An antimicrobial peptide and its application

Abstract

This invention relates to an antimicrobial peptide and its applications. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No. 1. The antimicrobial peptide exhibits excellent broad-spectrum antimicrobial activity, for example, against Staphylococcus spp. (…). Staphylococcus Acinetobacter spp. Acinetobacter ) and Bacillus spp. ( Bacillus This drug possesses antibacterial activity against bacteria. Against the backdrop of increasingly severe global bacterial resistance and the gradual ineffectiveness of traditional antibiotics against multidrug-resistant bacteria, this provides a new treatment option for bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii, and Bacillus subtilis, which pose a significant threat to public health. It also offers new possibilities for resolving the clinical dilemma of "no available drugs."

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to an antimicrobial peptide and its applications. Background Technology

[0002] Staphylococcus aureus ( 金黄色葡萄球菌 Methicillin-resistant Staphylococcus aureus (MRSA), a common Gram-positive opportunistic pathogen, can invade the human body through skin and mucous membrane lesions, the respiratory tract, and other routes, causing a variety of diseases ranging from skin and soft tissue infections (such as abscesses and cellulitis) to serious systemic infections (such as pneumonia, sepsis, endocarditis, and osteomyelitis). 金黄色葡萄球菌 MRSA, as its most threatening branch, is due to carrying... mecA The gene encoding penicillin-binding protein 2a (PBP2a) exhibits natural resistance to many commonly used antibiotics, including β-lactams (such as methicillin, penicillin, and cephalosporins), completely overturning traditional treatment logic. More seriously, some MRSA strains have shown reduced sensitivity to "last-line" drugs such as vancomycin (e.g., VISA / VRSA), resulting in extremely limited clinical treatment options. In terms of transmission scenarios, MRSA is divided into hospital-acquired MRSA (HA-MRSA) and community-acquired MRSA (CA-MRSA). HA-MRSA has an infection rate as high as 35% in intensive care units and postoperative wards, posing a particular threat to immunocompromised populations; CA-MRSA is also becoming increasingly prevalent, infecting healthy individuals through daily contact and causing rapidly progressing necrotizing pneumonia or sepsis.

[0003] Acinetobacter baumannii ( 鲍曼不动杆菌Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is renowned for its extremely strong environmental adaptability (it can survive on dry surfaces for more than 30 days) and its ability to colonize medical environments, earning it the nickname "nightmare bacterium" in the medical community. It is primarily transmitted through contact and can easily cause outbreaks in intensive care units, burn units, and neurosurgery departments, leading to ventilator-associated pneumonia (VAP), bloodstream infections, wound infections, urinary tract infections, and meningitis. It poses a particularly significant threat to mechanically ventilated patients, burn patients, and those with weakened immune systems. Its most worrying aspect is its drug resistance: listed by the WHO as a "critical priority pathogen requiring novel antibiotics," Acinetobacter baumannii can develop resistance to β-lactams, aminoglycosides, quinolones, and other antibiotics through multiple mechanisms, including acquiring resistance genes, altering membrane permeability, and producing inactivating enzymes. Furthermore, pan-drug-resistant strains (PDR-AB) resistant to almost all commonly used antibiotics have emerged. Global drug resistance surveillance data from 2024 showed that Acinetobacter baumannii had a resistance rate of 78% to carbapenems, and cases of resistance to "last resort" drugs such as colistin and tigecycline were also increasing year by year, leading to multiple clinical dilemmas of "no drugs available." In addition, its strong ability to spread drug resistance genes (such as through plasmid and transposon transfer) makes it a "number one challenge" in hospital infection control.

[0004] Bacillus subtilis ( 枯草芽孢杆菌 As a Gram-positive facultative anaerobic bacillus, Bacillus subtilis possesses a unique dual nature: on the one hand, it is widely distributed in soil, water, air, and on the surfaces of plants and animals. Due to its clear genetic background and ease of cultivation, it has become a "model bacterium" in microbiology and molecular biology research, and is often used as a primary screening model in antibacterial agent development (its cell wall structure and membrane permeability are highly similar to pathogenic Gram-positive bacteria such as MRSA, effectively reflecting the effect of compounds on Gram-positive bacterial targets); on the other hand, as an opportunistic pathogen, it can also pose an infection risk under certain conditions. Clinically, Bacillus subtilis infection is more common in individuals with severely compromised immune function, those receiving long-term broad-spectrum antibiotic treatment, or those with damaged skin and mucous membrane barriers, and can lead to sepsis, endocarditis, pneumonia, meningitis, and eye infections. In the medical environment, it may contaminate medical devices, biological products, or pharmaceuticals, causing iatrogenic infections. More importantly, it exhibits natural resistance to some traditional antibiotics, and its spore structure is highly resistant to physical and chemical disinfection, making it difficult to eradicate completely. This poses a challenge to treatment and also provides a potential vector for the horizontal transfer of resistance genes. Therefore, research on the inhibition of Bacillus subtilis has dual value in both clinical treatment and basic research. Summary of the Invention

[0005] One aspect of the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0006] The second invention provides a composition comprising an antimicrobial peptide as described in the first invention and a pharmaceutically acceptable carrier.

[0007] The third invention provides the use of the antimicrobial peptide according to the first invention or the composition according to the second invention in the preparation of antibacterial and / or bactericidal drugs.

[0008] In one specific embodiment, the application is the use of the antimicrobial peptide in the preparation of a drug for inhibiting and / or killing bacteria.

[0009] In one specific embodiment, the bacteria is Staphylococcus spp. ( 葡萄球菌属 Acinetobacter spp. 不动杆菌属 ) and Bacillus spp. ( 芽孢杆菌属 At least one of the bacteria in ).

[0010] In one specific embodiment, the bacteria is Staphylococcus aureus (Staphylococcus aureus). 金黄色葡萄球菌 Acinetobacter baumannii ( 鲍曼不动杆菌 ) and Bacillus subtilis ( 枯草芽孢杆菌 At least one of the bacteria in ).

[0011] In one specific embodiment, the bacteria is methicillin-resistant Staphylococcus aureus. 金黄色葡萄球菌 ).

[0012] In one specific embodiment, the bacteria is at least one of methicillin-resistant Staphylococcus aureus ATCC BAA-1026 strain, Acinetobacter baumannii ATCC BAA-1605 strain, and Bacillus subtilis CMCC (B) 63501 strain.

[0013] In one specific embodiment, the application is that the antimicrobial peptide is used in the preparation of a treatment for Staphylococcus aureus (Staphylococcus) 葡萄球菌属 ), especially Staphylococcus aureus ( 金黄色葡萄球菌 For example, methicillin-resistant Staphylococcus aureus (MRSA). 金黄色葡萄球菌 The drug is used for at least one of the following diseases: skin and soft tissue infections (such as abscesses, cellulitis), pneumonia, sepsis, endocarditis, and osteomyelitis caused by SARS-CoV-2.

[0014] In one specific embodiment, the application is that the antimicrobial peptide is used in the preparation of a treatment for Acinetobacter (Acinetobacter) 不动杆菌属 ), especially Acinetobacter baumannii ( 鲍曼不动杆菌 The drug is used for at least one of the following diseases: ventilator-associated pneumonia (VAP), bloodstream infection, wound infection, urinary tract infection, and meningitis caused by ventilator-associated pneumonia (VAP).

[0015] In one specific embodiment, the application is that the antimicrobial peptide is used in the preparation of a treatment for Bacillus subtilis (Bacillus) 芽孢杆菌属 ), especially Bacillus subtilis ( 枯草芽孢杆菌 The drug is used in the treatment of at least one of the following diseases: sepsis, endocarditis, pneumonia, meningitis, and eye infection caused by septicemia.

[0016] Beneficial effects of the present invention: The present invention has discovered that the antimicrobial peptides of the present invention have excellent broad-spectrum antimicrobial activity, for example, against Staphylococcus spp. ( 葡萄球菌属 (especially methicillin-resistant Staphylococcus aureus), Acinetobacter spp. ( 不动杆菌属 (especially Acinetobacter baumannii) and Bacillus spp. ( 芽孢杆菌属 The antimicrobial peptides (especially Bacillus subtilis) exhibit antibacterial activity, demonstrating micromolar-level killing efficacy against MRSA and carbapenem-resistant Acinetobacter baumannii. Against the backdrop of increasingly severe global bacterial resistance and the gradual ineffectiveness of traditional antibiotics against multidrug-resistant bacteria, this provides a new treatment option for bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii, and Bacillus subtilis, which pose a significant threat to public health, and offers new possibilities for overcoming the clinical dilemma of "no available drugs." Furthermore, the antimicrobial peptides also possess the excellent characteristic of low resistance risk. Attached Figure Description

[0017] Figure 1 The survival rates of methicillin-resistant Staphylococcus aureus ATCC BAA-1026 after treatment with various concentrations of antimicrobial peptides are shown.

[0018] Figure 2 The survival rates of Acinetobacter baumannii ATCC BAA-1605 after treatment with various concentrations of antimicrobial peptides are shown.

[0019] Figure 3 The survival rates of Bacillus subtilis CMCC (B) 63501 after treatment with various concentrations of antimicrobial peptides are shown.

[0020] Figure 4 The survival rates of *Staphylococcus aureus* infected with methicillin-resistant Staphylococcus aureus (MRSA) were shown after treatment with various concentrations of antimicrobial peptides.

[0021] Figure 5 The table shows the levels of methicillin-resistant Staphylococcus aureus (MRSA) in the hemolymph of *Staphylococcus aureus* infected with various concentrations of antimicrobial peptides. ***: P <0.001.

[0022] Figure 6 The survival rates of *Acinetobacter baumannii*-infected *Ceratophyllum demersum* were shown when treated with various concentrations of antimicrobial peptides.

[0023] Figure 7 The levels of Acinetobacter baumannii in the hemolymph of *Acinetobacter baumannii* infected with various concentrations of antimicrobial peptides are shown. ***: P <0.001. Detailed Implementation

[0024] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0025] Unless otherwise specified, the strains and reagents used in the embodiments of this invention can be purchased commercially.

[0026] The amino acid sequence of the antimicrobial peptide of the present invention is shown in SEQ ID No. 1, and was synthesized by Sangon Biotech Co., Ltd.

[0027] Methicillin-resistant Staphylococcus aureus 金黄色葡萄球菌 MRSA) ATCC BAA-1026 and Acinetobacter baumannii ( 鲍曼不动杆菌 The ATCC BAA-1605 strain was purchased from the China Industrial Microbial Culture Collection Center.

[0028] Bacillus subtilis ( 枯草芽孢杆菌 CMCC (B) 63501 was purchased from Beijing Solarbio Technology Co., Ltd.

[0029] Greater wax borer ( 大蜡螟 The feed was purchased from Qingdao Keyun Biotechnology Co., Ltd.

[0030] Tryptone-Soy Agar (TSA) medium: 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, 15 g agar, distilled water to a final volume of 1 L, adjust pH to 7.2 ± 0.2. Autoclave at 121°C for 20 minutes.

[0031] Tryptone-Soybean Broth (TSB) medium: 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, distilled water to a final volume of 1 L, adjust pH to 7.2 ± 0.2. Autoclave at 121°C for 20 minutes. Example 1

[0032] Antimicrobial peptides were dissolved in sterile distilled water to prepare aqueous solutions of antimicrobial peptides with concentrations of 1.25, 2.5, 5, 10, and 20 mmol / L.

[0033] Methicillin-resistant Staphylococcus aureus (ATCC) BAA-1026, stored at -80°C, was streaked onto TSA agar plates and incubated at 37°C for 16 hours to activate the strain. A single colony was picked and inoculated into an Erlenmeyer flask containing 20 ml of TSB medium, and cultured at 37°C and 180 rpm with shaking for 16 hours to prepare a seed culture. 1 ml of the seed culture was inoculated into 100 ml of TSB medium at a 1:100 volume ratio and cultured at 37°C and 180 rpm with shaking until the bacterial growth rate reached OD500. 600 When the pH reaches 0.8 (incubation time approximately 3 to 4 hours), the propagation bacterial suspension is prepared. The concentration of the propagation bacterial suspension is determined using a McFarland turbidimeter and diluted to 1×10⁻⁶ with TSB medium. 6 CFU / ml was used to obtain a diluted bacterial solution.

[0034] 999 μL of diluted bacterial culture was added to each well of a 48-well plate, followed by the addition of an antimicrobial peptide aqueous solution to achieve final concentrations of 1.25, 2.5, 5, 10, and 20 μmol / L, respectively. An equal volume of sterile distilled water was added as a blank control. The mixture was thoroughly mixed. Each treatment was replicated in 5 wells. After incubating the 48-well plate at 37°C for 16 h, the OD of the bacterial culture in each well was measured. 600 The value was then used to calculate the survival rate based on formula (1), and the results are shown in [the table]. Figure 1 .

[0035] Survival rate = OD of antimicrobial peptide treatment groups at different concentrations 600 Value × 100% / OD of blank control group 600 Value formula (1)

[0036] Based on the mortality rates of the antimicrobial peptide treatment groups at various concentrations, a regression equation between antimicrobial peptide concentration and mortality rate was obtained. Then, based on the regression equation, the antimicrobial peptide concentration (half-inhibitory concentration, IC50) at which the mortality rate was 50% was calculated. 50 And the concentration of antimicrobial peptides at which the mortality rate is 100% (minimum inhibitory concentration, MIC).

[0037] The results showed that the antimicrobial peptide had an IC50 of [missing information - likely related to methicillin-resistant Staphylococcus aureus]. 50 It is 1.3 μmol / L, and the MIC is 10 μmol / L. Example 2

[0038] In Example 1, methicillin-resistant Staphylococcus aureus ATCC BAA-1026 was replaced with Acinetobacter baumannii ATCC BAA-1605. The concentration of the antimicrobial peptide aqueous solution was adjusted appropriately, while everything else remained the same as in Example 1. After incubation at 37°C for 16 hours, the OD of each well was measured. 600 The value was then used to calculate the survival rate based on formula (1), and the results are shown in [the table]. Figure 2.

[0039] Based on the mortality rates of the antimicrobial peptide treatment groups at various concentrations, a regression equation between antimicrobial peptide concentration and mortality rate was obtained. Then, based on the regression equation, the antimicrobial peptide concentration (half-inhibitory concentration, IC50) at which the mortality rate was 50% was calculated. 50 The study also determined the concentration of the antimicrobial peptide at which the mortality rate was 100% (the minimum inhibitory concentration, MIC). The results showed that the antimicrobial peptide had an IC50 concentration of [missing information - likely related to Acinetobacter baumannii]. 50 It is 7.5 μmol / L, and the MIC is 18 μmol / L. Example 3

[0040] In Example 1, methicillin-resistant Staphylococcus aureus ATCC BAA-1026 was replaced with Bacillus subtilis CMCC (B) 63501. The concentration of the antimicrobial peptide aqueous solution was adjusted appropriately, while everything else remained the same as in Example 1. After incubation at 37°C for 16 hours, the OD of each well was measured. 600 The value was then used to calculate the survival rate based on formula (1), and the results are shown in [the table]. Figure 3 .

[0041] Based on the mortality rates of the antimicrobial peptide treatment groups at various concentrations, a regression equation between antimicrobial peptide concentration and mortality rate was obtained. Then, based on the regression equation, the antimicrobial peptide concentration (half-inhibitory concentration, IC50) at which the mortality rate was 50% was calculated. 50 ) and the concentration of antimicrobial peptides at which the mortality rate is 100% (minimum inhibitory concentration MIC).

[0042] The results showed that the antimicrobial peptide had an IC50 value against Bacillus subtilis. 50 It is 3 μmol / L, and the MIC is 5 μmol / L. Example 4

[0043] Antimicrobial peptides were dissolved in sterile distilled water to prepare aqueous solutions of 2, 10, and 50 micrograms per milliliter.

[0044] Vancomycin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was dissolved in sterile distilled water to prepare a 50 microgram / mL vancomycin aqueous solution.

[0045] A propagation broth of methicillin-resistant Staphylococcus aureus (ATCC) BAA-1026 was prepared using the same method as in Example 1. The concentration of the propagation broth was determined using a McFarland turbidimeter and diluted to 2.25 × 10⁻⁶ with TSB medium. 8 CFU / ml was used to obtain a diluted bacterial solution.

[0046] Each large wax moth was weighed, and those weighing 0.5 ± 0.02 g were selected. 40 μL of diluted bacterial solution was injected into the left leg of the last segment of each large wax moth, resulting in an injection volume of 9 × 10⁻⁶ bacteria per moth. 6CFU was used to infect the large wax moth at 37°C for 2 hours (during which the moths had free access to feed) to obtain large wax moths infected with MRSA. A blank control group consisting of large wax moths not infected with MRSA was prepared by injecting 40 μL of TSB culture medium.

[0047] Blank control group: 20 μL of sterile distilled water was injected into the left leg of the last segment of the large wax moth that was not infected with MRSA using a syringe.

[0048] The MRSA-infected giant wax moths were randomly divided into 5 groups, with 10 giant wax moths in each group (n=10).

[0049] NC group: Inject 20 microliters of sterile distilled water into the left leg of the last segment of the giant wax moth using a syringe.

[0050] Positive control group: 20 μL of vancomycin aqueous solution (50 μg / mL) was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of vancomycin to 2 mg / kg.

[0051] Treatment Group-1: 20 μL of an antimicrobial peptide aqueous solution at 2 μg / mL was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of the antimicrobial peptide to 0.08 mg / kg.

[0052] Treatment Group 2: 20 μL of an antimicrobial peptide aqueous solution at 10 μg / mL was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of the antimicrobial peptide to 0.4 mg / kg.

[0053] Treatment Group-3: 20 μL of 50 μg / mL antimicrobial peptide aqueous solution was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of the antimicrobial peptide to 2 mg / kg.

[0054] The blank control group, NC group, positive control group, treatment group-1, treatment group-2, and treatment group-3 were all placed in a 37°C constant temperature incubator (with free access to food). Photos were taken every 12 hours, and the survival rate of the *Hemiberlesia lataniae* in each group was recorded. The results are shown below. Figure 4 .

[0055] according to Figure 4 The results showed that, compared with the NC group, the antimicrobial peptide at a concentration of 0.08 mg / kg significantly improved the survival rate of MRSA-infected giant wax moths; when the concentration was 0.4 mg / kg, the survival rate of MRSA-infected giant wax moths reached 60% after 72 hours of antimicrobial peptide treatment, which was comparable to the positive control vancomycin. Example 5

[0056] Naphthylpyridinol acid solution: Naphthylpyridinol acid (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was dissolved in sterile physiological saline and filtered through a 0.22-micron bacterial filter membrane to obtain a 20 μg / mL naphthylpyridinol acid solution. It had no inhibitory effect on Staphylococcus aureus, but it could inhibit the growth of other bacteria.

[0057] The same procedures as in Example 4 were followed to obtain both uninfected and infected *Gnaphalium affine*. As in Example 4, blank control group, NC group, positive control group, treatment group-1, treatment group-2, and treatment group-3 were established. Each group of *Gnaphalium affine* was placed in a 37°C constant temperature incubator (with free access to food) and cultured for 36 hours.

[0058] Six live *Eriocheir sinensis* worms (n=6) were selected from each treatment group. The last segment of the tail of the worms was cut off with scissors, and the exudate (hemolymph) was collected. 100 μL of hemolymph was pipetted into 900 μL of naphthylpyridinic acid solution containing 20 μg / mL to obtain a hemolymph-naphthylpyridinic acid mixture. The hemolymph-naphthylpyridinic acid mixture was serially diluted with physiological saline to obtain each gradient dilution. 100 μL of each gradient dilution was plated onto TSA medium, with five plates for each gradient. The plates were incubated at 37°C for 18 h, and then colony counting was performed (preferably 30 to 300 CFU / plate). The MRSA content in the *Eriocheir sinensis* hemolymph was calculated based on the colony count and dilution gradient, and plotted using Graphpad. (See [link to Graphpad documentation]). Figure 5 (The target bacteria were not detected in the blank control group, so they are not shown in the figure.)

[0059] according to Figure 5 The results showed that, compared with the NC group, the antimicrobial peptides at a dosage concentration of 0.08 mg / kg or higher could significantly reduce the bacterial content in the hemolymph of *Eriocheir sinensis*, and were comparable to the positive control vancomycin. Example 6

[0060] Antimicrobial peptides were dissolved in sterile distilled water to prepare aqueous solutions of 2, 10, and 50 micrograms per milliliter.

[0061] Polymyxin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was dissolved in sterile distilled water to prepare a 50 μg / mL polymyxin aqueous solution.

[0062] Acinetobacter baumannii ATCC BAA-1605 was prepared using the same method as in Example 1. The concentration of the culture was determined using a McFarland turbidimeter and diluted to 2.25 × 10⁻⁶ with TSB medium. 8 CFU / ml was used to obtain a diluted bacterial solution.

[0063] Each large wax moth was weighed, and those weighing 0.5 ± 0.02 g were selected. 40 μL of diluted bacterial solution was injected into the left leg of the last segment of each large wax moth, resulting in an injection volume of 9 × 10⁻⁶ bacteria per moth. 6 CFU was used to infect the large wax moth at 37°C for 2 hours (during which the moths had free access to feed) to obtain large wax moths infected with Acinetobacter baumannii. A blank control group consisting of large wax moths not infected with Acinetobacter baumannii was prepared by injecting 40 μL of TSB medium.

[0064] Blank control group: 20 μL of sterile distilled water was injected into the left leg of the last segment of the large wax borer that was not infected with Acinetobacter baumannii using a syringe.

[0065] The large wax moths infected with Acinetobacter baumannii were randomly divided into 5 groups, with 10 large wax moths in each group (n=10).

[0066] NC group: Inject 20 microliters of sterile distilled water into the left leg of the last segment of the giant wax moth using a syringe.

[0067] Positive control group: 20 μL of 50 μg / mL polymyxin aqueous solution was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of polymyxin to 2 mg / kg.

[0068] Treatment Group-1: 20 μL of an antimicrobial peptide aqueous solution at 2 μg / mL was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of the antimicrobial peptide to 0.08 mg / kg.

[0069] Treatment Group 2: 20 μL of an antimicrobial peptide aqueous solution at 10 μg / mL was injected into the left leg of the last segment of the giant wax moth using a syringe to bring the final concentration of the antimicrobial peptide to 0.4 mg / kg.

[0070] Treatment Group 3: 20 μL of 50 μg / mL antimicrobial peptide aqueous solution was injected into the left leg of the last segment of the giant wax moth using a syringe to achieve a final concentration of 2 mg / kg of antimicrobial peptide.

[0071] The *Hemibarbus thuringiensis* from the blank control group, NC group, positive control group, treatment group-1, treatment group-2, and treatment group-3 were placed in a 37°C constant temperature incubator (with free access to food). Photos were taken every 12 hours, and the survival rate of the *Hemibarbus thuringiensis* in each group was calculated. The results are shown below. Figure 6 .

[0072] according to Figure 6 The results showed that, compared with the NC group, the antimicrobial peptide at a dosage concentration of 0.08 mg / kg could improve the survival rate of Acinetobacter baumannii infected with the giant wax borer; at a dosage concentration of 2 mg / kg, the survival rate of the giant wax borer after 72 hours reached 80%, which was much higher than the 50% survival rate of the positive control polymyxin. Example 7

[0073] Kanamycin solution: Kanamycin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) was dissolved in sterile physiological saline and filtered through a 0.22-micron bacterial membrane to obtain a 50 μg / mL kanamycin solution. It had no inhibitory effect on Acinetobacter baumannii, but it could inhibit the growth of other bacteria.

[0074] The same procedures as in Example 6 were followed to obtain both uninfected and infected *Acinetobacter baumannii* *Gnaphalium affine* borers. As in Example 6, blank control group, NC group, positive control group, treatment group-1, treatment group-2, and treatment group-3 were established. Each group of *Gnaphalium affine* borers was placed in a 37°C constant temperature incubator (with free access to food) and cultured for 36 hours.

[0075] Six live *Eriocheir sinensis* worms (n=6) were selected from each treatment group. The last segment of the tail of the worms was cut off with scissors, and the exudate (hemolymph) was collected. 100 μL of hemolymph was pipetted into 900 μL of a kanamycin solution containing 50 μg / mL to obtain a hemolymph-kanamycin mixture. The hemolymph-kanamycin mixture was serially diluted with physiological saline to obtain various dilutions. 100 μL of each dilution was plated onto TSA medium, with five plates for each dilution. The plates were incubated at 37°C for 18 h, and then colony counting was performed (preferably 30 to 300 CFU / plate). The bacterial content of *Acinetobacter baumannii* in the *Eriocheir sinensis* hemolymph was calculated based on the colony count and dilution gradient, and plotted using Graphpad. (See [link to Graphpad documentation]). Figure 7 (The target bacteria were not detected in the blank control group, so they are not shown in the figure.)

[0076] according to Figure 7 The results showed that, compared with the NC group, the antimicrobial peptide at a dosage of 0.08 mg / kg or higher could significantly reduce the bacterial content in the hemolymph of *Eriocheir sinensis*, and was comparable to the positive control polymyxin.

[0077] Bacillus subtilis is not pathogenic to the giant wax borer, therefore no infection model was constructed.

Claims

1. An antimicrobial peptide having the amino acid sequence shown in SEQ ID No.

1.

2. A composition comprising the antimicrobial peptide as described in claim 1 and a pharmaceutically acceptable carrier.

3. The use of the antimicrobial peptide according to claim 1 or the composition according to claim 2 in the preparation of a drug for inhibiting and / or killing bacteria, wherein the bacteria is Staphylococcus aureus (… Staphylococcus aureus Acinetobacter baumannii ( Acinetobacter baumannii ) and Bacillus subtilis ( Bacillus subtilis At least one of the bacteria in ).

4. The application according to claim 3, characterized in that, The bacteria were methicillin-resistant Staphylococcus aureus. Staphylococcus aureus ).

5. The application according to claim 3, characterized in that, The bacteria are at least one of methicillin-resistant Staphylococcus aureus ATCC BAA-1026 strain, Acinetobacter baumannii ATCC BAA-1605 strain, and Bacillus subtilis CMCC (B) 63501 strain.

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