A new type of antimicrobial peptide caprylic acid conjugate with high efficiency against protease hydrolysis and multidrug-resistant bacteria and its application
A technology of protease hydrolysis and peptide octanoic acid couple, applied in the field of biomedicine, can solve the problems of limited clinical development, poor serological stability of antibacterial peptides, and easy degradation by proteases, and achieve broad-spectrum high-efficiency antibacterial activity, good acid-base stability, The effect of strong inhibition
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Embodiment 1
[0020] This embodiment provides the preparation method of antibacterial peptide octanoic acid conjugate SAMP-A4-C8, specifically including the following contents:
[0021] In the solid-phase synthesis of the Fmoc protection system, first Rink Amide AMResin (1g) was installed in the reactor, 6 mL of dichloromethane was added to swell for 12 hours, and 20% piperidine solution was used for deprotection; the first amino acid Fmo- Ile-OH (5eq) was mixed with the deprotected Rink Amide-AM Resin in the reactor, and reacted for 2.5 hours under the catalysis of HOBU / DIC (5eq). Kaiser test, heating at 110°C for 1 min to observe that it does not turn blue. 20% piperidine solution to remove N-terminal Fmoc protection, and then according to the given amino acid sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu -OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Val-OH are coupled sequentially, extending from the C-terminal to the N-terminal one by one. After the coupling o...
Embodiment 2
[0025] This embodiment provides the antimicrobial spectrum determination of the antimicrobial peptide octanoic acid conjugate SAMP-A4-C8 prepared in Example 1, specifically including the following:
[0026] 1) Weigh and dissolve SAMP-A4-C8. To prepare PBS solution, if the initial concentration in the first column is 500 μg / mL, then 2 mg / mL SAMP-A4-C8 mother solution needs to be prepared. Dissolve SAMP-A4-C8 with 20mmol / L sodium phosphate buffer solution pH7.0, and filter the solution with a 0.22μm sterile filter membrane to sterilize.
[0027] 2) Using a pipette gun, add 100 μL of 20 mmol / L PBS buffer solution at pH 7.0 to each well of the 96-well plate.
[0028] 3) Pipette 100 μL of SAMP-A4-C8 stock solution (2 mg / mL) into each well of the first column of the 96-well plate.
[0029] 4) Repeatedly blow and suck the solution in the first row of the plate for 6-8 times, mix well without splashing.
[0030] 5) Aspirate 100 μL from the first column and add to the second column,...
Embodiment 3
[0043] This embodiment provides the hemolytic assay of the antimicrobial peptide octanoic acid conjugate SAMP-A4-C8 prepared in Example 1, specifically including the following:
[0044] After the freshly collected blood of healthy volunteers was centrifuged, the erythrocyte pellet was washed three times with normal saline, and a final concentration of 2% HRBCs suspension was prepared. Add 100 μL of SAMP-A4-C8 (0-500 μg / mL) that has been serially diluted twice to a 96-well plate. Add 100μL of 2% HRBCs to each well and incubate at 37°C for 30min. Take 100 μL of supernatant from each well, transfer it to a new 96-well plate, measure the absorbance at a wavelength of 540 nm with a microplate reader, and calculate the hemolysis rate. Red blood cells treated with 1% TritonX-100 were used as positive control (100% hemolysis); red blood cells not treated with SAMP-A4-C8 were used as negative control (no hemolysis). The equation for calculating the hemolysis rate is as follows:
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