Antimicrobial peptides with high antimicrobial activity and their applications

Abstract

The present invention discloses four antimicrobial peptides with high antimicrobial activity and their applications. The amino acid sequence of the peptides is SEQ ID NO. 2. All of the antimicrobial peptides of the present invention have antimicrobial activity. Preliminary toxicity analysis showed that they were non-hemolytic at a concentration of 50 g / mL and had no significant effect on the proliferation and cell morphology of 293T cells.

Description

[0001] Description of the case

[0002] This invention is a divisional application of the Chinese invention patent application with application number 2021113107558, application date 2021-11-8, and invention name “Antibacterial peptides with high antibacterial activity and their applications”. Technical Field

[0003] The present invention belongs to the technical field of biomedical engineering, and in particular relates to an antimicrobial peptide with high antimicrobial activity and applications thereof. Background Art

[0004] Penicillin was first discovered in 1928. Its use, along with that of sulfonamides as therapeutic drugs, gradually enriched the arsenal of drugs for treating microbial infections. The period from 1940 to 1960 marked the golden age of antimicrobial drug development, with the vast majority of antibiotics currently in use being discovered during this period. Antibiotics have become the most commonly prescribed medication, significantly contributing to reducing mortality and morbidity caused by microbial infections. However, due to the inappropriate use of antibiotics, bacteria are gradually developing resistance to them. Many bacteria, such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, and Pseudomonas aeruginosa, have evolved into multidrug-resistant bacteria, posing a significant threat to human health. In addition to the direct impacts of microbial infections, such as ineffective treatment, exacerbated illness, and increased mortality, antibiotic resistance also increases the cost of treatment and healthcare services. Furthermore, drug-resistant microorganisms and their genes can spread across regions and species. Resistance in one region or species can easily spread to other regions or affect other species, making antibiotic resistance a global problem. In this context, the search for new antimicrobial drugs is urgent. Compared to traditional antibiotics, antimicrobial peptides exhibit a broader spectrum of antibacterial activity, killing both Gram-negative and Gram-positive bacteria. They are fast in killing bacteria and are less likely to develop drug resistance. Therefore, they are considered potential alternatives to antibiotics and hold great promise for development in antimicrobial drug research and development. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the above-mentioned prior art and provide four antimicrobial peptides with high antimicrobial activity. These peptides can effectively inhibit bacterial activity, thereby achieving the effect of killing bacteria. The above-mentioned peptides or their derivative products can be used to prepare antimicrobial drugs.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] An antimicrobial peptide having antimicrobial activity, characterized in that it is selected from a polypeptide represented by any one of the following amino acid sequences:

[0008] AMP1:APEPRWKIFKRIEKVGRNVRDGVIKAGPAVAVLGQAKALGK(SEQ ID NO.1)

[0009] AMP2: KWKFGKKLERIGQNVFRAAEKVLPVATGYAQLPATLAG (SEQ ID NO. 2);

[0010] AMP3: RWKFGKKLERMGKRIFKATEKGLPVATGVAALARG (SEQ ID NO.3);

[0011] AMP4: PRWKGWKKIEKAGQRVFKAAEKTLPVAVGYVALAGK (SEQ ID NO. 4).

[0012] The antimicrobial peptide of the present invention is used in the preparation of antimicrobial drugs; preferably in the preparation of antimicrobial drugs against Gram-negative bacteria.

[0013] As a preferred embodiment of the present invention, the Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis.

[0014] The antimicrobial peptide of the present invention is used in the preparation of medical imaging reagents, preservatives, feed additives, daily chemical cleaning products, and medical devices with antimicrobial effects.

[0015] A medical imaging agent composition comprises any one or more polypeptides described in the present invention and an imaging agent.

[0016] A biological antibacterial agent comprises any one or more polypeptides described in the present invention, and other pharmaceutically acceptable excipients.

[0017] A preservative, characterized by comprising any one or more polypeptides described in the present invention.

[0018] An animal feed, characterized by comprising any one or more polypeptides described in the present invention and a basal diet.

[0019] A daily cleaning product, characterized by comprising any one or more polypeptides described in the present invention, and one or more surfactants.

[0020] The daily cleaning products also contain corresponding active substances, essences, pigments, etc.

[0021] A medical dressing characterized by comprising any one or more polypeptides described in the present invention and a matrix.

[0022] Beneficial effects:

[0023] The polypeptides of the present invention have good bacterial activity and can inhibit and kill bacteria, thereby being used to treat bacterial infections. They can also be used in a variety of scenarios where killing or inhibiting bacteria is required, such as for the preparation of medical imaging reagents, cosmetics or food preservatives, feed additives, daily chemical cleaning products, medical devices with antibacterial effects, etc.

[0024] The antimicrobial peptide of the present invention not only has a significant antibacterial effect, but also shows no toxicity to cells in in vitro cytotoxicity experiments, and has extremely high safety.

[0025] The preparation process of the antimicrobial peptide of the present invention is mature and the acquisition channels are convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the growth curve of the test strain in Example 1.

[0027] Figure 2 The following table shows the inhibition zone results for the antimicrobial peptides in Example 2. A, B, C, and D represent the inhibition zone results for the antimicrobial peptides and ampicillin against Escherichia coli K-12, Pseudomonas aeruginosa CGMCC1.10712, Staphylococcus aureus ATCC6538, and Bacillus subtilis 168, respectively. 1, 2, 3, 4, A, and C represent 50g drug-sensitive discs made with AMP1, AMP2, AMP3, AMP4, ampicillin, and cecropin-B, respectively. Band B represents a blank drug-sensitive disc. The presence of an inhibition zone indicates that the substance has an antibacterial effect against the corresponding bacteria.

[0028] Figure 3 This is the antimicrobial peptide PI staining result in Example 3.

[0029] Figure 4 This is the effect of the antimicrobial peptide AMP1 in Example 4 on the morphological changes of 293T cells.

[0030] Figure 5 This is the effect of the antimicrobial peptide AMP2 in Example 4 on the morphological changes of 293T cells.

[0031] Figure 6 This is the effect of the antimicrobial peptide AMP3 in Example 4 on the morphological changes of 293T cells.

[0032] Figure 7 This is the effect of the antimicrobial peptide AMP4 in Example 4 on the morphological changes of 293T cells.

[0033] Figure 8 This is the effect of the antimicrobial peptide cecropin-B in the control group of Example 4 on the morphological changes of 293T cells.

[0034] Figure 9 The effects of the four antimicrobial peptides and the control group in Example 4 on the proliferation of 293T cells. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to specific embodiments.

[0036] The polypeptide sequence of the present invention is as follows:

[0037] AMP1:APEPRWKIFKRIEKVGRNVRDGVIKAGPAVAVLGQAKALGK(SEQ ID NO.1)

[0038] AMP2:KWKFGKKLERIGQNVFRAAEKVLPVATGYAQLPATLAG(SEQ ID NO.2)

[0039] AMP3:RWKFGKKLERMGKRIFKATEKGLPVATGVAALARG(SEQ ID NO.3)

[0040] AMP4:PRWKGWKKIEKAGQRVFKAAEKTLPVAVGYVALAGK(SEQ ID NO.4)

[0041] The polypeptides of the present invention are all prepared by solid phase synthesis method.

[0042] The specific steps are as follows:

[0043] (1) Weigh an appropriate amount of Fmoc-Gly Wang Resin with a degree of substitution of 0.35 mmol / g, place it in a medium-sized reaction column, and soak it in DMF for 120 min;

[0044] (2) Drain the DMF and add 20% Pip / DMF (3 times the volume of the resin) and bubbling nitrogen for 30 min to remove Fmoc. Wash with DMF 5 times. Ninhydrin detection will reveal a dark blue color.

[0045] (3) Add the raw materials in proportion, add an appropriate amount of DMF, and react with nitrogen until the ninhydrin test is transparent;

[0046] (4) Repeat steps 2-3 to complete the sequence synthesis;

[0047] (5) Drain the DMF, add 20% Pip / DMF (3 times the volume of the resin), and blow nitrogen for 30 min to remove Fmoc, DMF*2, MeOH*2, DCM*2, and MeOH*2;

[0048] (6) Drain the resin in the reactor, transfer it to a cut tube, add 40 mL of liquid F, and shake at a controlled temperature for 2.5 h;

[0049] (7) Filter by suction, collect the cutting filtrate into a centrifuge tube, add 6 times the volume of glacial ether, and precipitate by low-speed centrifuge;

[0050] (8) Wash the precipitated crude product with ether three times to obtain the final crude product;

[0051] (9) Place the crude product in a drying pot, vacuum dry overnight, and send it to the purification department for purification to obtain the target purity polypeptide.

[0052] (10) The molecular weight and purity of the peptide were identified using high performance liquid chromatography-mass spectrometry (HPLC-MS).

[0053] To test the antibacterial activity of the polypeptides of the present invention, the size of the inhibition zone was used to determine their antibacterial activity in vitro. In the inhibition zone assay, the antimicrobial peptides AMP1, AMP2, AMP3, and AMP4 of the present invention all demonstrated good antibacterial effects. Furthermore, they showed no cytotoxicity in the in vitro cytotoxicity assay.

[0054] The antibacterial activity of the invented antimicrobial peptides was verified by inhibition zone experiments on Gram-negative bacteria such as Escherichia coli K-12 and Pseudomonas aeruginosa CGMCC 1.10712, and Gram-positive bacteria such as Staphylococcus aureus ATCC6538 and Bacillus subtilis 168. The results showed that all antimicrobial peptides had antibacterial effects on E. coli K-12, AMP1, AMP2, and AMP3 had antibacterial effects on P. aeruginosa CGMCC 1.10712 and B. subtilis 168, while all antimicrobial peptides had no antibacterial activity against S. aureus ATCC6538. The minimum inhibitory concentrations (MICs) of the antimicrobial peptides were determined using the broth microdilution method. The results showed that the four antimicrobial peptides exhibited high antibacterial activity against Gram-negative bacteria, particularly E. coli K12. With the exception of AMP4, the MICs of the remaining antimicrobial peptides against E. coli K12 were all less than 2 μg / mL, demonstrating strong antibacterial activity. Furthermore, the table shows that AMP2, AMP3, and the control group, Cecropin-B, also exhibited strong antibacterial activity against B. subtilis 168. However, none of the antimicrobial peptides had any inhibitory effect against S. aureus ATCC6538 or S. cerevisiae. A comprehensive comparison of the overall antibacterial activity of the five antimicrobial peptides against Gram-negative and Gram-positive bacteria revealed that all five antimicrobial peptides were more sensitive to Gram-negative bacteria, consistent with existing research on cecropin antimicrobial peptides. PI staining experiments were used to investigate the antibacterial mechanism of the invented antimicrobial peptides. The results suggest that these peptides likely exert their antimicrobial and bactericidal activities by disrupting cell membrane integrity. The cytotoxicity of these peptides was preliminarily assessed by analyzing the effects of four antimicrobial peptides, AMP1, AMP2, AMP3, and AMP4, as well as a control, Cecropin-B (H. cecropia), on sheep erythrocyte hemolysis, HEK293T cell proliferation, and cell morphological changes. The results showed that all antimicrobial peptides exhibited no hemolytic effect on sheep erythrocytes at the tested concentrations, indicating that none of the peptides exhibited a significant effect on 293T cell morphology within the tested concentration range. Furthermore, the peptides exhibited no significant effect on 293T cell morphology across the duration and concentration range of exposure. Combined with the cell morphology data and growth curve data, it was confirmed that none of the peptides exhibited cytotoxicity against 293T cells at a concentration of 50 μg / mL.

[0055] Example 1

[0056] Growth curve determination of experimental strains

[0057] The growth curve of the experimental strain was determined by turbidimetry. The specific steps are as follows:

[0058] (1) Strain activation: 20 L of bacterial solution stored in a glycerol tube was added to 100 mL of MHB medium and cultured at 37°C for 18 h.

[0059] (2) Numbering: Take 11 large test tubes containing meat extract peptone liquid culture medium and mark the culture time with a marker, i.e. 0, 1.5, 3, 4, 6, 8, 10, 12, 14, and 16 hours.

[0060] (3) Inoculation: Use a pipette to accurately draw 200L of E. coli culture medium and inoculate it into 11 numbered large test tubes of meat extract peptone liquid culture medium. After inoculation, shake to mix the bacteria.

[0061] (4) Incubation: Place the 11 inoculated test tubes on a shaker and incubate at 37°C. Remove the test tubes numbered with the corresponding time at 0, 1.5, 3, 4, 6, 8, 10, 12, 14, and 16 hours, and immediately store them in a refrigerator. Finally, measure the optical density by turbidimetric analysis.

[0062] (5) Turbidimetric determination: Use uninoculated meat extract peptone medium as a blank control and select a wavelength of 600 nm for photoelectric turbidimetric determination. Begin the determination with the most dilute bacterial suspension. For highly concentrated bacterial suspensions, dilute them appropriately with uninoculated meat extract peptone liquid medium and then determine their optical density (OD) within the range of 0.1-1.0. When recording the OD value, be sure to multiply it by the dilution factor.

[0063] The results are as follows Figure 1 As shown in the results, all strains entered the logarithmic growth phase after 5-6 hours of culture. After 14 hours, the number of colonies dropped sharply, entering the decay phase. Furthermore, the figure also shows that E. coli K12 and B. subtilis 168 grew rapidly, while S. aureus ATCC 6538 grew the slowest.

[0064] Example 2

[0065] 1. Antimicrobial peptide inhibition zone experiment

[0066] The antimicrobial peptide inhibition zone diameter was determined using the disc agar diffusion method, with synthetic Cecropin-B antimicrobial peptide and ampicillin as positive controls. The method was based on the Clinical and Laboratory Standards Institute (CLSI) of the United States. The specific experimental steps are as follows:

[0067] (1) Prepare antimicrobial peptide drug-sensitive paper sheets containing 50 g of drug.

[0068] (2) Isolation and purification of test strains;

[0069] (3) Preparation of inoculum: Pick three colonies with a diameter of approximately 1 mm from the top of the purified test bacteria plate and inoculate them into 3 mL of MH broth. Incubate at 35°C for 5-6 hours until the test bacteria reach the logarithmic growth phase. Then, calibrate the logarithmic growth phase of the bacterial solution with physiological saline to a concentration of 0.5 McFarland standard. After calibration, inoculate the solution within 15 minutes.

[0070] (4) Inoculation of bacterial solution onto the plate: Dip a sterile cotton swab into the bacterial solution, squeeze out the excess bacterial solution on the inner wall of the tube, and then evenly spread it on the surface of the MH agar medium three times, rotating the plate 60° each time, and finally spread it along the inner edge of the plate for a circle.

[0071] (5) After the plate was dried at room temperature for 3 min, the drug-containing paper was placed on the agar surface with tweezers. After incubation at 37°C for 18 h, the results were checked and the diameter of the inhibition zone was measured with a vernier caliper with an accuracy of 0.01 mm.

[0072] In this experiment, we used ampicillin and Cecropin-B as positive controls and blank drug-sensitive paper as negative controls. We applied 50g of antimicrobial peptides and ampicillin drug-sensitive paper to two Gram-negative bacteria and two Gram-positive bacteria. After 18 hours, we observed the antibacterial effect and measured the diameter of the inhibition zone. If an inhibition zone appeared, it was determined that the antimicrobial peptide had antibacterial activity against the corresponding bacteria. The inhibition zone results are as follows: Figure 2 ,As shown in Table 1, the results showed that all antimicrobial peptides had ,antibacterial activity against E. coli K-12, AMP2, AMP3, and AMP4 had antibacterial activity against P. aeruginosa CGMCC 1.10712 and B. subtilis 168, while all antimicrobial peptides had no antibacterial activity against S. aureus ATCC6538.

[0073] Table 1 Antimicrobial peptide inhibition zone diameter results

[0074]

[0075] 2. Determination of Minimum Inhibitory Concentration of Antimicrobial Peptides

[0076] The minimum inhibitory concentration of antimicrobial peptides was determined by the broth microdilution method, which was based on the Clinical and Laboratory Standards Institute (CLSI)

[127] . The specific experimental steps are as follows:

[0077] (1) Preparation of antimicrobial peptide stock solution: Prepare an antimicrobial peptide stock solution at a concentration of 5 mg / mL using PBS buffer solution (pH 7.4). After the stock solution is prepared, sterilize it by filtration through a microporous filter with a pore size of 0.25 μm and aliquot it for storage at -20°C.

[0078] (2) Isolation and purification of experimental bacteria

[0079] (3) The antimicrobial peptide stock solution was diluted in MH broth to a series of concentrations, with the highest concentration being twice the intended concentration. Then, 50 μL of the diluted antimicrobial peptide solution was added to wells 1 through 10 in descending order of concentration using a pipette. 50 μL of MHB medium was added to well 11, and 100 μL of MHB medium was added to well 12.

[0080] (4) Preparation of inoculum: Pick three colonies with a diameter of approximately 1 mm from the isolated and purified test bacteria plate and inoculate them into 3 mL of MH broth. Incubate at 35°C for 5-6 hours until the test bacteria reach the logarithmic growth phase. The bacteria in the logarithmic growth phase are then adjusted to a concentration of 0.5 McFarland standard with physiological saline and then diluted 100-fold with MH broth to a bacterial count of approximately 106 CFU / mL.

[0081] (5) Inoculation: Add 50 μL of the prepared bacterial solution to each of the wells containing different concentrations of antimicrobial peptides. Also add 50 μL of the prepared bacterial solution to the wells containing only 50 μL of MH broth without antimicrobial peptides as a positive control. The wells containing only 100 μL of MH broth served as blank controls.

[0082] (6) Culture in a constant temperature incubator at 37°C for 18 hours. After the culture is completed, the OD600 of each well is measured using a multifunctional microtiter plate. The concentration that significantly inhibits microbial growth is the minimum inhibitory concentration of the antimicrobial peptide.

[0083] Table 2 shows the minimum inhibitory concentrations (MICs) of the antimicrobial peptides against the test strains. As can be seen from the table, the five antimicrobial peptides exhibited high antibacterial activity against Gram-negative bacteria, particularly E. coli K12. With the exception of antimicrobial peptide AMP1, the remaining antimicrobial peptides exhibited MICs less than 2 μg / mL against E. coli K12, demonstrating strong antibacterial activity. Furthermore, the table shows that AMP3, AMP4, and the control group, Cecropin-B, also exhibited strong antibacterial activity against B. subtilis 168. However, none of the antimicrobial peptides had any inhibitory effect against S. aureus ATCC6538 or S. cerevisiae. A comprehensive comparison of the overall antimicrobial activity of the five antimicrobial peptides against Gram-negative and Gram-positive bacteria revealed a higher sensitivity against Gram-negative bacteria, consistent with existing research on cecropin antimicrobial peptides.

[0084] Table 2 Minimum inhibitory concentration of antimicrobial peptides

[0085]

[0086]

[0087] Example 3

[0088] PI staining test

[0089] The fluorescent dye PI (propidium iodide) is a nuclear staining reagent that can stain DNA and is commonly used for apoptosis detection. It is an analog of ethidium bromide and releases red fluorescence after being intercalated into double-stranded DNA. PI cannot pass through the membrane of living cells, but it can pass through damaged cell membranes and stain the nucleus. Based on this principle, we used Cecropin-B (H. cecropia) as a positive control and used PI staining to observe the damage to the cell membrane of E. coli K-12 after the action of antimicrobial peptides, in order to preliminarily explore its possible antibacterial mechanism. The specific operation method is as follows:

[0090] (1) Strain purification.

[0091] (2) Pick three colonies with a diameter of about 1 mm from the top of the purified test bacteria plate and inoculate them into 3 mL of MH broth medium. Culture at 35°C for 5-6 hours until the test bacteria reach the logarithmic growth phase.

[0092] (3) Centrifuge the bacterial solution in the logarithmic growth phase at 10,000 rpm / min for 3 minutes to collect the bacteria.

[0093] (4) Wash the cells with PBS buffer solution three times, and then resuspend the cells in MH broth to a concentration of 1×106 CFU / mL.

[0094] (5) Antimicrobial peptides were added to the resuspended bacterial solution to make the final concentration of each antimicrobial peptide 2 μg / mL and 4 μg / mL, respectively. The mixture was gently shaken. For the blank control group, an equal volume of PBS buffer solution was added to the resuspended bacterial solution and incubated at 37°C for 1 h.

[0095] (6) After incubation, centrifuge at 10,000 rpm / min for 3 min. Discard the supernatant and resuspend the bacterial pellet in 800 μL of cell staining buffer.

[0096] (7) Add 5 μL of PI staining solution to the bacterial resuspension, mix well, and incubate at 4°C for 30 min.

[0097] (8) After incubation, centrifuge at 10,000 rpm / min for 3 min, discard the supernatant, and then wash the bacterial pellet once with PBS buffer. Prepare a smear and observe the staining results under an inverted fluorescence microscope.

[0098] We used PI staining to preliminarily explore the antibacterial mechanism of three antimicrobial peptides with high antibacterial activity, AMP1, AMP2, and AMP3, against E. coli K12, with Cecropin-B antimicrobial peptide as the positive control group. Figure 5 As shown in the figure, we can see that after incubation for 1 hour, only a small number of cells in the blank control group emitted red fluorescence. Except for Cecropin-B, only a small number of cells in the 2g / mL experimental group and the antimicrobial peptide group emitted red fluorescence. However, more cells in the 4g / mL experimental group emitted red fluorescence, indicating that a large number of bacterial cell membranes were damaged at this time. Therefore, we speculate that these antimicrobial peptides are likely to exert their antibacterial and bactericidal activities by destroying the integrity of the cell membrane.

[0099] Example 4

[0100] 1. Antimicrobial peptide erythrocyte hemolysis test

[0101] In this experiment, PBS buffer solution was used as a negative control and 0.1% Trion X-100 was used as a positive control. The hemolytic activity of different concentrations of antimicrobial peptides on sheep red blood cells was detected by the cyanomethemoglobin method. The specific operation is as follows:

[0102] (1) Take 4 mL of defibrinated sheep blood and centrifuge it at 800 rpm / min for 10 min in a low-temperature high-speed centrifuge. After centrifugation, discard the supernatant to obtain the red blood cell pellet.

[0103] (2) Wash the red blood cell pellet with PBS buffer, centrifuge at 800 rpm / min for 10 min, and discard the supernatant. Repeat the wash 2-3 times until the supernatant no longer appears red.

[0104] (3) The washed red blood cells were diluted with PBS buffer to a red blood cell concentration of 2% (v / v).

[0105] (4) Prepare antimicrobial peptide solutions with concentrations of 1 μg / mL, 5 μg / mL, 20 μg / mL, and 50 μg / mL.

[0106] (5) Place 200 μL of antimicrobial peptide and 200 μL of diluted red blood cell suspension in a 1 mL EP tube, gently shake to mix, and incubate at 37°C for 1 h.

[0107] (6) After incubation, the mixed solution was centrifuged at 800 rpm / min for 10 min. After centrifugation, 200 μL of supernatant was pipetted into a 96-well cell culture plate. The absorbance of each well at 540 nm was then measured using a multi-functional microplate.

[0108] (7) The hemolysis rate of the antimicrobial peptide was calculated according to the formula: hemolysis rate = (Apeptide-APBS) / (A0.1% Trion X-100-APBS) x 100%.

[0109] The results showed that the hemolytic rates of all antimicrobial peptides were less than 5% at the tested concentrations, as shown in Table 3. Therefore, it can be concluded that at the tested concentrations, all antimicrobial peptides had no hemolytic effect on sheep erythrocytes.

[0110] Table 3 Results of hemolysis experiments on red blood cells by four antimicrobial peptides

[0111]

[0112]

[0113] 2. Antimicrobial peptide cytotoxicity test

[0114] The cytotoxicity of antimicrobial peptides is mainly evaluated by observing the effects of antimicrobial peptides on HEK293T cell proliferation and cell morphology changes. The specific procedures are as follows:

[0115] ①. Cell recovery (HEK293T cell line)

[0116] (1) Preparation before the experiment: Preheat the water bath to 37°C, wipe the clean bench with medical disinfectant alcohol, and place the sterilized centrifuge tubes, pipettes, and culture dishes in order on the clean bench. Turn on the ultraviolet lamp and sterilize with ultraviolet light for 15 minutes.

[0117] (2) Thawing cells: Take out the cells from the liquid nitrogen tank, clamp the cryotube with hemostatic forceps and quickly shake the cryotube in a preheated water bath in one direction to melt the liquid in the tube quickly. After the liquid in the cryotube is completely dissolved (about 2 minutes), wipe the outer wall of the cryotube with alcohol and then place the cryotube in the clean bench;

[0118] (3) Dilute cells: Slowly add the cells from the cryopreserved tube to a centrifuge tube containing 5 mL of complete culture medium (DMEM medium + 10% FBS + 1% double antibody) and gently pipette to mix. Then centrifuge at 1000 rpm for 5 minutes to obtain a cell pellet.

[0119] (4) Resuspend cells: discard the supernatant and add 1 mL of cell culture medium to resuspend the cells;

[0120] (5) Plating: Add cells to a culture dish containing culture medium, and place the culture dish in an incubator at 37°C and 5% CO2 for cell culture.

[0121] ②. Subculture

[0122] (1) Before cell passage, preheat the cell culture medium, PBS buffer, and trypsin digestion solution in a 37°C water bath. Spray the clean bench with alcohol spray and sterilize with UV for 15 minutes. Remove the cell culture dish from the incubator and observe the cell density under a microscope. When the cell confluence reaches 80%-90%, passage can be performed.

[0123] (2) Remove the cell culture medium with a vacuum pump, wash twice with PBS, add an appropriate amount of 0.25% trypsin digestion solution (the amount should just cover the cell monolayer), place in a cell culture incubator and digest for 1-2 minutes, and observe the cells under a microscope. When the cytoplasm shrinks, the cells become round, and the cells are no longer connected to form a sheet, it indicates that the cells are digested appropriately and the next step can be performed (avoid over-digestion to avoid damaging the cells);

[0124] (3) Add cell culture medium containing serum in an amount equal to that of the digestion solution to terminate the digestion;

[0125] (4) Use a gun to blow the cells to remove the undigested cells, then transfer the digested cells to a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 min;

[0126] (5) Aspirate the supernatant with a vacuum pump and discard it. Add 1 mL of cell culture medium and blow the cell pellet to form a cell suspension.

[0127] (6) Inoculate the cells into a new culture dish according to an appropriate ratio and place it in an incubator for further culture until the subculture is complete.

[0128] ③. Cell cryopreservation

[0129] (1) Select cells in the exponential growth phase (confluence of about 70%-80%) for cryopreservation;

[0130] (2) After digestion, centrifugation, and collection of cells according to the cell passaging procedure, the supernatant was discarded and 1 mL of freezing buffer (DMEM: FBS: DMSO = 6:3:1) was added to resuspend the cells;

[0131] (3) Add an appropriate amount of freezing solution according to the cell volume and mix thoroughly by pipetting. Then, dispense 1 mL into cryovials.

[0132] (4) The cell name, generation number, freezing date and person who saved the cell should be marked on the side of the cryotube before it is placed in the cryobox;

[0133] (5) After the cryopreservation box is placed at 4°C for 10 min, -20°C for 30 min, and -80°C for 16 h, the frozen cells are stored in liquid nitrogen.

[0134] ④. Determination of the effect of antimicrobial peptides on HEK293T cell growth

[0135] (1) When the confluence of HEK293T cells reaches the level required for passage, the cells are digested, centrifuged, and resuspended in culture medium according to the cell passage culture procedure.

[0136] (2) Take out 10 μL of cells and add them to 390 μL of PBS buffer and mix thoroughly. Then take out 10 μL of the diluted cells and mix thoroughly with trypan blue at a ratio of 1:1. Take out 10 μL and add it to a hemocytometer for counting.

[0137] (3) Based on the cell count results, the cell concentration was adjusted and different concentrations of antimicrobial peptides were added. 5 × 104 cells were plated per well of a 24-well culture plate in a final volume of 1 mL. Three time gradients of 24 h, 48 h, and 72 h were set. A blank control group was also set up with only PBS buffer without antimicrobial peptides. After plating, the culture plates were placed in an incubator at 37°C and 5% CO2.

[0138] (4) At the corresponding time points, the cells were first photographed to observe whether the antimicrobial peptides affected cell growth. Subsequently, the cells were digested and counted.

[0139] Cytotoxicity can usually be evaluated by indicators such as cell morphology, cell growth and biochemical changes. In this chapter, we preliminarily evaluated the cytotoxicity of antimicrobial peptides by analyzing the effects of antimicrobial peptides on the cell morphology and cell growth of 293T cells. After the four antimicrobial peptides acted on HEK293T cells, the cell morphological changes were as follows: Figure 4-8As shown. Comparing the cell morphology of the experimental group and the control group, it can be found that the 293T cells in the experimental group were typical epithelial cells during the culture period. The cells were relatively stretched and did not show obvious shrinkage. Comparing the cell growth density of the experimental group and the control group at different culture times, it can be seen that the density of 293T cells increased with the increase of culture time. Comparing the changes in cell morphology and cell density between the experimental groups with different concentrations of antimicrobial peptides during the culture time, it can be seen that they have the same change trend. This shows that the antimicrobial peptides have no significant effect on the morphological changes of 293T cells within the range of action time and concentration. After the four antimicrobial peptides acted on 293T cells, their growth curves are shown as follows. Figure 9 As shown. As can be seen from the figure, at the beginning of cell culture, the number of cells in both the experimental and control groups decreased, and the number of cells reached the lowest after 24 hours. Thereafter, as the culture time increased, the cells gradually entered the logarithmic growth phase, the number of cells increased sharply, and after 72 hours, all cells were still in the logarithmic growth phase. In addition, there was no significant difference in cell growth between the experimental and control groups (p>0.05), and there was no significant difference between the experimental groups with different concentrations (p>0.05). From this result, it can be concluded that all antimicrobial peptides will not affect the proliferation of 293T cells at the current test concentration. Combined with the data on cell morphology changes and growth curve data, we can determine that at a concentration of 50g / mL, all antimicrobial peptides did not show cytotoxic effects on 293T cells.

[0140] In summary, the antimicrobial peptides AMP1, AMP2, AMP3, and AMP4 of the present invention all exhibited good antibacterial effects and showed no cytotoxicity in in vitro cytotoxicity experiments.

Claims

1. An antimicrobial peptide having antimicrobial activity, characterized in that Selected from the polypeptide represented by the following amino acid sequence: AMP2: SEQ ID NO.

2.

2. Use of the antimicrobial peptide according to claim 1 in the preparation of antimicrobial drugs against Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis.

3. Use of the antimicrobial peptide according to claim 1 in the preparation of daily chemical cleaning products and medical devices with antimicrobial effects.

4. A biological antibacterial drug composition, characterized in that The invention comprises the antimicrobial peptide according to claim 1, and other pharmaceutically acceptable excipients.

5. A daily cleaning product composition, characterized in that The invention comprises the antimicrobial peptide according to claim 1, and one or more surfactants.

6. A medical dressing, characterized in that The invention comprises the antimicrobial peptide according to claim 1, and a matrix.

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