An antibacterial peptide RK-8 and application thereof in preparation of antibacterial drugs

Abstract

The application discloses an antibacterial peptide RK-8 and application thereof in preparation of antibacterial drugs, and belongs to the technical field of biological medicine. The amino acid sequence of the antibacterial peptide RK-8 is shown as SEQ ID NO. 2. The antibacterial peptide RK-8 provided by the application has high killing capacity on various gram-negative / positive bacteria, effectively retains antibacterial activity, greatly reduces cytotoxicity, and improves treatment effect. Meanwhile, the antibacterial peptide RK-8 can also produce synergistic antibacterial effect with various antibiotics, has significant application value in improving curative effect, overcoming antibiotic resistance, reducing drug dosage, delaying drug resistance, and coping with complex infections. The application provides a promising candidate substance resource for developing high-efficiency and low-toxicity antibacterial drugs, and provides an efficient, safe and sustainable solution for developing a new combined therapy and coping with the global drug resistance crisis, and has important clinical application value and industrialization potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antimicrobial peptide RK-8 and its application in the preparation of antimicrobial drugs. Background Technology

[0002] Globally, the widespread use and even abuse of antibiotics has triggered a severe drug resistance crisis. The emergence and spread of multidrug-resistant and pan-drug-resistant bacteria have led to a continuous decline in the effectiveness of traditional antibiotic treatments, resulting in a lack of effective drugs for clinical infection treatment and posing a significant challenge to public health security. Furthermore, traditional antibiotics often target specific molecular targets in bacteria, easily inducing resistance through gene mutations and the transfer of resistance genes. Additionally, some antibiotics suffer from low bioavailability, significant toxic side effects, and limited efficacy against complex infections, further restricting their clinical application.

[0003] Antimicrobial peptides, as core components of the innate immune system, possess unique cell membrane disruption mechanisms, making them less prone to inducing bacterial resistance. They also exhibit broad-spectrum antibacterial activity and immunomodulatory functions, making them an important research direction for replacing traditional antibiotics. However, natural and existing modified antimicrobial peptides still face numerous bottlenecks in industrialization and clinical translation: on the one hand, the long amino acid sequences of most antimicrobial peptides lead to high chemical synthesis costs, hindering large-scale production; on the other hand, insufficient cell selectivity is a key challenge, as many antimicrobial peptides, while exerting bactericidal effects, are prone to toxicity to normal human cells (such as erythrocytes and epithelial cells), causing adverse reactions such as hemolysis and tissue damage, severely limiting the development of systemic drug delivery routes. Furthermore, some antimicrobial peptides exhibit poor stability in physiological environments, easily degrading due to factors such as salt ions, serum enzymes, and temperature, leading to reduced antibacterial activity and further restricting the expansion of their practical applications.

[0004] Therefore, developing novel antimicrobial peptide molecules that combine high antibacterial activity with low cytotoxicity, and exploring synergistic application strategies with existing antibiotics to improve efficacy, reduce dosage, and delay the development of drug resistance, has become an urgent need in the biopharmaceutical field to address drug-resistant bacterial infections, and is of great significance to promoting the innovative development of antimicrobial drugs. Summary of the Invention

[0005] The purpose of this invention is to provide an antimicrobial peptide RK-8 and its application in the preparation of antimicrobial drugs, thereby solving the problems existing in the prior art. The antimicrobial peptide RK-8 provided by this invention exhibits highly efficient bactericidal activity against a variety of Gram-negative / positive bacteria, significantly reducing cytotoxicity while effectively retaining antimicrobial activity. This antimicrobial peptide RK-8 can also produce synergistic antimicrobial effects with various antibiotics, demonstrating significant application value in improving efficacy, overcoming antibiotic resistance, reducing drug dosage, delaying the development of resistance, and addressing complex infections.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] The present invention provides an antimicrobial peptide RK-8, the amino acid sequence of which is shown in SEQ ID NO.2.

[0008] The present invention also provides the application of the above-mentioned antimicrobial peptide RK-8 in the preparation of antimicrobial drugs.

[0009] Furthermore, the antibacterial drug has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria.

[0010] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis;

[0011] The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

[0012] The present invention also provides an antibacterial drug, wherein the antibacterial drug uses the above-mentioned antimicrobial peptide RK-8 as an active ingredient.

[0013] The present invention also provides the use of the above-mentioned antimicrobial peptide RK-8 in the combined preparation of an antimicrobial composition with an antibiotic, wherein the antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

[0014] Furthermore, the antibacterial composition has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria.

[0015] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis;

[0016] The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

[0017] The present invention also provides an antimicrobial composition comprising the above-mentioned antimicrobial peptide RK-8 and an antibiotic.

[0018] Furthermore, the antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

[0019] The present invention discloses the following technical effects:

[0020] This invention truncated the antimicrobial peptide BMAP-28 to obtain a novel antimicrobial peptide, RK-8, with the amino acid sequence RKILRAWK. Compared to its parent peptide BMAP-28, antimicrobial peptide RK-8 exhibits stronger antibacterial activity, lower cytotoxicity, and higher therapeutic potential, indicating that it possesses an extremely wide safety window for clinical application. It can achieve concentrations that completely kill pathogens with minimal cell damage, providing a new material resource for developing safer and more effective antimicrobial drugs. This antimicrobial peptide RK-8 also exhibits high thermal stability. Furthermore, experiments have confirmed that the antimicrobial peptide RK-8 of this invention can produce synergistic antimicrobial effects with various antibiotics, demonstrating significant application value in improving efficacy, overcoming antibiotic resistance, reducing dosage, delaying the development of resistance, and addressing complex infections. This invention provides a highly promising candidate material resource for developing highly effective and low-toxicity antimicrobial drugs, and also offers an efficient, safe, and sustainable solution for developing novel combination therapies and addressing the global drug resistance crisis, possessing significant clinical application value and industrialization potential. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The chromatogram of the antimicrobial peptide RK-8 is shown below.

[0023] Figure 2 This is the mass spectrum of the antimicrobial peptide RK-8;

[0024] Figure 3 Circular dichroism spectroscopy for antimicrobial peptide RK-8 in different buffer solutions;

[0025] Figure 4 The tertiary structure predictions are for the antimicrobial peptide RK-8; where A is the tertiary structure prediction for GG-28; and B is the tertiary structure prediction for RK-8. Detailed Implementation

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0029] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0030] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0031] Unless otherwise specified, the biological materials or reagents involved in the following embodiments can be purchased through conventional channels; unless otherwise specified, the experimental methods involved are conventional technical methods in this field.

[0032] The test strains involved in the following examples are shown in Table 1.

[0033] Table 1 Test strains

[0034]

[0035] Example 1

[0036] This embodiment uses the antimicrobial peptide BMAP-28 (hereinafter referred to as GG-28, GGLRSLGRKILRAWKKYGPIIVPIIRIG, SEQ ID NO.1) as the parent peptide. Its amino acid sequence was shortened by simultaneously truncating the N- and C-termini and selectively truncating the C-terminus to find antimicrobial peptides with better antimicrobial and physiological / biochemical properties. A truncated peptide RK-8 (RKILRAWK, SEQ ID NO.2) was obtained, and its structure and function were analyzed. The specific process is as follows:

[0037] 1. Experimental Methods

[0038] 1.1 Synthesis of antimicrobial peptides

[0039] The second amino acid located at the C-terminus of the designed sequence was added to the reaction column along with a condensing agent and N,N-dimethylformamide. After the reaction was completed, the reaction was detected using the ninhydrin method. The mixture was then washed with a protective solution and N,N-dimethylformamide, and the reaction was detected again. The above steps were repeated until the sequence was synthesized. The final product was precipitated by washing with dichloromethane, methanol, and diethyl ether.

[0040] 1.2 High-performance liquid chromatography purification

[0041] (1) Dissolve 200 mg of the synthesized sample in 15 mL of water and 5 mL of methanol in a beaker and sonicate until completely dissolved, then filter with a filter membrane;

[0042] (2) The solution obtained by filtration in step (1) was purified by gradient chromatography (Table 2). Samples were collected in the 0-30 min time period and the sample components were analyzed by high performance liquid chromatography (Pump A is 100% acetonitrile plus 0.1% trifluoroacetic acid, Pump B is 100% water plus 0.1% trifluoroacetic acid). After verifying the purity of the sample, it was concentrated by rotary evaporator. The sample was then freeze-dried and stored in the dark.

[0043] Table 2 Elution gradient times for antimicrobial peptide purification

[0044]

[0045] 1.3 Identification of antimicrobial peptides

[0046] The prepared antimicrobial peptide solution was collected and the target peak was identified using an LC6000 reversed-phase preparative chromatograph and a Waters 2000 mass spectrometer. The molecular weight of the antimicrobial peptide was calculated according to the following formula:

[0047] ;

[0048] In the formula, Mr is the actual molecular weight of the polypeptide; M is the molecular weight of the polypeptide after fragmentation; and n is the charge number of the fragmented polypeptide.

[0049] 1.4 Secondary Structure Determination

[0050] The secondary structure of antimicrobial peptides was detected using circular dichroism (CD) spectroscopy. Antimicrobial peptide solutions were prepared by adding 10 mM PBS buffer, 30 mM SDS, 50% TFE to simulate aqueous, negatively charged, and hydrophobic environments, respectively. The CD spectrometer parameters were: wavelength 190 nm–250 nm, resolution 0.5 nm, bandwidth 1.0 nm, scan speed 10 nm / min, and optical path length of the sample cell 0.1 cm. The test solutions were read in cuvettes, and the experiment was repeated three times. Finally, deconvolution analysis was performed using CDNN 2.1 software to calculate the secondary structure content of the tested peptides.

[0051] 1.5 Determination of antimicrobial peptide bioactivity

[0052] 1.5.1 Antibacterial activity assay

[0053] The minimal inhibitory concentration (MIC) method recommended by the Clinical and Laboratory Standards Institute (CLSI) was adopted, and the specific steps are as follows:

[0054] (1) Cell culture: The -20℃ frozen bacterial solution was inoculated into MHB medium at 2% (v / v) and cultured overnight in a shaker at 168 rpm and 37℃ until it reached the logarithmic growth phase. Then, a second-generation strain was inoculated and cultured again, and the second-generation strain was adjusted to OD using MHB. 600 =0.4, diluted 100 times as the bacterial solution to be tested.

[0055] (2) Peptide dilution: Add 95 μL and 50 μL of BSA solution to row A and other rows respectively. Then, add 5 μL of antimicrobial peptide solution to row A. Gradually dilute 50 μL from top to bottom. After gradient dilution in row G, discard 50 μL of the mixture.

[0056] (3) Inoculation of bacterial culture: 50 μL of diluted second-generation bacterial culture was inoculated into the AG row and the first 6 wells of the H row, and 50 μL of sterile MHB medium was added to the last 6 wells of the H row. The first 6 wells and the last 6 wells of the H row were used as positive and negative control groups, respectively. After 24 h of incubation, the negative control wells served as the contamination group of the 96-well plate. If no turbidity was observed, the absorbance was measured at 600 nm using a microplate reader. The concentration of antimicrobial peptide at which no obvious turbidity was observed in the solution in the wells was taken as the minimum inhibitory concentration. This experiment was independently repeated three times.

[0057] 1.5.2 Test for the thermal stability of antimicrobial peptides

[0058] The antimicrobial peptides were incubated at 40℃, 60℃, 80℃, and 100℃ for 1 h. The peptide solutions incubated at different temperatures were used instead of the peptide solution added in row A of section 1.5.1. The peptide solutions were diluted using the same method and then inoculated with bacterial culture.

[0059] 1.5.3 Hemolytic Activity Assay

[0060] Healthy human blood was centrifuged at 1000×g and 4℃ for 10 min, and the supernatant was discarded to obtain human erythrocytes. The cells were washed three times with PBS, and then resuspended in PBS solution. Equal volumes of human erythrocytes and different concentrations of peptides were mixed in 96-well plates and incubated at 37℃ for 4 h. The supernatant was collected by centrifugation, and its absorbance was measured at 570 nm using a microplate reader. Untreated human erythrocytes served as a negative control, and the 0.1% Triton X-100 treatment group served as a positive control. The minimum peptide concentration required to induce 10% hemolytic activity was defined as the minimum hemolytic concentration (MHC). This experiment was independently repeated three times. The formula for calculating hemolytic activity is as follows:

[0061]

[0062] In the formula, OD （样品测定值） The absorbance of human red blood cells treated with peptide solution; OD （阴性对照） Absorbance of untreated human erythrocytes without added peptide solution and Triton X-100; OD （阳性对照） The absorbance of human red blood cells treated with Triton X-100.

[0063] 1.5.4 Cytotoxicity Test

[0064] The specific operating procedure is the same as in Section 1.5.1, except that the bacterial culture is replaced with cell suspension. After incubating the treated 96-well plates in a CO2 incubator for 24 h, add 10 µL of CCK-8 solution and incubate for another 2 h. Use wells without cells as a blank control group and melitoxin as a positive control. Measure the absorbance at 450 nm using a microplate reader. Each experiment was independently repeated three times. Calculate cell viability using the following formula:

[0065]

[0066] In the formula, OD (样品测定值) The absorbance of the wells containing cells, culture medium, CCK-8 solution, and drug solution; OD (阴性对照)The absorbance of the pores containing culture medium, CCK-8 solution, and no cells; OD (阳性对照) The absorbance is the value of the well containing cells, culture medium, and CCK-8 solution, but no drug solution.

[0067] 2. Experimental Results

[0068] 2.1 Synthesis of antimicrobial peptides

[0069] Parental peptide GG-28 and truncated peptide RK-8 were obtained via Fmoc solid-phase synthesis. Subsequently, purification was performed to meet the purity requirements for the experiments. The antimicrobial peptides of the appropriate purity were obtained and analyzed using LC3000 reversed-phase chromatography to determine their purity. Figure 1 The analysis results for the antimicrobial peptides show a clear absorption peak, and the purity determination reaches 95%.

[0070] 2.2 Identification of antimicrobial peptides

[0071] The amino acid composition of the parental peptide GG-28 and the truncated peptide RK-8 was identified by electrospray mass spectrometry, and the results are as follows: Figure 2 As shown, the results indicate that the amino acid molecular weight of the truncated peptide is roughly consistent with the theoretical value, confirming that the final synthesized antimicrobial peptide has the same amino acid composition as the designed peptide, ultimately yielding the target designed peptide RK-8. The specific amino acid composition and parameters are shown in Table 3. RK-8 has a net positive charge of +4, enabling it to generate electrostatic interactions with the bacterial outer membrane, thus targeting the bacterial cell membrane. Although its GRAVY value is reduced, it still possesses a large number of hydrophobic amino acids (I, L, and W), providing the material basis for disrupting the physiological structure of the cell membrane.

[0072] Table 3 Amino acid composition and parameters of antimicrobial peptides

[0073]

[0074] Note: a Mass spectrometry for determining actual molecular mass; b The isoelectric point was calculated using an online tool (https: / / web.expasy.org / protparam / ); c is the overall average hydrophilicity value.

[0075] 2.3 Secondary structure of antimicrobial peptides

[0076] Circular dichroism (CD) spectroscopy was used to analyze the secondary structures of GG-28 and its truncated peptide in different simulated solution environments. 10 mM PBS was used to simulate an aqueous environment, 50% TFE to simulate a hydrophobic environment, and 30 mM SDS to simulate a negatively charged phospholipid bilayer environment. Results are as follows: Figure 3As shown in Table 4, in the aqueous environment simulated by PBS buffer, GG-28 and the truncated peptide have a negative absorption peak near 198 nm, and their conformation is irregularly coiled. In the negatively charged environment simulated by SDS, RK-8 is found to exhibit β-sheet by deconvolution fitting. In the hydrophobic environment simulated by TFE, RK-8 is found to exhibit β-sheet by deconvolution fitting.

[0077] Table 4. Secondary structure content of antimicrobial peptides in different buffer solutions

[0078]

[0079] Note: Different letter markers in the same row indicate significant differences (P < 0.05).

[0080] 2.4 Tertiary Structure Prediction of Antimicrobial Peptides

[0081] α-helices and β-turns are the most common structures in antimicrobial peptides. Tian proposed that alternating cationic and hydrophobic amino acids could yield highly potent amphiphilic α-helical antimicrobial peptides. However, perfectly amphiphilic α-helical antimicrobial peptides do not necessarily indicate optimal activity and cell selectivity. Bioinformatics was used to predict the three-dimensional structure of BMAP-28 and its truncated peptides. Figure 4 As shown, BMAP-28 tends to form an α-helix structure, and although RK-8 has a significantly shortened amino acid sequence, it still exhibits an α-helix tendency. The PG turn is due to the fact that the glycine (Gly) side chain group only has hydrogen ions, making it the simplest in structure and properties, while the proline (Pro) side chain group forms a heterocyclic structure with the amino group, becoming an amino acid that is difficult to rotate in the amino acid sequence. Because RK-8 has a shortened N-terminal 10-position proline (P), RK-8 does not tend to form a β-turn structure in structural predictions. Due to the loss of the β-turn, its cell selectivity may be weakened. However, the simultaneously reduced GRAVY value can reduce its cytotoxicity, and its cytotoxicity needs further investigation. In summary, the α-helix structure of RK-8 combined with its low GRAVY value suggests that it possesses certain antibacterial properties while further enhancing cell selectivity.

[0082] 2.5 Bioactivity of antimicrobial peptides

[0083] 2.5.1 Hemolytic activity

[0084] Using hemolytic activity of less than 10% as the standard for good blood safety, bacterial membrane selectivity and biocompatibility were evaluated through hemolysis tests on human erythrocytes using GG-28 and the truncated peptide. The hemolytic activity results are shown in Table 5. Even at the highest tested concentration (256 µM), the hemolytic activity of RK-8 remained less than 10%. In contrast, GG-28, as the parent peptide, and the positive control meliofemoral peptide exhibited significantly higher hemolytic activity. This indicates that the truncated peptide RK-8 possesses good blood safety.

[0085] Table 5 Hemolytic activity of antimicrobial peptides

[0086]

[0087] 2.5.2 Cytotoxicity

[0088] To evaluate the cytotoxicity of GG-28 and its truncated peptide against mouse macrophage RAW264.7 cells, a cytotoxicity assay was performed using a CCK-8 assay kit. The results are shown in Table 6. GG-28 reduced cell viability, but the truncated peptide RK-8 did not affect cell viability; at a high concentration (256 µM), cell viability remained above 100%, demonstrating low toxicity.

[0089] Table 6. Cytotoxicity (cell viability) of antimicrobial peptides

[0090]

[0091] 2.5.3 Antibacterial activity

[0092] The minimum inhibitory concentrations (MICs) of GG-28 and its truncated peptide against eight different bacterial strains are shown in Tables 7 and 8. The results indicate that both GG-28 and the truncated peptide RK-8 have inhibitory effects on both Gram-negative and Gram-positive bacteria, with the truncated peptide showing better inhibitory effects than the parent peptide.

[0093] Table 7. MIC values ​​(µM) of antimicrobial peptides against Gram-negative bacteria.

[0094]

[0095] Table 8. MIC values ​​(µM) of antimicrobial peptides against Gram-positive bacteria.

[0096]

[0097] This invention introduces the therapeutic index (TI) of antimicrobial peptides by calculating the ratio of GM to MHC, thus evaluating the targeting selectivity of antimicrobial peptides against pathogenic microorganisms and host cells. A smaller GM value indicates a lower minimum concentration of antimicrobial peptide required to kill bacteria. A larger TI value indicates higher cell selectivity. As shown in Table 9, after truncation, both the GM and TI values ​​of RK-8 against the tested bacteria were significantly improved. This indicates that it possesses good cell selectivity and antibacterial activity, demonstrating certain application potential.

[0098] Table 9. Therapeutic potential of peptides

[0099]

[0100] Note: a The peptide concentration that induces 10% hemolysis is the minimum hemolytic concentration (MHC). When the hemolytic concentration is >128 µM, 256 µM is used for calculation; when the hemolytic concentration is still greater than 10% at 2 µM, 2 µM is used to calculate the therapeutic index. b The geometric mean of the MIC values ​​of peptides. c The therapeutic index = MHC / GM, and the higher the value, the better the selectivity of the antimicrobial peptide.

[0101] 2.5.4 Stability Assessment

[0102] The process by which antimicrobial peptides kill bacteria, including during production, processing, and in vivo targeting, is influenced by complex physiological environments. This invention selects representative strains that can be significantly inhibited, *Cronobacter sakazakii* ATCC 12868 and *Staphylococcus albus* CGMCC1.490, and uses MIC to evaluate the stability characteristics of the antimicrobial peptides under heat conditions.

[0103] The results are shown in Tables 10 and 11. The MIC values ​​of GG-28 and RK-8 did not change under any temperature treatment. The test temperature may not have a strong effect on the structure of the antimicrobial peptides; therefore, the antimicrobial peptides can still exert their antimicrobial activity under the corresponding structures. Truncation leads to a decrease in the number of amino acids, but it does not cause instability in intermolecular interactions at the test temperatures. The truncated peptides possess a certain degree of thermal stability.

[0104] Table 10. MIC values ​​(µM) of antimicrobial peptides against Cronobacter sakazakii ATCC 12868 under different temperature treatments.

[0105]

[0106] Table 11. MIC values ​​(µM) of antimicrobial peptides against Staphylococcus albus CGMCC 1.490 under different enzyme treatments.

[0107]

[0108] Example 2 Combined drug susceptibility test

[0109] 1. Antibacterial activity assay of antibiotics

[0110] The method described in section 1.5.1 of Example 1 shall be followed.

[0111] 2. Antimicrobial peptide combined with antibiotic antimicrobial assay

[0112] Based on the modified Pankey method, the standard chessboard method is used for identification.

[0113] (1) Bacterial culture and preparation of bacterial inoculum: Same as step (1) in section 1.5.1 of Example 1.

[0114] (2) Drug preparation: In MHB medium, the antibiotic was diluted to twice the maximum detectable concentration, and the antimicrobial peptide to be tested was diluted to four times the maximum detectable concentration. The maximum detectable concentration for both the antibiotic and the antimicrobial peptide was 2 × MIC of the corresponding test bacteria.

[0115] (3) Drug dilution:

[0116] Antibiotic dilution: Add 50 µL of MHB medium to each well of the 96-well plate. Add 50 µL of the antibiotic solution prepared in (2) to wells A12-G12 in columns 11 and 12. Add an additional 50 µL of antibiotic solution to well H11. Mix the solution in column 11 thoroughly and aspirate 50 µL to column 10 for a 2-fold serial dilution. Continue this process until column 2 is reached. Aspirate 50 µL and discard the solution. At this point, the concentration of the antibiotic decreases by a factor of 2 along the X-axis from column 12 to column 2.

[0117] Dilution of antimicrobial peptides: Add 50 µL of the antimicrobial peptide solution prepared in (2) to wells H1-H11 in row H, mix well, and add 50 µL to row G. Repeat this process to make a 2-fold serial dilution to row B. Discard 50 µL. At this time, the concentration of antimicrobial peptides decreases by 2-fold along the Y-axis from row H to row B. The concentration of antibiotics in each column of the diluted 96-well plate is the same, and the concentration of antimicrobial peptides in each row is the same.

[0118] (4) Bacterial inoculation: Add 50 µL of the bacterial inoculation solution prepared in (1) (except for H12) to the 96-well plate. Add 100 µL of culture medium to the H12 well as a negative control. At this time, the A1 well of the 96-well plate does not contain antibiotics and antimicrobial peptides and serves as a positive control. After incubating the 96-well plate at 37℃ for 18-20 h, the negative control wells serve as the contamination group of the 96-well plate. If no turbidity is observed, the absorbance is measured at 600 nm using an ELISA reader. The concentration at which the solution in the wells does not show obvious turbidity is taken as the minimum inhibitory concentration when used in combination. This experiment was independently repeated three times.

[0119] (5) Results Analysis: The interaction between the tested drugs was evaluated by calculating the partial inhibitory concentration index (FIC index). The formula for calculating the FIC index is as follows:

[0120] .

[0121] Evaluation criteria: FIC index ≤0.5, 0.5~4 and >4 represent synergistic effect, no interaction and antagonistic effect, respectively.

[0122] 3. Results

[0123] 3.1 Antibacterial activity

[0124] The MIC values ​​of the selected test antibiotics against Cronobacter sakazakii ATCC 12868 were determined using the microdilution method, and the results are shown in Table 12. Ciprofloxacin, gentamicin, and cefixime showed good antibacterial effects.

[0125] Table 12 MIC values ​​of the drug against Cronobacter sakazakii ATCC 12868

[0126]

[0127] 3.2 Synergistic Effect

[0128] The checkerboard microdilution method was used to combine different antimicrobial peptides with different antibiotics in pairs to test the effect of combined antimicrobial peptides and antibiotics on the MIC value of *Cronobacter sakazakii* ATCC 12868, in order to further explore the synergistic effect of antimicrobial peptides and antibiotics. The experimental results are expressed using the Partially Known Concentration Index (FIC index), where FIC index ≤0.5, 0.5–4, and >4 represent synergistic effect, no interaction, and antagonistic effect, respectively. Table 13 shows that RK-8 exhibited synergistic effects with ciprofloxacin, gentamicin, and cefixime, but no interaction with polymyxin B sulfate. The synergistic bactericidal effect of antimicrobial peptides and antibiotics is beneficial for combined drug use, reducing bacterial resistance and the cost of using antimicrobial peptides / antibiotics.

[0129] Table 13 Synergistic effect of RK-8 combined with antibiotics against Cronobacter sakazakii ATCC 12868

[0130]

[0131] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An antimicrobial peptide RK-8, characterized in that, The amino acid sequence of the antimicrobial peptide RK-8 is shown in SEQ ID NO.

2.

2. The use of the antimicrobial peptide RK-8 according to claim 1 in the preparation of antimicrobial drugs.

3. The application according to claim 2, characterized in that, The antibacterial drug has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria.

4. The application according to claim 3, characterized in that, The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis; The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

5. An antibacterial drug, characterized in that, The antibacterial drug uses the antimicrobial peptide RK-8 as described in claim 1 as its active ingredient.

6. The use of the antimicrobial peptide RK-8 according to claim 1 in the combined preparation of an antimicrobial composition with an antibiotic, characterized in that, The antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

7. The application according to claim 6, characterized in that, The antibacterial composition has the effect of inhibiting or killing Gram-positive and Gram-negative bacteria.

8. The application according to claim 7, characterized in that, The Gram-positive bacteria include Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, and Mycobacterium abscessis; The Gram-negative bacteria include Escherichia coli, Cronobacter sakazakii, and Salmonella.

9. An antibacterial composition, characterized in that, The antimicrobial composition comprises the antimicrobial peptide RK-8 as described in claim 1 and an antibiotic.

10. The antibacterial composition according to claim 9, characterized in that, The antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

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