Micromolecule polypeptide RK12 and application thereof
A technology of small molecule polypeptide, RK12, applied in the field of biomedicine, can solve the problems of complex antibacterial peptide antibacterial mechanism, leakage of bacterial content, permeability change, etc., and achieve good market prospects, small molecular weight, and convenient artificial synthesis.
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Embodiment 1
[0017] The preparation of the small molecule polypeptide RK12 adopts the Fmoc solid-phase synthesis method, and the specific steps are as follows.
[0018] 1. Weigh 0.2g of the resin and place it in a dry and clean reaction tube, add an appropriate amount of N,N-dimethylamide (DMF), activate for 30 minutes, add 1mmol of the first amino acid residue and 150mg of DMAP to the reaction In the tube, react with DMF as a solvent for 3 hours; after the reaction, wash with DMF for 3 to 6 times, add appropriate pyridine and acetic anhydride at a volume ratio of 1:1, and react for 30 minutes; after the reaction, wash with DMF for 3 to 6 times; Then, piperidine was used to elute the protective group Fmoc of the amino acid, twice for 15 min each, followed by four washes with DMF and two washes with methanol.
[0019] 2. Weigh 3mmol of the second amino acid and 3mmol of HBTU into a reaction tube, add 0.5ml of DIEA, react for 40 minutes, wash with DMF for 3 to 6 times, add piperidine solutio...
Embodiment 2
[0023] Antibacterial Experiment of Small Molecule Peptide RK12
[0024] 1. Prepare Acinetobacter baumannii bacteria liquid, incubate at 37°C for 18 hours, and set aside;
[0025] 2. Prepare a small molecule polypeptide RK12 solution with a concentration of 10mg / ml, sterilize and dry a circular qualitative filter with a diameter of 5-7mm, and then immerse it in the above-mentioned small molecule polypeptide RK12 solution with different concentrations;
[0026] 3. Prepare LB medium, sterilize, and reserve;
[0027] 4. Melt LB medium, cool to 50°C, add 1ml of Acinetobacter baumannii bacteria solution, shake evenly, pour into a sterile plate, shake gently to spread the medium evenly on the plate;
[0028] 5. Use sterilized tweezers to neatly and orderly put the filter into the cooled plate, cover with a terracotta cover, mark it, and place it in a constant temperature incubator at 37°C for 24 hours;
[0029] 6. Use a caliper to measure the size of the inhibition zone, and compar...
Embodiment 3
[0035] Therapeutic Effect of Small Molecule Peptide RK12 in Animal Model
[0036] 1) Model construction
[0037] Cultivate Acinetobacter baumannii with LB medium to the logarithmic phase, then centrifuge at 3000rpm for 5min, wash 3 times with sterile normal saline, and finally resuspend with normal saline, and prepare 1×10 7 CFU / ml bacterial suspension, put in a 4°C refrigerator for later use.
[0038] The animal model of systemic infection with Acinetobacter baumannii was selected from male Kunming mice, which were divided into 7 groups, 6 mice in each group, and each group was injected with 1×10 7 The suspension of Acinetobacter baumannii of CFU / ml is grouped as follows: 1. Normal saline control group, RK12 (1,2,4mg / kg) and polymyxin E (0.25,0.5,1mg / kg) treatment group . Before inoculation, the immunosuppressive mouse model should be constructed first, and the construction method is as follows:
[0039] Prepare 20mg / ml cyclophosphamide solution with sterile normal saline...
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