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Hornet phallotoxin antitone analog WVD-II and preparation method and application thereof

A technology of WVD-II and wasp venom, which is applied in the preparation method of peptides, chemical instruments and methods, peptides, etc., can solve the problems of limiting clinical drug applications, and achieve significant bactericidal effect, good stability, and low hemolytic toxicity.

Active Publication Date: 2020-05-05
DALI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a large number of natural antimicrobial peptides have been found to be expected to replace the problem of drug resistance caused by the abuse of traditional antibiotics, there are still many shortcomings of natural antimicrobial peptides, which limit their application as clinical drugs, especially in terms of toxicity and Aspects of enzymatic stability

Method used

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  • Hornet phallotoxin antitone analog WVD-II and preparation method and application thereof
  • Hornet phallotoxin antitone analog WVD-II and preparation method and application thereof
  • Hornet phallotoxin antitone analog WVD-II and preparation method and application thereof

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preparation example Construction

[0022] The present invention also provides a preparation method of the venomin reverse analog WVD-II of the above scheme, which comprises the following steps:

[0023] 1) According to the amino acid sequence of the reverse analog WVD-II of the venomin peptide, the amino acids are sequentially coupled to the resin to obtain a peptide resin;

[0024] 2) The peptide resin described in step 1) is cleaved, a side chain protecting group scavenger is added, the target polypeptide is precipitated, and the precipitate is collected by centrifugation to obtain the venetin reverse analog WVD-II.

[0025] In the present invention, before the amino acids are sequentially coupled to the resin according to the amino acid sequence of the venetin reverse analog WVD-II, it preferably also includes the processes of washing the resin, soaking the resin, removing the amino protective group and washing the resin again The resin preferably includes Rink Amide MBHA resin (4-(2′,4′-dimethoxyphenyl-fluorenylme...

Embodiment 1

[0034] Using Fmoc protected amino acid for solid-phase synthesis, using RinkAmide MBHA resin as a carrier, 1-hydroxybenzotriazole and O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate Salt is used as coupling reagent. The cleavage of peptide from resin uses lysing solution trifluoroacetic acid and side chain protecting group scavenger (phenol / water / anisyl sulfide / 1,2-dimercaptoethanol), react at room temperature for 4~6h After removing most of the trifluoroacetic acid by rotary evaporation, ether was added dropwise at 0℃ to obtain a flocculent precipitate. After centrifugation, the crude peptide was obtained. The crude peptide was initially separated with a Sephadex G-15 column. The column was equilibrated with 5% acetic acid. 1mL·min -1 Next, eluted with 5% acetic acid, detected at 280nm, collected the first peak, and freeze-dried. A semi-preparative high performance liquid chromatograph was used for purification, and the most abundant peak was collected. The puri...

Embodiment 2

[0048] Example 2 Reverse High Performance Liquid Chromatography Purification and Mass Spectrometry Identification of Vespidin Reverse Analog WVD-Ⅱ

[0049] Purification was performed on a high performance liquid chromatograph, using Sepax Bio-C18 reverse phase column (10.0×250mm, 10μm, ). The oxidized crude peptide sample was dissolved in 1 mL of ultrapure water, centrifuged at 10,000 rpm for 5 min, and detected by W2998 detector with detection wavelengths of 215, 254, and 280 nm. The mobile phase is a four-phase elution system with a flow rate of 3mL·min -1 . The eluents were 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile solution (B). The column temperature was 25°C. The elution gradient of reversed-phase high performance liquid chromatography is shown in Table 1. Collect the elution peaks and use Q- TOF mass spectrometry analysis, the identification results are shown in Table 2. The molecular weight of the mutants analyzed by mass spectrometry is consistent with the...

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Abstract

The invention provides a hornet phallotoxin antitone analog WVD-II and a preparation method and application thereof, and belongs to and relates to the technical field of polypeptide synthesis. The amino acid sequence of the hornet phallotoxin antitone analog WVD-II is as shown in SEQID NO:1. The N end of the hornet phallotoxin antitone analog WVD-II disclosed by the invention has positive charges,so that the N end can be combined with a cell membrane having negative electricity; an alpha helical structure is formed from hydrophobic amino acids at a C end, and can bore a hole in the surface ofthe cell membrane, so that death of bacteria is caused. The in vitro antibacterial activity determination indicates that the hornet phallotoxin antitone analog WVD-II disclosed by the invention has significant sterilizing effects on gram-positive bacteria, gram-negative bacteria and fungi, has good stability, is low in hemolytic toxicity and has the characteristics of being broad-spectrum, efficient and safe. The hornet phallotoxin antitone analog WVD-II disclosed by the invention can be used as an active component clinically for replacing traditional antibiotics for treatment.

Description

Technical field [0001] The present invention relates to the technical field of polypeptide synthesis, in particular to a venetin reverse sequence analogue WVD-II and its preparation method and application. Background technique [0002] Antimicrobial peptides can resist microbial invasion, and have specific or non-specific effects on Gram-negative bacteria, positive bacteria, fungi, viruses, tumors and eukaryotes. In addition to antimicrobial effects, antimicrobial peptides have other functions. Antimicrobial peptides are mostly used in the form of polypeptide analogs or polypeptide derivatives. These substances can be obtained by modifying the amino acids of natural antimicrobial peptides. Although it has been found that a large number of natural antimicrobial peptides are expected to replace the drug resistance problem caused by the abuse of traditional antibiotics, there are still many shortcomings in the current natural antimicrobial peptides, which limit its application as c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/20C07K1/18C07K1/06C07K1/04A61P31/04A61P31/10
CPCC07K7/08A61P31/04A61P31/10Y02P20/55
Inventor 刘衡高孟婷陈壮志王音李超男巫秀美
Owner DALI UNIV
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