Method for expressing antimicrobial peptide apidaecin and preparing antimicrobial peptide apidaecin using Escherichia coli

Abstract

The invention relates to a method for expressing an antibacterial peptide apidaecin by using Escherichia coli and for preparing the antibacterial peptide apidaecin. The method comprises the following steps: cloning AP2 gene and a polyhedron gene sequence polh into a recombinant vector in order to form a recombinant vector with a polh-AP fusion gene fragment; cloning the polh-AP fusion gene fragment into an expression vector, converting the expression vector to an expression host cell, and culturing the expression host cell to express a fusion protein with an antibacterial peptide AP2; and culturing the expression host cell to induce the expression of the recombinant protein polh-AP, centrifuging induced bacterial strains, collecting, re-suspending, carrying out ultrasonic fragmentation, centrifuging, collecting the above obtained insoluble inclusion body, purifying, and collecting a recombinant protein sample. An expression system has the characteristics of simple expression system culture program, low production cost, efficient expression of the antibacterial peptide apidaecin, and realization of AP2 and polh fusion expression, so compared with the prior art, the method has the advantages of effective reduction of toxicity of the AP2 to Escherichia coli, improvement of the stability of the antibacterial peptide, realization of high level expression of the antibacterial peptide, convenient purification of the active antibacterial peptide, and improvement of the output of the antibacterial peptide apidaecin.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for expressing and preparing the antimicrobial peptide apidaecin through Escherichia coli. Background technique [0002] In recent years, the massive abuse of antibiotics has led my country's animal husbandry industry to a vicious circle, seriously threatening our food safety and human health. Antimicrobial peptides are a class of small molecular peptides that are encoded between organism genes and can resist external pathogenic infections. They have stable physical and chemical properties, no immunogenicity, and a wide antibacterial spectrum. Their antibacterial activity and antibacterial effect are very close to those of antibiotics. , showing great potential in replacing antibiotics. Apidaecin is a class of proline-rich antimicrobial peptides. It has attracted much attention due to its unique antibacterial mechanism. Studies have shown that apidaecin can specifical...

AI Technical Summary

Problems solved by technology

The above expressions all have the problems of low expression volume and high cost, which cannot meet the requirements of practical application

[0004] Escherichia coli expression system is a relatively mature prokaryotic expression system, and apidaecin protein can also be expressed in fusion in Escherichia coli expression system, but even with fusion expression, apidaecin in the fusion state still shows toxicity to the host, and the higher the expression level of apidaecin Higher, the more restricted the growth of the host cell, even the apidaecin expressed at the background level will pose a threat to the growth of the host cell. In addition, the apidaecin molecule is small, and the protease degradation of the host cell leads to a low absolute yield, and it is overexpressed in E. coli It exists in the form of inclusion bodies, so its renaturation and purification are also difficult problems

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