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Non-natural fully D-type snake venom cathelicidin antibacterial peptide and derivative, preparation method as well as application thereof

An antibacterial peptide and derivative technology, applied in the field of biomedicine, can solve problems such as affecting antibacterial activity, and achieve the effects of prolonging action time, maintaining antibacterial activity, and facilitating artificial synthesis.

Inactive Publication Date: 2012-01-11
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently discovered Cathelicidin antimicrobial peptide molecules of naturally occurring snake venoms 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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  • Non-natural fully D-type snake venom cathelicidin antibacterial peptide and derivative, preparation method as well as application thereof
  • Non-natural fully D-type snake venom cathelicidin antibacterial peptide and derivative, preparation method as well as application thereof
  • Non-natural fully D-type snake venom cathelicidin antibacterial peptide and derivative, preparation method as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1 Preparation of non-natural full D-type snake venom Cathelicidin antibacterial peptide and derivatives thereof:

[0025] 1. Use different D-type amino acid residues as raw materials to synthesize the full sequence of unnatural all-D-type snake venom Cathelicidin antimicrobial peptide and its derivatives with a fully automatic peptide synthesizer. Reversed phase C by HPLC 18 Desalted and purified by column chromatography.

[0026]2. Molecular weight was determined by Fast atom bombardment mass spectrometry (FAB-MS), with glycerol: m-nitrobenzyl alcohol: dimethyl sulfoxide (1:1:1, V:V:V, volume ratio ) as the substrate, Cs + As the bombardment particles, the current is 1μA and the emission voltage is 25Kv.

[0027] 3. The purity of the purified snake venom antimicrobial peptide was identified by high performance liquid chromatography (HPLC), and the molecular weight was determined by fast atom bombardment mass spectrometry.

[0028] The sequences of the sy...

Embodiment 2

[0039] The antibacterial growth inhibition effect of embodiment 2 non-natural full D-type snake venom Cathelicidin antibacterial peptide or derivatives thereof:

[0040] The antibacterial activity detection was carried out according to the conventional doubling dilution method. Add 90 μl of bacterial solution to each empty 96-well plate, (final concentration 10 6 CFU / ml), each hole is provided with 3 duplicate holes, with pH7.0LB medium (every liter contains 10 grams of tryptone, produced by Oxoid Company, 5 grams of yeast extract, produced by Oxoid Company and 10 grams of sodium chloride, domestic analysis Pure reagent) was used as negative control. Add 10 μl 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 600nm, and the lowest concentration with no change in absorbance is the minimum inhibitory concen...

Embodiment 3

[0050] Example 3 Non-natural full D-type snake venom Cathelicidin antibacterial peptide and its derivatives resist protease hydrolysis:

[0051] Experimental method: After mixing trypsin, chymotrypsin, pepsin and elastase purchased from Sigma at a ratio of enzyme:antibacterial peptide=1:10 (molar ratio), the antibacterial activity of Escherichia coli was carried out after incubation at 37°C for 24 hours The final concentration of antimicrobial peptides was determined to be 400 μg / ml. Antibacterial activity detection was carried out by agarose punching method, and the medium formula was: 1% low-melting point agarose (Sigma A6013), 0.3mg / ml tryptone (Oxoid product) dissolved in 10mM pH 7.4Na 2 HPO 4 -NaH 2 PO 4 in the buffer. Add 20ml of the prepared medium at 42°C to overnight culture of Escherichia coli ATCC 25922 in logarithmic growth phase (about 10 5 -10 6 CFU), shake well and spread evenly in a 76mm diameter petri dish. After solidification, 3 mm circular holes were...

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Abstract

The invention provides sequences of a snake venom cathelicidin antibacterial peptide consisting of non-natural fully D-type amino acid, which cannot be easily degraded by protease, and a derivative thereof and a preparation method thereof, and also provides application of the snake venom cathelicidin antibacterial peptide consisting of the non-natural fully D-type amino acid and the derivative thereof. The snake venom cathelicidin antibacterial peptide consisting of the non-natural fully D-type amino acid and the derivative thereof have an obvious effect of suppressing growth of bacteria and fungus, in particular to an excellent effect of suppressing clinical drug-resistant bacteria, have the beneficial characteristics of simple structure, convenience in artificial synthesis and wide antibacterial spectrum system, have the obvious characteristics of capability of resisting degradation of various protease, long antibacterial activity retention time and no drug residue, and can be applied to prevention and treatment of human or animal infected diseases, clinical artificial biomaterials, food preservative and fruit and vegetable refreshment, feed additives and daily chemical supplies.

Description

technical field [0001] The invention provides antibacterial peptides composed of unnatural full D-type snake venom Cathelicidin and derivatives thereof. The invention also relates to the preparation and application of these peptides and derivatives thereof, belonging 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 to 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, peptide antibiotics have many advantages: such as rapid and broad-spectrum killing of microorganisms at the minimum effective concentration; not easy to produce drug resistance; thermal stability, good water solubility; local...

Claims

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Application Information

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IPC IPC(8): C07K14/46C07K1/04A61K38/17A61K8/64A61P31/00A61P35/00A61Q1/00A61Q19/10A61Q9/02A61Q9/00A61Q11/00A61L15/44A61L27/22A61L31/04A23L3/3526A23B7/154A23K1/17A23K20/195
Inventor 李文辉张云李盛安赵峰
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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