Expression product in series of two fish antibacterial peptide genes and expression method thereof

Abstract

The invention discloses an expression product in series of two fish antibacterial peptide genes and an expression method thereof, relating to the gene engineering technology of marine organism, providing an expression product in series of the two fish antibacterial peptide genes and construction of an expression vector in series of two fish Hepcidin and a preparation method for the expression product in series. In the invention, two fish hepcidin genes are expressed in series by a pET32a prokaryocyte expression vector to realize the soluble expression of two antibacterial peptide hepcidin at the same time; the expression level of two antibacterial peptides in series is over 50mg / L, the soluble expression product in series is purified by a N-terminal His-Tag affinity chromatography, and then cuts off a protein label in series by enterokinase; and the cut mixture is purified by a Ni affinity chromatographic column to obtain a purified Hepcidin expression product in series. The purified expression product has obvious inhibiting effect on Staphylococcus aureus, Aeromonas hydrophila, micrococcus lysodeikticus and the like.

Description

technical field [0001] The present invention relates to a kind of genetic engineering technology of marine organisms, in particular to a method of serially connecting the genes of large yellow croaker antibacterial peptide hepcidin (abbreviated as PC-hepc) and black sea bream antibacterial peptide hepcidin (abbreviated as AS-hepc2) with antibacterial activity Expression, and activity identification of in vitro expression products. Background technique [0002] Antimicrobial peptides are a class of small molecular polypeptides with broad-spectrum antibacterial activity produced by animal and plant cells. Their production and release are part of the body's inflammatory response and an important barrier for the host to defend against the invasion of pathogenic microorganisms such as bacteria and fungi. Hepcidin (hepcidin) is a family of antibacterial peptides with a β-sheet structure that is rich in cysteine ​​residues at the C-terminal conservative site, and participates in ho...

AI Technical Summary

Problems solved by technology

In 2000, Krause et al. first discovered LEAP-1 (an antimicrobial peptide expressed in the liver, later called hepcidin) from human plasma, and tested the antibacterial activity of hepcidin by chemically synthesizing the polypeptide, and found that hepcidin can dose-dependently inhibit Growth of the gram-positive bacteria Micrococcus luteus, Staphylococcus carnosus, Bacillus megaterium, Bacillus subtilis and the gram-negative bacteria Neisseria griseus, but no activity against Escherichia coli and Pseudomonas fluorescens

Zhang et al. ([3] Zhang H, Yuan QP, Zhu YP, Ma RY. Expression and preparation of recombinant hepcidin in Escherichia coli [J]. Protein. Expr. Purif., 2005, 41: 409-416) used synthetic human hepcidin The nucleic acid fragment is recombined into the pGEX-hpc expression vector, and the fusion protein is expressed in Escherichia coli, but the expression product exists in the form of inclusion bodies, and it needs to undergo a renaturation process to obtain biologically active hepcidin, so pGEX-hpc is used The process of preparing hepcidin with expression vector is relatively complex and costly

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