Antimicrobial peptide H5-p5 and preparation method and application thereof
A h5-p5, antimicrobial peptide technology, applied in the field of biochemistry, can solve the problems of low antibacterial activity and host cell cytotoxicity, and achieve the effects of strong bactericidal effect, low eukaryotic cytotoxicity, and convenient artificial synthesis
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Embodiment 1
[0016] Example 1: Preparation of antibacterial peptide H5-p5
[0017] Ⅰ. Chemical synthesis method of antimicrobial peptide H5-p5: According to the amino acid sequence described in the content of the invention, the corresponding L-amino acid (purchased from Merck Sigma) and an automatic peptide synthesizer (433A, American Applied Biosystems) Synthesize its full sequence. Purified by HPLC reversed-phase column (WelchXB C18 4.6×150 mm) chromatographic desalination, the mobile phase is pure water (0.1% trifluoroacetic acid)-60% acetonitrile (0.1% trifluoroacetic acid), flow rate is 1mL / min, 220nm wavelength Next, the elution peak was collected and lyophilized. L-amino acids need not be preserved.
[0018] Ⅱ. Use electrospray mass spectrometry to identify the molecular weight of the synthesized antimicrobial peptide H5-p5. The sample was injected using a liquid phase system, the mobile phase was 50% H2O / 50% CAN, the flow rate was 0.2ml / min, the shielding gas flow rate was 1.5L / min, ...
Embodiment 2
[0025] Example 2: Antibacterial experiment of antibacterial peptide H5-p5:
[0026] Minimal inhibitory concentration (MIC): the lowest sample concentration at which no bacterial growth can be detected. Using the double dilution method, the specific method is as follows:
[0027] Bacteria were inoculated on Luria-Bertani (LB) solid medium and cultured upside down in an incubator at 37°C. After the colony grows, use an inoculating loop to pick a single colony and transfer it to the LB liquid medium, and culture it to the logarithmic growth phase in a 37°C incubator with shaking. Detect the OD of bacterial liquid on UV spectrophotometer 600 , According to OD 600 =1×10 8 CFU / ml Dilute the bacterial solution with liquid LB medium to 2×10 5 CFU / ml. Pre-add 100μL of LB liquid medium to each well of a sterile 96-well plate, then add 100μL of antimicrobial peptide sample diluted to a certain concentration through a 0.22μm microporous membrane filter sterilized in the first well, and mix i...
Embodiment 3
[0033] Example 3: Hemolytic activity experiment of antimicrobial peptide H5-p5:
[0034] Blood was collected from the heart of mice, and the collected blood was combined with Alsever Solution (8.0g sodium citrate, 0.55g citric acid, 20.5g glucose, 4.2g sodium chloride, add deionized water to 1L, adjust the pH to 6.1, After autoclaving, store at 4°C) mix in a 1:1 ratio, place in a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and wash with physiological saline until the supernatant is no longer red. Dilute the washed red blood cells with normal saline to 10 8 Concentration of suspension. The above-mentioned diluted red blood cell suspension was incubated with samples of different concentrations dissolved in physiological saline at 37°C for 30 minutes, and then centrifuged at 1000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm. The negative control used physiological saline, and the positive control used Triton X-100. The hemolytic activi...
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