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Cationic peptide C9 and application thereof

A cationic peptide, Gram-positive technology, applied in the field of protein engineering, can solve the problems of low protease stability and limited clinical application, and achieve increased depolarization level, good antibacterial activity in vitro, and anti-biofilm formation ability is not easy Effect

Pending Publication Date: 2022-06-17
GUIZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In general, antimicrobial peptides have a high rate of killing bacteria, killing bacteria within minutes, but if these killings are not fatal, bacteria can quickly respond to this stressful environment when exposed to antimicrobials. Its clinical application is also limited by factors such as low protease stability, in vivo efficacy, and pharmacokinetics.

Method used

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  • Cationic peptide C9 and application thereof
  • Cationic peptide C9 and application thereof
  • Cationic peptide C9 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: In vitro biological activity of C9

[0045] 1. Experimental strains

[0046] 34 clinical samples of Staphylococcus epidermidis were isolated, and duplicate samples were excluded from the same patient. Sample source: sputum, blood, urine, etc. Collection and use of clinical isolates have been approved by the Institutional Review Board (IRB) of Guizhou Medical University, and studies involving human participants have been reviewed and approved by Guizhou Medical University and the ethics committee affiliation. Bacterial identification and drug susceptibility tests were carried out by French bio-Mérieux VITEK-2 automatic microbiological analyzer. Staphylococcus epidermidis ATCC35984 was a positive strain for biofilm formation, and the above strains were preserved by the Laboratory of Pathogen Biology of Guizhou Medical University.

[0047] 2. Method

[0048] 2.1 Minimum inhibitory concentration (MIC) determination

[0049] 1) Preparation of bacterial cells:...

Embodiment 2

[0086] Example 2: Effect of C9 on Staphylococcus epidermidis biofilm

[0087] 1. Experimental strains

[0088] Staphylococcus epidermidis ATCC 35984 and clinical isolates (SE5).

[0089] 2. Method

[0090] 2.1 Biofilm forming ability

[0091] Take the logarithmic growth phase of Staphylococcus epidermidis and prepare the concentration of 2 × 10 in TSB medium. 6 CFU / mL bacterial suspension. 200 μL of bacterial solution was added to each well of a 96-well plate, TSB medium was used as a negative control, and three replicate wells were set up for each bacterial strain. After static culture at 37 °C for 24 h, the 96-well plate was taken out, non-adherent bacteria and cultures were discarded, the culture wells were washed three times with sterile PBS, the plate was placed in a 60 °C incubator to dry and fix for 30 min, and 200 μL was added to the wells after fixation. 0.1% crystal violet (CV), shaking at room temperature for 20 min; discarding the crystal violet solution, gent...

Embodiment 3

[0107] Example 3: Research on the bactericidal mechanism of C9

[0108] 1. Experimental strains

[0109] Staphylococcus epidermidis ATCC 35984 and clinical isolates (SE5).

[0110] 2. Experimental method

[0111] 2.1 Microscopic observation of morphological changes

[0112] 1) Laser confocal microscope

[0113] In order to verify whether C9 can bind to the cell membrane of S. epidermidis through electrostatic interaction, the labeling of S. epidermidis by FITC-C9 was observed by laser confocal microscopy.

[0114] Take log phase Staphylococcus epidermidis and prepare 1 mL with PBS to a concentration of 1 × 10 8 CFU / mL bacterial suspension was added with FITC-C9 (1×MIC) peptide solution and incubated in a 37°C incubator for 1 h. After the incubation, the cells were collected by centrifugation at 4000 rpm for 5 min, washed three times with PBS to remove unbound peptides, and finally resuspended in 500 μL of PBS. Coverslip and seal, and observe with a laser confocal microsc...

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Abstract

The invention belongs to the field of protein engineering, and particularly relates to a cationic peptide C9 and application thereof. The amino acid sequence of the cationic peptide C9 is as shown in SEQ ID NO. 1. The cationic peptide C9 provided by the invention has the characteristics of better in-vitro antibacterial activity, biofilm formation resistance, difficulty in generating drug resistance and the like, and by destroying a cell membrane structure, increasing the permeability and depolarization level of a membrane and disturbing the flowability of the membrane, bacteria cannot complete a necessary biological process and die, so that the cationic peptide C9 can be used for preparing the antibacterial peptide C9. Certain treatment potential is shown on in-vivo anti-infection treatment.

Description

technical field [0001] The invention belongs to the field of protein engineering, in particular to cationic peptide C9 and its application. Background technique [0002] Staphylococcus epidermidis belongs to coagulase-negative staphylococci and is a normal flora that colonizes the surface of human skin and mucous membranes. However, when the body is immunocompromised or transferred to other parts of the body by adhering to medical grafts such as indwelling venous catheters, artificial heart valves, and artificial joints, it can cause bacterial endocarditis, neonatal sepsis, and periprosthetic infections. , has become one of the important pathogens of hospital-acquired infection. [0003] For a long time, antibiotics have played an important role in the prevention and treatment of diseases. However, with the widespread use of antibiotics such as broad-spectrum penicillins, aminoglycosides and macrolides in clinical practice, the number of resistant strains of Staphylococcus ...

Claims

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Application Information

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IPC IPC(8): C07K7/08A61K38/10A61K45/06A61K38/14A61P31/04A61K31/496A61K31/431
CPCC07K7/08A61K38/10A61K45/06A61P31/04A61K38/14A61K31/496A61K31/431A61K38/00A61K2300/00
Inventor 仇志浪彭建毛成菊国果
Owner GUIZHOU MEDICAL UNIV
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