大口黑鲈抗菌肽hepcidin cDNA序列和结构分析

以大口黑鲈为材料,提取肝脏总RNA,经RT-PCR扩增出hepcidin cDNA的开放阅读框(ORF)及3′端非编码区序列,应用5′RACE方法得到大口黑鲈hepcidin cDNA5′末端。将所获得的两个片段分别克隆到T载体后进行测序,并拼接成大口黑鲈hepcidin全长cDNA。序列分析表明:大口黑鲈hepcidin全长cDNA为564bp,含有一个258bp的ORF,编码86个氨基酸残基,由信号肽(24个残基)、前肽(42个残基)和成熟肽(20个残基)3部分组成hepcidin前体。在前肽部分具有前肽转化酶典型的RX(K/R)R基元,成熟肽部分含有8个保守的半胱氨酸残基,可形成四个链内二硫桥,使β-折叠结构保持稳定。大口黑鲈Hepcidin与其他鱼类的同源性在29.7%~90.5%间,尤其是信号肽区域,与鳜、尼罗罗非鱼、真鲷、花鲈、黑鯛、金眼狼鲈仅有2~3个氨基酸的差别。

Cloning and Sequence Analysis of cDNA Encoding Hepcidin from Largemouth Bass, Microptorus salmonoidesi

Hepcidin is a antimicrobal peptide with a special character. To study its structure and characteristics further, the open reading frame (ORF) and 3′ untranslated region (3′-UTR) of hepcidin cDNA were amplified by RT-PCR, and the 5′-UTR was amplified by rapid amplification of 5′ complementary DNA ends (5′-RACE) from the total RNA of liver of the largemouth bass Microptorus salmonoidesi. The PCR products were cloned into pMD18-T vector and then sequenced. The full length cDNA fragment was determined to be 564 bp including an ORF coded 86-aa propeptide which consists of a signal peptide (24 amino acids), a prodomain (42 amino acids) and a mature peptide (20 amino acids). A RX(K/R)R motif typical of propeptide convertases was identified in the prodomain of hepcidin sequence, and in the mature peptide, there were 8 conservative cysteine residues forming four disulphide bonds, which can make the β-folding structure remain stable. Compared between largemouth bass hepcidin with that of other reported sequences in fish, they showed high homologous, especially in signal peptide, only 2 or 3 amino acids different from that of mandarin fish, turbot, Nile tilapia, black sea bream, red sea bream,white bass and Japanese sea perch.