Application of antimicrobial peptide RV-18-acy in the preparation of antifungal products

Abstract

The present invention relates to the technical field of antimicrobial peptides, in particular to the application of antimicrobial peptide RV-18-acy in the preparation of antifungal products. The antimicrobial peptide RV-18-acy of the present invention comprises polypeptide RV-18 having an amino acid sequence as shown in SEQ ID NO.1. The antimicrobial peptide RV-18-acy is a polypeptide that can form an amphiphilic α-helical structure, can form nanoparticles by self-assembly, has broad-spectrum antifungal activity, can target and destroy the fungal cell membrane to play a bactericidal role, shows good antibacterial effect against Candida albicans, with a minimum inhibitory concentration of 2.344-75 μM, and has no cytotoxicity and hemolytic effect; in addition, the antimicrobial peptide RV-18-acy contains 18 amino acids, and all amino acids are L-amino acids, which greatly reduces the production cost, is expected to become a new broad-spectrum antifungal candidate drug, and has good application prospects in the fight against "super fungi".

Description

Technical Field

[0001] The present invention relates to the technical field of antimicrobial peptides, and particularly to the application of antimicrobial peptide RV-18-acy in the preparation of antifungal products. Background Art

[0002] It is reported that about one million people die from fungal infections every year, but the number and types of effective antifungal drugs on the market are still limited. In addition, due to the overuse of antifungal drugs, the detection rate of drug-resistant "super fungi" has been increasing year by year. Candida albicans ( Candida albicans ) is the main culprit for infections caused by most fungal pathogens. However, antifungal drugs targeting the inhibition of Candida albicans have low bioavailability, high incidence of adverse reactions and drug interactions, and obvious off-target effects. Therefore, there is an urgent need in the market to develop antifungal drug molecules that can effectively inhibit Candida albicans and are not easily induced to develop drug resistance.

[0003] Antimicrobial peptides are a class of small polypeptide molecules with antibacterial activity, and have attracted worldwide attention due to their strong and broad-spectrum antibacterial activity. However, natural antimicrobial peptides are difficult to extract by proteases, have poor stability, and have a short half-life in vivo. Summary of the Invention

[0004] To solve the above problems, the present invention provides the application of antimicrobial peptide RV-18-acy in the preparation of antifungal products. The antimicrobial peptide RV-18-acy provided by the present invention has broad-spectrum antifungal activity, no cytotoxicity and obvious hemolytic effect, can be obtained by artificial synthesis, has low production cost, and good stability.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of antimicrobial peptide RV-18-acy in the preparation of antifungal products, and the antimicrobial peptide RV-18-acy includes polypeptide RV-18 with an amino acid sequence as shown in SEQ ID NO.1.

[0007] Preferably, the C-terminus or N-terminus of the polypeptide RV-18 is modified with a fatty acid; the fatty acid includes decanoic acid, dodecanoic acid, tetradecanoic acid or hexadecanoic acid.

[0008] Preferably, the linker between the fatty acid and the C-terminus or N-terminus of the polypeptide RV-18 is connected by a peptide bond; the linker at the N-terminus of the polypeptide RV-18 is glycine; the linker at the C-terminus of the antimicrobial peptide RV-18 is glycine-lysine.

[0009] Preferably, the fungus is a fungus of the genus Candida ( Candida )

[0010] Preferably, the fungus of the genus Candida is Candida albicans.

[0011] Preferably, the antifungal product includes an antifungal preparation or an antifungal drug.

[0012] Preferably, the antifungal drug is an anti-fungal infection drug.

[0013] The present invention provides an antifungal drug, the active ingredient of which includes an antimicrobial peptide RV-18-acy; the antimicrobial peptide RV-18-acy includes a polypeptide RV-18 having an amino acid sequence as shown in SEQ ID NO.1; the minimum inhibitory concentration of the antimicrobial peptide RV-18-acy in the antifungal drug is 2.344-75 μM.

[0014] Preferably, the antimicrobial peptide RV-18-acy includes one or more of GV1, GV2, GV3, GV4, RK1, RK2, RK3 and RK4; GV1 is a polypeptide RV-18 with decanoic acid modified at the N-terminus; GV2 is a polypeptide RV-18 with dodecanoic acid modified at the N-terminus; GV3 is a polypeptide RV-18 with tetradecanoic acid modified at the N-terminus; GV4 is a polypeptide RV-18 with hexadecanoic acid modified at the N-terminus; RK1 is a polypeptide RV-18 with decanoic acid modified at the C-terminus; RK2 is a polypeptide RV-18 with dodecanoic acid modified at the C-terminus; RK3 is a polypeptide RV-18 with tetradecanoic acid modified at the C-terminus; RK4 is a polypeptide RV-18 with hexadecanoic acid modified at the C-terminus;

[0015] The minimum inhibitory concentration of GV1 is 37.5-75 μM;

[0016] The minimum inhibitory concentration of GV2 is 9.375-37.5 μM;

[0017] The minimum inhibitory concentration of GV3 is 9.375-18.75 μM;

[0018] The minimum inhibitory concentration of GV4 is 2.344-4.688 μM;

[0019] The minimum inhibitory concentration of RK1 is 37.5-75 μM;

[0020] The minimum inhibitory concentration of RK2 is 9.375-18.75 μM;

[0021] The minimum inhibitory concentration of RK3 is 4.688-9.375 μM;

[0022] The minimum inhibitory concentration of RK4 is 2.344-4.688 μM.

[0023] Preferably, the fungus is Candida albicans.

[0024] Beneficial effects:

[0025] The present invention provides the application of the antimicrobial peptide RV-18-acy in the preparation of antifungal products. The antimicrobial peptide RV-18-acy comprises the polypeptide RV-18 with an amino acid sequence as shown in SEQ ID NO.1. The antimicrobial peptide RV-18-acy provided by the present invention is a polypeptide that can form an amphiphilic α-helical structure, can form nanoparticles by self-assembly, has broad-spectrum antifungal activity, can target and destroy the fungal cell membrane to play a bactericidal role, shows good antibacterial effects on the Candida albicans standard strain ATCC10231 and clinically-derived drug-resistant Candida albicans, with a minimum inhibitory concentration (MIC) of 2.344 - 75 μM, and has no cytotoxicity and hemolytic effect; in addition, the antimicrobial peptide RV-18-acy contains 18 amino acids, and all amino acids are L-amino acids, which greatly reduces the production cost, is expected to become a new type of broad-spectrum antifungal candidate drug, and has good application prospects in the treatment of "super fungi". Brief description of the drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0027] Figure 1 It is a helical wheel model diagram of the antimicrobial peptide RV-18;

[0028] Figure 2 It is a schematic diagram of fatty acid modification of the antimicrobial peptide RV-18-acy;

[0029] Figure 3 It is a circular dichroism spectrum diagram of different antimicrobial peptides in sodium dodecyl sulfate solution;

[0030] Figure 4 It is the transmission electron microscope image and particle size distribution results of the antimicrobial peptides GV1 - GV4;

[0031] Figure 5 It is the transmission electron microscope image and particle size distribution results of the antimicrobial peptides RK1 - RK4;

[0032] Figure 6 It is the determination result of the critical micelle concentration of the antimicrobial peptides GV1 - GV4;

[0033] Figure 7 It is the determination result of the critical micelle concentration of the antimicrobial peptides RK1 - RK4;

[0034] Figure 8 It is the determination result of the hemolysis rate of different antimicrobial peptides on rat red blood cells;

[0035] Figure 9Results of the effects of different antimicrobial peptides on the viability of human keratinocytes (HaCaT).

[0036] Figure 10 Results of the experiment on the membrane disruption of Candida albicans ATCC10231 by different antimicrobial peptides; among them, A shows the changes in fluorescence intensity after the action of antimicrobial peptides GV1 - GV4 and the control group on Candida albicans ATCC10231 at different times; B shows the changes in fluorescence intensity after the action of antimicrobial peptides RK1 - RK4 and the control group on Candida albicans ATCC10231 at different times; a refers to the addition of propidium iodide, and b refers to the addition of RV - 18 - acy sample or control sample. Detailed implementation mode

[0037] The present invention provides the application of antimicrobial peptide RV - 18 - acy in the preparation of antifungal products, and the antimicrobial peptide RV - 18 - acy includes polypeptide RV - 18 with an amino acid sequence shown in SEQ ID NO.1.

[0038] As an implementation mode, the C - terminus or N - terminus of the polypeptide RV - 18 is modified with fatty acids; the fatty acids include decanoic acid, dodecanoic acid, tetradecanoic acid or hexadecanoic acid. As another implementation mode, the fatty acid is dodecanoic acid. As an implementation mode, the linker between the fatty acid and the C - terminus or N - terminus of the polypeptide RV - 18 is connected by a peptide bond; the linker at the N - terminus of the polypeptide RV - 18 is glycine; the linker at the C - terminus of the antimicrobial peptide RV - 18 is glycine - lysine. By modifying the polypeptide RV - 18 with fatty acids, the present invention can improve the stability of the polypeptide RV - 18, thereby enhancing its antifungal activity.

[0039] As an implementation mode, all amino acids of the antimicrobial peptide RV - 18 - acy are of the L - type. The present invention uses L - type amino acids as raw materials to synthesize the antimicrobial peptide RV - 18 - acy, which can significantly reduce the production cost.

[0040] As an implementation mode, the fungus is a fungus of the genus Candida. As another implementation mode, the fungus of the genus Candida is Candida albicans. As an implementation mode, the antifungal product can be a product that inhibits the growth and / or kills fungi. As an implementation mode, the antifungal product includes an antifungal preparation or an antifungal drug. As another implementation mode, the antifungal drug is an antifungal infection drug.

[0041] The antibacterial peptide RV-18-acy provided by the present invention is a polypeptide that can form an amphiphilic α-helical structure, with a molecular weight of 2478.00 - 2690.33 Daltons. It can self-assemble into nanoparticles in solution, with an average particle size of 14.22 - 21.86 nm and a critical micelle concentration (CMC) of 8.5 - 1308 μM. The results of in vitro antibacterial experiments show that RV-18-acy has broad-spectrum antifungal activity, can target and destroy the fungal cell membrane to play a bactericidal role, and shows good antibacterial effects on the Candida albicans standard strain ATCC10231 and clinically-derived drug-resistant Candida albicans, with a minimum inhibitory concentration (MIC) of 2.344 - 75 μM. At the same time, the antibacterial peptide RV-18-acy has no cytotoxic effect and no obvious hemolytic effect at the MIC concentration.

[0042] Based on the above advantages, the present invention provides an antifungal drug, the active ingredient of which includes the antibacterial peptide RV-18-acy; the antibacterial peptide RV-18-acy includes the polypeptide RV-18 with the amino acid sequence shown in SEQ ID NO.1; the minimum inhibitory concentration of the antibacterial peptide RV-18-acy in the antifungal drug is 2.344 - 75 μM.

[0043] As an embodiment, the antibacterial peptide RV-18-acy includes one or more of GV1, GV2, GV3, GV4, RK1, RK2, RK3, and RK4; GV1 is the polypeptide RV-18 modified with decanoic acid at the N-terminus; GV2 is the polypeptide RV-18 modified with dodecanoic acid at the N-terminus; GV3 is the polypeptide RV-18 modified with tetradecanoic acid at the N-terminus; GV4 is the polypeptide RV-18 modified with hexadecanoic acid at the N-terminus; RK1 is the polypeptide RV-18 modified with decanoic acid at the C-terminus; RK2 is the polypeptide RV-18 modified with dodecanoic acid at the C-terminus; RK3 is the polypeptide RV-18 modified with tetradecanoic acid at the C-terminus; RK4 is the polypeptide RV-18 modified with hexadecanoic acid at the C-terminus;

[0044] The minimum inhibitory concentration of GV1 is 37.5 - 75 μM;

[0045] The minimum inhibitory concentration of GV2 is 9.375 - 37.5 μM;

[0046] The minimum inhibitory concentration of GV3 is 9.375 - 18.75 μM;

[0047] The minimum inhibitory concentration of GV4 is 2.344 - 4.688 μM;

[0048] The minimum inhibitory concentration of RK1 is 37.5 - 75 μM;

[0049] The minimum inhibitory concentration of RK2 is 9.375 - 18.75 μM;

[0050] The minimum inhibitory concentration of RK3 is 4.688 - 9.375 μM;

[0051] The minimum inhibitory concentration of RK4 is 2.344 - 4.688 μM.

[0052] As an implementation method, the fungus is a Candida fungus. As another implementation method, the Candida fungus is Candida albicans.

[0053] To further illustrate the present invention, the application of the antibacterial peptide RV-18-acy provided by the present invention in the preparation of antifungal products will be described in detail below in conjunction with the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0054] Example 1 Design and Preparation of Self-Assembled Antibacterial Peptide RV-18-acy

[0055] The amino acid sequence of antibacterial peptide RV-18 is RWRRFWGKAKRGIKKHGV (SEQ ID No.1), which contains 18 L-type amino acids and is a linear polypeptide. The helical wheel model of antibacterial peptide RV-18 was constructed through the website: https: / / heliquest.ipmc.cnrs.fr, and the results are shown in Figure 1 . The results show that antibacterial peptide RV-18 presents a typical amphiphilic structure.

[0056] To improve the stability and antibacterial activity of antibacterial peptide RV-18, fatty acids with different carbon chain lengths (decanoic acid, dodecanoic, tetradecanoic, hexadecanoic acid) were linked to the N-terminus or C-terminus of antibacterial peptide RV-18 to obtain a series of acylated polypeptides modified with fatty acids, and these acylated polypeptides were named RV-18-acy (see Figure 2 ). Among them, a glycine was added to the N-terminus of antibacterial peptide RV-18 as a linker, and then a peptide bond was formed through the amino group of glycine and the carboxyl group of fatty acids with different lengths to obtain antibacterial peptides GV1 - GV4; a glycine-lysine was added to the C-terminus of antibacterial peptide RV-18 as a linker, and then a peptide bond was formed through the side-chain amino group of lysine and the carboxyl group of fatty acids with different lengths to obtain antibacterial peptides RK1 - RK4.

[0057] Preparation of antibacterial peptide RV-18-acy: Entrusted to GL Biochem (Shanghai) Ltd., synthesized by solid-phase peptide synthesis method, and finally desalted and purified by HPLC reverse-phase column chromatography.

[0058] Example 2 Secondary Structure Analysis of RV-18-acy

[0059] The eight RV-18-acy in Example 1 were prepared into solutions with a concentration of 50 μM using an aqueous solution containing 30 mM sodium hexadecyl sulfate (SDS). The circular dichroism spectra of RV-18-acy in the wavelength range of 190-260 nm were scanned by a circular dichroism spectrometer (model Chirascan V100, Applied Photophysics, UK). The scanning conditions are as follows: scanning speed: 100 nm / min; bandwidth: 1 nm; sample cell length: 0.1 cm; reaction time: 1 s. Data processing: The original CD spectrum data was converted into mean residue ellipticity according to the following formula. Mean residue ellipticity = measured ellipticity (millidegrees) / (molar concentration of RV-18-acy × optical path length (mm) × number of residues).

[0060] The results are as follows Figure 3 As shown in the figure, the CD spectrum of RV-18-acy has positive bands in the range of 190-200 nm, and negative bands at 222 nm and 208 nm, that is, RV-18-acy can form an amphipathic α-helical structure in sodium hexadecyl sulfate solution. When in contact with the fungal cell membrane, due to the increase in hydrophobicity in the environment, RV-18-acy can form an amphipathic α-helical conformation to exert a fungicidal effect.

[0061] Example 3 Transmission electron microscopy observation of RV-18-acy

[0062] Drop the RV-18-acy sample solution on glossy paper, clamp a 300-mesh copper mesh with the carbon film facing down, cover and adsorb for 5 minutes, absorb excess liquid, use 2wt% uranyl acetate solution to uranium dye for 2 minutes, absorb excess dye, dry, and use a transmission electron microscope (model JEM-1400 Plus, JEOL Ltd.) to observe and take pictures. Data processing: Use Image J software to count the particle size of nanoparticles, and GraphPad Prism to draw a particle size distribution diagram. The results are shown in Figure 4 and Figure 5 As shown, the scale bars are all 200 nm.

[0063] Depend on Figure 4 and Figure 5 It can be seen that RV-18-acy appears in nanoparticle state under electron microscope, and the average particle size is 14.22~21.86 nm.

[0064] Example 4 Determination of critical micelle concentration (CMC) of RV-18-acy

[0065] 100 μL of RV-18-acy solution with gradient concentrations was added to a black-bottom transparent 96-well fluorescence microplate. Then, 100 μL of 25 μM nile red solution was added to each well. After mixing, the solution was left at room temperature for 30 min, and the emission spectrum of the solution was scanned using a multimode microplate reader. The buffer solution used for preparing the nile red solution and the RV-18-acy solution with gradient concentrations was 10 mM phosphate buffer solution (pH 7.4). Fluorescence spectrum scanning conditions: excitation wavelength was 550 nm, emission wavelength was 600 - 700 nm, and bandwidth was 1 nm. Data processing: Taking the logarithm of the RV-18-acy concentration as the abscissa and the maximum fluorescence intensity of nile red at each concentration as the ordinate, a nile red fluorescence curve in the presence of RV-18-acy with gradient concentrations was plotted (excitation wavelength was 550 nm, emission wavelength was 630 nm). The polypeptide concentration corresponding to the inflection point of the fluorescence curve was the minimum concentration at which RV-18-acy began to form micelles in aqueous solution, i.e., CMC.

[0066] The results are as Figure 6 and Figure 7 shown. When the concentration of RV-18-acy was higher than CMC, it could induce a significant increase in the fluorescence of nile red in the solution. Nile red is a lipophilic fluorescent dye, and it can produce strong fluorescence after inserting into the hydrophobic pocket of the micelle. The above results indicate that RV-18-acy undergoes self-assembly in aqueous solution to form amphiphilic micelles with hydrophilic periphery and hydrophobic interior. The data shows that the CMC of RV-18-acy is 8.5 - 1308 μM. In addition, with the increase in the fatty acid chain length, the CMC concentration of RV-18-acy decreases, and the ability to form micelles increases.

[0067] Example 5 Determination of the minimum inhibitory concentration (MIC) of RV-18-acy against different standard strains

[0068] The tested standard strains were the commercially available strain Candida albicans ATCC10231 and Candida albicans from clinical sources (numbered 5, 0063, and 0065).

[0069] The tested strains were inoculated onto YM solid plates. After the colonies grew, single colonies were picked and transferred to YM liquid medium, and cultured with shaking at 37°C and 180 rpm for 5 h. The OD of the bacterial solution was measured using an ultraviolet spectrophotometer. 600 According to the method of 1 OD 600 = 1×10 9 CFU / mL, the bacterial solution was diluted to 2×10 5Concentration of CFU / mL. Add 100 μL of the diluted bacterial solution to each well of a sterile 96-well plate, and then add 100 μL of the test sample diluted in a gradient manner with normal saline to each well. Use a pipette to aspirate and mix well, and then place it in a constant temperature incubator at 37°C for slow shaking culture overnight. After incubation at a constant temperature, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of the bacterial solution at OD 600 nm, and take the average value of the sample concentrations in the wells where no bacterial growth is detected and the adjacent wells as the MIC value. The results are shown in Table 1.

[0070] Table 1 Minimum inhibitory concentration (μM) of RV-18-acy against different standard fungal strains

[0071]

[0072] As can be seen from Table 1, RV-18-acy showed good antibacterial effects against Candida albicans standard strains (ATCC10231) and clinically derived strains (numbered 5, 0063, and 0065), and the overall MIC value was 2.34 - 75 μM. Among them, the MIC of RV-18-acy (GV1, RK1) linked to decanoic acid was 37.5 - 75 μM; the MIC of RV-18-acy (GV2, RK2) linked to dodecanoic acid was 9.375 - 37.5 μM; the MIC of RV-18-acy (GV3, RK3) linked to tetradecanoic acid was 4.688 - 18.75 μM; the MIC of RV-18-acy (GV4, RK4) linked to hexadecanoic acid was 2.344 - 4.688 μM. The above results suggest that with the increase in the length of the fatty acid chain, the antifungal activity of RV-18-acy gradually increases.

[0073] Example 6 Evaluation of the hemolytic effect of RV-18-acy on red blood cells

[0074] After anesthetizing the rats intraperitoneally, collect whole blood through the abdominal aorta. Mix the blood with Alsever's solution at a volume ratio of 1:1, centrifuge at 1000 rpm for 5 min, discard the supernatant, and wash the obtained red blood cells with normal saline multiple times until the supernatant no longer shows red. Resuspend and dilute the above-washed red blood cells with normal saline to a certain concentration (after treatment with 1% Triton X-100, measure OD 540nmValue is 1.6 - 2.0), reserved. Take an appropriate volume of the RV-18-acy stock solution and dilute it with physiological saline to 1.38 - 88 μM, reserved. Mix 125 μL of the diluted red blood cell suspension with an equal volume of the RV-18-acy sample solution, incubate at 37 °C for 30 min, then centrifuge at 1000 rpm for 5 min, and measure the absorbance value of 200 μL of the supernatant at 540 nm. The positive control (PC) and negative control (NC) are equal volumes of 1% Triton X-100 and physiological saline respectively. In the experiment, three parallel groups are set for each concentration gradient. Data processing: Define the hemolysis rate of the PC group as 100%, and calculate the hemolysis rate of the RV-18-acy sample relative to the PC group.

[0075] The results are as Figure 8 shown. At the MIC concentration, 8 RV-18-acy antimicrobial peptides have no hemolytic effect on red blood cells. Structure-activity relationship analysis shows that within the test concentration range (0.69 - 44 μM), RV-18-acy (GV1, GV2, RK1, and RK2) linked to decanoic acid and dodecanoic acid have no obvious hemolytic effect on red blood cells; as the length of the modified fatty acid chain increases, the hemolytic activity of RV-18-acy gradually increases. Among them, RV-18-acy modified with tetradecanoic acid (MIC is 4.688 - 18.75 μM) has no hemolytic effect on red blood cells within the concentration range below 22 μM, and RV-18-acy modified with hexadecanoic acid (MIC is 2.344 - 4.688 μM) has no significant hemolytic effect on red blood cells within the concentration range below 5.5 μM.

[0076] Example 7 Cytotoxicity Evaluation of RV-18-acy on Human Keratinocytes HaCaT

[0077] Regularly culture human immortalized keratinocytes (HaCaT) with DMEM medium (containing 10% fetal bovine serum and 1% double antibody). When the cells cover about 80% of the bottom of the culture flask, digest them with trypsin, disperse and count, and adjust the cell density to 5×10 4Cells were inoculated into a 96-well plate at a density of cells / mL and a volume of 100 μL per well. After culturing for 24 h, 10 μL of RV-18-acy sample solutions with different concentration gradients (final concentration: 1.38 - 44 μM) or an equal volume of DMEM medium (the background wells contained 110 μL of medium without cells and samples) were added respectively. After continuous culturing for 24 h, 10 μL of CCK8 reagent was added. After 1.5 - 2 h, the absorbance values of the solutions in each well were measured at a wavelength of 450 nm. Each sample concentration was set with three replicates. The background contained no cells and samples, only an equal volume of medium and CCK-8 reagent was added. Data processing: The cell viability in the absence of RV-18-acy sample was defined as 100%, the relative cell viability in the presence of RV-18-acy was calculated, and a bar graph of the relative cell viability corresponding to the RV-18-acy sample solution was plotted using GraphPad prism software.

[0078] The results are shown in Figure 9 Figure [Figure number], within the tested concentration range, RV-18-acy had no significant effect on the proliferation of HaCaT cells, that is, RV-18-acy had no toxic effect on HaCaT.

[0079] Example 8 Membrane-disrupting effect of RV-18-acy on Candida albicans

[0080] Candida albicans ATCC10231 was selected as the test strain to detect the membrane-disrupting effect of the antimicrobial peptide RV-18-acy on it. The bacterial solution in the logarithmic growth phase was centrifuged at 1500 g for 5 min, the supernatant was discarded, and the obtained cell pellet was washed twice with normal saline and then resuspended in normal saline to adjust the bacterial solution concentration to 2 × 10 8 CFU / mL. The prepared bacterial solution was added to a 96-well plate with a black transparent bottom, 100 μL per well, and the fluorescence intensity of the solution was continuously detected by a microplate reader within 15 min. Detection conditions: excitation wavelength Ex was 535 nm, emission wavelength Em was 615 nm, and the reading interval was 1 min. Then 10 μL of propidium iodide (PI) solution was added to make the final concentration 2.5 μg / mL, and the fluorescence intensity of the solution was continuously detected by a microplate reader within 15 min. Then 100 μL of RV-18-acy solution with gradient concentrations or an equal volume of control solution was added, and the fluorescence intensity of the solution was continuously detected by a microplate reader within 60 min. The final concentrations of the RV-18-acy samples were 5 times the minimum inhibitory concentration of different antimicrobial peptides (GV1 - GV4 and RK1 - RK4) against Candida albicans ATCC10231 (i.e., 5 × MIC), the final concentration of the positive control fluconazole was 5 × MIC, and the negative control was an equal volume of normal saline. The results are shown in Figure 10As shown, where a is the time of adding 10 μL of propidium iodide solution, and b is the time of adding the RV-18-acy sample or the control.

[0081] It can be seen from Figure 10 that as the concentration of RV-18-acy increases, the fluorescence intensity of PI in the solution gradually increases, indicating that RV-18-acy shows an obvious membrane-breaking effect on Candida albicans in a concentration-dependent manner.

[0082] In summary, the antibacterial peptide RV-18-acy provided by the present invention has antifungal activity, no cytotoxicity and obvious hemolytic effect, can be obtained by artificial synthesis, has low production cost and good stability.

[0083] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of the antibacterial peptide RV-18-acy in the preparation of an antifungal product, characterized in that, The antimicrobial peptide RV-18-acy is the polypeptide RV-18 with the amino acid sequence shown in SEQ ID NO.1; The antimicrobial peptide RV-18-acy is one or more of GV1, GV2, GV3, GV4, RK1, RK2, RK3, and RK4; GV1, GV2, GV3, and GV4 are obtained by adding a glycine as a linker to the N-terminus of the polypeptide RV-18, and then forming a peptide bond through the amino group of glycine and the carboxyl group of a fatty acid to obtain the antimicrobial peptides GV1, GV2, GV3, and GV4; RK1, RK2, RK3, and RK4 are obtained by adding a glycine-lysine as a linker to the C-terminus of the polypeptide RV-18, and then forming a peptide bond through the side-chain amino group of lysine and the carboxyl group of a fatty acid to obtain the antimicrobial peptides RK1, RK2, RK3, and RK4; The fatty acid is decanoic acid, dodecanoic acid, tetradecanoic acid, or hexadecanoic acid; The fungus is Candida albicans Candida albicans .

2. The application according to claim 1, wherein The antifungal product includes an antifungal preparation or an antifungal drug.

3. The application according to claim 2, characterized in that, The antifungal drug is an antifungal infection drug.

4. The application according to claim 2 or 3, characterized in that, The minimum inhibitory concentration of the antimicrobial peptide RV-18-acy in the antifungal drug is 2.344 - 75 μM.

5. The application according to claim 4, wherein The minimum inhibitory concentration of GV1 is 37.5 - 75 μM; The minimum inhibitory concentration of GV2 is 9.375 - 37.5 μM; The minimum inhibitory concentration of GV3 is 9.375 - 18.75 μM; The minimum inhibitory concentration of GV4 is 2.344 - 4.688 μM; The minimum inhibitory concentration of RK1 is 37.5 - 75 μM; The minimum inhibitory concentration of RK2 is 9.375 - 18.75 μM; The minimum inhibitory concentration of RK3 is 4.688 - 9.375 μM; The minimum inhibitory concentration of RK4 is 2.344 - 4.688 μM.

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