
2007年第7期765-770,共6页
Chinese High Technology Letters
基金
973计划（2004B117403）、863计划（2002AA626010）和国家自然科学基金（40376047）资助项目.
关键词
牙鲆
抗菌肽
HEPCIDIN
大肠杆菌
表达
活性
Paralichthys olivaceus, antimicrobial peptides, hepcidin, E. coli, exprssion, activity
