CN109553657B

CN109553657B - Non-perfect amphiphilic peptide W4 and preparation method and application thereof

Info

Publication number: CN109553657B
Application number: CN201811452528.7A
Authority: CN
Inventors: 单安山; 何诗琪; 杨占一; 王家俊
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2018-11-30
Filing date: 2018-11-30
Publication date: 2021-10-19
Anticipated expiration: 2038-11-30
Also published as: CN109553657A

Abstract

The invention provides a non-perfect amphiphilic peptide W4, and a preparation method and application thereof. The sequence of the imperfect amphipathic peptide W4 is shown in SEQ ID No. 1. The preparation method comprises the following steps: the invention selects 4 arginines as positive charge amino acid and 5 tryptophans as hydrophobic amino acid, two arginines are inserted into the tryptophans as a unit, the helical wheel projection forms a 'four-side' conformation, namely two hydrophobic surfaces and two hydrophilic surfaces, and the four arginines form two pairs of intramolecular hydrogen bonds, and the derived peptide is named as W4.

Description

Non-perfect amphiphilic peptide W4 and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a non-perfect amphiphilic peptide W4, and a preparation method and application thereof.

Background

The application of antibiotics as feed additives dates back to 1943, and Americans first found that some antibiotic fermentation residues can promote the growth of pigs. In 1949 American Cunha and Stokstad used penicillin for the first time to perform systematic comparison experiments in animals,

the results prove that the application of the antibiotic has better effects of preventing diseases and promoting growth of animals. In 1950, the U.S. food and drug administration officially approved the use of antibiotics such as penicillin in feed. Because the weight gain rate is improved by promoting the growth of animals; the reproductive performance of animals is improved; the survival rate is improved, and the morbidity is reduced; the utilization rate of the feed is improved, the cost is saved, and the like, and the antibiotic is rapidly and widely applied as a non-nutritive feed additive. While the use of antibiotics is of great interest to humans, its abuse is increasingly troublesome to humans. At present, all conventional antibiotics have corresponding drug-resistant pathogenic strains, and the drug resistance problem of pathogenic bacteria increasingly threatens the health of people. The search for new types of antibacterial substances is an effective way to solve the problem of drug resistance.

The antibacterial peptide is a micromolecular polypeptide for resisting the invasion of external microorganisms and clearing mutant cells in vivo of animal organisms, has the advantages of broad-spectrum antibacterial property, no toxicity, no drug resistance, no residue, no pollution and the like, has good thermal stability and small additive amount, completely meets the requirement of animal product safety, is suitable for being used in the feed production process, and has potential as a new generation feed additive. The antibacterial mechanism of the antibacterial peptide is different from that of antibiotics, and the antibacterial peptide kills cells by destroying cell membranes of bacteria to cause intracellular solutes to leak out, so that the bacteria are not easy to generate drug resistance. More importantly, the antibacterial peptide has little effect on eukaryotic cells, and only acts on prokaryotic cells and diseased eukaryotic cells. According to the reasons, the antibacterial peptide is completely possible to replace antibiotics to form a novel, efficient, low-toxicity and residue-free antibacterial substance, and has a wide application prospect. However, the natural cationic antibacterial peptide is not perfect, most of the natural antibacterial peptides have the defects of weak antibacterial activity, relatively narrow antibacterial spectrum, higher synthesis cost, certain toxicity to eukaryotes, high killing activity to pathogenic microorganisms, hemolysis to eukaryotes, sensitivity to protease and the like. Therefore, how to improve the activity and reduce the toxicity to the maximum becomes the aim of modifying the antibacterial peptide molecule, and is also the difficulty and hope of the development of the current antibacterial peptide drugs. Therefore, modifying antibacterial peptide to have higher antibacterial activity and reduce its toxicity has become a hot spot of research.

Disclosure of Invention

The invention aims to provide a non-perfect amphiphilic peptide W4, a preparation method and application thereof; the antibacterial peptide has high activity on gram-negative bacteria and relatively low cytotoxicity.

The purpose of the invention is realized by the following technology: a non-perfect amphiphilic peptide W4, the sequence of which is shown in SEQ ID No. 1.

The invention also has the following technical characteristics:

1. a novel non-perfect amphipathic peptide W4 as described above was prepared as follows: 4 arginines are selected as positive charge amino acid and 5 tryptophans are selected as hydrophobic amino acid, two arginines are inserted into the tryptophans as a unit, the projection of a spiral wheel forms a four-side conformation, namely two hydrophobic surfaces and two hydrophilic surfaces, and the four arginines form two pairs of intramolecular hydrogen bonds, the derived peptide is named as W4, and the sequence is shown as SEQ No. 1.

2. The application of the non-perfect amphipathic peptide W4 in preparing the medicine for treating gram-negative bacteria infectious diseases.

The invention has the following beneficial effects and advantages: the method has simple experimental technology, and performs antibacterial and hemolytic activity detection on the obtained antibacterial peptide, finds that W4 has obvious inhibiting effect on 3 gram-negative bacteria strains such as escherichia coli, salmonella typhimurium, pseudomonas aeruginosa and the like and positive bacteria such as staphylococcus aureus, staphylococcus epidermidis, methicillin-resistant staphylococcus aureus and the like, the treatment index of W4 is as high as 80.76, and the method has very low hemolytic activity, improves the selectivity of the antibacterial peptide between bacterial cells and mammalian cells, and improves the development potential of the antibacterial peptide as an antibiotic substitute. In conclusion, W4 is an antibacterial peptide with higher application value.

Drawings

FIG. 1 is a mass spectrum of antibacterial peptide W4.

FIG. 2 is a perspective view of the spiral wheel of antimicrobial peptide W4.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

Design of antimicrobial peptides

4 arginines are selected as positive charge amino acid and 5 tryptophans are selected as hydrophobic amino acid, two arginines are inserted into the tryptophans as a unit, the helical wheel projection forms a four-side conformation, namely two hydrophobic surfaces and two hydrophilic surfaces, and the four arginines form two pairs of intramolecular hydrogen bonds, the derived peptide is named as W4, and the sequence is shown in the following table.

TABLE 1 amino acid sequence of derived peptides

The charge numbers of W4 were +4, and the water repellency values were all 0.801. The carboxy terminus of W4 was amidated to increase one positive charge and increase the stability of the peptide. By adopting the method, the hemolytic activity of the antibacterial peptide is reduced under the condition of improving the bactericidal activity of the antibacterial peptide, the selectivity of the antibacterial peptide between bacterial cells and mammalian cells is improved, and the development potential of the antibacterial peptide as an antibiotic substitute is improved.

Example 2

Solid phase chemical synthesis method for synthesizing W4 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: when the antibacterial peptide obtained by the method is analyzed by electrospray mass spectrometry, the molecular weight (shown in figure 1) 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%.

The sample information parameters in fig. 1 are as follows:

interface: electrospray ion source voltage: +4.5kv

A dissolution method: gas flow rate for 15% acetonitrile + 85% water spray: 1.50L/min probe: -0.2kv

Radio frequency temperature: flow rate at 250 ℃: 0.2ml/min

Sample introduction amount: 1 μ l radio frequency voltage: 0v

Ratio of mobile phase: 50% Water/50% methanol

Blocking temperature: 200

The sequence is as follows: RWRWRWRWW-NH2

Modification: c-terminal amidation

Theoretical value: 1572.85

Detection value: 1572.00

Example 3: determination of antibacterial Activity of antibacterial peptides

1. Determination of antibacterial Activity: the peptides were prepared as a stock solution for use. 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 20h, and determining the minimum inhibitory concentration when no turbidity is observed at the bottom of the well. The results are shown in Table 2.

TABLE 2 bacteriostatic Activity of W4

As can be seen from the table 2, the antibacterial activity of W4 is higher, and the bactericidal activity for gram-positive bacteria is slightly better than that for gram-negative bacteria, which indicates that W4 has the potential to become a new generation of antibacterial drugs.

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 ℃; taking out after 1h, centrifuging at 4 ℃ 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 Table 3.

TABLE 3 determination of the hemolytic Activity of the antimicrobial peptides

As can be seen from Table 3, W4 showed no hemolytic activity in the detection range.

The above results show that the antibacterial activity of the compound is remarkably improved by forming a four-face structure and simultaneously forming W4 with two pairs of hydrogen bonds by four arginines. By comprehensively analyzing the bacteriostatic and hemolytic activities of the antibacterial peptides, the biological activity of each antibacterial peptide can be more comprehensively evaluated through a therapeutic index (the ratio of hemolytic concentration to bacteriostatic concentration). As can be seen from Table 3, W4 has a higher therapeutic index, indicating that the designed W4 antibacterial peptide has higher development potential of replacing antibiotics.

Sequence listing

<110> northeast university of agriculture

<120> non-perfect amphiphilic peptide W4, and preparation method and application thereof

<140> 201811452528.7

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<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial sequence

<220>

<221> MOD_RES

<222> (9)..(9)

<223> amidation

<400> 1

Arg Trp Arg Trp Arg Trp Arg Trp Trp-NH2

1 5

Claims

1. The application of the non-perfect amphipathic peptide W4 in preparing the medicine for treating gram-negative bacteria infectious diseases is disclosed, wherein the sequence of the non-perfect amphipathic peptide W4 is shown as SEQ ID No. 1.