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A plurality of reconstructing antibiotic peptide and preparation method and application thereof

An antibacterial peptide and gram-positive bacteria technology, applied in the field of modified antibacterial peptides and their preparation, can solve problems such as loss of binding ability, easy to cause hemolysis, etc., achieve easy preparation, enhance bactericidal power and LPS binding power, reduce Effects of side effects such as hemolysis

Inactive Publication Date: 2007-12-05
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Scholars such as Nagaoka I replaced all the negatively charged amino acid residues of LL-37, and increased the net charge of LL-37 to +11, then the bactericidal effect was significantly enhanced, and maintained the same under the condition of 175mmol / L NaCL Bactericidal, but lost the ability to combine with LPS, and easily lead to hemolysis (Clin Diagn Lab Immunol, 2002, 9 (5): 972); Kuwahara-Arai K and other scholars increased the positive charge in the LL-37 peptide chain The number of amino acids and hydrophobic amino acids can be used to increase the bactericidal power and the ability to antagonize endotoxin, but it will have a dose-responsive cytotoxic effect on the cells of the body (Inflamm Res, 2005, 54(2): 66)

Method used

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  • A plurality of reconstructing antibiotic peptide and preparation method and application thereof
  • A plurality of reconstructing antibiotic peptide and preparation method and application thereof
  • A plurality of reconstructing antibiotic peptide and preparation method and application thereof

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Experimental program
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Embodiment 1

[0058] As shown in Figure 1A and Figure 1B, Figure 1A is a spatial helical structure diagram of natural LL-37, and Figure 1B is a charge distribution diagram of natural LL-37. The three-dimensional structure of NMR of natural LL-37 is detected, and the spatial structure of LL-37 can be obtained, and the position of each amino acid residue and each atom in LL-37 in space, the connecting bonds between atoms, Angle, length and other data can accurately obtain the spatial helical structure of LL-37 and its charge distribution diagram. Peptide rLL-37, namely rLL-37-1~rLL-37-17, rLL-24-1~rLL-24-8, rLL-18-1~rLL-18-1, rLL-30-1~rLL- 30-10. The sequence listing is as follows:

[0059] (1) Reconstructed 37-peptide antibacterial peptide sequence (rLL-37):

[0060] rLL-37-1:

[0061] Leu Leu Gly Asn Phe Phe Arg Lys Ser Lys Asn Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg

[0062] Ile Lvs Asp Phe Leu Arg Asn Leu Val Pro Arg Thr Glu Ser

[0063] rLL-37-2:

[0064] Leu Leu Gly Asn P...

Embodiment 2

[0179] In order to prepare the rebuilt rLL-37 peptide antimicrobial peptide, the present invention also synthesized a set of negatively charged carrier protein sequence (Carrier protein molecule, CPM), which is based on the desired protein sequence. Designed for negative charge and high water solubility, it was detected on Gene Bank and has no identical gene sequence.

[0180] (1) The gene sequence of the stored protein is:

[0181] GAA GTT TGG AAC GCA CTT GAT GCA CTG GAG CTG GTA ATCCAA CAA GAG GAG GGT TCT AAT GGT ACT TCT ACT GGA TCC GAGGGC

[0182] (2) The amino acid sequence of the stored protein is:

[0183] Glu Val Trp Asn Ala Leu Asp Ala Leu Glu Leu Val Ile Gln Gln Glu Glu Glu Ser Asn Gly Thr Ser Thr Gly Ser Glu Gly

[0184] (3) The charge carrying the protein is: pHi=2.7; at pH7.4, the charge is -6.0

Embodiment 3

[0186] Preparation of 24-peptide series antimicrobial peptides and 18-peptide series antimicrobial peptides:

[0187] The antibacterial peptide sequence is relatively short, although it has a strong positive charge, by appropriately increasing the pH value of the synthesis solution to 8.35, the charge on the short peptide can be appropriately reduced, so that it can be directly synthesized by solid-phase chemical method.

[0188] (1) Preparation of rLL-24-1:

[0189] Synthesis of rLL-24-1 polypeptide by solid-phase chemical synthesis was carried out on an ABI 431A peptide synthesizer produced by PE Company in the United States. The standard Fmoc protocol method was used to couple arginine twice.

[0190] First, 0.25 mmol of HMP resin (hydroxymethylphenoxymethyl polystyrene resin, produced by PE Company) was selected, and the peptide chain was extended from the carboxyl terminal to the amino terminal one by one according to the polypeptide sequence.

[0191] After the synthes...

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Abstract

The present invention discloses one set of modified antibacterial peptides and their preparation process and application. The modified antibacterial peptides include 37 modified antibacterial peptides, i. e., rLL-37-1 to rLL-37-17 antibacterial peptides, rLL-24-1 to rLL-24-8 antibacterial peptides, rLL-18-1 to rLL-18-2 antibacterial peptides and rLL-30-1 to rLL-30-10 antibacterial peptides. The present invention also provides serial negatively charged protein carriers for preparing the modified antibacterial peptides. The modified antibacterial peptides are applied in medicine, can reduce hemolysis and other side reactions, and may be prepared easily.

Description

technical field [0001] The invention relates to the fields of protein information and gene recombination, in particular to a group of modified antimicrobial peptides and their preparation application. Background technique [0002] Antibacterial peptide (Antibacterial Peptide) (also known as cecropin, peptide antibiotic) is a small molecule polypeptide with biological activity induced by organisms. It is a class of polypeptides with endogenous bactericidal activity widely present in many organisms. It is an important part of the body's natural immunity (Innate Immunity). In 1980, it was first isolated from H. cecropia by Boman and other scholars, and later isolated antimicrobial peptides with very similar structures and functions from rats, monkeys and other species. There are more than 100 species in total. They have been shown to have broad-spectrum bactericidal activity against Gram-positive and negative bacteria, fungi, etc. [0003] So far, antimicrobial peptides in th...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/04C12N15/12C12N15/09A61K38/17A61P31/04
Inventor 葛晓冬
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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