CN111423501A

CN111423501A - Antibacterial peptide derived from scorpion venom as well as preparation method and application thereof

Info

Publication number: CN111423501A
Application number: CN202010234847.1A
Authority: CN
Inventors: 董娜; 薛宸宇; 方禹鑫
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-07-17
Anticipated expiration: 2040-03-30
Also published as: CN111423501B

Abstract

The invention provides an antibacterial peptide derived from scorpion venom, a preparation method and application thereof.A sequence of the antibacterial peptide R LL is shown as SEQ ID No. 1. A peptide RSN is obtained by intercepting 14 middle amino acids of the scorpion venom, and then, arginine with positive charge is replaced by aspartic acid with negative charge in the peptide RSN, and serine, glycine and asparagine in a peptide chain are replaced by leucine to obtain the antibacterial peptide R LL which has very low hemolytic activity and eukaryotic cytotoxicity, cannot cause 10% of erythrocyte hemolysis under the concentration of 128 mu mol/L, and the survival rate of mouse macrophage RAW264.7 reaches more than 80%, and becomes the development potential of an antibiotic substitute.

Description

Antibacterial peptide derived from scorpion venom as well as preparation method and application thereof

Technical Field

The invention belongs to the field of agricultural, livestock and veterinary application, and particularly relates to an antibacterial peptide derived from scorpion venom, and a preparation method and application thereof.

Background

The antibacterial peptide is an active polypeptide with antibacterial effect widely existing in organisms, is an immune response product of a biological non-specific defense system, and has biological activities of broad-spectrum antibiosis, antivirus, antifungal, antiparasitic, antitumor and the like. The antibacterial peptide mainly acts on the bacterial cell membrane through physical permeation to generate a bacteriostatic or bactericidal effect, and microorganisms such as bacteria and the like hardly change the cell membrane structure of the phospholipid bilayer of the microorganisms, so that the probability of generating drug resistance of the antibacterial peptide is greatly reduced. Therefore, the research of the antibacterial peptide becomes a research hotspot in the fields of genetic engineering, drug development and the like, and has extremely wide market application prospect.

At present, thousands of antibacterial peptides have been extracted from various animal and plant bodies, but few natural antibacterial peptides have been used in the medical field as antibiotic substitutes. One important reason that limits the natural antimicrobial peptides to be antibiotic substitutes is: since natural antimicrobial peptides also cause hemolysis of normal cells such as erythrocytes, it is imperative to find a method for improving the cell selectivity of antimicrobial peptides. Cell selectivity is in terms of the different selective effects of the antimicrobial peptide on different cells. The antibacterial peptide has the function of inhibiting or killing harmful microorganisms, and has high cell selectivity without toxicity to normal cells. The higher the cell selectivity of the antimicrobial peptide, the greater the feasibility of becoming an antibiotic alternative. The antibacterial peptide of scorpion venom buthin has over-high hemolytic value and high cytotoxicity, and is not favorable for being used as an antibacterial medicament.

Disclosure of Invention

The invention aims to simplify the scorpion toxin buthin and improve the cell selectivity, reduce the hemolytic activity of the antibacterial peptide under the condition of not reducing the bactericidal activity of the antibacterial peptide and improve the selectivity of the antibacterial peptide between bacterial cells and mammalian cells.

The aim of the invention is realized by that an antibacterial peptide R LL derived from scorpion toxin has a sequence shown in SEQ ID No. 1.

The invention is also characterized in that the preparation method of the scorpion toxin-derived antibacterial peptide R LL is characterized in that 14 amino acids in the middle of scorpion toxin Butinin are intercepted to obtain a polypeptide RSN with 5 positive charges and a hydrophobic value of-4.22, the amino acid sequence of the polypeptide RSN is shown as SEQ ID No.2, the negatively charged aspartic acid in the polypeptide RSN is replaced by the positively charged arginine, and the serine, the glycine and the asparagine in the peptide chain are replaced by the leucine to obtain the polypeptide R LL, the amino acid sequence of the polypeptide R LL is shown as SEQ ID No.1, the content of the positive charges is 6, the hydrophobic value is increased to 0.448, and the N end of the polypeptide R LL is acetylated to improve one positive charge and increase the stability of the peptide.

The invention also discloses application of the antibacterial peptide R LL in preparing a medicament for treating gram-positive bacteria or/and gram-negative bacteria infectious diseases.

The method has the advantages that the method simplifies the buthin and improves the cell selectivity, reduces the hemolytic activity of the antibacterial peptide under the condition of not reducing the bactericidal activity of the antibacterial peptide, improves the selectivity of the antibacterial peptide between bacterial cells and mammalian cells, and tests on the antibacterial activity and the hemolytic activity of the antibacterial peptide R LL prove that the antibacterial peptide has high-efficiency inhibiting effect on six strains such as escherichia coli, staphylococcus aureus, staphylococcus epidermidis, salmonella typhimurium and the like, has very low hemolytic activity and eukaryotic cytotoxicity, causes 2.24% of erythrocyte hemolysis under the concentration of 128 mu mol/L, fails to cause 10% of erythrocyte hemolysis and has the survival rate of mouse macrophage RAW264.7 reaching 93.17%, and becomes the development potential of antibiotic substitutes.

Drawings

FIG. 1 is a high performance liquid chromatogram of the antimicrobial peptide of the present invention.

FIG. 2 is a high performance liquid mass spectrum of the antimicrobial peptide of the present invention.

FIG. 3 is a graph of the hemolytic activity of peptide R LL and melittin ME.

FIG. 4 is a graph of the cytotoxic effect of peptide R LL and melittin ME on mouse macrophages.

Detailed Description

The invention is further illustrated by way of example in the accompanying drawings of the specification:

example 1

Design of antimicrobial peptides

The amino acid sequence of the scorpion venom buthin is as follows:

SIVPIRCRSNRDCRRFCGFRGGRCTYARQCLCGY；

the polypeptide containing 5 positive charges and the hydrophobic value of-4.22 is obtained by intercepting the middle 14 amino acids of the scorpion venom buthin. The obtained peptide RSN has the amino acid sequence as follows: RSNRDCRRFCGFRG, respectively;

replacing the negatively charged aspartic acid in the RSN with arginine with positive charge, and replacing serine, glycine and asparagine in a peptide chain with leucine to obtain R LL, wherein the amino acid sequence of the R LL is R LL RRCRRFC L FR L;

the positive charge content is 6, and the hydrophobic value is increased to 0.448.

The R LL obtained by modification has higher cell selectivity, the N end of R LL is acetylated to improve a positive charge and increase the stability of the peptide, and the sequence of the antibacterial peptide is shown in the table 1.

TABLE 1 amino acid sequence of derived peptides

Example 2

Solid phase chemical synthesis method for synthesizing R LL antibacterial peptide

1. The preparation of the antibacterial peptide is carried out one by one from the C end to the N end and is completed by a polypeptide synthesizer. Firstly, Fmoc-X (X is the first amino acid of the C end of each antibacterial peptide) is grafted to Wang resin, and then an Fmoc group is removed to obtain X-Wang resin; then Fmoc-Y-Trt-OH (9-fluorenylmethoxycarbonyl-trimethyl-Y, Y is the second amino acid at the C end of each antibacterial peptide); synthesizing the resin from the C end to the N end in sequence according to the procedure until the synthesis is finished to obtain the resin with the side chain protection of the Fmoc group removed;

2. adding a cutting reagent into the obtained peptide resin, reacting for 2 hours at 20 ℃ in a dark place, and filtering; washing precipitate TFA (trifluoroacetic acid), mixing washing liquor with the filtrate, concentrating by a rotary evaporator, adding precooled anhydrous ether with the volume about 10 times of that of the filtrate, precipitating for 3 hours at the temperature of-20 ℃, separating out white powder, centrifuging for 10min by 2500g, collecting precipitate, washing the precipitate by the anhydrous ether, and drying in vacuum to obtain polypeptide, wherein a cutting reagent is prepared by mixing TFA, water and TIS (triisopropylchlorosilane) according to the mass ratio of 95:2.5: 2.5;

3. performing column balance for 30min by using 0.2 mol/L sodium sulfate (pH is adjusted to 7.5 by phosphoric acid), dissolving polypeptide by using 90% acetonitrile water solution, filtering, performing C18 reverse phase normal pressure column, performing gradient elution (eluent is methanol and sodium sulfate water solution are mixed according to the volume ratio of 30: 70-70: 30), the flow rate is 1m L/min, the detection wave is 220nm, collecting a main peak, performing freeze-drying, further purifying by using a reverse phase C18 column, the eluent A is 0.1% TFA/water solution, the eluent B is 0.1% TFA/acetonitrile solution, the elution concentration is 25% B-40% B, the elution time is 12min, the flow rate is 1m L/min, collecting the main peak and performing freeze-drying;

4. identification of antibacterial peptides: the obtained antibacterial peptide is analyzed by electrospray mass spectrometry, the molecular weight (shown in figures 1 and 2) shown in a mass spectrogram is basically consistent with the theoretical molecular weight in table 1, and the purity of the antibacterial peptide is more than 95%.

Example 3:

determination of antibacterial Activity of antibacterial peptides

1. The antibacterial activity is measured by measuring the minimum inhibitory concentration of several antibacterial peptides by a trace broth dilution method, taking 0.01% acetic acid (containing 0.2% BSA) as a diluent, sequentially preparing a series of gradient antibacterial peptide solutions by a two-fold dilution method, placing the solutions 100 mu L in a 96-hole cell culture plate, and then respectively adding bacterial solutions (about 10) to be measured with the same volume⁵M L) in each well, respectively setting a positive control (containing bacterial liquid but not containing antibacterial peptide) and a negative control (containing neither bacterial liquid nor peptide), culturing at constant temperature of 37 ℃ for 14-18h, measuring the light absorption value at 492nm (OD492nm) by using an enzyme-labeling instrument, and determining the minimum inhibitory concentration and the detection result, which are shown in table 2.

TABLE 2 bacteriostatic Activity of antimicrobial peptides

As can be seen from table 2, R LL shows higher bacteriostatic activity against gram-negative and gram-positive bacteria.

TABLE 3 MHC (. mu.M), GM (. mu.M) and SI values of the short peptides

2. The hemolytic activity is measured by collecting 1m L fresh human blood, dissolving heparin after anticoagulation in 2m L PBS solution, centrifuging for 5min at 1000g, collecting erythrocytes, washing with PBS for 3 times, re-suspending with 10m L PBS, mixing 50 μ L erythrocyte suspension with 50 μ L antibacterial peptide solution dissolved in PBS with different concentrations, incubating at 37 deg.C for 1h, taking out l h, centrifuging for 5min at 4 deg.C and 1000g, taking out supernatant, measuring light absorption at 570nm with enzyme labeling instrument, averaging each group, and comparing for analysis, wherein 50 μ L erythrocytes plus 50 μ L PBS as negative control, 50 μ L erythrocytes plus 50 μ L0.1.1% Tritonx-100 as positive control, and the antibacterial peptide concentration when the minimum hemolytic concentration is 10% hemolytic rate caused by antibacterial peptide, is shown in FIG. 3.

As can be seen from FIG. 3, the antimicrobial peptide R LL showed no hemolytic activity within the detection range, and showed significant difference from the control melittin.

Determination of eukaryotic cytotoxicity: cytotoxicity assays were performed using MTT and by mouse macrophage RAW 264.7.

(1) Preparation of culture medium and culture of cells: DMEM (culture medium) and fetal calf serum 9:1 are mixed to prepare a complete culture medium, and mouse macrophage RAW264.7 in liquid nitrogen is recovered, preferably 80% -90% of cells are grown on the bottom of a bottle.

(2) Test treatment of the cells to be used, the cells were washed and resuspended 3 times with sterile PBS and digested with 0.25% trypsin solution to be detached at the bottom of the flask and rinsed with complete medium to obtain a single cell suspension, while a 96-well plate was filled with a 50. mu. L cell suspension at a final concentration of about 2 × 104.

(3) Antibacterial peptide treatment, namely adding 10 mu L antibacterial peptide into a first hole of an additional 96-well plate, diluting by multiple times, taking out 50 mu L peptide liquid, adding 1-10 holes of the original 96-well plate, adding 50 mu L complete culture medium into 11 holes, adding 100 mu L complete culture medium into 12 holes, and carrying out constant-temperature culture for 4 hours;

(4) the toxicity detection comprises adding 50 μ L5 mg/m L MTT solution into 96-well plate, culturing for 3-4 hr, adding 150 μ L DMSO (dimethyl sulfoxide), and labeling with enzyme OD_570nmMeasuring pipetteThe higher the luminosity. absorbance values, the weaker the toxicity was demonstrated and vice versa the results of the assay are shown in FIG. 4 it can be seen from FIG. 4 that R LL showed no toxicity to mouse macrophages in the range of the assay, with a significant difference in control melittin.

Sequence listing

<110> northeast university of agriculture

<120> an antibacterial peptide derived from scorpion venom, its preparation method and application

<160>1

<170>SIPOSequenceListing 1.0

<210>2

<211>14

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Arg Leu Leu Arg Arg Cys Arg Arg Phe Cys Leu Phe Arg Leu

1 5 10

Claims

1. An antibacterial peptide R LL derived from scorpion toxin, which is characterized in that the sequence is shown as SEQ ID No. 1.

2. The method for preparing the antibacterial peptide R LL derived from scorpion toxin according to claim 1, wherein 14 amino acids in the middle of buthin are intercepted to obtain a polypeptide RSN with 5 positive charges and a hydrophobic value of-4.22, the amino acid sequence of the polypeptide RSN is shown in SEQ ID No.2, the negatively charged aspartic acid in the polypeptide RSN is replaced by the positively charged arginine, and serine, glycine and asparagine in the peptide chain are replaced by leucine to obtain a polypeptide R LL, the amino acid sequence of the polypeptide R LL is shown in SEQ ID No.1, the content of the positive charges is 6, the hydrophobic value is increased to 0.448, and the N-terminal of the polypeptide R LL is acetylated to increase one positive charge and the stability of the peptide.

3. The use of the scorpion toxin-derived antibacterial peptide R LL according to claim 1 in the preparation of medicaments for treating gram-positive bacteria or/and gram-negative bacteria infectious diseases.