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Targeted antibacterial peptide against Gram-negative bacteria and preparation method and application

A gram-negative bacterium and a technology for making it, applied in the biological field, can solve the problems that gram-negative bacteria have no effect and no medicine is available, and achieve the effects of reduced screening costs, high application potential, and low hemolytic activity

Active Publication Date: 2019-01-18
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to resist the invasion of drug-resistant bacteria, countries all over the world are stepping up the research and development of new antibiotics. However, the new antibiotics (including vancomycin) discovered in recent years are only for Gram-positive bacteria, but have no effect on Gram-negative bacteria. Effect
In fact, since the discovery of fluoroquinolones in the 1960s, no new drug for the treatment of Gram-negative bacteria has been clinically used in the past 50 years, and human beings are facing a terrible situation where no drug is available

Method used

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  • Targeted antibacterial peptide against Gram-negative bacteria and preparation method and application
  • Targeted antibacterial peptide against Gram-negative bacteria and preparation method and application
  • Targeted antibacterial peptide against Gram-negative bacteria and preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Use the R language to statistically analyze the amino acid sequence characteristics of the natural antibacterial peptide library, and perform statistical analysis on the peptide chain parameters that affect the antibacterial activity of the peptide chains that only target Gram-negative bacteria, and screen to obtain the best amino acid composition, namely K(lys), G(Gly), L(Leu); number of positive charges: 4, hydrophobicity: 30%-50%. On this basis, combined with the study of structure-function relationship, tryptophan was inserted into the center of the screening sequence to design a centrosymmetric short peptide sequence; to obtain a short-chain narrow-spectrum antimicrobial peptide sequence with low toxicity, high efficiency, and less likely to cause immune regulation disorders.

[0019] Amino acid sequence of table 1 antimicrobial peptide

[0020]

Embodiment 2

[0022] Synthesis of pheromone-labeled antimicrobial peptides by solid-phase chemical synthesis

[0023] 1. The preparation of antimicrobial peptides is carried out one by one from the C-terminal to the N-terminal, and is completed by a peptide synthesizer. First, Fmoc-X (X is the first amino acid at the C-terminal of each antimicrobial peptide) is inserted into Wang resin, and then the Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9 -Fmoxy-trimethyl-Y, Y is the second amino acid at the C-terminus of each antimicrobial peptide); according to this procedure, it is synthesized from the C-terminus to the N-terminus until the synthesis is completed, and the side of the Fmoc group is removed chain protection resin;

[0024] 2. Add a cleavage reagent to the peptide resin obtained above, react for 2 hours at 20°C in the dark, filter; wash the precipitate with TFA (trifluoroacetic acid), mix the washing liquid with the above filtrate, concentrate with a rotary evap...

Embodiment 3

[0028] Determination of Antimicrobial Peptide Activity

[0029] 1. Determination of antibacterial activity: the antimicrobial peptides are configured into a certain storage solution for use. The minimum inhibitory concentrations of several antimicrobial peptides were determined by the broth microdilution method. Using 0.01% acetic acid (containing 0.2% BSA) as the diluent, a series of gradient antimicrobial peptide solutions were sequentially prepared using the double dilution method. Take 100 μL of the above solution and place it in a 96-well cell culture plate, then add an equal volume of the bacteria solution to be tested (~10 5 individual / mL) in each well. Positive controls (containing bacterial fluid but not antimicrobial peptides) and negative controls (neither bacterial fluid nor peptides) were set up. Incubate at a constant temperature of 37°C for 20 hours, and the minimum inhibitory concentration is the one where no turbidity is seen at the bottom of the well with ...

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Abstract

The invention provides a targeted antibacterial peptide against Gram-negative bacteria and a preparation method and an application. A sequence of the targeted antibacterial peptide is as shown in a sequence table SEQ ID No. 1, and in the preparation method, an R language is utilized for statistical analysis of amino acid sequence characteristics of a natural antimicrobial peptide library, variouspeptide chain parameters affecting antibacterial activity of a peptide chain having the antibacterial activity only against Gram-negative bacteria are statistically analyzed, and the optimal amino acid composition is obtained by screening, namely K, G, L, with the number of positive charges being 4, and the hydrophobicity being 30%-50%. Tryptophan is inserted into the center of a screening sequence to obtain a centrally symmetric short peptide sequence through design, so as to obtain a short-chain narrow-spectrum antibacterial peptide sequence which is low in toxicity and highly effective, andnot easy to cause disorder of immune regulation in a body. The targeted antibacterial peptide is beneficial to maintaining micro-ecological balance while killing harmful bacteria. The antibacterial peptide has relatively high activity, high stability, relatively low cytotoxicity, and substitute antibiotics, thereby having an application potential for becoming a safer and environmentally-friendlynovel antibacterial product.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a targeted antimicrobial peptide for Gram-negative bacteria, a production method and application thereof. Background technique [0002] Antibiotics have been used in animal husbandry since the 1950s. As feed additives, they have played an important historical role in increasing the output of animal products and promoting the development of aquaculture. However, long-term extensive use and even abuse of antibiotics have led to the widespread emergence of drug-resistant bacteria and superbugs around the world, causing great harm to human public health and environmental safety. According to incomplete statistics, at least 25,000 people in Europe die from drug-resistant bacterial infections every year, directly causing economic losses of 1.5 billion euros; nearly 2 million people in the United States are caught in the predicament of drug-resistant bacteria every year, and 23,0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/08C07K1/04C07K1/20A61K38/10A61P31/04
CPCA61K38/00A61P31/04C07K7/08Y02A50/30
Inventor 单安山丑淑丽王志华李丘轲
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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