Antimicrobial peptide AS-12W and its preparation method and use

Abstract

The present invention discloses an antimicrobial peptide AS-12W derived from Chinese alligator, wherein the amino acid sequence of the antimicrobial peptide AS-12W is as shown in SEQ ID NO: 1 or an amino acid sequence having more than 90% homology thereto. The present invention also discloses a preparation method of the antimicrobial peptide and its application. The antimicrobial peptide AS-12W of the present invention has good in vitro and in vivo antibacterial and bactericidal activity against drug-resistant bacteria, and can be used to treat diseases related to drug-resistant bacterial infection. It has the characteristics of a broad antimicrobial spectrum and low hemolytic activity, and can be applied to the fields of medicine, cosmetics, food, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to an antimicrobial peptide AS-12W and a preparation method and use thereof. Background Art

[0002] The emergence of antibiotics has not only saved the lives of patients, but also played a key role in achieving major advances in medicine and surgery. However, the increased use of antibiotics has led to the emergence of drug-resistant bacteria, and antibiotic resistance has become one of the biggest threats to global public health. In fact, at least more than 700,000 people die from drug-resistant bacterial infections every year worldwide. With the emergence of drug-resistant bacteria, many antibiotics have become ineffective and the antibiotic pipeline has been exhausted. Over the past three decades, the number of new antibiotics developed has steadily declined, antibiotic research has declined, but drug-resistant pathogens are increasing worldwide. This will lead to a future where drug-resistant bacteria will be untreatable, so new antibiotics are urgently needed to fight bacterial infections.

[0003] Antimicrobial peptides (AMPs) are a diverse group of small bioactive proteins that are part of the first line of defense for inactivating pathogens in the human body. They work by disrupting bacterial cell membranes, modulating immune responses, and regulating inflammation. Antimicrobial peptides are rapidly gaining attention as a new class of antimicrobial agents with far-reaching clinical potential. Antimicrobial peptides usually contain less than 100 amino acids, including many positively charged residues (such as lysine, arginine, and histidine) and most hydrophobic residues (>50%). Antimicrobial peptides interact with bacterial cell membranes through electrostatic interactions. The hydrophobic part can effectively interact with the hydrophobic interior of the bacterial cell membrane. After reaching a certain threshold concentration, AMPs can induce pore formation and destroy the membranes of bacterial organelles when entering the cell. In addition, AMPs are reported to inhibit protein and nucleic acid synthesis, block enzyme activity, or induce apoptosis-like cell death by producing reactive oxygen species. Because AMPs have multiple functions in killing microorganisms, unlike traditional antibiotics, it is difficult for bacteria to develop resistance. Summary of the invention

[0004] Purpose of the invention: The present invention mines the amino acid sequence of Cathelicidin in the biological proteome database by sequence alignment of the proteome database of the Yangtze alligator (Alligator sinensis). Further, after multiple sequence alignment, the mature peptide sequence of Cathelicidin was intercepted and analyzed to obtain AS-12 (IKKGFKKIFKRL), the full sequence of which is: Isoleucine-Lysine-Lysine-Glycine-Phenylalanine-Lysine-Lysine-Isoleucine-Phenylalanine-Lysine-Arginine-Leucine. Using AS-12 as a template, the glycine at the 4th position, which is not conducive to the formation of α-helix, is replaced with hydrophobic tryptophan, i.e., AS-12W, which is a peptide with good in vitro and in vivo antibacterial activity against drug-resistant bacteria. Therefore, the technical problem to be solved by the present invention is to provide a polypeptide AS-12W with good in vitro and in vivo antibacterial activity against drug-resistant bacteria.

[0005] The technical problem that the present invention also solves is to provide the use of the antimicrobial peptide AS-12W in preparing products for preventing and / or treating diseases caused by bacterial infections and in preparing drugs for preventing and / or treating sepsis, septicemia and related diseases.

[0006] Technical solution: In order to solve the above technical problems, the present invention provides an antimicrobial peptide AS-12W, the amino acid sequence of which is shown in SEQ ID: NO: 1 or an amino acid sequence having more than 90% homology thereto. It is composed of 12 amino acids, with an amino acid sequence of IKKWFKKIFKRL and a molecular weight of 1635.12. The full sequence is: isoleucine-lysine-lysine-tryptophan-phenylalanine-lysine-lysine-isoleucine-phenylalanine-lysine-arginine-leucine.

[0007] The present invention also includes a method for preparing the antimicrobial peptide AS-12W, which comprises the following steps:

[0008] 1) By comparing the sequence of the Yangtze alligator proteome database, the cathelicidin amino acid sequence in the biological proteome database was mined, and the mature peptide sequence of cathelicidin was further analyzed by multiple sequence alignment to obtain a peptide named AS-12, which was obtained by solid phase synthesis.

[0009] 2) Using AS-12 as a template, the glycine at position 4, which is not conducive to the formation of the α-helix, was replaced with a hydrophobic tryptophan. The modified peptide was AS-12W, which was obtained by solid phase synthesis.

[0010] The sequence of polypeptide AS-12 is shown in SEQ ID: NO: 2. Its sequence is: isoleucine-lysine-lysine-glycine-phenylalanine-lysine-lysine-isoleucine-phenylalanine-lysine-arginine-leucine.

[0011] The present invention also includes the use of the antimicrobial peptide AS-12W in preparing products for preventing and / or treating diseases caused by bacterial infections.

[0012] Wherein, the bacteria include Gram-negative bacteria, Gram-positive bacteria and / or fungi.

[0013] Wherein, the bacteria are drug-resistant bacteria.

[0014] Among them, the Gram-negative bacteria include one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Helicobacter pylori, the Gram-positive bacteria include one or more of Staphylococcus epidermidis and Staphylococcus aureus, and the fungi include Candida albicans.

[0015] Among them, the products include bactericidal, bacteriostatic or antibacterial products.

[0016] The in vitro antibacterial activity study of the present invention shows that the polypeptide AS-12W of the present invention replaces the glycine at the 4th position of the parent peptide AS-12, which is not conducive to the formation of α-helix, with hydrophobic tryptophan, which can significantly improve the antibacterial activity of the parent peptide and has a wider antibacterial spectrum. It shows good antibacterial effects on Gram-negative bacteria resistant to ciprofloxacin, ceftazidime, and meropenem and Gram-positive bacteria resistant to penicillin and ciprofloxacin.

[0017] The results of the in vitro bactericidal curve showed that the peptide AS-12W exhibited good bactericidal activity against clinical drug-resistant bacteria. It showed a good bactericidal effect on meropenem-resistant Pseudomonas aeruginosa, killing Pseudomonas aeruginosa in 0.5h. The clinical drug-resistant bacteria included Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) and Gram-positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis).

[0018] In vitro toxicity studies have shown that the hemolytic activity of the polypeptide AS-12W of the present invention on sheep erythrocytes at a concentration of 512 μg / ml is low, less than 5%. Within the effective range of AS-12W, the hemolytic activity can be ignored.

[0019] The present invention also includes the use of the antimicrobial peptide AS-12W in the preparation of drugs for preventing and / or treating sepsis, septicemia and related diseases.

[0020] Wherein, the concentration of the antimicrobial peptide AS-12W is 0.5-512 μg / ml.

[0021] The present invention uses a mouse abdominal infection bacteria to construct a sepsis model to study the in vivo antibacterial activity of the polypeptide AS-12W. The results show that the polypeptide AS-12W of the present invention can significantly improve the survival rate of sepsis mice. In addition, the polypeptide of the present invention can also significantly reduce the bacterial load in the blood and tissue organs.

[0022] Wherein, the drug-resistant strain is Pseudomonas aeruginosa, and the tissue organs are mouse lungs, liver and spleen.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the antimicrobial peptide AS-12W provided by the present invention has good in vitro and in vivo antibacterial activity, can kill a variety of drug-resistant bacteria, and can be used to prepare drugs for treating drug-resistant bacterial infections. The antimicrobial peptide AS-12W of the present invention has the characteristics of a broad antibacterial spectrum and low hemolytic activity, and can be used in the fields of medicine, cosmetics, food, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the HPLC spectrum of the antimicrobial peptide AS-12W of the present invention;

[0025] Figure 2 The mass spectrum of the antimicrobial peptide AS-12W of the present invention;

[0026] Figure 3 This is the bactericidal curve of the antimicrobial peptide AS-12W of the present invention against Escherichia coli ATCC25922;

[0027] Figure 4 The bactericidal curve of the antimicrobial peptide AS-12W of the present invention against ceftazidime-resistant Escherichia coli;

[0028] Figure 5 The bactericidal curve of the antimicrobial peptide AS-12W of the present invention against meropenem-resistant Pseudomonas aeruginosa;

[0029] Figure 6 The bactericidal curve of the antimicrobial peptide AS-12W of the present invention against ceftazidime-resistant Klebsiella pneumoniae;

[0030] Figure 7 This is the bactericidal curve of the antimicrobial peptide AS-12W of the present invention against Staphylococcus aureus ATCC29213;

[0031] Figure 8 This is the bactericidal curve of the antimicrobial peptide AS-12W of the present invention against ciprofloxacin-resistant Staphylococcus epidermidis;

[0032] Fig. 9 The bactericidal curve of the antimicrobial peptide AS-12W of the present invention against ciprofloxacin-resistant MRSA;

[0033] Fig.10The hemolytic activity of the antimicrobial peptide AS-12W of the present invention on sheep erythrocytes;

[0034] Fig.11 The antimicrobial peptide AS-12W of the present invention protects the survival of septic mice;

[0035] Fig.12 The effect of the antimicrobial peptide AS-12W of the present invention on the number of bacteria in the blood of septic mice;

[0036] Fig.13 The effect of the antimicrobial peptide AS-12W of the present invention on the number of bacteria in the lung tissue of septic mice;

[0037] Fig.14 The effect of the antimicrobial peptide AS-12W of the present invention on the bacterial count in the liver tissue of septic mice;

[0038] Fig.15 The figure shows the effect of the antimicrobial peptide AS-12W of the present invention on the number of bacteria in the spleen of septic mice. DETAILED DESCRIPTION

[0039] Example 1: Design and synthesis of peptide AS-12W

[0040] (1) Using the classic cathelicidin family antimicrobial peptide precursor sequence OH-CATH from cobra as a template, the online tool BLAST was used on the NCBI (https: / / www.ncbi.nlm.nih.gov) website to perform sequence comparison with the proteome database of Chinese alligator. The cathelicidin family antimicrobial peptide precursor sequence from this species was obtained as follows:

[0041]

[0042] (2) The cathelicidin family antimicrobial peptide precursor sequences from different species such as humans, pigs, sheep, and Bungarus bungarus were downloaded from UniProt (https: / / www.uniprot.org) protein data, and imported into MEGA7 software for multiple sequence alignment with the antimicrobial peptide precursor sequences obtained by BLAST, to verify the conservative sequence of the cathelin region and predict the amino acid residue cleavage site of elastase, i.e., valine at position 134 (V134). Then, the mature peptide sequence can be determined, i.e., between arginine at position 135 and arginine at position 172 (R135-R172). The sequence between isoleucine at position 146 and leucine at position 157 (I146-L157) in the mature peptide sequence was intercepted, and the sequence of polypeptide AS-12 was obtained as IKKGFKKIFKRL;

[0043] (3) Using AS-12 as a template, in order to improve the antibacterial activity, the glycine at position 4, which is not conducive to the formation of the α-helix, was replaced with a hydrophobic tryptophan. The sequence of the modified peptide AS-12W was IKKWFKKIFKRL.

[0044] Example 2: Synthesis and structural confirmation of the polypeptide AS-12W of the present invention

[0045] The polypeptide of the present invention was artificially synthesized by conventional solid phase synthesis method. The specific experimental steps are as follows:

[0046] The prepared AS-12W polypeptide sequence is: isoleucine-lysine-lysine-tryptophan-phenylalanine-lysine-lysine-isoleucine-phenylalanine-lysine-arginine-leucine

[0047] (1) Synthesis of polypeptides: The synthesis of polypeptides is carried out one by one from the C-terminus to the N-terminus. Soak Fmoc-Val-Wang Resin in dichloromethane for 15 minutes, wait for the resin to swell, and remove the dichloromethane; add a 1:4 volume ratio of hexahydropyridine / DMF solution, use nitrogen to agitate, and react twice, for 5 minutes and 15 minutes. After the reaction, wash the resin with DMF 9 times. Take a small amount of resin and add 2-3 drops of color test reagent A solution, B solution and C solution (A solution: ninhydrin / anhydrous ethanol solution; B solution: pyridine; C solution: phenol / anhydrous ethanol solution) and heat at 100°C for 3 minutes. The color of the solution and resin will turn blue to remove the amino protection. Add Fmoc-Val-OH and HOBT, dissolve with appropriate amount of DMF, add DIC and Collidine, agitate with nitrogen, react for 1 hour, wash the resin with DMF 6 times after the reaction, repeat the condensation reaction, connect each Fmoc protected amino acid in sequence, complete the synthesis of the linear sequence, soak the resin with dichloromethane and ether and then drain. Add TFA, react in a constant temperature shaker for 2h, the shaker speed is 110r / min, and the temperature is 25℃. Filter out the resin, add anhydrous ether to the filtrate, centrifuge to obtain a solid, add anhydrous ether to wash, centrifuge again, repeat several times and dry to obtain the crude AS-12W polypeptide.

[0048] (2) Purification: Weigh a certain amount of crude product, add an appropriate amount of acetonitrile, sonicate until clarified, and use a filter to remove large particle impurities. At the same time, pass through a preparative liquid chromatograph to collect samples in sections. Use an analytical chromatograph to perform gradient analysis and retain the samples that reach the required purity. Then freeze-dry. The purified polypeptide is identified by mass spectrometry and high-performance liquid chromatography. The above method was completed by Shanghai Jier Biochemical Co., Ltd.; batch number: P210927-LR934538.

[0049] Liquid chromatography analysis conditions: C18 column (4.6×250 mm, 5 μm); mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile solution containing 0.1% trifluoroacetic acid. Detection wavelength is 220 nm; flow rate is 1.0 ml / min; injection volume is 10 μl, and gradient elution is performed. The gradient elution conditions are shown in Table 1.

[0050] Table 1 Gradient elution conditions

[0051]

[0052] Depend on Figure 1 It can be seen that the purity of the polypeptide of the present invention is greater than 96.09% as determined by HPLC.

[0053] Depend on Figure 2 It can be determined that the molecular weight is 1634.10, which is consistent with the theoretical value of 1635.12.

[0054] Example 3: Determination of in vitro antibacterial activity of the polypeptide AS-12W of the present invention

[0055] (1) Recovery and activation of bacterial strains

[0056] Transfer the test bacteria (see Table 2) stored at -20℃ from the glycerol tube to the corresponding agar slant medium. Incubate the bacteria at 37℃ for 24h, and the fungi at 28℃ for 48h, and place them in a refrigerator at 4℃ for later use.

[0057] (2) Preparation of bacterial solution

[0058] Inoculate a small amount of the bacteria in the slant, transfer to 2 ml of the corresponding liquid culture medium, culture at 37°C or 25°C for 18 hours, and dilute with liquid culture medium to 10 5 CFU / ml bacterial suspension for later use.

[0059] (3) Preparation of peptide drugs

[0060] Weigh appropriate amounts of polypeptides AS-12W and AS-12, dissolve them in physiological saline, prepare 1024 μg / ml stock solutions, filter through a 0.22 μm aqueous filter, sterilize, aliquot, and store at -20°C for later use.

[0061] (4) Determination of minimum inhibitory concentration (MIC)

[0062] After the peptide drug was diluted to different concentration gradients with liquid culture medium, 50 μl was taken in a 96-well plate, and then an equal volume was inoculated with the liquid culture medium diluted to 10 5CFU / ml bacterial suspension, the final drug concentration is 128, 64, 32, 16, 8, 4, 2, 1, 0.5μg / ml, and no drug is set as a control group. The bacteria are cultured at 37℃ for 16-18h, and the test results are observed and recorded. The lowest drug concentration that does not grow bacteria is the MIC value.

[0063] The results are shown in Table 2:

[0064] Table 2 MIC or MIC of peptides against Gram-negative bacteria and Gram-positive bacteria 50 value

[0065]

[0066]

[0067] Note: IS: clinical isolate; CRE: carbapenem-resistant Escherichia coli; CRPA: carbapenem-resistant Pseudomonas aeruginosa; MRSA: methicillin-resistant Staphylococcus aureus; ND: not detected.

[0068] As can be seen from Table 2, AS-12W has a broad spectrum of antibacterial activity, showing good antibacterial activity against a variety of standard and clinical bacteria, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, and Candida albicans. The MIC value is in the range of 4 to 16 μg / ml. However, AS-12 has poor antibacterial activity, has no obvious antibacterial activity against a variety of standard and clinical bacteria, and has poor antibacterial activity against the tested Gram-negative bacteria, Gram-positive bacteria and fungi, with a MIC of ≥128 μg / ml.

[0069] (5) Bactericidal curve of peptide AS-12W against drug-resistant bacteria

[0070] The test strains of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus epidermidis, and MRSA were diluted to about 1×10 5 CFU / ml, add polypeptide AS-12W to make its concentration 4×MIC of the corresponding strain, and culture the bacteria at 37℃. Take out 200μl of mixed culture regularly at 0, 0.5, 1, 2, 4, 8, 12 and 24h, and count the live bacteria on the plate after gradient dilution. At the same time, set the normal saline group as the blank control group, draw the bactericidal curve with the polypeptide action time as the horizontal axis and the colony logarithm as the vertical axis.

[0071] Results Figure 3-9 : Figure 3-7They are the bactericidal effects of AS-12W on bacteria (including: Escherichia coli ATCC 25922, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis, MRSA). It can be seen from the figure that AS-12W can kill all meropenem-resistant Pseudomonas aeruginosa within 0.5 hours, kill all ceftazidime-resistant Klebsiella pneumoniae within 2 hours, and kill all ciprofloxacin-resistant Staphylococcus epidermidis within 4 hours, indicating that AS-12W has a rapid killing effect on drug-resistant bacteria.

[0072] Example 4: Effect of the polypeptide AS-12W of the present invention on the hemolytic activity of sheep erythrocytes

[0073] Fresh defibrinated sheep blood (Nanjing Shenghang Biotechnology Co., Ltd.) was centrifuged (3000rpm, 10min), the supernatant was discarded, the lower red blood cells were washed 3 times with PBS, 300μl of sheep red blood cells were aspirated and resuspended in 10ml of PBS to prepare a 3% volume fraction of sheep red blood cell suspension. The 3% sheep red blood cell suspension was added to a 96-well cell culture plate, 50μl per well, and then 50μl of the peptide AS-12W diluted in multiples was added to each well, so that the final concentration of the peptide AS-12W was 8, 16, 32, 64, 128, 256, 512μg / ml, respectively. The positive control was a final concentration of 0.1% Triton X-100, and the negative control was PBS. Three replicate wells were set for each group. After adding the drug, the culture plate was placed at 37℃ for 30min, centrifuged, and the supernatant was taken. The wavelength at 450nm was measured using an enzyme marker. The hemolysis rate of the peptide on sheep red blood cells was calculated by taking the positive control group as 100% hemolysis rate and the negative control group as 0% hemolysis rate. 450 - Negative control group OD 450 ) / (OD of positive control group 450 - Negative control group OD 450 )×100%.

[0074] Results Fig.10 ,Depend on Figure 8 It can be seen that when the peptide AS-12W is at the highest concentration of 512 μg / ml, the hemolytic rate of Mianyang erythrocytes does not exceed 5%. Combined with the MIC value range, it can be concluded that the hemolytic activity of the peptide AS-12W can be ignored within the effective range.

[0075] Example 5: Therapeutic effect of the polypeptide AS-12W of the present invention on mice with sepsis caused by Pseudomonas aeruginosa infection

[0076] Healthy ICR mice (Yangzhou University) weighing 20-22 g, half male and half female, were randomly divided into 6 groups, with 12 mice in each group. Five groups of mice were intraperitoneally inoculated with MLD at a concentration of 1×108 CFU / ml) of bacterial suspension (0.5ml / mouse) was used for infection to establish a sepsis model, and a non-infected and non-administered group was set as a blank control group. The mice in the infection groups were given 10mg / kg AS-12W (high-dose group), 5.0mg / kg AS-12W (medium-dose group), 2.5mg / kg AS-12W (low-dose group), 5mg / kg meropenem (positive control group), and normal saline (model control). The polypeptide administration group and the normal saline group were intraperitoneally administered twice 0.5h and 2h after infection, respectively, and the meropenem group was administered once by tail vein 2h after infection. The survival of the mice was observed and recorded for 7 consecutive days.

[0077] 12 hours after infection, about 300 μl of blood was collected from the orbital venous plexus of mice and anticoagulated with sodium heparin. The samples were diluted in multiples with sterile saline to obtain different concentration gradients (10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 At the same time, 12 hours after infection, the lung, liver, and spleen tissues of mice were taken and placed in sterile EP tubes. 9 volumes of normal saline were added (V tissue: V normal saline = 1:9). The homogenate was prepared by electric homogenizer. Part of the tissue homogenate was taken and diluted gradiently to obtain tissue homogenate dilutions of different concentration gradients (with 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 5 Take 200 μl of each dilution and add it to a sterile plate. Use the pouring plate method to add about 10 ml of nutrient agar medium at a constant temperature of 56°C. Mix thoroughly. After solidification, incubate at 37°C for 18 to 24 hours. Observe and record the number of colonies on the plate.

[0078] Results Figure 11-15 ,Depend on Fig.11 It can be seen that all mice in the model group died due to bacterial infection, while the survival rates of mice in the low-, medium- and high-dose peptide administration groups were increased by 22.2%, 44.4% and 77.8% respectively compared with the model group, proving that the peptide has a great protective effect on the survival of mice infected with bacteria, and the in vivo efficacy of the peptide changes in a dose-dependent manner within a certain concentration range.

[0079] Depend on Fig.12 It can be seen that 12 hours after infection, the number of bacteria in the blood of mice in the model group was about 10 7CFU / ml, the bacterial count in the medium-dose group of the polypeptide was reduced by 1 log value compared with the model group, while the high-dose group was reduced by nearly 3 log values, which was significantly different from the model group. Since Pseudomonas aeruginosa was resistant to meropenem, meropenem could not significantly reduce the bacterial load in the blood of mice.

[0080] Figure 13-15 They are the number of bacteria in the lung, liver, and spleen tissues of infected mice. It can be seen from the figure that although the low dose of AS-12W (2.5 mg / kg) has no obvious effect on clearing bacteria, the high dose (10 mg / kg) can significantly reduce the number of live bacteria in the tissues and organs of infected mice, with a decrease of about 2 to 3 log values, and there is a significant difference compared with the model group. At the same time, meropenem cannot significantly reduce the bacterial load in the lungs, liver, and spleen of mice.

[0081] comprehensive Figure 11-15 It can be seen that peptide AS-12W exhibits strong bacterial clearance, which may be related to the improved survival rate of infected mice.

Claims

1. Antimicrobial peptide AS-12W, characterized in that: The amino acid sequence of the antimicrobial peptide AS-12W is shown in SEQ ID NO:

1.

2. The method for preparing the antimicrobial peptide AS-12W according to claim 1, characterized in that: The preparation method is to synthesize the antimicrobial peptide AS-12W by a solid phase synthesis method.

3. The use of the antimicrobial peptide AS-12W according to claim 1 in the preparation of an in vitro bactericidal, bacteriostatic or antibacterial product, wherein the bacteria include Gram-negative bacteria and / or Gram-positive bacteria, the bacteria are drug-resistant bacteria, the Gram-negative bacteria are one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Helicobacter pylori, and the Gram-positive bacteria are one or more of Staphylococcus epidermidis, Staphylococcus aureus, and MRSA.

4. The use of the antimicrobial peptide AS-12W according to claim 1 in the preparation of antibacterial drugs, wherein the bacteria include Gram-negative bacteria, Gram-positive bacteria and / or fungi, the bacteria are drug-resistant bacteria, the Gram-negative bacteria are one or more of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, and Helicobacter pylori, and the Gram-positive bacteria are one or more of Staphylococcus epidermidis, Staphylococcus aureus, and MRSA.

5. Use of the antimicrobial peptide AS-12W according to claim 1 in the preparation of a medicament for treating sepsis or septicemia.

6. The use according to claim 5, characterized in that: The concentration of the antimicrobial peptide AS-12W is 0.5-512 μg / ml.

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