Litopenaeus vannamei antibacterial peptide GPCR10 capable of cooperating with NaCl and application thereof
An antibacterial peptide, vanabine technology, applied in the direction of antibacterial drugs, medical preparations containing active ingredients, peptides, etc.
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Embodiment 1
[0034] Example 1 Identification and Screening of Litopenaeus vannamei NaCl Synergistic Antimicrobial Peptides
[0035] Firstly, samples were prepared from Litopenaeus vannamei, and each healthy Litopenaeus vannamei was injected with 0.1 mL of Staphylococcus aureus (1.0 × 10 6-7 CFU / mL) or an equivalent amount of sodium chloride, live shrimp were collected 24 hours after infection and immediately minced with PBS buffer to form a suspension, 1 mL was placed in a centrifuge tube, centrifuged for 15 minutes, and the supernatant was taken, filtered through 3000Da Membranes were stored at -20°C. The amino acid sequence of the polypeptide was identified and analyzed by ultra-high performance liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: mobile phase A was ultrapure aqueous solution containing 0.1% methanol; B was acetonitrile, and the injection volume was 5 μL. Gradient elution is shown in Table 1. The resolution of the primary scannin...
Embodiment 2
[0041] Embodiment 2 Minimum Inhibitory Concentration (MIC) Determination
[0042] Dissolve 0.36g of nutrient broth powder in 20mL of distilled water, add 1.5% NaCl, 2% NaCl, and 2.5% NaCl respectively, sterilize at 121°C for 20 minutes, then inoculate 200μL of Staphylococcus aureus (ATCC 29213), shake at 37°C The bed was shaken for 12 hours to the logarithmic growth phase of the bacteria, and the bacteria were diluted to 10 with phosphate buffer (0.01M, pH 7.2). 6-7 CFU / mL. Mix the diluted antimicrobial peptide GPCR10 with Staphylococcus aureus in equal proportions so that the final concentrations are 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL, mix well and place in Incubate at 37°C for 2 hours in a biochemical incubator. After fully reacting, take 20 μL sample and add it to the petri dish for coating, and start to count the total number of colonies after incubating at 37 °C for 20 h. Such as figure 2 Shown, the minimum inhibitory concentration (MIC) ...
Embodiment 3
[0043] Embodiment 3 time-killing curve (Time-kill)
[0044] The bactericidal efficiency of antimicrobial peptide GPCR10 synergistically with NaCl on Staphylococcus aureus was evaluated by plate colony counting method. Dissolve 0.36g of nutrient broth powder in 20mL of distilled water, add 1.5% NaCl, 2% NaCl, 2.5% NaCl respectively, sterilize at 121°C for 20min, take 200μL Staphylococcus aureus (ATCC 29213) into the sterilized liquid In the culture medium, shake at 37°C for 12h to the logarithmic growth phase of the bacteria, and dilute the bacterial concentration to 10 with phosphate buffer (0.01M, pH 7.2). 6-7 CFU / mL. The bacterial solution under different salt addition conditions was mixed with Staphylococcus aureus in equal proportions so that the final concentration of antimicrobial peptides was 1×MIC. Take it out after 2 and 2.5 hours, draw 20μL sample and add it to the petri dish to coat, place the petri dish at 37°C for 20 hours and start colony counting, the bacteric...
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