A self-assembling antimicrobial peptide Lau-IL-9-C2 and its applications

Abstract

The present invention belongs to the technical field of antimicrobial peptides, and specifically relates to a self-assembling antimicrobial peptide Lau-IL-9-C2 and its application. The self-assembling antimicrobial peptide Lau-IL-9-C2 provided by the present invention is a dendritic polypeptide with one main chain and two side chains. By connecting lauric acid, it provides a self-assembling driving force relying on its strong hydrophobicity. Moreover, the two side chains can increase the surface polypeptide density, increase the activity, and enhance the stability. The results of the examples show that the antimicrobial peptide Lau-IL-9-C2 provided by the present invention can undergo self-assembly, presenting a nanoparticle state under the electron microscope, with a particle size of 10-16 nm, a critical micelle concentration of 229.1 μM, good stability, no obvious cytotoxicity and hemolytic effect, and strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria. The minimum inhibitory concentrations against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus are 1.17-4.69 μM.

Description

Technical Field

[0001] The present invention belongs to the technical field of antimicrobial peptides, and particularly relates to a self-assembled antimicrobial peptide Lau-IL-9-C2 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 class of small molecule polypeptides that can inhibit the growth of bacteria, fungi, viruses, etc. They are widely present in organisms and are an important part of the innate immune system. Antimicrobial peptides usually have a certain net positive charge and an amphiphilic α-helical structure. Such a structure enables antimicrobial peptides to target and bind to the negatively charged bacterial plasma membrane on the one hand, and on the other hand, the amphiphilic characteristics can promote the interaction between antimicrobial peptides and the bacterial plasma membrane, facilitating the penetration and destruction of the bacterial cell membrane by antimicrobial peptides, ultimately leading to the death of bacteria. Antimicrobial peptides are not only not easily induced to produce drug resistance in bacteria, but also exhibit advantages such as broad-spectrum antibacterial activity, high efficiency at low doses, and immunomodulation. However, not any small molecule polypeptide has antibacterial activity. Even if it has antibacterial activity, there are many limitations in actual production and application. The essence of antimicrobial peptides is a polypeptide, which results in the sensitivity of antimicrobial peptides to the environment. The ionic concentration and the presence of enzymes will affect the structure and bactericidal effect of the polypeptide, resulting in instability in actual applications, as well as unknown hemolytic activity and cytotoxicity, etc., which severely limit the development of antimicrobial peptides as antibacterial drugs, and there are very few antimicrobial peptide drugs that can enter phase III clinical research. The self-assembly of polypeptides can improve the structural stability, enhance biocompatibility, and increase the local action concentration. However, not all self-assembly behaviors can improve the performance of antimicrobial peptides. Summary of the Invention

[0004] The purpose of the present invention is to provide a self-assembled antimicrobial peptide Lau-IL-9-C2 with good stability, no obvious cytotoxicity and hemolytic effect, having potent and broad-spectrum antibacterial activity and a low minimum inhibitory concentration.

[0005] The present invention provides a self-assembled antimicrobial peptide Lau-IL-9-C2, and the self-assembled antimicrobial peptide Lau-IL-9-C2 includes lauric acid, a main chain, a first side chain, and a second side chain;

[0006] The main chain includes the amino acid sequence shown in SEQ ID NO:1;

[0007] The first side chain and the second side chain respectively comprise the amino acid sequence shown in SEQ ID NO:2;

[0008] The N-terminus of the main chain is connected to the lauric acid through an amide bond;

[0009] The C-terminus of the first side chain is connected to the side chain amino group of the third amino acid of the amino acid sequence shown in SEQ ID NO:1 through an amide bond; the C-terminus of the second side chain is connected to the side chain amino group of the fourth amino acid of the amino acid sequence shown in SEQ ID NO:1 through an amide bond.

[0010] The present invention also provides the application of the self-assembled antibacterial peptide Lau-IL-9-C2 described in the above technical solution in the preparation of antibacterial products.

[0011] Preferably, the antibacterial product includes antibacterial infection products.

[0012] Preferably, the bacteria include Gram-negative bacteria and / or Gram-positive bacteria.

[0013] Preferably, the Gram-negative bacteria include Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), and Escherichia coli ( Escherichia coli ), and one or more of them.

[0014] Preferably, the Gram-positive bacteria include Staphylococcus aureus ( Staphylococcus aureus ).

[0015] Preferably, the product includes a reagent or a drug

[0016] The present invention provides an antibacterial product, and the antibacterial product includes the self-assembled antibacterial peptide Lau-IL-9-C2 described in the above technical solution.

[0017] Preferably, the antibacterial product includes an antibacterial reagent or an antibacterial drug.

[0018] Beneficial effects:

[0019] The present invention provides a self-assembling antimicrobial peptide Lau-IL-9-C2, which comprises lauric acid, a main chain, a first side chain and a second side chain; the main chain comprises the amino acid sequence shown in SEQ ID NO:1; the first side chain and the second side chain respectively comprise the amino acid sequence shown in SEQ ID NO:2; the N-terminus of the main chain is connected to the lauric acid through an amide bond; the C-terminus of the first side chain is connected to the side-chain amino group of the third amino acid of the amino acid sequence shown in SEQ ID NO:1 through an amide bond; the C-terminus of the second side chain is connected to the side-chain amino group of the fourth amino acid of the amino acid sequence shown in SEQ ID NO:1 through an amide bond. The self-assembling antimicrobial peptide Lau-IL-9-C2 of the present invention is a dendritic polypeptide with one main chain and two side chains. By connecting lauric acid, it provides a self-assembling driving force relying on its strong hydrophobicity. Moreover, the two side chains can increase the surface polypeptide density, increase the activity and enhance the stability. The results of the examples show that the antimicrobial peptide Lau-IL-9-C2 provided by the present invention can self-assemble, presenting a nanoparticle state under the electron microscope, with a particle size of 10-16 nm and a critical micelle concentration of 229.1 μM; and the self-assembling antimicrobial peptide Lau-IL-9-C2 obtained by the present invention has good stability, no obvious cytotoxicity and hemolysis effect, has strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria, and has a minimum inhibitory concentration of 1.17-4.69 μM against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, showing strong broad-spectrum antibacterial property. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 It is a schematic diagram of the synthesis of the self-assembling antimicrobial peptide Lau-IL-9-C2;

[0022] Figure 2 It is a transmission electron microscope observation result and particle size statistical result diagram of the self-assembling antimicrobial peptide Lau-IL-9-C2; wherein, A is the transmission electron microscope image, and B is the particle size statistical result diagram;

[0023] Figure 3 It is a critical micelle concentration determination result diagram of the self-assembling antimicrobial peptide Lau-IL-9-C2; wherein, A is the statistical result diagram of the fluorescence intensity of the gradient concentration self-assembling antimicrobial peptide Lau-IL-9-C2 at different wavelengths, and B is the Nile red fluorescence curve diagram of the gradient concentration self-assembling polypeptide Lau-IL-9-C2;

[0024] Figure 4Figure showing the results of detecting the viability of human embryonic kidney cells HEK293T with self - assembled antimicrobial peptide Lau - IL - 9 - C2 at different concentrations;

[0025] Figure 5 Figure showing the results of detecting the viability of human embryonic kidney cells HEK293T with linear polypeptide IL - 9 at different concentrations;

[0026] Figure 6 Figure showing the evaluation results of the hemolytic effect of self - assembled antimicrobial peptide Lau - IL - 9 - C2 at different concentrations;

[0027] Figure 7 Figure showing the evaluation results of the hemolytic effect of linear polypeptide IL - 9 at different concentrations. Detailed implementation manners

[0028] The present invention provides a self - assembled antimicrobial peptide Lau - IL - 9 - C2, and the self - assembled antimicrobial peptide Lau - IL - 9 - C2 includes lauric acid, a main chain, a first side chain and a second side chain; the main chain includes the amino acid sequence shown in SEQ ID NO:1; the first side chain and the second side chain respectively include the amino acid sequence shown in SEQ ID NO:2; the N - terminal of the main chain is connected with the lauric acid through an amide bond; the C - terminal of the first side chain is connected with the side - chain amino group of the third amino acid of the amino acid sequence shown in SEQ ID NO:1 through an amide bond; the C - terminal of the second side chain is connected with the side - chain amino group of the fourth amino acid of the amino acid sequence shown in SEQ ID NO:1 through an amide bond.

[0029] As an implementation manner, from the N - end to the C - end, the main chain is composed of a linker and a linear polypeptide; the linear polypeptide and the linker are connected through an amide bond; the amino acid sequence of the linear polypeptide is shown in SEQ ID NO:3; the amino acid sequence of the linker is G. As an implementation manner, the N - terminal of the connection is connected with the lauric acid through an amide bond. The present invention utilizes lauric acid, and the length of lauric acid is appropriate. It can not only increase the activity and promote self - assembly, but also the toxicity will not increase significantly. Compared with other types of fatty acid chains with strong hydrophobicity, it is safer, and the toxicity of the self - assembled antimicrobial peptide is smaller after self - assembly. The self - assembled antimicrobial peptide Lau - IL - 9 - C2 of the present invention is a dendritic polypeptide with one main chain and two side chains, with a molecular weight of 1955.41 daltons, and all amino acids are of L - type. The self - assembled antimicrobial peptide Lau - IL - 9 - C2 presents a nanoparticle state under the electron microscope, with a particle size of 10 - 16 nm, a critical micelle concentration of 229.1 μM, good stability, no obvious cytotoxicity and hemolytic effect, and has strong antibacterial activity against both Gram - negative bacteria and Gram - positive bacteria. The minimum inhibitory concentrations against Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus are 1.17 - 4.69 μM, and it has strong broad - spectrum property.

[0030] The present invention has no strict requirements for the preparation method of the antibacterial 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.

[0031] The present invention also provides the application of the self-assembled antibacterial peptide Lau-IL-9-C2 described in the above technical solution in the preparation of antibacterial products.

[0032] 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.

[0033] As an embodiment, the Acinetobacter baumannii described in the present invention includes one or more of Acinetobacter baumannii ATCC 19606, 10769, and 0357; as another embodiment, the Acinetobacter baumannii described in the present invention is Acinetobacter baumannii ATCC 19606. In the examples of the present invention, Acinetobacter baumannii ATCC 19606, 10769, and 0357 are taken as examples for illustration, but it should not be understood that this is the entire protection scope of the present invention.

[0034] As an embodiment, the Escherichia coli described in the present invention includes one or more of Escherichia coli ATCC 8739, 0894, and 5017; as another embodiment, the Escherichia coli described in the present invention is Escherichia coli ATCC 8739. In the examples of the present invention, Escherichia coli ATCC 8739, 0894, and 5017 are taken as examples for illustration, but it should not be understood that this is the entire protection scope of the present invention.

[0035] 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. 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.

[0036] As an implementation mode, the Staphylococcus aureus described in the present invention includes one or more of Staphylococcus aureus ATCC 6538, 220823, and 15775; as another implementation mode, 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 scope of protection of the present invention.

[0037] In the present invention, the strains corresponding to the numbers 10769, 0357, 0894, 5017, 90068, 170682, 20823, and 15775 are known strains in the art, preserved 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 Amphipathicity for Superior Activity and Selectivity. J Med Chem. 2024 Nov 14;67(21):19561-19572.

[0038] As an implementation mode, the product includes a reagent or a drug.

[0039] The present invention provides an antibacterial product, and the antibacterial product includes the self-assembled antibacterial peptide Lau-IL-9-C2 described in the above technical solution; the antibacterial product includes an antibacterial reagent or an antibacterial drug. As an implementation mode, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is ≥1.17 μM; as another implementation mode, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 1.17 - 4.69 μM; as another implementation mode, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 1.17 - 2.34 μM; as another implementation mode, the minimum inhibitory concentration of the antibacterial peptide in the antibacterial product is 1.17 μM.

[0040] To further illustrate the present invention, the following describes in detail a self-assembled antibacterial peptide Lau-IL-9-C2 and its application provided by the present invention with reference to the drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] Entrusted GIL Biochemicals (Shanghai) Co., Ltd., through solid-phase peptide synthesis method, according to Figure 1A dendritic polypeptide is synthesized according to a flow chart, and is desalted and purified by HPLC reverse phase column chromatography to obtain the dendritic polypeptide, which is named Lau-IL-9-C2; the dendritic polypeptide Lau-IL-9-C2 has a main chain and two side chains, and the N-terminus of the main chain is connected to lauric acid (Lau) through an amide bond; wherein the main chain amino acid sequence is GIKKGIKKLL (SEQ ID NO: 1), which consists of 10 amino acids; the amino acid sequences of the two side chains are both KGIKKLL (SEQ ID NO: 2), which consist of 7 amino acids, and are respectively connected to the side chain amino groups of the third lysine (K) and the fourth lysine (K) of the main chain through amide bonds; the third lysine is the third amino acid residue from the N-terminus to the C-terminus of the main chain, and the fourth lysine is the fourth amino acid residue from the N-terminus to the C-terminus of the main chain; the molecular weight of Lau-IL-9-C2 is 2841.9 Daltons, and all amino acids are L-type.

[0043] Comparative Example 1

[0044] We commissioned Gill Biochemical (Shanghai) Co., Ltd. to synthesize a linear polypeptide IL-9 with only 9 amino acids through peptide solid phase synthesis. Its amino acid sequence is IKKGIKKLL (SEQ ID NO: 3), all of which are L-type amino acids.

[0045] Test Example 1

[0046] Transmission electron microscopy observation

[0047] (1) Drop the solution containing the dendritic peptide Lau-IL-9-C2 obtained in Example 1 onto glossy paper, clamp a 300-mesh copper mesh, with the carbon film facing down, cover and adsorb for 5 minutes, and absorb the excess liquid; after uranium staining (2%wt uranyl acetate) for 2 minutes, absorb the excess dye, dry it, and observe and take pictures using a transmission electron microscope (model JEM-1400 Plus, JEOL Ltd.). The results showed that the dendritic peptide Lau-IL-9-C2 was in the form of nanoparticles under the transmission electron microscope, indicating that the dendritic peptide Lau-IL-9-C2 can form nanoparticles by self-assembly, which is a self-assembling peptide Lau-IL-9-C2 ( Figure 2 Middle A).

[0048] (2) Data processing: Image J software was used to calculate the particle size of nanoparticles, and GraphPad Prism was used to plot the particle size distribution. The results showed that the particle size of the self-assembling peptide Lau-IL-9-C2 was 10-16 nm ( Figure 2 Middle B).

[0049] Test Example 2

[0050] Critical micelle concentration (CMC) determination

[0051] (1) Add 100 μL of self-assembled polypeptide Lau-IL-9-C2 solution with gradient concentrations to a black-bottom transparent 96-well fluorescence microplate, and then add 100 μL of 25 μM nile red solution to each well. After mixing, let it stand at room temperature for 30 min, and scan the emission spectrum of the solution with a multifunctional microplate reader ( Figure 3 in A).

[0052] Among them, the buffer solution used to prepare the nile red solution and the self-assembled polypeptide Lau-IL-9-C2 solution with gradient concentrations is 10 mM phosphate buffer solution (pH value is 7.4); Fluorescence spectrum scanning conditions: excitation wavelength is 550 nm, emission wavelength is 600 - 700 nm, and bandwidth is 1 nm.

[0053] (2) Data processing: Take the logarithm of the concentration of self-assembled polypeptide Lau-IL-9-C2 as the abscissa, and the maximum fluorescence intensity of nile red at each concentration as the ordinate, and plot the nile red fluorescence curve in the presence of self-assembled polypeptide Lau-IL-9-C2 with gradient concentrations (excitation wavelength is 550 nm, emission wavelength is 630 nm). The polypeptide concentration corresponding to the inflection point of the fluorescence curve is the minimum concentration at which self-assembled polypeptide Lau-IL-9-C2 begins to form micelles in aqueous solution, that is, the CMC value ( Figure 3 in B).

[0054] According to Figure 3 It can be seen that the CMC value of self-assembled polypeptide Lau-IL-9-C2 is 229.1 μM. Nile red is a lipophilic fluorescent dye, and it can produce strong fluorescence after inserting into the hydrophobic pocket of the micelle. When the concentration of self-assembled polypeptide Lau-IL-9-C2 is higher than the CMC, it can induce a significant increase in the fluorescence of nile red in the solution, indicating that self-assembled polypeptide Lau-IL-9-C2 undergoes self-assembly in aqueous solution to form amphiphilic micelles with hydrophilic periphery and hydrophobic interior.

[0055] Test Example 3

[0056] Antibacterial activity analysis

[0057] 1. Test strains

[0058] (1) Acinetobacter baumannii: ATCC 19606, 10769, and 0357;

[0059] (2) Escherichia coli: ATCC 8739, 0894, and 5017;

[0060] (3) Pseudomonas aeruginosa: ATCC 27853, 90068, and 17068;

[0061] (4)Staphylococcus aureus: ATCC 6538, 220823, and 15775.

[0062] 2. Minimum Inhibitory Concentration (MIC) Detection

[0063] First, inoculate the test strains onto LB solid plates. After colonies grow, pick single colonies and transfer them to LB liquid medium. Incubate with shaking at 37°C and 180 rpm for 5 h. Measure the OD of the bacterial solution using an ultraviolet spectrophotometer. 600 , according to the ratio of 1 OD 600 = 1×10 9 CFU / mL, dilute the bacterial solution to a concentration of 2×10 5 CFU / mL with LB liquid medium; Add 100 μL of the diluted bacterial solution to each well of a sterile 96-well plate. Then, add 100 μL of the test samples diluted in gradient with normal saline to each well. Mix well with a pipette. After mixing, place it in a 37°C constant temperature incubator and incubate with slow shaking overnight; The test samples are Lau-IL-9-C2 obtained in Example 1 and the linear polypeptide IL-9 obtained in Comparative Example 1; The concentration of the diluted test samples is 0 - 200 μg / mL; After incubation, measure the absorbance of the bacterial solution at OD 600 nm. Take the average value of the sample concentrations in the wells where no bacterial growth is detected and the adjacent wells as the MIC value. The results are shown in Table 1.

[0064] Table 1 Minimum Inhibitory Concentration Values of Test Samples against Test Strains

[0065]

[0066] As can be seen from Table 1, Lau-IL-9-C2 obtained in Example 1 showed significant antibacterial effects against all the tested strains, with MIC values of 1.17 - 4.69 μM. Compared with the linear polypeptide IL-9 in Comparative Example 1, the antibacterial activity changed from none (MIC > 50 μM) to having.

[0067] Test Example 4

[0068] Plasma Stability Analysis

[0069] 1. Test Strains

[0070] Escherichia coli: ATCC 8739; Staphylococcus aureus: ATCC 6538; Acinetobacter baumannii: ATCC 19606; Pseudomonas aeruginosa: ATCC 27853.

[0071] 2. Take a 1.5 mL EP tube and add 100 μL of 15 mg / mL EDTA-K2 anticoagulant. Draw blood from the mouse's eyeball into the above tube, immediately invert and mix well, centrifuge at 3500 rpm for 10 min at 4 °C, carefully aspirate the supernatant to obtain mouse plasma.

[0072] 3. Use Lau-IL-9-C2 obtained in Example 1 as the test sample, dissolve it in sterile physiological saline, and the final concentration is 400 μg / mL; mix the test sample solution and mouse plasma according to a volume ratio of 1:1, incubate in a 37 °C incubator, and take out 200 μL of the sample at incubation times of 0, 0.5, 1, 2, 4, 6, and 8 h respectively 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 shown in Table 2.

[0073] Table 2 Effect of plasma on the antibacterial activity of Lau-IL-9-C2 (MIC, μM)

[0074]

[0075] It can be seen from Table 2 that the antibacterial activity of Lau-IL-9-C2 against four standard strains has no change after incubation with plasma for 8 h, indicating its excellent plasma stability.

[0076] Test Example 5

[0077] Cytotoxicity analysis

[0078] Routinely culture human embryonic kidney cells (HEK293T) with DMEM medium (containing 10% fetal bovine serum and 1% double antibody). When the cells grow to 80% of the culture flask, wash them 3 times with phosphate buffer first, then digest the cells with 0.25% trypsin, and prepare a cell suspension with a concentration of 5×10 5 cells / mL. Add 200 μL of the cell suspension to each well of a sterile 96-well plate and continue to culture overnight; the next day, add test samples with different concentration gradients (final concentration 0 - 100 μM) or the same volume of DMEM medium. Set 3 replicates for each sample concentration, continue to culture for 24 h, then add 15 μL of MTT solution with a concentration of 5 mg / mL to each well, continue to culture in the dark for 4 h, then aspirate the culture solution in the plate wells, add 200 μL of DMSO to each well, place the culture plate on a shaker and gently shake for 10 min to dissolve the crystals, and then measure the absorbance value of each well at 450 nm with an enzyme-linked immunosorbent assay reader. The results are as Figure 4 and Figure 5 shown; among them, the test samples are Lau-IL-9-C2 obtained in Example 1 and the linear polypeptide IL-9 obtained in Comparative Example 1.

[0079] According to Figure 4 andFigure 5 It can be seen that the linear polypeptide IL-9 obtained in Comparative Example 1 is not toxic to human keratinocyte HaCaT within the test concentration range; Lau-IL-9-C2 obtained in Example 1 is not toxic to human keratinocyte HaCaT within the concentration range of 50 μM, and only shows significant cytotoxicity at a concentration of 100 μM (** P <0.01). Through the self-assembly method, the present invention enables the antibacterial activity of the linear polypeptide IL-9 to change from none (MIC > 50 μM) to having, while not having cytotoxicity and having good safety.

[0080] Test Example 6

[0081] Hemolysis analysis

[0082] 1. Mix fresh human whole blood and Alsever's solution according to a volume ratio of 1:1, centrifuge at 1000 rpm for 5 min, discard the supernatant, wash the obtained red blood cells with physiological saline, and repeat 3 times until the supernatant no longer shows red, to obtain washed red blood cells.

[0083] 2. Dilute the washed red blood cells with physiological saline to a density of 1×10 7 cells / mL, incubate the red blood cell suspension with the test samples dissolved in physiological saline at different concentrations (0 - 100 μM) at 37 °C for 30 min, then centrifuge at 1000 rpm for 5 min, and measure the absorbance of the supernatant at 540 nm. The positive control uses the same volume of Triton X-100 solution (PC), and the volume concentration of the Triton X-100 solution is 10%; the negative control uses physiological saline; wherein, the test samples are Lau-IL-9-C2 obtained in Example 1 and the linear polypeptide IL-9 obtained in Comparative Example 1.

[0084] 3. Data processing: Measure the absorbance value at 540 nm, define the hemolysis rate (Hemolysis) of the positive control (PC group) as 100%, and calculate the hemolysis rate of the test sample group relative to the PC group according to the following formula, and the results are as Figure 6 and Figure 7 shown.

[0085] 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%.

[0086] According to Figure 6 and Figure 7It can be seen that the linear polypeptide IL-9 obtained in Comparative Example 1 and Lau-IL-9-C2 obtained in Example 1 showed high safety against human red blood cells and had no significant hemolytic effect within the concentration range of 100 μM. Through self-assembly, the present invention enables the antibacterial activity of the linear polypeptide IL-9 to change from none (MIC > 50 μM) to having, while having no significant hemolytic effect and good safety.

[0087] It can be seen from the above that the self-assembled antibacterial peptide Lau-IL-9-C2 provided by the present invention has strong antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria, good stability, strong broad-spectrum property, and extremely high safety, without cytotoxicity or hemolytic effect.

[0088] 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 these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A self-assembling antimicrobial peptide Lau-IL-9-C2, characterized in that: The self-assembling antimicrobial peptide Lau-IL-9-C2 consists of lauric acid, a main chain, a first side chain and a second side chain; The amino acid sequence of the main chain is shown in SEQ ID NO: 1; The amino acid sequences of the first side chain and the second side chain are respectively shown in SEQ ID NO: 2; The N-terminus of the main chain is connected to the lauric acid via an amide bond; The C-terminus of the first side chain is connected to the side chain amino group of the third amino acid in the amino acid sequence shown in SEQ ID NO: 1 through an amide bond; the C-terminus of the second side chain is connected to the side chain amino group of the fourth amino acid in the amino acid sequence shown in SEQ ID NO: 1 through an amide bond.

2. Use of the self-assembling antimicrobial peptide Lau-IL-9-C2 according to claim 1 in the preparation of antibacterial products; The bacteria are 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 reagents.

5. An antibacterial product, characterized in that: The antibacterial product comprises the self-assembling antimicrobial peptide Lau-IL-9-C2 according to claim 1.

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

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