Hybrid peptide expressed via Bacillus subtilis, and preparation method and application thereof

A Bacillus subtilis, hybrid peptide technology, applied in hybrid peptides, biochemical equipment and methods, cationic antibacterial peptides, etc., can solve the problems of difficult purification and separation, inactivity, etc., achieve high cell selection, increase solubility, promote The effect of protein expression

Inactive Publication Date: 2017-03-15
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the antimicrobial peptide expression system, the Escherichia coli expression system occupies an important position, but there are also some disadvantages, such as the formation of inactive inclusion bodies of secreted proteins, and the secretion contains cell wall components of Gram-negative bacteria such as pyrogenic lipopolysaccharide, which is harmful to the antibacterial peptide expression system. Difficulties in later purification and separation

Method used

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  • Hybrid peptide expressed via Bacillus subtilis, and preparation method and application thereof
  • Hybrid peptide expressed via Bacillus subtilis, and preparation method and application thereof
  • Hybrid peptide expressed via Bacillus subtilis, and preparation method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0026] The gene synthesis of embodiment 1 hybrid peptide PR-FO

[0027] Antimicrobial peptide PR-FO is a peptide obtained by hybridization of PRW4 and antimicrobial peptide α-helical Fowlicidin-2. PRW4 was obtained by replacing the seventh and eleventh lysines in the truncated linear PMAP-36's 16 N-terminal amino acid residues with tryptophan. Fowlicidin-2 (FO) is an α-helical antibacterial peptide extracted from chicken bone marrow cells. The hybrid peptide PR-FO is obtained by hybridizing two antibacterial peptides. Its amino acid sequence is RFRRLRWKTRWRLKKIRFGRFLRKIRRFRPK. Artificially design the gene fragment encoding PR-FO, and add EcoRI restriction site and signal peptide SP at the 5' end amyQ (SP sacB ), 6×His tag, SUMO, plus a stop codon and a BamHI restriction site at the 3’ end. Gene synthesis was completed by Shanghai Sangon Bioengineering Technology Service Company.

Embodiment 2

[0028] Construction of embodiment 2 expression plasmid

[0029] The target gene and the expression vector pGJ148 plasmid were digested with EcoRI and BamHI respectively, and then subjected to agarose gel electrophoresis, and the gel containing the target band was excised and recovered with a gel recovery kit. The recovered target gene and expression vector fragments were connected with T4 ligase, and the two constructed plasmids pGJ148-(SP AmyQ )SUMO-PR-FO and pGJ148-(SP SacB ) SUMO-PR-FO were respectively transformed into Bacillus subtilis competent cells prepared in advance. The sequencing results of the expression plasmids were as follows: figure 1 , figure 2 shown.

Embodiment 3

[0030] Example 3 induced expression

[0031] Single colonies of positive recombinants were picked and inoculated in 10 mL of LB medium containing 10 μg / mL of chloramphenicol and 10 μg / mL of neomycin, and cultured with shaking at 37 °C and 225 r / m overnight. Transfer to 50mL fresh LB medium according to 1% inoculum amount, shake culture under the condition of 37°C and 225r / m. After 3 hours of fermentation, maltose with a final concentration of 1%, 3%, 5%, 7%, and 9% was added to induce expression, and the bacterial supernatant was collected after 48 hours. Two plasmids pGJ148-(SP AmyQ )SUMO-PR-FO and pGJ148-(SP SacB ) SUMO-PR-FO optimum fermentation maltose concentrations were 3% and 5%.

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Abstract

The objective of the invention is to provide a hybrid peptide expressed via Bacillus subtilis, and a preparation method and application thereof. The sequence of the hybrid peptide is as shown in SEQ ID No. 1 in a sequence table. The preparation method comprises the following steps: artificially designing a fusion gene of SP<AmyQ>-SUMO-PR-FO and SP<SacB>-SUMO-PR-FO; inserting EcoRI and BamHI enzyme sites at two terminals; linking a target gene with an expression vector pGJ148; transferring a plasmid into the competent cell of Bacillus subtilis by using a chemical approach and inducing fermentation via maltose so as to produce a fusion expression product; and carrying SUMO digestion so as to obtain the hybrid peptide PR-FO. The antibacterial peptide PR-FO is obtained by subjecting PRW4 and alpha-helical antibacterial peptide fowlicidin-2 to heterozygosis and presents strong bactericidal activity and high stability and cell selectivity compared with the original peptide PRW4.

Description

technical field [0001] The invention relates to a hybrid peptide expressed by Bacillus subtilis and its preparation method and application. Background technique [0002] There is no doubt that the invention and use of antibiotics is a milestone in the history of human science. In the early days, the therapeutic effect of antibiotics was very good, and at the same time it brought great benefits to the livestock industry. However, the abuse of antibiotics has led to the emergence of many drug-resistant strains and residues in the body at the same time, which will cause a series of safety problems. Therefore, people have to re-examine antibiotics. Therefore, it is imminent to develop a new drug that can replace antibiotics without causing these safety problems. Antimicrobial peptides (AMPs) are just this new type of drug, which has attracted the attention of scientists because of its little or no drug resistance and no residue in the body. [0003] The antimicrobial peptide...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/75A61K38/17A61P31/04
CPCC07K14/465A61K38/00C07K14/4723C07K2319/02C07K2319/21C12N15/75C12N2800/101
Inventor 单安山魏丹丹李晓丹
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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