Antibacterial peptide CN-2 and application thereof

Abstract

The invention provides an antibacterial peptide CN-2 and application thereof, and belongs to the technical field of peptide inhibitors. The amino acid sequence of the antibacterial peptide CN-2 is RGNYDAAQRGPGGAWAAK, and the amino acid sequence of the antibacterial peptide CN-2 is as shown in SEQ ID NO. 1. The minimum inhibitory concentration (MIC) of the antibacterial peptide CN-2 to staphylococcus aureus and escherichia coli is 5.0 mg / mL. According to the antibacterial peptide CN-2 and the application thereof, the antibacterial peptide CN-2 has staphylococcus aureus and escherichia coli bacteriostatic activity, and plays a role by destroying bacterial cell membranes; the antibacterial peptide CN-2 is not easy to generate drug resistance, low in cytotoxicity and high in cell selectivity, can be used for preparing an antibacterial composition for treating staphylococcus aureus and escherichia coli infection, and can also be used as a peptide inhibitor to be applied to the fields of foods and medicines.

Description

Technical Field

[0001] The present invention relates to the technical field of peptide inhibitors, in particular to antimicrobial peptide CN-2 and applications thereof. Background Art

[0002] With the rapid development of the global economy and the acceleration of urbanization, the complexity of the food supply chain has increased significantly, resulting in an increasing risk of foodborne diseases year by year. According to statistics from the World Health Organization, the number of deaths caused by foodborne diseases worldwide continues to rise, becoming a major threat to public health. Among them, Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) and Escherichia coli ( Escherichia coli , E. coli ) as the main pathogen, not only causes a wide range of gastrointestinal symptoms, but may also lead to serious complications, especially in individuals with low immunity.

[0003] In the current medical environment, the widespread use of traditional antibiotics has led to an increase in drug resistance, which poses an unprecedented challenge to clinical treatment. At the same time, the public's growing concern about the potential health risks of chemical preservatives has further promoted the demand for natural antimicrobial agents. In this context, antimicrobial peptides, as an emerging natural antimicrobial agent, have shown great potential in the treatment of E. coli and S. aureus Good inhibitory activity, low potential for the development of drug resistance, and good biocompatibility make antimicrobial peptides promising for application in ensuring food safety and preventing foodborne diseases, and more systematic and in-depth research is urgently needed to verify their effectiveness and safety. Summary of the invention

[0004] The purpose of the present invention is to provide an antimicrobial peptide CN-2 and its application. The antimicrobial peptide CN-2 has antibacterial activity against Staphylococcus aureus and Escherichia coli, and exerts its effect by destroying the surface structure of bacteria. The antimicrobial peptide CN-2 is not easy to produce drug resistance, has low cytotoxicity and high cell selectivity, can be used to prepare an antimicrobial composition for treating Staphylococcus aureus and Escherichia coli infections, and can also be used as a peptide inhibitor in the food and medical fields.

[0005] To achieve the above object, the present invention provides an antimicrobial peptide CN-2, whose amino acid sequence is RGNYDAAQRGPGGAWAAK, as shown in SEQ ID NO.1.

[0006] The present invention also provides the use of the antimicrobial peptide CN-2 in the preparation of antimicrobial agents.

[0007] Preferably, the minimum inhibitory concentration of the antimicrobial peptide CN-2 against Staphylococcus aureus and Escherichia coli is 5.0 mg / mL.

[0008] The present invention also provides the use of the antimicrobial peptide CN-2 in the preparation of drugs against Staphylococcus aureus and Escherichia coli.

[0009] The present invention also provides a composition, wherein the active ingredient of the composition is the antimicrobial peptide CN-2.

[0010] The present invention also provides an antibacterial additive, wherein the active ingredient of the antibacterial additive is the antibacterial peptide CN-2.

[0011] Therefore, the present invention adopts the above-mentioned antimicrobial peptide CN-2 and its application, and the beneficial technical effects are as follows: (1) The antimicrobial peptide CN-2 of the present invention has antibacterial activity against Staphylococcus aureus and Escherichia coli, and exerts its effect by destroying the surface structure of bacteria.

[0012] (2) The antimicrobial peptide CN-2 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 Staphylococcus aureus and Escherichia coli infections.

[0013] (3) The antimicrobial peptide CN-2 of the present invention can be used as a peptide inhibitor in the food and pharmaceutical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Antimicrobial peptide CN-2 S. aureus and E.coil Time-killing curve; Figure 2 The antimicrobial peptide CN-2 was treated with enzyme S. aureus Changes in inhibition rate; Figure 3 The antimicrobial peptide CN-2 was treated with enzyme E.coil Changes in inhibition rate; Figure 4 for and SEM images of Figure 4 (A) is the one not treated with antimicrobial peptides ; Figure 4 (B) is the sample treated with 0.5×MIC antimicrobial peptide CN-2. ; Figure 4 (C) shows the results after treatment with 1×MIC antimicrobial peptide CN-2 S. aureus ; Figure 4 (D) is the normal E.coil ; Figure 4 (E) is after treatment with 0.5×MIC antimicrobial peptide CN-2 E.coil ; Figure 4(F) is the result of treatment with 1×MIC antimicrobial peptide CN-2 . DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0016] 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.

[0017] Embodiment 3

[0018] Safety evaluation of the antimicrobial peptide CN-2.

[0019] Using the online software CAMPR 3 The antibacterial activity of peptides was predicted. The physicochemical properties of peptides, including net charge, GRAVY overall average hydrophilicity and Wimley-White all-residue hydrophobicity scale, and protein binding potential (Boman index) were calculated using the online software APD 3 Calculations were performed; 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. The SMILES structural formula of the antimicrobial peptides was first obtained using the web-based tool PepSMI. The SMILES structural formula was then entered 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, half-life, etc. The peptides predicted by ADMET were chemically synthesized by solid phase synthesis.

[0020] Through the above indicators, the peptide CN-2 (RGNYDAAQRGPGGAWAAK, shown in SEQ ID NO.1) was obtained, with a Boman index of 0.625, indicating that the peptide has a medium protein binding potential (usually the higher the Boman index, the greater the possibility of binding to the protein). Net charge number: +2, indicating that the peptide has a positive charge, which helps to interact with the bacterial membrane, thereby enhancing the antibacterial activity. GRAVY index: -0.28, indicating that the peptide is hydrophilic as a whole, which helps with stability and activity in a water-soluble environment. Molecular weight (MW): 2070.26 Da, which is a medium-sized peptide and is suitable for antibacterial applications. Isoelectric point (pI): 9.5, indicating that the peptide is alkaline.

[0021] In summary, the CN-2 peptide has a positive charge and moderate hydrophilicity, has potential in antibacterial and other biological activities, and has a suitable molecular weight and ideal protein binding potential (as shown in Table 1).

[0022] Table 1 Prediction of physicochemical properties and antibacterial activity of peptides ; Table 2 shows the ADMET scores of peptide CN-2 obtained based on the ADMETlab platform. The CN-2 peptide is a peptide with significant antibacterial activity and performs well in ADMET properties. The peptide has efficient intestinal absorption capacity (HIA "+++"), but low permeability in the Caco-2 cell model. Its volume distribution is in the optimal range (0.04–20 L / kg), indicating that the peptide has moderate distribution in the body and cannot cross the blood-brain barrier (BBB). The plasma protein binding rate of the peptide is less than 90%, which is conducive to the biological activity. The peptide does not inhibit or substrate effect on the CYP1A2 and CYP3A4 enzyme systems, showing good metabolic safety. In addition, the peptide has a high clearance rate and a short half-life, which is suitable for high-frequency administration to maintain in vivo concentrations. In the toxicity assessment, the peptide has no hERG channel blocking effect, low hepatotoxicity, and no mutagenicity and carcinogenicity risks. In summary, the CN-2 peptide has ideal pharmacokinetic and safety characteristics and is suitable for the development of antibacterial drugs.

[0023] Table 2 ADMET prediction scores of antimicrobial peptide CN-2

[0024] Embodiment 2

[0025] Activity analysis of antimicrobial peptide CN-2.

[0026] 1. Determination of minimum inhibitory concentration.

[0027] The indicator bacteria S. aureus and E.coil After activation and passage to the third generation, the cells were cultured at 37°C with shaking for 18 h until the logarithmic growth phase. The concentration of the indicator bacteria was adjusted to 1 × 10 6 CFU / mL. Take 10 mg of antimicrobial peptide CN-2 sample and dissolve it in 1 mL 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 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 h to allow the antimicrobial peptide CN-2 to fully function, take 30 mL of the reaction solution for coating and place it in a 37°C incubator for 18 h.

[0028] Antimicrobial peptide CN-2 S. aureus and E. coil The antibacterial activity results showed that the antimicrobial peptide CN-2 S. aureus and E. coil The minimum inhibitory concentration (MIC) of both drugs was 2.42 mM.

[0029] 2. Time killing curve.

[0030] 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 0 h, 0.5 h, 1 h, 1.5 h, 2 h, and 3 h, and the live bacteria were counted using the plate pouring method. After culturing at 37°C for 24 h, 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-2 S. aureus and E.coil It has concentration-dependent antibacterial activity, and the antibacterial effect increases with the extension of treatment time.

[0031] 3. Protease treatment.

[0032] 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-2 at a final concentration of 1 MIC; at the same time, the antimicrobial peptide CN-2 at 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°C for 4 h to allow for full reaction. Then, mix the reacted sample with an equal volume of bacterial suspension for 2 h, and measure the OD 600 The antibacterial rate was calculated to verify its antibacterial effect.

[0033] The results are as follows Figure 2 and Figure 3 As 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-2 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-2 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.

[0034] Embodiment 3

[0035] Research on the antibacterial mechanism of antimicrobial peptides.

[0036] 1. Place indicator bacteria S. aureus and E.coil After the culture reached the logarithmic growth phase, the bacterial sludge was collected by low-speed centrifugation at 4000 r / min for 5 min, and the sludge was washed three times with PBS to remove impurities attached to the bacterial surface. S. aureus The experimental group was treated with antimicrobial peptide CN-2 at a concentration of 1 MIC. E.coil The indicator bacteria were fully mixed with the antimicrobial peptide CN-2 solution, incubated at 37°C for 2h, and then centrifuged at 4000r / min for 5 min. The supernatant was discarded and the bacterial sludge was retained. 2.5% glutaraldehyde electron microscope fixative was added to fix at room temperature for 2 h, then placed at 4°C overnight, and then dehydrated with ethanol for 15 min. 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.

[0037] Antimicrobial peptide CN-2 S. aureus and E. coli To explore its in vitro mechanism of action, scanning electron microscopy (SEM) was used to observe the microstructural changes of the strain at different concentrations. Figure 4 The morphological characteristics of the two indicator bacteria under different treatment conditions are shown. Figure 4 As shown in (A), the S. aureus The cells showed a complete spherical structure with smooth cell walls and clear edges. In contrast, after being treated with the antimicrobial peptide CN-2 at 0.5× minimum inhibitory concentration (MIC), The cell surface showed obvious wrinkles and depressions, losing its original plump appearance ( Figure 4 (B)). When the concentration of antimicrobial peptide CN-2 increased to 1×MIC, The cells even ruptured. Figure 4 (C) in the figure.

[0038] Likewise, Figure 4 (D) shows that E. coli without antimicrobial peptide CN-2 treatment presents a typical full rod-shaped structure with clear and intact cell wall texture. After treatment with 0.5×MIC antimicrobial peptide CN-2, The surface becomes wrinkled and concave, and some cells have abnormal morphology ( Figure 4 (E)); Under 1×MIC conditions, The cells are severely shrunken ( Figure 4 (F) in the figure.

[0039] Based on these results, it is speculated that the antibacterial mechanism of antimicrobial peptide CN-2 is to destroy and The cell wall and cell membrane structure of the indicator bacteria lead to leakage of cell contents, thus achieving a bactericidal effect. This mechanism shows that the antimicrobial peptide CN-2 can effectively damage the membrane integrity of the indicator bacteria, leading to leakage of cell contents, and thus exerting a significant antibacterial effect on them.

[0040] Therefore, the present invention adopts the above-mentioned antimicrobial peptide CN-2 and its application. The antimicrobial peptide CN-2 has antibacterial activity against Staphylococcus aureus and Escherichia coli, and exerts its effect by destroying the surface structure of bacteria. The antimicrobial peptide CN-2 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. It can also be used as a peptide inhibitor in the food and medical fields.

[0041] 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.

[0042] 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-2, characterized in that: The amino acid sequence thereof is RGNYDAAQRGPGGAWAAK, as shown in SEQ ID NO.

1.

2. Use of the antimicrobial peptide CN-2 as claimed in claim 1 in the preparation of antimicrobial agents.

3. The use according to claim 2, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide CN-2 against Staphylococcus aureus and Escherichia coli is 5.0 mg / mL.

4. Use of the antimicrobial peptide CN-2 as claimed in claim 1 in the preparation of drugs against Staphylococcus aureus and Escherichia coli.

5. A composition, characterized in that The active ingredient of the composition is the antimicrobial peptide CN-2 as claimed in claim 1.

6. An antibacterial additive, characterized in that: The active ingredient of the antibacterial additive is the antibacterial peptide CN-2 as claimed in claim 1.

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