An antibacterial peptide and its application

Abstract

The present invention belongs to the technical field of antimicrobial peptides, and specifically relates to an antimicrobial peptide and its application. The amino acid sequence of the antimicrobial peptide provided by the present invention is as shown in SEQ ID NO: 1. It has a small number of amino acids, a low synthesis cost, and strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria. The minimum inhibitory concentration against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus is 4.68 - 37.5 μg / mL, showing broad-spectrum antibacterial properties, good stability, no obvious cytotoxicity and hemolytic effect. Moreover, by forming a cyclic amide bond at the head and tail in the present invention, the antibacterial activity is further enhanced, and the antibacterial effect against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus is enhanced by 2 - 4 times, and the minimum inhibitory concentration is reduced to 2.34 - 9.38 μg / mL. After forming the cyclic amide bond at the head and tail, the plasma stability and safety of the antimicrobial peptide are further improved, and the cytotoxicity and hemolytic effect are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and particularly relates to an antimicrobial peptide and its application. Background Art

[0002] Microbial infections, including bacteria, fungi, viruses, etc., can cause many very serious human diseases including hepatitis, pneumonia, AIDS, tuberculosis, etc. Infectious epidemics caused by pathogenic microorganisms have always been one of the important causes of human death. Since the invention of antibiotics, countless lives of patients with bacterial infections have been saved. However, the extensive use of antibiotics has given rise to a large number of strains resistant to antibiotics.

[0003] Antimicrobial peptides (AMPs) are a general term for short peptides with antibacterial activity. In 1975, Swedish scientists G. Boman et al. induced and isolated a bactericidal peptide from the pupae of Hyalophora cecropia and named it cecropin. Since then, people have successively discovered and isolated a variety of polypeptides with antibacterial activity from bacteria, fungi, amphibians, insects, higher plants, mammals and even humans.

[0004] However, the existing natural antimicrobial peptides generally have the following problems: (1) They have a long sequence and a high synthesis cost. For example, in the prior art (Hu Lili. Research on the mechanism of neutralizing LPS by polypeptides derived from fish vitelline phosphoprotein and the anti-inflammatory mechanism of salidroside [D]. Ocean University of China, 2015.), site-directed point mutations were carried out on the amino acid sequence of the Pt5 polypeptide of zebrafish Pv to obtain Pt5e, which showed stronger antibacterial performance than Pt5 and was an antimicrobial peptide with good antibacterial effect. However, Pt5e has a sequence length of 55 amino acids, and the increase in its sequence length has led to an increase in the production cost of the antimicrobial peptide. (2) The antibacterial activity of truncated antimicrobial peptides needs to be improved. For example, Chinese Patent CN108570102A discloses a polypeptide Pv26-3 with antibacterial activity. The polypeptide Pv26-3 is a mutant of the parent molecule Pv26 truncated based on the vitelline phosphoprotein sequence. Compared with the above antimicrobial peptide Pt5e, the amino acid sequence length of Pv26-3 is significantly shortened. However, the minimum inhibitory concentration of truncated Pv26-3 against Gram-positive bacteria and Gram-negative bacteria reaches more than 1.2 mM, and there are deficiencies in antibacterial activity. Summary of the Invention

[0005] The purpose of the present invention is to provide a short-sequence antimicrobial peptide with strong and broad-spectrum antibacterial activity, low synthesis cost, low minimum inhibitory concentration, good stability, no obvious cytotoxicity and hemolytic effect.

[0006] The present invention provides an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO:1.

[0007] Preferably, the amino acid sequence of the antibacterial peptide forms a ring with the head and tail amide bonds.

[0008] The present invention also provides the application of the antibacterial peptide described in the above technical solution in the preparation of antibacterial products.

[0009] Preferably, the antibacterial product includes antibacterial infection products.

[0010] Preferably, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria.

[0011] Preferably, the Gram-negative bacteria include Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), and Escherichia coli ( Escherichia coli ) or more of them.

[0012] Preferably, the Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

[0013] Preferably, the product includes a reagent or a drug

[0014] The present invention provides an antibacterial product, and the antibacterial product includes the antibacterial peptide described in the above technical solution.

[0015] Preferably, the antibacterial product includes an antibacterial reagent or an antibacterial drug.

[0016] Beneficial effects:

[0017] The present invention provides an antibacterial peptide, and the amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO:1. The antibacterial peptide provided by the present invention has a small number of amino acids, low synthesis cost, and strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria. The results of the examples show that the minimum inhibitory concentration (MIC) of the antibacterial peptide provided by the present invention against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus is 4.68 - 37.5 μg / mL, has broad-spectrum antibacterial activity, good stability, and no obvious cytotoxicity and hemolysis effect.

[0018] Based on the linear amino acid sequence shown in SEQ ID NO:1, the present invention forms a cyclic amide bond at the head and tail to obtain a cyclic antibacterial peptide, further enhancing the antibacterial activity. The results of the examples show that the antibacterial activity of the antibacterial peptide with a cyclic amide bond at the head and tail against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus is enhanced by 2 to 4 times, and the minimum inhibitory concentration (MIC) is reduced to 2.34 to 9.38 μg / mL. Moreover, after forming the cyclic amide bond at the head and tail, the plasma stability of the antibacterial peptide is further improved, and the cytotoxicity and hemolysis are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0020] Figure 1 Schematic diagram of cyclization of RK-15 to obtain RK-15-C;

[0021] Figure 2 Results graph of plasma stability analysis of RK-15 and RK-15-C based on Staphylococcus aureus;

[0022] Figure 3 Results graph of plasma stability analysis of RK-15 and RK-15-C based on Pseudomonas aeruginosa;

[0023] Figure 4 Results graph of plasma stability analysis of RK-15 and RK-15-C based on Escherichia coli;

[0024] Figure 5 Results graph of plasma stability analysis of RK-15 and RK-15-C based on Acinetobacter baumannii;

[0025] Figure 6 Results graph of hemolysis evaluation of different concentrations of RK-15;

[0026] Figure 7 Results graph of hemolysis evaluation of different concentrations of RK-15-C;

[0027] Figure 8 Results graph of the effect of different concentrations of RK-15 on the viability of human embryonic kidney cells HEK293T;

[0028] Figure 9 Results graph of the effect of different concentrations of RK-15 on the viability of human keratinocytes HaCaT

[0029] Figure 10 Results graph of the effect of different concentrations of RK-15-C on the viability of human embryonic kidney cells HEK293T;

[0030] Figure 11 Results of the effects of different concentrations of RK-15-C on the viability of human keratinocyte HaCaT cells Detailed implementation mode

[0031] The present invention provides an antimicrobial peptide, and the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1. As an implementation mode, the amide bonds at the head and tail of the amino acid sequence of the antimicrobial peptide form a ring.

[0032] The antimicrobial peptide of the present invention contains 15 amino acids, with a molecular weight of 1955.41 daltons, and all amino acids are of the L type. The present invention has no strict requirements for the preparation method of the antimicrobial peptide, and conventional methods in the art can be used, such as the solid-phase synthesis method of polypeptides. The present invention has no special requirements for the solid-phase synthesis method of polypeptides, and methods well-known to those skilled in the art can be used.

[0033] The present invention also provides the application of the antimicrobial peptide described in the above technical solution in the preparation of antibacterial products.

[0034] As an implementation mode, the antibacterial product is an antibacterial infection product. As an implementation mode, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria. As an implementation mode, the Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; as another implementation mode, the Gram-positive bacteria include Staphylococcus aureus.

[0035] As an implementation mode, the Acinetobacter baumannii of the present invention includes one or more of Acinetobacter baumannii ATCC 19606, 10769, and 0357; as another implementation mode, the Acinetobacter baumannii of the present invention is Acinetobacter baumannii ATCC 19606. The present invention takes Acinetobacter baumannii ATCC 19606, 10769, and 0357 as examples in the embodiments, but it should not be understood as the entire protection scope of the present invention only.

[0036] As an implementation mode, the Escherichia coli of the present invention includes one or more of Escherichia coli ATCC 8739, 0894, and 5017; as another implementation mode, the Escherichia coli of the present invention is Escherichia coli ATCC 8739. The present invention takes Escherichia coli ATCC 8739, 0894, and 5017 as examples in the embodiments, but it should not be understood as the entire protection scope of the present invention only.

[0037] As an implementation manner, the Pseudomonas aeruginosa described in the present invention includes one or more of Pseudomonas aeruginosa ATCC 27853, 90068, and 17068; as another implementation manner, the Pseudomonas aeruginosa described in the present invention is Pseudomonas aeruginosa ATCC 27853. In the examples of the present invention, Pseudomonas aeruginosa ATCC 27853, 90068, and 17068 are taken as examples for illustration, but it should not be understood that this is the entire protection scope of the present invention.

[0038] As an implementation manner, the Staphylococcus aureus described in the present invention includes one or more of Staphylococcus aureus ATCC 6538, 220823, and 15775; as another implementation manner, the Staphylococcus aureus described in the present invention is Staphylococcus aureus ATCC 6538. In the examples of the present invention, Staphylococcus aureus ATCC 6538, 220823, and 15775 are taken as examples for illustration, but it should not be understood that this is the entire protection scope of the present invention.

[0039] The strains corresponding to the numbers 10769, 0357, 0894, 5017, 90068, 170682, 20823, and 15775 described in the present invention are known strains in the art, which are preserved in the Institute of Medical Biology, Chinese Academy of Medical Sciences and are disclosed in the prior art Development of α-Helical Antimicrobial Peptides with Imperfect Amphipathicity for Superior Activity and Selectivity. J Med Chem.. 2024 Nov 14;67(21):19561-19572.

[0040] As an implementation manner, the product includes a reagent or a drug.

[0041] The above-mentioned antibacterial peptide sequence is obtained by truncating the cathelicidin-related polypeptide predicted from the gecko genome, and has antibacterial activity against both Gram-negative bacteria and / or Gram-positive bacteria, with strong broad-spectrum property. The results of the examples show that the minimum inhibitory concentration (MIC) of the linear polypeptide sequence shown in SEQ ID NO:1 against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus is 4.68 - 37.5 μg / mL; based on the linear polypeptide sequence shown in SEQ ID NO:1, the head and tail amide bonds are cyclized to obtain a cyclic antibacterial peptide, which can further enhance the antibacterial activity. The antibacterial effect of the antibacterial peptide with cyclized head and tail amide bonds against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus is enhanced by 2 - 4 times, and the minimum inhibitory concentration (MIC) is reduced to 2.34 - 9.38 μg / mL. Moreover, after cyclizing the head and tail amide bonds, the plasma stability of the antibacterial peptide is further improved, and the cytotoxicity and hemolysis are reduced. The results of the examples show that after incubating the cyclized antibacterial peptide with plasma for 8 h, its activity remains basically unchanged, showing extremely high safety, neither cytotoxic nor hemolytic.

[0042] The present invention provides an antibacterial product, which includes the antibacterial peptide described in the above technical solution; the antibacterial product includes an antibacterial reagent or an antibacterial drug. As an implementation manner, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is ≥2.34 μg / mL; as another implementation manner, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34 - 37.5 μg / mL; as another implementation manner, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34 - 18.75 μg / mL; as another implementation manner, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34 - 9.38 μg / mL; as another implementation manner, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34 - 4.68 μg / mL.

[0043] To further illustrate the present invention, an antibacterial peptide and its application provided by the present invention will be described in detail below with reference to the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] 1. Using the cathelicidin-related polypeptide (NCBI Reference Sequence: XP_015277841.1) predicted from the gecko (Gekko japonicus) genome as a template, a short sequence containing 15 amino acids (RWRRFWGKAKRGIKK, SEQ ID NO:1) is truncated.

[0046] 2. Entrust Gil Biochemical (Shanghai) Co., Ltd. to synthesize the amino acid sequence described in SEQ ID No:1 by solid-phase peptide synthesis method, and desalt and purify it by HPLC reverse-phase column chromatography to obtain a linear polypeptide, named RK-15. RK-15 consists of 15 amino acids, with a molecular weight of 1973.41 Daltons, and all amino acids are of L-type.

[0047] 3. Also entrust Gil Biochemical (Shanghai) Co., Ltd. to carry out cyclization modification on RK-15 obtained in step (2) by forming another amide bond between the head and tail amino acids, and desalt and purify it by HPLC reverse-phase column chromatography to obtain a cyclic polypeptide, denoted as RK-15-C. RK-15-C still consists of 15 amino acids, with a molecular weight of 1955.41 Daltons, and all amino acids are of L-type. The schematic diagram of cyclizing RK-15 to obtain RK-15-C is as Figure 1 shown.

[0048] Example 2

[0049] Antibacterial activity analysis

[0050] 1. Test strains

[0051] (1) Acinetobacter baumannii: ATCC 19606, 10769 and 0357;

[0052] (2) Escherichia coli: ATCC 8739, 0894 and 5017;

[0053] (3) Pseudomonas aeruginosa: ATCC 27853, 90068 and 17068;

[0054] (4) Staphylococcus aureus: ATCC 6538, 220823 and 15775.

[0055] 2. Minimum inhibitory concentration (MIC) detection

[0056] First, inoculate the test strains onto LB solid plates. After the colonies grow, pick single colonies and transfer them to LB liquid medium, and shake culture at 37 °C and 180 rpm for 5 h. Measure the OD of the bacterial solution under an ultraviolet spectrophotometer 600 , according to the ratio of 1 OD 600 = 1×10 9 CFU / mL, dilute the bacterial solution to 2×10 5 with LB liquid mediumConcentration of CFU / mL; Add 100 μL of the diluted bacterial solution to each well of a sterile 96-well plate, and then add 100 μL of the test sample diluted in physiological saline at different gradients to each well. Use a pipette to blow and mix well, and then place it in a constant temperature incubator at 37 °C for slow shaking culture overnight; the test sample is RK-15 and RK-15-C obtained in Example 1; the concentration of the diluted test sample is 0-200 μg / mL; after incubation, use a microplate reader to measure the absorbance of the bacterial solution at OD 600 at the absorption value at nm, and use the average value of the sample concentrations of the wells where no bacterial growth is detected and the adjacent wells as the MIC value. The results are shown in Table 1.

[0057] Table 1 Minimum inhibitory concentration values of different test samples against the test strains

[0058]

[0059] It can be seen from Table 1 that both RK-15-C and RK-15 showed significant antibacterial effects against the tested strains. The MIC value of RK-15-C was 2.34-9.38 μg / mL (i.e., 1.20-4.80 μM); while the MIC value of RK-15 was 4.68-37.5 μg / mL (i.e., 2.37-19.01 μM). It can be seen that the antibacterial activity of RK-15-C was stronger, increasing by 2-4 times. The cyclization modification of RK-15 can significantly increase its antibacterial activity.

[0060] Example 3

[0061] Plasma stability analysis

[0062] 1. Test strains

[0063] Acinetobacter baumannii: ATCC 19606; Escherichia coli: ATCC 8739; Pseudomonas aeruginosa: ATCC 27853; Staphylococcus aureus: ATCC 6538.

[0064] 2. Take a 1.5 mL EP tube and add 100 μL of 15 mg / mL EDTA-K2 anticoagulant. Collect blood from the mouse's eye socket into the above tube, immediately invert and mix well, centrifuge at 4 °C and 3500 rpm for 10 min, and carefully aspirate the supernatant to obtain mouse plasma.

[0065] Using the RK-15 and RK-15-C obtained in Example 1 as the test samples, they were dissolved in sterile physiological saline to a final concentration of 400 μg / mL. The test sample solution was mixed with mouse plasma at a volume ratio of 1:1 and incubated in an incubator at 37°C. At incubation times of 0, 0.5, 1, 2, 4, 6, and 8 h, 200 μL of the sample was taken out for MIC experiment detection to observe the change in antibacterial activity after incubation with plasma. The MIC inspection method is as described in Example 2, and the specific detection results are as Figures 2 - 5 shown.

[0066] According to Figures 2 - 5 it can be seen that compared with RK-15, RK-15-C has better plasma stability. After incubation with plasma for 8 h, the activity of RK-15-C remains basically unchanged, while the activity of its uncyclized linear polypeptide RK-15 decreases significantly after incubation with plasma, and the MIC increases by 8 - 30 times.

[0067] Example 4

[0068] Analysis of hemolytic effect

[0069] 1. After anesthetizing the rats intraperitoneally, whole blood was collected from the abdominal aorta. The blood was mixed with Alsever's solution at a volume ratio of 1:1 and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the obtained red blood cells were washed with physiological saline multiple times until the supernatant no longer showed red.

[0070] 2. The washed red blood cells were diluted with physiological saline to a density of 1×10 7 cells / mL. The red blood cell suspension was incubated with different concentrations of the test samples (RK-15 and RK-15-C obtained in Example 1) dissolved in physiological saline at 37°C for 30 min, and then centrifuged at 1000 rpm for 5 min. The absorbance of the supernatant was measured at 540 nm. The positive control used the same volume of Triton X-100 solution (PC), and the volume concentration of the Triton X-100 solution was 10%; the negative control used physiological saline.

[0071] 3. Data processing: Based on the absorbance measured at 540 nm, the hemolysis rate (Hemolysis) of the positive control (PC group) was defined as 100%, and the hemolysis rate of the test sample relative to the PC group was calculated according to the following formula. The results are as Figure 6 and Figure 7 shown.

[0072] Hemolysis rate (%) = (OD value of positive control - OD value of negative control) / (OD value of test sample group - OD value of negative control) × 100%. 540 value - OD value of negative control 540 value) / (OD value of test sample group 540 value - OD value of negative control 540 value) × 100%.

[0073] According to Figure 6 and Figure 7 It can be seen that within the test concentration range (1.6 - 100 μg / mL), neither RK-15 nor RK-15-C has an obvious hemolytic effect on red blood cells, and the hemolytic effect is further reduced after RK-15 is cyclized to form RK-15-C.

[0074] Example 5

[0075] Cytotoxicity analysis

[0076] 1. Human embryonic kidney cells (HEK293T) and human keratinocytes (HaCaT) were routinely cultured with DMEM medium (containing 10% fetal bovine serum and 1% double antibiotics). When the cells covered about 80% of the bottom of the culture flask, they were digested with trypsin, dispersed and counted, and the cell density was adjusted to 5×10 4 cells / mL, and inoculated into a 96-well plate at a volume of 100 μL per well.

[0077] 2. After culturing the cells for 24 h, 10 μL of the test samples with different concentration gradients (RK-15 and RK-15-C obtained in Example 1) solutions (final concentration 0 - 200 μg / mL) or an equal volume of DMEM medium were added respectively. After continuing to culture for 24 h, 10 μL of CCK8 reagent was added. After 1.5 - 2 h, the absorbance value of each well solution at a wavelength of 450 nm was measured. Three replicates were set for each sample concentration. The background contained no cells and samples, and only an equal volume of medium and CCK-8 reagent was added.

[0078] 3. Data processing: According to the absorption value detected at 450 nm, the cell viability in the absence of the test sample was defined as 100%, and the relative cell viability in the presence of the test sample was calculated. A bar chart of the relative cell viability corresponding to the RK-15-C sample solution was drawn using GraphPad prism software, and the results are as Figures 8 - 11 shown. According to Figures 8 - 11 It can be seen that within the test concentration range, neither RK-15 nor RK-15-C has an obvious toxic effect on human embryonic kidney cells and human keratinocytes, laying a foundation for the further development of the antimicrobial peptide RK-15-C.

[0079] It can be seen from the above content that the antimicrobial peptide provided by the present invention has a small number of amino acids, low synthesis cost, strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria, good stability, strong broad-spectrum property, extremely high safety, and neither cytotoxicity nor hemolytic effect.

[0080] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1; the amino acid sequence of the antimicrobial peptide forms a ring through amide bonds at the head and tail.

2. Use of the antimicrobial peptide according to claim 1 in the preparation of antibacterial products; The bacteria include Gram-negative bacteria and / or Gram-positive bacteria; The Gram-negative bacteria are Acinetobacter baumannii ( Acinetobacter baumannii )、Pseudomonas aeruginosa( Pseudomonas aeruginosa ) and Escherichia coli ( Escherichia coli ) one or more; The Gram-positive bacteria is Staphylococcus aureus ( Staphylococcus aureus ).

3. The use according to claim 2, characterized in that: The anti-bacterial products include anti-bacterial infection products.

4. The use according to claim 2 or 3, characterized in that: The product includes a reagent or a drug.

5. An antibacterial product, characterized in that: The antibacterial product comprises the antimicrobial peptide according to claim 1.

6. The antibacterial product according to claim 5, characterized in that The antibacterial product includes an antibacterial agent or an antibacterial drug.

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