Antimicrobial peptide AMP3 with high antimicrobial activity and its application

Abstract

This invention discloses the antimicrobial peptide AMP3 with high antibacterial activity and its applications. The amino acid sequence of the peptide is shown in SEQ ID NO.3. All antimicrobial peptides of this invention exhibit antibacterial activity. Preliminary toxicity analysis shows that they are non-hemolytic at a concentration of 50 g / mL and have no significant effect on the proliferation or 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 invention belongs to the technical field of biomedical engineering, and in particular relates to an antimicrobial peptide AMP3 with high antimicrobial activity and an application thereof. Background Art

[0004] Penicillin was first discovered in 1928 and used together with sulfonamides as therapeutic drugs, which gradually enriched the drug library for treating microbial infections. The golden age of antimicrobial drug development was from 1940 to 1960, and most of the antibiotics currently used were discovered during this period. So far, antibiotics have become the most commonly prescribed drugs, which have made a great contribution to reducing the mortality and morbidity caused by microbial infections. However, due to the irregular use of antibiotics, bacteria gradually develop resistance to them. Many bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, etc. have evolved into multi-drug resistant bacteria, posing a great threat to human health. In addition to the direct impacts such as the inability to effectively treat microbial infections, worsening of illness, and increased mortality, antibiotic resistance will also increase the cost of treatment and medical services, leading to national and global economic losses. Some studies predict that by 2050, antibiotic resistance will cause 10 million deaths each year and cause 100 trillion economic losses worldwide. In addition, 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, both in developed and developing countries. Therefore, antibiotic resistance is a global problem. If antibiotics continue to be abused as antibacterial drugs, it will pose a great threat to global public health, food safety, and food security. In this case, it is urgent to find new antibacterial drugs. Compared with traditional antibiotics, antimicrobial peptides exhibit a broader spectrum of antibacterial activity, have a killing effect on both Gram-negative and Gram-positive bacteria, have a fast sterilization rate and are not easy to develop drug resistance. Therefore, they are regarded as potential antibiotic substitutes and have broad development prospects in the research and development of antimicrobial drugs. 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] The antimicrobial peptide having antimicrobial activity is characterized by being selected from any one of the following amino acid sequences: AMP1: APEPRWKIFKRIEKVGRNVRDGVIKAGPAVAVLGQAKALGK (SEQ ID NO.1)

[0008] AMP2: KWKFGKKLERIGQNVFRAAEKVLPVATGYAQLPATLAG (SEQ ID NO. 2);

[0009] AMP3: RWKFGKKLERMGKRIFKATEKGLPVATGVAALARG (SEQ ID NO.3);

[0010] AMP4: PRWKGWKKIEKAGQRVFKAAEKTLPVAVGYVALAGK (SEQ ID NO. 4).

[0011] 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.

[0012] As a preferred embodiment of the present invention, the Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis.

[0013] The antimicrobial peptide of the present invention is used in the preparation of medical imaging reagents, daily chemical cleaning products, and medical devices with antimicrobial effects.

[0014] A medical imaging agent composition comprises any one or more polypeptides described in the present invention and an imaging agent.

[0015] A biological antibacterial agent comprises any one or more polypeptides described in the present invention, and other pharmaceutically acceptable excipients.

[0016] A daily cleaning product, characterized by comprising any one or more polypeptides described in the present invention, and one or more surfactants.

[0017] The daily cleaning products also contain corresponding active substances, essences, pigments and the like.

[0018] A medical dressing characterized by comprising any one or more polypeptides described in the present invention and a matrix.

[0019] Beneficial effects:

[0020] The polypeptide of the present invention has good bacterial activity, can inhibit and kill bacteria and thus be used in the treatment of bacterial infections. It can also be applied to a variety of scenarios where bacteria need to be killed or inhibited, such as for the preparation of medical imaging agents, cosmetics or daily chemical cleaning products, medical devices with antibacterial effects, etc.

[0021] The antimicrobial peptide of the present invention not only has a significant antibacterial effect, but also shows no toxicity to cells in an in vitro cytotoxicity experiment, and has extremely high safety.

[0022] The preparation process of the antimicrobial peptide of the present invention is mature and the acquisition channel is convenient. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the growth curve of the test strain in Example 1.

[0024] Figure 2 The results of the antimicrobial peptide inhibition zone in Example 2. A, B, C, and D respectively represent the inhibition zone results of antimicrobial peptides and ampicillin against Escherichia coli K-12, Pseudomonas aeruginosa CGMCC1.10712, Staphylococcus aureus ATCC6538, and Bacillus subtilis 168. 1, 2, 3, 4, A, and C represent AMP1, AMP2, AMP3, AMP4, ampicillin, and cecropin-B, respectively, with a drug content of 50 g. B represents a blank drug-sensitive paper. The appearance of an inhibition zone indicates that the substance has an antibacterial effect on the corresponding bacteria.

[0025] Figure 3 This is the antimicrobial peptide PI staining result in Example 3.

[0026] Figure 4 This is the effect of the antimicrobial peptide AMP1 in Example 4 on the morphological changes of 293T cells.

[0027] Figure 5 This is the effect of the antimicrobial peptide AMP2 in Example 4 on the morphological changes of 293T cells.

[0028] Figure 6 This is the effect of the antimicrobial peptide AMP3 in Example 4 on the morphological changes of 293T cells.

[0029] Figure 7 This is the effect of the antimicrobial peptide AMP4 in Example 4 on the morphological changes of 293T cells.

[0030] 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.

[0031] Fig. 9 The effects of the four antimicrobial peptides and the control group in Example 4 on the proliferation of 293T cells. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific embodiments.

[0033] The polypeptide sequence of the present invention is as follows:

[0034] AMP1:APEPRWKIFKRIEKVGRNVRDGVIKAGPAVAVLGQAKALGK(SEQ ID NO.1)

[0035] AMP2:KWKFGKKLERIGQNVFRAAEKVLPVATGYAQLPATLAG(SEQ ID NO.2)

[0036] AMP3:RWKFGKKLERMGKRIFKATEKGLPVATGVAALARG(SEQ ID NO.3)

[0037] AMP4:PRWKGWKKIEKAGQRVFKAAEKTLPVAVGYVALAGK(SEQ ID NO.4)

[0038] The polypeptides of the present invention are all prepared by solid phase synthesis method.

[0039] The specific steps are as follows:

[0040] (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;

[0041] (2) Drain DMF, add 20% Pip / DMF in an amount three times the volume of the resin, and blow nitrogen for 30 min to remove Fmoc. Wash with DMF five times, and the color will turn dark blue when tested with ninhydrin.

[0042] (3) Add raw materials in proportion, add appropriate amount of DMF, and react with nitrogen until the ninhydrin test is transparent;

[0043] (4) Repeat steps 2-3 to complete the synthesis of the sequence;

[0044] (5) Drain 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;

[0045] (6) Drain the resin in the reactor, transfer it to a cutting tube, add 40 mL of liquid F, and shake at a controlled temperature for 2.5 h;

[0046] (7) Filter by suction, collect the cutting filtrate into a centrifuge tube, add 6 volumes of icy ether, and centrifuge at a low speed for precipitation;

[0047] (8) Wash the precipitated crude product with ether three times to obtain the final crude product;

[0048] (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.

[0049] (10) High performance liquid chromatography-mass spectrometry (HPLC-MS) was used to identify the molecular weight and purity of the peptide.

[0050] In order to detect the antibacterial activity of the polypeptide of the present invention, the antibacterial activity was determined by the size of the inhibition zone in vitro. In the inhibition zone experiment, the antimicrobial peptides AMP1, AMP2, AMP3, and AMP4 of the present invention all showed good antibacterial effects. And they did not show toxicity to cells in the in vitro cytotoxicity experiment.

[0051] 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 concentration of antimicrobial peptides was determined by the microbroth dilution method. The results showed that the four antimicrobial peptides showed high antibacterial activity against Gram-negative bacteria, especially against E. coli K12. Except for the antimicrobial peptide AMP4, the minimum inhibitory concentrations of the other antimicrobial peptides against E. coli K12 were less than 2g / mL, showing a strong antibacterial effect. In addition, it can be seen from the table that AMP2, AMP3 and the control group Cecropin-B also have strong antibacterial activity against B. subtilis168, but all antimicrobial peptides have no antibacterial effect on S. aureus ATCC6538 and S. cerevisiae. A comprehensive comparison of the overall antibacterial effects of the five antimicrobial peptides on Gram-negative and Gram-positive bacteria showed that the five antimicrobial peptides were more sensitive to Gram-negative bacteria, which is consistent with the existing research results on Cecropin antimicrobial peptides. The antibacterial mechanism of the invented antimicrobial peptides was explored through PI staining experiments. According to the results, it is speculated that these antimicrobial peptides are very likely to exert antibacterial and bactericidal activity by destroying the integrity of the cell membrane. The cytotoxic effects of these antimicrobial peptides were preliminarily evaluated by analyzing the effects of the four antimicrobial peptides AMP1, AMP2, AMP3, and AMP4 and the control group Cecropin-B (H.cecropia) on the hemolysis of sheep erythrocytes, the proliferation of HEK293T cells, and cell morphological changes. The results showed that the hemolysis rate of all antimicrobial peptides was less than 5% at the tested concentration. Therefore, it can be judged that at the tested concentration, all antimicrobial peptides have no hemolytic effect on sheep erythrocytes. And the antimicrobial peptides have no significant effect on the morphological changes of 293T cells within the action time and concentration range. Combined with the data of cell morphological changes and growth curve data, it can be determined that at the action concentration of 50g / mL, all antimicrobial peptides have no cytotoxic effect on 293T cells.

[0052] Example 1

[0053] Growth curve determination of experimental strains

[0054] The growth curve of the experimental strain was determined by turbidimetry. The specific steps are as follows:

[0055] (1) Strain activation: Take 20 L of bacterial solution stored in a glycerol tube and add it to 100 mL of MHB medium and culture at 37°C for 18 h.

[0056] (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.

[0057] (3) Inoculation: Use a pipette to accurately draw up 200 L 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.

[0058] (4) Cultivation: Place the 11 inoculated test tubes on a shaker and culture at 37°C. Take out the test tubes numbered 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 turbidimetry.

[0059] (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 from the most dilute bacterial suspension in sequence. For the bacterial suspension with a high concentration, dilute it appropriately with uninoculated meat extract peptone liquid medium and then determine its optical density value within 0.1-1.0. When recording the OD value, be sure to multiply it by the dilution multiple.

[0060] The results are as follows Figure 1 As shown in the figure, it can be seen from the results that after 5-6 hours of culture, all strains are in the logarithmic growth phase, and after 14 hours, the number of colonies drops sharply and enters the decay phase. In addition, it can be seen from the figure that E. coli K12 and B. subtilis 168 grow rapidly, while S. aureus ATCC6538 grows the slowest.

[0061] Example 2

[0062] 1. Antimicrobial peptide inhibition zone experiment

[0063] The antimicrobial peptide inhibition zone diameter was determined by the paper 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:

[0064] (1) Prepare antimicrobial peptide drug-sensitive paper sheets containing 50 g of drug.

[0065] (2) Isolation and purification of test strains;

[0066] (3) Preparation of inoculated bacterial solution: Pick 3 colonies with a diameter of about 1 mm from the purified test bacterial 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. Then, calibrate the bacterial solution in the logarithmic growth phase with physiological saline to a concentration of 0.5 McFarland standard. After calibration, inoculate the solution within 15 minutes.

[0067] (4) Inoculation of bacterial solution onto plates: 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 apply it on the surface of the MH agar medium three times, rotating the plate 60° each time, and finally apply it along the inner edge of the plate for a circle.

[0068] (5) After the plate was dried at room temperature for 3 min, the drug-containing paper was pressed against the agar surface of the plate 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.

[0069] 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 appears, it is determined that the antimicrobial peptide has antibacterial activity against the corresponding bacteria. The inhibition zone results are shown in Figure 1. Figure 2 As shown in Table 1, the results showed that all antimicrobial peptides had an antibacterial effect on E. coli K-12, AMP2, AMP3, and AMP4 had an antibacterial effect on P. aeruginosa CGMCC1.10712 and B. subtilis 168, while all antimicrobial peptides had no antibacterial activity against S. aureus ATCC6538.

[0070] Table 1 Antimicrobial peptide inhibition zone diameter results

[0071]

[0072] 2. Determination of minimum inhibitory concentration of antimicrobial peptides

[0073] 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) of the United States

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

[0074] (1) Preparation of antimicrobial peptide stock solution: Use PBS buffer solution with pH = 7.4 as solvent to prepare an antimicrobial peptide stock solution with a concentration of 5 mg / mL. After the stock solution is prepared, filter it with a microporous filter membrane with a pore size of 0.25 μm for sterilization, and store it in small amounts for standby use at -20°C.

[0075] (2) Isolation and purification of experimental bacteria

[0076] (3) The stock solution of antimicrobial peptide was diluted with MH broth medium to form a series of concentrations, with the highest concentration being twice the concentration to be tested. Then, 50 μL of the diluted antimicrobial peptide solution was added from well 1 to well 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.

[0077] (4) Preparation of inoculated bacterial solution: Pick 3 colonies with a diameter of about 1 mm from the isolated and purified test bacteria plate and inoculate them into 3 mL of MH broth medium. Culture at 35°C for 5-6 hours to allow the test bacteria to reach the logarithmic growth phase. Then, calibrate the bacterial solution in the logarithmic growth phase with physiological saline to a concentration of 0.5 McFarland standard, and then dilute it 100 times with MH broth medium to make the inoculated bacterial solution contain about 106 CFU / mL.

[0078] (5) Inoculation: Add 50 μL of the prepared bacterial solution to the wells containing different concentrations of antimicrobial peptides. Add 50 μL of the prepared bacterial solution to the wells containing only 50 μL MH broth medium without antimicrobial peptides as a positive control. The wells containing only 100 μL MH broth medium serve as blank controls.

[0079] (6) Culture in a constant temperature incubator at 37°C for 18 h. After the culture is completed, the OD600 of each well is measured using a multifunctional microplate. The concentration that significantly inhibits the growth of microorganisms is the minimum inhibitory concentration of the antimicrobial peptide.

[0080] The results of the minimum inhibitory concentration of antimicrobial peptides against the test strains are shown in Table 2. As can be seen from the table, the five antimicrobial peptides showed high antibacterial activity against Gram-negative bacteria, especially against E. coli K12. Except for the antimicrobial peptide AMP1, the minimum inhibitory concentrations of the remaining antimicrobial peptides against E. coli K12 were all less than 2 g / mL, showing a strong antibacterial effect. In addition, it can be seen from the table that AMP3, AMP4 and the control group Cecropin-B also had strong antibacterial activity against B. subtilis 168, but all antimicrobial peptides had no antibacterial effect on S. aureus ATCC6538 and S. cerevisiae. A comprehensive comparison of the overall antibacterial effects of the five antimicrobial peptides on Gram-negative and Gram-positive bacteria showed that the five antimicrobial peptides were more sensitive to Gram-negative bacteria, which is consistent with the existing research results on Cecropin antimicrobial peptides.

[0081] Table 2 Results of minimum inhibitory concentration of antimicrobial peptides

[0082]

[0083]

[0084] Example 3

[0085] PI staining assay

[0086] The fluorescent dye PI (propidium iodide) is a nuclear staining reagent that can stain DNA and is often used for apoptosis detection. It is an analog of ethidium bromide and releases red fluorescence after being embedded in double-stranded DNA. PI cannot pass through the membrane of living cells, but it can pass through damaged cell membranes to stain the nucleus. Based on this principle, we used Cecropin-B (H.cecropia) as a positive control and used PI staining to observe the cell membrane damage of E.coliK-12 after the action of antimicrobial peptides, in order to preliminarily explore its possible antibacterial mechanism. The specific operation method is as follows:

[0087] (1) Strain purification.

[0088] (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 to allow the test bacteria to reach the logarithmic growth phase.

[0089] (3) Centrifuge the bacterial solution in the logarithmic growth phase at 10,000 rpm / min for 3 min to collect the bacteria.

[0090] (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.

[0091] (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.

[0092] (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.

[0093] (7) Add 5 μL of PI dye solution to the bacterial resuspension, mix well, and incubate at 4°C for 30 min.

[0094] (8) After incubation, centrifuge at 10,000 rpm / min for 3 min, discard the supernatant, then wash the bacterial pellet once with PBS buffer, make a smear, and observe the staining results under an inverted fluorescence microscope.

[0095] 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, and Cecropin-B antimicrobial peptide was used 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. In the 2g / mL experimental group, except for Cecropin-B, only a small number of cells in the antimicrobial peptide group emitted red fluorescence, while in the 4g / mL experimental group, more cells 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 very likely to exert their antibacterial and bactericidal activity by destroying the integrity of the cell membrane.

[0096] Example 4

[0097] 1. Antimicrobial peptide red blood cell hemolysis test

[0098] In this experiment, PBS buffer solution was used as negative control, 0.1% TrionX-100 was used as positive control, and the cyanide methemoglobin method was used to detect the hemolytic activity of different concentrations of antimicrobial peptides on sheep red blood cells. The specific operation is as follows:

[0099] (1) Take 4 mL of defibrinated sheep blood and centrifuge it at 800 rpm / min in a low-temperature high-speed centrifuge for 10 min. After centrifugation, discard the supernatant to obtain the red blood cell pellet.

[0100] (2) Wash the red blood cell pellet with PBS buffer, centrifuge at 800 rpm / min for 10 min, and discard the supernatant. Repeat the washing 2-3 times until the supernatant no longer appears red.

[0101] (3) The washed red blood cells were diluted with PBS buffer to a red blood cell concentration of 2% (v / v).

[0102] (4) Prepare antimicrobial peptide solutions with concentrations of 1 μg / mL, 5 μg / mL, 20 μg / mL, and 50 μg / mL.

[0103] (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.

[0104] (6) After the incubation, the mixed solution was centrifuged at 800 rpm / min for 10 min. After the centrifugation, 200 L of the supernatant was pipetted into a 96-well cell culture plate using a pipette, and then the absorbance of each well at a wavelength of 540 nm was measured using a multifunctional microplate.

[0105] (7) The hemolysis rate of the antimicrobial peptide was calculated according to the formula: hemolysis rate = (Apeptide-APBS) / (A0.1%TrionX-100-APBS) x 100%.

[0106] The results showed that the hemolytic rates of all antimicrobial peptides at the tested concentrations were less than 5%, as shown in Table 3. Therefore, it can be determined that at the tested concentrations, all antimicrobial peptides had no hemolytic effect on sheep erythrocytes.

[0107] Table 3 Results of hemolysis experiments on red blood cells by four antimicrobial peptides

[0108]

[0109] 2. Antimicrobial peptide cytotoxicity test

[0110] 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 operation is as follows:

[0111] ①. Cell recovery (HEK293T cell line)

[0112] (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.

[0113] (2) Thawing cells: Take out the cells from the liquid nitrogen tank, clamp the cryotube with hemostats and quickly shake the cryotube in a preheated water bath in one direction to melt the liquid in the tube quickly. When 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 a clean bench;

[0114] (3) Dilute cells: slowly add the cells in the cryopreservation tube into 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;

[0115] (4) Resuspend cells: discard the supernatant and add 1 mL of cell culture medium to resuspend the cells;

[0116] (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.

[0117] ②. Subculture

[0118] (1) Before cell subculturing, 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 min, remove the cell culture dish from the incubator and observe the cell density under a microscope. Subculturing can be performed when the cell confluence reaches 80%-90%;

[0119] (2) Vacuum pump to suck out the cell culture medium, wash twice with PBS, add an appropriate amount of 0.25% trypsin digestion solution (the amount should just cover the cell monolayer), put it into the cell culture incubator for digestion 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 piece, it indicates that the cells are digested appropriately and the next step can be performed (avoid over-digestion to avoid damaging the cells);

[0120] (3) Adding an equal amount of cell culture medium containing serum to the digestion solution to terminate the digestion;

[0121] (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;

[0122] (5) Use a vacuum pump to discard the supernatant and add 1 mL of cell culture medium to blow the cell pellet into a cell suspension;

[0123] (6) Inoculate the cells into a new culture dish at an appropriate ratio and place it in an incubator for continued culture to complete the subculture.

[0124] ③. Cell cryopreservation

[0125] (1) Select cells in the exponential growth phase (confluence of about 70%-80%) for cryopreservation;

[0126] (2) After digestion, centrifugation, and collection of cells according to the cell passaging procedure, the supernatant was discarded and 1 mL of freezing solution (DMEM: FBS: DMSO = 6:3:1) was added to resuspend the cells;

[0127] (3) Add an appropriate amount of freezing solution according to the cell volume and mix thoroughly by pipetting, then dispense into cryopreservation tubes at a rate of 1 mL per tube;

[0128] (4) The cell name, generation number, freezing date and person who made the cryopreservation should be marked on the side of the cryopreservation tube before it is placed in the cryopreservation box;

[0129] (5) After the cryopreservation box is placed at 4°C for 10 min, at -20°C for 30 min, and at -80°C for 16 h, the frozen cells are stored in liquid nitrogen.

[0130] ④. Determination of the effect of antimicrobial peptides on the growth of HEK293T cells

[0131] (1) When the confluence of HEK293T cells reaches the level required for subculturing, the cells are digested, centrifuged, and resuspended in culture medium according to the cell subculturing procedure.

[0132] (2) Take out 10 μL of cells and add them to 390 μL PBS buffer and mix thoroughly. Then take out 10 μL of the diluted cells and mix thoroughly with trypan blue in a 1:1 ratio, and take out 10 μL and add it to the hemocytometer for counting.

[0133] (3) According to the cell counting results, the cell concentration was adjusted and different concentrations of antimicrobial peptides were added. 5×104 cells were plated in each well of a 24-well culture plate with a final volume of 1 mL. Three time gradients of 24 h, 48 h and 72 h were set. At the same time, a blank control group was set with only PBS buffer added without antimicrobial peptides. After the plating was completed, the culture dish was placed in an incubator at 37°C and 5% CO2 for culture.

[0134] (4) At the corresponding time point, the cells are first photographed to observe whether the antimicrobial peptide affects cell growth. Subsequently, the cells are digested and counted.

[0135] Cytotoxicity can usually be evaluated based on 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 changes in cell morphology were as follows: Figure 4-8 As shown. Comparing the cell morphology of the experimental group and the control group, it can be found that during the culture period, the 293T cells in the experimental group were typical epithelial cells, 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 is also increasing with the increase of culture time. Comparing the changes in cell morphology and cell density between 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 action time and concentration range. After the four antimicrobial peptides acted on 293T cells, their growth curves are shown as follows. Fig. 9As shown. As can be seen from the figure, at the beginning of cell culture, the number of cells in both the experimental group and the control group 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 all cells were still in the logarithmic growth phase after 72 hours. In addition, there was no significant difference in cell growth between the experimental group and the control group (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 of cell morphology changes and growth curve data, we can determine that at the action concentration of 50g / mL, all antimicrobial peptides have no cytotoxic effect on 293T cells.

[0136] In summary, the antimicrobial peptides AMP1, AMP2, AMP3 and AMP4 of the present invention all exhibited good antibacterial effects and showed no toxicity to cells in in vitro cytotoxicity experiments.

Claims

1. An antimicrobial peptide having antimicrobial activity, characterized in that Selected from the following polypeptides represented by the amino acid sequence: AMP3: SEQ ID NO.

3.

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 polypeptide as claimed in claim 1, and other pharmaceutically acceptable excipients.

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

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

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