Antibacterial peptide RF-18 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 RF-18 and application thereof in preparation of antibacterial products. The amino acid sequence of the antibacterial peptide RF-18 provided by the invention is as shown in SEQ ID NO. 1. The antibacterial peptide RF-18 provided by the invention is an antibacterial peptide capable of being artificially synthesized and only contains 18 amino acids, and all the amino acids are L-type amino acids, so that the production cost is greatly reduced; besides, the antibacterial peptide RF-18 provided by the invention is a novel antibacterial peptide with an imperfect amphiphilic structure, not only can exert efficient and broad-spectrum antibacterial activity, but also has extremely high safety, has no cytotoxicity or hemolysis effect in a concentration range of 800 mu g / mL, and can be used for preparing antibacterial infection drugs.

Description

Technical Field

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

[0002] The discovery and clinical application of antibiotics are considered one of the most important breakthroughs in medicine. Their use has greatly increased human life expectancy. However, due to the extensive use or abuse of antibiotics and antibacterial drugs, bacteria have become increasingly resistant to antibiotics, giving rise to a large number of "super bacteria", such as the extensively drug-resistant Acinetobacter baumannii ( Acinetobacter baumannii ) and Staphylococcus aureus ( Staphylococcus aureus ) etc. In order to cope with the increasingly serious problem of antibiotic resistance in the world and avoid the arrival of the post-antibiotic era without effective antibiotics, it is urgent to develop new antibacterial drug molecules.

[0003] Antimicrobial peptides, also known as host defense peptides, are an important part of the body's innate immune system. They can help multicellular organisms such as plants, animals or humans resist microbial infections. They have the characteristics of fast bactericidal speed and broad antibacterial spectrum. Unlike antibiotics, antimicrobial peptides exert their antibacterial effects mainly by targeting the cell membrane of bacteria, which makes it almost impossible for bacteria to develop resistance to antimicrobial peptides. Therefore, antimicrobial peptides can be developed as new antimicrobial candidate drugs. However, the sequences of natural antimicrobial peptides are relatively long and the synthesis cost is relatively high. At the same time, antimicrobial peptides often have poor selectivity and are prone to produce certain toxicity to mammalian cells. This characteristic greatly limits the development of antimicrobial peptides as antimicrobial candidate drugs. Summary of the invention

[0004] In order to solve the above problems, the present invention provides an antimicrobial peptide RF-18 and its application in the preparation of antibacterial products. The antimicrobial peptide RF-18 of the present invention is an antimicrobial peptide that can be artificially synthesized, contains only 18 amino acids, and all amino acids are L-type amino acids, which greatly reduces the production cost; at the same time, the antimicrobial peptide RF-18 of the present invention also has extremely high safety, and has neither cytotoxicity nor hemolytic effect within the concentration range of 800 μg / mL.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides an antimicrobial peptide RF-18, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Preferably, the C-terminus of the antimicrobial peptide RF-18 is amidated.

[0007] The present invention provides the use of the antimicrobial peptide RF-18 described in the above technical solution in the preparation of antibacterial products.

[0008] Preferably, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

[0009] 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 ).

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

[0011] The present invention provides an antibacterial drug, the active ingredient of which includes the antibacterial peptide RF-18 described in the above technical solution.

[0012] Preferably, the minimum inhibitory concentration (MIC) of the antimicrobial peptide RF-18 in the antibacterial drug is 1.03-3.81 μg / mL.

[0013] Preferably, the bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

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

[0015] Beneficial effects: The present invention provides an antimicrobial peptide RF-18, the amino acid sequence of which is shown in SEQ ID NO.1. The antimicrobial peptide RF-18 provided by the present invention is an artificially synthesized antimicrobial peptide, which contains only 18 amino acids, and all amino acids are L-type amino acids, which greatly reduces the production cost; in addition, the antimicrobial peptide RF-18 provided by the present invention is a new antimicrobial peptide with an imperfect amphiphilic structure, which can not only exert efficient and broad-spectrum antimicrobial activity, but also has extremely high safety, has neither cytotoxicity nor hemolytic effect within a concentration range of 800 μg / mL, and can be used to prepare antibacterial infection drugs. 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 RF-18; Figure 2The figure shows the cytotoxicity test results of the antimicrobial peptide RF-18 on human keratinocytes (HaCaT); Figure 3 This is the experimental result of the hemolytic effect of the antimicrobial peptide RF-18 on human red blood cells. DETAILED DESCRIPTION

[0018] The present invention provides an antimicrobial peptide RF-18, the amino acid sequence of which is shown in SEQ ID NO.1, specifically as follows: RRKVKKVIKAIKKGIKKF. As an embodiment, the C-terminus of the antimicrobial peptide RF-18 is amidated. The present invention improves the stability of the antimicrobial peptide RF-18 by amidating the C-terminus of the antimicrobial peptide RF-18. As an embodiment, all amino acids of the antimicrobial peptide RF-18 are L-type. The present invention uses L-type amino acids as raw materials to synthesize the antimicrobial peptide RF-18, which can greatly reduce production costs.

[0019] As an embodiment, the synthesis method of the antimicrobial peptide RF-18 can be a polypeptide solid phase synthesis method. The present invention has no special requirements for the polypeptide solid phase synthesis method, and a method well known to those skilled in the art can be used.

[0020] The antimicrobial peptide RF-18 provided by the present invention contains 18 amino acids, a molecular weight of 2167.83 Daltons, an isoelectric point of 12.06, a straight-chain polypeptide, and all amino acids are L-type. In vitro antibacterial experiments show that RF-18 has a broad-spectrum antibacterial activity, and exhibits good antibacterial effects on standard strains and clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, with a minimum inhibitory concentration (MIC) of 1.03~3.81 μg / mL. At the same time, RF-18 can form an imperfect amphipathic α-helical structure, showing extremely high safety, and has neither cytotoxicity nor hemolytic effect within a concentration range of 800 μg / mL.

[0021] Based on the above advantages, the present invention provides the use of the antimicrobial peptide RF-18 described in the above technical solution in the preparation of antibacterial products. 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.

[0022] 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.

[0023] Based on the above advantages, the present invention provides an antibacterial drug, the active ingredient of which includes the antibacterial peptide RF-18 described in the above technical solution.

[0024] As an embodiment, the minimum inhibitory concentration of the antimicrobial peptide RF-18 in the antibacterial drug is 1.03~3.81μg / mL.

[0025] 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.

[0026] As an embodiment, the antibacterial drug further comprises a pharmaceutically acceptable excipient.

[0027] In order to further illustrate the present invention, an antimicrobial peptide RF-18 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.

[0028] Example 1 Gill Biochemical (Shanghai) Co., Ltd. was commissioned to synthesize the polypeptide shown in SEQ ID No. 1 by peptide solid phase synthesis, and amidation modification was performed on its C-terminus. Finally, the antimicrobial peptide RF-18 was obtained by desalting and purifying by HPLC reverse phase column chromatography.

[0029] The helical wheel model of the antimicrobial peptide RF-18 was constructed through the website: https: / / heliquest.ipmc.cnrs.fr. The results are as follows Figure 1 The results showed that the antimicrobial peptide RF-18 exhibited imperfect amphipathic characteristics.

[0030] Example 2 The antimicrobial activity analysis of the antimicrobial peptide RF-18 is as follows: The strains to be tested are commercially available standard strains and clinically derived strains. The clinically derived strains are clinically derived drug-resistant strains, which are disclosed in Chinese patent CN116675740A. The specific strains to be tested and their numbers are as follows: The tested Acinetobacter baumannii strains were: the standard strain of Acinetobacter baumannii ATCC19606 and the strains with clinical source numbers 10769 and 0357; The Escherichia coli strains to be tested are: Escherichia coli standard strain ATCC8739 and strains with clinical source numbers 0894 and 5017; The Pseudomonas aeruginosa strains to be tested are: the standard strain of Pseudomonas aeruginosa ATCC27853 and the strains with clinical source numbers 90068 and 17068; The strains of Staphylococcus aureus to be tested are: the standard strain of Staphylococcus aureus ATCC6538 and the strains with clinical source numbers 220823 and 15775.

[0031] The strain to be tested was inoculated onto the LB solid plate. After the colonies grew, a single colony was picked and transferred to the LB 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 1OD 600 =1×10 9 CFU / mL ratio, dilute the bacterial solution to 2×10 5 CFU / mL concentration; 100 μL of diluted bacterial solution was added to each sterile 96-well plate, and then 100 μL of the sample to be tested diluted with physiological saline in a gradient manner was added to each well, and the mixture was pipetted and mixed with a pipette, and then placed in a 37°C constant temperature incubator for slow shaking and culture overnight; the sample to be tested was the antimicrobial peptide RF-18; the concentration of the diluted sample to be tested was 0-200 μg / mL; after constant temperature culture, the bacterial solution was measured at OD using an enzyme marker 600 The absorbance value at nm was taken as the minimum inhibitory concentration (MIC) according to the average of the sample concentrations of the well where no bacterial growth was detected and the adjacent wells. The results are shown in Table 1.

[0032] Table 1 Minimum inhibitory concentration of antimicrobial peptide RF-18 against test strains

[0033] As shown in Table 1, RF-18 showed significant antibacterial effects on the standard strain of Acinetobacter baumannii (ATCC19606) and clinical strains (numbered 10769 and 0357), with MIC values ​​of 2.64~3.81 μg / mL; RF-18 also showed significant antibacterial effects on the standard strain of Escherichia coli (ATCC8739) and clinical strains (numbered 0894 and 5017), with MIC values ​​of 1.03~1.76 μg / mL; RF-18 also showed significant antibacterial effects on the standard strain of Pseudomonas aeruginosa (ATCC27853) and clinical strains (numbered 90068 and 17068), with MIC values ​​of 2.34 μg / mL; RF-18 showed significant antibacterial effects on the standard strain of Staphylococcus aureus (ATCC6538) and clinical strains (numbered 220823 and 15775), with MIC values ​​of 1.17~1.76 μg / mL.

[0034] Example 3 The cytotoxicity test of antimicrobial peptide RF-18 is as follows: When the HaCaT cells covered about 80% of the bottom of the culture flask containing DMEM medium, they were washed three times with phosphate buffer, and then digested with 0.25% trypsin. The cells were diluted with DMEM medium to 5 × 10 5 100 μg / mL cell suspension was added to each well of a sterile 96-well plate, and 200 μL of the cell suspension was added to each well, and the culture was continued overnight. On the second day, RF-18 samples with different concentration gradients (0-1600 μg / mL) were added, and 3 replicates were set for each concentration, and the culture was continued for 24 h. After the culture was completed, 15 μL of MTT solution with a concentration of 5 mg / mL was added to each well, and the culture was continued for 4 h in the dark. Then, the culture medium in the plate wells was aspirated and 200 μL of dimethyl sulfoxide (DMSO) was added to each well. The culture plate was placed on a shaker and gently shaken for 10 min to dissolve the crystals, and then the absorbance value of each well was measured at 450 nm using an enzyme reader. According to the absorption value detected at 450nm, the cell viability in the absence of the sample to be tested was defined as 100%, and the relative cell viability in the presence of the sample to be tested was calculated, and the bar graph of the relative cell viability of the RF-18 sample solution was drawn using GraphPad prism software. The results are shown in Figure 2 .

[0035] Depend on Figure 2 It can be seen that RF-18 has no toxicity to human keratinocytes HaCaT within the concentration range of 800 μg / mL, and only exhibits weak cytotoxicity at a concentration of 1600 μg / mL.

[0036] Example 4 The hemolytic effect test of antimicrobial peptide RF-18 is as follows: Fresh human whole blood was mixed with Aldrich solution at a volume ratio of 1:1, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the red blood cells were washed with physiological saline. This was repeated 3 times until the supernatant no longer appeared red, and the washed red blood cells were obtained. The antimicrobial peptide RF-18 prepared in Example 1 was dissolved in physiological saline, and RF-18 samples to be tested with different concentrations (3.125-1600 μg / mL) were prepared; The washed red blood cells were diluted with saline to 1×10 7 The density of each sample was 100 / mL. The diluted red blood cell suspension was incubated with different concentrations of the RF-18 sample at 37°C for 30 min, and then centrifuged at 1000 rpm for 5 min. The absorbance of the supernatant was detected at 540 nm. The positive control used the same volume of Triton X-100 solution (denoted as PC), in which the volume concentration of Triton X-100 in the Triton X-100 solution was 10%; the negative control used the same volume of physiological saline. The hemolysis rate of the RF-18 sample with different concentrations was calculated. The hemolysis rate calculation method is shown in Formula I: Formula I; Among them, A is the positive control OD 540 value, B is the negative control OD 540 Value, C is the OD of the sample group to be tested 540 value.

[0037] The hemolysis rate results are shown in Figure 3 .Depend on Figure 3 It can be seen that the antimicrobial peptide RF-18 showed extremely high safety to human red blood cells, and only showed a weak hemolytic effect (hemolytic rate was 9%) at a concentration of 1600 μg / mL. There was no hemolytic effect within the concentration range of 800 μg / mL.

[0038] In summary, the antimicrobial peptide RF-18 provided by the present invention is a new antimicrobial peptide with an imperfect amphiphilic structure. The antimicrobial peptide not only can exert efficient and broad-spectrum antimicrobial activity, but also has extremely high safety. It has neither cytotoxicity nor hemolytic effect within the concentration range of 800 μg / mL, and can be used to prepare antibacterial infection drugs.

[0039] 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 RF-18, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The antimicrobial peptide RF-18 according to claim 1, characterized in that: The C-terminus of the antimicrobial peptide RF-18 is amidated.

3. Use of the antimicrobial peptide RF-18 according to claim 1 or 2 in the preparation of antibacterial products.

4. The use according to claim 3, characterized in that: The bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

5. The use according to claim 4, 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 .

6. The use according to claim 3, characterized in that: The antibacterial products include antibacterial preparations or antibacterial drugs.

7. An antibacterial drug, characterized in that: The active ingredient comprises the antimicrobial peptide RF-18 according to claim 1 or 2.

8. The antibacterial drug according to claim 7, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide RF-18 in the antibacterial drug is 1.03-3.81 μg / mL.

9. The antibacterial drug according to claim 7 or 8, characterized in that: The bacteria are Gram-negative bacteria and / or Gram-positive bacteria.

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

Images