关键词: Hepcidin, 成熟肽, cDNA克隆, 真核表达, 毕赤酵母

Abstract: Hepcidin is a small cysteine-rich cationic antimicrobial peptide with the regulative function for iron metabolism. It is expressed predominantly in the liver of animals. Hepcidin plays an important role in the host’s immune response against microbial invasion. Thus, it is considered to be good substitutes for traditional antibiotics. TH1-5, one of the three different hepcidin cDNAs from tilapia（Oreochromis mossambicus）. The cDNA encoding hepcidin mature peptide（mTH）containing 22 residues was cloned from hepcidin full-length cDNA of tilapia（Oreochromis niloticus）liver by PCR. The forward and reverse primers were designed with reference to the nucleotide sequence of #br#O. mossambicus hepcidin TH1-5. This cDNA fragment carrying Xba I and Xho I sites was inserted into pGAPZ-A plasmid with the same restriction sites to construct a recombinant expression plasmid “pGAPZ-A-mTH”. The colony PCR, Xba I and Xho I restriction endonuclease digestion and DNA sequencing demonstrated that the “pGAPZ-A-mTH” recombinant expression plasmid was constructed successfully. In addition, the recombinant expression plasmid was transformed into Pichia pastoris GS115 by an electroporation system. PCR using yeast DNA as template demonstrated that the recombinant expression plasmid was inserted into the yeast chromosome.

Key words: Hepcidin, Mature peptide, cDNA cloning, Eukaryotic expression, Pichia pastoris