Antibacterial peptide and application thereof

Abstract

The invention belongs to the technical field of antibacterial peptides, and particularly relates to an antibacterial peptide and application thereof. The amino acid sequence of the antibacterial peptide is shown as SEQ ID NO: 1, the number of amino acids is small, the synthesis cost is low, the antibacterial peptide has high antibacterial activity on gram-negative bacteria and gram-positive bacteria, the minimum inhibitory concentration of the antibacterial peptide on acinetobacter baumannii, escherichia coli, pseudomonas aeruginosa and staphylococcus aureus is 4.68-37.5 mu g / mL, and the antibacterial peptide has the advantages of broad-spectrum antibacterial property, good stability and good application prospect. No obvious cytotoxicity and hemolysis effect exist; moreover, end-to-end amido bond cyclization is carried out, so that the antibacterial activity is further enhanced, the antibacterial effect on 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 mu g / mL; after the end-to-end amido bonds are cyclized, the plasma stability and safety of the antibacterial peptide are further improved, and the cytotoxicity and hemolysis are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and in particular relates to an antimicrobial peptide and an application thereof. 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 death. Since the invention of antibiotics, countless lives of patients with bacterial infections have been saved. However, with the extensive use of antibiotics, a large number of antibiotic-resistant strains have emerged.

[0003] Antimicrobial peptides (AMPs) are a general term for short peptides with antimicrobial activity. In 1975, Swedish scientist G. Boman and others induced and isolated a bactericidal peptide from the pupa of Cecropinus cecropinus and named it cecropin. Since then, people have discovered and isolated a variety of peptides with antimicrobial activity from bacteria, fungi, amphibians, insects, higher plants, mammals and even humans.

[0004] However, existing natural antimicrobial peptides generally have the following problems: (1) The sequence is long and the synthesis cost is high. For example, the existing technology (Hu Lili. Study on the neutralization of LPS by fish yolk phosphoprotein-derived peptides and the anti-inflammatory mechanism of salidroside [D]. Ocean University of China, 2015.) conducted a targeted point mutation on the amino acid sequence of the zebrafish Pv Pt5 peptide to obtain Pt5e, which showed stronger antibacterial properties than Pt5 and is an antimicrobial peptide with good antibacterial effect. However, Pt5e has a sequence length of 55 amino acids, and the increase in its sequence length leads to an increase in the production cost of antimicrobial peptides. (2) The antimicrobial activity of truncated antimicrobial peptides needs to be improved. For example, Chinese patent CN108570102A discloses a polypeptide Pv26-3 with antimicrobial activity. The polypeptide Pv26-3 is a mutant of the parent molecule Pv26 obtained by truncating the phosphoprotein sequence. Compared with the above-mentioned antimicrobial peptide Pt5e, the amino acid sequence length of Pv26-3 is significantly shortened. However, the minimum inhibitory concentration of the truncated Pv26-3 against Gram-positive bacteria and Gram-negative bacteria is above 1.2 mM, which is insufficient in terms of antibacterial activity. Summary of the invention

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

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

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

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

[0009] Preferably, the anti-bacterial product comprises an anti-bacterial infection product.

[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 )

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

[0013] Preferably, the product comprises a reagent or a drug The present invention provides an antibacterial product, which comprises the antibacterial peptide described in the above technical solution.

[0014] Preferably, the antibacterial product comprises an antibacterial agent or an antibacterial drug.

[0015] Beneficial effects: The present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID NO: 1. The antimicrobial peptide provided by the present invention has a small number of amino acids, a low synthesis cost, and has strong antimicrobial activity against both Gram-negative and Gram-positive bacteria. The results of the examples show that the minimum inhibitory concentration (MIC) of the antimicrobial peptide provided by the present invention against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus is 4.68-37.5 μg / mL, has a broad-spectrum antimicrobial activity, good stability, and no obvious cytotoxicity and hemolytic effect.

[0016] The present invention forms a ring by forming a head-to-tail amide bond on the basis of the linear amino acid sequence shown in SEQ ID NO: 1 to obtain a cyclic antimicrobial peptide, which further enhances the antibacterial activity. The results of the embodiment show that the antimicrobial peptide formed by forming a ring by forming a head-to-tail amide bond has a 2-4-fold enhancement in antibacterial effect on Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, and the minimum inhibitory concentration (MIC) is reduced to 2.34-9.38 μg / mL. And after the ring by forming a head-to-tail amide bond, the plasma stability of the antimicrobial peptide is further improved, and the cytotoxicity and hemolysis are reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 Schematic diagram of the cyclization of RK-15 to obtain RK-15-C; Figure 2 The graph is a plasma stability analysis result of RK-15 and RK-15-C based on Staphylococcus aureus; Figure 3 The figure is the result of the plasma stability analysis of RK-15 and RK-15-C based on Pseudomonas aeruginosa; Figure 4 The graph is a plasma stability analysis result of RK-15 and RK-15-C based on E. coli; Figure 5 The figure is the result of plasma stability analysis of RK-15 and RK-15-C based on Acinetobacter baumannii; Figure 6 This is the result diagram of the hemolytic effect evaluation of different concentrations of RK-15; Figure 7 This is the result diagram of the hemolytic effect evaluation of different concentrations of RK-15-C; Figure 8 This is the result diagram of the effect of different concentrations of RK-15 on the viability of human embryonic kidney cells HEK293T; Fig. 9 The results of the effects of different concentrations of RK-15 on the viability of human keratinocytes HaCaT cells Fig.10 This is the result diagram of the effect of different concentrations of RK-15-C on the viability of human embryonic kidney HEK293T cells; Fig.11 This is a graph showing the effects of different concentrations of RK-15-C on the viability of human keratinocytes HaCaT cells. DETAILED DESCRIPTION

[0019] The present invention provides an antimicrobial peptide, the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1. As an embodiment, the amino acid sequence of the antimicrobial peptide forms a ring with amide bonds at the head and tail.

[0020] The antimicrobial peptide of the present invention comprises 15 amino acids, has a molecular weight of 1955.41 Daltons, and all amino acids are L-type. The present invention has no strict requirements on the preparation method of the antimicrobial peptide, and conventional methods in the art can be used, such as solid phase synthesis of polypeptides. The present invention has no special requirements on the solid phase synthesis of polypeptides, and methods well known to those skilled in the art can be used.

[0021] The present invention also provides the use of the antimicrobial peptide described in the above technical solution in the preparation of antibacterial products.

[0022] As an embodiment, the antibacterial product is an antibacterial infection product. As an embodiment, the bacteria include 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; as another embodiment, the Gram-positive bacteria include Staphylococcus aureus.

[0023] As an embodiment, the Acinetobacter baumannii of the present invention includes one or more of Acinetobacter baumannii ATCC 19606, 10769 and 0357; as another embodiment, the Acinetobacter baumannii of the present invention is Acinetobacter baumannii ATCC 19606. The present invention is described in the embodiments using Acinetobacter baumannii ATCC 19606, 10769 and 0357 as examples, but they should not be understood as the entire protection scope of the present invention.

[0024] As an embodiment, the Escherichia coli of the present invention includes one or more of Escherichia coli ATCC 8739, 0894 and 5017; as another embodiment, the Escherichia coli of the present invention includes Escherichia coli ATCC 8739. The present invention is described by taking Escherichia coli ATCC 8739, 0894 and 5017 as examples in the embodiments, but they cannot be understood as the entire protection scope of the present invention.

[0025] As an embodiment, the Pseudomonas aeruginosa described in the present invention includes one or more of Pseudomonas aeruginosa ATCC 27853, 90068 and 17068; as another embodiment, the Pseudomonas aeruginosa described in the present invention is Pseudomonas aeruginosa ATCC 27853. The present invention is described in the embodiments using Pseudomonas aeruginosa ATCC 27853, 90068 and 17068 as examples, but they should not be construed as the entire protection scope of the present invention.

[0026] As an embodiment, the Staphylococcus aureus of the present invention includes one or more of Staphylococcus aureus ATCC 6538, 220823 and 15775; as another embodiment, the Staphylococcus aureus of the present invention is Staphylococcus aureus ATCC 6538. The present invention is described in the embodiments using Staphylococcus aureus ATCC 6538, 220823 and 15775 as examples, but they cannot be understood as the entire protection scope of the present invention.

[0027] The strains corresponding to the numbers 10769, 0357, 0894, 5017, 90068, 170682, 20823 and 15775 of the present invention are respectively known strains in the art, deposited in the Institute of Medical Biology, Chinese Academy of Medical Sciences, and disclosed in the prior art Development of α-Helical Antimicrobial Peptides with Imperfect Amphipathicityfor Superior Activity and Selectivity. J Med Chem.. 2024 Nov 14;67(21):19561-19572.

[0028] As an embodiment, the product includes a reagent or a drug.

[0029] The present invention obtains the above antimicrobial peptide sequence by intercepting the cathelicidin-related polypeptide predicted from the gecko genome, and has antimicrobial activity against Gram-negative bacteria and / or Gram-positive bacteria, and has a strong broad spectrum. The results of the embodiment show that the minimum inhibitory concentration (MIC) of the linear polypeptide sequence shown in SEQ ID NO:1 for Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus is 4.68~37.5μg / mL; the first and last amide bonds are cyclized on the basis of the linear polypeptide sequence shown in SEQ ID NO:1 to obtain a cyclic antimicrobial peptide, which can further enhance the antimicrobial activity. The antimicrobial peptide with the first and last amide bonds cyclized has a 2~4-fold enhancement in antimicrobial effect on Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, and the minimum inhibitory concentration (MIC) is reduced to 2.34~9.38μg / mL. And after the first and last amide bonds are cyclized, the plasma stability of the antimicrobial peptide is further improved, and the cytotoxicity and hemolysis are reduced. The results of the embodiment show that the activity of the cyclized antimicrobial peptide remains basically unchanged after incubation with plasma for 8 hours, showing extremely high safety and having neither cytotoxicity nor hemolytic effect.

[0030] The present invention provides an antibacterial product, which includes the antibacterial peptide described in the above technical solution; the antibacterial product includes an antibacterial agent or an antibacterial drug. As one embodiment, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is ≥2.34μg / mL; as another embodiment, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34~37.5μg / mL; as another embodiment, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34~18.75μg / mL; as another embodiment, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34~9.38μg / mL; as another embodiment, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 2.34~4.68μg / mL.

[0031] In order to further illustrate the present invention, an antimicrobial peptide and its application provided by the present invention 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.

[0032] Example 1 1. Using the cathelicidin-related polypeptide predicted in the gecko (Gekko japonicus) genome (NCBI Reference Sequence: XP_015277841.1) as a template, a short sequence containing 15 amino acids (RWRRFWGKAKRGIKK, SEQ ID NO: 1) was extracted.

[0033] 2. Entrust Jier Biochemical (Shanghai) Co., Ltd. to synthesize the amino acid sequence described in SEQ ID No: 1 by peptide solid phase synthesis method, and desalt and purify 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 L-type.

[0034] 3. Similarly, Jier Biochemical (Shanghai) Co., Ltd. was commissioned to perform cyclization modification on RK-15 obtained in step (2) by forming an amide bond between the head and tail amino acids, and then desalting and purifying by HPLC reverse phase column chromatography to obtain a cyclic polypeptide, which was recorded 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 L-type. The schematic diagram of RK-15 cyclization to obtain RK-15-C is shown in the following figure Figure 1 shown.

[0035] Example 2 Antimicrobial activity analysis 1. Test strains (1) Acinetobacter baumannii: ATCC 19606, 10769 and 0357; (2) Escherichia coli: ATCC 8739, 0894 and 5017; (3) Pseudomonas aeruginosa: ATCC 27853, 90068 and 17068; (4) Staphylococcus aureus: ATCC 6538, 220823 and 15775.

[0036] 2. Minimum inhibitory concentration (MIC) test First, the test strain 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 hours. The OD of the bacterial solution was measured under 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 mixed by pipetting, and then placed in a 37°C constant temperature incubator for slow shaking and culture overnight; the sample to be tested was RK-15 and RK-15-C obtained in Example 1; 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 MIC value according to the average of the sample concentrations of the wells where no bacterial growth was detected and the adjacent wells. The results are shown in Table 1.

[0037] Table 1 Minimum inhibitory concentration values ​​of different samples for the tested strains

[0038] According to Table 1, both RK-15-C and RK-15 showed significant antibacterial effects on the tested strains. The MIC value of RK-15-C was 2.34~9.38μg / mL (i.e. 1.20~4.80μM), while the MIC value of RK-15 was 4.68~37.5μg / mL (i.e. 2.37~19.01μM). 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.

[0039] Example 3 Plasma stability analysis 1. Test strains Acinetobacter baumannii: ATCC 19606; Escherichia coli: ATCC 8739; Pseudomonas aeruginosa: ATCC 27853; Staphylococcus aureus: ATCC 6538.

[0040] 2. Take a 1.5mL EP tube and add 100μL 15mg / mL EDTA-K2 anticoagulant. Remove the eyeball of the mouse and collect blood into the above tube. Immediately invert to mix. Centrifuge at 4℃ and 3500rpm for 10min. Carefully aspirate the supernatant to obtain mouse plasma.

[0041] RK-15 and RK-15-C obtained in Example 1 were used as test samples, respectively, and dissolved in sterile physiological saline to a final concentration of 400 μg / mL; the test sample solution was mixed with mouse plasma in a volume ratio of 1:1, and incubated in a 37°C incubator. 200 μL of the sample was taken out at 0, 0.5, 1, 2, 4, 6, and 8 hours of incubation to perform MIC experimental detection to see the changes in its antibacterial activity after incubation with plasma. The MIC test method is as described in Example 2, and the specific test results are as follows: Figure 2~Figure 5 shown.

[0042] according to Figure 2~Figure 5 It can be seen that compared with RK-15, RK-15-C has better plasma stability. After incubation with plasma for 8 hours, the activity of RK-15-C remains basically unchanged, while the activity of its uncyclized linear peptide RK-15 is significantly reduced after incubation with plasma, and the MIC increases by 8 to 30 times.

[0043] Example 4 Hemolysis assay 1. After intraperitoneal anesthesia, whole blood was collected from rats through the abdominal aorta. The blood was mixed with Aldrich's solution in a volume ratio of 1:1 and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the red blood cells were washed several times with saline until the supernatant was no longer red.

[0044] 2. Dilute the washed red blood cells to 1×10 7 The red blood cell suspension was incubated with different concentrations of the test sample (RK-15 and RK-15-C obtained in Example 1) dissolved in physiological saline at 37°C for 30 minutes, and then centrifuged at 1000 rpm for 5 minutes. The absorbance of the supernatant was detected 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.

[0045] 3. Data processing: Based on the absorption value detected at 540nm, the hemolysis rate of the positive control (PC group) is defined as 100%, and the hemolysis rate of the test sample relative to the PC group is calculated according to the following formula. The results are as follows: Figure 6 and Figure 7 shown.

[0046] Hemolysis rate (%) = (positive control OD 540 Value - negative control OD 540 value) / (OD of the sample group to be tested 540 Value - negative control OD 540 value) × 100%.

[0047] according to Figure 6 and Figure 7 It can be seen that within the test concentration range (1.6~100μg / mL), RK-15 and RK-15-C had no obvious hemolytic effect on red blood cells, and the hemolytic effect was further reduced after RK-15 was cyclized to generate RK-15-C.

[0048] Example 5 Cytotoxicity assay 1. Human embryonic kidney cells (HEK293T) and human 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 4 100 μL per well was inoculated into a 96-well plate.

[0049] 2. After 24 hours of cell culture, 10 μL of the sample to be tested (RK-15 and RK-15-C obtained in Example 1) solution with different concentration gradients (final concentration of 0-200 μg / mL) or an equal volume of DMEM medium were added. After 24 hours of continuous culture, 10 μL of CCK8 reagent was added, and after 1.5-2 hours, the absorbance value of each well solution at a wavelength of 450 nm was measured. Three replicates were set for each sample concentration, and the background did not contain cells and samples, and only the same volume of culture medium and CCK-8 reagent were added.

[0050] 3. Data processing: Based on the absorption value detected at 450nm, 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. The GraphPad prism software was used to draw a bar graph of the relative cell viability of the RK-15-C sample solution. The results are shown in Figure 2. Figure 8~Figure 11 As shown. Figure 8~Figure 11 It can be seen that within the tested concentration range, RK-15 and RK-15-C had no obvious toxic effects on human embryonic kidney cells and human keratinocytes, laying the foundation for the further development of the antimicrobial peptide RK-15-C.

[0051] From the above content, it can be seen that the antimicrobial peptide provided by the present invention has a small number of amino acids, a low synthesis cost, strong antibacterial activity against both Gram-negative and Gram-positive bacteria, good stability, strong broad spectrum, extremely high safety, and no cytotoxicity or hemolytic effect.

[0052] 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, 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.

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, characterized in that: The bacteria include 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 Escherichia coli ( Escherichia coli ) 6. The use according to claim 4, characterized in that: The Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

7. The use according to any one of claims 2 to 6, characterized in that: The product includes a reagent or a drug.

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

9. The antibacterial product according to claim 8, characterized in that The antibacterial product includes an antibacterial agent or an antibacterial drug.

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