Antibacterial peptide and application thereof in prevention and treatment of multi-drug-resistant bacteria
An antibacterial peptide, drug-resistant bacteria technology, applied in the direction of antibacterial drugs, resistance to vector-borne diseases, medical preparations containing active ingredients, etc., can solve the problem of lack of extensive research, and achieve the effect of obvious antibacterial effect
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Embodiment 1
[0013] Example 1: Screening and detection of antimicrobial peptides
[0014] The antibacterial peptide with the amino acid sequence of VFILSKYRWVMRFVPWCKALLKRFGKYKKHAK (SEQ ID NO: 1) was obtained by screening from chicken bursa, named AUDap. After Oxford Cup experiment, it was found that the antimicrobial peptide AUDAP has obvious bacteriostatic effect on non-resistant gram-negative bacteria Escherichia coli and gram-positive bacteria Bacillus subtilis; However, the antibacterial effect of Escherichia coli 577 (ATCC 577, E.coli 577) and Klebsiella pneumoniae strain Klebsiellapneumoniae 2182 (ATCC 2182, K.pneumonia 2182) with drug-resistant bacteria was not significant.
[0015] The minimum inhibitory concentration (MIC) results of AUDAP antimicrobial peptides against the above four strains are shown in Table 1.
[0016] Table 1: The minimum inhibitory concentration values MIC (μg / mL) of AUDAP antimicrobial peptides against four strains
[0017]
[0018] Therefore, the A...
Embodiment 2
[0019] Example 2: Cytotoxicity detection of AUDAP antimicrobial peptide
[0020] The survival rate of mouse macrophage cells under the action of AUDAP antimicrobial peptide was determined by MTT colorimetry. The results showed that AUDAP antimicrobial peptide did not show obvious toxicity to RAW264.7 mouse macrophages at a concentration of 15 μg / ml. Injury effect, indicating that AUDAP antimicrobial peptide has no significant toxicity to mammalian normal tissue cells.
[0021] To determine the erythrocyte hemolysis of the antimicrobial peptide AUDAP, the specific steps are as follows:
[0022] 1) Fresh mouse whole blood, anticoagulated with heparin, centrifuged at 3000 r / min at 4°C for 10 min, washed with normal saline, and resuspended at 8% (v / v);
[0023] 2) Mix 100 μL of red blood cell suspension with 100 μL of antimicrobial peptide solutions of different concentrations. The mixed system was placed in a 37°C incubator and incubated for 1h, taken out and centrifuged at 300...
Embodiment 3
[0025] Example 3: Combined use of AUDAP antimicrobial peptides with antibiotics
[0026] The tested strains were Escherichia coli 577 (ATCC 577, E.coli 577), Klebsiella pneumoniae 2182 (ATCC 2182, K.pneumonia 2182) and methicillin-resistant Staphylococcus aureus USA500 ( S. aureus USA500).
[0027] The AUDAP antimicrobial peptide was synthesized by a biological company, and the carboxyl terminal was amidated during synthesis, and the purity of the synthesized product was greater than 98%.
[0028] The synthesized AUDAP antibacterial peptide was dissolved in 1% (v / v) trifluoroethanol solution to prepare a mother solution with a final concentration of 5 mg / ml, and then stored at -80° C. for later use.
[0029] Inoculate the strain to be detected in LB liquid medium for expanded culture, until the bacteria are cultured to the logarithmic growth phase, after centrifugation to collect the bacterial cells, the bacterial precipitate is repeatedly washed with PBS solution, and the ob...
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