| 摘要: |
| 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。
|
| 关键词: 三疣梭子蟹, 抗菌肽, I 型Crustin, cDNA 克隆, 真核表达 |
| DOI：10.11693/hyhz201003012012 |
| 分类号: |
| 基金项目:国家科技支撑计划项目, 2007BAD43B08; 浙江省重大科技专项重大项目, 2007C02001; 浙江省面上项目, 2009C32019 号 |
|
| cDNA CLONING OF TYPE I CRUSTIN FROM THE BLUE CRAB PORTUNUS TRITUBERCULATUS AND CONSTRUCTION OF THE RECOMBINANT EUKARYOTIC EXPRESSION SYSTEM |
|
SHEN Wang, YE Mao, SHI Ge, WANG Ri-Xin
|
|
Laboratory of Marine Biology Resources and Molecular Engineering, Ocean Science College,Zhejiang Ocean University
|
| Abstract: |
| 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.
|
| Key words: Portunus trituberculatus, Antimicrobial peptide, Type I Crustin, cDNA cloning, Eukaryotic expression |
