Expression carrier for black porgy antibiotic peptide Hepcidin and expression product and constructing preparation method

Abstract

The invention discloses an expressing carrier of black porgy antibiotic peptide Hepcidin, expressing product and preparing method, which is characterized by the following: incorporating E. coliTrc promoter, protein project improving CKS gene and procaryotic expressing carrier pTrc-CKS of histidine tag (His-Tag); connecting to black porgy Hepcidin gene; constructing pTrc-CKS / hepcidin expressing plasmid; possessing P3C enzyme cutting site; fusing six histidine on C end; getting CKS-hepcidin; purifying through C end His-Tag affinity chromatography; getting the purified product; cutting CKS with P3C enzyme in fuse protein; getting the purified Hepcidin.

Description

technical field [0001] The invention relates to fish gene engineering in the field of biotechnology, in particular to the construction of an expression vector of black sea bream antibacterial peptide Hepcidin with antibacterial activity and its preparation method. Background technique [0002] Hepcidin is a family of antimicrobial peptides rich in cysteine ​​residues at the C-terminal conserved site. Their C-terminals all retain the enrichment region formed by cysteine, and CD (circular dichroism, circular dichroism) spectroscopic characteristics studies have confirmed that hepcidin has two stable β-sheet structures in phosphate buffer. It is very similar to the cystine (cystin-eknot) structure of the antibacterial polypeptide. Cysteine-rich antimicrobial peptides have been isolated from fat bodies of insects, hemolymph of molluscs and crustaceans, epithelial cells of mammals, and circulating cells such as neutrophils and macrophages. These antimicrobial peptides have acti...

AI Technical Summary

Problems solved by technology

2000, Krause et al(Krause A, Neitz S, MagertHJ, Schulz A, Forssmann WG, Knappe PS, Adermann K. LEAP-1, a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity[J]. FEBS Lett. , 2000, 480: 147-150) first discovered LEAP-1 (later called hepcidin) from human plasma. The antibacterial activity of hepcidin was detected by chemically synthesizing the polypeptide, and it was found that hepcidin can dose-dependently (Dose- dependent) against Gram-positive bacteria Micrococcus luteus, Staphylococcus carnosus, Bacillus megaterium, B. subtilis and Gram-negative bacteria Gray Nai growth of Neisseria cinerea, but no activity against Escherichia coli (E.coli BL21 G-) and Pseudomonas fluorescens G-, the half inhibitory concentration (half- maximal inhibitory activity, IC50) is 40μg / ml (14.4μM)

[0004] Since the hepcidin polypeptide contains 8 cysteines and has a special structure of 4 disulfide bonds, the chemical synthesis of the polypeptide is very difficult and expensive, so it cannot meet the requirements of industrialization. The feasible and effective way is to pass the gene in vitro Engineering bacteria express this antimicrobial peptide, but there are technical difficulties and risks, so there are few research reports on this aspect

Zhang et al (Zhang H, Yuan QP, Zhu YP, Ma RY. Expression and preparation of recombinanthepcidin in Escherichia coli [J]. Protein. Expr. Purif., 2005, 41: 409-416.) once 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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