Preparation method and application of human antibacterial peptide
By truncating and optimizing the amino acid sequence of LL-37, the novel antimicrobial peptide GK-19 was obtained, which solved the problems of insufficient antimicrobial activity and cytotoxicity of the natural antimicrobial peptide LL-37, and achieved efficient antimicrobial effect and stability.
Patent Information
- Application Number
- CN202411560250.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-04
- Publication Date
- 2025-02-07
AI Technical Summary
The existing natural antibacterial peptide LL-37 has disadvantages such as poor antibacterial activity, hemolysis and cytotoxicity, which limits its application in actual production, and in actual applications its anti-enzymatic, cytotoxic and anti-drug-resistant bacterial activities. There are still many challenges in the problem.
By truncating the amino acids at positions 14-32 of LL-37, the novel antimicrobial peptide GK-19 was obtained, and its sequence was optimized by substitution of basic amino acids and hydrophobic amino acids, and finally antibacterial activity, cytotoxicity, hemolytic activity and Stability detection.
The obtained novel antimicrobial peptide GK-19 and its derived peptide showed high antimicrobial activity, reduced cytotoxicity and hemolytic activity, and had good stability, effectively solving the problems of natural antimicrobial peptides in practical applications.
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Figure CN119390811A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of preparation of human antimicrobial peptides, and in particular to a preparation method of human antimicrobial peptides and application thereof. Background Art
[0002] Antimicrobial peptides, also known as host defense peptides, are a class of small molecule peptide substances expressed and secreted by tissue cells of animals and plants. They have broad-spectrum antibacterial, antiviral, anticancer and immunomodulatory properties. They are an important component of the body's innate immune system and play an important role in maintaining the body's health. Therefore, the development of antimicrobial peptides as a new type of alternative antibiotic product has great potential. Compared with antibiotics, antimicrobial peptides have specific antibacterial mechanisms and are not prone to drug resistance. Studies have shown that antimicrobial peptides mainly bind to bacterial cell membranes through electrostatic adsorption, penetrate the cell membrane through conformational changes, causing the outflow of cell contents, or bind to cell ribosomes to hinder DNA synthesis, thereby causing bacterial death.
[0003] However, most natural antimicrobial peptides have disadvantages such as weak antimicrobial activity, hemolysis and cytotoxicity, which limit their application in actual production. In addition, there are still many challenges in their anti-enzymatic activity, cytotoxicity and anti-resistant bacteria activity in actual applications. Summary of the invention
[0004] The purpose of the present invention is to provide a method for preparing a human antimicrobial peptide and its application, aiming to solve the shortcomings of the natural antimicrobial peptide LL-37 in the prior art, such as poor antimicrobial activity, hemolysis and cytotoxicity, which limit its application in actual production, and in actual application, there are still many challenging technical problems in its anti-enzymatic hydrolysis, cytotoxicity and anti-resistant bacteria activity.
[0005] To achieve the above object, the present invention adopts a method for preparing a human antimicrobial peptide, comprising the following steps:
[0006] First, the truncation method was used to truncate the 14-32 amino acids of LL-37 to obtain the novel antimicrobial peptide GK-19;
[0007] Secondly, aspartic acid and glutamic acid in the GK-19 sequence were replaced with the basic amino acid arginine;
[0008] Thirdly, glutamine and asparagine in the GK-19 sequence were replaced with the hydrophobic amino acid isoleucine;
[0009] Finally, the antibacterial activity test, cytotoxicity test, hemolytic activity test and stability test of the derivative peptides were carried out in sequence.
[0010] The method for detecting the antibacterial activity of the derivative peptides is as follows: the test bacteria frozen at -20°C are cultured in a shaker at 37°C and 300 rpm / min, and when they grow to the logarithmic phase, they are diluted to 10 using LB medium. 6 cfu / ml; prepare 0.2% BSA solution with sterile water, draw 50μL from it and add it to wells 2 to 12 of a 96-well plate, draw 90μL and add it to well 1, and at the same time add 10μL of 2560μmol·L to well 1. -1 The antimicrobial peptide solution was pipetted and mixed, and 50 μL of the solution was aspirated and added to well 2. The solution was diluted to well 10 and discarded. Then, 50 μL of the diluted bacterial suspension was added to wells 1 to 10, so that the final concentration of the antimicrobial peptide was 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μmol·L -1 ; Incubate at 37℃ for 18-24h, observe the clarity of the solution with naked eyes, and the antimicrobial peptide concentration corresponding to the clear wells without turbidity is the MIC value.
[0011] Among them, the detection method of the derived peptide cytotoxicity detection is as follows: the cytotoxicity of the new antimicrobial peptide to Vero cells is detected by a cell proliferation and toxicity detection method; Vero cells are subcultured using a DMEM medium containing 10% serum, and when the cells grow to 80-90% by attachment, the medium is discarded, PBS is washed 1-2 times, trypsin is added for digestion and centrifugation, and the supernatant is discarded, DMEM medium is added, and blown and mixed to make a single cell suspension, 100 μL per well is added to a 96-well plate, and cultured in an incubator at 37°C and 5% CO2 for 12 hours, after the cells are attached, the old medium is discarded, and the medium is replaced after culturing in a constant temperature incubator for 24 hours, 10 μL of CCK-8 solution is added to each well, and incubated in an incubator for 1 hour; OD is detected 450 .
[0012] The method for detecting the hemolytic activity of the derived peptide is as follows: fresh mouse eyeball blood is collected, centrifuged at 1000×g for 5 minutes, the supernatant is discarded, red blood cells are collected, and washed three times with phosphate buffer, centrifuged at 1000×g for 5 minutes after each wash, and PBS is added thereto to prepare an 8% red blood cell suspension; 50 μL of PBS is added to wells 2 to 10 of a 96-well plate, and 95 μL of PBS and 5 μL of 2560 μmol·L are added to one well. -1 The antimicrobial peptide solution was diluted to 8 wells, and 50 μL of red blood cell suspension was added to each well. The 0.2% TritonX-100 treatment group was used as a positive control, and the PBS treatment group was used as a negative control. The cells were cultured at 37°C for 1 hour, and 90 μL of the supernatant was aspirated to a 96-well plate after centrifugation. The OD 562 , calculate the hemolysis rate.
[0013] The stability test method of the derivative peptide is as follows: using 1 mol·L-1 The pH value of the aqueous solution was adjusted to 2, 4, 6, 8, and 10 by adding HCl and NaOH, and the antimicrobial peptide solution was mixed with the solutions of different pH values in equal volumes. After being treated in a water bath at 37°C for 30 min, the MIC values of the antimicrobial peptides against E. coli MG1655 under different pH conditions were detected according to the subsequent steps of the detection method for the antibacterial activity of the derivative peptides; the antimicrobial peptides were treated in a water bath at 25, 37, 55, and 100°C for 30 min, and the MIC values of the antimicrobial peptides against E. coli MG1655 under different temperature conditions were detected according to the subsequent steps of the antibacterial activity detection of the derivative peptides; pepsin, trypsin, and proteinase K solutions with a reaction concentration of 1 mg / mL were added to the antimicrobial peptide solution, respectively, so that the final concentration of the antimicrobial peptides was 256 μmol·L -1 After 1 h in a 37°C water bath, 100 μL was aspirated and added to 1 well of a 96-well plate. The MIC value of the antimicrobial peptide against E. coli MG1655 was detected according to the subsequent steps of the antibacterial activity detection of the derivative peptides. The antimicrobial peptides were diluted with NaCl solution to prepare final concentrations of 50 and 150 mmol·L -1 NaCl and 256 μmol·L -1 The antimicrobial peptide solution was incubated in a 37°C water bath for 30 min, and 100 μL was added to one well of a 96-well plate. The MIC values of the antimicrobial peptides against E. coli MG1655 in different salt ion environments were detected according to the subsequent steps of the antibacterial activity detection of the derived peptides.
[0014] The hemolysis rate is equal to the sample OD 562 Subtract the negative control OD 562 Divide the difference by the positive control OD 562 Subtract the negative control OD 562 The difference.
[0015] The present invention also provides a preparation method using human antimicrobial peptides, which is applied in clinical or animal husbandry production.
[0016] The invention discloses a preparation method and application of a humanized antimicrobial peptide. The method comprises the following steps: by shortening the sequence length of the natural antimicrobial peptide LL-37, increasing the number of positive charges and the hydrophobicity, etc., the natural antimicrobial peptide LL-37 is molecularly designed to obtain different antimicrobial peptide sequences. The physical and chemical properties of the improved antimicrobial peptides are predicted by online software, and the obtained four antimicrobial peptides are all cationic peptides, and all contain 19 amino acid residues. In terms of hydrophobicity, the hydrophobicity of GK-19 and GK-19R2 is less than that of GK-19I2 and GK-19R2I2. In terms of stability, the stability of GK-19 and GK-19I2 is higher than that of GK-19R2 and GK-19R2I2. In terms of antimicrobial activity, the four antimicrobial peptides all exhibit high antimicrobial activity, thereby effectively solving the disadvantages of the natural antimicrobial peptide LL-37, such as low antimicrobial activity, hemolysis and cytotoxicity, and realizing its application in actual production. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
[0018] Figure 1 It is a line graph showing the cytotoxic effects of the derivative peptides of the present invention.
[0019] Figure 2 It is a flow chart of the steps of the present invention. DETAILED DESCRIPTION
[0020] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.
[0021] See also Figure 2 The present invention provides a method for preparing a human antimicrobial peptide, comprising the following steps:
[0022] First, the truncation method was used to truncate the 14-32 amino acids of LL-37 to obtain the novel antimicrobial peptide GK-19;
[0023] Secondly, aspartic acid and glutamic acid in the GK-19 sequence were replaced with the basic amino acid arginine;
[0024] Furthermore, glutamine and asparagine in the GK-19 sequence were replaced with the hydrophobic amino acid isoleucine;
[0025] Finally, the antibacterial activity test, cytotoxicity test, hemolytic activity test and stability test of the derivative peptides were carried out in sequence.
[0026] Furthermore, the method for detecting the antibacterial activity of the derivative peptide is as follows: the test bacteria frozen at -20°C are cultured in a shaker at 37°C and 300 rpm / min, and when they grow to the logarithmic phase, they are diluted to 10 using LB medium. 6 cfu / ml; prepare 0.2% BSA solution with sterile water, draw 50μL from it and add it to wells 2 to 12 of a 96-well plate, draw 90μL and add it to well 1, and at the same time add 10μL of 2560μmol·L to well 1. -1 The antimicrobial peptide solution was pipetted and mixed, and 50 μL of the solution was aspirated and added to well 2. The solution was diluted to well 10 and discarded. Then, 50 μL of the diluted bacterial suspension was added to wells 1 to 10, so that the final concentration of the antimicrobial peptide was 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μmol·L -1 ; Incubate at 37℃ for 18-24h, observe the clarity of the solution with naked eyes, and the antimicrobial peptide concentration corresponding to the clear wells without turbidity is the MIC value.
[0027] Table 1 Amino acid sequences of LL-37 derived peptides
[0028]
[0029] Table 2 Prediction of physicochemical properties of LL-37 derived peptides
[0030]
[0031] Table 3 Minimum inhibitory concentration of LL-37 derived peptides against three bacteria
[0032]
[0033] In the present invention, as shown in Table 1, except for GK-19I2, the other three antimicrobial peptides all showed certain antibacterial activity against Escherichia coli MG1655, Salmonella typhimurium SL1344 and Staphylococcus aureus ATCC6538; among them, GK-19R2 had the best comprehensive inhibitory effect on the three pathogens.
[0034] Furthermore, the detection method of the derived peptide cytotoxicity detection is as follows: the cytotoxicity of the novel antimicrobial peptide to Vero cells is detected by a cell proliferation and toxicity detection method; Vero cells are subcultured using a DMEM medium containing 10% serum, and when the cells grow to 80-90% by attachment, the medium is discarded, PBS is washed 1-2 times, trypsin is added for digestion and centrifugation, and the supernatant is discarded, DMEM medium is added, and mixed by blowing to make a single cell suspension, 100 μL per well is added to a 96-well plate, and cultured in an incubator at 37°C and 5% CO2 for 12 hours, after the cells are attached, the old medium is discarded, and the medium is replaced after culturing in a constant temperature incubator for 24 hours, 10 μL of CCK-8 solution is added to each well, and incubated in an incubator for 1 hour; OD is detected 450 .
[0035] In the present invention, the toxic effects of LL-37 derived peptides on Vero cells, such as Figure 1 As shown in the figure, except for GK-19I2, the other three antimicrobial peptides showed certain cytotoxicity with the increase of concentration, reducing the survival rate of Vero cells. Among them, when the concentration of GK-19, GK-19R2, and GK-19R2I2 was 32μg / mL, the survival rate of cells was below 60%, which showed obvious cytotoxicity.
[0036] Furthermore, the hemolytic activity detection method of the derivative peptide is as follows: fresh mouse eyeball blood is collected, centrifuged at 1000×g for 5 min, the supernatant is discarded, red blood cells are collected, and washed three times with phosphate buffer, centrifuged at 1000×g for 5 min after each wash, and PBS is added thereto to prepare an 8% red blood cell suspension; 50 μL of PBS is added to wells 2 to 10 of a 96-well plate, and 95 μL of PBS and 5 μL of 2560 μmol·L are added to one well. -1 The antimicrobial peptide solution was diluted to 8 wells, and 50 μL of red blood cell suspension was added to each well. The 0.2% TritonX-100 treatment group was used as a positive control, and the PBS treatment group was used as a negative control. The cells were cultured at 37°C for 1 hour, and 90 μL of the supernatant was aspirated to a 96-well plate after centrifugation. The OD 562 , calculate the hemolysis rate.
[0037] In the present invention, the in vitro hemolytic activity of LL-37 derived peptides was tested by adding 1 to 256 μmol·L -1 After the four derivative peptides with different concentration gradients were mixed with the red blood cell suspension for 1 h, the OD was measured. 562 The experimental results showed that the negative control group OD 562 The average value was 0.049, and the positive control OD 562 The average value is 0.5658, 256 μmol·L -1The OD562 values of GK-19 and GK-19R2 were between 0.045 and 0.07, and the hemolysis rates of mouse erythrocytes were less than 5%. The OD562 values of GK-19I2 and GK-19IR2I2 were between 256 μmol·L -1 At high concentrations, the hemolysis rate was less than 8%, showing only slight hemolysis.
[0038] Furthermore, the stability test method of the derivative peptide is as follows: using 1 mol·L -1 The pH value of the aqueous solution was adjusted to 2, 4, 6, 8, and 10 by adding HCl and NaOH, and the antimicrobial peptide solution was mixed with the solutions of different pH values in equal volumes. After being treated in a water bath at 37°C for 30 min, the MIC values of the antimicrobial peptides against E. coli MG1655 under different pH conditions were detected according to the subsequent steps of the detection method for the antibacterial activity of the derivative peptides; the antimicrobial peptides were treated in a water bath at 25, 37, 55, and 100°C for 30 min, and the MIC values of the antimicrobial peptides against E. coli MG1655 under different temperature conditions were detected according to the subsequent steps of the antibacterial activity detection of the derivative peptides; pepsin, trypsin, and proteinase K solutions with a reaction concentration of 1 mg / mL were added to the antimicrobial peptide solution, respectively, so that the final concentration of the antimicrobial peptides was 256 μmol·L -1 After 1 h in a 37°C water bath, 100 μL was aspirated and added to 1 well of a 96-well plate. The MIC value of the antimicrobial peptide against E. coli MG1655 was detected according to the subsequent steps of the antibacterial activity detection of the derivative peptides. The antimicrobial peptides were diluted with NaCl solution to prepare final concentrations of 50 and 150 mmol·L -1 NaCl and 256 μmol·L -1 The antimicrobial peptide solution was incubated in a 37°C water bath for 30 min, and 100 μL was added to one well of a 96-well plate. The MIC values of the antimicrobial peptides against E. coli MG1655 in different salt ion environments were detected according to the subsequent steps of the antibacterial activity detection of the derived peptides.
[0039] Table 4 Thermal stability of LL-37 derived peptides
[0040]
[0041] In the present invention, the changes in the antibacterial activity of LL-37-derived peptides against E. coli MG1655 after treatment at different temperatures are shown in Table 4. Except for GK-19I2, the other three antimicrobial peptides showed good stability after treatment at different temperatures. Among them, the MIC values of GK-19 and GK-19R2 against E. coli MG1655 remained stable at room temperature and under heating conditions. Compared with room temperature conditions, the MIC of GK-19R2I2 increased by 4 times after heating treatment, but remained stable at heating temperatures of 55, 75, and 100°C.
[0042] Furthermore, the hemolysis rate is equal to the sample OD 562 Subtract the negative control OD 562 Divide the difference by the positive control OD 562 Subtract the negative control OD 562 The difference.
[0043] Table 5 pH stability of LL-37 derived peptides
[0044]
[0045] In the present invention, the changes in the antibacterial activity of LL-37 derivative peptides against E.coli MG1655 under different pH environments are shown in Table 5. Except for GK-19I2, the other three antibacterial peptides have strong antibacterial activity under the conditions of pH 2 to 10. Among them, GK-19 has the smallest MIC value under the neutral environment of pH 6 to 8, that is, the highest antibacterial activity, and the antibacterial activity decreases under the alkaline environment of pH 10; the antibacterial activity of GK-19R2 remains stable at pH 2 to 8, and the antibacterial activity decreases slightly under the alkaline environment of pH 10; the antibacterial activity of GK-19R2I2 gradually decreases at pH 2 to 10, and the antibacterial activity is the highest at pH 2; indicating that the LL-37 derivative peptide has a certain acid and alkali resistance, and the antibacterial effect is best in an acidic or neutral environment.
[0046] Table 6 LL-37 derivative peptide protease stability
[0047]
[0048] Table 7 LL-37 derivative peptide salt ion stability
[0049]
[0050] In the present invention, the changes in the antibacterial activity of LL-37-derived peptides against E. coli MG1655 after treatment with three different proteases are shown in Table 6. Except for the antibacterial activity of GK-19R2I2 after hydrolysis with pepsin, which maintained a high level, the antibacterial activity of the other antimicrobial peptides after treatment with different proteases was significantly reduced compared with the control group.
[0051] In the present invention, LL-37 derived peptides are + The changes of the antibacterial activity against E.coli MG1655 under the following conditions are shown in Table 7. -1 Na + The concentration had little effect on the antibacterial activity of antimicrobial peptides, except for GK-19R2I2 at 150mmol·L -1 Na + The MIC values under the following conditions increased slightly, while the others remained unchanged.
[0052] The present invention also provides a preparation method using human antimicrobial peptides, which is applied in clinical or animal husbandry production.
[0053] In summary, the preparation method and application of a human antimicrobial peptide of the present invention are studied. The biological activity and antibacterial stability of four molecularly improved LL-37 derivative peptides are studied. Except for the antimicrobial peptide GK-19I2, GK-19, GL-19R2, and GK-19R2I2 all show strong antibacterial activity against Staphylococcus aureus ATCC6538, Escherichia coli MG1655, and Salmonella typhimurium SL1344, and have good antibacterial stability and anti-hemolytic activity. Among them, GV-R2 has the best comprehensive antibacterial effect. By shortening the antimicrobial peptide sequence length, replacing amino acid residues, increasing the number of positive charges and hydrophobicity of the peptide chain, the molecular improvement design is carried out to obtain four new LL-37 derivative peptides, and their antibacterial activity, cytotoxicity and stability are tested, in order to provide a certain reference basis for the development and utilization of natural antimicrobial peptides.
[0054] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.
Claims
1. A method for preparing a human antimicrobial peptide, characterized in that: The steps include: First, the truncation method was used to truncate the 14-32 amino acids of LL-37 to obtain the novel antimicrobial peptide GK-19; Secondly, aspartic acid and glutamic acid in the GK-19 sequence were replaced with the basic amino acid arginine; Thirdly, glutamine and asparagine in the GK-19 sequence were replaced with the hydrophobic amino acid isoleucine; Finally, the antibacterial activity test, cytotoxicity test, hemolytic activity test and stability test of the derivative peptides were carried out in sequence.
2. The method for preparing a human antimicrobial peptide according to claim 1, characterized in that: The method for detecting the antibacterial activity of the derivative peptides is as follows: the test bacteria frozen at -20°C are cultured in a shaker at 37°C and 300 rpm / min, and when they grow to the logarithmic phase, they are diluted to 10 using LB medium. 6 cfu / ml; prepare 0.2% BSA solution with sterile water, draw 50μL from it and add it to wells 2 to 12 of a 96-well plate, draw 90μL and add it to well 1, and at the same time add 10μL of 2560μmol·L to well 1. -1 The antimicrobial peptide solution was pipetted and mixed, and 50 μL of the solution was aspirated and added to well 2. The solution was diluted to well 10 and discarded. Then, 50 μL of the diluted bacterial suspension was added to wells 1 to 10, so that the final concentration of the antimicrobial peptide was 256, 128, 64, 32, 16, 8, 4, 2, 1, and 0.5 μmol·L -1 ; Incubate at 37℃ for 18-24h, observe the clarity of the solution with naked eyes, and the antimicrobial peptide concentration corresponding to the clear wells without turbidity is the MIC value.
3. The method for preparing a human antimicrobial peptide according to claim 1, characterized in that: The detection method of the derived peptide cytotoxicity detection is as follows: the cytotoxicity of the novel antimicrobial peptide to Vero cells is detected by a cell proliferation and toxicity detection method; Vero cells are subcultured using a DMEM medium containing 10% serum, and when the cells grow to 80-90% by adhesion, the medium is discarded, PBS is washed 1-2 times, trypsin is added for digestion and centrifugation, the supernatant is discarded, DMEM medium is added, and mixed by blowing to prepare a single cell suspension, 100 μL per well is added to a 96-well plate, and cultured in an incubator at 37° C. and 5% CO2 for 12 hours, after the cells are attached, the old medium is discarded, and the medium is replaced after culturing in a constant temperature incubator for 24 hours, 10 μL of CCK-8 solution is added to each well, and the suspension is incubated in an incubator for 1 hour; OD is detected 450 .
4. The method for preparing a human antimicrobial peptide according to claim 1, characterized in that: The hemolytic activity detection method of the derivative peptide is as follows: fresh mouse eyeball blood is collected, centrifuged at 1000g for 5 minutes, the supernatant is discarded, red blood cells are collected, and washed with phosphate buffer for 3 times, centrifuged at 1000g for 5 minutes after each wash, and PBS is added thereto to prepare an 8% red blood cell suspension; 50 μL of PBS is added to wells 2 to 10 of a 96-well plate, and 95 μL of PBS and 5 μL of 2560 μmol·L -1 The antimicrobial peptide solution was diluted to 8 wells, and 50 μL of red blood cell suspension was added to each well. The 0.2% TritonX-100 treatment group was used as a positive control, and the PBS treatment group was used as a negative control. The cells were cultured at 37°C for 1 hour, and 90 μL of the supernatant was aspirated to a 96-well plate after centrifugation. The OD 562 , calculate the hemolysis rate.
5. The method for preparing a human antimicrobial peptide according to claim 2, characterized in that: The stability test method of the derivative peptide is as follows: using 1 mol·L -1 The pH value of the aqueous solution was adjusted to 2, 4, 6, 8, and 10 by adding HCl and NaOH, and the antimicrobial peptide solution was mixed with the solutions of different pH values in equal volumes. After being treated in a water bath at 37°C for 30 minutes, the MIC values of the antimicrobial peptides against E. coli MG1655 under different pH conditions were detected according to the subsequent steps of the detection method for the antibacterial activity of the derivative peptides; the antimicrobial peptides were treated in a water bath at 25, 37, 55, and 100°C for 30 minutes, and the MIC values of the antimicrobial peptides against E. coli MG1655 under different temperature conditions were detected according to the subsequent steps of the antibacterial activity detection of the derivative peptides; pepsin, trypsin, and proteinase K solutions with a reaction concentration of 1 mg / mL were added to the antimicrobial peptide solution, respectively, so that the final concentration of the antimicrobial peptides was 256 μmol·L -1 After 1 h in a 37°C water bath, 100 μL was aspirated and added to 1 well of a 96-well plate. The MIC value of the antimicrobial peptide against E. coli MG1655 was detected according to the subsequent steps of the antibacterial activity detection of the derivative peptides. The antimicrobial peptides were diluted with NaCl solution to prepare final concentrations of 50 and 150 mmol·L -1 NaCl and 256 μmol·L -1 The antimicrobial peptide solution was incubated in a 37°C water bath for 30 min, and 100 μL was added to one well of a 96-well plate. The MIC values of the antimicrobial peptides against E. coli MG1655 in different salt ion environments were detected according to the subsequent steps of the antibacterial activity detection of the derived peptides.
6. The method for preparing a human antimicrobial peptide according to claim 3, characterized in that: The hemolysis rate is equal to the sample OD 562 Subtract the negative control OD 562 Divide the difference by the positive control OD 562 Subtract the negative control OD 562 The difference.
7. A method for preparing a human antimicrobial peptide, wherein the prepared human antimicrobial peptide is used in clinical or animal husbandry production.