Artificially synthesized antimicrobial peptide, preparation method and application thereof
A technology for artificially synthesizing and antibacterial peptides, which is applied in the preparation methods, applications, and antibacterial drugs of peptides. It can solve the problems of small number of disulfide bonds, low content of antibacterial peptides, and small molecular weight. Strong antibacterial activity and small molecular weight
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Embodiment 1
[0037] Example 1: Solid-phase chemical synthesis, separation, purification and identification of antimicrobial peptides
[0038] 1. Artificial design of novel antimicrobial peptides
[0039]Using the structural simulation software ESyPred3D (Lambert C, Leonard N, De Bolle X, Depiereux E. ESyPred3D: Prediction of proteins 3D structures. Bioinformatics. 2002 Sep; 18(9): 1250-1256.), the Mediterranean mussel (Mytilus galloprovincialis ) mytilin B (Protein Structure Database No.: 2EEM) was used as a template to simulate the spatial structure of mytilin-1 from the thick-shelled mussel. According to the results of structural simulation, we found that mytilin-1 also has two β-sheets and a loop connecting the two β-sheets, and there are basic amino acid residues distributed on the rings. According to mytilin- 1, select the decapeptide fragment between alanine 20 (Ala20) and cysteine 29 (Cys29), and replace Ala20 and Ser22 with Cys to obtain a new polypeptide named MDP -1, the N-te...
Embodiment 2
[0047] Example 2: Functional detection of antimicrobial peptides
[0048] 1. Antibacterial experiments and antibacterial spectrum of new antimicrobial peptides
[0049] The antimicrobial activities of MDP-1 and MDP-2 were determined by medium dilution method. Bacteria were cultured in LB liquid medium to logarithmic growth phase (A 630nm 0.001), add 90 μL bacterial solution to each well of the 96-well plate. The peptide was pre-dissolved in PBS, the highest concentration was 1mM, and then double-diluted, the lowest concentration was 6.25μM. And the peptide solutions of various concentrations were added to 96-well plates, 10 μL / well, and PBS was used as a negative control.
[0050] Gently shake the 96-well plate on a shaker to mix the sample and the bacterial solution thoroughly, and then culture it at 37°C for 16-24h. The OD value of each well was measured by a microplate reader to measure the inhibitory effect of the polypeptide on bacterial growth. The minimum inhibitor...
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