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A method for expressing antibacterial peptides by using sumo fusion

A technology of antimicrobial peptides and fusion proteins, which is applied in cationic antimicrobial peptides, hybrid peptides, fusion with degradation motifs, etc., can solve problems such as degradation, small molecular weight of antimicrobial peptides, and toxicity of host bacteria, so as to improve folding rate and reduce Toxicity, the effect of increasing expression yield

Active Publication Date: 2022-06-28
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the genetic engineering expression of antimicrobial peptides is currently facing two problems. One is that the sterilization of antimicrobial peptides is toxic to the host bacteria, which is not conducive to the normal growth of bacteria; degraded

Method used

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  • A method for expressing antibacterial peptides by using sumo fusion
  • A method for expressing antibacterial peptides by using sumo fusion
  • A method for expressing antibacterial peptides by using sumo fusion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: Design and Screening of Antimicrobial Peptides

[0026] The present invention takes the antibacterial peptide LL-37 as the starting polypeptide, and designs a new antibacterial peptide LLv by adding hydrophobic residues and positively charged amino acids. Compared to LL-37, LLv increases stability and elongates the α-helical structure. The amino acid sequence of the modified antimicrobial peptide is as follows (SEQ ID NO: 1): LLGDFFKKSKEK IGKEFKRIVQRIKDFLRNLVWKTEK.

[0027] By adding hydrophobic residues and positively charged amino acids, the new antimicrobial peptide LLv increases thermal stability compared to LL-37. Antibacterial peptide LLv has certain activity at pH 2.0~12.0. The antibacterial activity did not change significantly after 40min of boiling water bath, and the structure and antibacterial activity of antimicrobial peptide LLv could not be destroyed by high pressure at 121℃ for 21min.

Embodiment 2

[0028] Example 2: Analysis of physicochemical properties of antimicrobial peptide LLv Antibacterial test

[0029] 1. The antibacterial properties of the antimicrobial peptide LLv were detected by the dilution method antibacterial test. The specific steps are as follows:

[0030] 1) Bacterial liquid preparation, inoculate experimental bacteria (Escherichia coli ATCC25922, avian-derived Escherichia coli O1, O2, Pasteurella, Bacillus subtilis, Staphylococcus aureus ATCC25923) into MH broth, place a shaker incubator, and incubate at 37°C 12-18h, so that the bacteria are in the logarithmic growth phase. Dilute the bacterial solution with sterile normal saline to the desired bacterial count.

[0031] 2) Determination by microdilution method

[0032] Take a sterile 96 polystyrene microplate test tube, and add the filter-sterilized chromatographically purified antimicrobial peptide solution to the MH broth medium in turn, so that the final concentrations of the first to the tenth we...

Embodiment 3

[0041] Example 3: Scanning Electron Microscopy of Antimicrobial Peptide LLv Treated Bacteria

[0042]The antimicrobial peptide LLv at a concentration of 2 μg / ml was incubated with Escherichia coli ATCC25922 for 60 min at 37°C, then the bacterial liquid was collected and centrifuged at 3000 r / min for 10 min for three times, the supernatant was discarded, and 0.06 mol / L phosphate buffer (PH7.2) rinsed 3 times, retained the precipitate, added 2.5% glutaraldehyde to make a bacterial suspension, fixed at 4°C for 4 h, and centrifuged at 3000 r / min, 10 min each time, three times. The detailed method for the preparation of normal bacteria control samples is the same as that of the antimicrobial peptide group. Finally, the bacterial suspension was dispersed with PBS to prepare a suspension. The bacteria were then allowed to sink freely, dried, and sputtered with gold under vacuum. Finally, the bacterial structure was observed by scanning electron microscope, and the working voltage w...

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Abstract

The present invention provides a method for expressing antimicrobial peptides through fusion of SUMO, that is, the ubiquitin-like protein modification molecule SUMO and antimicrobial peptides are made into a fusion protein, so that the antimicrobial peptides can be recombined and expressed more effectively. The amino acid sequence of the antimicrobial peptide is SEQ ID NO:1. The invention adopts the sumo fusion expression method to express the antibacterial peptide LLv with strong antibacterial activity in Escherichia coli. By fusing with sumo, the toxicity to the host bacteria is reduced, the folding rate and expression yield are improved, and soluble expression is successfully achieved.

Description

technical field [0001] The invention belongs to the technical field of preparation and application of biological products, and in particular relates to a method for expressing antibacterial peptides by using SUMO fusion. Background technique [0002] Antimicrobial peptides are small molecular substances with a certain immune function extracted from the tissues and cells of various organisms such as insects, tunicates, amphibians, birds, fish, mammals, plants and even humans. Do peptide antibiotics or antimicrobial peptides. Its unique amino acid composition and the amphiphilic and cationic characteristics of its structure enable the polypeptide to combine with macromolecules in the nucleus such as nucleic acids, proteins, etc., as well as negatively charged components on the surface of viruses or bacteria, thereby destroying the cell membrane structure or intracellular macromolecules. molecules that disrupt the normal function of cells and lead to cell death. [0003] In r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70
CPCC07K14/4723C12N15/70C07K2319/95
Inventor 刘晓东刘旭董旭峰王述柏秦志华
Owner QINGDAO AGRI UNIV
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