Novel broad-spectrum antibacterial peptide SAMP1-A3 and preparation method thereof
An antimicrobial peptide and broad-spectrum technology, applied in the field of new antimicrobial peptides and their preparation, can solve problems such as unstable expression products, low product yields, and difficult purification
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Embodiment 1
[0018] Example 1 Preparation of Antimicrobial Peptide SAMP1-A3
[0019] The target peptide was synthesized by the solid-phase Fmoc method from the C-terminus to the N-terminus. After activating the carboxyl group of the first Fmoc-Ile-OH at the C-terminus of the polypeptide, bind to the resin, remove the Fmoc-protecting group, combine with the Fmoc-Leu-OH activated by the second carboxyl group, and then remove the Fmoc-protecting group, The cycle is repeated until all amino acids are added. Cut the peptide on the resin with cutting solution, collect the cutting solution, dry it in a centrifuge tube, wash the precipitate with ice ether 2~3 times, centrifuge to remove the supernatant, and dry the precipitate. The obtained crude product was purified by HPLC.
Embodiment 2
[0020] Example 2 Determination of antimicrobial spectrum of antimicrobial peptide SAMP1-A3
[0021] 1) Weigh and dissolve protein. Prepare the SAMP1-A3 phosphoric acid solution, set the initial concentration in the first column to 500 μg / mL, and set the final concentration to 0.975 μg / mL. It is necessary to prepare 2mg / mL SAMP1-A3 stock solution. Dissolve SAMP1-A3 with 20 mmol / L sodium phosphate buffer solution pH6.0, and filter the solution through a 0.22 μm sterile filter membrane to sterilize.
[0022] 2) Using a pipette, add 100 μL of 20 mmol / L pH6.0 sodium phosphate buffer to each well of the 96-well plate to dilute SAMP1-A3.
[0023] 3) Use a pipette gun to pipette 100 μL of 2 mg / mL SAMP1-A3 stock solution into each well of the first column of the 96-well plate.
[0024] 4) Repeatedly blow and aspirate the solution in the first column of the plate for 6-8 times, mix well without splashing.
[0025] 5) Aspirate 100 μL from the first column and add it to the second col...
Embodiment 3
[0037] Example 3 Determination of antibacterial peptide SAMP1-A3 bactericidal and antibacterial activity
[0038] The bactericidal and antibacterial activity of SAMP1-A3 was detected by measuring the number of viable bacteria / fungi after the action of antimicrobial peptides. Candida tropicalis is the representative fungus, and Listeria monocytogenes is the representative bacterial strain.
[0039] Candida tropicalis: culture Candida tropicalis at 28°C with SD liquid medium to OD 600 ≈0.6-0.8. Add the bacterial suspension into the solution containing 1×MIC 100 In the SD liquid medium of SAMP1-A3, the bacterial concentration was about 1×10 5 CFU / mL. At the same time, a blank SD medium without SAMP1-A3 was set as a control. Cultivate at a constant temperature in a shaker at 28°C. After culturing for 4 hours, absorb 100 μL of the bacterial solution, spread it on an SD agar plate after dilution, and culture overnight at 28°C for colony counting.
[0040] Listeria monocytoge...
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