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Antibacterial peptides and application of antibacterial peptides to preparation of medicament resisting drug-resistant bacteria

A technology of antimicrobial peptides and drug-resistant bacteria, which is applied in the direction of antibacterial drugs, peptides, and decapeptides, and can solve the problems of hemolytic side effects, low antibacterial activity, and hemolytic side effects of antibacterial peptides, and achieve the solution of selection problems. The preparation method is simple, The effect of improving the survival rate

Inactive Publication Date: 2012-12-05
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few attempts to directly use natural antimicrobial peptides as antibacterial drugs
The main reasons are: the antibacterial activity of many natural antimicrobial peptides is not high, and the yield is low; many antimicrobial peptides have strong hemolytic side effects
For example, melittin extracted from bee venom has strong antibacterial activity, but it also has hemolytic side effects, which limits the application of melittin as an antibacterial drug.

Method used

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  • Antibacterial peptides and application of antibacterial peptides to preparation of medicament resisting drug-resistant bacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Solid Phase Synthesis of Antimicrobial Peptide Derivatives

[0027] Antimicrobial peptides were synthesized by solid-phase synthesis using FMOC (9-fluorenylmethoxycarbonyl) as a protecting group. It was cleaved from the resin with 95% trifluoroacetic acid, 2.5% water and 2.5% triisopropylsilane (TIA). After repeated precipitation with diethyl ether, the polypeptide was purified by reverse-phase high-performance liquid chromatography. A C18 reverse-phase column was used in the purification: 0%-60% acetonitrile containing 0.05% trifluoroacetic acid was used as the mobile phase, and gradient elution was performed at a flow rate of 3 mL / min. Then the peptide was dissolved in oxidation buffer (100 mmol / L ammonium acetate, pH 8.5) at a concentration of 1 mg / mL, and stirred continuously at room temperature for 3 days to fully oxidize and fold to form disulfide bonds. Finally, it was purified to more than 95% by reverse-phase HPLC, and freeze-dried for future use. The seque...

Embodiment 2

[0036] Determination of Antibacterial Activity

[0037] A peptide sample solution with a concentration of 6 mg / mL was prepared with sterile saline. The bacteria used in the test were inoculated in the nutrient broth, cultured at 37°C for 24 hours, diluted 1:105 times with sterile saline before use, and 12 bacterial culture tubes were taken and numbered, and nutrient meat was added to the first tube Add 1.8mL broth, and add 1.0mL broth to the remaining 11 tubes. Add 0.2mL of polypeptide solution to the first tube, mix well, take out 1.0mL and add it to the second tube, repeat this process, and dilute to the 12th tube in turn, add 0.2mL of bacterial solution to each tube, shake gently, place at 37 Cultivate for 24 hours. The lowest concentration of peptides for sterile growth is the minimum sample concentration (MIC) that inhibits bacterial growth. Table 2 shows the minimum inhibitory concentrations of antimicrobial peptide derivatives prepared in Example 1 to several bacte...

Embodiment 3

[0041] Determination of polypeptide hemolytic activity: Suspend human red blood cells in phosphate buffer (pH 7.4) to obtain red blood cell suspension (5% v / v). Dissolve the peptide in phosphate buffer to make about 5mg / mL stock solution, take 14 1.5mL centrifuge tubes, add 1mL peptide stock solution to the first centrifuge tube, add 0.5mL phosphate buffer to the other tubes solution, take 0.5mL peptide stock solution from the first tube and add it to the second tube, mix evenly with a micro mixer, then take 0.5mL solution from the second tube, add it to the third tube and mix evenly, and so on, That is, dilute to the 14th tube sequentially by doubling dilution method, discard 0.5mL, add 0.5mL prepared 5% red blood cell suspension to each tube until the final volume is 1.0mL, shake gently, and incubate in a 37°C incubator After 60 min, it was centrifuged at 4000 rpm for 10 minutes, and the supernatant was taken for colorimetry at 414 nm. Red blood cells suspended in phosphate ...

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Abstract

The invention discloses antibacterial peptides and application of the antibacterial peptides to preparation of a medicament resisting drug-resistant bacteria. The antibacterial peptides consist of 21 amino acids, wherein the amino acid sequence is shown as SEQIDNO.1. The in-vivo and in-vitro research shows that six kinds of antibacterial peptides have a strong inhibiting and killing action for the mostly clinically-separated drug-resistant bacteria and can remarkably reduce the mortality of a septicemia model caused by the drug-resistant bacteria; and a hemolytic test and an acute local stimulus test results show that six kinds of derivates have no hemolytic test or acute toxic stimulus reaction when the concentration is 2.5mg / mL.

Description

[0001] technical field [0002] The invention relates to an antimicrobial peptide and its derivatives, in particular to a group of antimicrobial peptides and their application in the preparation of drugs against drug-resistant bacteria. Background technique [0003] The discovery of antibiotics is of great significance in the history of human medicine. Its use has saved countless lives and increased human life expectancy by more than ten years. But with the widespread use of antibiotics, bacteria have gradually adapted and developed resistance to them. Although there are hundreds of antibiotics in clinical use at present, they are all small chemical molecules, and the newly developed antibiotics are generally structural modifications of them. It is difficult to solve the problem of bacterial resistance to antibiotics. With the emergence of drug-resistant bacteria, the development of new antibiotics against drug-resistant bacteria has become an international concern. [000...

Claims

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Application Information

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IPC IPC(8): C07K14/00A61K38/16A61P31/04
Inventor 吴国球
Owner SOUTHEAST UNIV
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