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Tryptophan tension chain-beta-hairpin antibacterial peptide and preparation method and application thereof

A tryptophan zipper and antibacterial peptide technology, applied in the biological field, can solve the problems of increasing synthesis difficulty, production cost, loss, biological activity decline, etc.

Inactive Publication Date: 2017-05-31
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current research on antibacterial peptides with β-hairpin structure is still insufficient. The main reason is that it is difficult for short peptides to form a stable β-hairpin structure. Usually, more than one pair of cysteines are required to form internal disulfide bonds in the molecule to make antimicrobial peptides Molecular cyclization stabilizes its secondary structure, which not only increases the difficulty and production cost of peptide synthesis, but also disulfide bonds are easily cleaved by substances containing thiol groups in organisms, resulting in a decrease or even loss of biological activity

Method used

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  • Tryptophan tension chain-beta-hairpin antibacterial peptide and preparation method and application thereof
  • Tryptophan tension chain-beta-hairpin antibacterial peptide and preparation method and application thereof
  • Tryptophan tension chain-beta-hairpin antibacterial peptide and preparation method and application thereof

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Effect test

Embodiment 1

[0015] Molecular Design of Antimicrobial Peptides

[0016] according to figure 1 The molecular structure of the water-transporting tryptophan W is arranged in pairs on the non-hydrogen bond formation sites of the two arms of the hairpin to form a cross-chain W-W interaction to stabilize the β-hairpin structure. The positively charged tryptophan is selected Lysine K is arranged on the hydrogen bond formation site to make the antibacterial peptide cationic, so that the length of the two arms of the hairpin is extended in pairs with WK and KW as repeating units, and the WK series antimicrobial peptides are designed: (WK) n D PG(KW) n -NH 2 , n=2, 3, 4; when n=2, the antimicrobial peptide was named WK2; when n=3, the antimicrobial peptide was named WK3, and when n=4, the antimicrobial peptide was named WK4. Its amino acid sequence is shown in Table 1.

[0017] Table 1 Amino Acid Sequence of Derived Peptides

[0018]

[0019] The carboxyl termini of the three peptides WK2,...

Embodiment 2

[0021] Synthesis of Three Antimicrobial Peptides WK2, WK3 and WK4 by Solid Phase Chemical Synthesis

[0022] 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;

[0023] 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...

Embodiment 3

[0027] Determination of antimicrobial activity of antimicrobial peptides

[0028] 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 naked eye....

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Abstract

The invention provides a tryptophan tension chain-beta-hairpin antibacterial peptide and a preparation method and application thereof. A sequence of the antibacterial peptide is as shown in a sequence table SEQ ID No.1. The preparation method comprises the following steps: by taking a tryptophan tension chain structure as a template, combining amino acid composition and structural characteristics of the antibacterial peptide; and by using interaction of cross-chain W-W as a structural stable factor, symmetrically and circularly arranging an amino acid lysine with charges and a hydrophobic amino acid tryptophan as repeated binary sequence units on two arms of the beta-hairpin to obtain the antibacterial peptide WK3. The antibacterial peptide has relatively high bacteriostatic activity and cell selectivity, and the treatment index reaches up to 161.27. The antibacterial peptide designed by the method needs not to bind a disulfide bond but has extremely high structural stability, so that the selectivity of the antibacterial peptide between bacterial cells and mammalian cells is improved, and the antibacterial peptide has an application potential as an antibiotic substituent. The antibacterial peptide is relatively high in activity and relatively low in cytotoxicity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a tryptophan zipper β-hairpin antibacterial peptide and its preparation method and application. Background technique [0002] The rapid development of the feed industry is inseparable from the important role and contribution of antibiotic feed additives. However, the long-term overuse or even abuse of antibiotics has led to the widespread emergence of drug residues and drug-resistant strains around the world, posing a direct threat to public health and environmental safety. Antimicrobial peptides have the characteristics of high antibacterial activity, no residue, and easy production, and their antibacterial mechanism is different from traditional antibiotics, making bacteria less resistant to antibiotics, so they have the potential to be developed into a new generation of antibacterial drugs. Among them, β-hairpin structure antimicrobial peptides have received widesprea...

Claims

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Application Information

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IPC IPC(8): C07K7/08C07K7/06C07K1/06C07K1/04A61K38/10A61K38/08A61P31/04
CPCA61K38/00C07K7/06C07K7/08
Inventor 单安山徐林丑淑丽马清泉董娜
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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