CN111518187B

CN111518187B - Antibacterial peptide DN6NH2 and application thereof

Info

Publication number: CN111518187B
Application number: CN202010301218.6A
Authority: CN
Inventors: 王建华; 王秀敏; 王振龙; 李婷; 韩卉卉; 毛若雨; 杨娜; 郝娅; 马炫炫
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2022-05-10
Anticipated expiration: 2040-04-16
Also published as: CN111518187A

Abstract

The invention relates to a novel antibacterial peptide DN6NH₂And applications thereof. Antibacterial peptide DN6NH₂Is obtained by amidation modification of carboxyl at the C terminal of antibacterial peptide N6, and the other amino acids except glycine are D-type amino acids. Antibacterial peptide DN6NH₂Can obviously improve the parent peptide N6NH₂Has the characteristics of quick sterilization and no rebound sterilization on multiple drug-resistant Aeromonas veronii, and has low cytotoxicity and low hemolysis. DN6NH₂Can inhibit the formation of aeromonas veronii biofilm in vitro; in vivo, the composition can obviously improve the phenomenon of back skin abscess necrosis of mice caused by infection of Aeromonas veronii biomembranes, and the effect is superior to that of parent peptide and ciprofloxacin. The experimental result shows that DN6NH₂Obviously improves N6NH₂The enzymatic stability and the biological activity of the compound. Antibacterial peptide DN6NH₂Is a micromolecular polypeptide with great application value and has wide application prospect.

Description

Antibacterial peptide DN6NH2And uses thereof

Technical Field

The invention relates to the field of biological medicine, in particular to antibacterial peptide DN6NH₂And applications thereof.

Background

Antimicrobial peptides (AMPs) are mostly cationic amphiphilic molecules of linear or cyclic structure, generally less than 100 amino acids in length, present in almost all organisms and being part of the host innate immune system. The number of synthetic peptide mimetics using natural antimicrobial peptides as templates has reached thousands. They have been shown to possess a wide range of biological activities (antibacterial, fungal, protozoan, viral, parasitic, and tumor, etc.). Compared with the traditional antibiotics, the antibacterial peptide has the characteristics of multiple action targets, low drug resistance and the like on pathogenic bacteria, and is considered to be one of ideal substitutes of the antibiotics. The antibacterial peptide has an antibacterial function, and can inhibit the formation of bacterial biofilms and neutralize bacterial endotoxins; meanwhile, the traditional Chinese medicine composition has the functions of immunoregulation, cell apoptosis inhibition, angiogenesis promotion, wound healing and the like, and plays an important role in the health of human and animals.

Aeromonas veronii is a common pathogenic bacterium of human, livestock and fish. Relevant research reports indicate that Aeromonas veronii is one of the important pathogenic sources causing the fish infection. It can infect various fishes, and the clinical symptoms are mainly manifested by blackening the whole body of the fish, severe bleeding on the body surface and fin rays and forming an ulcer focus; the gill filaments of seriously ill fishes are seriously necrotic, are grey brown and rot, and have a large amount of light yellow ascites in abdominal cavities. In recent years, the drug resistance of Aeromonas veronii is continuously enhanced due to the abuse of antibiotics, which poses a potential threat to national health and aquaculture in China. Therefore, the development of novel antibacterial agents is urgently required.

Disclosure of Invention

The invention aims to provide a novel antibacterial peptide DN6NH₂And applications thereof.

To realize the present inventionIn a first aspect, the invention provides an antibacterial peptide DN6NH₂Antibacterial peptide DN6NH₂Is obtained by amidation modification of carboxyl at the C terminal of antibacterial peptide N6, and the other amino acids except glycine are D-type amino acids.

Antibacterial peptide DN6NH₂The structure of (A) is as follows:

the antibacterial peptide DN6NH of the invention₂An active polypeptide designed to be synthesized artificially, comprising 21 amino acid residues. Antibacterial peptide DN6NH₂The molecular weight of (A) is 2474.88Da, and the amino acid sequence is shown in SEQ ID NO. 1.

In a second aspect, the invention provides a peptide containing the antibacterial peptide DN6NH₂The broad spectrum antibacterial drug or composition of (1).

In a third aspect, the invention provides a peptide containing antibacterial DN6NH₂The preservative or the bactericide of (1).

In a fourth aspect, the invention provides the antimicrobial peptide DN6NH₂Any of the following uses:

1) for preparing broad-spectrum antibacterial drugs or compositions;

2) for the preparation of preservatives;

3) is used for preparing bactericide.

The bacteria include gram positive bacteria and gram negative bacteria.

Such bacteria include, but are not limited to, Staphylococcus (Staphylococcus), Escherichia (Escherichia), Salmonella (Salmonella), Pseudomonas (Pseudomonas), Aeromonas (Aeromonas) bacteria.

Preferably, the bacteria include Staphylococcus suis (Staphylococcus hyicus), Staphylococcus aureus (Staphylococcus aureus), Escherichia coli (Escherichia coli), Salmonella typhimurium (Salmonella typhimurium), Salmonella pullorum (Salmonella pullulum), Salmonella enteritidis (Salmonella enteritidis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Aeromonas veronii (Aeromonas veronii), and the like.

In a fifth aspect, the invention provides the antimicrobial peptide DN6NH₂The application in preparing biological products for treating or preventing infection caused by Aeromonas veronii and biomembrane thereof and related diseases caused by the infection.

The disease includes subcutaneous abscesses caused by biofilms produced by aeromonas veronii.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

the antibacterial peptide DN6NH provided by the invention₂Can obviously improve the parent peptide N6NH₂(the C-terminal carboxyl group of the antimicrobial peptide N6 was amidated and modified, GFAWNVCVYRNGVRVCHRRAN-NH)₂) Has the characteristics of quick sterilization and no rebound sterilization on multiple drug-resistant Aeromonas veronii, and has low cytotoxicity and low hemolysis. DN6NH₂Can inhibit the formation of aeromonas veronii biofilm in vitro; in vivo, the composition can obviously improve the phenomenon of abscess necrosis of back skin of a mouse caused by infection of an aeromonas veronii biofilm, and the effect is superior to that of parent peptide and ciprofloxacin. The experimental result shows that DN6NH₂Obviously improves N6NH₂The enzymatic stability and the biological activity of the compound. DN6NH₂The molecular weight is small, the artificial synthesis is easy, the polypeptide is a small molecular polypeptide with great application value, and the polypeptide can be used for preparing novel antibacterial agents and has wide application prospect.

Drawings

FIG. 1 shows the antibacterial peptide DN6NH in example 1 of the invention₂Mass spectrum of (2).

FIG. 2 shows the antibacterial peptide DN6NH in example 3 of the invention₂The bactericidal power curve for the clinical isolate strain Aeromonas veronii L1.

FIG. 3 shows the antibacterial peptide DN6NH in example 5 of the invention₂The result of the serum stability test.

FIG. 4 shows the antibacterial peptide DN6NH in example 6 of the invention₂The result of the cytotoxicity assay of (1).

FIG. 5 shows the antibacterial peptide DN6NH in example 7 of the invention₂The result of the hemolytic assay.

FIG. 6 is the bookAntibacterial peptide DN6NH in inventive example 8₂Influence on primary membranes of Aeromonas veronii.

FIG. 7 shows the antibacterial peptide DN6NH in example 9 of the invention₂Influence on the mature membrane of Aeromonas veronii.

FIG. 8 shows the antibacterial peptide DN6NH in example 10 of the present invention₂Influence on the number of Aeromonas veronii in mice.

FIG. 9 shows the antibacterial peptide DN6NH in example 11₂The curative effect on the biomembrane in the body of the mouse.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1 antimicrobial peptide DN6NH₂Design and synthesis of

(1) With N6NH₂Based on the parent peptide sequence, antimicrobial peptide N6NH₂Wherein amino acids other than glycine are substituted for the D form of the amino acid to obtain the D form of N6NH₂So as to improve the stability of the antibacterial peptide to the protease.

(2) The antibacterial peptide is synthesized by a solid-phase synthesis method through a twelve-channel semi-automatic polypeptide synthesizer. Purity of synthetic peptide measured by reverse phase high performance liquid chromatography C18 column>90%), ESI-MS mass spectrum confirmation of derivative peptide DN6NH₂Has a molecular weight of 2474.88Da, DN6NH₂The mass spectrum of (A) is shown in FIG. 1.

Example 2 antimicrobial peptide DN6NH₂Bacteriostasis experiment of

In the examples, the pathogens were selected from China veterinary culture Collection center (CVCC), China Industrial culture Collection center (CICC), American Type Culture Collection (ATCC) and clinical isolates, and the specific strains are shown in Table 1.

Determination of Minimum Inhibitory Concentration (MIC) of antimicrobial peptides reference is made to the methods established by the Clinical and Laboratory Standards Institute (CLSI) (WIEGAND et al, adhesive and broth concentrations (MIC) of antimicrobial sub-sites Nature protocols,2008,3(2):163-175), with minor modifications as the case may be:

picking a single colony of a tested strain into an MHB liquid culture medium, shaking at 37 ℃ and 250rpm, culturing overnight for activation, transferring into the MHB liquid culture medium for culture to logarithmic phase, and preparing into 10⁵CFU/ml of the culture broth was added to a 96-well sterile cell culture plate in an amount of 90. mu.l/well. The antimicrobial peptides were diluted 2-fold by dilution with PBS to give final concentrations of 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 and 0.0625. mu.g/ml antimicrobial peptides at 10. mu.l/well, negative control being PBS test broth and blank control being sterile MH medium. Three replicates were processed each. And placing the culture plate in a constant-temperature incubator at 37 ℃ for incubation for 16-18 h until visible obvious turbid bacterial liquid appears in the negative control hole, wherein the minimum concentration capable of completely inhibiting bacterial growth is the MIC value of the antibacterial peptide on the tested strain. And if the hole jumping or the result inconsistency among the parallel samples occurs, retesting.

The results are shown in Table 1, antimicrobial peptide DN6NH₂The MIC value for Aeromonas veronii is 1.62-25.86 mu M, and the MIC value for Escherichia coli and salmonella is 0.81-6.46 mu M; MIC value for Pseudomonas aeruginosa of 3.23. mu.M; the MIC value of the complex is 1.62-6.46 mu M for staphylococcus aureus and staphylococcus suis, which indicates DN6NH₂Has broad-spectrum antibacterial performance. DN6NH₂With the parent peptide N6NH₂The bacteriostatic effect on gram-negative bacteria is equivalent; DN6NH₂The antibacterial effect on gram-positive bacteria is better than that of parent peptide N6NH₂。

TABLE 1 antimicrobial peptide DN6NH₂MIC value determination of

Note: a is a liver tissue clinical separation strain of micropterus salmoides at a south oral laboratory test base of Chinese agricultural academy of sciences; b is a Tianjin pig farm clinical separation strain; the Aeromonas veronii L1 shows drug resistance to antibiotics such as imipenem, meropenem, tetracycline, trimethoprim and the like, and is highly sensitive to quinolone antibiotics such as ciprofloxacin, norfloxacin, ofloxacin and the like.

Example 3 antimicrobial peptide DN6NH₂In vitro bactericidal kinetics assay for Aeromonas veronii L1

Selecting Aeromonas veronii L1 single colony, inoculating to MHB liquid culture medium, culturing overnight, transferring to 10ml fresh MHB liquid culture medium at 1%, culturing to logarithmic phase (37 deg.C, 250rpm), diluting to 1 × 10⁵CFU/ml, spare. An appropriate amount of the above-mentioned bacterial liquid was taken, and antimicrobial peptide solutions were added to make the final concentrations 1 × MIC, 2 × MIC and 4 × MIC, respectively, and then PBS and Ciprofloxacin (CIP) having the final concentrations 2 × MIC equal in volume were set as negative and positive controls, respectively, and the cells were cultured in a shaker (37 ℃ C., 250 rpm). Then, 100 mu l of bacterial liquid samples are respectively taken at time points of 0 hour, 0.5 hour, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 20 hours and 24 hours, and are coated on a solid agar plate after being diluted according to gradient, inverted and cultured at 37 ℃ for 16-18 hours, colonies are counted, and a time-sterilization curve is drawn.

The result is shown in FIG. 2, DN6NH₂The antibacterial effect of (b) has time and concentration dependence. Wherein 2 × MIC DN6NH₂All the aeromonas veronii L1 were killed within 1 h; by comparison, 2 × MIC N6NH₂All bacteria were killed within 8h, 2 × MIC ciprofloxacin started to rebound after 2h, failing to kill bacteria, indicating DN6NH₂The sterilization efficiency is better than N6NH₂And ciprofloxacin.

Example 4 antimicrobial peptide DN6NH₂Proteolytic stability assay of

Dissolving pepsin (3000U/mg, pH 2.0, Huamei organism), trypsin (250U/mg, pH 8.0, Huamei organism) and proteinase K (40mAU/mg, pH 7.0, Huamei organism) with lyophilized powder of antibacterial peptide at a ratio of 1:10(w/w) in the optimal buffer solution corresponding to the above enzymes, standing at 37 deg.C for incubation for 3h, adding 10 μ l90 μ l log phase (1X 10)⁵CFU/ml) Aeromonas veronii L1 bacterial liquid, and the final concentration of the antibacterial peptide is 128-0.0625 mu g/ml; taking the corresponding buffer solution as a control, incubating in a constant-temperature incubator at 37 ℃ for 16-18 h until obvious turbid bacteria liquid visible to naked eyes appears in negative control holes, and observing the MIC change condition of the peptide after different protease treatments.

The results are shown in Table 2, DN6NH₂After being treated by Pepsin (Pepsin), Trypsin (Trypsin) and proteinase K (protease K), the MIC value of the composition is not changed; parent peptide N6NH₂After being treated by trypsin and proteinase K, the antibacterial activity is lost, which shows that the D-type amino acid replacement modification can obviously improve the parent peptide N6NH₂The capability of resisting protease obviously improves the clinical application value of the antibacterial peptide.

Table 2 antimicrobial peptide DN6NH₂Protease stability assay of

Example 5 antimicrobial peptide DN6NH₂Serum stability assay of

Adding the antibacterial peptide solution into mouse serum (37 ℃) for incubation, respectively incubating for 0min, 30min, 60min, 120 min, 270 min and 360min, and sampling, and detecting the content of the antibacterial peptide remained in the serum by a reversed phase high performance liquid chromatography (RP-HPLC) method.

As a result, as shown in FIG. 3, the serum stability of the antimicrobial peptide was inversely proportional to the incubation time, and DN6NH was found after incubating the antimicrobial peptide in the mouse serum for 6h₂、N6NH₂The peptide content of (A) was 84.25% and 41.93%, respectively, which indicated DN6NH₂The stability in serum is obviously higher than that of the parent peptide N6NH₂。

Example 6 antimicrobial peptide DN6NH₂Determination of cytotoxicity

RAW 264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂And culturing in an incubator under the saturated humidity condition. When the cells grew to log phase, a monolayer of cells was digested with 0.25% trypsin and thenCells were resuspended in DMEM medium containing 10% calf serum at 5X 10⁴The cells were seeded in 96-well plates at a density of 100. mu.l per well; 3-5 auxiliary holes are arranged, and CO is put into the auxiliary holes₂Removing the culture medium after culturing for 24 hours in a constant-temperature incubator; after washing twice with PBS, 100. mu.l of antimicrobial peptide solutions at concentrations of 0.5, 1, 2, 4, 8, 16, 32, 64 and 128. mu.g/ml were added to each well in concentration gradients. Cells without the antimicrobial peptide served as positive controls, and cells without the antimicrobial peptide served as negative controls. After the cells were incubated for 24h, the well medium was aspirated, washed twice with PBS, and 20. mu.l MTT was added to each well at a concentration of 5mg/ml, and placed in an incubator for further incubation for 4 h. The MTT solution is slightly absorbed and discarded, 150 mu l DMSO is added, the micro oscillator oscillates for 10min, and after the crystals at the bottom of the wells are completely dissolved, the absorbance (OD value) of each well is measured at the wavelength of 570nm by the microplate reader. The cell proliferation inhibition Index (IR) was calculated according to the following formula:

survival (%) ═ OD_{Antibacterial peptide}/OD_{Negative control}×100％

As shown in FIG. 4, the cytotoxicity was almost nil at a concentration < 64. mu.g/ml, and DN6NH at a concentration of 128. mu.g/ml₂And N6NH₂The cell survival rates of the cells were 87.33% and 94.38%, respectively, indicating that they had little toxic effect on normal tissue cells of animals at low concentrations.

Example 7 antimicrobial peptide DN6NH₂Measurement of hemolytic activity

Taking a 6-week-old SPF-grade ICR female mouse, taking blood from eyeballs, and collecting by using a heparin sodium anticoagulation tube. The collected blood was centrifuged at 1500rpm at 4 ℃ for 10min, and red blood cells were washed repeatedly three times with 10mM PBS (pH7.3) until the supernatant was colorless and transparent, and diluted to an 8% red blood cell suspension. The antibacterial peptide is dissolved in sterile 0.9% physiological saline to prepare mother liquor with the concentration of 512 mu g/ml, and the mother liquor is diluted to the final concentration of 2 mu g/ml according to the 2-fold ratio. 100 mul of the erythrocyte suspension and antimicrobial peptide solutions with different gradient concentrations are respectively added into a 96-well plate, so that the final concentration of the erythrocytes is 4%. Placing the mixed solution in a constant-temperature incubator at 37 ℃ for standing incubation for 1h, then centrifuging for 5min at 4 ℃ and 1500rpm, absorbing the supernatant into a 96-well plate, and detecting the ultraviolet absorbance value at 540nm by using an enzyme-labeling instrument. Saline and 0.1% Triton X-100 were used as controls for 0% and 100% hemolysis, respectively. The degree of hemolysis is calculated as follows:

hemolysis rate (%) - (Abs540 nm)_{Antibacterial peptide}-Abs540 nm_{Physiological saline})/(Abs540 nm _0.1％Triton _X-100-Abs540 nm_{Physiological saline})]×100％

The results are shown in FIG. 5, DN6NH with a concentration in the range of 1-256. mu.g/ml₂And N6NH₂Has little effect on red blood cells; DN6NH at a concentration of 256. mu.g/ml₂And N6NH₂The hemolysis rates are 1.45% and 0.24%, which shows that the novel antibacterial peptide DN6NH₂And parent peptide N6NH₂Has little hemolytic activity and is safe for intravenous injection.

Example 8 antimicrobial peptide DN6NH₂Determination of Effect on Primary Membrane of Aeromonas veronii L1

Aeromonas veronii L1 was diluted in TSB medium (concentration 1X 10)⁸CFU/ml) and cultured in 96-well plates (200. mu.l of the bacterial suspension per well), followed by addition of DN6NH diluted 2-fold₂、N6NH₂And ciprofloxacin to give final concentrations of 0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC, 8 × MIC, and 16 × MIC, respectively. Wells without added antimicrobial were negative control wells and fresh TSB medium wells were blank control wells. After the culture plate was placed in an incubator at 37 ℃ for 24 hours, the supernatant in each well was carefully aspirated and discarded by a pipette gun, followed by washing with PBS 3 times and air-drying. Subsequently, 200. mu.l of 2.5% glutaraldehyde fixative was added to each well, and after 90min the fixative was discarded and washed twice with PBS. Staining with 0.1% crystal violet 100 μ l for 15min, rinsing off the staining solution with distilled water, drying at room temperature, finally dissolving with 200 μ l 95% ethanol for 30min, and determining the OD of each well at 570 nm.

As a result, as shown in FIG. 6, the primary membrane formation of Aeromonas veronii L1 was reduced to 28.67% (DN6 NH) respectively after the treatment with 16 XMIC antimicrobial peptide₂) And 42.41% (N6 NH)₂) Indicating DN6NH₂The ability to inhibit early membrane formation was higher than the parent peptide, but slightly lower than the antibiotic (19.25%). This indicates DN6NH₂Can obviously inhibit the formation of the primary membrane of the aeromonas veronii L1.

Example 9 antimicrobial peptide DN6NH₂Determination of the Effect on the mature Membrane of Aeromonas veronii L1

Aeromonas veronii L1 was diluted to a concentration of 1X 10 in TSB medium⁸CFU/ml, culturing in 96-well plate, culturing at 37 deg.C for 24 hr to form mature biofilm, and adding DN6NH diluted by 2 times₂、 N6NH₂And ciprofloxacin to final concentrations of 0.5 × MIC, 1 × MIC, 2 × MIC, 4 × MIC, 8 × MIC, and 16 × MIC, respectively, the wells to which the antibacterial substance was not added were negative control wells, and the fresh TSB medium wells were blank control wells, followed by further incubation in an incubator at 37 ℃ for 24 hours. After incubation, the supernatant in each well was carefully aspirated off with a pipette, washed 3 times with PBS, and air dried. Subsequently, 200. mu.l of 2.5% glutaraldehyde fixing solution was added to each well, and after fixing for 90min, the fixing solution was discarded and washed twice with PBS. Staining with 0.1% crystal violet 100 μ l for 15min, rinsing off the staining solution with distilled water, drying at room temperature, finally dissolving with 200 μ l 95% ethanol for 30min, and determining the OD of each well at 570 nm.

The results are shown in FIG. 7, using 16 × MIC DN6NH₂And N6NH₂After the treatment of the mature membranes of the Aeromonas veronii L1, the mature membranes of the Aeromonas veronii L1 were respectively reduced to 8.10% (DN6 NH)₂) And 8.43% (N6 NH)₂). After treatment with 16 × MIC ciprofloxacin, the mature membrane of aeromonas veronii L1 decreased to 10.12%. This indicates DN6NH₂Can inhibit mature biomembrane of Aeromonas veronii L1, and has effect superior to that of parent peptide and ciprofloxacin.

Example 10 antimicrobial peptide DN6NH₂Effect on the amount of Aeromonas veronii in mice

ICR female mice (SPF grade, 20g) were used to construct in vivo biofilm models: firstly, anesthetizing a mouse by using isoflurane, implanting catheters in which a biological membrane of Aeromonas veronii L1 is incubated on two sides of the back of the mouse, suturing wounds by using a suture needle, and infecting the mouse for 24 hours to obtain an in-vivo biological model. Subsequently, the mice were divided into four treatment groups of 6 mice each, and 5. mu. mol/kg DN6NH was injected through the backs of the mice, respectively₂、N6NH₂With ciprofloxacin; the non-treated group of the challenge bacteria was used as a negative control and implanted withoutThe ductal group of bacteria served as a blank (CK). After 7 days, the mice were sacrificed by anesthesia, catheters in the backs of the mice were collected, sufficiently shaken in sterile PBS, and the antimicrobial peptide DN6NH was evaluated by colony counting method₂Influence on the amount of Aeromonas veronii in mice.

The results are shown in FIG. 8, using 5. mu. mol/kg DN6NH₂After treatment, bacterial cells in the catheters were reduced by about 4 orders of magnitude compared to the negative control group, DN6NH₂、N6NH₂The ciprofloxacin treatment group and the negative control group have significant difference, and DN6NH₂Has better sterilization effect than N6NH₂Ciprofloxacin gave the worst bactericidal effect. These results show that the antimicrobial peptide DN6NH₂Not only can inhibit the formation of a biological membrane, but also can kill aeromonas veronii in a mouse body, and the effect is superior to that of parent peptide and ciprofloxacin.

Example 11 antimicrobial peptide DN6NH₂Therapeutic effect on biofilm in mice

The same in vivo biofilm model and drug treatment as in example 10 was constructed using ICR female mice (SPF grade, 20 g). After 7 days, the mice are anesthetized and killed, the obtained skin tissues are fixed by 4% paraformaldehyde, the fixed skin tissues are washed by distilled water for 10-20 min to remove the paraformaldehyde, then ethanol is selected for gradient dehydration, then a mixed solution of absolute ethanol and xylene in a ratio of 1:1 is used for treating for 30min, then the xylene is used for transparentization, the tissues wrapped by the transparentizing agent are subjected to wax dipping, the temperature is reduced to-20 ℃, and the tissues are buried in wax blocks after paraffin is solidified. Slicing the wax block by using a slicer, controlling the thickness to be about 4 mu m, placing the naturally flattened slice on a glass slide, and baking for 15-30 min at 60 ℃ in an oven; placing the slide glass into dimethylbenzene-absolute ethyl alcohol-96% ethyl alcohol-90% ethyl alcohol-80% ethyl alcohol-70% ethyl alcohol in sequence to carry out dewaxing treatment for different times, then carrying out water washing and eosin dyeing by using distilled water, and finally carrying out serial dehydration. And (3) selecting neutral gum for sealing the slices, and drying in an oven at 35-38 ℃. The prepared slides were placed under an OLYMPUS BX43 microscope and the morphology of the mouse skin was observed at magnifications of 100 and 200, respectively.

The results are shown in the figure9, the skin tissue of the mice in the blank Control (CK) group is not abnormal, which indicates that the implantation of the sterile catheter does not influence the skin tissue of the mice; in the negative control group implanted with the Aeromonas veronii L1 biomembrane duct, intradermal lymphocyte infiltration occurs, obvious inflammation appears at local parts, the defect of connection among epidermal cells is obvious, mild sweat gland and sebaceous gland inflammation can be seen locally, fibrous tissues under epidermis are proliferated slightly, the inflammation range is relatively large, and deep soft tissue inflammation appears, which indicates that the Aeromonas veronii biomembrane seriously damages the skin tissues of mice. Using antibacterial peptide DN6NH₂After treatment, the skin condition of the back of the mouse is obviously improved, only a small amount of lymphocyte infiltration (slight inflammation in epidermis) exists in the epidermis, the inflammation is only limited to hypodermal tissue of the epidermis, and deep soft tissue inflammation is not seen; via N6NH₂After treatment, the condition of the skin on the back of the mouse is also improved, only a small amount of lymphocyte infiltration (slight inflammation in epidermis) is seen in the epidermis, the connection between local epidermal cells is damaged, the inflammation is limited in the hypodermal tissue of the epidermis, and the deep soft tissue inflammation is not seen; in the ciprofloxacin treatment group, the skin of the back of the mouse is also improved, only a small amount of lymphocyte infiltration (slight inflammation in epidermis) is seen in the epidermis, the connection between local epidermal cells is damaged, fibrous tissues under the epidermis are slightly proliferated, the inflammation is limited to dermal tissues under the epidermis, and deep soft tissue inflammation is not seen. Thus, DN6NH for in vivo experiments with Aeromonas veronii biofilms₂Not only has better bactericidal effect, but also can obviously improve the inflammatory condition of the back skin of the mouse, and the effect is better than that of the parent peptide and the antibiotic.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> institute of feed of Chinese academy of agricultural sciences

<120> antimicrobial peptide DN6NH2 and application thereof

<130> KHP201111121.0

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gly Phe Ala Trp Asn Val Cys Val Tyr Arg Asn Gly Val Arg Val Cys

1 5 10 15

His Arg Arg Ala Asn

20

Claims

1. Antibacterial peptide DN6NH₂The application in preparing biological products for treating or preventing infection caused by Aeromonas veronii (Aeromonas veronii) and biomembrane thereof and related diseases caused by the infection;

the antibacterial peptide DN6NH₂The antibacterial peptide is obtained by amidating and modifying a carboxyl group at the C terminal of antibacterial peptide N6, and the rest amino acids except glycine are D-type amino acids;

antibacterial peptide DN6NH₂The structure of (A) is as follows:

2. the use according to claim 1, wherein the disease comprises subcutaneous abscesses caused by biofilms produced by aeromonas veronii.