点带石斑鱼抗菌肽hepcidin基因克隆及其成熟肽的原核融合表达  

Cloning and expression of antimicrobial peptide hepcidin from Epinephelelus malabaricus

作　　者：陈大玮[1] 邓利[1] 李荔[1] 彭永鹤 刘志刚[3] 

机构地区：[1]深圳大学生命科学学院,广东深圳518060 [2]深圳市圣西马生物技术有限公司,广东深圳518057 [3]深圳大学医学院,广东深圳518060

出　　处：《热带医学杂志》2011年第2期117-121,共5页Journal of Tropical Medicine

基　　金：国家863项目(2006AA100308);深圳市科技计划项目(200326);深圳大学科技创新团队项目(200904)

摘　　要：目的克隆点带石斑鱼hepcidin前体cDNA序列,构建其成熟肽pET-32a融合表达载体。结论根据已报道的硬骨鱼类hepcidin cDNA序列设计简并引物,以点带石斑鱼(Epinephelelus malabaricus)肝脏为材料,通过RT-PCR扩增hepcidin cDNA序列,用比对推导的氨基酸序列预测其成熟肽段。再将成熟肽cDNA序列亚克隆至pET-32a构建原核表达并进行原核融合表达。结果获得点带石斑鱼hepcidin-like抗菌肽前体cDNA序列1条,GenBank登录号为HM474788。DNA序列的Blast分析以及推导氨基酸序列NJ进化树分析表明,HM474788是点带石斑鱼第1条报道的抗菌肽hepcidin cDNA序列。成功构建了该序列的成熟肽原核融合表达载体pET-32a-hepcidin(EM1),并成功在大肠杆菌origami(DE3)中表达。结论成功克隆点带石斑鱼hepcidin-like抗菌肽前体cDNA序列,成熟肽成功在大肠杆菌中融合表达,为后续有关石斑鱼hepcidin的功能研究与实践应用奠定重要基础。Objective This project aims to perform molecular cloning of hepcidin precursor from Epinephelelus malabaricus and to construct the fusion expression vector pET-32a-hepcidin. Methods Total RNA was prepared from the liver of Epinephelelus malabaricus. The hepcidin precursor was amplified by RT-PCR with degenerate primers based on the homology of the previously reported hepcidins in teleost.The mature peptide was predicted from the deduced amino acid sequence.The cDNA sequence of the mature peptide was subcloned into the expression vector pET-32a. Results The hepcidin-like antimicrobial peptide precursor cDNA of Epinephelelus malabaricus was amplified and sequenced. The sequence of Epinephelelus malabaricus was submitted to GenBank and an accession number HM474788 was assigned.Blast analysis with sequence of DNA and the phylogenetic neighbor-joining tree of the deduced amino acid sequence suggested that sequence of HM474788 is the first reported hepcidin-like antimicrobial peptide cDNA of Epinephelelus malabaricus. The predicted mature peptide of this sequence was subcloned intto pET-32a to construct a prokaryotic fusion expression vector pET-32a-hepcidin（EM1）, which was expressed in E.coli （DE3）. Conclusion The hepcidin-like antimicrobial peptide precursor cDNA was cloned. The fusion mature hepcidin peptide was expressed in E.coli. The present results laid the foundation for the future functional study and application of grouper hepcidin in aquaculture.

关 键 词：点带石斑鱼 抗菌肽 HEPCIDIN 分子克隆 原核融合表达 

分 类 号：R392.11[医药卫生—免疫学]