CN112724201B

CN112724201B - Antibacterial peptide and application thereof

Info

Publication number: CN112724201B
Application number: CN202110170639.4A
Authority: CN
Inventors: 崔鹏飞; 谭蓉; 张晓娜; 汝少国
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2022-07-26
Anticipated expiration: 2041-02-08
Also published as: CN112724201A

Abstract

The method discloses an antibacterial peptide, the amino acid sequence of which is SEQ ID NO. 1, and simultaneously provides the antibacterial peptide with specific targeting property and carries out related functional verification. The invention further provides application of the antibacterial peptide in preparing a medicament or feed additive for preventing and treating bacterial infection. The antibacterial peptide provided by the invention can kill gram-negative bacteria and gram-positive bacteria by mechanisms such as causing cell membrane damage and the like, but has no toxicity to mammalian cells.

Description

Antibacterial peptide and application thereof

Technical Field

The invention belongs to the technical field of aquaculture epidemic prevention, and relates to an antibacterial peptide for aquaculture and a preparation method and application thereof.

Background

In recent years, the aquaculture industry develops rapidly, the problem of aquatic animal diseases caused by bacterial diseases becomes more serious, antibiotic drugs are widely applied to the treatment of bacterial diseases, the abuse of antibiotics not only improves the feeding cost, but also leads the drug resistance and the pathogenicity of pathogenic microorganisms to be obviously enhanced, the microecological balance of the digestive tract of animals is seriously damaged, the antibiotic drugs are remained and enriched in the bodies of the animals after long-term use, the health level of animal organisms is weakened due to the toxic and side effects of the drugs, and the immunity and the disease resistance are obviously reduced. Such vicious circle results in the destruction of the water environment and the structure of the animal intestinal microflora, which in turn results in the frequent occurrence of various virulent infectious diseases in the aquaculture of aquatic animals, and is difficult to control and treat. On the other hand, the drug residue and the increasingly serious food safety problem of the livestock and poultry products not only directly threaten the health of human beings, but also hinder the development of the breeding industry. Therefore, a new strategy for effectively controlling drug-resistant bacteria is found, and safe, reliable, nontoxic and harmless medicaments are developed to prevent and treat diseases of aquaculture animals, so that the method is a pressing research target in all countries in the world at present.

Among many antibiotic substitutes, Antimicrobial peptides (AMPs) are receiving wide attention due to their strong Antimicrobial activity and new mechanism of action. The antibacterial mechanism of the antibacterial peptide is completely different from that of antibiotics, and in the reported action mechanism of the antibacterial peptide, some antibacterial peptides cause the exosmosis of bacterial contents to die by destroying the cell membrane structure of bacteria; some antibacterial peptides can inhibit bacteria specific enzyme or DNA transcription and protein translation, influence intracellular protein interaction and enzymatic cascade and cytosol signal transduction pathways, and the action mechanisms are not easy to cause bacterial drug resistance, so the antibacterial peptides are expected to replace antibiotics to solve the problem of bacterial drug resistance and a series of problems caused by abuse of antibiotics. However, natural antibacterial peptide has the problems of high production cost, generally weak antibacterial activity and the like, and a part of antibacterial peptide still has certain cytotoxicity due to positive charges.

Disclosure of Invention

Aiming at the defects of the natural antibacterial peptide and the demand for antibacterial drugs in aquaculture, the invention designs and reforms antibacterial peptide molecules by a bioinformatics technology and a computer-aided drug design technology, and further constructs specific targeted antibacterial peptide by connecting targeted peptide segments, thereby verifying the pathogenic bacteria activity, the related action mechanism, the cytotoxicity and the influence on animal intestinal flora of the anti-drug-resistant aquatic animals, and simultaneously screening out the efficient, stable and nontoxic specific targeted antibacterial peptide, providing inspection indexes and schemes for the creation of novel antibiotic substitutes for aquatic products and the development of related new drugs, and providing a solution for controlling the bacterial drug resistance caused by antibiotic abuse and the large-scale diffusion of drug resistance genes in the aquaculture process.

The technical problem of the invention can be solved by the following technical scheme:

an antibacterial peptide containing an antibacterial functional segment, wherein the antibacterial functional segment is SEQ ID NO 1WKKWSKX ₁ WX ₂ HWIPQCKKFGX ₃ X ₄ (ii) a Wherein, X ₁ Selected from any one of K, R or H, X ₂ Selected from any one of K, R or H, X ₃ Selected from any one of K, R or H, X4 is selected from any one of K, R or H, and X is ₁ 、X ₂ 、X ₃ And X ₄ Not simultaneously R. The invention found at X1X ₂ X ₃ And X ₄ When the amino acids are respectively the same or different basic groups, the formed polypeptides can form alpha helical conformation, have amphipathy and have stronger antibacterial activity.

In one embodiment according to the invention, the amino acid sequence of the antibacterial functional segment is SEQ ID NO 2.

In one embodiment of the invention, the antibacterial functional segment further comprises a targeting peptide segment connected to the C terminal or the N terminal of the antibacterial functional segment. The invention also discovers that the specificity of the corresponding antibacterial peptide can be effectively improved by connecting the targeting peptide segment at the tail end of the antibacterial functional segment, so that the antibacterial peptide has better effect on killing and preventing the corresponding bacterial infection. The targeting peptide segment aiming at specific bacteria enables the antibacterial peptide to be more efficient in identifying specific bacteria and has more remarkable killing effect.

In one embodiment according to the invention, the targeting peptide stretch amino acid sequence is SEQ ID NO 4.

In one embodiment according to the present invention, the amino acid sequence of the antimicrobial peptide is SEQ ID NO 3.

The invention also provides application of the antibacterial peptide in preparing antibacterial drugs.

In one embodiment according to the invention, the antibacterial agent is a drug for the treatment of bacterial infections that are resistant to antibiotics.

In one embodiment according to the present invention, the drug-resistant bacterium is a bacterium that is resistant to multiple antibiotics.

The invention further provides a pharmaceutical composition for treating bacterial infection, which comprises the antibacterial peptide and pharmaceutically acceptable auxiliary materials.

The invention further provides the application of the antibacterial peptide in preparing a feed additive for preventing and treating bacterial infection of cultured animals; preferably, the farmed animals are aquatic animals, more preferably the aquatic animals are selected from one or more of fish, shrimp or crab.

In a further aspect of the present invention, there is provided a feed additive for controlling bacterial infections in animals, comprising the antimicrobial peptide according to any one of claims 1 to 6; preferably, the antimicrobial peptide is encapsulated in a membrane that prevents the antimicrobial peptide from being digested and degraded by digestive juices and allows for administration.

The invention has the beneficial effects that:

the invention designs two antibacterial peptides which can effectively realize antibacterial and bacteriostatic effects, and performs related functional verification. The antibacterial peptides 1-6 and L-6 provided by the invention can kill gram-negative bacteria and gram-positive bacteria by mechanisms such as causing cell membrane damage, but have no toxicity to mammalian cells. Provides inspection indexes and schemes for creation of antibiotic substitutes and development of related new drugs.

Drawings

FIG. 1 is a schematic of the three-dimensional structure of the L-6 conformation, in which the light portion is the added targeting fragment and the dark portion is the AMP1-6 portion;

FIG. 2a is a schematic diagram of a 1-6 helical wheel structure in which, in helical wheel projection, hydrophilic residues appear in the form of circles, hydrophobic residues appear in the form of diamonds, residues that may be negatively charged appear in the form of triangles, and residues that may be positively charged appear in the form of squares;

FIG. 2b is a schematic L-6 helix structure in which, in helix wheel projection, hydrophilic residues appear in the form of circles, hydrophobic residues appear in the form of diamonds, residues that may be negatively charged appear in the form of triangles, and residues that may be positively charged appear in the form of squares;

FIG. 3 shows the bactericidal effect of the antimicrobial peptides 1-6 and L-6 provided in example 1 of the present invention on Vibrio anguillarum and Micrococcus luteus (the percentage indicates the bactericidal rate);

FIG. 4 is a graph showing the bactericidal activity of antimicrobial peptides 1-6 and L-6 provided in example 1 of the present invention, and antimicrobial peptide mBjAMP1 disclosed in the prior art; wherein, a) to f) are respectively the time-sterilization curves of the antimicrobial peptides 1 to 6 or L-6 of example 1 of the present invention against three bacteria (positive control: and (3) ANB: ampicillin sodium KNA: kanamycin sulfate); g) and h) is the time-kill curve for mBjAMP1 for both bacteria;

FIG. 5 is a schematic diagram showing the detection results of the effect of antibacterial peptides 1-6 and L-6 on the survival rate of mouse macrophage RAW264.7 provided in example 1 of the present invention;

FIG. 6 is a photograph showing the morphology of each of several typical bacteria treated with the antimicrobial peptide of the present invention, which is observed using a transmission electron microscope; wherein a), b) and c) are photomicrographs of the morphology of the untreated bacteria; a1) b1) and c1) are the bacterial shape micrographs after the antibacterial peptide 1-6 treatment; a2) b2) and c2) are the bacterial shape micrographs after the antibacterial peptide L-6 treatment;

FIG. 7 is a graph showing the influence of the antimicrobial peptides 1 to 6 and L to 6 on the membrane permeability of Vibrio anguillarum, wherein the percentage represents the proportion of dead cells;

FIG. 8 is a graph of the effect of antimicrobial peptides 1-6, L-6 on membrane permeability of Micrococcus luteus, where the percentages represent the proportion of dead cells;

FIG. 9 is a graph of the membrane permeability effect of antimicrobial peptides 1-6, L-6 on Listeria, where the percentages represent the proportion of dead cells;

FIG. 10 is a graph of the effect of antimicrobial peptides 1-6, L-6 on the membrane permeability of Pseudomonas bacteria, where the percentage represents the proportion of dead cells;

FIG. 11 is a graph showing the influence of antimicrobial peptides 1 to 6 and L to 6 on the membrane permeability of Vibrio parahaemolyticus, in which percentages represent the proportion of dead cells.

FIG. 12 is a graph of the effect on reactive oxygen species levels in bacterial cells after treatment with antimicrobial peptides 1-6 and L-6; wherein a) is the influence of the antibacterial peptide 1-6 and L-6 on the level of active oxygen of vibrio anguillarum, b) is the influence graph of the antibacterial peptide 1-6 and L-6 on the level of active oxygen of micrococcus luteus, c) is the influence of the antibacterial peptide 1-6 and L-6 on the level of active oxygen of listeria, d) is the influence of the antibacterial peptide 1-6 and L-6 on the level of active oxygen of pseudomonas, and e) is the influence of the antibacterial peptide 1-6 and L-6 on the level of active oxygen of vibrio parahaemolyticus.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

Example 1 antimicrobial peptide AMP1-6(1-6) and Listeria targeting AMP1-6(L-6)

This example provides two exemplary antimicrobial peptides. The antibacterial peptide AMP1-6(1-6) is composed of 21 amino acid residues, the amino acid sequence is SEQ ID NO:2WKKWSKKWRHWIPQCKKFGKK, and a spiral wheel in figure 1 shows that the antibacterial peptide AMP1-6 has an amphiphilic structure. In the invention, Listeria target AMP1-6(L-6, SEQ ID NO:3) is obtained by connecting Listeria pheromone ASSLLLVG (SEQ ID NO:4) target fragments and embedding into specific target antibacterial peptide, the physical and chemical properties of the Listeria target AMP1-6(L-6, SEQ ID NO:3) are analyzed by using analytical informatics technology and computer-assisted drug molecular design software, as shown in Table 1, and the antibacterial peptide (L-6) is subjected to three-dimensional structure modeling analysis through a website (https:// zhanglab. ccmb. med. umic. edu /), as shown in FIG. 1, the antibacterial peptide (L-6) is shown to have an alpha helical structure. FIGS. 2a and 2b are helical wheel structures of antimicrobial peptide AMP1-6 and antimicrobial peptide L-6, respectively, showing that both antimicrobial peptides are amphiphilic. As the antibacterial peptides are all shorter in sequence, mature in synthesis technology and low in cost, the antibacterial peptides in the embodiment are synthesized by GL biochemistry (Shanghai) limited company, and all the antibacterial peptides are subjected to terminal amidation modification, so that the purity is higher than 95%. The antibacterial peptide has stable physicochemical properties, has broad-spectrum antibacterial action and no cytotoxicity, is an ideal molecular design and modification template, has higher antibacterial activity than natural antibacterial peptide (figure 3, table 3), and has weakened hemolytic activity.

TABLE 1 antimicrobial peptide sequences and physicochemical Properties

Example 2 antibacterial experiment:

adjusting the concentration of bacteria to 4 × 10 ⁴ One/ml for subsequent experiments. The final effect concentrations of the peptides were 3. mu.g/ml, 6. mu.g/ml, 12. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml, respectively. PBS was used as blank control. After half an hour incubation at 25 ℃, the protein bacteria mixture was averaged for each concentrationDivided into three portions, spread on three glass plates containing LB solid medium, and cultured overnight at 37 ℃.

The bactericidal rate of the protein was counted by colony counting based on the number of colonies on each plate. The calculation method of the sterilization efficiency comprises the following steps: the number of colonies on the plates of 3 replicates was averaged to give the number of colonies per sample. The number of colonies in the control group was defined as 100%, and the bactericidal activity was defined as (number of colonies in the control group-number of colonies in the experimental group)/number of colonies in the control group × 100%.

The Minimum Bactericidal Concentration (MBC) is shown in table 2: the minimum drug concentration required to kill 99.9% of the bacteria.

According to the invention, a gram-negative bacterium Vibrio anguillarum (Vibrio anguillarum) and a gram-positive bacterium Micrococcus luteus (Micrococcus luteus) are respectively selected to study the bactericidal activity of the antibacterial peptides, as shown in figure 3, the percentage represents the bactericidal rate, the bactericidal experiment result shows that the two designed antibacterial peptides can directly kill bacteria and have higher bactericidal rate, and the antibacterial peptide L-6 has stronger bactericidal effect than the antibacterial peptides 1-6.

TABLE 2 Minimum Bactericidal Concentration (MBC) of antimicrobial peptides

EXAMPLE 3 bacteriostatic experiments

Centrifuging the bacteria at room temperature of 6000 Xg for 5 min, removing upper culture medium, sterilizing with sterilized PBS (pH7.4), repeating for three times, resuspending the bacteria, and adjusting the concentration to 10 ⁶ One/ml for subsequent experiments.

Mixing LB liquid culture medium 160 μ L, diluted bacterial liquid 10 μ L, and antibacterial peptide dilution (with PBS to adjust antibacterial peptide concentration) of different concentrations, culturing in 96-well plate at 37 deg.C for 8h, wherein the final volume of each well is 200 μ L, and taking out every 1 hr, and measuring the absorbance at 595nm with microplate reader. And drawing a bacterial growth curve according to the obtained result, wherein the Minimum Concentration capable of completely inhibiting the bacterial growth is the Minimum Inhibitory Concentration (MIC), and the Inhibitory activity and the Minimum Inhibitory Concentration (MIC) of the specific targeting antibacterial peptide to different bacteria are counted.

The results of bacteriostatic experiments show (fig. 4) that the antibacterial peptides designed by the antibacterial peptide provided in example 1 of the present invention have significant inhibitory effect on Vibrio parahaemolyticus (Vibrio parahaemolyticus), Pseudomonas (Pseudomonas adaceae) and Listeria (Listeria monocytogens), table 3 shows that the minimum inhibitory concentration MIC of the two antibacterial peptides of the present invention on six typical bacteria is shown, the antibacterial effect of the antibacterial peptide L-6 with the Listeria targeting fragment added thereto is more targeted than that of the antibacterial peptides 1-6, and compared with the antibacterial peptide mBjAMP1 disclosed in the prior art, the antibacterial peptides 1-6 provided in the present invention have lower MIC for inhibiting Staphylococcus aureus (Staphylococcus aureus, s.aureus), acinetobacter baumannii (a.baumannii) and escherichia coli (e.coli), i.e. the corresponding antibacterial effects are better. On the whole, the antibacterial effect of the antibacterial peptide L-6 added with the targeting fragment is equivalent to that of the original antibacterial peptide 1-6. The amino acid sequence of the Antimicrobial Peptide mBjAMP1 disclosed in the prior art is SEQ ID NO 5NLCASLRARHTIPQCKKFGRR ("Structural and Functional Association of mBjAMP1, an Antimicrobial Peptide from branched sphingosoma japonica, modified a Novel alpha-hairpin-like scanned with Membrane Permeable and DNA Binding Activity", Liu et al 2015).

TABLE 3 Minimum Inhibitory Concentration (MIC) of antimicrobial peptides against different bacteria

Example 4 cytotoxicity assay

In this example, the activity of the cells was tested by MTT assay to determine whether the antimicrobial peptides were toxic to mammalian cells. The animal cell used in the experiment was mouse macrophage RAW 264.7.

The experimental principle is as follows: MTT entering living cells can be acted on by intracellular enzymes to produce bluish-purple crystals which are precipitated in the cells. While dead cells cannot form bluish-purple crystals. The organic solvent DMSO is able to dissolve the blue-violet precipitate formed. And detecting the light absorption value of the blue-violet crystal in the sample by using a spectrophotometer to reflect the activity of the cell. The light absorption value of the blue-violet crystal in the sample is positively correlated with the number of living cells.

The method comprises the following specific steps:

1) recovering cells, recovering mouse macrophage RAW264.7 stored in liquid nitrogen, culturing in DMEM medium (containing 10% fetal calf serum), culturing in a carbon dioxide incubator at 37 deg.C, and subculturing for 2-3 times to prepare for subsequent experiments.

2) Adherent cells were treated with PE (PBS solution containing EDTA), and then resuspended in serum-free medium to adjust the cell concentration to 1X 10 ⁶ One per ml.

3) The cell suspension was added to the wells of a 96-well plate, 180. mu.l was added to each well, and cultured in a carbon dioxide incubator at 37 ℃ for 2 hours to allow adherent growth of the cells.

4) Thereafter, antimicrobial peptide 1-6 and L-6 solutions were added to each well at different concentrations to give a final concentration of protein of 12.5. mu.g/ml, 25. mu.g/ml, 50. mu.g/ml and 100. mu.g/ml PBS (pH7.4) as a blank. 3 replicates of each sample were set up, and the 96-well plates were then incubated in a 37 ℃ carbon dioxide incubator for 4 hours.

5) After 4 hours, 20. mu.l of MTT solution (5mg/ml) was added to each well, and the 96-well plate was placed in a 37 ℃ carbon dioxide incubator for another 4 hours.

6) After 4 hours, the liquid in each well was aspirated off by a pipette, 150. mu.l of DMSO solvent was added to each well, and the absorbance of the sample at 492nm was measured.

7) The cell viability was calculated as (treatment OD/control OD) × 100%, and the results were expressed as mean ± standard deviation.

The results are shown in fig. 5, and toxicity experiments show that the antibacterial peptides with different concentrations set in the experiments have no obvious influence on the survival rate of mouse macrophage RAW264.7, and the experimental results primarily show that the antibacterial peptide provided in example 1 of the present invention has no toxicity to mammalian cells.

Example 5 transmission electron microscopy experiments:

1) sample preparation in the experiment

Culturing Micrococcus luteus, Vibrio anguillarum and Listeria monocytogenes to logarithmic growth stage. Centrifuging at room temperature of 5000 Xg for 3 min, removing upper culture medium, washing bacteria with sterilized PBS, repeating for three times, resuspending bacteria, and adjusting bacteria concentration to 10 ⁹ One/ml for subsequent experiments.

2) Mixing the bacterial liquid with the protein solution

50. mu.l of Micrococcus luteus bacterial liquid was combined with 50. mu.l of antimicrobial peptide to set the final protein concentration to 25. mu.g/ml. PBS group served as blank control. The protein and bacterial solution were incubated at 25 ℃ for half an hour.

3) Sample fixation

The bacterial protein mixture was mixed with an equal volume of 2.5% glutaraldehyde (in PBS) solution to immobilize the bacterial cells. Thereafter, 50. mu.l of the bacterial fixing solution was pipetted onto the plate, and the carrier net was immersed in the mixed solution for 10 minutes to allow the bacteria to be adsorbed on the carrier net. Subsequently, excess liquid was aspirated off with filter paper and left to stand.

4) And placing the carrier net into a transmission electron microscope for observation and taking a picture.

The transmission electron microscope experiment result shows (figure 6), after the antibacterial peptide 1-6 or L-6 of the invention is incubated, the shape of the bacteria is obviously changed (as shown in a1, b1) and c1), a2), b2) and c2) in the invention), the shape of the bacteria body becomes irregular after the antibacterial peptide is treated, the cell content flows out, and holes appear on the surface of individual bacteria cells.

Example 6 cell Membrane Permeability assay

The fluorescent dye Propidium Iodide (PI) is a fluorescent dye that binds to DNA and is unable to enter living cells. But can enter into cells with increased permeability or dead cells, and can be combined with DNA molecules to emit red fluorescence. Thus, the PI fluorescence intensity can be used to indirectly reflect the ratio of dead cells to live cells in the cell. The proportion of bacteria that fluoresced PI was significantly increased, indicating that the bacterial permeability following the action of the antimicrobial peptide was significantly increased (FIGS. 7-11).

The method comprises the following specific steps:

1) sample preparation in the experiment

The bacteria were cultured to log-extended periods. Centrifuging at room temperature of 6000 Xg for 5 min, removing upper layer culture medium, washing bacteria with sterilized PBS (pH7.4), repeating for three times, resuspending bacteria, and adjusting concentration to 1X 10 ⁸ One/ml for subsequent experiments.

2) The bacterial solution and the protein solution were mixed, and the final concentration of the antimicrobial peptide was 12.5. mu.g/ml and 25. mu.g/ml (diluted with PBS), PBS (pH7.4) was used as a blank, and the final volume of the ep tube was 600. mu.L. The protein and the bacterial solution were incubated at 37 ℃ for 1 hour.

3) After incubation, Propidium Iodide (PI) fluorescent dye was added to the protein-bacteria mixture so that the dye concentration was 12.5. mu.g/ml (in the dark and in the dark), and then the sample was incubated at 4 ℃ for 15 minutes.

4) After the dye had entered the cells sufficiently, the fluorescence of the PI was measured by flow cytometry. 10000 cells were detected per sample.

5) The resulting data were analyzed with flow cytometry analysis software.

The invention takes vibrio anguillarum, micrococcus luteus, listeria monocytogenes, pseudomonas and vibrio parahaemolyticus as typical pathogenic bacteria respectively for the detection of the antibacterial effect, the results are respectively shown in figures 7-11,

FIG. 7 is a graph showing the influence of the antimicrobial peptides 1 to 6 and L to 6 on the membrane permeability of Vibrio anguillarum, wherein the percentage represents the proportion of dead cells; specifically, a) is PBS control group, the ratio of dead cells of vibrio anguillarum is 3.45%,

b) 1-6 of 12.5 mu g/ml and 23.54 percent of dead cells after the action of the vibrio anguillarum,

c) 1-6 of 25 microgram/ml and vibrio anguillarum, the dead cell ratio is 37.73 percent,

d) after the L-6 with the concentration of 12.5 mu g/ml is acted with the vibrio anguillarum, the dead cell ratio is 46.98 percent,

e) after 25 mu g/ml L-6 is acted with the vibrio anguillarum, the dead cell rate is 55.51 percent.

FIG. 8 is a graph of the membrane permeability effect of antimicrobial peptides 1-6, L-6 on Micrococcus luteus, where the percentages represent the proportion of dead cells; specifically, a) is a PBS control group, the dead cell proportion of micrococcus luteus is 4.49%,

b) 12.5 mu g/ml 1-6, the dead cell ratio is 56.69 percent after the micrococcus luteus acts on the cell,

c) 25 mu g/ml 1-6, the dead cell ratio is 74.89 percent after the micrococcus luteus acts on the cell,

d) after 12.5 mu g/ml L-6 is acted with micrococcus luteus, the proportion of dead cells is 63.84 percent,

e) after 25. mu.g/ml L-6 had reacted with Micrococcus luteus, the proportion of dead cells was 90.38%.

FIG. 9 is a graph of the membrane permeability effect of antimicrobial peptides 1-6, L-6 on Listeria, where the percentages represent the proportion of dead cells; specifically, a) is PBS control group, the ratio of the dead cells of the listeria is 2.33%,

b) after 12.5 mu g/ml 1-6 acts with Listeria, the dead cell proportion is 15.14 percent,

c) 25 mu g/ml 1-6, the ratio of dead cells is 38.99 percent after the action with the listeria monocytogenes,

d) after 12.5 mu g/ml L-6 acts with the listeria, the dead cell ratio is 44.43 percent,

e) after L-6 at 25. mu.g/ml acted on Listeria, the proportion of dead cells was 74.70%.

FIG. 10 is a graph of the effect of antimicrobial peptides 1-6, L-6 on the membrane permeability of Pseudomonas bacteria, wherein the percentage represents the proportion of dead cells; specifically, a) is a PBS control group, the proportion of dead cells of pseudomonas is 12.08 percent,

b) 12.5 mu g/ml 1-6 and pseudomonas, the dead cell ratio is 60.46 percent,

c) 1-6 of 25 mu g/ml and the proportion of dead cells after the pseudomonas is acted is 71.51 percent,

d) after 12.5 mu g/ml L-6 is acted with pseudomonas, the dead cell ratio is 61.48 percent,

e) after 25. mu.g/ml L-6 had reacted with Pseudomonas, the proportion of dead cells was 83.88%.

FIG. 11 is a graph showing the effect of antimicrobial peptides 1 to 6 and L to 6 on the membrane permeability of Vibrio parahaemolyticus, wherein percentages represent the proportion of dead cells; specifically, a) is PBS control group, the ratio of dead cells of the vibrio parahaemolyticus is 10.48%,

b) 12.5 mu g/ml 1-6, and 61.71% of dead cells after the action of the vibrio parahaemolyticus,

c) 25 mu g/ml 1-6, and 68.62% of dead cells after the vibrio parahaemolyticus acts on the cells,

d) 12.5 mu g/ml L-6 and the ratio of dead cells is 57.03 percent after the action of the vibrio parahaemolyticus,

e) 25. mu.g/ml L-6 reacted with Vibrio parahaemolyticus, the dead cell ratio was 63.77%.

As shown in FIGS. 7-11, the proportion of PI-fluorescing bacteria in b), c), d) and e) of FIGS. 7-11, respectively, was significantly increased compared to control a), indicating that the bacterial permeability was significantly increased after the action of antimicrobial peptides 1-6 and L-6.

Example 7 detection of Reactive Oxygen Species (ROS) levels in bacterial cells

The fluorescent probe DCFH2-DA was used as an indicator of intracellular reactive oxygen species levels. DCFH2-DA is a lipophilic molecule that passes freely through the cell membrane and is not itself fluorescent. DCFH2-DA was enzymatically hydrolyzed in the cells and deacetylated to form DCFH 2. DCFH2 itself also did not fluoresce, but it could not freely pass through the cell membrane, and probe DCFH2-DA was therefore loaded into the cells. DCFH2 produces fluorescent DCF under the action of Reactive Oxygen Species (ROS) in cells, and the level of fluorescence in DCF is detected to reflect the level of reactive oxygen species in cells. The mixture of Rosu is a mixture capable of raising the active oxygen level significantly in a short time.

Carrying out the step

1) The bacteria were cultured to logarithmic growth phase with the probe loaded. Centrifuging at room temperature of 6000 Xg for 5 min, removing upper layer culture medium, resuspending the bacterial solution with culture medium containing DCFH2-DA fluorescent probe (DCFH2-DA concentration of 10 μ M), and adjusting the bacterial concentration to 1X 10 ⁸ Cells/ml were incubated at 37 ℃ for 30 minutes.

2) Thereafter, the bacterial suspension was centrifuged at 6000 Xg for 10 minutes at room temperature, and the bacteria were washed with sterilized PBS (pH7.4) three times to wash off the probes which had not entered the bacterial cells.

3) Finally, resuspending the bacteria, resuspending the experimental group with different antibacterial peptide solutions, wherein the final concentration of the antibacterial peptide is 12.5 mug/ml; positive control H formulated with PBS (pH7.4) ₂ O ₂ The solution is used for resuspending bacteria liquid; blank control bacteria were resuspended in PBS (pH7.4). The mixed solution of the above groups was incubated at 25 ℃ for 1 hour. And then, measuring the fluorescence value by using a microplate reader, wherein the excitation light wavelength set by the microplate reader is 488nm, and the emission light wavelength is 525 nm.

The results are shown in FIG. 12, where the fluorescence intensity represents the intracellular reactive oxygen species level

a) The intracellular active oxygen level of the antibacterial peptide 1-6 and the antibacterial peptide L-6 after acting on the vibrio anguillarum is obtained;

b) the intracellular active oxygen level of the micrococcus luteus after the antibacterial peptides 1-6 and L-6 act on the micrococcus luteus;

c) the intracellular reactive oxygen species level of the antibacterial peptides 1-6 and L-6 after acting on the listeria monocytogenes;

d) the intracellular active oxygen level of the antibacterial peptide 1-6 and the antibacterial peptide L-6 after acting on pseudomonas is shown;

e) the active oxygen level in cells after the antibacterial peptides 1-6 and L-6 act on the vibrio parahaemolyticus.

If the active oxygen level in the cells is high, the cells can be necrotized or apoptotic. The results of the intracellular reactive oxygen species level detection experiment shown in FIG. 12 indicate that the test was compared with the positive control H ₂ O ₂ Compared with the antibacterial peptides 1-6 and L-6, the antibacterial peptides can obviously improve the reactive oxygen species level in five bacterial cells, thereby inducing cell necrosis or apoptosis.

The above examples are provided for illustrative purposes only and are not intended to limit the present invention; it should be noted that various changes and modifications can be made by those skilled in the art without departing from the scope of the inventive concept, which fall within the scope of the invention; therefore, all equivalent changes and modifications within the scope of the claims of the present invention should be covered by the claims of the present invention.

Sequence listing

<110> China oceanic university

<120> antibacterial peptide and application thereof

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1 5 10 15

Lys Phe Gly Lys Lys

20

<210> 3

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ala Ser Ser Leu Leu Leu Val Gly Trp Lys Lys Trp Ser Lys Lys Trp

1 5 10 15

Lys His Trp Ile Pro Gln Cys Lys Lys Phe Gly Lys Lys

20 25

<210> 4

<211> 8

<212> PRT

<213> Listeria monocytogenes

<400> 4

Ala Ser Ser Leu Leu Leu Val Gly

1 5

<210> 5

<211> 21

<212> PRT

<213> Branchiostoma californiense

<400> 5

Asn Leu Cys Ala Ser Leu Arg Ala Arg His Thr Ile Pro Gln Cys Lys

1 5 10 15

Lys Phe Gly Arg Arg

20

Claims

1. The antibacterial peptide is characterized in that the amino acid sequence of the antibacterial peptide is SEQ ID NO. 2 or SEQ ID NO. 3.

2. Use of an antimicrobial peptide according to claim 1 for the preparation of an antimicrobial medicament for the treatment of a bacterial infection which is resistant to an antibiotic.

3. A pharmaceutical composition for treating bacterial infection comprising the antimicrobial peptide of claim 1, and a pharmaceutically acceptable excipient.

4. Use of an antimicrobial peptide according to claim 1 for the preparation of a feed additive for the control of bacterial infections in farmed animals.

5. The use of claim 4, wherein the farmed animals are aquatic animals.

6. The use of claim 5, wherein the aquatic animal is selected from one or more of fish, shrimp, or crab.

7. A feed additive for controlling bacterial infections in animals comprising the antimicrobial peptide of claim 1.

8. The feed additive of claim 7 wherein the antimicrobial peptide is encapsulated in a membrane that prevents digestion and degradation of the antimicrobial peptide by digestive juices and allows administration of the antimicrobial peptide.