A kind of antibacterial peptide and its preparation method and application
A technology of antimicrobial peptides and active polypeptides, applied in the field of peptides, can solve the problems of restricting the popularization and application of antimicrobial peptides, animal cytotoxic side effects, and low antimicrobial activity, and achieves the effects of improving intestinal health, facilitating artificial synthesis, and improving growth performance.
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Embodiment 1
[0022] Preparation of antimicrobial peptides
[0023] 1. The chemical synthesis method of the antimicrobial peptide: according to the amino acid sequence shown in SEQ ID NO.1, the full sequence of the antimicrobial peptide was synthesized with a fully automatic peptide synthesizer (ABI433), and desalted and purified by HPLC reverse-phase column chromatography;
[0024] 2. The molecular weight of the purified antimicrobial peptide was determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), the isoelectric point was determined by isoelectric focusing electrophoresis, and the amino acid sequence was analyzed by an automatic amino acid sequencer.
[0025] The antimicrobial peptide obtained in this example contains 15 amino acid residues, the molecular weight is 1483.88 Da, and the isoelectric point is 10.30. PD21.
Embodiment 2
[0027] Antimicrobial peptide PD21 antibacterial experiment
[0028] The antibacterial effect was reflected by measuring the minimum inhibitory concentration (MIC) of the antimicrobial peptide PD21, which was the lowest sample concentration at which no bacterial growth could be detected. The two-fold dilution method was adopted, and the specific method was as follows: the test bacteria were inoculated on LB solid medium, and then cultured upside down in a 37°C incubator. After the colonies grow out, use a disposable inoculation loop to pick a single colony of each test bacterium into LB liquid medium, and culture it in a 37°C incubator with shaking until the logarithmic growth phase. Detect the OD600 of the bacterial solution on the ultraviolet spectrophotometer, according to 1 OD600=1×10 9 CFU / mL Dilute the test bacteria liquid with liquid LB medium to 2×10 5 CFU / mL. Then add 100 μL of LB liquid medium to each well of a sterile 96-well plate, and then add 100 μL of antimicr...
Embodiment 3
[0030] Cytotoxicity experiment of antimicrobial peptide PD21
[0031] Pig small intestinal epithelial cells IPEC-1 were cultured in HAM'S / F12 medium containing 5% bovine serum, 200U / mL double antibody (penicillin and streptomycin each 100U / mL), 5ug / L epidermal growth factor (EGF) After reaching the logarithmic phase, wash three times with phosphate buffer (HyClone), add 0.25% trypsin to digest to a single cell, add HAM'S / F12 medium and centrifuge at 400g for 4 minutes, discard the supernatant and use fresh HAM'S / F12 Suspend cells in F12 medium and adjust to a cell density of 1×10 6 cells / mL, and then add 200uL to each well of the 96-well cell culture plate, and add different concentrations of antimicrobial peptide PD21 after the cells adhere to the wall, as for the carbon dioxide incubator (37 ° C, 5% CO 2 ) after culturing for 24 hours, add 20uL CCK-8 (Beiyuntian) CCK-8 solution to each well, continue culturing for 2 hours, and then use a microplate reader to detect the abso...
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