Antibacterial peptide with antibacterial activity in acidic environment, preparation method and application
A technology of antibacterial activity and antimicrobial peptides, which is applied in the field of antimicrobial peptides and its preparation to achieve high application value
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Embodiment 1
[0012] Antimicrobial Peptide Design:
[0013] The amino acid sequence of a pH-responsive antimicrobial peptide F5 is:
[0014]
[0015] Using the scorpion toxin antimicrobial peptide AaeAP2a as a template, select histidine with imidazolyl pKa=6.0 to replace the arginine and lysine residues on the side chain, so as to provide the net positive charge responsiveness of the polypeptide to pH. In addition, the introduction of glutamic acid at the active site of the sequence weakens the helical conformation through the electrostatic attraction of the anionic carboxylic acid group of glutamic acid and the cationic amine group of histidine, and the electrostatic attraction is influenced by the net effect of histidine residues. The effect of a change in positive charge. The amino acid sequences of the derived peptides are shown in Table 1.
[0016] Table 1 Amino Acid Sequence of Derived Peptides
[0017]
[0018] By introducing histidine and glutamic acid to increase the pH re...
Embodiment 2
[0019] Embodiment 2: the mensuration of antimicrobial peptide antibacterial activity
[0020] 1. Determination of antibacterial activity: Utilize the micro broth dilution method to measure the minimum inhibitory concentration of the antimicrobial peptide, to adjust the pH value of the diluent (0.2% BSA) with glacial acetic acid, and use the doubling dilution method to dilute the concentration of the peptide successively. Take 100 μL of the above solution and place it in a 96-well cell culture plate, then add an equal volume of the bacteria solution to be tested (~10 5 individual / mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the bottom of the well with the naked eye. The test results are shown in Table 2.
[0021] An...
Embodiment 3
[0026] Example 3: Antimicrobial Peptide Hemolytic Activity and Therapeutic Index
[0027] Determination of hemolytic activity of antimicrobial peptides:
[0028] 1ml of fresh human blood (from volunteers) was collected, the fresh plasma was centrifuged at 1000g for 5min, washed 3 times with PBS buffer of pH 74 and pH 6.5, and resuspended in 10ml of PBS buffer of different pH. The antimicrobial peptides were diluted to 96-well cell culture plates. Then add equal volumes of red blood cell suspensions with different pH to each well. A positive control (blood cells+0.2% Tritonx-100) and a negative control (blood cells+PBS buffer) were set up respectively. Incubate at a constant temperature of 37° C. for 1 h, centrifuge to take the supernatant into a new 96-well plate, and measure the absorbance at 570 nm.
[0029] Table 3 The geometric mean of the minimum antibacterial activity of antimicrobial peptides, hemolytic activity and therapeutic index
[0030]
[0031] a GM, the ...
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