Antibacterial peptide CN-3 and application thereof

Abstract

The invention provides an antibacterial peptide CN-3 and application thereof, and relates to the technical field of peptide inhibitors. The amino acid sequence of the antibacterial peptide CN-3 is GPGGAWAAKVIS, and is as shown in SEQ ID NO. 1. The minimum inhibitory concentration (MIC) of the antibacterial peptide CN-3 to staphylococcus aureus and escherichia coli is 5.0 mg / mL. According to the antibacterial peptide CN-3 and the application thereof, the CN-3 has staphylococcus aureus and escherichia coli bacteriostatic activity, and plays a role by destroying bacterial cell membranes; cN-3 is not easy to generate drug resistance, low in cytotoxicity and high in cell selectivity, and can be used for preparing an antibacterial composition for treating staphylococcus aureus and escherichia coli infection.

Description

Technical Field

[0001] The present invention relates to the technical field of peptide inhibitors, and in particular to an antibacterial peptide CN-3 and its applications. Background Art

[0002] With the acceleration of globalization and the continuous growth of the population, food safety issues have become increasingly severe, and the incidence of foodborne diseases has risen significantly. According to statistics, the number of infection cases caused by foodborne diseases worldwide exceeds 600 million annually, having a major impact on public health. Among them, Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), as the main pathogenic bacteria, can cause a variety of health problems ranging from mild diarrhea to severe infections, posing a greater threat especially to children and the elderly.

[0003] In today's medical system, the widespread use of traditional antibiotics has led to an exacerbation of the drug resistance problem, presenting an unprecedented challenge to treatment. At the same time, consumers are increasingly concerned about the safety of chemical preservatives and additives in food, which has driven the demand for natural alternatives. Against this background, antibacterial peptides, as a new type of biological antibacterial agent, are receiving extensive attention due to their significant inhibitory effects on E. coli and Salmonella and the relatively low risk of drug resistance. This enables antibacterial peptides to show good application potential in ensuring food safety and preventing foodborne diseases, and there is an urgent need to conduct in-depth research and development. Summary of the Invention

[0004] The object of the present invention is to provide an antibacterial peptide CN-3 and its applications. The antibacterial peptide CN-3 has antibacterial activities against Staphylococcus aureus and Escherichia coli, and exerts its effects by destroying the surface structure of bacteria; the antibacterial peptide CN-3 is not easily resistant, has low cytotoxicity and high cell selectivity, and can be used to prepare antibacterial compositions for treating Staphylococcus aureus and Escherichia coli infections.

[0005] To achieve the above object, the present invention provides an antibacterial peptide CN-3, whose amino acid sequence is GPGGAWAAKVIS, as shown in SEQ ID NO.1.

[0006] The present invention also provides the application of the antibacterial peptide CN-3 in the preparation of antibacterial agents.

[0007] Preferably, the minimum inhibitory concentration of the antibacterial peptide CN-3 against both Staphylococcus aureus and Escherichia coli is 5.0 mg / mL.

[0008] The present invention also provides the application of the antibacterial peptide CN-3 in the preparation of drugs against Staphylococcus aureus and Escherichia coli.

[0009] The present invention also provides a composition, and the active ingredient of the composition is the antibacterial peptide CN-3 as described above.

[0010] The present invention also provides a bacteriostatic additive, and the active ingredient of the bacteriostatic additive is the antibacterial peptide CN-3 as described above.

[0011] Therefore, the present invention adopts the above-mentioned antibacterial peptide CN-3 and its applications, and the beneficial technical effects are as follows:

[0012] (1) The antibacterial peptide CN-3 in the present invention has antibacterial activities against Staphylococcus aureus and Escherichia coli, and exerts its effects by destroying the surface structures of bacteria;

[0013] (2) The antibacterial peptide CN-3 in the present invention is not prone to drug resistance, has low cytotoxicity and high cell selectivity, and can be used to prepare antibacterial compositions for treating Staphylococcus aureus and Escherichia coli infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the time-kill curve of the antibacterial peptide CN-3 in the present invention against S. aureus and E. coli;

[0015] Figure 2 is the change in the antibacterial rate of the antibacterial peptide CN-3 against S. aureus after enzyme treatment in the present invention;

[0016] Figure 3 is the change in the antibacterial rate of the antibacterial peptide CN-3 against E. coli after enzyme treatment in the present invention;

[0017] Figure 4 is the SEM image of S. aureus and E. coli in the examples of the present invention; wherein, Figure 4 in (a) is S. aureus without treatment with the antibacterial peptide; Figure 4 in (b) is S. aureus after treatment with 0.5×MIC of the antibacterial peptide CN-3; Figure 4 in (c) is S. aureus after treatment with 1×MIC of the antibacterial peptide CN-3; Figure 4 in (d) is normal E. coli without treatment with the antibacterial peptide; Figure 4 in (e) is E. coli after treatment with 0.5×MIC of the antibacterial peptide CN-3; Figure 4 in (f) is E. coli after treatment with 1×MIC of the antibacterial peptide CN-3. DETAILED DESCRIPTION OF THE INVENTION

[0018] The technical solutions of the present invention will be further described below with reference to the drawings and examples.

[0019] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings as understood by those of ordinary skill in the art to which the present invention pertains.

[0020] Example 1

[0021] The online software CAMPR3 was used to predict the antibacterial activity of the peptide segments. The physicochemical properties of the peptide segments, including the net charge number, the total average hydrophilicity of GRAVY, the Wimley-White whole residue hydrophobicity scale, and the protein binding potential (Boman index), were calculated using the online software APD3; the isoelectric point and instability index were predicted using ProtParam in ExPASy. The peptide segments were subjected to ADMET scoring, which included the absorption, distribution, metabolism, excretion, and toxicity characteristics of the peptide segments. First, the SMILES structural formula of the antimicrobial peptide was obtained through the web-based tool PepSMI. Subsequently, the SMILES structural formula was 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 rate, half-life, etc. The peptide segments predicted by ADMET were chemically synthesized by solid-phase synthesis.

[0022] Based on the above indicators, the peptide segment CN-3 (GPGGAWAAKVIS, shown in SEQ ID NO.1) was obtained, with the Boman index in the range of 0-1; the net charge number > 0, the GRAVY index < 0, and the molecular weight was 1564.66 Da; the pI was basic (shown in Table 1).

[0023] Table 1 Prediction of the physicochemical properties and antibacterial activity of the peptide segments

[0024]

[0025] Table 2 shows the ADMET scores of the peptide segment CN-3 obtained based on the ADMET lab platform. The ADMET spectrum evaluates that the antimicrobial peptide CN-3 is safe and reliable.

[0026] Table 2 ADMET prediction scores of the antimicrobial peptide

[0027]

[0028]

[0029] Example 2

[0030] Activity analysis of the antimicrobial peptide.

[0031] 1. Determination of the minimum inhibitory concentration.

[0032] Activate the indicator bacteria S. aureus and E. coli and transfer them to the third generation. Incubate them at 37 °C with shaking for 18 h until the logarithmic growth phase, and adjust the concentration of the indicator bacteria to 1×10 6 CFU / mL using phosphate buffer (PBS). Dissolve 10 mg of the antimicrobial peptide CN-3 sample in 1 mL of PBS, sterilize it by passing through a 0.22 μm filter membrane, and mix it with an equal volume of the bacterial suspension respectively 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 the mixture of PBS and an equal volume of the bacterial suspension. After incubating at 37 °C for 2 h to allow the antimicrobial peptide to fully exert its effect, take 30 mL of the reaction solution for coating and place it in a 37 °C incubator for 18 h.

[0033] The antibacterial activity results of the antimicrobial peptide CN-3 against E. coli and S. aureus show that the minimum inhibitory concentration (MIC) of the antimicrobial peptide CN-3 against S. aureus and E. coli is both 5.0 mg / mL.

[0034] 2. Time-kill curve.

[0035] Culture the S. aureus and E. coli indicator bacteria by the same method as above. Mix the bacterial suspension and the antimicrobial peptide solution or PBS at a volume ratio of 1:1 to make the final concentration of the antimicrobial peptide 0×MIC, 1×MIC, and 0 MIC as the blank control. Take samples at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 3 h, and use the plate pouring method to count the viable bacteria. After culturing at 37 °C for 24 h, count the viable bacteria and draw the time-kill curve of the antimicrobial peptide against the indicator bacteria. The results are as Figure 1 shown, indicating that the antimicrobial peptide CN-3 has a concentration-dependent antibacterial activity against S. aureus and E. coli, and at the same time, the antibacterial effect is enhanced with the extension of the treatment time.

[0036] 3. Protease treatment.

[0037] Culture the S. aureus and E. coli indicator bacteria by the same method as above. Weigh 1 mg / mL of trypsin, pepsin, and papain respectively at the final concentration, and mix them fully with the antimicrobial peptide CN-3 at the final concentration of 1 MIC; at the same time, the antimicrobial peptide CN-3 with a concentration of 1 MIC but not treated with protease is used as the control group for the enzyme treatment experiment group. Incubate at 37 °C for 4 h to allow them to react fully. Then mix the reaction sample with an equal volume of the bacterial suspension and treat it for 2 h, and verify its antibacterial effect by measuring OD 600 and calculating its antibacterial rate.

[0038] The results are as Figure 2 and Figure 3As shown in the figure, the control group was treated with indicator bacteria plus 1MIC antimicrobial peptide, and the other experimental groups were treated with indicator bacteria plus antimicrobial peptide CN-3 treated with protease. The higher the number of viable bacteria of the indicator bacteria in the experimental results, the worse the antibacterial activity of the sample. After antimicrobial peptide CN-3 was treated with trypsin, pepsin and papain, the antibacterial activity of S. aureus and E. coil was reduced to varying degrees. The enzyme treatment experiment showed that the substance that produced the antibacterial activity was the antimicrobial peptide, and this sensitivity to proteases can prevent the accumulation of antimicrobial peptides in the body and improve their safety.

[0039] Embodiment 3

[0040] Research on the antibacterial mechanism of antimicrobial peptides.

[0041] 1. Cultivate the indicator bacteria S.aureus and E.coil to the logarithmic growth phase, collect the mung bean-sized bacterial mud by low-speed centrifugation at 4000r / min for 5min, and wash the bacterial mud three times with PBS to remove impurities attached to the surface of the bacteria. Use 1MIC concentration of CN-3 to treat S.aureus as the experimental group, and use 1MIC concentration of CN-3 to treat E.coil as the experimental control group. Mix the indicator bacteria with the antimicrobial peptide CN-3 solution thoroughly, incubate at 37℃ for 2h, then centrifuge at 4000r / min for 5min, discard the supernatant, and retain the bacterial mud precipitate. Continue to add 2.5% glutaraldehyde electron microscope fixative to fix at room temperature for 2h, then place it at 4℃ overnight for fixation, then dehydrate with ethanol for 15min, and after sufficient drying, spray gold on the bacterial cells on the IB-3 ion gold plating instrument, and observe the sample by SEM.

[0042] The antimicrobial peptide CN-3 has antibacterial activity against S. aureus and E. coil, so the changes in the microstructure of the indicator bacteria were observed by SEM to explore the in vitro mechanism of action of the antimicrobial peptide CN-3 against S. aureus and E. coil. Figure 4 As shown, Figure 4 (a) is S. aureus without antimicrobial peptide CN-3 treatment. It can be observed that the bacteria have a complete spherical cell shape, a smooth cell wall surface, and clear edges. Figure 4 As shown in (b), wrinkles and depressions appeared on the surface of S. aureus; after being treated with 1×MIC antimicrobial peptide CN-3, S. aureus Figure 4 The cells of some bacteria shown in (c) are severely shrunk and have lost their normal cell morphology. Figure 4 (d) is a normal E.coil without antimicrobial peptide treatment. The cell morphology is full rod-shaped, and the complete and clear texture structure of the cell wall surface can be observed. The E.coil treated with 0.5×MIC antimicrobial peptide CN-3 is shown in Figure 2.Figure 4 As shown in (e) of Figure 4 , wrinkles and depressions appeared on the surface of E. coli, and the cells lost their normal cell morphology; after treatment with 1×MIC antibacterial peptide CN-3, the cells of some bacteria of E. coli were damaged as shown in (f) of

[0043] It should be noted that the content not elaborated in detail in the present invention is prior art and well-known to those skilled in the art.

[0044] Therefore, the present invention adopts the above antibacterial peptide CN-3 and its application. The antibacterial peptide CN-3 has antibacterial activity against Staphylococcus aureus and Escherichia coli, and exerts its effect by destroying the surface structure of bacteria; the antibacterial peptide CN-3 is not easy to produce drug resistance, has low cytotoxicity and high cell selectivity, and can be used to prepare antibacterial compositions for treating Staphylococcus aureus and Escherichia coli infections.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Antibacterial peptide CN-3, characterized in that, Its amino acid sequence is GPGGAWAAKVIS, as shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide CN-3 according to claim 1 in the preparation of an antibacterial agent.

3. The application according to claim 2, wherein The minimum inhibitory concentration of the antimicrobial peptide CN-3 against Staphylococcus aureus and Escherichia coli is 5.0 mg / mL for both.

4. Use of the antimicrobial peptide CN-3 according to claim 1 in the preparation of a drug against Staphylococcus aureus and Escherichia coli.

5. A composition, characterized in that, The active ingredient of the composition is the antimicrobial peptide CN-3 according to claim 1.

6. An antibacterial additive, characterized in that, The active ingredient of the antibacterial additive is the antimicrobial peptide CN-3 according to claim 1.

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