Antibacterial peptide RV-18-acy and application thereof in preparation of antibacterial products

Abstract

The invention relates to the technical field of antibacterial peptides, in particular to an antibacterial peptide RV-18-ack and application thereof in preparation of antibacterial products. The antibacterial peptide RV-18-ack provided by the invention comprises a main chain and fatty acid modified at a C end or an N end of the main chain, the amino acid sequence of the main chain is as shown in SEQ ID NO. 1. The antibacterial peptide RV-18-ack provided by the invention is a polypeptide capable of forming an amphiphilic alpha helical structure, has broad-spectrum antibacterial activity, shows a better antibacterial effect on acinetobacter baumannii, pseudomonas aeruginosa, escherichia coli and staphylococcus aureus, has the minimum inhibitory concentration of 1.17-4.69 mu M, and is free of cytotoxicity and hemolysis; all amino acids are L-type amino acids, so that the production cost is greatly reduced, and the compound is expected to become a novel broad-spectrum antibacterial candidate drug and has a good application prospect in the aspect of resisting super bacteria.

Description

Technical Field

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

[0002] Antibiotic-induced bacterial resistance has become a major public health issue that threatens human life and health worldwide. Humans may face the advent of a post-antibiotic era without effective antibiotics. Antimicrobial peptides, also known as host defense peptides, are polypeptide substances with broad-spectrum antimicrobial activity and are considered a new alternative to traditional antibiotics.

[0003] Antimicrobial peptides are a class of small polypeptide molecules with antimicrobial activity. They are widely distributed in insects, animals and plants, and show good and broad-spectrum antimicrobial activity. At the same time, due to their small molecular weight, strong thermal stability, good water solubility, non-immunogenicity, low resistance to drug resistance, and broad antimicrobial spectrum, antimicrobial peptides have become the best antibiotic substitute molecules. However, there are still many limitations in the development and application of antimicrobial peptides, such as the difficulty in extracting antimicrobial peptides from organisms, high production costs, and instability in vivo. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an antimicrobial peptide RV-18-acy and its use in the preparation of antibacterial products. The antimicrobial peptide RV-18-acy provided by the present invention has broad-spectrum antibacterial 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 invention provides an antimicrobial peptide RV-18-acy, comprising a main chain and a fatty acid modified at the C-terminus or the N-terminus of the main chain; the amino acid sequence of the main chain is shown in SEQ ID NO.1.

[0006] Preferably, the fatty acid is 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 main chain via a peptide bond; the linker at the N-terminus of the main chain is glycine; and the linker at the C-terminus of the main chain is glycine-lysine.

[0008] The present invention provides the use of the antimicrobial peptide RV-18-acy described in the above technical solution in the preparation of antibacterial products.

[0009] Preferably, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

[0010] Preferably, the Gram-negative bacteria include Acinetobacter baumannii (Acinetobacter baumannii )、Pseudomonas aeruginosa( Pseudomonas aeruginosa ) and Escherichia coli ( Escherichia coli ) one or more; the Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

[0011] Preferably, the antibacterial product comprises an antibacterial preparation or an antibacterial drug.

[0012] The present invention provides an antibacterial drug, the active ingredient of which includes the antibacterial peptide RV-18-acy described in the above technical solution.

[0013] Preferably, the minimum inhibitory concentration of the antimicrobial peptide RV-18-acy in the antibacterial drug is 1.17~4.69 μM.

[0014] Preferably, the bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.

[0015] Beneficial effects: The present invention provides an antimicrobial peptide RV-18-acy, comprising a main chain and a fatty acid modified at the C-terminus or N-terminus of the main chain; the amino acid sequence of the main chain is 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 antibacterial activity, and exhibits good antibacterial effects on Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 1.17~4.69 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 antibacterial candidate drug, and has good application prospects in the fight against "super bacteria". 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 keratinocyte HaCaT cells. DETAILED DESCRIPTION

[0018] The invention provides an antimicrobial peptide RV-18-acy, comprising a main chain and a fatty acid modified at the C-terminus or the N-terminus of the main chain; the amino acid sequence of the main chain is shown in SEQ ID NO.1.

[0019] As an embodiment, the fatty acid can be decanoic acid, dodecanoic acid, tetradecanoic acid or hexadecanoic acid. As another embodiment, the fatty acid is dodecanoic acid. As an embodiment, the fatty acid is connected to the linker at the C-terminus or N-terminus of the main chain through a peptide bond; the linker at the N-terminus of the main chain is glycine; the linker at the C-terminus of the main chain 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 antibacterial 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] 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 antibacterial activity, and exhibits good antibacterial effects on Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 1.17~4.69 μM. At the same time, the antimicrobial peptide RV-18-acy has no cytotoxic effect and no obvious hemolytic effect at the MIC concentration.

[0022] Based on the above advantages, the present invention provides the use of the antimicrobial peptide RV-18-acy described in the above technical solution in the preparation of antibacterial products.

[0023] As an embodiment, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria. As an embodiment, the Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa and Escherichia coli; the Gram-positive bacteria include Staphylococcus aureus.

[0024] As an embodiment, the antibacterial product can be a product that inhibits bacterial growth and / or kills bacteria. As an embodiment, the antibacterial product includes an antibacterial preparation or an antibacterial drug. As another embodiment, the antibacterial drug is an antibacterial infection drug.

[0025] Based on the above advantages, the present invention provides an antibacterial drug, the active ingredient of which includes the antimicrobial peptide RV-18-acy described in the above technical solution. As an embodiment, the bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.

[0026] As an embodiment, the minimum inhibitory concentration of the antimicrobial peptide RV-18-acy in the antibacterial drug is 1.17~4.69 μM.

[0027] 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 2.34 μM; The minimum inhibitory concentration of GV2 was 1.17 μM; The minimum inhibitory concentration of GV3 is 2.34~4.69 μM; The minimum inhibitory concentration of GV4 was 2.34 μM; The minimum inhibitory concentration of RK1 is 2.34~4.69 μM; The minimum inhibitory concentration of RK2 is 1.17 μM; The minimum inhibitory concentration of RK3 is 1.17~2.34 μM; The minimum inhibitory concentration of RK4 is 4.69 μM.

[0028] As an embodiment, the antibacterial drug further comprises a pharmaceutically acceptable excipient.

[0029] In order to further illustrate the present invention, the antimicrobial peptide RV-18-acy provided by the present invention and its application in the preparation of antibacterial products are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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).

[0034] 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 bacterial cell membrane, due to the increase in hydrophobicity in the environment, RV-18-acy can form an amphipathic α-helical conformation to play a bactericidal role.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Example 5 Minimum inhibitory concentration (MIC) test of RV-18-acy against different standard strains The standard bacterial strains used for testing are all commercially available strains, specifically: Acinetobacter baumannii ATCC19606, Escherichia coli ATCC8739, Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC6538.

[0040] The strain to be tested was inoculated from a single colony on the LB solid culture plate into LB liquid culture medium for shaking culture. The bacterial solution in the logarithmic growth phase was centrifuged (1500 g × 5 min), the supernatant was discarded, and the bacterial precipitate was adjusted to a bacterial solution concentration of 2×10 5 CFU / mL concentration. By microdilution method, 100 μL of diluted bacterial solution was mixed with gradient concentration RV-18-acy sample solution in a sterile 96-well plate, and then the mixture was placed in a constant temperature incubator at 37°C. After 24 h, the bacterial solution was measured at OD 600nmThe absorbance value at the well where no bacterial growth was detected and the average of the sample concentrations of the adjacent wells were used as the MIC value. The results are shown in Table 1.

[0041] Table 1 Minimum inhibitory concentration of RV-18-acy against different standard bacterial strains (μM)

[0042] As shown in Table 1, RV-18-acy showed good antibacterial effects against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, with MICs ranging from 1.17 to 4.69 μM. The results showed that the MIC of RV-18-acy (GV2, RK2) linked to dodecanoic acid was 1.17 μM, and the MICs of the two RV-18-1cy (GV4, RK4) linked to hexadecanoic acid were 2.34 and 4.69 μM, respectively. It can be seen that the fatty acid chain length has an effect on the antibacterial activity of RV-18-acy: with the increase of fatty acid chain length, the antibacterial activity of RV-18-acy first increases and then weakens. Among them, when the fatty acid chain length is 12 carbon atoms, RV-18-acy has the best antibacterial activity.

[0043] 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). 540nm value 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.

[0044] The results are as follows Figure 8As 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 1.17~4.69 μM) had no hemolytic effect on erythrocytes within the concentration range below 22 μM, and RV-18-acy modified with hexadecanoic acid (MIC of 2.34~4.69 μM) had no significant hemolytic effect on erythrocytes within the concentration range below 5.5 μM.

[0045] 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.

[0046] 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.

[0047] In summary, the antimicrobial peptide RV-18-acy provided by the present invention has a broad-spectrum antibacterial activity, has no cytotoxicity and obvious hemolytic effect, can be obtained by artificial synthesis, has low production cost and good stability.

[0048] 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. An antimicrobial peptide RV-18-acy, characterized in that It comprises a main chain and a fatty acid modified at the C-terminus or N-terminus of the main chain; the amino acid sequence of the main chain is shown in SEQ ID NO.

1.

2. The antimicrobial peptide RV-18-acy according to claim 1, characterized in that The fatty acid is decanoic acid, dodecanoic acid, tetradecanoic acid or hexadecanoic acid.

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

4. Use of the antimicrobial peptide RV-18-acy according to any one of claims 1 to 3 in the preparation of antibacterial products.

5. The use according to claim 4, characterized in that: The bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

6. The use according to claim 5, characterized in that: The Gram-negative bacteria include Acinetobacter baumannii Acinetobacter baumannii Pseudomonas aeruginosa Pseudomonas aeruginosa and E. coli Escherichia coli One or more of; the Gram-positive bacteria include Staphylococcus aureus Staphylococcus aureus .

7. The use according to claim 4, characterized in that: The antibacterial products include antibacterial preparations or antibacterial drugs.

8. An antibacterial drug, characterized in that: The active ingredient comprises the antimicrobial peptide RV-18-acy according to any one of claims 1 to 3.

9. The antibacterial drug according to claim 8, characterized in that The minimum inhibitory concentration of the antimicrobial peptide RV-18-acy in the antibacterial drug is 1.17~4.69 μM.

10. The antibacterial drug according to claim 8 or 9, characterized in that: The bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus.

Images