Method for expressing antibacterial peptide apidaecin by using Escherichia coli and for preparing antibacterial peptide apidaecin

A technology of Escherichia coli and antibacterial peptides, applied in the field of genetic engineering, can solve the problems of low expression, high cost, and low absolute yield, and achieve the effects of simple culture procedures, improved stability, and low production costs

Active Publication Date: 2015-07-29
福建旭牧联生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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  • Method for expressing antibacterial peptide apidaecin by using Escherichia coli and for preparing antibacterial peptide apidaecin
  • Method for expressing antibacterial peptide apidaecin by using Escherichia coli and for preparing antibacterial peptide apidaecin
  • Method for expressing antibacterial peptide apidaecin by using Escherichia coli and for preparing antibacterial peptide apidaecin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction of pET-polh-AP expression vector

[0025] Chemically synthesize the AP2 gene fragment, its nucleotide sequence is as described in SEQ ID NO.1, the amino acid sequence of the AP2 protein expressed by the gene is as described in SEQ ID NO.2, and an EcoR I site is added at its 5' end during synthesis And hydroxylamine cleavage site coding sequence (Asn-Gly), 3' end added Xho I site. like figure 1 As shown, the above fragment was inserted into pUC57 to obtain the recombinant plasmid pUC57-AP containing the AP gene (provided by Shenggong). The polyhedron gene sequence polh (Gene ID: 7872889) was obtained by PCR cloning from the BmNPV genome, and the forward primer for PCR cloning was 5'-AAC GAATTC ATATGCCGGATTATTCATAC-3' (Nco I), reverse primer is 5'-TCGT GAATTC ATACGCCGGACCAGTG-3' (EcoR I).

[0026] The amplification system is:

[0027]

[0028] The amplification conditions are: pre-denaturation at 94°C for 5 minutes, and then enter the...

Embodiment 2

[0032] Embodiment 2: Expression of polh-AP fusion protein

[0033] The expression vector pET-polh-AP obtained in Example 1 was transformed into E. coli BL21 competent cells, and cultured overnight at 37°C on LB culture plates containing kanamycin (50 μg / ml). Pick monoclonal plaques, place them in 5ml LB culture tubes, and culture them with shaking at 37°C for 10h, as the seeds of expression culture. According to the scale-up culture of 1:100, the conditions were 37°C and 150rpm shaking culture. When the OD600 reached 0.6, IPTG with a final concentration of 1mM was added to induce the expression of the recombinant protein polh-AP. 37°C, 150rpm shaking culture for 5h to end the expression, and SDS detection, the results are as follows figure 2 As shown, lane 1 is the expression profile 5h after IPTG induction, lane 2 is the expression profile without induction, and lane 3 is the expression profile of Mark protein, where a is the polh-AP fusion protein.

Embodiment 3

[0034] Embodiment 3: Purification and cracking of polh-AP fusion protein

[0035] Centrifuge at 5000rpm for 10min at 4°C to collect the E.coliBL21 cells expressing polh-AP fusion protein obtained in Example 2, resuspend in PBS (pH7.8), sonicate and centrifuge at high speed (15000rpm, 20min) to separate insoluble inclusion bodies . The resulting precipitate was then resuspended with PBS (pH9), shaken on ice to dissolve for 30 minutes, and the insoluble inclusion bodies were collected by high-speed centrifugation (15,000 rpm, 20 minutes). Finally, adjust the pH of the protein solution to pH 7.8 with 1M hydrochloric acid, let it stand for 2 hours, and centrifuge (15000rpm, 20min) to collect the precipitated protein.

[0036] Dissolve the obtained recombinant protein sample with PBS (pH12) to a final concentration of 15mg / ml, mix the recombinant protein solution with 3 times the volume of hydroxylamine lysis reaction buffer, and incubate at 55°C for 24h. Under these conditions, P...

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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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/62C07K19/00C07K7/08
Inventor 曹翠平李卫芬相兴伟陈琳吴小锋
Owner 福建旭牧联生物科技有限公司
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