Linear polypeptide and synthetic method and application thereof
A linear polypeptide and linear technology, applied in the field of antibacterial peptides, can solve the problems of narrow antibacterial spectrum, easy generation of drug resistance, ineffectiveness, etc., and achieve the effects of low hemolytic toxicity, less resistance to drug resistance, and easy and safe operation.
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Embodiment 1
[0025] Preparation and separation and purification of embodiment 1 antimicrobial peptide HR-1
[0026] The amino acid sequence of the antimicrobial peptide HR-1 is shown in Table 1.
[0027] This embodiment adopts the solid-phase peptide synthesis method. The specific steps are as follows: the assembly of amino acids is carried out one by one from the C-terminal to the N-terminal, and is controlled manually. First weigh 0.1 mmol of Wang resin (purchased from Jill Biochemical (Shanghai) Co., Ltd.) combined with the first amino acid, namely Asn, pack it into a column, and use 20% volume ratio of dichloromethane (DCM) dimethylformamide (DMF ) solution was swollen for 30 min, and then the protecting group (Fmoc) was deprotected with 30% by volume piperidine dimethylformamide solution, and washed 3 times with DMF. The amino acid Fmoc-D-Phe-OH protected by 9-fluorenylmethoxycarbonyl (Fmoc) was dissolved in tripyrrolylphosphineoxybenzotriazole hexafluorophosphorus (PyBOP), hydroxyb...
Embodiment 2
[0030] The antibacterial activity detection of embodiment 2 synthetic antimicrobial peptides
[0031] Various bacterial strains used in the following examples were purchased from China Institute of Biological Products.
[0032] The antibacterial activity of the synthesized antimicrobial peptide was detected by using the 96-well plate method. The specific steps were: resuscitate the strain, inoculate the slant and cultivate overnight at 37°C, pick the bacteria in MH medium, cultivate overnight at 37°C, dilute the bacterial solution to make Its concentration is 10 4 ~10 5 CFU / ml, inoculate 100 μl of bacterial solution per well in a 96-well plate, then dilute the polypeptide in a certain proportion, add 10 μl to each well, and culture the 96-well plate at 37°C overnight, and detect the OD with a microplate reader 570 value. The test results are listed in Table 2.
[0033] Growth concentration of bacteria containing antimicrobial peptides (OD 570 ) and the growth concentratio...
Embodiment 3
[0038] Example 3 In Vitro Hemolytic Activity Detection
[0039] This embodiment is used to detect the hemolytic activity of the synthetic linear antimicrobial peptide on human red blood cells, and the blood samples used are taken from normal human blood.
[0040] The specific detection steps are: human red blood cells are washed with PBS buffer solution (35mM phosphate buffer solution / 0.15mol / L NaCl, pH 7.0), take 100 μl of 8% human red blood cell suspension in a 96-well plate, and add 100 μl of antimicrobial peptide solution was shaken at 37°C for 1 hour, centrifuged at 1500 rpm for 5 minutes, and 100 μl of supernatant was transferred to a new 96-well plate, and the absorption at 414nm wavelength was detected by a microplate reader. The positive control used 0.1% Triton X-100, and the negative control used PBS. The test results are listed in Table 3.
[0041] Table 3 Hemolytic activity of five antimicrobial peptides
[0042]
[0043] The smaller the hemolytic rate value...
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