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Method for fusion expression of antibacterial peptide by using SUMO

A technology of antimicrobial peptides and fusion proteins, 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 reduce toxicity and improve folding Effect of rate and expression yield

Active Publication Date: 2021-03-09
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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

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  • Method for fusion expression of antibacterial peptide by using SUMO
  • Method for fusion expression of antibacterial peptide by using SUMO
  • Method for fusion expression of antibacterial peptide by using SUMO

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0026] The invention uses 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 with LL-37, LLv has increased stability and elongated 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 has increased thermostability compared with LL-37. Antimicrobial peptide LLv has certain activity at pH 2.0-12.0. The antibacterial activity did not change significantly after boiling water bath for 40 minutes, and the structure and antibacterial activity of antimicrobial peptide LLv could not be destroyed under high pressure at 121°C for 21 minutes.

Embodiment 2

[0028] Embodiment 2: Physicochemical property analysis antibacterial test of antimicrobial peptide LLv

[0029] 1. Use the dilution method antibacterial test to detect the antibacterial performance of the antimicrobial peptide LLv. The specific steps are as follows:

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

[0031] 2) Determination by microdilution method

[0032] Take a sterile 96 polystyrene microwell plate test tube, add the antimicrobial peptide solution sterilized by filtration to the MH broth medium in sequence, so that the final concentrations of the first hole to the tenth hole are 276 ...

Embodiment 3

[0041] Embodiment 3: the scanning electron microscope examination of the antimicrobial peptide LLv processed bacterium

[0042]Incubate the antimicrobial peptide LLv at a concentration of 2 μg / ml with Escherichia coli ATCC25922 at 37°C for 60 minutes, then collect the bacterial solution, centrifuge at 3000 r / min for 10 minutes, three times, discard the supernatant, and replace with 0.06 mol / L phosphate buffer (PH7.2) rinse 3 times, retain the precipitate, add 2.5% glutaraldehyde to make bacterial suspension, fix at 4°C for 4h, centrifuge at 3000r / min, 10min each time, three times. The detailed method of preparing samples of normal bacteria control group is the same as that of antimicrobial peptide group. Finally, PBS was used to suspend the bacteria to make a suspension. The bacteria were then allowed to sink freely, dried, and sputtered with gold under vacuum. Finally, the bacteria structure was observed by scanning electron microscope, and the working voltage was 10KV.

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Abstract

The invention provides a method for fusion expression of an antibacterial peptide by using SUMO. According to the method, ubiquitin-like protein modified molecules SUMO and the antibacterial peptide are prepared into a fusion protein, so that the antibacterial peptide is more effectively recombined and expressed. The amino acid sequence of the antibacterial peptide is SEQ ID NO: 1. The antibacterial peptide LLv with relatively strong antibacterial activity is expressed in escherichia coli by adopting a sumo fusion expression method. By virtue of fusion with sumo, the toxicity to host bacteriais reduced, the folding rate and the expression yield are increased, and soluble expression is successfully realized.

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 antimicrobial peptides by using SUMO fusion. Background technique [0002] Antimicrobial peptides are small molecular substances with certain immune effects 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 amphipathic and cationic characteristics in the 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 cause cell death. [0003] In recent yea...

Claims

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Application Information

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Patent Type & Authority Applications(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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