斜带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达  

Cloning of antimicrobial peptide hepcidin from Epinephelus coioides and prokaryotic fusion expression of its predicted mature peptide

作　　者：陈大玮[1] 邓利[1] 刘志刚[1] 孟铂渊[1] 马一见[1] 

机构地区：[1]深圳大学生命科学学院,广东深圳518060

出　　处：《南方水产科学》2011年第1期1-7,共7页South China Fisheries Science

基　　金：国家高技术研究发展计划(863计划)项目(2006AA100308);深圳市重点实验室提升项目"石斑鱼抗菌肽基因表达研究"

摘　　要：文章根据已报道的硬骨鱼类hepcidin cDNA序列设计简并引物，通过RT—PCR从重要海水经济鱼类斜带石斑鱼（Epinephelus coioides）肝脏中扩增出1条具有完整开放阅读框的246bpcDNA序列，Blast分析表明该序列是鱼类hepcidin基因家族的一员，被命名为斜带石斑鱼hepcidin样抗菌肽前体，该cDNA所推导的氨基酸序列有如下特点：1）信号肽序列与多数鱼类hepcidin信号肽的同源性在67％～87％之间；2）C端20个氨基酸序列具有绝大多数动物hepcidin成熟肽的共同典型保守序列特征，即在对应位置具有8个Cys；3）序列中不具有已报道的hepcidin前体肽转化酶作用典型基序[RX（K／R）R]；4）与GenBank中已注册的2条斜带石斑鱼hepcidincDNA序列（GU391241和GU391242）所推导的氨基酸序列同源性分别为36％和33％，且NJ进化树显示不与这2条序列聚为一枝，表明该序列是斜带石斑鱼hepcidin基因家族中一种新亚型，其预测成熟肽融合表达载体成功在大肠杆菌（Escherichiacoli）中表达，为后续研究奠定基础。Based on the known hepcidin cDNA sequences in teleosts, we designed some degenerate primers and cloned a 246 bp cDNA sequence with complete open reading frame from the liver of Epinephelus coioides, an important commercial marine fish. According to Blast analysis, the obtained sequence, named as E. coioides hepcidin-like antimicrobial peptide precursor, is a member of fish hepci- din gene family. The deduced amino acid sequence has such features as follows ： 1 ） the similarity of hepcidins between the present sig- nal peptide and that of most other fish is 67% - 87% ; 2） twenty animo acids in the C-terminal region contain 8 cysteines, which is the typical feature of conserved sequence of mature peptide in most animals＇ hepeidins ; 3 ） [ RX （K/R） R ] motif for propeptide eonvertases of the reported hepcidins is not found ; 4 ） the similarity of the deduced amino acids between the present sequence and other 2 hepcidin sequences of E. coioides （GU391241 and GU391242） registered in GenBank is 36% and 33% , respectively. Phylogenetie neighbor- joining tree indicates that the deduced amino acid sequences of above 3 hepeidins of E. coioides do not cluster into one clade. Thus, the hepeidin obtained in the present study is presumably a new isoform of hepeidin gene family in E. coioides. The prokaryotic fusion ex- pression vector of its predicted mature hepcidin peptide has been constructed and expressed successfully in Escherichia coli BL21 （ DE3 ） , which lays foundation for future study.

关 键 词：斜带石斑鱼 抗菌肽 HEPCIDIN 分子克隆 原核融合表达 

分 类 号：Q516[生物学—生物化学]