Artificially synthesized antimicrobial peptide, preparation method and application thereof

A technology for artificially synthesizing and antibacterial peptides, which is applied in the preparation methods, applications, and antibacterial drugs of peptides. It can solve the problems of small number of disulfide bonds, low content of antibacterial peptides, and small molecular weight. Strong antibacterial activity and small molecular weight

Inactive Publication Date: 2011-06-01
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problem that the structure of the existing antimicrobial peptides is unstable, easy to inactivate, the content of the antibacterial peptides in the existing organisms is extremely low, the method of expression through gene recombination contains multiple pairs of disulfide bonds, and the method of artificial synthesis is used because of the natural antibacterial peptides. Due to the shortcomings of long sequence and many disulfide bonds, the synthesis cost is too high. According to the active region of mytilin, a new type of antibacterial peptide is designed, which has small molecular weight, stable structure, strong antibacterial activity, short sequence, disulfide The number of keys is small and the cost is low

Method used

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  • Artificially synthesized antimicrobial peptide, preparation method and application thereof
  • Artificially synthesized antimicrobial peptide, preparation method and application thereof
  • Artificially synthesized antimicrobial peptide, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Solid-phase chemical synthesis, separation, purification and identification of antimicrobial peptides

[0038] 1. Artificial design of novel antimicrobial peptides

[0039]Using the structural simulation software ESyPred3D (Lambert C, Leonard N, De Bolle X, Depiereux E. ESyPred3D: Prediction of proteins 3D structures. Bioinformatics. 2002 Sep; 18(9): 1250-1256.), the Mediterranean mussel (Mytilus galloprovincialis ) mytilin B (Protein Structure Database No.: 2EEM) was used as a template to simulate the spatial structure of mytilin-1 from the thick-shelled mussel. According to the results of structural simulation, we found that mytilin-1 also has two β-sheets and a loop connecting the two β-sheets, and there are basic amino acid residues distributed on the rings. According to mytilin- 1, select the decapeptide fragment between alanine 20 (Ala20) and cysteine ​​29 (Cys29), and replace Ala20 and Ser22 with Cys to obtain a new polypeptide named MDP -1, the N-te...

Embodiment 2

[0047] Example 2: Functional detection of antimicrobial peptides

[0048] 1. Antibacterial experiments and antibacterial spectrum of new antimicrobial peptides

[0049] The antimicrobial activities of MDP-1 and MDP-2 were determined by medium dilution method. Bacteria were cultured in LB liquid medium to logarithmic growth phase (A 630nm 0.001), add 90 μL bacterial solution to each well of the 96-well plate. The peptide was pre-dissolved in PBS, the highest concentration was 1mM, and then double-diluted, the lowest concentration was 6.25μM. And the peptide solutions of various concentrations were added to 96-well plates, 10 μL / well, and PBS was used as a negative control.

[0050] Gently shake the 96-well plate on a shaker to mix the sample and the bacterial solution thoroughly, and then culture it at 37°C for 16-24h. The OD value of each well was measured by a microplate reader to measure the inhibitory effect of the polypeptide on bacterial growth. The minimum inhibitor...

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Abstract

The invention discloses an artificially synthesized antimicrobial peptide, which belongs to the field of biological pharmaceutical technology. The amino acid sequence of the artificially synthesized antimicrobial peptide is Cys-Val-Cys-Phe-Gly-Arg-ARG-Cys-Ile-Cys or Cys-Ile-Cys-Arg-Arg-Gly-Phe-Cys-Val-Cys. The invention also discloses a method for artificially synthesizing the antimicrobial peptide and application of the artificially synthesized antimicrobial peptide in development of preparing medicaments for treating Gram-positive bacteria and Gram-negative bacteria or feed additives. Compared with the prior art, the artificially synthesized antimicrobial peptide has the following advantages and effects, such as small molecular weight as compared with the natural ones, stable structure, strong antimicrobial activity, short sequence, less disulfide bond number, low cost and the like.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to artificially synthesized antibacterial peptides and applications thereof. Background technique [0002] Antimicrobial peptides, as an important part of the innate immune system, play an indispensable role in protecting biological organisms. So far, more than 800 antimicrobial peptides have been isolated and identified. According to their amino acid composition and spatial structure, they can be divided into (i) α-helical cationic linear antimicrobial peptides, (ii) antimicrobial peptides containing disulfide bonds, (iii) antimicrobial peptides rich in certain amino acid residues, and (iv) Anionic antimicrobial peptides. The antibacterial activity of antimicrobial peptides has the characteristics of high selectivity, rapid sterilization, broad spectrum of action and difficulty in forming resistance, making them ideal candidates for research and development of new an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K1/20C07K1/06C07K1/04A61K38/08A61P31/04A61P31/10A23K1/16A23K20/195
Inventor 廖智王日昕石戈刘梅鲁涛
Owner ZHEJIANG OCEAN UNIV
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