三疣梭子蟹(Portunus trituberculatus)Ⅰ型Crustin抗菌肽的基因克隆与真核重组表达

cDNA CLONING OF TYPE I CRUSTIN FROM THE BLUE CRAB PORTUNUS TRITUBERCULATUS AND CONSTRUCTION OF THE RECOMBINANT EUKARYOTIC EXPRESSION SYSTEM

Crustin是目前报道广泛存在于甲壳类中的一类具有广谱抗菌活性的抗菌肽,主要由甲壳类血淋巴细胞合成。本研究采用RACE技术和真核表达技术,进行了三疣梭子蟹Crustin基因的克隆和重组表达研究。结果表明,在三疣梭子蟹血细胞Crustin基因cDNA全序列中,开放阅读框编码110个氨基酸残基的Crustin前体,Crustin前体由N端21个氨基酸残基的信号肽、富含Cys结构域和C端WAP结构域三部分组成,属于I型Crustin,构建的分泌型真核表达质粒pVT102U/α-crustin转化酿酒酵母S78,经RT-PCR和SDS-PAGE电泳验证,表明重组Crustin表达成功。

Crustin are a group of antimicrobial peptides （AMPs） ubiquitously found in crustaceans.In this study,a full-lenth cDNA of type I Crustin had been cloned from blue crab Portunus trituberculatus by the RACE technology.This cDNA contains an open reading frame of 330bp encoding a precursor of 110 amino acids;21 of which consist of the signal peptide,and the remaining 89 amino acids the mature peptide.The putative mature peptides consist of two different domains： a Cys-rich region at the N-terminus and a WAP domain at the C-terminal region.The secretive eukaryotic expression plasmid pVT102U/α-Crustin was constructed and transformed into Saccharomyces cerevisiae S-78.The recombinant Crustin was expressed successfully in S.cerevisiae S-78 as confirmed by RT-PCR and SDS-PAGE analysis.