棕点石斑鱼Piscidin样肽基因的克隆及其成熟肽的原核表达  

Cloning of antimicrobial piscidin-like peptide and prokaryotic expression of the putative mature peptide in Epinephelus fuscoguttatus

作　　者：陈大玮[1] 邓利[1] 何凡[1] 邱聪龄[1] 马一见[1] 刘志刚[2] 

机构地区：[1]深圳大学生命科学学院,518060 [2]深圳大学医学院,518060

出　　处：《渔业科学进展》2011年第4期126-133,共8页Progress in Fishery Sciences

基　　金：国家高技术研究发展计划(863计划)项目(2006AA100308);广东省大学生创新实验项目(1059010021)共同资助

摘　　要：以简并引物通过RT-PCR从重要经济海水鱼类棕点石斑鱼Epinephelus fuscoguttatus白细胞中扩增出一条388bp具有完整编码框的cDNA序列。Blast分析初步显示,该序列属于鱼类抗菌肽Piscidin家族的一员,将其命名为棕点石斑鱼Piscidin样肽（GU592793）,推导氨基酸序列具有界定鱼类Piscidin的以下共同特征：（1）预测的信号肽序列与其他Piscidin的同源性较高,在60%~95%之间;（2）推测的成熟肽N端6个氨基酸残基富含Phe、Ile及His;（3）成熟肽第8个和第13个氨基酸位点均为Gly。NJ进化树分析表明,该cDNA推导的氨基酸序列与赤点石斑鱼Epinephelusakaarapiscidin-like peptide（ACE78290）、斜带石斑鱼Epinephelus coioidesPiscidin-like peptide（ACE78291）以高达81%的支持率聚为一支。也表明本研究成功克隆到棕点石斑鱼首条PiscidincDNA序列。推测的成熟肽为两亲性阳离子肽,等电点12.48,Schiffer-Edmunson预测其二级结构呈α螺旋构型。将推测的成熟肽序列亚克隆至pET-32a构建融合表达载体pET-32a-pis（EF）,并在大肠杆菌E.coliOrigami（DE3）中成功表达出融合蛋白,以1mmol/ml IPTG于30℃进行诱导表达,4 h可获得大量表达,目的蛋白以包涵体形式存在。A 388bp cDNA sequence with complete open reading frame was amplified by RT-PCR with degenerate primers from leucocytes of Epinephelus fuscoguttatus,an important commercial marine fish species.Blast analysis preliminarily suggested that the obtained sequence,which was named as Epinephelus fuscoguttatus piscidin-like peptide（GU592793）,is a member of fish antimicrobial peptide piscidin family.The deduced amino acid sequence was found to have the common features for defining the fish piscidin,including：（1） signal peptide sequence had 60%~95% similarity to other piscidins;（2） six amino acid residues in N-terminal part of the putative mature peptide are rich in isoleucine,phenylalanine,and histidine;（3） the amino acid residues both at position 8 and 13 of the mature peptide are glycines.The phylogenetic neighbor-joining tree showed that the present sequence cluster within the clade of piscidin-like peptide of Epinephelus akaara and Epinephelus coioides（ACE78290,ACE78291） with 81% bootstrap support.Accordingly,the first piscidin precursor cDNA from Epinephelus fuscoguttatus was amplified and reported here.The present putative mature peptide is an amphiphilic and cationic peptide with the calculated isoelectric points of 12.48.A Schiffer-Edmunson plot suggested the peptide had the potential to form an amphipathic α-helix.The prokaryotic fusion expression vector of predicted mature peptide（pET-32a-pis（EF）） was constructed and expressed in E.coli Origami（DE3）.The fusion protein containing the present mature peptide was efficiently expressed in the form of inclusion body after 1mmol/ml IPTG induction at 30℃ for 4h.

关 键 词：抗菌肽 Piscidin 基因克隆 原核表达 棕点石斑鱼 

分 类 号：S917[农业科学—水产科学]