Antimicrobial drug and antimicrobial peptide MRSA12 thereof

Abstract

The present invention discloses an antibacterial drug and the antibacterial peptide MRSA12, belonging to the fields of biotechnology and biomedicine technology. The broad-spectrum antibacterial peptide provided by the present invention is named MRSA12, and the sequence is shown in SEQ ID No. 1, which can significantly inhibit the growth of Bacillus subtilis and methicillin-resistant Staphylococcus aureus. The antibacterial peptide has a small molecular weight and a simple amino acid composition, and is easy to synthesize on a large scale. It uses a non-receptor membrane lysis mechanism to reduce the resistance of bacteria to antibacterial peptides and is not easy to develop drug resistance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an antibacterial drug and the antibacterial peptide MRSA12 thereof. Background Art

[0002] Antimicrobial peptides are short peptides with a length of 2 to 50 amino acids, which have a broad spectrum of biological activities, can quickly kill or inhibit the growth of microorganisms (within minutes), are not affected by antibiotic resistance mutations, and show synergistic effects with traditional antibiotics. In general, most antimicrobial peptides contain more positively charged residues (such as lysine, arginine and histidine) and hydrophobic residues (40-50%). As host defense molecules widely present in the innate immune system of invertebrates and vertebrates, they are still effective after millions of years and are therefore regarded as an important template for the development of a new generation of antibiotics to combat antibiotic-resistant superbugs, viruses and tumors. More importantly, resistant pathogens show a high frequency of incidental sensitivity to antimicrobial peptides, while cross-resistance is relatively rare. In addition, compared with non-peptide drugs, peptide drugs have lower toxicity and higher specificity, and are easier to synthesize and modify. Therefore, they have great potential in controlling bacterial contamination. The diversity and effectiveness of antimicrobial peptides make them attractive candidates for translational applications, and several have entered the clinical trial stage. Therefore, there is an urgent need to develop a new antimicrobial peptide for infections caused by antibiotic-resistant strains. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to inhibit or kill antibiotic-resistant strains. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0004] To solve the above technical problems, the present invention first provides an antibacterial drug, which comprises C1) and C2): C1) is an antibacterial peptide or a pharmaceutically acceptable salt thereof; C2) a pharmaceutically acceptable carrier or excipient; the antibacterial peptide is MRSA12, which is a polypeptide with an amino acid sequence of SEQ ID No. 1 in the sequence list.

[0005] The aforementioned antibacterial drugs have at least one of the following functions:

[0006] F1) Antibacterial;

[0007] F2) Treatment and / or prevention and / or auxiliary treatment of diseases caused by bacterial infection;

[0008] F3) destroy the integrity of bacterial cell membrane;

[0009] F4) Reduce the permeability of bacterial cell outer membrane;

[0010] F5) disrupts bacterial membrane potential.

[0011] Specifically, the bacteria in the above antibacterial drugs include Bacillus subtilis and / or Staphylococcus aureus.

[0012] Furthermore, the Staphylococcus aureus in the above antibacterial drug is drug-resistant Staphylococcus aureus.

[0013] Furthermore, the drug-resistant Staphylococcus aureus in the above-mentioned antibacterial drug is methicillin-resistant Staphylococcus aureus.

[0014] The present invention also provides an antimicrobial peptide or a pharmaceutically acceptable salt thereof. The antimicrobial peptide is MRSA12, which is a polypeptide having an amino acid sequence of SEQ ID No. 1 in the sequence table.

[0015] The present invention also provides any of the following uses of the antimicrobial peptide MRSA12 or a pharmaceutically acceptable salt thereof:

[0016] A1) destroy the integrity of bacterial cell membrane;

[0017] A2) preparing products that disrupt the integrity of bacterial cell membranes;

[0018] A3) Reduce the permeability of bacterial cell outer membrane;

[0019] A4) preparing products that reduce the permeability of bacterial cell outer membranes;

[0020] A5) destroy bacterial membrane potential;

[0021] A6) preparing products that disrupt bacterial membrane potential;

[0022] A7) inhibiting bacterial activity;

[0023] A8) Preparation of products with inhibitory activity against bacteria.

[0024] In practical applications, the cyclic polypeptide of the present invention, its derivative, or its pharmaceutically acceptable salt, the polymer, the composition or the pharmaceutical compound can be directly administered to a patient as a drug, or mixed with a suitable carrier or excipient and administered to a patient to achieve the purpose of treating and / or preventing infections caused by various bacteria. The carrier materials here include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Among them, water-soluble carrier materials are preferred. These materials can be made into a variety of dosage forms, including but not limited to tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, freeze-dried powder injections, etc. Among them, the suppository can be a vaginal suppository, a vaginal ring, or an ointment, cream or gel suitable for vaginal application. It can be a common preparation, a sustained-release preparation, a controlled-release preparation and various microparticle delivery systems. In order to prepare the unit dosage form into tablets, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, white clay, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and binders, such as water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinyl pyrrolidone, etc.; disintegrants , such as dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid esters, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, hydrogenated oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. The tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets. In order to make the unit dosage form into a pill, various carriers known in the art can be widely used. Examples of carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin, talc, etc.; binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste or flour paste, etc.; disintegrants, such as agar powder, dry starch, alginate, sodium lauryl sulfate, methyl cellulose, ethyl cellulose, etc. In order to prepare a unit dosage form as a suppository, various carriers known in the art can be widely used. Examples of carriers include, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.In order to prepare the unit dosage form into an injection preparation, such as a solution, emulsion, lyophilized powder injection and suspension, all diluents commonly used in the art can be used, for example, water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, etc. In addition, in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerol can be added to the injection preparation. In addition, conventional cosolvents, buffers, pH adjusters, etc. can also be added. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparation.

[0025] The above dosage form can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracisternal injection or infusion, etc.; cavity administration, such as rectal, vaginal and sublingual; respiratory administration, such as nasal cavity; mucosal administration. The above administration route is preferably injection, and the preferred injection route is subcutaneous injection.

[0026] Specifically, the bacteria in the above application include Bacillus subtilis and / or Staphylococcus aureus.

[0027] Furthermore, the Staphylococcus aureus in the above application is drug-resistant Staphylococcus aureus.

[0028] Furthermore, the drug-resistant Staphylococcus aureus in the above application is methicillin-resistant Staphylococcus aureus.

[0029] The antimicrobial peptide MRSA12 disclosed in the present invention has a significant inhibitory effect on Bacillus subtilis and methoxy-resistant Staphylococcus aureus; and the antimicrobial peptide has a small molecular weight and a simple amino acid composition, which is convenient for large-scale synthesis. Therefore, it has good application potential in the prevention and control of animal and plant infections caused by the above pathogens. In addition, the antimicrobial peptide targets bacteria and causes their death by destroying the integrity of the bacterial cell membrane, the permeability of the cell outer membrane, and the membrane potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the predicted three-dimensional structure of the antimicrobial peptide MRSA12;

[0031] Figure 2 The antibacterial effect of antimicrobial peptide MRSA12 on Bacillus subtilis and methicillin-resistant Staphylococcus aureus at 200 μM;

[0032] Figure 3 The time-killing curve of the antimicrobial peptide MRSA12 against Bacillus subtilis at 200 μM;

[0033] Figure 4To observe the effect of antimicrobial peptide MRSA12 on the morphological changes of Bacillus subtilis by transmission electron microscopy, the scale bars in the upper and lower figures are 1 μm and 500 nm, respectively;

[0034] Figure 5 To observe the effect of antimicrobial peptide MRSA12 on the cell membrane of Bacillus subtilis by confocal microscopy, the scale bar is 5 μm;

[0035] Figure 6 To analyze the proportion of damaged cells of Bacillus subtilis after treatment with antimicrobial peptide MRSA12 by flow cytometry;

[0036] Figure 7 The effect of antimicrobial peptide MRSA12 on the cell membrane integrity of Bacillus subtilis;

[0037] Figure 8 The effect of antimicrobial peptide MRSA12 on the outer membrane permeability of Bacillus subtilis cells;

[0038] Fig. 9 The effect of antimicrobial peptide MRSA12 on the membrane potential of Bacillus subtilis;

[0039] Fig.10 The effect of antimicrobial peptide MRSA12 on the cell wall of Bacillus subtilis. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way. Based on the antimicrobial peptide MRSA12 of the present invention, all other experimental results obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0041] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0042] In the following examples, Bacillus subtilis (ATCC35021) and methicillin-resistant Staphylococcus aureus (ATCC BAA-1026) are both products of ATCC (American Type Culture Collection, website: http: / / www.atcc.org / ).

[0043] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation. Unpaired t-test was used, and P < 0.05 (*) indicated a significant difference, and P < 0.001 (***) and P < 0.0001 (****) indicated a very significant difference.

[0044] Example 1

[0045] 1. Determination of the antibacterial activity of antimicrobial peptide MRSA12

[0046] 1.1 Preparation of antimicrobial peptide MRSA12

[0047] The amino acid sequence of the antimicrobial peptide MRSA12 is as follows:

[0048] NH2-Arg-Val-Pro-Gln-Gln-Ala-Cys-Arg-Glu-Pro-Phe-Leu-COOH

[0049] The single-letter amino acid sequence of the antimicrobial peptide MRSA12 is shown in SEQ ID No. 1:

[0050] SEQ ID No. 1: RVPQQACREPFL

[0051] The three-dimensional structure of the antimicrobial peptide MRSA12 was predicted using PEP-FOLD3 software. Figure 1 As shown. Figure 1 It can be seen that MRSA12 presents a typical α-helical structure.

[0052] The antimicrobial peptide MRSA12 was commissioned to Shanghai Nuoyou Biotechnology Co., Ltd. and Guoping Pharmaceutical Co., Ltd. for synthesis through solid phase synthesis. The purity of the synthesized antimicrobial peptide is greater than 90% and it is easily soluble in pure water.

[0053] 1.2 Determination of antimicrobial activity of antimicrobial peptide MRSA12

[0054] LB medium: Tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, the rest is water, and the pH is adjusted to 7.4 with NaOH.

[0055] LB agar medium: Dissolve 15 g agar in 1 L LB medium and sterilize at 121°C for 15 min.

[0056] The freeze-dried antimicrobial peptide powder synthesized in 1.1 was dissolved in sterilized pure water to prepare a 400 μM stock solution for later use, thereby obtaining an antimicrobial peptide MRSA12 solution.

[0057] Pick a single colony of Bacillus subtilis (or MRSA) into 5 mL LB medium, shake at 220 rpm, 37°C overnight to obtain a Bacillus subtilis suspension (or Staphylococcus aureus suspension). Dilute the suspension 10 times with fresh LB medium to OD 600nm =0.1, and then diluted 1,000-fold again with LB medium to obtain Bacillus subtilis bacterial liquid (or Staphylococcus aureus bacterial liquid).

[0058] The antibacterial activity experiment was performed in a 96-well plate as follows:

[0059] (1) Blank control group: 200 μL LB solution was added to each well and cultured at 37°C for 16-18 h. The OD was measured using a UV spectrophotometer. 600nm The experiment was repeated three times with at least three replicate wells each time.

[0060] (2) Bacterial control group (Control): 100 μL of sterile water and 100 μL of bacterial solution were added to each well, and the rest of the operation was the same as the blank control group; the bacterial solution was Bacillus subtilis (referred to as B. subtilis) and methicillin-resistant Staphylococcus aureus (referred to as MRSA).

[0061] (3) Antimicrobial peptide experimental group (MRSA12): 100 μL of Bacillus subtilis solution (or Staphylococcus aureus solution) and 100 μL of antimicrobial peptide MRSA12 solution were added to each well, and the rest of the operation was the same as the blank control group. The bacterial solution was Bacillus subtilis solution (referred to as B. subtilis) or methicillin-resistant Staphylococcus aureus solution (referred to as MRSA).

[0062] The results are as follows Figure 2 As shown in Figure 2, compared with the bacterial control group (Control), the addition of antimicrobial peptide MRSA12 solution can significantly reduce the OD of Bacillus subtilis and MRSA. 600nm , which means that the proliferation of Bacillus subtilis (P<0.0001) and MRSA (P<0.0001) was extremely significantly inhibited.

[0063] 1.3 Duration of action of antimicrobial peptide MRSA12

[0064] CaMHB medium: purchased from Qingdao Rishui Biotechnology Co., Ltd., catalog number 11865.

[0065] Prepare the antimicrobial peptide MRSA12 solution according to the method shown in 1.2.

[0066] Experimental group (MRSA12): Pick a single colony of Bacillus subtilis in 5 mL LB medium and shake overnight at 220 rpm and 37°C. Dilute with fresh CaMHB medium to OD600nm =0.1, 220rpm, 37℃2h shake to logarithmic growth phase, obtain Bacillus subtilis suspension. Mix the antimicrobial peptide MRSA12 solution with an equal amount of Bacillus subtilis suspension and incubate at 37℃. Take 100μL sample (liquid after the antimicrobial peptide MRSA12 solution is mixed with an equal amount of Bacillus subtilis suspension) for 10 times dilution and inoculate into LB agar medium at 0, 0.5, 1, 2, 4 and 6 hours of incubation respectively. Incubate at 37℃ overnight and count the colony forming units. The control group (Control) replaces the antimicrobial peptide MRSA12 solution with an equal amount of sterile water.

[0067] The results are as follows Figure 3 As shown, compared with the control group Control, the growth of Bacillus subtilis was inhibited 2 hours after the addition of antimicrobial peptide MRSA12, the colony count of Bacillus subtilis was significantly lower than that of the control group Control 1 hour after the addition of antimicrobial peptide MRSA12 (P<0.05), and the colony count of Bacillus subtilis was extremely significantly lower than that of the control group Control 2 hours after the addition of antimicrobial peptide MRSA12 (P<0.01).

[0068] 2. Study on the antibacterial mechanism of antimicrobial peptide MRSA12

[0069] 2.1 Effects of antimicrobial peptide MRSA12 on the morphological changes of Bacillus subtilis

[0070] Sample preparation: After culturing Bacillus subtilis to the logarithmic growth phase, the cells were collected and washed three times in PBS to obtain Bacillus subtilis cells. The antimicrobial peptide MRSA12 solution with a final concentration of 200 μM (the preparation method is as shown in 1.2, that is, Figure 4 200 μM) and 2 mM antimicrobial peptide MRSA12 solution (preparation method refers to the method shown in 1.2, i.e. Figure 4 2mM) was mixed evenly with the aforementioned Bacillus subtilis cells and incubated for 2h; the control group (Control) used pure water instead of the antimicrobial peptide MRSA12 solution. Then prepare a sealing film of appropriate size, with the clean side facing up, absorb a drop of fresh sample and place it on the film, place the copper mesh digital side down on the sample droplet, and let it stand for adsorption for 5min. Then wash it twice with deionized water, then place the copper mesh on a drop of 0.5% uranyl acetate, negatively stain for 10s, and then continue to wash it twice with deionized water, and absorb the excess liquid with filter paper. Adjust the appropriate focus and grayscale for observation under a transmission electron microscope.

[0071] The results are as follows Figure 4 As shown, the antimicrobial peptide MRSA12 caused the morphology of Bacillus subtilis to change, and the morphology of Bacillus subtilis after adding the antimicrobial peptide MRSA12 was significantly different from that of the control group without adding the antimicrobial peptide MRSA12.

[0072] 2.2 Effects of antimicrobial peptide MRSA12 on Bacillus subtilis cell membrane

[0073] Propidium iodide (PI) is a membrane-impermeable probe that cannot pass through intact cell membranes, but can only pass through damaged cell membranes to stain nuclear DNA. 4',6-diamidino-2-phenylindole (DAPI) is a DNA-binding fluorescent dye with strong penetration, which can stain both live and dead cells. Therefore, the effect of antimicrobial peptides on the cell membrane of Bacillus subtilis was observed by using a confocal microscope. The specific experimental method is as follows:

[0074] After the Bacillus subtilis was cultured to the logarithmic growth phase, the cells were collected and washed three times in PBS. The antimicrobial peptide MRSA12 solution with a final concentration of 200 μM (preparation method refers to the method shown in 1.2, i.e. Figure 4 200μM in PBS) was mixed with Bacillus subtilis cells and incubated for 1h. The control group (Control) used pure water instead of antimicrobial peptide MRSA12 solution. PI with a final concentration of 20μM was added and incubated in the dark for 30min, followed by washing three times with PBS (analyzed by flow cytometry). DAPI was then added and incubated in the dark for 20min, followed by washing three times with PBS. Finally, the bacteria were suspended with 100μL PBS, mixed by pipetting, and observed using a confocal microscope.

[0075] The results are as follows Figure 5 As shown in the figure, compared with the control group, the cell membrane of Bacillus subtilis was damaged after adding the antimicrobial peptide MRSA12, allowing PI to pass through the damaged cell membrane and stain the nuclear DNA of Bacillus subtilis. It can be seen that the antimicrobial peptide MRSA12 can significantly damage the integrity of the cell membrane of Bacillus subtilis. And as the concentration of the added antimicrobial peptide MRSA12 increases, the proportion of damaged bacteria gradually increases (such as Figure 6 shown).

[0076] 2.3 Effect of antimicrobial peptide MRSA12 on Bacillus subtilis cell membrane integrity

[0077] Pick a single colony of Bacillus subtilis and culture it in LB medium until the logarithmic growth phase. Collect the bacteria, wash them three times in PBS, resuspend them in PBS and adjust the OD to 600nm=1, and obtain bacterial solution. Then add antimicrobial peptide MRSA12 solution with final concentrations of 15, 30, 60 and 120 μM to the bacterial solution (refer to the method shown in 1.2 for preparation method), incubate at 37°C for 1 hour, and use pure water instead of antimicrobial peptide MRSA12 solution in the control group. Add 20 μM PI to each well and incubate at 37°C in the dark for 30 minutes. Use an ELISA reader to measure the fluorescence signal at excitation and emission wavelengths of 535 nm and 615 nm, respectively.

[0078] The results are as follows Figure 7 As shown, with the increase of the concentration of antimicrobial peptide MRSA12, the cell damage rate of Bacillus subtilis increased significantly (P<0.0001).

[0079] 2.4 Effect of antimicrobial peptide MRSA12 on the outer membrane permeability of Bacillus subtilis

[0080] N-Phenyl-1-naphthylamine (NPN) can be used as a fluorescent probe. When it binds to the hydrophobic region of the cell membrane, it will emit fluorescence. It is usually used as a hydrophobic probe to study the phase transition of membrane lipids in whole cells. Using this property, it is possible to detect how much they enter the cell to evaluate their effect on the permeability of the bacterial outer membrane.

[0081] After Bacillus subtilis was cultured to the logarithmic growth phase, the cells were collected and washed three times in PBS, resuspended in PBS and adjusted to OD 600nm =0.5 to obtain bacterial solution. Then, the bacterial solution was incubated with the fluorescent probe NPN at a final concentration of 10 μM at 37°C for 30 min. Then, 50 μL of antimicrobial peptide MRSA12 solution (preparation method refers to the method shown in 1.2) at a final concentration of 0.125, 0.25, 0.5 and 1 mM and 150 μL of probe (PI / ALP / NPN / DISC3(5)) were added to a 96-well plate and the labeled bacteria were incubated for 30 min. The fluorescence signal was measured using an ELISA reader at excitation and emission wavelengths of 350 nm and 420 nm, respectively.

[0082] The results are as follows Figure 8 As shown in the figure, with the increase of the concentration of antimicrobial peptide MRSA12, the fluorescence value of NPN increased significantly (P<0.0001). Since NPN can enter the cell and produce fluorescence only after the outer membrane is damaged, it can be seen that the antimicrobial peptide MRSA12 can destroy the membrane structure of Bacillus subtilis and change the permeability of the outer membrane of Bacillus subtilis.

[0083] 2.5 Effect of antimicrobial peptide MRSA12 on the cell membrane potential of Bacillus subtilis

[0084] 3,3'-Dipropylthiadicarbocyanine iodide (DiSC3(5)) is a fluorescent probe that can accumulate in the cytoplasmic membrane and self-quench its own fluorescence. It is often used as a tracer dye to assess the cell membrane potential. However, when the membrane potential is disrupted, it is released into the culture medium. Therefore, the ability of antimicrobial peptides to depolarize bacterial cell membranes can be assessed by measuring the intensity of its fluorescence signal.

[0085] After Bacillus subtilis was cultured to the logarithmic growth phase, the cells were collected and washed three times in PBS, resuspended in PBS and adjusted to OD 600nm =1.0 to obtain bacterial solution. 0.5 μM DiSC3(5) was added to the bacterial solution and incubated at room temperature in the dark for 30 min. Then, 150 μL of DiSC3(5)-labeled bacteria were treated with 50 μL of antimicrobial peptide MRSA12 solution with a final concentration of 0.125, 0.25, 0.5 and 1 mM (preparation method refers to the method shown in 1.2) at 37°C for 1 h. The fluorescence signal was measured using an ELISA reader at excitation and emission wavelengths of 622 nm and 670 nm, respectively.

[0086] The results are as follows Fig. 9 As shown, 1 mM antimicrobial peptide MRSA12 can significantly destroy the membrane potential of Bacillus subtilis cell membrane (P<0.0001), causing DiSC3(5) accumulated in Bacillus subtilis to be released into the culture medium.

[0087] 2.6 Effect of antimicrobial peptide MRSA12 on the cell wall of Bacillus subtilis

[0088] Since alkaline phosphatase (ALP) is located in the periplasmic space, its leakage reflects the dissolution of the cell wall. The leakage of ALP was detected by an alkaline phosphatase assay kit to evaluate the ability of antimicrobial peptides to disrupt the bacterial cell wall.

[0089] Pick a single colony of Bacillus subtilis and culture it in LB medium until the logarithmic growth phase. Collect the bacteria, wash them three times in PBS, resuspend them in PBS and adjust the OD to 600nm =0.5 to obtain bacterial solution. Then add antimicrobial peptide MRSA12 solution with final concentrations of 0.125, 0.25, 0.5 and 1 mM (refer to the method shown in 1.2 for preparation method) to the bacterial solution, and incubate at 37°C for 1 hour. Take 50 μL of the reaction solution and add it to another 96-well plate, add 50 μL of color substrate to mix, and incubate at 37°C for 30 minutes. After adding 100 μL of stop solution, use an enzyme reader to measure the absorbance value at 405 nm.

[0090] The results are as follows Fig.10As shown, 1 mM antimicrobial peptide MRSA12 had no significant effect on the cell wall lysis of Bacillus subtilis.

[0091] In summary, the antimicrobial peptide MRSA12 provided by the present invention has a small molecular weight, a simple amino acid composition, and is convenient for large-scale synthesis. It has been verified by experiments that the antimicrobial peptide targets bacteria by destroying the integrity of the bacterial cell membrane, the permeability of the outer cell membrane, and the membrane potential, thereby significantly inhibiting the growth of Bacillus subtilis and methicillin-resistant Staphylococcus aureus. Because it has positive charge and amphipathicity, it can be selectively combined with negatively charged components (such as lipopolysaccharides and phospholipids) of the bacterial cell membrane by electrostatic action, and then its hydrophobic domain is inserted into the plasma membrane, and holes are formed on the bacterial membrane to cause leakage of its contents, thereby quickly and efficiently killing bacteria. Based on specific physicochemical properties, this non-receptor membrane lysis mechanism makes bacteria less resistant to antimicrobial peptides, and is not easy to develop drug resistance. Therefore, the antimicrobial peptide MRSA12 has a good application prospect as a new antibacterial drug to treat infections caused by various Gram-positive bacteria including methicillin-resistant Staphylococcus aureus.

[0092] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An antibacterial drug, characterized in that The antibacterial drug comprises C1) and C2, C1) is an antibacterial peptide or a pharmaceutically acceptable salt thereof; C2) a pharmaceutically acceptable carrier or excipient; the antibacterial peptide in C1) is MRSA12, and its amino acid sequence is shown in SEQ ID No. 1 in the sequence list.

2. An antimicrobial peptide or a pharmaceutically acceptable salt thereof, characterized in that: The antimicrobial peptide is MRSA12, and its amino acid sequence is shown in SEQ ID No. 1 in the sequence table.

3. Use of the antimicrobial peptide or a pharmaceutically acceptable salt thereof according to claim 2 in the preparation of a product for inhibiting bacterial activity, wherein the bacteria are Bacillus subtilis and / or Staphylococcus aureus.

4. The use according to claim 3, characterized in that: The Staphylococcus aureus is drug-resistant Staphylococcus aureus.

5. The use according to claim 4, characterized in that: The drug-resistant Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

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