Study On Swine Hepcidin Gene Expression In Pichia Pastoris

Hepcidin is a liver-expressed,low-molecular-weight, highly disulfide bonded peptide, which exhibits obvious antibacterial and antifungal activity. Expect for its antimicrobial function, Hepcidin plays an important role in regulating iron absorption in small intestine and body iron homeostasis. Recently, its potential medical value has become a hot research topic. Althouth Hepcidin can be separated from cells, its native concentration is very low. In addition, the chemical synthesis of Hepcidin is very difficult. So it is necessary to produce enough Hepcidin through DNA recombinant technique. Up to the present, the expression of recombinant Hepcidin from porcine in eukaryotes has not been reported.Pichia pastoris (P. pastoris) is currently one of the most widely systems to be used to express heterologous protein. P. pastoris is easy to culture and convenient to manipulate, and its post-translational processing capability of eukaryotic proteins is also attractive. Futhermore, it has interesting characteristics of high expression, high stability and high secretion, and so on.According to codon preference in yeast and amino acid sequence of Hepcidin, S-Hepcidin was designed and synthesized for coding pig Hepcidin. It was connected with pGAPZaA plasmid by ligase to construct a eukaryotic expression vector-pGAPZaA-Hepcidin. After transformed into DH5a, the recombinant was confirmed by PCR detection and enzymes digested by EcoR I and Xba I. DNA sequencing revealed that the correct insert of Hepcidin into pGAPZaA was confirmed. The recombinant plasmid was linearized by Bln I and transformed into Pichia pastoris (X-33) by electroporation. The target colonies were selected by zeocin and PCR detection. In order to improve the expression, the high copy inserts were selected by different dose of zeocin. Tricine-SDS-PAGE showed that Hepcidin peptide was successfully expressed as secretory protein at molecular weight of2.76kDa in P. pastoris.Bacteriostasis experiment indicated that the expression supernatant could inhibit growth of Staphyloccocus aureus and Bacillus subtilis. Therefore, the study provided a basis for veterinary drugs and feed additives.