大菱鲆hepcidin抗菌肽基因的克隆及在大肠杆菌中的高效表达

Cloning and High-level Expression of A Turbot Hepcidin Antimicrobial Peptide Gene

通过同源克隆结合RACE的方法从大菱鲆(Scophthal mus maximus)肝脏中克隆了大菱鲆hepcidin抗菌肽基因全长cDNA(GenBank accession number AY994074)。大菱鲆hepcidin基因cDNA全长778bp,包含有1个273bp的开放阅读框,编码1个长90氨基酸的前体肽。将大菱鲆hepcidin抗菌肽基因的成熟肽序列重组至原核表达载体pGEX-4T-1中,用IPTG诱导后,SDS-PAGE电泳显示在29ku处有特异性蛋白条带出现,表达的融合蛋白主要以包涵体形式存在,融合蛋白约占总蛋白的26%。Western-blotting分析发现表达的融合蛋白可以特异性地与GST抗体相识别。这些结果表明大菱鲆hepcidin抗菌肽基因成功实现了原核表达。

The molecular cloning of a hepcidin antimicrobial peptide gene from the liver of turbot using RACE were studied.The full-length cDNA of turbot hepcidin is 778 bases long and contains an ORF of 273 bases encoding a prepropeptide of 90 amino acid residues.The hepcidin mature peptide sequence was amplified and inserted into pGEX-4T-1 and fusion expression plasmid was constructed.The plasmid was transferred into E.coli BL21(DE3)plyS and induced by IPTG.SDS-PAGE and western-blotting analysis showed that the recombinant had expressed a 29 ku specific protein successfully.