CN111518168A

CN111518168A - Antibacterial peptide derived from carnivorous bacteriocin and preparation method and application thereof

Info

Publication number: CN111518168A
Application number: CN202010234601.4A
Authority: CN
Inventors: 董娜; 方禹鑫; 汪陈思
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2020-08-11
Anticipated expiration: 2040-03-30
Also published as: CN111518168B

Abstract

The invention provides an antibacterial peptide derived from carnivorous bacitracin and a preparation method and application thereof, wherein the sequence of the antibacterial peptide WRW is shown as SEQ ID No. 1. The invention obtains a peptide WGW by intercepting 17 amino acids at the carboxyl terminal of carnivorous bacitracin, and neutral glycine, negatively charged glutamic acid, and glutamine and asparagine with uncharged polarity in the peptide WGW are replaced by arginine with positive charge; then serine, threonine and valine in the peptide chain are replaced by leucine to obtain peptide WRW; the antibacterial peptide has high-efficiency antibacterial action and very low hemolytic activity and eukaryotic cytotoxicity, causes 4.07 percent of erythrocyte hemolysis under the concentration of 128 mu mol/L, fails to cause 10 percent of erythrocyte hemolysis and leads the survival rate of mouse macrophage RAW264.7 to reach 89.57 percent, and improves the development potential of becoming an antibiotic substitute.

Description

Antibacterial peptide derived from carnivorous bacteriocin and preparation method and application thereof

Technical Field

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

Background

Antibiotics are always powerful weapons for human beings to treat diseases caused by pathogenic microorganism infection, but with the massive use of traditional antibiotics, the drug resistance of pathogenic bacteria is gradually enhanced, and the antibiotic age is gradually coming to the end. The best antibiotics at present are also gradually losing effectiveness, so that the search for new antibacterial drugs has become a focus of much attention. Especially, the recent report on the superbacteria suggests that the police bell is knocked. The search for a new green feed supplement to replace antibiotics has become an urgent task.

The antibacterial peptides are various in types and are widely distributed in nature, but the examples of the antibacterial peptides really applied to the fields of medicine and livestock husbandry are few, and the factors limiting the application of the antibacterial peptides are many, wherein the low antibacterial activity of some antibacterial peptides is a main influence factor limiting the application of the antibacterial peptides. Therefore, it is imperative to find a method for effectively improving the antibacterial activity of the antibacterial peptide on the premise of ensuring no toxicity to normal cells. Hybrid design is a good strategy to optimize these antimicrobial peptide molecules. This method is capable of retaining the biological activity of the desired fragment well without being affected by a change in a parameter. Carnobacterin Carnobacteriocin X has a low MIC value of antibacterial activity, has poor inhibition effect on bacteria, and is not favorable for being used as an antibacterial drug.

Disclosure of Invention

The invention aims to disclose an antibacterial peptide derived from carnobacteriaceae element Carnobacteriocin X and a preparation method thereof. So that it possesses the advantages of high activity and low toxicity.

The purpose of the invention is realized as follows: a WRW derived from carnivorous bacitracin, and the sequence of the WRW is shown as SEQ ID No. 1.

The invention also has the following features: a process for the preparation of WRW derived from carnivosine as described above, characterized in that it comprises: obtaining a polypeptide WGW which contains 1 positive charge and has a hydrophobic value of-0.43 by intercepting 17 amino acids at the carboxyl terminal of carnobacteriaceae element Carnobacteriocin X, wherein the amino acid sequence of the polypeptide WGW is shown as SEQ ID No. 2; replacing neutral glycine, negatively charged glutamic acid, and polar uncharged glutamine and asparagine in the polypeptide WGW with positively charged arginine; and then replacing serine, threonine and valine in the peptide chain with leucine to obtain polypeptide WRW, wherein the sequence of the polypeptide WRW is shown as SEQ ID No.1, the positive charge content of the polypeptide WRW is 6, and the hydrophobic value is increased to 0.65.

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

The invention has the advantages that: the method of the invention simplifies carnivorous bacitracin by intercepting or replacing, reserves the active center, reduces the hemolytic activity of the antibacterial peptide under the condition of not reducing the bactericidal activity of the antibacterial peptide, and improves the selectivity of the antibacterial peptide between bacterial cells and mammalian cells. The antibacterial peptide RLL is subjected to antibacterial and hemolytic activity detection, and found to have a high-efficiency inhibiting effect on six strains such as escherichia coli, staphylococcus aureus, staphylococcus epidermidis, salmonella typhimurium and the like, and have very low hemolytic activity and eukaryotic cytotoxicity, the antibacterial peptide causes 4.07% of erythrocyte hemolysis under the concentration of 128 mu mol/L, 10% of erythrocyte hemolysis cannot be caused, the survival rate of mouse macrophage RAW264.7 reaches 89.57%, and the development potential of the antibacterial peptide RLL becoming an antibiotic substitute is improved.

Drawings

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

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

FIG. 3 is a graph showing hemolytic activity of the antibacterial peptide WRW and melittin ME of the present invention.

FIG. 4 is a graph showing the cytotoxic effect of the antibacterial peptides WRW and melittin ME of the present invention 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 carnobacterium element Carnobacteriocin X is as follows:

WGWKEVVQNGQTIFSAGQKLGNMVGKIVPLPFG；

by intercepting the 17 amino acids at the carboxy terminus of Carnobacteriocin X, a polypeptide containing 1 positive charge with a hydrophobic value of-0.43 was obtained.

The obtained polypeptide WGW has the amino acid sequence as follows:

WGWKEVVQNGQTIFSAG-NH₂；

the neutral glycine, negatively charged glutamic acid and polar uncharged glutamine and asparagine in polypeptide WGW are replaced with positively charged arginine.

And then substituting serine, threonine and valine in the peptide chain with leucine to obtain polypeptide WRW, wherein the amino acid sequence of the polypeptide WRW is as follows: WRWKRLLRRGRLIFLAG-NH₂；

The positive charge content is 6, and the hydrophobic value is increased to 0.65.

The WRW polypeptide obtained by modification has high cell selectivity. The N-terminus of WRW is acetylated to increase one positive charge and increase the stability of the peptide. The sequence of antimicrobial peptide WRW is shown in table 1.

TABLE 1 amino acid sequence of derived peptides

Example 2

Solid phase chemical synthesis method for synthesizing WRW 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 equilibrium with 0.2mol/L sodium sulfate (pH is adjusted to 7.5 by phosphoric acid) for 30min, dissolving polypeptide with 90% acetonitrile water solution, filtering, performing C18 reversed-phase normal pressure column, performing gradient elution (eluent is methanol and sodium sulfate water solution are mixed according to a volume ratio of 30: 70-70: 30), the flow rate is 1mL/min, the detection wave is 220nm, collecting main peak, and freeze-drying; further purifying with reverse phase C18 column, wherein eluent A is 0.1% TFA/water solution; 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 1mL/min, and then the main peak is collected and freeze-dried as above;

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. Determination of antibacterial Activity: the minimum inhibitory concentrations of several antimicrobial peptides were determined using the broth dilution method. Serial gradients of antimicrobial peptide solutions were prepared sequentially using a two-fold dilution method with 0.01% acetic acid (containing 0.2% BSA) as the diluent. Taking 100 mu L of the solution, placing the solution into a 96-hole cell culture plate, and then respectively adding the bacterial liquid to be detected (10-10) with the same volume⁵one/mL) in each well. Positive controls (containing the bacterial solution but not the antimicrobial peptide) and negative controls (containing neither the bacterial solution nor the peptide) were set separately. Culturing at 37 deg.C for 14-18h, and measuring light at 492nm (OD492nm) with microplate readerAnd (4) determining an absorption value and determining the minimum inhibitory concentration. The results are shown in Table 2.

TABLE 2 bacteriostatic Activity of antimicrobial peptides

As can be seen from table 2, WRW exhibits high bacteriostatic activity against gram-negative and positive bacteria.

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

2. Determination of hemolytic Activity: collecting 1mL of fresh human blood, dissolving heparin in a 2mLPBS solution after anticoagulation, centrifuging for 5min at 1000g, and collecting erythrocytes; washed 3 times with PBS and resuspended in 10mL PBS; uniformly mixing 50 mu L of erythrocyte suspension with 50 mu L of antibacterial peptide solution dissolved by PBS and having different concentrations, and incubating for 1h at constant temperature in an incubator at 37 ℃; l h taking out, centrifuging at 4 deg.C for 5min at 1000 g; taking out the supernatant, and measuring the light absorption value at 570nm by using an enzyme-labeling instrument; the average value of each group is taken and compared and analyzed. Wherein 50 μ L red blood cells plus 50 μ LPBS served as negative controls; 50 μ L of red blood cells plus 50 μ L of 0.1% Tritonx-100 served as a positive control. The minimum hemolytic concentration is the concentration of antimicrobial peptide at which the antimicrobial peptide causes a 10% hemolytic rate. The results are shown in FIG. 3. As can be seen from FIG. 3, WRW showed no hemolytic activity in the detection range, caused 4.07% of erythrocytic hemolysis at the concentration of 128. mu. mol/L, failed to cause 10% of erythrocytic hemolysis, and showed significant difference from 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) Experimental treatment of the cells to be used: the cells were washed and resuspended 3 times in sterile PBS and digested with 0.25% trypsin solution to detach them from the bottom of the flask, rinsed with complete medium to obtain a single cell suspension, while filling a 96-well plate with 50 μ L of cell suspension at a final concentration of about 2 × 104.

(3) And (3) antibacterial peptide treatment: after 10. mu.L of antibacterial peptide is added into the first hole of the additional 96-well plate and diluted in multiple proportion, 50. mu.L of peptide liquid is taken out and added into 1-10 holes of the original 96-well plate, 50. mu.L of complete culture medium is added into 11 holes, and 100. mu.L of complete culture medium is added into 12 holes. Culturing for 4h at constant temperature;

(4) and (3) toxicity detection: adding 50 μ L of 5mg/mL MTT solution into 96-well plate, culturing for 3-4h, adding 150 μ L DMSO (dimethyl sulfoxide), and OD (OD) with microplate reader_570nmThe absorbance was measured. Higher absorbance values demonstrate less toxicity and vice versa. The results are shown in FIG. 4. As can be seen from FIG. 4, WRW showed no toxicity to mouse macrophage in the detection range, and the survival rate of mouse macrophage RAW264.7 at 128. mu. mol/L concentration reached 89.57%, which is significantly different from the control group melittin.

Sequence listing

<110> northeast university of agriculture

<120> an antibacterial peptide derived from carnobacterine, preparation method and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Trp Arg Trp Lys Arg Leu Leu Arg Arg Gly Arg Leu Ile Phe Leu Ala

1 5 10 15

Gly

<210>2

<211>17

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Trp Gly Trp Lys Glu Val Val Gln Asn Gly Gln Thr Ile Phe Ser Ala

1 5 10 15

Gly

Claims

1. An antibacterial peptide WRW derived from carnivorous bacitracin, characterized in that the sequence thereof is shown as SEQ ID No. 1.

2. The preparation method of antibacterial peptide WRW derived from carnobacterine according to claim 1, which is characterized in that: obtaining a polypeptide WGW which contains 1 positive charge and has a hydrophobic value of-0.43 by intercepting 17 amino acids at the carboxyl terminal of carnobacteriaceae element Carnobacteriocin X, wherein the amino acid sequence of the polypeptide WGW is shown as SEQ ID No. 2; replacing neutral glycine, negatively charged glutamic acid, and polar uncharged glutamine and asparagine in the polypeptide WGW with positively charged arginine; and then replacing serine, threonine and valine in the peptide chain with leucine to obtain polypeptide WRW, wherein the sequence of the polypeptide WRW is shown as SEQ ID No.1, the positive charge content of the polypeptide WRW is 6, and the hydrophobic value of the polypeptide WRW is increased to 0.65.

3. Use of an antibacterial peptide WRW derived from carnobacterine according to claim 1 in the preparation of a medicament for treating infectious diseases of gram-positive bacteria or/and gram-negative bacteria.