Wood frog antibacterial peptide and application thereof in antibiosis, anti-inflammation and antioxidation

Abstract

The invention discloses a wood frog antibacterial peptide and application thereof in antibiosis, anti-inflammation and antioxidation, and belongs to the technical field of biology. The method comprises the following steps: firstly, analyzing skin mucus of wood frogs in Northeast by utilizing a peptidomics technology to obtain a large amount of peptide fragment information; and then, comparing the peptide fragment information with a known antibacterial peptide database, and screening out peptide fragments which possibly have antibacterial activity. Furthermore, through experiments, an antibacterial peptide which is extremely low in hemolytic activity and highly stable to pH and temperature and has anti-inflammatory and anti-oxidation effects is screened out. The amino acid sequence of the antibacterial peptide is as shown in SEQ ID NO: 3, and the antibacterial peptide lays a foundation for enriching the types of antibacterial peptides and adds new advantages for the application of the antibacterial peptide.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a wood frog antibacterial peptide and its application in antibacterial, anti-inflammatory and anti-oxidation. Background Art

[0002] In today's era, the widespread use of antibiotics has brought about serious consequences, that is, the increasing drug resistance of bacteria. Antibiotics, which were once powerful, now often seem helpless in the face of some drug-resistant bacteria. This grim reality makes the search for new antibiotics a top priority. Antimicrobial peptides, as a new type of antimicrobial substance, have received widespread attention in recent years. Compared with traditional antibiotics, antimicrobial peptides have many advantages. First, the mechanism of action of antimicrobial peptides is unique. It can quickly destroy the cell membrane of bacteria, causing bacterial death, and it is difficult for bacteria to develop drug resistance. Secondly, antimicrobial peptides have broad-spectrum antimicrobial activity and can inhibit a variety of bacteria, fungi and even viruses. In addition, antimicrobial peptides are metabolized quickly in organisms, will not remain in the environment, and have little impact on the ecological environment.

[0003] As a species endemic to the Northeast, the wood frog secretes rich antimicrobial peptides in its skin mucus. These antimicrobial peptides have high biological activity and stability and are a good source for extracting antimicrobial peptides. However, traditional methods for extracting antimicrobial peptides have great drawbacks. The traditional method mainly obtains antimicrobial peptide sequences through continuous separation and purification, a process that is not only time-consuming and labor-intensive, but also inefficient. In addition, the traditional method also requires a large number of samples and complex instruments and equipment, which is costly. Therefore, there is an urgent need for simple, low-cost rapid screening of new antimicrobial peptides to enrich the types of antimicrobial peptides and provide new antimicrobial peptides for clinical treatment. Summary of the invention

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

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

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

[0007] The present invention also provides application of the forest frog antibacterial peptide in preparing antibacterial products.

[0008] Optionally, the antibacterial product includes but is not limited to products that inhibit Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella.

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

[0010] The present invention also provides application of the forest frog antibacterial peptide in preparing anti-inflammatory products.

[0011] Optionally, the anti-inflammatory product includes but is not limited to an anti-inflammatory drug. Optionally, the anti-inflammatory drug is an anti-inflammatory drug for human or veterinary use.

[0012] The present invention also provides application of the forest frog antibacterial peptide in preparing antioxidant products.

[0013] Optionally, the antioxidant products include but are not limited to products for removing DPPH free radicals. They also include foods and feed additives that are helpful for antioxidants, and the foods include but are not limited to health foods and functional foods.

[0014] The invention also provides an antibacterial product containing the forest frog antibacterial peptide.

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

[0016] The present invention also provides an anti-inflammatory product, which contains the forest frog antimicrobial peptide. The anti-inflammatory product includes but is not limited to anti-inflammatory drugs. Optionally, the anti-inflammatory drugs are anti-inflammatory drugs for human or animal use.

[0017] The present invention also provides an antioxidant product containing the Rana sylvatica antimicrobial peptide, the antioxidant product including but not limited to drugs for removing DPPH free radicals and food and feed additives that are helpful for antioxidant effects, and the drugs including but not limited to drugs for human or animal use.

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

[0019] The present invention first uses peptide omics technology to analyze the skin mucus of the wood frog and obtains a large amount of peptide information. Then, the peptide information is compared with the known antimicrobial peptide database to screen out peptides that may have antimicrobial activity. The present invention combines peptide omics technology with the antimicrobial peptide database, which can greatly improve the speed and accuracy of antimicrobial peptide screening and provide a new approach for the research and development of antimicrobial peptides. This method can not only quickly and accurately screen out peptides with antimicrobial activity, but also has low cost and great application prospects.

[0020] The present invention screens out an antimicrobial 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 antimicrobial peptide means that it has very little destructive effect on human red blood cells during use, which greatly improves its safety. At the same time, its high stability to pH and temperature enables it to maintain good antimicrobial activity under different environmental conditions, expanding its scope of application. In addition, its anti-inflammatory and antioxidant effects add new advantages to its application. The antimicrobial peptide can also inhibit a variety of Gram-positive and Gram-negative bacteria, enriching the types of antimicrobial peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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 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.

[0022] Figure 1 This is the flow chart of crude separation of antimicrobial peptides;

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

[0024] Figure 3 Structural characterization of the antimicrobial peptide GK19; A: primary structure of the antimicrobial peptide; B: helical wheel diagram of the antimicrobial peptide; C: tertiary structure of the antimicrobial peptide; D: circular dichroism spectrum characterization of the antimicrobial peptide;

[0025] Figure 4 The biosafety assessment results of the antimicrobial peptide GK19; A: Cytotoxicity test of the antimicrobial peptide GK19; BC: Hemolysis test of the antimicrobial peptide GK19; SC, NC, and PC represent the experimental group, negative control group, and positive control group, respectively;

[0026] Figure 5 The time-inhibition curve and time-killing curve of the antimicrobial peptide GK19 are measured; A and B are the time-inhibition curves of the antimicrobial peptide GK19 against ATCC25922 and ATCC25923, respectively; C and D are the time-killing curves of the antimicrobial peptide GK19 against ATCC25922 and ATCC25923, respectively;

[0027] Figure 6 The results of anti-inflammatory and antioxidant tests of the antimicrobial peptide GK19; A: NO release under different concentrations of peptide treatment; B: DPPH clearance rate corresponding to different concentrations of peptide;

[0028] Figure 7 The results of the stability experiment of the antimicrobial peptide GK19; A: The antibacterial effect of the antimicrobial peptide on ATCC25922 and ATCC25923 is affected by temperature; B: The antibacterial effect of the antimicrobial peptide on ATCC25922 and ATCC25923 is affected by Na + C: The antibacterial effect of antimicrobial peptides on ATCC25922 and ATCC25923 was affected by Ca 2+ The effect of concentration; D: The antibacterial effect of antimicrobial peptides on ATCC25922 and ATCC25923 is affected by simulated pancreatic juice; E: The antibacterial effect of antimicrobial peptides on ATCC25922 and ATCC25923 is affected by simulated gastric juice; F: The antibacterial effect of antimicrobial peptides on ATCC25922 and ATCC25923 is affected by pH;

[0029] Figure 8 The antibacterial mechanism of antimicrobial peptide GK19; A: gel retardation experiment; B: nucleic acid and protein leakage experiment of ATCC25922; C: nucleic acid and protein leakage experiment of ATCC25923. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a 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 back skin of the forest frog with a voltage of 25V to produce mucus on the back skin of the forest frog. Use running water to wash and collect the mucus on the back skin of the forest frog. Then centrifuge at 4℃ and 3000rpm for 30min 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] A single colony of purified bacteria was inoculated into fresh Luria broth (LB) medium and cultured overnight at 37°C and transferred to another fresh LB medium for 4-6 hours during the logarithmic growth phase. The bacteria were washed and resuspended in PBS to prepare a concentration equivalent to approximately 10 5CFU / mL of bacterial suspension was used. ATCC 25922 and ATCC 25923 were used as representative strains of Gram-positive and Gram-negative strains in subsequent experiments.

[0043] 1.3 Ultrafiltration of Antibacterial Crude Extract

[0044] The collected mucus was separated using ultrafiltration tubes with a molecular weight cutoff of 10KD and 3KD, so that the crude antimicrobial peptide extract was divided into three parts: <3KD, 3-10KD, and >10KD. 50μL of antimicrobial peptide and 50μL of bacteria (1×10 5 CFU / mL) and placed in a bacterial incubator and cultured at 37°C for 18 h. 600nm Determine the absorbance. The calculation method of the inhibition rate is as follows:

[0045] Inhibition rate = 1-(OD 600nm Experimental group-OD 600nm Blank group) / OD 600nm Control group × 100%;

[0046] The experimental group consisted of 50 μL of antimicrobial peptide and 50 μL of bacteria mixed at OD 600nm The blank group was 100 μL ddH2O at OD 600nm The control group was mixed with 50 μL of ddH2O and 50 μL of bacteria at OD 600nm Output the measured value.

[0047] Test results such as Figure 2 As shown in the figure, the ultrafiltration fraction with a diameter of <3 KD had the best antibacterial effect on both ATCC25922 and ATCC25923. Therefore, the ultrafiltration fraction with a diameter of <3 KD was selected for further experiments.

[0048] 1.4 LC-MS / MS identification of peptide sequences

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

[0050] 1.5 Antimicrobial peptide screening

[0051] First, CAMPR3 (http: / / www.camp.bicnirrh.res.in / ) was used to predict the probability that the peptide was an antimicrobial peptide. In short, the sequences obtained by mass spectrometry identification were batch-entered into CAMPR3 and analyzed using its integrated algorithm and database. The computational analysis followed the instructions provided by the CAMP3 tool. At the same time, 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 peptide. Finally, the secondary structure of the peptide was predicted using an online website. The screening results are shown in Table 1.

[0052] Table 1 Antimicrobial peptide screening results

[0053]

[0054]

[0055] 1.6 Peptide synthesis

[0056] The 10 peptides in the above table were chemically synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd.

[0057] 1.7 MIC determination

[0058] The MIC determination method was based on the National Committee for Clinical Laboratory Standards (NCCLS) with some modifications. The antimicrobial peptides were dissolved in sterile water to a concentration of 1024 μg / mL and then diluted to 32 μg / mL. 50 μL of different concentrations of antimicrobial peptides were mixed with an equal number of bacteria (1×10 5 CFU / mL) were mixed in a 96-well plate and then cultured at 37°C for 18 h. 600nm Measure the absorbance, OD 600nm ≤0.1 was taken as MIC. Through MIC experiments, 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 value of Gram-positive bacteria was greater than that of Gram-negative bacteria, which may be related to the thicker cell wall of Gram-positive bacteria.

[0059] Table 2 MIC of antimicrobial peptide GK19 against different strains

[0060]

[0061]

[0062] Note: In the table a Gram-negative bacteria,b Gram-positive bacteria.

[0063] 1.8 Structural characterization

[0064] The primary structure of the peptide 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. At the same time, the secondary structure of the antimicrobial peptide was detected using circular dichroism spectroscopy.

[0065] Structural characterization of antimicrobial peptides Figure 3 As shown, Figure 3 A in the figure is the primary structure diagram of the antimicrobial peptide GK19. Figure 3 As shown in Figure 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 the antimicrobial peptide has amphipathic properties. Figure 3 As shown in Figure C, the surface charge distribution in the three-dimensional structure shows that GK19 is a cationic peptide. This is consistent with the structural characteristics of antimicrobial peptides. Figure 3 As shown in D, GK19 maintains 100% random coil structure in aqueous solution.

[0066] 1.9 Biosafety Testing

[0067] Hemolysis experiment: The rabbit's red blood cells were collected in a test tube containing heparin, then washed three times with 1×PBS, and finally centrifuged at 1,000×g for 5 minutes at 4°C. The blood cells were resuspended in PBS to reach a final concentration of 1% (v / v) red blood cells. 50 μL of red blood cell suspension was incubated with 50 μL of peptide components of different concentrations at 37°C for 1 hour. Red blood suspensions incubated with PBS and 10% Triton-X-100 were used as blanks and controls, respectively. The samples were centrifuged at 1,000×g for 5 minutes at 4°C, and the supernatant was transferred to a 96-well microtiter plate. The absorbance at 570nm was read using a microplate reader. The percentage of hemolysis was calculated using the following formula:

[0068] Hemolysis rate (%) = (OD 570nm Experimental-OD 570nm blank) / (OD 570nm Control-OD 570nm blank).

[0069] Cytotoxicity assay: RAW264.7 cells were diluted to 1×10 in DMEM medium containing 10% fetal bovine serum.5 cell / mL, 100 μL of cell suspension was cultured in a sterile 96-well cell culture plate and cultured overnight at 37°C and 5% CO2. Different concentrations of antimicrobial peptide GK19 were added and incubated for 24 h, and the toxic effect of the drug on the cells was detected using CCK-8 reagent.

[0070] like Figure 4 As shown in A, the results showed that even at 400 μg / mL, there was still no obvious cytotoxicity to RAW264.7. Figure 4 The BC showed that even under the condition of 16×MIC of ATCC25922, the hemolytic activity of GK19 was still less than 1%.

[0071] 1.10 Determination of time-inhibition curve, growth curve and time-killing curve

[0072] Time-inhibition curve assay: ATCC25922 and ATCC25923 were selected for bacterial growth curve assay. 50 μL of bacteria with a concentration of 1×10 5 50 μL of antimicrobial peptide GK19 at concentrations of 0.25×MIC, 0.5×MIC, and 1×MIC were added to the suspensions of ATCC25922 and ATCC5923 at CFU / mL, respectively, and cultured in a 96-well plate at 37°C. The absorbance at 600 nm was measured at intervals of 2 h for 16 h using a microplate reader.

[0073] Time-kill curve assay: ATCC25922 and ATCC25923 were selected for bacterial growth curve assay. 50 μL of bacteria with a concentration of 1 × 10 5 50 μL of antimicrobial peptide GK19 at concentrations of 1×MIC, 2×MIC, 4×MIC, and 8×MIC was added to the suspension of ATCC25922 and ATCC5923 at CFU / mL and cultured in a 96-well plate at 37°C. At 0, 1, 2, 4, 8, 12, and 16 h, 10 μL aliquots of the sample were diluted with 90 μL LB culture medium, and 5 μL aliquots were spread on LB agar plates. After incubation at 37°C for 24 h, the colony counts were determined and the time-kill curve was plotted.

[0074] Figure 5 AB in the figure is the time-inhibition curve of the antimicrobial 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 has an inhibitory effect on bacteria, and even after 16 hours, it still cannot reach the plateau phase of the blank group. Under the condition of 0.25×MIC, ATCC25923 is close to the plateau phase, which may be due to the fact that the cell wall of Gram-positive bacteria is too thick, inhibiting the antimicrobial peptide from exerting its effect.

[0075] Figure 5 CD in the middle is the time-killing curve of the antimicrobial peptide. The experimental results show that 1×MIC can only inhibit the growth of bacteria, but cannot effectively kill the bacteria. Under the condition of 4×MIC, the bacteria can be completely killed. Under the condition of 8×MIC, the bacteria can be quickly killed within 1 hour.

[0076] 1.11 Determination of anti-inflammatory and antioxidant activities

[0077] Inflammatory reactions can lead to changes in the content of nitric oxide (NO) in the body, and measuring the NO content can be used as an indicator to prove whether there is an anti-inflammatory effect. Therefore, the present invention verifies whether it has antibacterial activity through the content of NO.

[0078] RAW 264.7 cells were induced with LPS and then treated with different concentrations of GK19 for 24 h. Afterwards, the supernatant was collected and NO was measured using a NO assay kit. Figure 6 Figure A shows that when the concentration of antimicrobial peptide GK19 reached 400 μg / mL, the release of NO was significantly reduced. When the concentration reached 1000 μg / mL, the NO release of RAW 264.7 reached the level before LPS induction.

[0079] from Figure 6 It can be seen from Figure B that in addition to the antibacterial and anti-inflammatory effects, the antimicrobial peptide also has a certain antioxidant activity, and the antioxidant activity increases with the increase of peptide concentration. When the concentration of the antimicrobial peptide GK19 is 8 mg / mL, the scavenging rate of the DPPH free radical of the antimicrobial peptide exceeds 50%.

[0080] 1.12 Stability Test

[0081] To evaluate the stability of GK19, the antimicrobial peptides at 1×MIC were treated under different conditions, and the antimicrobial activities of the antimicrobial peptides treated under different conditions were evaluated with reference to the method in the above MIC determination.

[0082] Effect of temperature on the antibacterial activity of GK19: The antimicrobial peptides were treated at 60°C, 80°C, and 100°C for 30 min and 60 min, respectively. Then, 50 μL of the antimicrobial peptides treated under different conditions were mixed with 50 μL of bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37°C for 18 h.

[0083] Effect of pH on the antibacterial activity of GK19: The antimicrobial peptides were 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 antimicrobial peptides treated under different conditions were mixed with 50 μL of bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h.

[0084] Effect of salt ions on the antibacterial activity of GK19: The antimicrobial peptides were treated in 1%, 2%, 3%, 4% and 5% NaCl and CaCl2 for 1 h, and then 50 μL of antimicrobial peptides treated under different conditions were mixed with 50 μL of bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h;

[0085] Antibacterial activity of GK19 in simulated gastric fluid: The antimicrobial peptides were dissolved in simulated gastric fluid and treated for 10, 30, 60, 90 and 120 min, respectively, and then boiled for 10 min to inactivate the enzyme, and then the pH was adjusted back to pH = 6-7, and then 50 μL of the antimicrobial peptides treated under different conditions were mixed with 50 μL of bacterial suspension (ATCC25922 and ATCC25923), and incubated in a 96-well plate at 37 °C for 18 h;

[0086] Antibacterial activity of GK19 in simulated pancreatic juice: The antimicrobial peptides were dissolved in simulated pancreatic juice and treated for 10, 30, 60, 90, and 120 min, then boiled for 10 min to inactivate the enzyme. Then, 50 μL of the antimicrobial peptides treated under different conditions were mixed with 50 μL of bacterial suspension (ATCC25922 and ATCC25923) and incubated in a 96-well plate at 37 °C for 18 h.

[0087] Formula for simulated gastric fluid: Take 2.0g sodium chloride and 3.2g pepsin (labeled as 800-2500 activity units per mg), add 7.0mL hydrochloric acid and water to dissolve to 1000mL. The pH value of the solution should be 1.2.

[0088] Formula for simulated pancreatic juice: Take 6.8g of potassium dihydrogen phosphate, add 250mL of water to dissolve it, add 77mL of 0.2mol / L sodium hydroxide solution and 500mL of water, then add 10g of trypsin to dissolve it, adjust the pH value to 6.8±0.1 with 0.2mol / L sodium hydroxide solution or 0.2mol / L hydrochloric acid solution, and then add water to dilute to 1000mL.

[0089] Depend on Figure 7As shown in Figure A, when the temperature is 60°C, whether it is treated for 30 minutes or 60 minutes, there is no effect on the antibacterial effect of ATCC25922. There is a slight effect on the antibacterial effect of ATCC25923, but the inhibition rate can still be maintained at more than 90%. When the temperature is 80°C or 100°C, the antibacterial effect on ATCC25922 and ATCC25923 decreases with the treatment time, but can still be maintained at more than 50%.

[0090] Depend on Figure 7 As shown in Figure B, the antibacterial effect of antimicrobial peptides increases with Na + The concentration of Na + When the concentration of saline is 0.9%, the antibacterial effect is reduced to 70%, which still has a high antibacterial activity. Therefore, under physiological conditions, antimicrobial peptides still have a good antibacterial effect. Figure 7 As shown in C, the results show that the antibacterial effect of antimicrobial peptides increases with Ca 2+ The concentration of Na + The trend is consistent.

[0091] Depend on Figure 7 As shown in Figure D, after the antimicrobial peptide was treated with simulated pancreatic juice, the antimicrobial activity decreased to more than 50% after 10 minutes, and then the antimicrobial activity did not decrease further with the increase of treatment time, which may be due to the fact that the antimicrobial peptide was completely degraded within 10 minutes. The remaining antimicrobial activity was caused by the degraded peptide fragments.

[0092] Depend on Figure 7 As shown in Figure E, after the antimicrobial peptides were treated with simulated gastric fluid for 10 minutes, their antimicrobial activities against ATCC25922 and ATCC25923 were only 40% and 50%, respectively. As the treatment time was further extended, the antimicrobial activity further decreased.

[0093] Depend on Figure 7 As can be seen from Figure F, when the pH range is 3-11, the antimicrobial activity of the antimicrobial peptide is not affected. Therefore, the antimicrobial peptide GK19 is an antimicrobial peptide that is very stable to pH changes.

[0094] 1.13 Experiments on the mechanism of action of antimicrobial peptides

[0095] Gel retardation experiment: DNA of two bacteria, ATCC25922 and ATCC25923, was extracted and mixed in equal proportions at the same concentration. The mixed DNA was then mixed in equal proportions with different concentrations of antimicrobial peptides (32, 64, 128 μg / mL) in 20 μL 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. Afterwards, the mobility of the mixture was checked by electrophoresis on a 1% agarose gel.

[0096] Nucleic acid and protein leakage experiment: ATCC25922 and ATCC25923 were cultured in the logarithmic phase and diluted at OD 600nm =0.1, and then diluted 10 times, mixed with 0.5×MIC, 1×MIC and 2×MIC in equal proportions. Place in a 37°C incubator for 1 hour. Take the supernatant and 260nm and OD 280nm Test

[0097] Depend on Figure 8 As shown in Figure A, when the peptide concentration was 128 μg / mL, the antimicrobial peptide significantly inhibited the migration of nucleic acids, indicating that the antimicrobial peptide may play an antibacterial role by binding to DNA. Figure 8 From the BC, it can be seen that for both ATCC25922 and ATCC25923, incubation with antimicrobial peptides led to an increase in the content of nucleic acids and proteins in the supernatant, indicating that antimicrobial peptides can rupture the bacterial cell membrane and thus play a bactericidal role. In summary, GK19 can play a bactericidal role in multiple ways, including destroying the cell membrane and binding to DNA.

[0098] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A forest frog antimicrobial peptide, characterized in that: The amino acid sequence of the forest frog antibacterial peptide is: GLFSVVKGVLKAVGKNVAK.

2. Use of the forest frog antibacterial peptide as claimed in claim 1 in the preparation of antibacterial products.

3. The use according to claim 2, characterized in that The antibacterial products include products that inhibit Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Salmonella.

4. Use of the Rana sylvatica antimicrobial peptide as claimed in claim 1 in the preparation of anti-inflammatory products.

5. Use of the Rana sylvatica antimicrobial peptide as claimed in claim 1 in the preparation of antioxidant products.

6. The use according to claim 5, characterized in that The antioxidant products include products for scavenging DPPH free radicals.

7. An antibacterial product, characterized in that: Contains the Rana sylvatica antimicrobial peptide according to claim 1.

8. The antibacterial product according to claim 7, characterized in that The bacteria include Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella.

9. An anti-inflammatory product, characterized in that: Contains the Rana sylvatica antimicrobial peptide according to claim 1.

10. An antioxidant product, characterized in that: The invention contains the wood frog antimicrobial peptide according to claim 1, wherein the antioxidant product comprises a product for removing DPPH free radicals.

Images