Larimichthys crocea hemoglobin antibacterial peptide and application thereof
A technology of hemoglobin and antimicrobial peptides, applied in the field of large yellow croaker hemoglobin antimicrobial peptides, can solve the problems of discovering antimicrobial peptides and achieve low toxicity
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[0037] Example 1: Screening of antimicrobial peptides derived from hemoglobin of large yellow croaker
[0038] Because fish hemoglobin is rich in alpha helix structure, which is the most common characteristic structure of antibacterial peptides, we use AntiBP server to predict potential antibacterial peptides with alpha helix structure from large yellow croaker hemoglobin, and use CAMP software to predict the reliability of peptides. Evaluation. Six antimicrobial peptides with alpha helix potential are predicted, as shown in Table 1.
[0039] Table 1 Prediction of potential antimicrobial peptides in hemoglobin of large yellow croaker
[0040]
[0041] It can be seen from Table 1 that the peptide LCH4 scored the highest. At the same time, the peptide LCH4 also has the characteristics of a typical antimicrobial peptide, that is, the number of amino acids ( 30%). The first 4 peptides (LCH1-LCH4) with the highest scores were synthesized, and antibacterial experiments were carried out ...
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[0042] Example 2: Determination of Minimum Inhibitory Concentration (MIC)
[0043] Staphylococcus aureus, Escherichia coli and Streptococcus dolphinus were cultured at 37°C for 12 hours to the logarithmic growth phase, and diluted to 10 in 0.01M pH 7.2 phosphate buffer 6-7 CFU / mL. The peptide was dissolved in phosphate buffer and mixed with bacteria in equal volume at 37°C for 2h. The minimum inhibitory concentration (MIC) refers to the lowest concentration of antimicrobial peptide at which no bacterial growth can be seen from the microtiter plate after overnight incubation at 37°C. Such as figure 1 , figure 2 with image 3 As shown, the minimum inhibitory concentration (MIC) of LCH4 against Staphylococcus aureus and Escherichia coli are both 15.6 μg / mL, and the minimum inhibitory concentration (MIC) of LCH4 against Streptococcus dolphin is 62.5 μg / mL.
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[0044] Example 3: Transmission electron microscopy analysis
[0045] By 10 6-7 CFU / mL bacteria were treated with 2×MIC LCH4 at 37°C for 2h, then centrifuged at 2700g for 10min and washed twice with phosphate buffer (pH 7.2). After fixing with 1% osmic acid, dehydrating with 95% ethanol, and then treating with acetone for 20 min. The samples were baked at 70°C for 24h, and 70-90nm slices were prepared on a copper grid, and then stained with lead citrate and uranyl acetate. The H-7650 transmission electron microscope was used to observe and capture the ultrastructure.
[0046] Such as Figure 4 , Figure 5 with Image 6 As shown, for the untreated bacteria, the intracellular tissue and structural integrity of the bacterial cells are good. However, after treatment with the peptide LCH4, it can be seen that the structure of the bacterial cell membrane begins to blur as the cell vacuoles, the shape of the cell becomes irregular, the cell membrane is completely collapsed, and the cytop...
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