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Antimicrobial peptide derived from scorpion venom as well as preparation method and application of antimicrobial peptide

A technology of antimicrobial peptides and self-scorpion toxins, which is applied in the direction of antibacterial drugs, medical preparations containing active ingredients, peptides, etc., can solve the problems of unfavorable use of antibacterial drugs, high cytotoxicity, and high hemolytic value, and achieve low hemolytic activity and Effects of eukaryotic cytotoxicity, reduced hemolytic activity, and high inhibitory effect

Active Publication Date: 2020-07-17
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The antibacterial peptide of scorpion venom Buthinin has too high hemolysis value and high cytotoxicity, which is not conducive to the use as an antibacterial drug

Method used

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  • Antimicrobial peptide derived from scorpion venom as well as preparation method and application of antimicrobial peptide
  • Antimicrobial peptide derived from scorpion venom as well as preparation method and application of antimicrobial peptide
  • Antimicrobial peptide derived from scorpion venom as well as preparation method and application of antimicrobial peptide

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Design of Antimicrobial Peptides

[0016] The amino acid sequence of scorpion venom Buthinin is:

[0017] SIVPIRCRSNRDCRRFCGFRGGRCTYARQCLCGY;

[0018] By intercepting the 14 amino acids in the middle of the scorpion venom Buthinin, a polypeptide containing 5 positive charges and a hydrophobic value of -4.22 was obtained. The amino acid sequence of a peptide RSN obtained is: RSNRDCRRFCGFRG;

[0019] Replace negatively charged aspartic acid in RSN with positively charged arginine, and then replace serine, glycine and asparagine in the peptide chain with leucine to obtain RLL, whose amino acid sequence is: RLLRRCRRFCLFRL;

[0020] Its positive charge content is 6, and its hydrophobic value is increased to 0.448.

[0021] The RLLs obtained through transformation all have high cell selectivity. The N-terminus of RLL is acetylated to raise a positive charge and increase the stability of the peptide. The sequences of the antimicrobial peptides are shown in Table 1.

[00...

Embodiment 2

[0025] Synthesis of RLL Antimicrobial Peptides by Solid Phase Chemical Synthesis

[0026] 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;

[0027] 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 evaporator, and th...

Embodiment 3

[0031] Determination of antimicrobial activity of antimicrobial peptides

[0032] 1. Determination of antibacterial activity: The minimum inhibitory concentration of several antibacterial peptides was determined by the micro broth dilution 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. Cultivate at a constant temperature of 37°C for 14-18h, measure the light absorption value at 492nm (OD492nm) with a microplate reader, and determine the minimum inhibitory concentration. The test results are shown in Table 2.

[00...

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Abstract

The invention provides an antimicrobial peptide derived from scorpion venom as well as a preparation method and application of the antimicrobial peptide. The antimicrobial peptide RLL has a sequence shown as SEQ ID No.1. A peptide RSN is obtained by intercepting 14 amino acids in the middle of the scorpion toxin, negatively charged aspartic acid in the peptide RSN is replaced with positively charged arginine, and serine, glycine and asparagine in a peptide chain are replaced with leucine to obtain the antimicrobial peptide RLL; and the antimicrobial peptide RLL has very low hemolytic activityand eukaryotic cytotoxicity, the antimicrobial peptide cannot cause 10% erythrocyte hemolysis at a concentration of 128 [mu]mol / L, the survival rate of mouse macrophages RAW 264.7 reaches 80% or more,and the antimicrobial peptide has the development potential of becoming an antibiotic substitute.

Description

technical field [0001] The invention belongs to the application field of agricultural animal husbandry and veterinary medicine, and in particular relates to an antibacterial peptide derived from scorpion venom, a preparation method and application thereof. Background technique [0002] Antimicrobial peptides are active polypeptides with antibacterial effects that widely exist in organisms. They are immune response products of biological nonspecific defense systems and have broad-spectrum antibacterial, antiviral, antifungal, antiparasitic and antitumor biological activities. Antimicrobial peptides produce antibacterial or bactericidal effects mainly through physical penetration on bacterial cell membranes, and it is difficult for microorganisms such as bacteria to change the cell membrane structure of their own phospholipid bilayers, thus making antimicrobial peptides resistant to drugs. Greatly reduced. Therefore, the study of antimicrobial peptides has become a research h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435A61K38/17A61P31/04
CPCC07K14/43522A61P31/04A61K38/00
Inventor 董娜薛宸宇方禹鑫
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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