Application of modified peptide of human α-defensin 5 in the preparation of neutralizing endotoxin medicine

A defensin and endotoxin technology, applied in the biological field, can solve the problem of inability to neutralize endotoxin

Active Publication Date: 2020-09-08
ZHEJIANG LONGTERM MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have confirmed that oxidized HD5 can neutralize some exotoxins, such as protein toxins of Vibrio cholerae (Immunity.2014,41(5):709-21.), but cannot neutralize endotoxins (SciRep.2016, 6:22875.)
In the previous research, our research group modified human α-defensin 5, and modified the C-terminus of human α-defensin 5 with myristic acid, which significantly enhanced its antibacterial activity, especially against methicillin-resistant Staphylococcus aureus (patent document CN 106146643A), but it has not been reported whether it plays a role in neutralizing endotoxin

Method used

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  • Application of modified peptide of human α-defensin 5 in the preparation of neutralizing endotoxin medicine
  • Application of modified peptide of human α-defensin 5 in the preparation of neutralizing endotoxin medicine
  • Application of modified peptide of human α-defensin 5 in the preparation of neutralizing endotoxin medicine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Effect of HD5-myr on endotoxin LPS-mediated inflammatory response

[0028] (1) The macrophage cell line Raw264.7 was planted in a 24-well plate, 5×10 per well 5 cells.

[0029] (2) Mix 10 μl of LPS at a concentration of 50 ng / ml with 10 μl of HD5 or HD5-myr at a concentration of 100 μg / ml to form a 20 μl system, and incubate at 37° C. for 30 minutes.

[0030] (3) Add the above system into a 24-well plate, so that the final volume in the 24-well plate is 400 μl. After adding the sample, place the 24-well plate at 37°C with 5% CO 2 In a constant temperature cell incubator, cultured for 3 h.

[0031] (4) After 3 hours, take the following operations: ①Pipe the cell supernatant into a 1.5ml EP tube, and detect the content of tumor necrosis factor α (tumor necrosis factor α, TNF-α) in the cell supernatant by ELISA method; ②The cells in the 24-well plate Wash 3 times with phosphate buffered saline, add 1ml TRIzol to each well, pipette evenly, aspirate and place in 1.5ml EP...

Embodiment 2

[0035] Improvement effect of HD5-myr on endotoxin LPS-induced acute lung injury in septic mice

[0036] (1) Take a C57Bl / 6 mouse, weighing 21-23g, aged 6-8 weeks.

[0037] (2) Prepare LPS solution with PBS, and prepare HD5 solution and HD5-myr solution with ultrapure water.

[0038] (3) Design experimental groups: WT group, Vehicle group, HD5 group, HD5-myr group. The mice in the Vehicle group, HD5 group, and HD5-myr group were intraperitoneally injected with LPS at a dose of 10 mg / kg, and the mice in the WT group were intraperitoneally injected with the same volume of solvent PBS.

[0039] (4) After the LPS-induced sepsis model was established, mice in the HD5 group and HD5-myr group were injected with 60 μg of the same volume of solvent H 2 O.

[0040] (5) The mice were reared in a suitable environment; after 24 hours, the lungs of the mice were taken for tissue section and hematoxylin-eosin staining, and the injury of the lungs of the mice was observed.

[0041] The res...

Embodiment 3

[0044] Transmission electron microscope image of LPS and HD5-myr mixture

[0045] (1) Dissolve LPS, HD5 and HD5-myr in ultrapure water.

[0046] (2) Prepare LPS, LPS+HD5 mixed solution, LPS+HD5-myr mixed solution, the final concentration of polypeptide (HD5 and HD5-myr) in the above solution is 50 μg / ml, and the final concentration of LPS is 1 μg / ml.

[0047] (3) Dip a small amount of sample with the coated copper mesh, after it is fully infiltrated, absorb the excess liquid with filter paper, after drying for 30 seconds, add 2% uranyl acetate solution dropwise, and after it is fully dyed, absorb the excess liquid with filter paper , dry at room temperature (try to avoid heating and drying to prevent the copper mesh from breaking).

[0048] (4) Observe and take pictures with a transmission electron microscope after drying.

[0049] The result is as image 3 As shown, after mixing LPS and HD5-myr, nano-hybrid micelles formed, and the particle size increased significantly; ho...

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Abstract

The invention discloses the application of a human alpha defensin 5 modified peptide in preparation of endotoxin neutralization drugs, and belongs to the technical field of biology. The human alpha defensin 5 modified peptide is obtained in the step that amino acid residues are added on the C terminal of human alpha defensin 5, modified and connected with myristic acid. The human alpha defensin 5modified peptide can form nano hybrid micelles with endotoxins, inhibits the release of inflammatory factors caused by the endotoxins and reduces the damage to tissue and the organs through neutralization with the endotoxins, and is of great value for the treatment of sepsis. Therefore, the human alpha defensin 5 modified peptide can be applied to preparation of the endotoxin neutralization drugsand drugs for the treatment of sepsis caused by the endotoxins.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of a modified peptide of human alpha defensin 5 in the preparation of neutralizing endotoxin drugs. Background technique [0002] Endotoxin, also known as lipopolysaccharide (lipopolysaccharide, LPS), is a lipopolysaccharide substance in the outer membrane of the Gram-negative bacterial cell wall, which is composed of lipid A, core polysaccharide and O-specific side chain. An important component in the development of sepsis. The endotoxin produced by bacterial disintegration is released into the blood, and activates downstream signals by recognizing Toll like receptor 4 (TLR4) on the surface of immune cells or entering the cell through mouse Caspase11 (human Caspase4 / 5) pathway, which mediates a wide range of immune inflammatory responses, leading to multiple organ damage and septic shock. Plasma endotoxin levels are significantly elevated in patients with sepsis, a...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): A61K38/17A61P31/04C07K14/47
CPCA61K38/1729A61P31/04C07K14/4723
Inventor方向明孙亚奇程宝莉罗赣侯金超
OwnerZHEJIANG LONGTERM MEDICAL TECH CO LTD