Application of antibacterial peptide RV-18-ack in preparation of antifungal product

Abstract

The invention relates to the technical field of antibacterial peptides, in particular to application of an antibacterial peptide RV-18-ack in preparation of an antifungal product. The antibacterial peptide RV-18-acy disclosed by the invention comprises a polypeptide RV-18 of which the amino acid sequence is shown as SEQ ID NO. 1. The antibacterial peptide RV-18-ack is a polypeptide capable of forming an amphiphilic alpha helical structure, can form nanoparticles in a self-assembly manner, has broad-spectrum antifungal activity, can damage fungal cell membranes in a targeted manner to play a bactericidal role, shows a relatively good antibacterial role on candida albicans, has the minimum inhibitory concentration of 2.344-75 mu M, and is free of cytotoxicity and hemolysis; besides, the antibacterial peptide RV-18-ack contains 18 amino acids, and all the amino acids are L-type amino acids, so that the production cost is greatly reduced, and the antibacterial peptide RV-18-ack is expected to become a novel broad-spectrum antifungal candidate drug and has a good application prospect in the aspect of resisting super fungi.

Description

Technical Field

[0001] The invention relates to the technical field of antimicrobial peptides, and in particular to 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 increased year by year. Candida albicans ) is the culprit of most infections caused by fungal pathogens. However, antifungal drugs that target and inhibit Candida albicans have low bioavailability, high incidence of adverse reactions and drug interactions, and significant off-target effects. Therefore, the market is in urgent need of developing antifungal drug molecules that can effectively inhibit Candida albicans and are not prone to inducing drug resistance.

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

[0004] In order to solve the above problems, the present invention provides the use 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] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides use of an antimicrobial peptide RV-18-acy in the preparation of an antifungal product. The antimicrobial peptide RV-18-acy comprises a polypeptide RV-18 having an amino acid sequence as shown in SEQ ID NO.1.

[0006] 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.

[0007] Preferably, the fatty acid is connected to the linker at the C-terminus or N-terminus of the polypeptide RV-18 via a peptide bond; the linker at the N-terminus of the polypeptide RV-18 is glycine; and the linker at the C-terminus of the antimicrobial peptide RV-18 is glycine-lysine.

[0008] Preferably, the fungus is Candida ( Candida ) fungi.

[0009] Preferably, the Candida fungus is Candida albicans.

[0010] Preferably, the antifungal product comprises an antifungal preparation or an antifungal drug.

[0011] Preferably, the antifungal drug is an antifungal infection drug.

[0012] The present invention provides an antifungal drug, wherein the active ingredient comprises an antimicrobial peptide RV-18-acy; the antimicrobial peptide RV-18-acy comprises a polypeptide RV-18 whose amino acid sequence is shown in SEQ ID NO.1; and the minimum inhibitory concentration of the antimicrobial peptide RV-18-acy in the antifungal drug is 2.344-75 μM.

[0013] Preferably, the antimicrobial peptide RV-18-acy includes one or more of GV1, GV2, GV3, GV4, RK1, RK2, RK3 and RK4; the GV1 is a polypeptide RV-18 with a decadecanoic acid modified at the N-terminus; the GV2 is a polypeptide RV-18 with a dodecanoic acid modified at the N-terminus; the GV3 is a polypeptide RV-18 with a tetradecanoic acid modified at the N-terminus; the GV4 is a polypeptide RV-18 with a hexadecanoic acid modified at the N-terminus; the RK1 is a polypeptide RV-18 with a decadecanoic acid modified at the C-terminus; the RK2 is a polypeptide RV-18 with a dodecanoic acid modified at the C-terminus; the RK3 is a polypeptide RV-18 with a tetradecanoic acid modified at the C-terminus; the RK4 is a polypeptide RV-18 with a hexadecanoic acid modified at the C-terminus; 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.

[0014] Preferably, the fungus is Candida albicans.

[0015] Beneficial effects: The present invention provides an application of an antimicrobial peptide RV-18-acy in the preparation of an antifungal product, wherein the antimicrobial peptide RV-18-acy includes a polypeptide RV-18 having 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 amphipathic α-helical structure, can form nanoparticles by self-assembly, has a broad-spectrum antifungal activity, can target and destroy fungal cell membranes to exert a bactericidal effect, and exhibits a good antibacterial effect on the standard strain of Candida albicans 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-type amino acids, which greatly reduces the production cost, and is expected to become a new broad-spectrum antifungal candidate drug, and has a good application prospect in the fight against "super fungi". BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 This is the helical wheel model diagram of the antimicrobial peptide RV-18; Figure 2 Schematic diagram of fatty acid modification of antimicrobial peptide RV-18-acy; Figure 3 The circular dichroism spectra of different antimicrobial peptides in sodium hexadecyl sulfate solution; Figure 4 Transmission electron microscope images and particle size distribution results of antimicrobial peptides GV1~GV4; Figure 5 Transmission electron microscope images and particle size distribution results of antimicrobial peptides RK1~RK4; Figure 6 The results of the determination of the critical micelle concentration of antimicrobial peptides GV1~GV4; Figure 7 The results of the determination of critical micelle concentration of antimicrobial peptides RK1~RK4; Figure 8 The results of the hemolysis rate of different antimicrobial peptides on rat erythrocytes; Fig. 9 The results show the effects of different antimicrobial peptides on the viability of human keratinocytes (HaCaT); Fig.10These are the experimental results of different antimicrobial peptides on the cell membrane disruption of Candida albicans ATCC10231; A is the change in fluorescence intensity of the antimicrobial peptides GV1~GV4 and the control group after acting on Candida albicans ATCC10231 for different time periods; B is the change in fluorescence intensity of the antimicrobial peptides RK1~RK4 and the control group after acting on Candida albicans ATCC10231 for different time periods; a refers to the addition of propidium iodide, and b refers to the addition of RV-18-acy samples or control samples. DETAILED DESCRIPTION

[0018] The present invention provides use of an antimicrobial peptide RV-18-acy in the preparation of an antifungal product. The antimicrobial peptide RV-18-acy comprises a polypeptide RV-18 having an amino acid sequence as shown in SEQ ID NO.1.

[0019] As one embodiment, 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. As another embodiment, the fatty acid is dodecanoic acid. As one embodiment, the fatty acid is connected to the linker at the C-terminus or N-terminus of the polypeptide RV-18 through 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. The present invention can improve the stability of the polypeptide RV-18 by modifying the polypeptide RV-18 with fatty acids, thereby improving its antifungal activity.

[0020] As an embodiment, all amino acids of the antimicrobial peptide RV-18-acy are 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 production costs.

[0021] As an embodiment, the fungus is a Candida fungus. As another embodiment, the Candida fungus is Candida albicans. As an embodiment, the antifungal product can be a product that inhibits fungal growth and / or kills fungi. As an embodiment, the antifungal product includes an antifungal preparation or an antifungal drug. As another embodiment, the antifungal drug is an antifungal infection drug.

[0022] The antimicrobial peptide RV-18-acy provided by the present invention is a polypeptide that can form an amphipathic α-helical structure, has a molecular weight of 2478.00~2690.33 Daltons, can self-assemble in solution to form nanoparticles, has 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 a broad-spectrum antifungal activity, can target and destroy fungal cell membranes to exert a bactericidal effect, and exhibits good antibacterial effects on the standard strain of Candida albicans ATCC10231 and clinically resistant Candida albicans, with a minimum inhibitory concentration (MIC) of 2.344~75 μM. At the same time, the antimicrobial peptide RV-18-acy has no cytotoxic effect and no obvious hemolytic effect at the MIC concentration.

[0023] Based on the above advantages, the present invention provides an antifungal drug, the active ingredient of which includes antimicrobial peptide RV-18-acy; the antimicrobial peptide RV-18-acy includes a polypeptide RV-18 with 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.

[0024] As an embodiment, the antimicrobial peptide RV-18-acy includes one or more of GV1, GV2, GV3, GV4, RK1, RK2, RK3 and RK4; the GV1 is a polypeptide RV-18 with a decadecanoic acid modified at the N-terminus; the GV2 is a polypeptide RV-18 with a dodecanoic acid modified at the N-terminus; the GV3 is a polypeptide RV-18 with a tetradecanoic acid modified at the N-terminus; the GV4 is a polypeptide RV-18 with a hexadecanoic acid modified at the N-terminus; the RK1 is a polypeptide RV-18 with a decadecanoic acid modified at the C-terminus; the RK2 is a polypeptide RV-18 with a dodecanoic acid modified at the C-terminus; the RK3 is a polypeptide RV-18 with a tetradecanoic acid modified at the C-terminus; the RK4 is a polypeptide RV-18 with a hexadecanoic acid modified at the C-terminus; 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.

[0025] In one embodiment, the fungus is a Candida fungus. In another embodiment, the Candida fungus is Candida albicans.

[0026] In order to further illustrate the present invention, the application of the antimicrobial peptide RV-18-acy provided by the present invention in the preparation of antifungal products is described in detail below in combination with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 Design and preparation of self-assembling antimicrobial peptide RV-18-acy The amino acid sequence of the antimicrobial peptide RV-18 is RWRRFWGKAKRGIKKHGV (SEQ ID No. 1), which contains 18 L-type amino acids and is a straight-chain polypeptide. The helical wheel model of the antimicrobial peptide RV-18 was constructed through the website: https: / / heliquest.ipmc.cnrs.fr. The results are shown in Figure 1 The results showed that the antimicrobial peptide RV-18 exhibited a typical amphipathic structure.

[0028] In order to improve the stability and antibacterial activity of the antimicrobial peptide RV-18, fatty acids with different carbon chain lengths (decadecanoic acid, dodecadecanoic acid, tetradecanoic acid, and hexadecanoic acid) were linked to the N-terminus or C-terminus of the antimicrobial peptide RV-18 to obtain a series of fatty acid-modified acylated peptides, which were named RV-18-acy (see Figure 2 ). Among them, a glycine was added as a linker at the N-terminus of the antimicrobial peptide RV-18, and a peptide bond was formed through the amino group of glycine and the carboxyl group of fatty acids of different lengths to obtain antimicrobial peptides GV1~GV4; a glycine-lysine was added as a linker at the C-terminus of the antimicrobial peptide RV-18, and a peptide bond was formed through the side chain amino group of lysine and the carboxyl group of fatty acids of different lengths to obtain antimicrobial peptides RK1~RK4.

[0029] Preparation of antimicrobial peptide RV-18-acy: It was commissioned to Jier Biochemical (Shanghai) Co., Ltd. and synthesized by peptide solid phase synthesis method, and finally purified by desalting by HPLC reverse phase column chromatography.

[0030] Example 2 Secondary structure analysis of RV-18-acy 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).

[0031] 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.

[0032] Example 3 Transmission electron microscopy observation of RV-18-acy 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.

[0033] 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.

[0034] Example 4 Determination of critical micelle concentration (CMC) of RV-18-acy 100 μL of gradient concentration RV-18-acy solution was added to a 96-well fluorescent ELISA plate with a black bottom, and 100 μL of 25 μM Nile red solution was added to each well. After mixing, it was placed at room temperature for 30 min, and the emission spectrum of the solution was scanned with a multifunctional ELISA reader. The buffer solution used to prepare Nile red solution and gradient concentration RV-18-acy solution was 10 mM phosphate buffer solution (pH 7.4). Fluorescence spectrum scanning conditions: excitation wavelength of 550 nm, emission wavelength of 600~700 nm, bandwidth of 1 nm. Data processing: The logarithm of RV-18-acy concentration was used as the horizontal axis, and the maximum fluorescence intensity of Nile red at each concentration was used as the vertical axis. The Nile red fluorescence curve in the presence of gradient concentration RV-18-acy was plotted (excitation wavelength of 550 nm, emission wavelength of 630 nm). The peptide concentration corresponding to the inflection point of the fluorescence curve is the minimum concentration at which RV-18-acy begins to form micelles in aqueous solution, that is, CMC.

[0035] The results are as follows Figure 6 and Figure 7 As shown in the figure, when the concentration of RV-18-acy is higher than the CMC, it can induce a significant increase in the fluorescence of Nile red in the solution. Nile red is a lipophilic fluorescent dye, which can produce strong fluorescence after being inserted into the hydrophobic pocket of the micelle. The above results indicate that RV-18-acy self-assembles in aqueous solution to form amphiphilic micelles with hydrophilic periphery and hydrophobic interior. The data show that the CMC of RV-18-acy is 8.5~1308 μM. In addition, as the fatty acid chain length increases, the CMC concentration of RV-18-acy decreases and the ability to form micelles increases.

[0036] Example 5 Minimum inhibitory concentration (MIC) test of RV-18-acy against different standard strains The standard strains used for testing were the commercially available strain Candida albicans ATCC10231 and the Candida albicans from clinical sources (numbers 5, 0063 and 0065).

[0037] The strain to be tested was inoculated onto a YM solid plate. After the colonies grew, a single colony was picked and transferred to a YM liquid medium. The culture was placed at 37°C and 180 rpm for 5 h. The OD of the bacterial solution was measured using a UV spectrophotometer. 600 , according to 1 OD 600 = 1 × 10 9 CFU / mL method, the bacterial solution was diluted to 2×10 5CFU / mL concentration. Add 100 μL of diluted bacterial solution to each sterile 96-well plate, and then add 100 μL of the sample to be tested diluted with physiological saline in a gradient manner to each well. Use a pipette to blow and aspirate evenly, mix well, and place in a 37°C constant temperature incubator for slow shaking and incubation overnight. After constant temperature incubation, use an enzyme marker to measure the bacterial solution at OD 600 The absorbance value at nm was taken as the MIC value according to the average of the sample concentrations of the wells where no strain growth was detected and the adjacent wells. The results are shown in Table 1.

[0038] Table 1 Minimum inhibitory concentration of RV-18-acy against different standard fungal strains (μM)

[0039] As shown in Table 1, RV-18-acy showed good antibacterial effect on the standard strain of Candida albicans (ATCC10231) and clinical strains (No. 5, 0063 and 0065), with an overall MIC value of 2.34~75 μM. Among them, the MIC of RV-18-acy (GV1, RK1) linked to deca-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 indicate that the antifungal activity of RV-18-acy gradually increases with the increase of fatty acid chain length.

[0040] Example 6 Evaluation of the hemolytic effect of RV-18-acy on red blood cells After intraperitoneal anesthesia, whole blood was collected from the abdominal aorta of rats. The blood was mixed with Aldrich's solution at a volume ratio of 1:1, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. The red blood cells were washed with saline several times until the supernatant no longer appeared red. The washed red blood cells were resuspended and diluted with saline to a certain concentration (OD was measured after treatment with 1% Triton X-100). 540nmvalue is 1.6~2.0) for later use. Take an appropriate volume of RV-18-acy mother solution, dilute it to 1.38~88 μM with normal saline, and set aside. Mix 125 μL of diluted red blood cell suspension with an equal volume of RV-18-acy sample solution, incubate at 37°C for 30 min, and then centrifuge at 1000 rpm for 5 min, and detect the absorbance value of 200 μL supernatant at 540 nm. The positive control (PC) and negative control (NC) were equal volumes of 1% Triton X-100 and normal saline, respectively. In the experiment, three parallel groups were set for each concentration gradient. Data processing: The hemolysis rate of the PC group was defined as 100%, and the hemolysis rate of the RV-18-acy sample relative to the PC group was calculated.

[0041] The results are as follows Figure 8 As shown in the figure, at the MIC concentration, the eight RV-18-acy antimicrobial peptides had no hemolytic effect on erythrocytes. Structure-activity relationship analysis showed that within the test concentration range (0.69~44 μM), RV-18-acy (GV1, GV2, RK1 and RK2) linked to decadecanoic acid and dodecanoic acid had no obvious hemolytic effect on erythrocytes; with the increase of the length of the modified fatty acid chain, the hemolytic activity of RV-18-acy gradually increased, among which RV-18-acy modified with tetradecanoic acid (MIC of 4.688~18.75 μM) had no hemolytic effect on erythrocytes within the concentration range below 22 μM, and RV-18-acy modified with hexadecanoic acid (MIC of 2.344~4.688 μM) had no significant hemolytic effect on erythrocytes within the concentration range below 5.5 μM.

[0042] Example 7 Evaluation of RV-18-acy cytotoxicity against human keratinocytes HaCaT Human immortalized keratinocytes (HaCaT) were routinely cultured in DMEM medium (containing 10% fetal bovine serum and 1% double antibody). When the cells covered about 80% of the bottom of the culture flask, they were digested with trypsin, blown away, and counted. The cell density was adjusted to 5×10 4 / mL, and 100 μL per well was inoculated into a 96-well plate. After 24 h of culture, 10 μL of RV-18-acy sample solution with different concentration gradients (final concentration of 1.38~44 μM) or an equal volume of DMEM culture medium (the background well contains 110 μL of culture medium without cells and samples) was added. Continue to culture for 24 h, add 10 μL of CCK8 reagent 1.5~2 h later, and measure the absorbance value of each well solution at a wavelength of 450nm. Three replicates were set for each sample concentration, the background did not contain cells and samples, and only the same volume of culture medium and CCK-8 reagent were 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 the bar graph of the relative cell viability of the RV-18-acy sample solution was drawn using GraphPad prism software.

[0043] The results are as follows Fig. 9 As shown, 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.

[0044] Example 8: Membrane disruption effect of RV-18-acy on Candida albicans Candida albicans ATCC10231 was selected as the test strain to detect the membrane-breaking effect of the antimicrobial peptide RV-18-acy. The bacterial solution in the logarithmic growth phase was centrifuged at 1500 g for 5 min, the supernatant was discarded, the obtained bacterial precipitate was washed twice with saline, and the bacterial solution was resuspended with saline to adjust the bacterial solution concentration to 2 × 10 8 CFU / mL. Add the prepared bacterial solution to a 96-well plate with a black transparent bottom, 100 μL per well, and use an enzyme reader to continuously detect the fluorescence intensity of the solution within 15 minutes. Detection conditions: excitation wavelength Ex is 535 nm, emission wavelength Em is 615 nm, and reading interval is 1 min. Then add 10 μL of propidium iodide (PI) solution to make the final concentration 2.5 μg / mL, and use an enzyme reader to continuously detect the fluorescence intensity of the solution within 15 minutes. Continue to add 100 μL of gradient concentration antimicrobial peptide RV-18-acy solution or an equal volume of control solution, and use an enzyme reader to continuously detect the fluorescence intensity of the solution within 60 minutes. The final concentration of the RV-18-acy sample is 5 times the minimum inhibitory concentration of different antimicrobial peptides (GV1~GV4 and RK1~RK4) against Candida albicans ATCC10231 (ie, 5 × MIC), the final concentration of the positive control fluconazole is 5 × MIC, and the negative control is an equal volume of normal saline. The results are as follows Fig.10As shown, a is the time for adding 10 μL of propidium iodide solution, and b is the time for adding RV-18-acy sample or control.

[0045] Depend on Fig.10 It can be seen that with the increase of RV-18-acy concentration, the fluorescence intensity of PI in the solution gradually increased, indicating that RV-18-acy showed obvious membrane-breaking effect on Candida albicans in a concentration-dependent manner.

[0046] In summary, the antimicrobial 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.

[0047] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of antimicrobial peptide RV-18-acy in the preparation of antifungal products, characterized in that: The antimicrobial peptide RV-18-acy comprises a polypeptide RV-18 whose amino acid sequence is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: 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.

3. The use according to claim 2, characterized in that: The fatty acid is connected to the linker at the C-terminus or N-terminus of the polypeptide RV-18 via a peptide bond; the linker at the N-terminus of the polypeptide RV-18 is glycine; and the linker at the C-terminus of the antimicrobial peptide RV-18 is glycine-lysine.

4. The use according to any one of claims 1 to 3, characterized in that: The fungus is a Candida fungus.

5. The use according to claim 4, characterized in that: The Candida fungus is Candida albicans Candida albicans .

6. The use according to claim 5, characterized in that: The antifungal products include antifungal preparations or antifungal drugs.

7. The use according to claim 6, characterized in that: The antifungal drug is an antifungal infection drug.

8. An antifungal drug, characterized in that: The active ingredient includes the antimicrobial peptide RV-18-acy; the antimicrobial peptide RV-18-acy includes the polypeptide RV-18 whose amino acid sequence is 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.

9. The antifungal drug according to claim 8, characterized in that The antimicrobial peptide RV-18-acy includes one or more of GV1, GV2, GV3, GV4, RK1, RK2, RK3 and RK4; the GV1 is a polypeptide RV-18 with a decadecanoic acid modified at the N-terminus; the GV2 is a polypeptide RV-18 with a dodecanoic acid modified at the N-terminus; the GV3 is a polypeptide RV-18 with a tetradecanoic acid modified at the N-terminus; the GV4 is a polypeptide RV-18 with a hexadecanoic acid modified at the N-terminus; the RK1 is a polypeptide RV-18 with a decadecanoic acid modified at the C-terminus; the RK2 is a polypeptide RV-18 with a dodecanoic acid modified at the C-terminus; the RK3 is a polypeptide RV-18 with a tetradecanoic acid modified at the C-terminus; the RK4 is a polypeptide RV-18 with a hexadecanoic acid modified at the C-terminus; 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.

10. The antifungal drug according to claim 8 or 9, characterized in that: The fungus is a Candida fungus.

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