A tail-anchored α-helix antimicrobial peptide gw4a and preparation method and application thereof
An antibacterial peptide and α-helix technology, applied in the field of α-helix antibacterial peptide GW4A and its preparation, can solve the problems of inactivated clinical application, poor stability, unsatisfactory antibacterial activity, etc.
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Embodiment 1
[0017] Design of Antimicrobial Peptides
[0018] Taking W4 as the active center, the sequence of peptide W4 is: RWRWWWRWR, anchored with different hydrophobic amino acids (Ala, Trp, Val, Ile, Phe, Leu) at its tail, and designing a cap structure with glycine at the N-terminus. A series of antimicrobial peptides; when glycine is added to the N-terminal and alanine is anchored at the tail, it is named GW4A. The amino acid sequences of GW4A and W4A are shown in Table 1 when only glycine is anchored at the tail end.
[0019] Table 1 Amino acid sequences of GW4A and W4A
[0020] peptide amino acid sequence Molecular weight (Dalton) wxya Gly Arg Trp Arg Trp Trp Trp Arg Trp Arg Ala-NH2 1700.99 W4A Arg Trp Arg Trp Trp Trp Arg Trp Arg Ala-NH2 1643.94
[0021] The charge numbers of GW4A and W4A are +5, respectively, and the average hydrophobicity values are 0.684 and 0.752, respectively. The carboxyl termini of both peptides, GW4A and W4A, were amid...
Embodiment 2
[0023] Synthesis of Two Antimicrobial Peptides GW4A and W4A by Solid Phase Chemical Synthesis
[0024] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminal to the N-terminal, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminal of each antimicrobial peptide) is inserted into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9 -Fmoxy-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus until the synthesis is completed, and the side of the Fmoc group is removed Resin for chain protection.
[0025] 2. Add a cleavage reagent to the peptide resin obtained above, react for 2 hours at 20°C in the dark, filter; wash the precipitate with TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate with a rotary e...
Embodiment 3
[0028] Embodiment 3: Determination of biological activity of antimicrobial peptides
[0029] 1. Determination of antibacterial activity: Prepare the peptide as a storage solution for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. Take 100 μL of the above solution and place it in a 96-well cell culture plate, then add an equal volume of the bacteria solution to be tested (~10 5 individual / mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the bottom of the well with the...
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