Cation antibacterial peptide
A technology of antimicrobial peptides and cations, which is applied in the fragments of melittin and its analogue polypeptides, cationic antimicrobial peptides as antibiotics, and can solve problems such as melittin blockage
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Embodiment l
[0022] Taking Mel(12-24) as an example, the method for synthesizing peptides by solid-phase method is illustrated:
[0023] Add 1g of Rinkamide MBHA resin (0.74mmol N / g) to a 50ml round-bottomed flask with a branch pipe on the side and a sand plate filter element in the branch pipe, add 15ml DMF to swell for 10 minutes, and remove the solvent by suction filtration. Then 15ml of 20% piperidine / DMF solution was added, stirred for 30 minutes, and suction filtered. The resin was washed 6 times with DMF, and the solvent was removed by suction filtration. Add 1.44g (2.22mmol) Fmoc-Arg(Pbf)-OH (Pbf is 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), 0.46g ( 2.22mmol) DCC (dicyclohexylcarbodiimide) and 0.30g (2.22mmol) HOBt (1-hydroxybenzotriazole) and 15ml DMF were stirred at room temperature for condensation reaction. After 2 hours of reaction, a small amount of resin was taken for ninhydrin color test, and the result showed that the condensation reaction was complete. Suction...
Embodiment 2
[0027]Determination of the antibacterial activity of the polypeptide: a polypeptide sample solution with a concentration of ~1 mg / ml was prepared with sterilized physiological saline. The bacteria used in the test were inoculated in the nutrient broth, cultured at 370C for 18-24 hours, diluted with physiological saline 1:105 before use, and 12 bacterial culture tubes were taken and numbered, and 1.8 nutrient broth was added to the first tube. ml, and 1.0ml broth was added to the remaining 11 tubes. Add 0.2ml of polypeptide solution to the first tube, mix well, take out 1.0ml and add it to the second tube, repeat this process, dilute to the 12th tube in turn, discard the remaining 1ml, add 0.2ml of the prepared bacteria solution to each tube, Gently shake evenly, culture at 370C for 18-24 hours, and observe. After the culture, the culture tube is clear, and it is still clear after shaking, and the tube is considered aseptic growth; the culture tube is turbid, which indicates t...
Embodiment 3
[0031] Determination of the hemolytic activity of the polypeptide: Suspend human red blood cells in phosphate buffer (pH=7.4) to obtain red blood cell suspension (5% v / v). Dissolve the peptide in phosphate buffer solution to make about 5mg / ml stock solution, take 14 1.5ml centrifuge tubes, add 1ml peptide stock solution to the first centrifuge tube, add 0.5ml phosphate buffer to the remaining tubes solution, take 0.5ml of peptide stock solution from the first tube and add it to the second tube, mix it evenly with a micro mixer, then take 0.5ml solution from the second tube, add it to the third tube and mix it evenly, and so on, That is, dilute to the 14th tube sequentially by the double-half dilution method, discard 0.5ml, add 0.5ml of prepared 5% red blood cell suspension to each tube to a final volume of 1.0ml, shake gently, and keep warm in a 37°C incubator After 60 minutes, centrifuge at 4000 rpm for 10 minutes, take the supernatant and perform colorimetry at 414nm, take r...
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