Antibacterial peptide and application thereof in bacteriostasis and sterilization

Abstract

The application provides an antibacterial peptide and application thereof in bacteriostasis and sterilization, and belongs to the technical field of active peptides.The application provides an antibacterial peptide RG14, which comprises an amino acid sequence shown in SEQ ID No.1.The antibacterial peptide RG14 has the characteristics of wide antibacterial spectrum and strong bacteriostatic activity, low hemolytic activity, no cytotoxicity and high stability, and has obvious advantages and wide application prospect.The antibacterial peptide RG14 can inhibit a plurality of microorganisms, such as gram-negative bacteria, and can be applied to preparation of products with bacteriostatic and sterilizing effects, so that the application range of the antibacterial peptide RG14 is increased.

Description

Technical Field

[0001] This invention belongs to the field of active peptide technology, specifically relating to an antimicrobial peptide and its application in inhibiting and killing bacteria. Background Technology

[0002] Antibiotic resistance is occurring worldwide, making more and more infectious diseases more difficult to treat, and sometimes even untreatable. The development of new antibacterial drugs has become an urgent problem to be solved.

[0003] Antimicrobial peptides are a class of polypeptides containing 5-50 amino acids with antibacterial activity. Initially isolated from insects, they have since been found in other plants, animals, and humans. These are immunologically active molecules developed during long-term evolution to adapt to their environment and seek survival. They are important mediators of natural immunity, playing a crucial role in the host's immune defense against pathogen invasion, and are figuratively called "natural antibacterial agents." To date, 3940 antimicrobial peptides with different structures have been identified. Compared to traditional small-molecule antibiotics, antimicrobial peptides have advantages such as rapid bactericidal action, low resistance rate, and multiple anti-infective mechanisms, showing broad application prospects in the development of novel antibiotics.

[0004] However, existing antimicrobial peptides, while exhibiting good biological activity, suffer from drawbacks such as hemolysis, cytotoxicity, and poor in vivo stability, hindering their clinical application. Currently, many drugs are derived from peptide molecules through structural optimization, such as the antihypertensive drug captopril and the hepatitis C drug telaprevir. Structural modification of naturally derived antimicrobial peptides is also an important direction for future drug design and disease treatment development. Summary of the Invention

[0005] This invention provides an antimicrobial peptide and its application in inhibiting and killing bacteria. The antimicrobial peptide has high antimicrobial activity, as well as low hemolytic activity, no cytotoxicity, and high stability.

[0006] This invention provides an antimicrobial peptide RG14, wherein the antimicrobial peptide RG14 has any of the following amino acid sequences:

[0007] 1) SEQ ID No. 1;

[0008] 2) An amino acid sequence of SEQ ID No. 1 that has undergone substitution, deletion and / or addition of one or more amino acids and has the same function as SEQ ID No. 1;

[0009] 3) An amino acid sequence that has more than 90% homology with SEQ ID No. 1 and has the same function as SEQ ID No. 1.

[0010] In one specific embodiment of the present invention, the antimicrobial peptide RG14 is further modified.

[0011] In one specific embodiment of the present invention, the C-terminus and / or N-terminus of the antimicrobial peptide RG14 are amidated.

[0012] The present invention also provides the application of the above-mentioned antimicrobial peptide RG14 in the preparation of antibacterial and bactericidal agents.

[0013] In one specific embodiment of the present invention, the antibacterial spectrum of the bacteriostatic agent includes Gram-negative bacteria.

[0014] In one specific embodiment of the present invention, the Gram-negative bacteria include at least one of the following: Escherichia coli, Pseudomonas aeruginosa, Shigella castellani, Acinetobacter baumannii, and Clostridium perfringens.

[0015] The present invention also provides an antibacterial and bactericidal product, the active ingredient of which includes the above-mentioned antimicrobial peptide RG14, and also includes excipients.

[0016] In one specific embodiment of the present invention, the active ingredient also includes other antibacterial and bactericidal components.

[0017] In one specific embodiment of the present invention, the other antibacterial and bactericidal components include at least one of the following: antibiotics, proteins, polypeptides, plant extracts, and cytokines.

[0018] In one specific embodiment of the present invention, the types of antibacterial and bactericidal products include pharmaceuticals and skin care products.

[0019] Beneficial effects: This invention provides an antimicrobial peptide RG14 with an amino acid sequence as shown in SEQ ID No. 1. The antimicrobial peptide RG14 has the characteristics of broad antimicrobial spectrum and strong antibacterial activity. At the same time, it has been verified that the antimicrobial peptide RG14 has low hemolytic activity, no cytotoxicity and high stability compared with existing antimicrobial peptides, with significant advantages and broad application prospects.

[0020] The antimicrobial peptide RG14 described in this invention can inhibit a variety of microorganisms. For example, in the examples, the MIC values ​​of antimicrobial peptide RG14 against Escherichia coli CMCC44102, Pseudomonas aeruginosa CMCC10104, Shigella castellani ATCC12022, Acinetobacter baumannii ATCC19606, and Clostridium perfringens ATCC13124 were 2.34 μg / mL, 4.69 μg / mL, 4.69 μg / mL, 2.34 μg / mL, and 9.38 μg / mL, respectively. It can be applied to the preparation of products with antibacterial and bactericidal effects, thus expanding the application scope of antimicrobial peptide RG14. Attached Figure Description

[0021] Figure 1 The results of the bactericidal kinetics of antimicrobial peptide RG14 against Escherichia coli in Example 2 are shown.

[0022] Figure 2 The results are the hemolytic activity assay results of the antimicrobial peptide RG14 in Example 3;

[0023] Figure 3 The results are the cytotoxicity assay results of the antimicrobial peptide RG14 in Example 3. Detailed Implementation

[0024] This invention provides an antimicrobial peptide RG14, wherein the antimicrobial peptide RG14 has any of the following amino acid sequences:

[0025] 1) SEQ ID No. 1;

[0026] 2) An amino acid sequence of SEQ ID No. 1 that has undergone substitution, deletion and / or addition of one or more amino acids and has the same function as SEQ ID No. 1;

[0027] 3) An amino acid sequence that has more than 90% homology with SEQ ID No. 1 and has the same function as SEQ ID No. 1.

[0028] The antimicrobial peptide RG14 referred to in this invention is a linear polypeptide, and its amino acid sequence can be as shown in SEQ ID No. 1: RLFGRLRWSLRRWG. Point mutations can also be made to the amino acid sequence shown in SEQ ID No. 1, such as substitution, deletion and / or addition of one or more amino acids, as long as it has the same function as SEQ ID No. 1. Alternatively, it can be an amino acid sequence with more than 90% homology to SEQ ID No. 1 and with the same function as SEQ ID No. 1, such as having 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology.

[0029] In one specific embodiment of the present invention, the C-terminus and / or N-terminus of the antimicrobial peptide RG14 may be modified. For example, in one embodiment, the C-terminus is modified with amidation, specifically with the sequence: RLFGRLRWSLRRWG-NH2, a molecular weight of 1859.22 Da, and an isoelectric point of 12.60.

[0030] This invention does not specifically limit the preparation method of the antimicrobial peptide RG14. It can be prepared using various methods such as direct synthesis, prokaryotic expression, and eukaryotic expression. The prokaryotic and eukaryotic expression methods are not particularly limited; expression can be performed using conventional methods in the art, such as inserting the encoding gene of the antimicrobial peptide RG14 into an expression vector, transforming it into prokaryotic or eukaryotic expression cells, and then inducing expression. In one embodiment of this invention, the antimicrobial peptide RG14 is prepared using a solid-phase synthesis method, followed by purification using high-performance liquid chromatography.

[0031] The present invention also provides the application of the above-mentioned antimicrobial peptide RG14 in the preparation of antibacterial and bactericidal agents.

[0032] This invention confirms that the antibacterial and bactericidal agent has an antibacterial spectrum that includes Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Shigella castellani, Acinetobacter baumannii, and Clostridium perfringens. In this embodiment of the invention, the antimicrobial peptide RG14 exhibits high antimicrobial activity against Escherichia coli CMCC44102, Pseudomonas aeruginosa CMCC10104, Shigella castellani ATCC12022, Acinetobacter baumannii ATCC19606, and Clostridium perfringens ATCC13124, with MIC values ​​of 2.34 μg / mL, 4.69 μg / mL, 4.69 μg / mL, 2.34 μg / mL, and 9.38 μg / mL, respectively.

[0033] The present invention also provides an antibacterial and bactericidal product, the active ingredient of which includes the above-mentioned antimicrobial peptide RG14, and also includes excipients.

[0034] In this invention, the antimicrobial peptide can kill Escherichia coli at lower concentrations and in a shorter time; moreover, the antimicrobial peptide exhibits low hemolytic activity at different concentrations and is non-cytotoxic to various cell types, demonstrating high safety. The antimicrobial peptide RG14 provided by this invention can directly kill pathogenic bacteria, exhibiting significant and rapid action. Furthermore, RG14 has low hemolytic activity, no cytotoxicity, and high stability, making it suitable for the preparation of antibacterial and bactericidal products, such as antibacterial infection drugs or skincare products, especially medical aesthetic products.

[0035] In one specific embodiment of the antibacterial and bactericidal product of the present invention, the antimicrobial peptide RG14 or a reagent containing the antimicrobial peptide RG14 is used as the sole active ingredient; in other embodiments, multiple active ingredients may be used, such as including the antimicrobial peptide RG14 or a reagent containing the antimicrobial peptide RG14, as well as other antimicrobial and bactericidal ingredients, such as at least one of antibiotics, proteins, peptides, plant extracts and cytokines.

[0036] The antibacterial and bactericidal product of the present invention further includes excipients, which may be one or more of fillers, wetting agents, binders, disintegrants, lubricants, and surfactants; wherein the fillers include one or more of starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, and mannitol; the wetting agents and binders include distilled water, ethanol, starch paste, sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose and ethyl cellulose, hydroxypropyl methyl cellulose, gelatin solution, sucrose solution, and aqueous or alcoholic solutions of polyvinylpyrrolidone (pVp); the disintegrants include one or more of dry starch, sodium methyl starch, low-substituted hydroxypropyl cellulose, croscarmellose, and croscarmellose sodium; the lubricants include one or more of magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, and magnesium lauryl sulfate; and the surfactants include sodium dodecyl sulfate.

[0037] The present invention does not specifically limit the preparation method of the antibacterial and bactericidal product. Based on the type of active ingredient and the selection of excipients, it can be prepared according to conventional methods in the field.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes an antimicrobial peptide provided by the present invention and its application in inhibiting and killing bacteria, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Example 1

[0040] Synthesis and purification steps of antimicrobial peptide RG14:

[0041] 1. Based on the designed amino acid sequence: RLFGRLRWSLRRWG-NH2, a crude polypeptide was synthesized using a solid-phase synthesis method.

[0042] 2. The crude polypeptide was desalted and purified by HPLC reversed-phase column chromatography to determine its purity. The specific method is as follows:

[0043] Dissolve 0.1 mg of the crude peptide prepared in step 1 in 1 mL of ultrapure water containing 0.1% trifluoroacetic acid. If there are insoluble impurities, filter through a 0.45 μm filter membrane. Mobile phase A is 0.1% trifluoroacetic acid-water, and mobile phase B is 0.1% trifluoroacetic acid-acetonitrile. After the baseline stabilizes, start loading the sample with a loading volume of 50 μL. The chromatographic column is a silica alkyl bonded phase C18 column (4.6 mm × 300 mm, particle size 5 μm, pore size 100 Å). A binary mobile phase gradient elution system is used for gradient elution, that is, the content of mobile phase B in the eluent increases linearly from 0% to 80% within 30 min. The flow rate is 1 mL / min, the detection wavelength is 215 nm, and the determination is carried out at 25 °C until the purity of the peptide is not less than 95%, and the purified peptide, namely antimicrobial peptide RG14, is obtained.

[0044] 3. The antimicrobial peptide RG14 obtained in step 2 was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF). The specific method is as follows:

[0045] The antimicrobial peptide RG14 obtained in step 2 was dissolved in deionized water to prepare a 1 μmol / mL solution. 10 μL of the solution was mixed with an equal volume of saturated matrix solution (α-cyano-4-hydroxycinnamic acid was dissolved in 50% acetonitrile solution containing 0.1% trifluoroacetic acid to prepare a saturated solution, centrifuged, and the supernatant was collected) and the molecular weight of the antimicrobial peptide RG14 was determined to be 1859.22 Da.

[0046] 4. The isoelectric point of the antimicrobial peptide RG14 was determined to be 11.29 by isoelectric focusing electrophoresis, and the amino acid sequence structure of the purified peptide was determined by an automated amino acid sequencer, which identified it as Arg-Leu-Phe-Gly-Arg-Leu-Arg-Trp-Ser-Leu-Arg-Arg-Trp-Gly-NH2.

[0047] Example 2

[0048] Antimicrobial activity of antimicrobial peptide RG14

[0049] Definition of minimum inhibitory concentration: the lowest drug concentration that can inhibit bacterial growth and reproduction.

[0050] 2.1 Quantitative antibacterial analysis was performed using a two-fold serial dilution method. The specific procedures are as follows:

[0051] Prepare 1 mg / mL RG14 samples using sterile ultrapure water. Prepare fresh bacterial suspensions separately, according to 1 OD... 600 =1×10 9 CFU / mL, the above bacterial suspension concentration was adjusted to 2×10⁻⁶ CFU / mL using fresh LB liquid medium. 5 CFU / mL.

[0052] Subsequently, 90 μL of physiological saline was added to a sterile 96-well plate beforehand. The prepared RG14 sample (with penicillin and meropenem as positive controls) was added to the first well. The samples were then serially diluted twofold, and 100 μL of a 2×10⁻⁶ concentration was added to each well. 5 The bacterial suspension at CFU / mL was mixed by pipetting and placed in a constant temperature incubator at 37℃ overnight. Finally, the absorbance of the bacterial suspension at 600nm was detected by an enzyme-linked immunosorbent assay (ELISA) reader. The minimum inhibitory concentration (MIC) was the average of the concentrations of the wells in which no bacterial growth was detected and the concentrations of the samples in adjacent wells. The antimicrobial activity of the antimicrobial peptide RG14 is shown in Table 1.

[0053] Table 1 Minimum inhibitory concentration of antimicrobial peptide RG14

[0054]

[0055] 2.2 Inoculate *Escherichia coli* CMCC44102 onto LB agar plates and incubate at 37°C inverted until colonies appear. Use an inoculation loop to pick a single colony and inoculate it into LB liquid medium, then incubate at 37°C in a shaking incubator until the logarithmic growth phase. Dilute the bacterial culture to 1×10⁻⁶ with fresh LB liquid medium. 6 CFU / mL, add the sample to the diluted bacterial solution to a final concentration of 1×, 2×, and 4×MIC. The positive control is the corresponding volume of 2×MIC ampicillin, and the negative control is the corresponding volume of sterile ultrapure water. Immediately place the bacterial solution containing the sample into a 37℃ shaking incubator and incubate with shaking at 150 rpm. At 0 min, 10 min, 30 min, 60 min, 120 min, and 180 min, take 10 μL of the bacterial solution and dilute it with sterile physiological saline to 1×10⁻⁶. 3 Double the volume of the culture medium (50 μL) and spread it onto an LB agar plate. Incubate the plate upside down at 37°C for 16 hours, then count the colonies. Perform three replicates per group. Results are as follows: Figure 1 As shown:

[0056] Depend on Figure 1 It can be seen that the antimicrobial peptide RG14 has a rapid bactericidal effect. At a concentration of 2×MIC, RG14 can kill Escherichia coli within 60 minutes and at a concentration of 4×MIC, it can kill Escherichia coli within 30 minutes. In contrast, the positive control ampicillin requires 120 minutes to kill Escherichia coli.

[0057] Example 3

[0058] The hemolytic activity and cytotoxicity assay of antimicrobial peptide RG14 were performed using the following steps:

[0059] 3.1 Dilute the washed mouse red blood cells to 10 with physiological saline. 7 -10 8 The red blood cell suspension was collected at a concentration of [number] cells / mL, and then mixed with peptide samples of different concentrations. The mixture was incubated at 37°C for 30 min. After centrifugation at 1000 rpm for 5 min, the supernatant was transferred to a 96-well plate, and the UV absorbance was measured at 540 nm. The negative control was physiological saline, and the positive control was Triton X-100. As a positive control, hemolytic activity was directly proportional to the absorbance at 540 nm; hemolytic activity is shown in [details omitted]. Figure 2 ;

[0060] Depend on Figure 2It can be seen that the light absorption value of the antimicrobial peptide RG14 at 540nm is significantly lower than that of the positive control TritonX-100, but very close to that of the negative control. Therefore, it can be concluded that the hemolytic activity of the antimicrobial peptide RG14 is low at different concentrations.

[0061] 3.2 Activated HaCAT cells (purchased from Guangzhou Saiku Biotechnology Co., Ltd.) were seeded in culture dishes. When the cells covered approximately 80% of the bottom area of ​​the dish, they were digested with 0.25% trypsin. The cells were gently scraped off, pipetted until single cells were formed, and then transferred to sterile centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in RPMI 1640 cell culture medium. After rehydration, the cells were counted using a hemocytometer. After counting, the cells were diluted to the required concentration for the experiment, and these were then used as seed cells.

[0062] Take a sterile 96-well plate and add 10,000 seed cells to each well. Adjust the total volume of each well to 100 μL with RPMI 1640. After the cells adhere, add peptide samples of various concentration gradients, and set up a plate control and a positive control group. Incubate at 37℃ for 24 h, then add 20 μL of LTT solution (5 mg / mL, freshly prepared and stored in the dark), and continue incubation for 4 h. Then discard the cell culture medium in each well, add 150 μL of DMSO (dimethyl sulfoxide), and gently shake to dissolve the crystals. Measure the absorbance at 490 nm using a full-wavelength microplate reader. The results are as follows: Figure 3 As shown, compared with the control group NC, the antimicrobial peptide RG14 at different concentrations did not exhibit cytotoxicity, indicating that the antimicrobial peptide RG14 described in this invention has high safety.

[0063] Example 4

[0064] Stability of antimicrobial peptide RG14

[0065] After supplementing the culture medium with sodium chloride (150 mM), potassium chloride (4.5 mM), ammonium chloride (6 μM), magnesium chloride (1 mM), calcium chloride (2.5 mM), and ferric chloride (4 μM), the minimum inhibitory concentration (MIC) was tested again. The blank control was LB medium without added salt ions. The results are shown in Table 2. RG14 showed good stability in potassium chloride, ammonium chloride, magnesium chloride, and ferric chloride ions.

[0066] Table 2 Salt Ion Stability of RG14

[0067]

[0068] The antimicrobial peptide RG14 provided by this invention can directly kill pathogens, with significant and rapid effects. Furthermore, RG14 has low hemolytic activity, no cytotoxicity, and high stability, and can be used in the preparation of antibacterial drugs or skin care products, especially medical aesthetic products.

[0069] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide RG14, characterized in that, The amino acid sequence of the antimicrobial peptide RG14 is shown in SEQ ID No.

1.

2. The antimicrobial peptide RG14 according to claim 1, characterized in that, It also includes modifying the antimicrobial peptide RG14 by amidation modification of the C-terminus and / or N-terminus of the antimicrobial peptide RG14.

3. The application of the antimicrobial peptide RG14 according to claim 1 or 2 in the preparation of antibacterial and bactericidal agents, characterized in that, The antibacterial and bactericidal agent targets at least one of the following: Escherichia coli (Escherichia coli) Escherichia coli ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa Shigella ( Shigella castellani Acinetobacter baumannii ( Acinetobacter baumnnii ) and Clostridium perfringens ( Clostridium perfringens ).

4. A bactericidal and antibacterial product, characterized in that, The active ingredient includes the antimicrobial peptide RG14 as described in claim 1 or 2, and also includes excipients.

5. The antibacterial and bactericidal product according to claim 4, characterized in that, The active ingredients also include other antibacterial and bactericidal components.

6. The antibacterial and bactericidal product according to claim 5, characterized in that, The other antibacterial and bactericidal ingredients include at least one of the following: antibiotics, peptides, and plant extracts.

7. The antibacterial and bactericidal product according to claim 4, characterized in that, The types of antibacterial and bactericidal products are selected from pharmaceuticals and skin care products.

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