Bloody noun antibacterial peptide temporin-Lb, genes thereof and use in pharmacy
An antimicrobial peptide and bullfrog technology, applied in the field of biomedicine, can solve the problems of limited research on skin active peptides, achieve the effect of inhibiting the growth of bacteria and fungi, simple structure, and wide antibacterial spectrum
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Embodiment 1
[0046] Bullfrog antimicrobial peptide gene cloning:
[0047] I. Extraction of Total RNA from Bullfrog Skin
[0048] A. The living bullfrog was cleaned with double distilled water, and the sterilized needle was paralyzed from the medullary cavity of the bullfrog. Take 50-100 mg bullfrog skin tissue and put it into a dry-baked mortar, add 1 mL TRIZOL Reagent (product of Invitrogen Company) Grind in an ice water bath.
[0049] B. After fully mixing, transfer to a 1.5mL centrifuge tube (DEPC tube) and place at 15-30°C for 5min, add an equal volume of chloroform, vortex mix for 15s, and centrifuge at 12,000×g for 15min at 4°C.
[0050] C. Add 500 μL of isopropanol to the supernatant, place at 15-30°C for 5 minutes, vortex for 15 seconds to mix, and centrifuge at 12,000×g for 10 minutes at 4°C. Discard the supernatant, add 1 mL of 75% ethanol to rinse the precipitate, centrifuge at 7500×g for 5 min, and repeat once. Dry in an ultra-clean workbench for 90 seconds, dissolve with 30...
Embodiment 2
[0118] Preparation of bullfrog antimicrobial peptide
[0119] I. Sample preparation method: deduce the amino acid sequence of the bullfrog antimicrobial peptide according to the gene encoding the bullfrog antimicrobial peptide, and synthesize its full sequence with an automatic peptide synthesizer APEX396. Reversed phase C by HPLC 18 Desalted and purified by column chromatography.
[0120] II. Molecular weight was determined by electrospray ionization (ESI) with an emission voltage of 4.5Kv.
[0121] III, the bullfrog antimicrobial peptide of purification is identified its purity (flow velocity 1.0ml / min with high performance liquid chromatography HPLC method; Mobile phase acetonitrile+water+TFA0.01%, gradient elution; Chromatographic column C 18 , the detection wavelength is 220nm), the molecular weight is determined by fast atom bombardment mass spectrometry, the isoelectric point is determined by isoelectric focusing electrophoresis, and the amino acid sequence structure ...
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