lipopeptide lin-lf4nh 2 and lin-lf5nh 2 and its application
A technology of lin-lf4nh2 and lfcinb4, applied in the fields of peptide/protein components, specific peptides, chemical instruments and methods, etc., to achieve high antibacterial activity and thermal stability, small MIC value, and reduce skin tissue damage.
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Embodiment 1
[0036] Example 1 Lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 design and synthesis
[0037] The amino acid sequences of the polypeptides LfcinB4 and LfcinB5 are shown in SEQ ID NOs: 1 and 2, respectively. In order to further improve the antibacterial activity of LfcinB4 and LfcinB5, the structures of the two polypeptides were optimized and designed respectively.
[0038] Fmoc-Phe-OH, HOBT, HBTU, and DIEA are dissolved and mixed in DMF, and subjected to a condensation reaction with MBHA resin from which the Fmoc protecting group has been removed to obtain Fmoc-Phe-resin; the subsequent amino acids are sequentially condensed and reacted in the same way, Fmoc-Lf4-resin and Fmoc-Lf5-resin were obtained, respectively, and the side chain protecting group was removed with DCM solution containing 1% TFA to obtain Lf4NH 2-resin and Lf5NH 2 -resin; respectively dissolve and mix the fatty acids Lin, HOBT, HBTU and DIEA in DMF, and mix with Lf4NH 2 -resin, Lf5NH 2 -resin undergoes condens...
Embodiment 2
[0040] Example 2 Lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 Antibacterial activity testing of
[0041] According to the antimicrobial peptides and lipopeptides obtained in Example 1 (Lf4NH 2 ,Lf5NH 2 ,Lin-Lf4NH 2 and Lin-Lf5NH 2 ), prepare an antimicrobial peptide solution with a concentration of 2560 μg / mL in ultrapure water, dilute 2-fold to a final concentration of 2 μg / mL, and add the antimicrobial peptide solutions of different concentrations to sterile 96-well cell culture plates. 10 μL, three parallels for each sample, the same amount (10 μL) of PBS was used as a negative control, and the blank control group was sterile MH medium. Prepare MIC plates. The strains were cultured in MH culture liquid medium and shaken at 37°C to grow logarithmic phase (OD). 600nm = 0.4 to 0.6), prepare the bacterial liquid into a bacterial suspension with a concentration equivalent to 0.5 McFarland turbidity standard, and dilute the sterile MH liquid medium to 10 after incubation at 37 ...
Embodiment 3
[0045] Example 3 Lipopeptide Lin-Lf4NH 2 and Lin-Lf5NH 2 cytotoxicity assay
[0046] Detection of antimicrobial peptides and lipopeptides (Lf4NH) by MTT 2 , Lf5NH 2 , Lin-Lf4NH 2 and Lin-Lf5NH 2 ), was cytotoxic to Hacat. MTT can be reduced to insoluble blue-violet crystalline formazan by succinate dehydrogenase in the mitochondria of living cells and deposited in the cells, but this reaction does not occur in dead cells. DMSO can dissolve the blue-violet crystalline formazan in cells, and the absorbance value of the solution can indirectly reflect the number of living cells after dissolution. Within a certain range of cell numbers, the amount of crystal formation is proportional to the number of cells.
[0047] 37°C, 5% CO 2 and saturated humidity conditions, Hacat cells were cultured in MEM complete medium. Pipette the cells with a pipette, resuspend the cells in MEM complete medium at 2.5 × l0 5 Cells / mL density was seeded in 96-well plates, 100 μL per well, and ...
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