Antimicrobial peptide CN-1 and its application

Abstract

The present invention provides an antimicrobial peptide CN-1 and its application, belonging to the technical field of peptide inhibitors. The amino acid sequence of the antimicrobial peptide CN-1 is TILPKRKVMPFL, as shown in SEQ ID NO.1. The minimum inhibitory concentration (MIC) of the antimicrobial peptide CN-1 against Staphylococcus aureus and Escherichia coli is 10.0 mg / mL and 5.0 mg / mL, respectively. The present invention adopts the above-mentioned antimicrobial peptide CN-1 and its application. The antimicrobial peptide CN-1 has antibacterial activity against Escherichia coli and Staphylococcus aureus, and exerts its effect by destroying the bacterial cell membrane; the antimicrobial peptide CN-1 is not easy to produce drug resistance, has low cytotoxicity and high cell selectivity, and can be used to prepare an antimicrobial composition for treating Escherichia coli and Staphylococcus aureus infections.

Description

Technical Field

[0001] The present invention relates to the technical field of peptide inhibitors, in particular to antimicrobial peptide CN-1 and applications thereof. Background Art

[0002] As the global population continues to grow, the incidence of foodborne diseases is also increasing year by year. Staphylococcus aureus, S.aureus ) and Escherichia coli ( Escherichia coli, E. coli ) are two common and important foodborne pathogens. Escherichia coli can not only cause diarrhea and food poisoning, but also lead to serious consequences such as kidney failure; while Staphylococcus aureus, with its multiple toxins, can destroy the host's immune system and cause widespread infection.

[0003] At present, traditional treatments are facing the problem of increasing antibiotic resistance, and the use of chemical preservatives has also caused public health concerns. Therefore, the development of new antimicrobial agents is imperative. S. aureus and E. coli All of them showed good inhibitory activity, and they were not easy to develop drug resistance and could be rapidly degraded, which made them a hot topic of research. This made antimicrobial peptides show broad application prospects in preventing and treating foodborne diseases caused by these bacteria. Summary of the invention

[0004] The purpose of the present invention is to provide an antimicrobial peptide CN-1 and its application. The antimicrobial peptide CN-1 has antibacterial activity against Escherichia coli and Staphylococcus aureus, and exerts its effect by destroying the surface structure of bacteria. The antimicrobial peptide CN-1 is not easy to produce drug resistance, has low cytotoxicity and high cell selectivity, and can be used to prepare an antimicrobial composition for treating Staphylococcus aureus and Escherichia coli infections.

[0005] To achieve the above object, the present invention provides an antimicrobial peptide CN-1, whose amino acid sequence is TILPKRKVMPFL, as shown in SEQ ID NO.1.

[0006] The present invention also provides the use of the antimicrobial peptide CN-1 in the preparation of antimicrobial agents.

[0007] Preferably, the minimum inhibitory concentrations of the antimicrobial peptide CN-1 against Staphylococcus aureus and Escherichia coli are 10.0 mg / mL and 5.0 mg / mL, respectively.

[0008] The present invention also provides the use of the antimicrobial peptide CN-1 in the preparation of drugs against Escherichia coli and Staphylococcus aureus.

[0009] The present invention also provides the above-mentioned composition, wherein the active ingredient of the composition is the above-mentioned antimicrobial peptide CN-1.

[0010] The present invention also provides the above-mentioned antibacterial additive, wherein the active ingredient of the antibacterial additive is the above-mentioned antibacterial peptide CN-1.

[0011] Therefore, the present invention adopts the above antimicrobial peptide CN-1 and its application, and the beneficial technical effects are as follows:

[0012] (1) The antimicrobial peptide CN-1 of the present invention has antibacterial activity against Staphylococcus aureus and Escherichia coli, and exerts its effect by destroying the surface structure of bacteria.

[0013] (2) The antimicrobial peptide CN-1 of the present invention is not prone to drug resistance, has low cytotoxicity and high cell selectivity, and can be used to prepare an antimicrobial composition for treating Escherichia coli and Staphylococcus aureus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Antimicrobial peptide CN-1 S. aureus and E.coil Time-killing curve;

[0015] Figure 2 The antimicrobial peptide CN-1 was treated with enzyme S. aureus and E.coil Changes in inhibition rate;

[0016] Figure 3 for S. aureus and E.coil SEM images of Figure 3 (A) is the one not treated with antimicrobial peptides S. aureus ; Figure 3 (B) is the sample after treatment with 0.5×MIC CN-1 S. aureus ; Figure 3 (C) shows the cells treated with 1×MIC antimicrobial peptide CN-1. S. aureus ; Figure 3 (D) is the normal E.coil ; Figure 3 (E) is the sample treated with 0.5×MIC antimicrobial peptide CN-1. E.coil ; Figure 3 (F) is the result of treatment with 1×MIC antimicrobial peptide CN-1 E.coil . DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0018] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0019] Embodiment 1

[0020] Safety evaluation of the antimicrobial peptide CN-1.

[0021] The antibacterial activity of peptides was predicted using the online software CAMPR3. The physicochemical properties of peptides, including net charge number, GRAVY total average hydrophilicity and Wimley-White all-residue hydrophobicity scale, and protein binding potential (Boman index), were calculated using the online software APD3; the isoelectric point and instability index were predicted in ExPASy using ProtParam. The peptides were scored by ADMET, which included the absorption, distribution, metabolism, excretion, and toxicity characteristics of the peptides. First, the SMILES structural formula of the antimicrobial peptides was obtained using the web-based tool PepSMI. The SMILES structural formula was then input into the ADMET lab platform to evaluate the absorption, distribution, metabolism, excretion, and toxicity characteristics of key parameters such as human intestinal absorption, mutagenicity, carcinogenicity, central nervous system permeability, drug-induced liver injury, cytochrome P450 enzyme inhibition, clearance, and half-life. The peptides predicted by ADMET were chemically synthesized by solid phase synthesis.

[0022] Through the above indicators, it was found that the peptide CN-1 (TILPKRKVMPFL, shown in SEQ ID NO.1) had a CAMPR3 score > 0.5, which predicted that it had antibacterial activity; the net charge number was 2, the GRAVY index was < 0, the molecular weight was 1441.88 Da; and the pI was basic (shown in Table 1).

[0023] Table 1 Prediction of physicochemical properties and antibacterial activity of peptides

[0024]

[0025] Table 2 shows the ADMET score of peptide CN-1 obtained based on the ADMETlab platform. The ADMET spectrum evaluated that the antimicrobial peptide CN-1 was safe and reliable.

[0026] Table 2 ADMET prediction scores of antimicrobial peptides

[0027]

[0028] Embodiment 2

[0029] Activity analysis of antimicrobial peptides.

[0030] 1. Determination of minimum inhibitory concentration.

[0031] The indicator bacteria S. aureus and E.coil After activation, the cells were cultured at 37°C for 18 h until the logarithmic growth phase. The concentration of the indicator bacteria was adjusted to 1 × 106 CFU / mL. Take 10 mg of the antimicrobial peptide CN-1 sample and dissolve it in 1 mL of PBS, sterilize it through a 0.22 μm filter membrane, and mix it with an equal volume of bacterial suspension to make the final concentration of the antimicrobial peptide 10.0 mg / mL, 5.0 mg / mL, 2.0 mg / mL, 1.0 mg / mL, 0.5 mg / mL and 0.25 mg / mL. The blank control is PBS mixed with an equal volume of bacterial suspension. After incubating at 37°C for 2 hours to allow the antimicrobial peptide to fully take effect, take 30 mL of the reaction solution for coating and place it in a 37°C incubator for 18 hours.

[0032] Antimicrobial peptide CN-1 E.coil and S. aureus The antibacterial activity results showed that the antimicrobial peptide CN-1 S. aureus and E.coil The minimum inhibitory concentration (MIC) of the two drugs was 10.0mg / mL and 5.0mg / mL, respectively.

[0033] 2. Time killing curve.

[0034] The same method as above was used to culture S. aureus and E.coil Indicator bacteria. The indicator bacteria suspension was mixed with the antimicrobial peptide solution or PBS in a volume ratio of 1:1, so that the final concentration of the antimicrobial peptide was 0 MIC and 1 MIC, and 0 MIC was used as a blank control. Samples were taken at 0h, 0.5h, 1h, 1.5h, 2h, and 3h, and the live bacteria were counted using the plate pouring method. After culturing at 37℃ for 24h, the number of live bacteria was counted, and the time-killing curve of the antimicrobial peptide against the indicator bacteria was drawn. The results are shown in Figure 1 As shown, it shows that the antimicrobial peptide CN-1 S. aureus and E.coil It has concentration-dependent antibacterial activity, and the antibacterial effect increases with the extension of treatment time.

[0035] 3. Protease treatment.

[0036] The same method as above was used to culture S. aureus and E.coil Indicator bacteria. Weigh trypsin, pepsin and papain at a final concentration of 1 mg / mL respectively and mix them thoroughly with the antimicrobial peptide CN-1 at a final concentration of 1 MIC; at the same time, the antimicrobial peptide CN-1 with a concentration of 1 MIC but not treated with protease was used as the control group of the enzyme treatment experimental group. Incubate at 37℃ for 4 hours to allow for full reaction. Then, mix the reacted sample with an equal volume of bacterial suspension for 2 hours, and measure the OD 600 The antibacterial rate was calculated to verify its antibacterial effect.

[0037] The results are as follows Figure 2As shown in the figure, the control group was treated with indicator bacteria plus 1 MIC antimicrobial peptide, and the experimental group was treated with indicator bacteria plus antimicrobial peptide CN-1 treated with protease. The higher the number of viable bacteria in the experimental results, the worse the antibacterial activity of the sample. After antimicrobial peptide CN-1 was treated with trypsin, pepsin, and papain, S. aureus and E.coil The antibacterial activity of the samples was reduced to varying degrees. The enzyme treatment experiment showed that the substance producing the antibacterial activity was antimicrobial peptide, and this sensitivity to protease could prevent the accumulation of antimicrobial peptide in the body and improve its safety.

[0038] Embodiment 3

[0039] Research on the antibacterial mechanism of antimicrobial peptides.

[0040] The indicator bacteria S. aureus and E.coil After culture to the logarithmic growth phase, the mung bean-sized bacterial sludge was collected by low-speed centrifugation at 4000r / min for 5min, and the bacterial sludge was washed three times with PBS to remove impurities attached to the surface of the bacteria. The bacteria were treated with antimicrobial peptide CN-1 at a concentration of 1 MIC as the experimental group, and the bacteria were treated with antimicrobial peptide CN-1 at a concentration of 1 MIC as the experimental control group. The indicator bacteria were fully mixed with the antimicrobial peptide CN-1 solution, incubated at 37°C for 2h, and then centrifuged at a low speed of 4000r / min for 5min, the supernatant was discarded, and the bacterial sludge precipitate was retained. Continue to add 2.5% glutaraldehyde electron microscope fixative to fix at room temperature for 2h, then place it at 4°C overnight for fixation, and then dehydrate with ethanol for 15min. After sufficient drying, the bacterial cells were sprayed with gold on the IB-3 ion gold plating instrument, and the samples were observed by SEM.

[0041] Antimicrobial peptide CN-1 S. aureus and E.coil Therefore, the changes in the microstructure of the indicator bacteria were observed by SEM to explore the effect of antimicrobial peptide CN-1 on S. aureus and E.coil The results showed that Figure 3 As shown, Figure 3 (A) is the one not treated with antimicrobial peptide CN-1 S. aureus It can be observed that the bacteria have a complete spherical cell shape, a smooth cell wall surface, and clear edges. S. aureus like Figure 3 As shown in (B), S. aureus The surface was wrinkled and concave; after being treated with 1×MIC antimicrobial peptide CN-1 S. aureus like Figure 3 As shown in (C), the cell walls of some bacteria are damaged and the cells have lost their normal cell morphology. Figure 3 (D) is the normal E.coil The cell morphology was full rod-shaped, and the complete and clear texture structure of the cell wall surface could be observed; after treatment with 0.5×MIC antimicrobial peptide CN-1 E.coil like Figure 3 As shown in (E), E.coil The surface was wrinkled and concave; after being treated with 1×MIC antimicrobial peptide CN-1 E.coil like Figure 3 The cell wall of some bacteria shown in (F) was damaged. Based on the above results, it is speculated that the antimicrobial peptide CN-1 has S. aureus and E.coil The antibacterial mechanism is to destroy the cell wall and cell membrane structure of the indicator bacteria, causing the leakage of cell contents, thereby achieving a bactericidal effect.

[0042] It is worth noting that the contents not elaborated in detail in the present invention are all prior art and are well known to those skilled in the art.

[0043] Therefore, the present invention adopts the above-mentioned antimicrobial peptide CN-1 and its application. The antimicrobial peptide CN-1 has antibacterial activity against Escherichia coli and Staphylococcus aureus, and exerts its effect by destroying the cell wall of bacteria; the antimicrobial peptide CN-1 is not easy to produce drug resistance, has low cytotoxicity and high cell selectivity, and can be used to prepare an antimicrobial composition for treating Escherichia coli and Staphylococcus aureus infections.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Antimicrobial peptide CN-1, characterized in that: Its amino acid sequence is TILPKRKVMPFL, as shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide CN-1 according to claim 1 in the preparation of an antimicrobial agent, wherein the antimicrobial agent is used to inhibit Staphylococcus aureus and Escherichia coli, and the minimum inhibitory concentrations of the antimicrobial peptide CN-1 against Staphylococcus aureus and Escherichia coli are 10.0 mg / mL and 5.0 mg / mL, respectively.

3. Use of the antimicrobial peptide CN-1 as claimed in claim 1 in the preparation of drugs against Escherichia coli and Staphylococcus aureus.

4. A composition, characterized in that The active ingredient of the composition is the antimicrobial peptide CN-1 as claimed in claim 1.

5. An antibacterial additive, characterized in that: The active ingredient of the antibacterial additive is the antimicrobial peptide CN-1 as claimed in claim 1.

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