Antibacterial peptide RV15 and application thereof

Abstract

The application discloses an antibacterial peptide RV15 and application thereof, and belongs to the technical field of biology. The amino acid sequence of the antibacterial peptide RV15 is shown as SEQ ID NO. 1. The antibacterial peptide RV15 provided by the application has broad-spectrum antibacterial activity, has a significant bacteriostatic effect on gram-positive bacteria and gram-negative bacteria, and has a long bactericidal duration. The hemolytic activity is very low, and the biological safety is good. The antibacterial peptide RV15 can be used for treating bacterial infection, and can also be applied to other scenes requiring bactericidal action or bacterial growth inhibition. The antibacterial peptide RV15 provided by the application has a short sequence, a small molecular weight and low chemical synthesis difficulty, and can save the production cost on a large scale.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to an antimicrobial peptide RV15 with high antimicrobial activity and its applications. Background Technology

[0002] Through prolonged and repeated exposure to antibiotics and other drugs, the majority of susceptible bacterial strains are continuously killed off, while drug-resistant strains proliferate and replace them, leading to a continuous increase in bacterial resistance rates. Simultaneously, resistance genes spread among microorganisms, exacerbating bacterial and fungal resistance. Drug-resistant bacteria also evolve and mutate, acquiring resistance to various antimicrobial agents. Bacteria gradually progress from single-drug resistance to multidrug resistance and even pan-drug resistance, ultimately becoming drug-resistant superbugs, posing a serious threat to human and animal health.

[0003] Antimicrobial peptides (AMPs) are a class of basic polypeptides with antimicrobial activity produced in vivo through induction, and are an important component of the innate immune system of organisms. Natural antimicrobial peptides are typically small cationic polypeptides composed of 12–60 amino acids. Rich in basic amino acids such as lysine, arginine, and histidine, antimicrobial peptides usually carry 2–7 positive charges, with an isoelectric point greater than 7, exhibiting strong cationic characteristics. Antimicrobial peptides generally have an amphiphilic structure, with one hydrophobic region binding to lipids and one positively charged hydrophilic region binding to water or negatively charged residues. These properties enable antimicrobial peptides to bind well to amphiphilic molecules, especially electronegative cell membranes, which is the structural basis for the interaction between antimicrobial peptides and bacterial cell membranes.

[0004] Because antimicrobial peptides have broad-spectrum antibacterial activity, they also inhibit and kill some viruses, parasites, and tumor cells, and are not prone to developing drug resistance. As a possible alternative to antibiotics, they have good application prospects in research fields such as medicine, veterinary medicine, and life sciences.

[0005] Natural AMPs are widely distributed in specific tissues of various animals; data shows that more than 2,000 AMPs have been reported in the literature to date. However, compared with antibiotics, antimicrobial peptides have limitations such as lower antimicrobial activity, uncertain toxicity characteristics, and easy inactivation during production and transportation, hindering their widespread application. To address these challenges, current research focuses on developing antimicrobial peptides with enhanced activity, reduced toxicity, and improved hydrolysis resistance.

[0006] In recent years, researchers have used computer-aided bioinformatics prediction and other related software technologies to design and optimize the coding sequences of antimicrobial proteins, obtaining a series of improved recombinant antimicrobial peptides / proteins. To address issues such as the spatial instability of some AMPs and their hemolytic activity, researchers have attempted to enhance their antimicrobial activity and reduce their immune response by replacing certain amino acids within the AMP molecule and modifying its molecular structure. Alternatively, altering the content of certain amino acids in AMPs can further enhance their bactericidal activity and reduce hemolytic reactions.

[0007] Most current research on antimicrobial peptides is still in the preclinical stage, with insufficient research on toxicity and stability, and relatively few antimicrobial peptides have been developed into finished drugs. Therefore, developing more novel antimicrobial peptides with good antimicrobial activity and fewer side effects remains a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to provide a novel antimicrobial peptide with strong antibacterial activity and low toxicity.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] This invention provides an antimicrobial peptide RV15, the amino acid sequence of which is shown in SEQ ID NO.1. Specifically, it is Arg-Val-Leu-Trp-Ile-Lys-Arg-Trp-Ile-Lys-Arg-Phe-Phe-Arg-Pro.

[0011] In this invention, the antimicrobial peptide RV15 can be prepared by chemical synthesis or genetic engineering.

[0012] This invention provides the use of the antimicrobial peptide RV15 in the preparation of antimicrobial drugs. The bacteria inhibited by the antimicrobial drugs include Gram-positive and Gram-negative bacteria.

[0013] Furthermore, the antibacterial drug is used in the preparation of drugs that inhibit Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa.

[0014] Furthermore, the minimum inhibitory concentration (MIC) of the antimicrobial peptide RV15 is 8 μg / mL against Enterococcus faecalis; 8 μg / mL against Staphylococcus aureus; 8 μg / mL against Acinetobacter baumannii; 8 μg / mL against Escherichia coli; 16 μg / mL against Klebsiella pneumoniae; and 16 μg / mL against Pseudomonas aeruginosa.

[0015] The bacteria inhibited by the antimicrobial drug include both drug-resistant and non-drug-resistant bacteria. This invention demonstrates that the antimicrobial peptide RV15 exhibits good antimicrobial activity against both drug-resistant and non-drug-resistant Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae.

[0016] Compared to polymyxin E, the antimicrobial peptide RV15 provided by this invention has a longer duration of bactericidal effect against Escherichia coli and can effectively prevent the development of bacterial resistance.

[0017] The antimicrobial peptide RV15 provided by this invention has low hemolytic activity and is biosafe.

[0018] The antimicrobial peptide RV15 provided by this invention can also be applied to various scenarios where bacteria need to be killed or inhibited. Specifically, this invention also provides the application of antimicrobial peptide RV15 in the preparation of preservatives, daily chemical detergents, and medical devices with antimicrobial effects.

[0019] This invention provides a biological antimicrobial agent whose antimicrobial component includes the aforementioned antimicrobial peptide RV15. The antimicrobial peptide RV15 is combined with pharmaceutically or food-acceptable excipients to prepare the corresponding biological antimicrobial agent.

[0020] The present invention provides a preservative comprising the aforementioned antimicrobial peptide RV15.

[0021] Furthermore, the preservative is a preservative used in food or a preservative used in cosmetics.

[0022] The present invention also provides a daily chemical detergent composition comprising the aforementioned antimicrobial peptide RV15.

[0023] Furthermore, the composition may be, but is not limited to, hand sanitizer, soap, shower gel, shampoo, toothpaste, laundry detergent, laundry powder, etc.

[0024] The present invention provides a medical dressing comprising the aforementioned antimicrobial peptide RV15.

[0025] The beneficial effects of this invention are as follows:

[0026] (1) The antimicrobial peptide RV15 provided by the present invention has broad-spectrum antimicrobial activity, and has significant antibacterial effects against both Gram-positive and Gram-negative bacteria, and its bactericidal effect lasts for a long time; it has extremely low hemolytic activity and good biosafety, and can be used for the treatment of bacterial infections, and can also be applied to other scenarios that require sterilization or inhibition of bacterial growth.

[0027] (2) The antimicrobial peptide RV15 provided by the present invention has a short sequence, small molecular weight, and low chemical synthesis difficulty, which can save the cost of large-scale production. Attached Figure Description

[0028] Figure 1The time-killing curves of RV15 and polymyxin E (col) against ATCC 25922 are shown.

[0029] Figure 2 The hemolysis rate of 100 μg / mL antimicrobial peptide RV15. Detailed Implementation

[0030] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0032] 1. Strains

[0033] The standard strains used in this experiment were: Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922.

[0034] Clinically resistant strains include: pan-drug resistant (PDR) Klebsiella pneumoniae 418015, extensive-drug resistant (XDR) Klebsiella pneumoniae 325016, multi-drug resistant (MDR) Klebsiella pneumoniae 327004, MDR Escherichia coli 103231, MDR Pseudomonas aeruginosa 304238, and MDR Acinetobacter baumannii 316039. Their drug susceptibility results are shown in Table 1.

[0035] Table 1. Antimicrobial susceptibility results of clinical strains

[0036]

[0037] Note: R: drug resistance; S: sensitivity; I: intermediate.

[0038] Example 1

[0039] 1. Preparation of antimicrobial peptide RV15

[0040] The antimicrobial peptide RV15 provided in this embodiment was designed using artificial intelligence technology, and its amino acid sequence is: RVLWIKRWIKRFFRP (SEQ ID NO.1). The antimicrobial peptide was synthesized by a commissioned biotechnology company using solid-phase synthesis.

[0041] 2. MIC determination of antimicrobial peptides

[0042] The MIC assay was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, using the microbroth dilution method. First, the antimicrobial peptide was dissolved in sterile distilled water to an initial concentration of 5120 μg / mL and stored at 4°C. Before use, the antimicrobial peptide was diluted to 512 μg / mL with cationic-adjusted Mueller-Hinton broth (CAMHB), followed by a two-fold serial dilution in 96-well plates. The bacterial culture was adjusted to a 0.5 McFarland concentration, diluted 100-fold with fresh CAMHB, and 100 μL was added to each well of the 96-well plate containing the antimicrobial peptide to achieve a final bacterial concentration of 5 × 10⁻⁶. 5 CFU / mL. After incubation at 37°C for 18 hours, the MIC value was the minimum concentration of the antimicrobial peptide at which no obvious bacterial growth was observed visually. The results are shown in Tables 2 and 3.

[0043] Table 2. Antimicrobial activity of antimicrobial peptide RV15 against 6 standard bacterial strains

[0044] <![CDATA[MIC a (μg / mL)]]> <![CDATA[ATCC 29212 b ]]> 8 <![CDATA[ATCC 29213 c ]]> 8 <![CDATA[ATCC 700603 d ]]> 16 <![CDATA[ATCC 19606 e ]]> 8 <![CDATA[ATCC 27853 f ]]> 16 <![CDATA[ATCC 25922 g ]]> 8

[0045] In the table, a: minimum inhibitory concentration; b: Enterococcus faecalis; c: Staphylococcus aureus; d: Klebsiella pneumoniae; e: Acinetobacter baumannii; f: Pseudomonas aeruginosa; g: Escherichia coli; all quality controls were within the allowable range, all growth control wells were turbid, and all blank control wells were clear.

[0046] Table 3. MIC of RV15 against drug-resistant bacteria

[0047]

[0048]

[0049] As shown in Table 2, the MICs of RV15 against standard Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae ATCC 700603, Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922 were all between 8 and 16 μg / mL.

[0050] As shown in Table 3, RV15 also exhibits good antibacterial activity against clinically resistant bacteria, with MICs ranging from 8 to 16 μg / mL.

[0051] 3. Time-based sterilization experiment

[0052] The bactericidal kinetics of RV15 against standard *Escherichia coli* ATCC 25922 were studied. *E. coli* culture was adjusted to a 0.5 McFarland concentration, diluted 100-fold with fresh CAMHB medium, and incubated at 37°C and 150 rpm for 3-4 hours to induce the logarithmic growth phase. Four shake tubes were prepared by adding 10 mL of 1×MIC polymyxin E (0.5 μg / mL) and RV15 (8 μg / mL), and 4×MIC polymyxin E (2 μg / mL) and RV15 (32 μg / mL), respectively, to CAMHB medium. Another shake tube was prepared by adding 10 mL of CAMHB medium. The *E. coli* culture in the logarithmic growth phase was adjusted to 1 MCF, diluted 100-fold, and added to five shake tubes to achieve a final bacterial concentration of 3×10⁻⁶. 6 CFU / mL was incubated at 37°C and 150 rpm. At 0, 2, 4, 6, 18, and 24 hours, the medium was serially diluted 10-fold with fresh CAMHB medium and dropped onto Mueller-Hinton agar (MHA) plates, incubated at 37°C and 150 rpm for 18–24 hours, and the results were read. No antimicrobial peptide was used as a blank control, and polymyxin E was used as a positive control.

[0053] The results are as follows Figure 1 As shown, RV15 with a concentration of 1×MIC can kill all E. coli within 2 hours, and no E. coli can revive until 24 hours. While polymyxin E with a concentration of 4×MIC can exert a rapid bactericidal effect within 2 hours, bacteria gradually revive after 4 hours of co-incubation. This indicates that RV15 has a similar bactericidal rate to polymyxin E against standard E. coli, but with a longer duration of action.

[0054] 4. Antimicrobial peptide hemolysis test

[0055] Fresh human red blood cells (RBCs) were washed three times with sterile PBS and then added to 96-well U-shaped plates containing 100 μg / mL antimicrobial peptides to achieve a RBC concentration of 2%, with a final volume of 200 μL per well. RBCs treated with PBS alone served as a negative control, and RBCs treated with 0.5% Triton X-100 served as a positive control. After incubation at 37°C for 1 hour, the plates were centrifuged at 1200g for 15 minutes at 4°C. The supernatant was collected in new flat-bottomed 96-well plates, and OD was measured. 570 The experiment was repeated three times, and the results were as follows: Figure 2 As shown. Calculate the hemolysis rate using the following formula.

[0056]

[0057] like Figure 2As shown, the hemolysis rate of RV15 at 100 μg / mL was only 6.5%, indicating that RV15 has low hemolytic activity and high blood compatibility.

[0058] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. For example, based on the antibacterial activity of the antimicrobial peptide RV15, it can be used to prepare antimicrobial biological products. Therefore, all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide RV15, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide RV15 as described in claim 1 in the preparation of a medicament for inhibiting Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, or Pseudomonas aeruginosa.

3. The application of the antimicrobial peptide RV15 as described in claim 1 in the preparation of preservatives, daily chemical detergents, and medical devices with antimicrobial effects.

4. A biological antibacterial agent, characterized in that, Its antibacterial component is the antimicrobial peptide RV15 as described in claim 1.

5. A preservative, characterized in that, Includes the antimicrobial peptide RV15 as described in claim 1.

6. The preservative as described in claim 5, characterized in that, The preservative is a preservative used in food or a preservative used in cosmetics.

7. A daily chemical detergent composition, characterized in that, Includes the antimicrobial peptide RV15 as described in claim 1.

8. A medical dressing, characterized in that, Includes the antimicrobial peptide RV15 as described in claim 1.

Images