Broad-spectrum antibacterial peptide and application thereof in skin injury repair
An antimicrobial peptide and broad-spectrum technology, applied in the field of peptides, can solve the problems of insufficient separation and functional research of antimicrobial peptides, harsh natural living conditions, poor stability, etc., and achieve good medicinal value and strong antibacterial activity. Effect
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Embodiment 1
[0015] Example 1 Design of Antimicrobial Peptide PD-1m Series
[0016] The antimicrobial peptide PD-1m series is based on the amino acid sequence (SEQ ID NO.1) of the natural antimicrobial peptide from Periplaneta americana for a series of mutation design. In order to improve the stability of antimicrobial peptides, according to the amino acid sequence composition, charge and hydrophobicity of natural antimicrobial peptides, try to replace part or all of the 8 lysines (Lys) in natural antimicrobial peptides with histidine (His), the specific substitution site and the physicochemical property analysis of each mutant are shown in Table 1.
[0017] Table 1
[0018]
[0019] As shown in Table 1, by mutating the lysine site of the natural antibacterial peptide sequence, the stability of the five polypeptide sequences of the mutant polypeptides PD-1m1, PD-1m7, PD-1m8, PD-1m9, and PD-1m10 performance was significantly improved.
Embodiment 2
[0020] Example 2 Synthesis of antimicrobial peptide mutants PD-1m1, PD-1m7, PD-1m8, PD-1m9, PD-1m10
[0021] Weigh 0.23g of Rink Amide MBHA resin, place it in a PSI300 peptide synthesizer reactor, add 10mL of DMF, soak for 2h, then add 10mL of 20% PIP / DMF solution, mix for 30min to remove the amino protecting group, wash the resin 7 times with DMF, Then add Fmoc-L-3-(2-Thienyl) L-alanine-OH, equimolar coupling reagent DIC (0.3mol / L) and HOBt (0.3mol / L) in the reactor to react, the reaction temperature is At room temperature, the reaction process was monitored by ninhydrin reaction. If no color was detected, the reaction was completed. The resin was washed 5 times with DMF. After the first amino acid is coupled to the resin, the coupling reaction of the next amino acid can be continued according to the above method, and this cycle is repeated until all amino acid couplings are completed. After the synthesis, the resin was vacuum dried and weighed. Add the lysis reagent accord...
Embodiment 3
[0026] Example 3 Detection of the Minimum Inhibitory Concentration of Serial Mutant Antimicrobial Peptides (MIC Detection)
[0027] In this test, the minimum inhibitory concentration was detected by the microdilution method, and the detected bacteria were Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), methicillin-resistant Staphylococcus aureus (ATCC6538), Staphylococcus epidermidis (ATCC12228), Bacillus subtilis (ATCC6638) and Klebsiella pneumoniae (ATCC700603). The specific method is: resuscitate each strain, spread it on MH solid medium, place it in a constant temperature incubator at 37°C overnight, pick a single colony and inoculate it in fresh liquid LB medium, and place it in a constant temperature incubator at 37°C overnight; Then dilute the 5 bacterial suspensions to 105 CFU / mL with fresh LB liquid medium by means of a McFarland turbidimeter. Filter the purified AMP-1 with a known concentration through a 0.22 μm filter membrane, and then use fresh l...
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