Derived peptide IR2 of pig-derived antibacterial peptide as well as preparation method and application thereof
A technology of derivatized peptides and antimicrobial peptides, applied in the field of porcine-derived antimicrobial peptide IR2 and its preparation, can solve the problems of unsatisfactory antibacterial activity and high cytotoxicity of natural antimicrobial peptides
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Embodiment 1
[0017] Design of Antimicrobial Peptides
[0018] The amino acid sequence of porcine antimicrobial peptide PG-1 is:
[0019] Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val Gly Arg-NH 2
[0020] 1 5 10 15 18
[0021] By intercepting the amino acid sequence CRRRFC (8-13 amino acids) of the β-turn position of PG-1, the charged arginine R and the hydrophobic amino acid I are selected to extend the sheets at both ends of the PG-1 turn position. By intercepting the amino acid sequence of the PG-1β-turn, and connecting the two ends of the turn with IR or RI respectively, the IR series peptides were designed: (IR) n H(RI) n -NH 2 , (n=1, 2; H is the β-turn amino acid sequence CRRRFC of PG-1), when n=1, the derived peptide is named IR1; when n=2, the derived peptide is named IR2. The amino acid sequences of the derived peptides are shown in Table 1.
[0022] Table 1 Amino Acid Sequence of Derived Peptides
[0023]
[0024] The charge numbers of IR1 and IR2 a...
Embodiment 2
[0026] Synthesis of Two Antimicrobial Peptides IR1 and IR2 by Solid Phase Chemical Synthesis
[0027] 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 chain protection resin;
[0028] 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 evapor...
Embodiment 3
[0031] Embodiment 3: the mensuration of antimicrobial peptide antibacterial activity
[0032] 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 th...
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