Anti-inflammatory hybrid antimicrobial peptide with alpha-helical structure, and preparation method and application thereof
A hybrid antibacterial peptide and α-helix technology, which is applied in the field of anti-inflammatory hybrid antibacterial peptide and its preparation, can solve the problems of high hemolytic activity, low antibacterial activity, and limited application prospects of antibacterial peptides, so as to reduce costs and shorten peptide chains length, the effect of enhancing cell selectivity
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Embodiment 1
[0015] Design and Synthesis of Antimicrobial Peptides
[0016] CA: the 1-8 amino acid functional sequence of the cecropin antimicrobial peptide Cecropin A, this fragment is a peptide chain with an amphipathic helical structure rich in cations. The shortest sequence identified as having antibacterial and anti-inflammatory activities can effectively bind to anionic phospholipids of bacterial cell membranes and exhibit good selectivity.
[0017] FO: The 1-15th amino acid functional sequence of the antibacterial peptide Fowlicidin-2. This fragment is a peptide chain with an amphipathic helical structure rich in cations and has good antibacterial activity.
[0018] The α-helical antimicrobial peptide CA and FO were hybridized to obtain the hybrid antimicrobial peptide CA-FO. The helical wheel prediction diagram of the hybrid peptide CA-FO is as follows figure 1 shown. Using a peptide synthesizer, the above three antimicrobial peptides were synthesized by solid-phase synthesis. ...
Embodiment 2
[0022] Physicochemical Index Analysis of Antimicrobial Peptides
[0023] The secondary structure of the antimicrobial peptide was analyzed by CD spectrum, and the results showed that both the hybrid peptide and the original peptide showed a random coil structure in the water environment, while in the hydrophobic environment (50% TFE) of the simulated microbial membrane and negative In the charged prokaryotic membrane environment (30mM SDS), the original peptide CA, FO and the hybrid peptide CA-FO all showed a certain α-helical structure.
[0024] The stability analysis of antimicrobial peptides showed that under different physiological concentrations of salt ions, the hybrid peptide CA-FO had ideal resistance to Gram-positive bacteria, and its antibacterial activity was slightly reduced.
Embodiment 3
[0026] Bacteriostatic, Cytotoxic and Endotoxin Neutralizing Activities of Antimicrobial Peptides
[0027] 1 Antibacterial activity
[0028] The peptide was configured as a 2.56mM / L storage solution for future use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. 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 18 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the ...
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