Protein structure characteristics of antibacterial peptide and research method thereof

Abstract

The invention relates to the technical field of biology, and discloses a protein structure characteristic of an antibacterial peptide and a research method thereof.The protein structure characteristic of the antibacterial peptide is obtained by collecting a biological sample, pretreating the biological sample, conducting separation according to the charge property of the antibacterial peptide through the ion exchange chromatography technology, conducting gel filtration chromatography on eluent obtained through ion exchange chromatography, and conducting high-purity protein structure characteristics of the antibacterial peptide. Carrying out dialysis or ultrafiltration on eluent obtained by gel filtration chromatography, and removing salt and small molecular impurities, so as to obtain purified antibacterial peptide; the amino acid sequence of the antibacterial peptide is determined by mass spectrometry, the grade structure of the antibacterial peptide is determined by circular dichroism, and the three-dimensional structure of the antibacterial peptide is analyzed by nuclear magnetic resonance; the antibacterial activity of the antibacterial peptide is determined by adopting an agar diffusion method, and the antibacterial performance of the antibacterial peptide is evaluated by determining the inhibition effect of the antibacterial peptide on bacteria under different concentrations; analyzing the influence of the antibacterial peptide on the physiological process in bacterial cells, and determining the antibacterial action mechanism of the antibacterial peptide; the antibacterial peptide disclosed by the invention has broad-spectrum antibacterial activity and has an inhibiting effect on various bacteria.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a protein structural characteristic of an antimicrobial peptide and a research method thereof. Background Art

[0002] With the widespread use of antibiotics, the problem of bacterial resistance is becoming increasingly serious, and finding new antibacterial drugs has become a top priority. Antimicrobial peptides, as a natural antibacterial substance, have the advantages of broad-spectrum antibacterial activity and low resistance, and have received widespread attention. Summary of the invention

[0003] The purpose of the present invention is to solve the above problems and to design a protein structural characteristic of an antimicrobial peptide and a research method thereof.

[0004] The first aspect of the present invention provides a protein structural property of an antimicrobial peptide and a research method thereof. The protein structural property research method of the antimicrobial peptide comprises the following steps:

[0005] S1. Collect biological samples, pre-treat the biological samples, separate the antimicrobial peptides according to their charge properties using ion exchange chromatography, perform gel filtration chromatography on the eluate obtained by the ion exchange chromatography, perform dialysis or ultrafiltration on the eluate obtained by the gel filtration chromatography to remove salt and small molecule impurities, and obtain purified antimicrobial peptides;

[0006] S2. Determine the amino acid sequence of the purified antimicrobial peptide by mass spectrometry, determine the secondary structure of the purified antimicrobial peptide by circular dichroism, and analyze the three-dimensional structure of the purified antimicrobial peptide by nuclear magnetic resonance;

[0007] S3. The antibacterial activity of antimicrobial peptides was determined by agar diffusion method. The antibacterial properties of antimicrobial peptides were evaluated by measuring the inhibitory effects of antimicrobial peptides on various bacteria at different concentrations.

[0008] S4. Use fluorescent probe technology to study the interaction mechanism between antimicrobial peptides and bacterial cell membranes, analyze the effects of antimicrobial peptides on physiological processes within bacterial cells, and determine the antibacterial mechanism of antimicrobial peptides.

[0009] Optionally, in a first implementation of the first aspect of the present invention, step S1 specifically includes the following steps: collecting a biological sample, pretreating the biological sample, selecting a cation exchange resin according to the charge properties of the antimicrobial peptide, separating the antimicrobial peptide by ion exchange chromatography, separating the antimicrobial peptide by ion exchange chromatography according to the charge properties of the antimicrobial peptide, using a 20 mM, pH 7.0 phosphate buffer as the buffer system, eluting by gradually increasing the salt concentration from 0 M NaCl and gradually increasing it to 0-1 M NaCl, controlling the elution rate at 1-5 ml / min, and collecting the eluate containing the antimicrobial peptide;

[0010] During gel filtration chromatography, Sephadex G-75 dextran gel was selected as the filtration medium, the buffer system used a 20 mM, pH 7.0 phosphate buffer, the elution rate was 0.5-2 ml / min, and the eluate was collected;

[0011] Select a dialysis bag with a molecular weight cutoff of 1000-5000Da, put the gel filtration chromatography eluate containing the antimicrobial peptide into the dialysis bag, put it into a 20mM phosphate buffer with a pH of 7.0, and perform dialysis, replace the dialysis buffer every 6 hours, and the dialysis time is 12-48 hours;

[0012] An ultrafiltration membrane with a molecular weight cutoff of 1000-5000Da is selected, and the gel filtration chromatography eluate containing the antimicrobial peptide is passed through an ultrafiltration device to remove salt and small molecular impurities, and finally a purified antimicrobial peptide is obtained.

[0013] Optionally, in a second implementation of the first aspect of the present invention, the biological sample is from animal tissue, microbial fermentation broth or plant extract; the pretreatment process includes: centrifuging the biological sample at 3000-10000 rpm for 10-20 minutes, and filtering the centrifuged biological sample using a 0.22-0.45 μm microporous filter membrane.

[0014] Optionally, in a third implementation of the first aspect of the present invention, the mass spectrometry determination in step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in an acetonitrile / water mixed solution containing 0.1% formic acid, with a sample concentration of 0.5 mg / ml, selecting electrospray ionization mass spectrometry, setting the spray voltage to 3.5 kV, the capillary temperature to 250°C, the mass analyzer resolution to 10000, the mass range to 500-2000 Da, the detector gain to 100, the threshold to 10 counts, performing three mass spectrometry analyses, and obtaining the amino acid sequence of the antimicrobial peptide by analyzing the mass spectrum fragments and using de novo sequencing combined with comparison with a protein database, wherein the amino acid sequence of the antimicrobial peptide includes at least lysine and arginine.

[0015] Optionally, in a fourth implementation of the first aspect of the present invention, the secondary structure determination of step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in 10 mM, pH 7.4 phosphate buffer with a sample concentration of 0.1-1 mg / ml, using a circular dichroism spectrometer equipped with a polarized light source and a detector, setting the wavelength range to 190-260 nm, the scanning speed to 50 nm / min, the bandwidth to 1 nm, and the number of data acquisition points to 1024, performing five circular dichroism analyses, and using circular dichroism data analysis software to determine the secondary structure of the antimicrobial peptide according to the characteristics of the circular dichroism curve, wherein the antimicrobial peptide exists in an α-helical structure.

[0016] Optionally, in a fifth implementation of the first aspect of the present invention, the three-dimensional structure determination in step S2 specifically comprises the following steps: dissolving the purified antimicrobial peptide sample in heavy water, adjusting the sample concentration to 0.8 mg / ml, and using 15 The amino acids were labeled with N-labeled amino acids, and an 800 MHz high-field strength NMR instrument was selected with an acquisition time of 0.5 s. Fourier transform combined with phase correction was used for four NMR analyses. The three-dimensional structure of the antimicrobial peptide was determined by attribution and analysis of the NMR signals using ab initio calculation combined with distance geometry.

[0017] Optionally, in a sixth implementation of the first aspect of the present invention, step S3 specifically includes the following steps: culturing Staphylococcus aureus in a 37° C. constant temperature incubator using a tryptone soy broth medium, culturing Escherichia coli in a 37° C. constant temperature incubator using an LB medium, and the culturing time is 18-24 hours;

[0018] Prepare nutrient agar medium, dissolve peptone, beef extract, sodium chloride, and agar in distilled water according to a certain ratio, adjust the pH to 7.2-7.4, sterilize by high pressure at 120-130℃ for 20-25 minutes, and pour the medium into a plate when it cools to 45-55℃;

[0019] The purified antimicrobial peptides were dissolved in phosphate buffer to prepare antimicrobial peptide solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, and 10 μg / ml;

[0020] Staphylococcus aureus and Escherichia coli were inoculated on nutrient agar medium respectively, and cultured in a constant temperature incubator at 37°C for 18 hours to allow the bacteria to form uniform colonies on the medium, and a hole puncher with a diameter of 6-8 mm was used to punch small holes on the medium that was already full of bacteria, with a distance of 20-25 mm between the holes; antimicrobial peptide solutions of different concentrations were added to different small holes using a micropipette, and 40-50 μl was added to each well, and a penicillin solution with a concentration of 10 μg / ml was set as a positive control, and a phosphate buffer solution with a pH of 7.4 was set as a negative control, and the culture medium after the addition was placed in a constant temperature incubator at 37°C for further culture for 24 hours, and whether an inhibition zone appeared around the small hole was observed, and the diameter of the inhibition zone was measured using a vernier caliper;

[0021] The diameter of the inhibition zone corresponding to each concentration of the antimicrobial peptide solution was recorded, the experiment was repeated three times and the average value was taken. The relationship curve between the diameter of the inhibition zone and the concentration of the antimicrobial peptide was plotted. It was found from the curve that with the increase of the concentration of the antimicrobial peptide, the diameter of the inhibition zone gradually increased. The inhibition rate of the antimicrobial peptide on various bacteria at different concentrations was calculated, and it was found that the antimicrobial peptide had an antibacterial spectrum.

[0022] Optionally, in a seventh implementation of the first aspect of the present invention, step S4 specifically includes the following steps: selecting a lipophilic fluorescent dye DiI to mark the bacterial cell membrane, culturing Escherichia coli in LB medium to a logarithmic growth phase at 37°C for 15-30 hours; adding DiI to the Escherichia coli culture solution to a final concentration of 5 μM, incubating at 37°C for 15-30 minutes to allow the fluorescent probe to fully mark the bacterial cell membrane, mixing the Escherichia coli marked with the fluorescent probe with antimicrobial peptide solutions of different concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, and incubating at 37°C for 15-60 minutes; observing changes in fluorescence signals using a fluorescence microscope; comparing changes in fluorescence intensity under treatment with different concentrations of antimicrobial peptides, calculating the rate of change of fluorescence intensity, and judging whether the antimicrobial peptides cause changes in the cell membrane structure by observing changes in fluorescence distribution. If the fluorescence distribution becomes uneven or aggregation occurs, it indicates that the cell membrane structure is damaged.

[0023] In the technical scheme provided by the present invention, biological samples are collected, pretreated, separated according to the charge properties of antimicrobial peptides by ion exchange chromatography, the eluate obtained by ion exchange chromatography is subjected to gel filtration chromatography, and the eluate obtained by gel filtration chromatography is dialyzed or ultrafiltered to remove salt and small molecule impurities to obtain purified antimicrobial peptides; the amino acid sequence of the purified antimicrobial peptides is determined by mass spectrometry, the secondary structure of the purified antimicrobial peptides is determined by circular dichroism, and the three-dimensional structure of the purified antimicrobial peptides is analyzed by nuclear magnetic resonance; the antimicrobial peptides are determined by agar diffusion method. The antimicrobial activity of the antimicrobial peptides was evaluated by measuring the inhibitory effects of the antimicrobial peptides on various bacteria at different concentrations; the interaction mechanism between the antimicrobial peptides and the bacterial cell membrane was studied by using fluorescent probe technology, the effects of the antimicrobial peptides on the physiological processes in bacterial cells were analyzed, and the antimicrobial action mechanism of the antimicrobial peptides was determined; the antimicrobial peptides of the present invention have broad-spectrum antimicrobial activity and have inhibitory effects on a variety of bacteria. Compared with traditional antibiotics, the antimicrobial peptides are not easy to develop drug resistance and have higher safety. The research method is simple, reliable, and highly operable, which provides an effective technical means for the research of antimicrobial peptides and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0025] Figure 1 A flow chart of the protein structural characteristics and research methods of antimicrobial peptides provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The terms "first", "second", "third", "fourth", etc. (if any) in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein. In addition, the terms "including" or "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, device, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0027] For ease of understanding, the specific process of the embodiment of the present invention is described below. Figure 1 The present invention provides a flowchart of the protein structure characteristics of antimicrobial peptides and a research method thereof, the method specifically comprising the following steps:

[0028] S1. Collect biological samples, pre-treat the biological samples, separate the antimicrobial peptides according to their charge properties using ion exchange chromatography, perform gel filtration chromatography on the eluate obtained by the ion exchange chromatography, perform dialysis or ultrafiltration on the eluate obtained by the gel filtration chromatography to remove salt and small molecule impurities, and obtain purified antimicrobial peptides;

[0029] S2. Determine the amino acid sequence of the purified antimicrobial peptide by mass spectrometry, determine the secondary structure of the purified antimicrobial peptide by circular dichroism, and analyze the three-dimensional structure of the purified antimicrobial peptide by nuclear magnetic resonance;

[0030] S3. The antibacterial activity of antimicrobial peptides was determined by agar diffusion method. The antibacterial properties of antimicrobial peptides were evaluated by measuring the inhibitory effects of antimicrobial peptides on various bacteria at different concentrations.

[0031] S4. Use fluorescent probe technology to study the interaction mechanism between antimicrobial peptides and bacterial cell membranes, analyze the effects of antimicrobial peptides on physiological processes within bacterial cells, and determine the antibacterial mechanism of antimicrobial peptides.

[0032] In this embodiment, step S1 specifically includes the following steps: collecting biological samples, pre-treating the biological samples, selecting cation exchange resins according to the charge properties of antimicrobial peptides, separating by ion exchange chromatography, separating by ion exchange chromatography according to the charge properties of antimicrobial peptides, using 20 mM, pH 7.0 phosphate buffer as the buffer system, eluting by gradually increasing the salt concentration from 0 M NaCl and gradually increasing to 0-1 M NaCl, controlling the elution rate at 1-5 ml / min, and collecting the eluate containing antimicrobial peptides;

[0033] During gel filtration chromatography, Sephadex G-75 dextran gel was selected as the filtration medium, the buffer system used a 20 mM, pH 7.0 phosphate buffer, the elution rate was 0.5-2 ml / min, and the eluate was collected;

[0034] Select a dialysis bag with a molecular weight cutoff of 1000-5000Da, put the gel filtration chromatography eluate containing the antimicrobial peptide into the dialysis bag, put it into a 20mM phosphate buffer with a pH of 7.0, and perform dialysis, replace the dialysis buffer every 6 hours, and the dialysis time is 12-48 hours;

[0035] An ultrafiltration membrane with a molecular weight cutoff of 1000-5000Da is selected, and the gel filtration chromatography eluate containing the antimicrobial peptide is passed through an ultrafiltration device to remove salt and small molecular impurities, and finally a purified antimicrobial peptide is obtained.

[0036] In this embodiment, the biological sample comes from animal tissue, microbial fermentation broth or plant extract; the pretreatment process includes: centrifuging the biological sample at 3000-10000 rpm for 10-20 minutes, and filtering the centrifuged biological sample using a 0.22-0.45 μm microporous filter membrane.

[0037] In this embodiment, the mass spectrometry determination in step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in an acetonitrile / water mixed solution containing 0.1% formic acid, with a sample concentration of 0.5 mg / ml, selecting electrospray ionization mass spectrometry, setting the spray voltage to 3.5 kV, the capillary temperature to 250° C., the mass analyzer resolution to 10000, the mass range to 500-2000 Da, the detector gain to 100, the threshold to 10 counts, performing three mass spectrometry analyses, and obtaining the amino acid sequence of the antimicrobial peptide by analyzing the mass spectrum fragments and using a de novo sequencing method combined with comparison with a protein database, wherein the amino acid sequence of the antimicrobial peptide includes at least lysine and arginine.

[0038] In this embodiment, the secondary structure determination of step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in 10 mM, pH 7.4 phosphate buffer with a sample concentration of 0.1-1 mg / ml, using a circular dichroism spectrometer equipped with a polarized light source and a detector, setting the wavelength range to 190-260 nm, the scanning speed to 50 nm / min, the bandwidth to 1 nm, and the number of data acquisition points to 1024, performing five circular dichroism analyses, and using circular dichroism data analysis software to determine the secondary structure of the antimicrobial peptide according to the characteristics of the circular dichroism curve, wherein the antimicrobial peptide exists in an α-helical structure.

[0039] In this embodiment, the three-dimensional structure determination in step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in heavy water, adjusting the sample concentration to 0.8 mg / ml, and using 15 The amino acids were labeled with N-labeled amino acids, and an 800 MHz high-field strength NMR instrument was selected with an acquisition time of 0.5 s. Fourier transform combined with phase correction was used for four NMR analyses. The three-dimensional structure of the antimicrobial peptide was determined by attribution and analysis of the NMR signals using ab initio calculation combined with distance geometry.

[0040] In this embodiment, step S3 specifically includes the following steps: culturing Staphylococcus aureus in a 37°C constant temperature incubator using a tryptone soy broth medium, culturing Escherichia coli in a 37°C constant temperature incubator using an LB medium for 18-24 hours;

[0041] Prepare nutrient agar medium, dissolve peptone, beef extract, sodium chloride, and agar in distilled water according to a certain ratio, adjust the pH to 7.2-7.4, sterilize by high pressure at 120-130℃ for 20-25 minutes, and pour the medium into a plate when it cools to 45-55℃;

[0042] The purified antimicrobial peptides were dissolved in phosphate buffer to prepare antimicrobial peptide solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, and 10 μg / ml;

[0043] Staphylococcus aureus and Escherichia coli were inoculated on nutrient agar medium respectively, and cultured in a constant temperature incubator at 37°C for 18 hours to allow the bacteria to form uniform colonies on the medium, and a hole puncher with a diameter of 6-8 mm was used to punch small holes on the medium that was already full of bacteria, with a distance of 20-25 mm between the holes; antimicrobial peptide solutions of different concentrations were added to different small holes using a micropipette, and 40-50 μl was added to each well, and a penicillin solution with a concentration of 10 μg / ml was set as a positive control, and a phosphate buffer solution with a pH of 7.4 was set as a negative control, and the culture medium after the addition was placed in a constant temperature incubator at 37°C for further culture for 24 hours, and whether an inhibition zone appeared around the small hole was observed, and the diameter of the inhibition zone was measured using a vernier caliper;

[0044] Record the diameter of the inhibition zone corresponding to each concentration of the antimicrobial peptide solution, repeat the experiment three times and take the average value. For example, for Staphylococcus aureus, the diameter of the inhibition zone corresponding to the antimicrobial peptide solution with a concentration of 0.5μg / ml is 8.5mm, 8.3mm, 8.7mm, and the average value is 8.5mm. For Escherichia coli, the diameter of the inhibition zone corresponding to the antimicrobial peptide solution with a concentration of 1μg / ml is 7.2mm, 7.4mm, 7.1mm, and the average value is 7.2mm. Record the results of the positive control and negative control at the same time;

[0045] The relationship curve between the diameter of the inhibition zone and the concentration of the antimicrobial peptide was drawn, with the concentration of the antimicrobial peptide as the horizontal axis and the diameter of the inhibition zone as the vertical axis. The curves for Staphylococcus aureus and Escherichia coli were drawn respectively. It can be seen from the curves that with the increase of the concentration of the antimicrobial peptide, the diameter of the inhibition zone gradually increased; the inhibition rate of the antimicrobial peptide on various bacteria at different concentrations was calculated. For example, for Staphylococcus aureus, when the concentration of the antimicrobial peptide was 2 μg / ml, the colony diameter of the control group was 15 mm, and the colony diameter of the experimental group was 7 mm, then the inhibition rate = (15-7) / 15×100% = 53.3%; based on the data analysis results, the antibacterial performance of the antimicrobial peptide was evaluated. A comprehensive evaluation was carried out from the aspects of minimum inhibitory concentration (MIC), inhibition zone size, antibacterial spectrum, etc. The experiment found that the antimicrobial peptide had certain antibacterial activity against Staphylococcus aureus and Escherichia coli, with minimum inhibitory concentrations of 5μg / ml and 10μg / ml, respectively. The size of the inhibition zone increased with the increase of the antimicrobial peptide concentration, indicating that the antibacterial activity was positively correlated with the concentration. At the same time, the antimicrobial peptide had a certain antibacterial spectrum and had inhibitory effects on both Gram-positive and Gram-negative bacteria.

[0046] In this embodiment, step S4 specifically includes the following steps: using a lipophilic fluorescent dye DiI to mark the bacterial cell membrane, culturing Escherichia coli in LB medium to a logarithmic growth phase at 37°C for 15-30 hours; adding DiI to the Escherichia coli culture solution to a final concentration of 5 μM, incubating at 37°C for 15-30 minutes to allow the fluorescent probe to fully mark the bacterial cell membrane, mixing the Escherichia coli marked with the fluorescent probe with antimicrobial peptide solutions of different concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, and incubating at 37°C for 15-60 minutes; Use a fluorescence microscope to observe the changes in fluorescence signals; compare the changes in fluorescence intensity under treatment with different concentrations of antimicrobial peptides, calculate the fluorescence intensity change rate, and judge whether the antimicrobial peptides cause changes in the cell membrane structure by observing the changes in fluorescence distribution. For example, the fluorescence intensity is 100 when no antimicrobial peptides are added, and the fluorescence intensity becomes 80 after adding 10 μg / ml antimicrobial peptides. The fluorescence intensity change rate is (100-80) / 100×100%=20%. By observing the changes in fluorescence distribution, it is judged whether the antimicrobial peptides cause changes in the cell membrane structure. If the fluorescence distribution becomes uneven or aggregation occurs, it may indicate that the cell membrane structure is damaged.

[0047] Protein synthesis was selected as an indicator of intracellular physiological processes, and changes in protein synthesis were evaluated by detecting ribosome activity. Escherichia coli was mixed with antimicrobial peptide solutions of different concentrations and incubated at 37°C for 60 minutes. The ribosome activity was detected by radioisotope labeling. Radioactively labeled amino acids are added to the culture medium. After a period of culture, the intensity of radioactivity incorporated into the protein is measured to reflect the ribosome activity. The changes in ribosome activity under treatment with different concentrations of antimicrobial peptides are compared to calculate the rate of change in ribosome activity. For example, the ribosome activity is 100 when no antimicrobial peptide is added, and the ribosome activity becomes 70 after adding 15 μg / ml antimicrobial peptide. The rate of change in ribosome activity is (100-70) / 100×100%=30%. The degree of influence of antimicrobial peptides on protein synthesis is determined based on the changes in ribosome activity. The results of the fluorescent probe technology and the analysis of intracellular physiological processes are combined. It is found from the fluorescent probe experiment that with the increase in the concentration of antimicrobial peptides, the fluorescence intensity of the bacterial cell membrane gradually weakens and is unevenly distributed, indicating that the antimicrobial peptides may destroy the cell membrane structure. In the protein synthesis analysis, the ribosome activity is significantly reduced after antimicrobial peptide treatment, indicating that the antimicrobial peptides inhibit protein synthesis. In summary, antimicrobial peptides may exert their antibacterial effects by destroying the bacterial cell membrane structure and then affecting intracellular protein synthesis.

[0048] Use a specific cell membrane stabilizer, such as cholesterol, to treat E. coli at the same time as the antimicrobial peptide. If the inhibitory effect of the antimicrobial peptide on bacteria is weakened after the addition of the cell membrane stabilizer, it means that the antimicrobial effect of the antimicrobial peptide is related to the destruction of the cell membrane structure. Perform a gene mutation experiment to mutate the ribosome gene of E. coli and observe the changes in the sensitivity of the mutated bacteria to the antimicrobial peptide. If the resistance of the bacteria to the antimicrobial peptide is enhanced after the mutation, it means that the effect of the antimicrobial peptide is related to the ribosome.

[0049] Comprehensive analysis and verification experimental results show that this antimicrobial peptide exerts its antibacterial effect mainly by destroying the cell membrane structure of Escherichia coli and then inhibiting intracellular protein synthesis.

[0050] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A protein structural property of an antimicrobial peptide and a research method thereof, characterized in that: The method for studying the protein structural characteristics of the antimicrobial peptide comprises the following steps: S1. Collect biological samples, pre-treat the biological samples, separate the antimicrobial peptides according to their charge properties using ion exchange chromatography, perform gel filtration chromatography on the eluate obtained by the ion exchange chromatography, perform dialysis or ultrafiltration on the eluate obtained by the gel filtration chromatography to remove salt and small molecule impurities, and obtain purified antimicrobial peptides; S2. Determine the amino acid sequence of the purified antimicrobial peptide by mass spectrometry, determine the secondary structure of the purified antimicrobial peptide by circular dichroism, and analyze the three-dimensional structure of the purified antimicrobial peptide by nuclear magnetic resonance; S3. The antibacterial activity of antimicrobial peptides was determined by agar diffusion method. The antibacterial properties of antimicrobial peptides were evaluated by measuring the inhibitory effects of antimicrobial peptides on various bacteria at different concentrations. S4. Use fluorescent probe technology to study the interaction mechanism between antimicrobial peptides and bacterial cell membranes, analyze the effects of antimicrobial peptides on physiological processes within bacterial cells, and determine the antibacterial mechanism of antimicrobial peptides.

2. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 1, characterized in that: Step S1 specifically includes the following steps: collecting biological samples, pre-treating the biological samples, selecting cation exchange resins according to the charge properties of antimicrobial peptides, separating by ion exchange chromatography, separating by ion exchange chromatography according to the charge properties of antimicrobial peptides, using 20 mM, pH 7.0 phosphate buffer as the buffer system, eluting by gradually increasing the salt concentration from 0 M NaCl to 0-1 M NaCl, controlling the elution rate at 1-5 ml / min, and collecting the eluate containing antimicrobial peptides; During gel filtration chromatography, Sephadex G-75 dextran gel was selected as the filtration medium, the buffer system used a 20 mM, pH 7.0 phosphate buffer, the elution rate was 0.5-2 ml / min, and the eluate was collected; Select a dialysis bag with a molecular weight cutoff of 1000-5000Da, put the gel filtration chromatography eluate containing the antimicrobial peptide into the dialysis bag, put it into a 20mM phosphate buffer with a pH of 7.0, and perform dialysis, replace the dialysis buffer every 6 hours, and the dialysis time is 12-48 hours; An ultrafiltration membrane with a molecular weight cutoff of 1000-5000Da is selected, and the gel filtration chromatography eluate containing the antimicrobial peptide is passed through an ultrafiltration device to remove salt and small molecular impurities, and finally a purified antimicrobial peptide is obtained.

3. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 2, characterized in that: The biological sample is derived from animal tissue, microbial fermentation broth or plant extract; The pretreatment process includes: centrifuging the biological sample at 3000-10000 rpm for 10-20 minutes, and filtering the biological sample after centrifugation with a 0.22-0.45 μm microporous filter membrane.

4. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 1, characterized in that: The mass spectrometry determination in step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in an acetonitrile / water mixed solution containing 0.1% formic acid, with a sample concentration of 0.5 mg / ml, selecting electrospray ionization mass spectrometry, setting the spray voltage to 3.5 kV, the capillary temperature to 250° C., the mass analyzer resolution to 10000, the mass range to 500-2000 Da, the detector gain to 100, the threshold to 10 counts, performing three mass spectrometry analyses, and obtaining the amino acid sequence of the antimicrobial peptide by analyzing the mass spectrum fragments and using a de novo sequencing method combined with comparison with a protein database, wherein the amino acid sequence of the antimicrobial peptide includes at least lysine and arginine.

5. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 1, characterized in that: The secondary structure determination of step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in 10 mM, pH 7.4 phosphate buffer with a sample concentration of 0.1-1 mg / ml, using a circular dichroism spectrometer equipped with a polarized light source and a detector, setting the wavelength range to 190-260 nm, the scanning speed to 50 nm / min, the bandwidth to 1 nm, and the number of data acquisition points to 1024, performing five circular dichroism analyses, and using circular dichroism data analysis software to determine the secondary structure of the antimicrobial peptide based on the characteristics of the circular dichroism curve, wherein the antimicrobial peptide exists in an α-helical structure.

6. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 1, characterized in that: The three-dimensional structure determination of step S2 specifically includes the following steps: dissolving the purified antimicrobial peptide sample in heavy water, adjusting the sample concentration to 0.8 mg / ml, and using 15 The amino acids were labeled with N-labeled amino acids, and an 800 MHz high-field strength NMR instrument was selected with an acquisition time of 0.5 s. Fourier transform combined with phase correction was used for four NMR analyses. The three-dimensional structure of the antimicrobial peptide was determined by attribution and analysis of the NMR signals using ab initio calculation combined with distance geometry.

7. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 1, characterized in that: Step S3 specifically includes the following steps: culturing Staphylococcus aureus in a 37°C constant temperature incubator using a tryptone soy broth medium, culturing Escherichia coli in a 37°C constant temperature incubator using an LB medium for 18-24 hours; Prepare nutrient agar medium, dissolve peptone, beef extract, sodium chloride, and agar in distilled water according to a certain ratio, adjust the pH to 7.2-7.4, sterilize by high pressure at 120-130℃ for 20-25 minutes, and pour the medium into a plate when it cools to 45-55℃; The purified antimicrobial peptides were dissolved in phosphate buffer to prepare antimicrobial peptide solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 5 μg / ml, and 10 μg / ml; Staphylococcus aureus and Escherichia coli were inoculated on nutrient agar medium respectively, and cultured in a constant temperature incubator at 37°C for 18 hours to allow the bacteria to form uniform colonies on the medium, and a hole puncher with a diameter of 6-8 mm was used to punch small holes on the medium that was already full of bacteria, with a distance of 20-25 mm between the holes; antimicrobial peptide solutions of different concentrations were added to different small holes using a micropipette, and 40-50 μl was added to each well, and a penicillin solution with a concentration of 10 μg / ml was set as a positive control, and a phosphate buffer solution with a pH of 7.4 was set as a negative control, and the culture medium after the addition was placed in a constant temperature incubator at 37°C for further culture for 24 hours, and whether an inhibition zone appeared around the small hole was observed, and the diameter of the inhibition zone was measured using a vernier caliper; The diameter of the inhibition zone corresponding to each concentration of the antimicrobial peptide solution was recorded, the experiment was repeated three times and the average value was taken. The relationship curve between the diameter of the inhibition zone and the concentration of the antimicrobial peptide was plotted. It was found from the curve that with the increase of the concentration of the antimicrobial peptide, the diameter of the inhibition zone gradually increased. The inhibition rate of the antimicrobial peptide on various bacteria at different concentrations was calculated, and it was found that the antimicrobial peptide had an antibacterial spectrum.

8. The protein structural characteristics of an antimicrobial peptide and the research method thereof according to claim 1, characterized in that: Step S4 specifically includes the following steps: using lipophilic fluorescent dye DiI to mark bacterial cell membrane, culturing Escherichia coli in LB medium to logarithmic growth phase, the temperature is 37°C, and the culturing time is 15-30 hours; adding DiI to the Escherichia coli culture solution to make a final concentration of 5 μM, incubating at 37°C for 15-30 minutes to allow the fluorescent probe to fully mark the bacterial cell membrane, mixing the Escherichia coli marked with the fluorescent probe with antimicrobial peptide solutions of different concentrations of 0 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml, and incubating at 37°C for 15-60 minutes; observing the change of fluorescence signal using a fluorescence microscope; comparing the change of fluorescence intensity under treatment with different concentrations of antimicrobial peptides, calculating the fluorescence intensity change rate, and judging whether the antimicrobial peptides cause changes in the cell membrane structure by observing the change in fluorescence distribution. If the fluorescence distribution becomes uneven or aggregation occurs, it indicates that the cell membrane structure is damaged.

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