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Artificially synthesized antibacterial peptide and application thereof

An antibacterial peptide, artificial technology, applied in the field of biomedicine, can solve problems such as affecting antibacterial activity

Active Publication Date: 2021-08-31
江苏亢钧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Most of the naturally occurring antimicrobial peptide molecules discovered so far are composed of L-type amino acid residues. Because the molecules contain more basic amino acid residues, they are susceptible to degradation by various proteases, which affects their antibacterial activity.

Method used

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  • Artificially synthesized antibacterial peptide and application thereof
  • Artificially synthesized antibacterial peptide and application thereof
  • Artificially synthesized antibacterial peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1 Preparation of artificially designed synthetic antimicrobial peptides:

[0014] 1. Synthesize all D-type antimicrobial peptides, all L-type antimicrobial peptides and basic amino acid lysine residues as D-type amino acid residues and the rest Amino acid residues are the complete sequence of synthetic molecules composed of L-type amino acid residues. Desalted and purified by HPLC reverse phase C18 column chromatography.

[0015] 2. The molecular weight is determined by Fast atom bombardment mass spectrometry (FAB-MS), using glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1:1:1, V:V:V, volume ratio) as substrate, Cs ＋ As the bombardment particles, the current is 1 electrical microampere, and the emission voltage is 25 Kv.

[0016] 3. The purity of the purified antimicrobial peptide was identified by high performance liquid chromatography (HPLC), and the molecular weight was determined by fast atom bombardment mass spectrometry.

[0017] The sequence of ...

Embodiment 2

[0019] Example 2 Antimicrobial peptides of different configurations inhibit bacterial growth:

[0020] The antibacterial activity detection was carried out according to the conventional doubling dilution method. Add 90 microliters of bacterial solution to each space of the 96-well plate (final concentration 2×104CFU / ml), set 3 duplicate wells in each well, and use pH 7.0 LB medium (10g tryptone per liter, produced by Oxoid Company; 5g yeast Extract, produced by Oxoid Company; 10g sodium chloride, domestic analytical reagent) was used as negative control. Add 10 microliters of antimicrobial peptides prepared with sterile ultrapure water at different concentrations, so that the final concentration decreases exponentially. Incubate at 37°C for 14-16 hours, measure the absorbance of each well at 600 nm, and the lowest concentration with no change in absorbance is the minimum inhibitory concentration (MIC). In order to test the effect of antimicrobial peptides on marine bacteria ...

Embodiment 3

[0031] Example 3 Artificially designed synthetic antimicrobial peptides against protease hydrolysis:

[0032] Experimental method: trypsin, chymotrypsin, pepsin and elastase purchased from Sigma were mixed according to the ratio of enzyme: antimicrobial peptide = 1:10 (molar ratio), incubated at 37 °C for 24 h, and the antibacterial activity of Escherichia coli was tested Determination, the final concentration of antimicrobial peptides were 400 ug / ml. The antibacterial activity was tested by the agarose punching method, and the medium formula was: 1% low-melting point agarose (Sigma A6013), 0.3 mg / ml trypsin jelly (Oxoid product) dissolved in 10 mM pH 7.4Na 2 HPO 4 -NaH 2 PO 4 in the buffer. Add 20 ml of the prepared culture medium at 42°C to culture Escherichia coli ATCC 25922 in the logarithmic phase overnight (approximately 10 5 -10 6CFU), shake well to spread evenly in the 76mm diameter petri dish. After solidification, 3 mm round holes were punched on the culture m...

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Abstract

The invention provides a sequence of an artificially designed and synthesized antibacterial peptide and a preparation method thereof, and further provides application of the antibacterial peptide with different configurations to microorganism resistance. The antibacterial peptide disclosed by the invention has a remarkable effect of inhibiting the growth of bacteria and fungi. The antibacterial peptide disclosed by the invention has the beneficial characteristics of simple structure, artificial synthesis convenience and wide antibacterial spectrum; the full D-type and basic amino acids are composed of D-type amino acids, and the molecules have the characteristics of tolerance on degradation of various proteases, long antibacterial activity retention time and no drug residue; and the compound can be applied to prevention and treatment of infectious diseases of human or animals, artificial biological materials, food preservation, fruit and vegetable preservation and feed additives.

Description

technical field [0001] The invention provides an artificially designed and synthesized antibacterial peptide. The invention also relates to the preparation and application of the antibacterial peptide, which belongs to the field of biomedicine. Background technique [0002] Antimicrobial peptides are a class of small molecule polypeptides that exist in organisms and can resist external microorganisms. They are an important part of the innate immune system. Antimicrobial peptides usually consist of 15-45 amino acid residues, most of which are positively charged; antimicrobial peptides are widely distributed in amphibians, insects, plants and mammals. Compared with antibiotics widely used at present, antimicrobial peptides have many advantages: such as rapid and broad-spectrum killing of microorganisms at the minimum concentration of action; not easy to produce drug resistance; thermal stability, good water solubility; local infection and systemic Infection is effective, etc....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K1/04C07K1/06A61K38/16A61K45/06A61P31/04A61P31/10A23B7/154A23L3/3526
CPCC07K14/001A61K38/16A61K45/06A61P31/04A61P31/10A23L3/3526A23B7/154A23V2002/00A61K38/00A23V2250/55Y02A50/30
Inventor 李文辉张云钱晓明朱新鹏
Owner 江苏亢钧生物科技有限公司
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