A Rana chensinensis antimicrobial peptide and its applications in antibacterial, anti-inflammatory and antioxidant aspects

Abstract

The present invention discloses a Rana chensinensis antimicrobial peptide and its applications in antibacterial, anti-inflammatory and antioxidant aspects, belonging to the field of biotechnology. First, the present invention uses peptidomics technology to analyze the skin mucus of Rana chensinensis, obtaining a large amount of peptide segment information. Then, these peptide segment information are compared with the known antimicrobial peptide database to screen out the peptide segments that may have antibacterial activity. Further through experiments, an antimicrobial peptide with extremely low hemolytic activity, highly stable to pH and temperature, and having anti-inflammatory and antioxidant effects is screened out. The amino acid sequence of this antimicrobial peptide is shown as SEQ ID NO: 3. This antimicrobial peptide lays a foundation for enriching the types of antimicrobial peptides and adds new advantages to the application of antimicrobial peptides.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a Rana chensinensis antimicrobial peptide and its applications in antibacterial, anti-inflammatory and antioxidant activities. Background Art

[0002] In today's era, the widespread use of antibiotics has brought serious consequences, namely the increasing drug resistance of bacteria. Once-powerful antibiotics often appear helpless when facing some drug-resistant bacteria. This severe reality makes it urgent to find new antibacterial substances. As a new type of antibacterial substance, antimicrobial peptides have received extensive attention in recent years. Compared with traditional antibiotics, antimicrobial peptides have many advantages. First, the mechanism of action of antimicrobial peptides is unique. They can rapidly disrupt the cell membrane of bacteria, leading to bacterial death, and it is difficult for bacteria to develop resistance to them. Second, antimicrobial peptides have broad-spectrum antibacterial activity and can inhibit a variety of bacteria, fungi and even viruses. In addition, the metabolic rate of antimicrobial peptides in living organisms is relatively fast, and they do not remain in the environment, having less impact on the ecological environment.

[0003] As an endemic species in Northeast China, the skin mucus secreted by Rana chensinensis contains abundant antimicrobial peptides. These antimicrobial peptides have high biological activity and stability and are good sources for extracting antimicrobial peptides. However, traditional methods for extracting antimicrobial peptides have great drawbacks. Traditional methods mainly obtain antimicrobial peptide sequences through continuous separation and purification. This process is not only time-consuming and laborious but also inefficient. In addition, traditional methods require a large amount of samples and complex instruments and equipment, resulting in high costs. Therefore, there is an urgent need for simple and low-cost methods for rapidly screening new antimicrobial peptides to enrich the types of antimicrobial peptides and provide new antimicrobial peptides for clinical treatment. Summary of the Invention

[0004] The object of the present invention is to provide a Rana chensinensis antimicrobial peptide and its applications in antibacterial, anti-inflammatory and antioxidant activities to solve the problems existing in the above-mentioned prior art. This antimicrobial peptide can inhibit a variety of Gram-negative and Gram-positive bacteria and also has anti-inflammatory and antioxidant effects, enriching the types of antimicrobial peptides for clinical treatment.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a Rana chensinensis antimicrobial peptide, and the amino acid sequence of the Rana chensinensis antimicrobial peptide is: GLFSVVKGVLKAVGKNVAK.

[0007] The present invention also provides the application of the above-mentioned Rana chensinensis antimicrobial peptide in the preparation of antibacterial drugs.

[0008] Optionally, the antibacterial drugs include but are not limited to drugs that inhibit Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella.

[0009] Optionally, the antibacterial agent includes, but is not limited to, antibacterial drugs and antibacterial agents. Optionally, the antibacterial agent is an anti-inflammatory drug for human or veterinary use.

[0010] The present invention also provides the application of the Rana chensinensis antibacterial peptide in the preparation of anti-inflammatory drugs.

[0011] Optionally, the anti-inflammatory drug is an anti-inflammatory drug for human or veterinary use.

[0012] The present invention also provides the application of the Rana chensinensis antibacterial peptide in the preparation of antioxidant products.

[0013] Optionally, the antioxidant product includes, but is not limited to, products for scavenging DPPH free radicals. It also includes food and feed additives that contribute to antioxidant activity, and the food includes, but is not limited to, health foods and functional foods.

[0014] The present invention also provides an antibacterial agent containing the Rana chensinensis antibacterial peptide.

[0015] Optionally, the antibacterial agent includes, but is not limited to, antibacterial drugs and antibacterial agents for inhibiting Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella. Optionally, the antibacterial agent is an anti-inflammatory drug for human or veterinary use.

[0016] The present invention also provides an anti-inflammatory drug containing the Rana chensinensis antibacterial peptide. Optionally, the anti-inflammatory drug is an anti-inflammatory drug for human or veterinary use.

[0017] The present invention also provides an antioxidant product containing the Rana chensinensis antibacterial peptide. The antioxidant product includes, but is not limited to, drugs for scavenging DPPH free radicals and food and feed additives that contribute to antioxidant activity. The drugs include, but are not limited to, drugs for human or veterinary use.

[0018] The present invention discloses the following technical effects:

[0019] The present invention first analyzes the skin mucus of Rana chensinensis using proteomics technology to obtain a large amount of peptide information. Then, these peptide information are compared with a known antibacterial peptide database to screen out peptide segments that may have antibacterial activity. The present invention combines proteomics technology with an antibacterial peptide database, which can greatly improve the speed and accuracy of antibacterial peptide screening, providing a new approach for the research and development of antibacterial peptides. This method can not only quickly and accurately screen out peptide segments with antibacterial activity, but also has a low cost and great application prospects.

[0020] The present invention has screened out an antibacterial peptide with extremely low hemolytic activity, high stability to pH and temperature, and anti-inflammatory and antioxidant effects. The extremely low hemolytic activity of this antibacterial peptide means that it causes very little damage to human red blood cells during use, greatly improving its safety. At the same time, its high stability to pH and temperature enables it to maintain good antibacterial activity under different environmental conditions, expanding its application scope. In addition, its anti-inflammatory and antioxidant effects add new advantages to its application. This antibacterial peptide can also inhibit a variety of Gram-positive and Gram-negative bacteria, enriching the types of antibacterial peptides. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0022] Figure 1 Flow chart for the rough separation of antibacterial peptides;

[0023] Figure 2 Bacteriostatic rates of crude extracts of antibacterial peptides in different molecular weight ranges; A: ATCC25922, B: ATCC25923;

[0024] Figure 3 Structural characterization of antibacterial peptide GK19; A: Primary structure diagram of antibacterial peptide; B; Helical wheel diagram of antibacterial peptide; C: Tertiary structure diagram of antibacterial peptide; D: Circular dichroism spectrum characterization of antibacterial peptide;

[0025] Figure 4 Results of the biosafety assessment of antibacterial peptide GK19; A: Cytotoxicity experiment of antibacterial peptide GK19; B-C; Hemolysis experiment of antibacterial peptide GK19; SC, NC, PC represent the experimental group, negative control group, and positive control group respectively;

[0026] Figure 5 Results of the determination of the time-bacteriostatic curve and time-bactericidal curve of antibacterial peptide GK19; A, B are the time-bacteriostatic curves of antibacterial peptide GK19 against ATCC25922 and ATCC25923 respectively; C, D are the time-bactericidal curves of antibacterial peptide GK19 against ATCC25922 and ATCC25923 respectively;

[0027] Figure 6 Results of the anti-inflammatory and antioxidant determination of antibacterial peptide GK19; A: NO concentration release under treatment with peptides at different concentrations; B: DPPH scavenging rate corresponding to peptides at different concentrations;

[0028] Figure 7 Results of the stability experiment of antibacterial peptide GK19; A: The antibacterial effects of the antibacterial peptide against ATCC25922 and ATCC25923 are affected by temperature; B: The antibacterial effects of the antibacterial peptide against ATCC25922 and ATCC25923 are affected by the concentration of Na + concentration; C: The antibacterial effects of the antibacterial peptide against ATCC25922 and ATCC25923 are affected by the concentration of Ca 2+ concentration; D: The antibacterial effects of the antibacterial peptide against ATCC25922 and ATCC25923 are affected by simulated pancreatic juice; E: The antibacterial effects of the antibacterial peptide against ATCC25922 and ATCC25923 are affected by simulated gastric juice; F: The antibacterial effects of the antibacterial peptide against ATCC25922 and ATCC25923 are affected by pH;

[0029] Figure 8 Antibacterial mechanism of antibacterial peptide GK19; A: Gel retardation assay; B: Nucleic acid and protein leakage assays of ATCC25922; C: Nucleic acid and protein leakage assays of ATCC25923. Detailed implementation manners

[0030] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0031] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] Example 1

[0036] 1. Experimental methods

[0037] 1.1 Crude separation of antimicrobial peptides

[0038] Thirty healthy male forest frogs were purchased from the Changchun Farmer's Market. The skin on the back of the forest frog was gently wiped with physiological saline to remove impurities and dirt on the surface. The forest frog was carefully removed from the appropriate breeding environment and placed in a clean container to allow it to adapt to the experimental environment for a period of time to reduce stress. All animal experiments were approved and supervised by the Experimental Animal Center of Jilin University and were conducted in accordance with the "Guidelines for the Ethical Use of Animals" of the Animal Welfare and Research Ethics Committee of Jilin University.

[0039] Stimulate the skin of the back of the forest frog with a voltage point of 25 V to produce mucus on the skin of the back of the forest frog. Use running water to wash and collect the mucus on the skin of the back of the forest frog. Then centrifuge at 4 °C and 3000 rpm for 30 min to collect the supernatant. Add an equal amount of acetonitrile and place in a cool place. The antimicrobial peptides are concentrated by the volatilization of acetonitrile (see Figure 1 The concentrated antimicrobial peptides were stored at 4°C.

[0040] 1.2 Strain culture

[0041] The bacteria involved in the present invention include Escherichia coli ATCC 25922, Staphylococcus aureus ATCC25923, Staphylococcus aureus ATCC 25913, ATCC44102, ATCC43300, Pseudomonas aeruginosa (product number CMCC10116), EHEC O157:H7, ETEC K88, Salmonella pullorum, and Salmonella typhi. Except for ETEC K88, which was kindly donated by Mr. Jin Mingliang of the College of Animal Science, Zhejiang University, all other strains were kept for future use in this experiment.

[0042] Inoculate a single colony of purified bacteria into fresh Luria broth (LB) medium and grow overnight at 37 °C and transfer to another fresh LB medium for 4-6 h during the logarithmic growth phase. Wash the bacteria and resuspend them in PBS to prepare a concentration equivalent to approximately 10 5A bacterial suspension of CFU / mL was used. ATCC 25922 and ATCC 25923 were used as representative strains of Gram-positive and Gram-negative strains for subsequent experiments.

[0043] 1.3 Ultrafiltration of the antibacterial crude extract

[0044] The collected mucus was separated using ultrafiltration tubes with a molecular weight cut-off of 10 KD and 3 KD, so that the crude extract of antibacterial peptides was divided into three fractions: <3 KD, 3 - 10 KD, and >10 KD. 50 μL of antibacterial peptides and 50 μL of bacteria (1×10 5 CFU / mL) were mixed and incubated in a bacterial incubator at 37 °C for 18 h, and the absorbance was measured at OD 600nm The method for calculating the antibacterial rate is as follows:

[0045] Antibacterial rate = 1 - (OD 600nm experimental group - OD 600nm blank group) / OD 600nm control group × 100%;

[0046] The experimental group was the value measured at OD 600nm after mixing 50 μL of antibacterial peptides and 50 μL of bacteria. The blank group was the value at OD 600nm for 100 μL of ddH2O. The control group was the value measured at OD 600nm after mixing 50 μL of ddH2O and 50 μL of bacteria.

[0047] The test results are as Figure 2 shown. For both ATCC25922 and ATCC25923, the antibacterial effect of the <3 KD ultrafiltration fraction was the best. Therefore, the <3 KD ultrafiltration fraction was selected for further experiments.

[0048] 1.4 Identification of peptide sequences by LC-MS / MS

[0049] The ultrafiltration fraction with the best antibacterial effect was identified by mass spectrometry. The LC-MS / MS identification was completed by Sci-go (www.sci-go.com) on behalf. By constructing a fasta database containing the protein sequences of Rana, a total of 2303 peptide sequences were identified.

[0050] 1.5 Screening of antibacterial peptides

[0051] First, the probability of a peptide being an antimicrobial peptide was predicted using CAMPR3 (http: / / www.camp.bicnirrh.res.in / ). Briefly, the sequences obtained by mass spectrometry identification were batch-input into CAMPR3, and analyzed using its integrated algorithms and databases. The computational analysis followed the instructions provided by the CAMP3 tool. Meanwhile, peptides with a score higher than 0.9 were selected for further prediction. Specifically, ADP3 (https: / / aps.unmc.edu / AP / ) was used to identify the charge, hydrophobicity, and relative molecular mass of the peptides. Finally, the secondary structure of the peptides was predicted using an online website. The screening results are shown in Table 1.

[0052] Table 1 Screening results of antimicrobial peptides

[0053]

[0054] 1.6 Peptide synthesis

[0055] The 10 peptides in the above table were chemically synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.

[0056] 1.7 MIC determination

[0057] The MIC determination method was based on the National Committee for Clinical Laboratory Standards (NCCLS) with appropriate modifications. The antimicrobial peptide was dissolved in sterile water to a concentration of 1024 μg / mL and then serially diluted to 32 μg / mL. 50 μL of antimicrobial peptides at different concentrations was mixed with an equal volume of bacteria (1×10 5 CFU / mL) in a 96-well plate and then incubated at 37 °C for 18 h. After incubation, the absorbance was measured at OD 600nm and OD 600nm ≤ 0.1 was used as the MIC. Through the MIC experiment, it was found that GLFSVVKGVLKAVGKNVAK (named GK19, i.e., FAMP-3 in Table 1) had good antibacterial activity. The MIC test results of GK19 are shown in Table 2. The MIC values were between 64 - 256 μg / mL. The MIC values for Gram-positive bacteria were greater than those for Gram-negative bacteria, which may be related to the thicker cell wall of Gram-positive bacteria.

[0058] Table 2 MIC of antimicrobial peptide GK19 against different strains

[0059]

[0060] Note: In the table a Gram-negative bacteria, b Gram-positive bacteria.

[0061] 1.8 Structural characterization

[0062] The primary structure diagram of the polypeptide was obtained through the online website (https: / / tool.allpeptide.com / jiegoutu.html). The helical wheel diagram of the peptide was predicted through the online website (https: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParams.py). The three-dimensional structure of the antimicrobial peptide GK19 was predicted by I-TASSER. Meanwhile, the secondary structure of the antimicrobial peptide was detected using circular dichroism spectroscopy.

[0063] The structural characterization of the antimicrobial peptide is as Figure 3 shown. Figure 3 In [A], it is the primary structure diagram of the antimicrobial peptide GK19. As Figure 3 shown in [B], valine (Val, V), leucine (Leu, L), and phenylalanine (Phe, F), as hydrophobic amino acids, are all located on one side of the helical wheel diagram, indicating that this antimicrobial peptide has amphiphilicity. As Figure 3 shown in [C], the surface charge distribution in the three-dimensional structure indicates that GK19 is a cationic polypeptide, which conforms to the structural characteristics that an antimicrobial peptide should have. As Figure 3 shown in [D], GK19 maintains a 100% random coil structure in aqueous solution.

[0064] 1.9 Biosafety test

[0065] Hemolysis experiment: Rabbit red blood cells were collected in a heparin-containing test tube, then washed 3 times with 1×PBS, and finally centrifuged at 1,000×g for 5 min at 4 °C. The blood cells were resuspended in PBS to achieve a final concentration of 1% (v / v) red blood cells. 50 μL of the red blood cell suspension was incubated with 50 μL of peptide components at different concentrations at 37 °C for 1 h. The red blood cell suspension incubated with PBS and 10% Triton-X-100 was used as the blank and control, respectively. The samples were centrifuged at 1,000 × g for 5 min at 4 °C, and the supernatant was transferred to a 96-well microtiter plate. The absorbance at 570 nm was read using a microplate reader. The hemolysis percentage was calculated using the following formula:

[0066] Hemolysis rate (%) = (OD 570nm experimental - OD 570nm blank) / (OD 570nm control - OD 570nm blank).

[0067] Cytotoxicity experiment: RAW 264.7 cells were diluted to 1×10 5cells / mL. Take 100 μL of the cell suspension and culture it in a sterile 96-well cell culture plate. Incubate overnight at 37 °C and 5% CO2. Add antibacterial peptide GK19 at different concentrations and incubate for 24 h. Use the CCK-8 reagent to detect the cytotoxic effect of the drug on the cells.

[0068] As Figure 4 shown in A, the results indicate that there is no obvious cytotoxicity to RAW 264.7 even at 400 μg / mL. Figure 4 B-C show that even under the condition of 16×MIC of ATCC25922, the hemolytic activity of GK19 is still less than 1%.

[0069] 1.10 Determination of time-bacteriostatic curve growth curve and time-kill curve

[0070] Determination of time-bacteriostatic curve: ATCC25922 and ATCC25923 were selected for the determination of bacterial growth curves. Add 50 μL of antibacterial peptide GK19 at concentrations of 0.25×MIC, 0.5×MIC, and 1×MIC to 50 μL of the suspensions of ATCC25922 and ATCC5923 with a bacterial concentration of 1×10 5 CFU / mL respectively, and culture in a 96-well plate at 37 °C. Continuously measure the absorbance at 600 nm every 2 h for 16 h using a microplate reader.

[0071] Determination of time-kill curve: ATCC25922 and ATCC25923 were selected for the determination of bacterial growth curves. Add 50 μL of antibacterial peptide GK19 at concentrations of 1×MIC, 2×MIC, 4×MIC, and 8×MIC to 50 μL of the suspensions of ATCC25922 and ATCC5923 with a bacterial concentration of 1×10 5 CFU / mL, and culture in a 96-well plate at 37 °C. At 0, 1, 2, 4, 8, 12, and 16 h, dilute 10 μL of the aliquot sample with 90 μL of LB culture medium, and spread 5 μL of the aliquot on an LB agar plate. After incubation at 37 °C for 24 h, determine the colony count and plot the time-kill curve.

[0072] Figure 5 A-B are the time-bacteriostatic curves of the antibacterial peptide. The experimental results show that under the condition of 1×MIC, GK19 can significantly inhibit the growth of bacteria. Even under the conditions of 0.25×MIC and 0.5×MIC, it still has an inhibitory effect on bacteria. Even after 16 h, it still cannot reach the plateau of the blank group. Under the condition of 0.25×MIC, the reason why ATCC25923 is close to the plateau may be that the cell wall of Gram-positive bacteria is too thick, which inhibits the action of the antibacterial peptide.

[0073] Figure 5 In C-D, it is the time-kill curve of the antibacterial peptide. The experimental results show that 1×MIC can only inhibit the growth of bacteria and cannot effectively kill the bacteria. When at 4×MIC, the bacteria can be completely killed. When at 8×MIC, rapid bactericidal action can occur within 1 h.

[0074] 1.11 Determination of anti-inflammatory and antioxidant activities

[0075] Inflammatory reactions can lead to changes in the content of nitric oxide (NO) in the body. Measuring the content of NO can be used as an indicator to prove whether there is an anti-inflammatory effect. Therefore, the present invention verifies its antibacterial activity through the content of NO.

[0076] RAW 264.7 was induced with LPS, and then RAW 264.7 cells were treated with different concentrations of GK19 for 24 h. After that, the supernatant was collected, and NO was measured using a NO assay kit. Figure 6 In A, it shows that when the concentration of the antibacterial peptide GK19 reaches 400 μg / mL, the release amount of NO is significantly reduced. When the concentration reaches 1000 μg / mL, the NO release amount of RAW 264.7 reaches the level before being induced by LPS.

[0077] From Figure 6 In B, it can be seen that in addition to antibacterial and anti-inflammatory effects, this antibacterial peptide also has certain antioxidant activity, and the antioxidant activity increases with the increase of the peptide concentration. When the concentration of the antibacterial peptide GK19 is 8 mg / mL, the scavenging rate of DPPH free radicals of the antibacterial peptide exceeds 50%.

[0078] 1.12 Stability test

[0079] To evaluate the stability of GK19, the antibacterial peptide at 1×MIC was treated under different conditions, and the antibacterial activity of the antibacterial peptide treated under different conditions was evaluated with reference to the method in the above MIC determination.

[0080] Effect of temperature on the antibacterial activity of GK19: The antibacterial peptide was treated at 60 °C, 80 °C, and 100 °C for 30 min and 60 min respectively. Then, 50 μL of the antibacterial peptide treated under different conditions was mixed with 50 μL of the bacterial suspension (ATCC25922 and ATCC25923), and incubated in a 96-well plate at 37 °C for 18 h;

[0081] Effect of pH on the antibacterial activity of GK19: The antibacterial peptide was treated at pH = 3, 5, 7, 9, and 11 for 30 min, and then the pH was adjusted back to pH = 6 - 7. Then, 50 μL of the antibacterial peptide treated under different conditions was mixed with 50 μL of the bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h;

[0082] Effect of salt ions on the antibacterial activity of GK19: The antibacterial peptide was treated in 1%, 2%, 3%, 4%, and 5% NaCl and CaCl2 for 1 h. Then, 50 μL of the antibacterial peptide treated under different conditions was mixed with 50 μL of the bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h;

[0083] Antibacterial activity of GK19 in simulated gastric juice: The antibacterial peptide was dissolved in simulated gastric juice and treated for 10, 30, 60, 90, and 120 min, and then boiled for 10 min to inactivate the enzyme. Then, the pH was adjusted back to pH = 6 - 7. Then, 50 μL of the antibacterial peptide treated under different conditions was mixed with 50 μL of the bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h;

[0084] Antibacterial activity of GK19 in simulated pancreatic juice: The antibacterial peptide was dissolved in simulated pancreatic juice and treated for 10, 30, 60, 90, 120 min, and then boiled for 10 min to inactivate the enzyme. Then, 50 μL of the antibacterial peptide treated under different conditions was mixed with 50 μL of the bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h.

[0085] Formula for simulated gastric juice: Take 2.0 g of sodium chloride and 3.2 g of pepsin (the label should indicate 800 - 2500 activity units per milligram), add 7.0 mL of hydrochloric acid and water to dissolve to 1000 mL. The pH value of this solution should be 1.2.

[0086] Formula for simulated pancreatic juice: Take 6.8 g of potassium dihydrogen phosphate, add 250 mL of water to dissolve, add 77 mL of 0.2 mol / L sodium hydroxide solution and 500 mL of water, then add 10 g of trypsin to dissolve, adjust the pH value to 6.8 ± 0.1 with 0.2 mol / L sodium hydroxide solution or 0.2 mol / L hydrochloric acid solution, and then dilute with water to 1000 mL.

[0087] By Figure 7As can be seen from Figure A, when the temperature is 60 °C, whether the treatment time is 30 min or 60 min, it has no effect on the antibacterial effect against ATCC25922. It has a slight effect on the antibacterial effect against ATCC25923, but the bacteriostatic rate can still be maintained above 90%. When the temperature is 80 °C or 100 °C, the antibacterial effects against ATCC25922 and ATCC25923 decrease with the treatment time, but can still be maintained above 50%.

[0088] As can be seen from Figure 7 Figure B, the antibacterial effect of the antimicrobial peptide decreases with the increase of Na + concentration. When there is 1% Na + , the antibacterial effect decreases to 70%, and it still has a high antibacterial activity. The concentration of physiological saline is 0.9%. Therefore, under physiological conditions, the antimicrobial peptide still has a good antibacterial effect. As can be seen from Figure 7 Figure C, the results show that the antibacterial effect of the antimicrobial peptide decreases with the increase of Ca 2+ concentration, which is consistent with the trend of Na + .

[0089] As can be seen from Figure 7 Figure D, after the antimicrobial peptide is treated with simulated pancreatic juice, the antibacterial activity decreases to more than 50% after 10 min. Then, with the increase of the treatment time, the antibacterial activity is not further reduced, which may be because the antimicrobial peptide has been completely degraded within 10 min. The remaining antibacterial activity is due to the peptide segments after degradation.

[0090] As can be seen from Figure 7 Figure E, after the antimicrobial peptide is treated with simulated gastric juice for 10 min, the antibacterial activities against ATCC25922 and ATCC25923 only remain 40% and 50% respectively. With the further extension of the treatment time, the antibacterial activity is further reduced.

[0091] As can be seen from Figure 7 Figure F, when the pH range is 3 - 11, the antibacterial activity of the antimicrobial peptide is not affected. Therefore, the antimicrobial peptide GK19 is an antimicrobial peptide that is very stable to pH changes.

[0092] 1.13 Antimicrobial Peptide Action Mechanism Experiment

[0093] Gel retardation assay: DNA of two bacteria, ATCC25922 and ATCC25923, was extracted and the DNA of the two bacteria was mixed in equal proportions at the same concentration. Then the mixed DNA was mixed in equal proportions with different concentrations of antimicrobial peptides (32, 64, 128 μg / mL) in 20 μL of binding buffer (1 mM EDTA, 10 mM Tris-HCl (pH = 8.0), 1 mM dithiothreitol, 5% glycerol, 20 mM KCl, and 50 μg / mL BSA). Next, the mixture was incubated at 37 °C for 1 h. After that, the mobility of the mixture was examined by electrophoresis on a 1% agarose gel.

[0094] Nucleic acid and protein leakage assay: After culturing ATCC25922 and ATCC25923 to the logarithmic phase, they were diluted to OD 600nm = 0.1 and then serially diluted 10-fold. They were mixed in equal proportions with 0.5×MIC, 1×MIC, and 2×MIC and placed in a 37 °C incubator for 1 h. The supernatant was taken and measured at OD 260nm and OD 280nm .

[0095] As can be seen from Figure 8 A, when the peptide concentration was 128 μg / mL, the antimicrobial peptide significantly inhibited the migration of nucleic acid, indicating that the antimicrobial peptide might play an antibacterial role by binding to DNA. As can be seen from Figure 8 B-C, for both ATCC25922 and ATCC25923, after incubation with the antimicrobial peptide, it led to an increase in the content of nucleic acid and protein in the supernatant, indicating that the antimicrobial peptide could rupture the bacterial cell membrane and thus play a bactericidal role. In summary, GK19 could play a bactericidal role through multiple ways including disrupting the cell membrane and binding to DNA.

[0096] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A Rana chensinensis antimicrobial peptide, characterized in that, The amino acid sequence of the Rana chensinensis antimicrobial peptide is: GLFSVVKGVLKAVGKNVAK.

2. Use of the Rana chensinensis antimicrobial peptide according to claim 1 in the preparation of an antibacterial drug, characterized in that, The antimicrobial agent is an agent that inhibits Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella.

3. The use of the Rana chensinensis antimicrobial peptide according to claim 1 in the preparation of an anti-inflammatory drug.

4. The use of the Rana chensinensis antimicrobial peptide according to claim 1 in the preparation of an antioxidant product.

5. The application according to claim 4, characterized in that, The antioxidant product includes a product for scavenging DPPH free radicals.

6. An antibacterial drug, characterized in that, Contains the Rana chensinensis antimicrobial peptide according to claim 1.

7. An anti-inflammatory drug, characterized in that, Contains the Rana chensinensis antimicrobial peptide according to claim 1.

8. An antioxidant product, characterized in that, Contains the Rana chensinensis antimicrobial peptide according to claim 1, and the antioxidant product includes a product for scavenging DPPH free radicals.

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