A kind of antimicrobial peptide rf-cath1 derived from horseshoe bat and its application
A RF-CATH1 and antimicrobial peptide technology, applied in the field of biomedicine, can solve the problems of dissolution of cell contents, cell death, damage to cell membrane integrity, etc., and achieve the effect of small molecular weight, low hemolytic activity, and broad-spectrum high-efficiency antibacterial effect
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Embodiment 1
[0017] Discovery of the natural antimicrobial peptide RF-CATH1
[0018] 1) Extraction of total RNA from the lung tissue of the horsetail bat:
[0019] ①Take 100mg of the lung tissue of the horsetail chrysanthemum bat, put it in a mortar and add liquid nitrogen to grind it into powder, transfer it to an EP tube, add 1ml of total RNA extraction buffer (Trizol, a product of Life Company, USA), mix well, and then Centrifuge at 12000 rpm for 10 min at 4°C.
[0020] ② Take the supernatant by centrifugation, add 0.2 ml of chloroform solution, mix vigorously, leave at room temperature for 10 minutes, then centrifuge at 4°C and 12000 rpm for 10 minutes, and discard the precipitate.
[0021] ③Add an equal volume of isopropanol to the supernatant, place it at room temperature for 10 minutes, centrifuge at 4°C and 12000rpm for 10 minutes, collect the precipitate, wash it once with 75% (V / V) ethanol, and dry it. The sediment at the bottom of the tube is Wuyi Tuan Frog skin total RNA.
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Embodiment 2
[0037] Chemical Synthesis of Antimicrobial Peptide RF-CATH1
[0038] (1) Synthesize the complete sequence of RF-CATH1 with an automatic peptide synthesizer (433A, Applied Biosystems), and then HPLC C 18 Reversed-phase column chromatography for desalting purification. (2) The molecular weight was determined by conventional matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). (3) Purified RF-CATH1 was identified by high performance liquid chromatography (HPLC) with a purity of >95%, isoelectric point determined by isoelectric focusing electrophoresis was 10.16, and its amino acid sequence structure was determined to be consistent with natural RF-CATH1 by automatic amino acid sequencer.
Embodiment 3
[0040] Antibacterial activity detection of antimicrobial peptide RF-CATH1
[0041] (1) The test strains stored on the slant were picked and evenly spread on the MH solid medium (purchased from Qingdao Haibo Biotechnology Co., Ltd.) flat plate, and the sterilized 0.5cm diameter filter paper was placed on the medium On the surface, 10 μl of RF-CATH1 sample solution of 2 mg / ml dissolved in sterilized deionized water was added dropwise, and incubated upside down at 37°C for 18-20 hours to observe whether the inhibition zone was formed. If the sample has antibacterial activity, a clear and transparent antibacterial circle will be formed around the filter paper, and the larger the antibacterial circle, the stronger the antibacterial activity of the sample. The results showed that the antimicrobial peptide RF-CATH1 had antibacterial activity against the strains listed in Table 1.
[0042] (2) The minimum inhibitory concentration (Minimum Inhibitory Concentration) was determined by t...
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