Antibacterial peptide Loong-3 and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an antibacterial peptide Loong-3 and application thereof. The sequence of the antibacterial peptide Loong-3 is as follows: lysine, arginine, isoleucine, alanine, lysine, leucine, alanine, leucine, lysine, alanine, isoleucine, arginine, lysine, isoleucine, leucine, arginine, leucine and isoleucine. The antibacterial peptide has better antibacterial activity on gram-positive bacteria (staphylococcus aureus), gram-negative bacteria (escherichia coli and salmonella) and fungi (candida albicans), and has the characteristics of heat resistance, salt resistance, acid and alkali resistance, repeated freezing and thawing resistance, serum resistance and organic solvent resistance. Therefore, the antibacterial peptide Loong-3 has wide application prospects in the fields of preparation of drugs for resisting bacterial and / or fungal infection, animal husbandry development, prevention and treatment of human diseases, food preservation, detergents, cosmetics, antibacterial molecular materials and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an antimicrobial peptide Loong-3 and an application thereof. Background Art

[0002] Antibiotics have been used to treat bacterial diseases since they were discovered, and have saved countless human and animal lives. However, the abuse of antibiotics has led to the emergence of drug-resistant bacteria, which has brought troubles to the treatment of drug-resistant bacterial infections. For this reason, it has become more important to find alternatives to antibiotics. At present, there are many types of antibiotic alternatives, among which antimicrobial peptides are considered to be the "leader" of antibiotic alternatives.

[0003] Antimicrobial peptides (AMPs) are an important part of the immune system of biological organisms and participate in building the body's defense against infection. Antimicrobial peptides have diverse biological activities (antimicrobial activity, antitumor activity, immunomodulatory activity, etc.), heat resistance, acid and alkali resistance, and are not easy to induce drug resistance. They also have different bactericidal mechanisms from antibiotics. Based on these advantages, antimicrobial peptides have been widely concerned by academia and industry as the "leader" of antibiotic alternatives, and are expected to solve the problem of bacterial resistance.

[0004] At present, many antimicrobial peptides have been discovered. The Antimicrobial Peptide Database APD3 is the most authoritative database in the world that specifically collects antimicrobial peptides, and it contains 3,940 antimicrobial peptides (as of December 12, 2024). Although there are many antimicrobial peptides discovered so far, there are not many antimicrobial peptides that have been developed and can be used clinically. To this end, it is necessary to continuously enrich the antimicrobial peptide library to provide more "candidates" for the development of antimicrobial peptide drugs for clinical use.

[0005] Antimicrobial peptides can be divided into natural antimicrobial peptides and artificially designed antimicrobial peptides. The process of obtaining natural antimicrobial peptides is relatively cumbersome, time-consuming, labor-intensive, and costly, and the success rate is not very high. However, based on the structure-activity relationship of antimicrobial peptides and with the help of bioinformatics tools, artificially designed antimicrobial peptides are simpler, faster, more economical, and have a high success rate. They have become a hot topic in antimicrobial peptide-related research. Therefore, artificially designed antimicrobial peptides can provide more antimicrobial peptides with high antimicrobial activity and stability, providing an important guarantee for solving the problem of bacterial resistance. Summary of the invention

[0006] The present invention provides an antimicrobial peptide Loong-3 and its application. The antimicrobial peptide Loong-3 is an artificially designed antimicrobial peptide with a spiral structure. It has good antimicrobial activity against a variety of animal pathogenic bacteria. Heat, acid and alkali, salt ions, repeated freezing and thawing, serum, and organic solvents have almost no effect on its antimicrobial activity, and it has a certain degree of safety. Therefore, the antimicrobial peptide Loong-3 provided by the present invention has broad application prospects in the fields of anti-pathogenic infection, animal breeding, food preservation and antiseptic, cosmetics, detergents, disinfectants, environmental biological hazard prevention and control, antimicrobial molecular materials, etc.

[0007] The present invention is achieved through the following technical solutions: The present invention provides an antimicrobial peptide Loong-3. The amino acid sequence of the antimicrobial peptide Loong-3 is as follows: lysine-arginine-isoleucine-alanine-lysine-leucine-alanine-leucine-lysine-alanine-isoleucine-arginine-lysine-isoleucine-leucine-arginine-leucine-isoleucine (KRIAKLALKAIRKILRLI), as shown in SEQ ID NO: 1.

[0008] Furthermore, the antimicrobial peptide Loong-3 has 18 amino acid residues, a molecular weight of 2117.75 Da, an isoelectric point of 12.4, 7 positively charged amino acid residues, and a helical structure.

[0009] The present invention also provides the use of the antimicrobial peptide Loong-3 in the preparation of drugs for resisting bacterial and / or fungal infections.

[0010] The present invention has found through experiments that the antimicrobial peptide Loong-3 product of the present invention has a certain hemolytic property, a broad antimicrobial spectrum, good heat resistance, salt resistance, acid and alkali resistance, repeated freezing and thawing resistance, organic solvent resistance and serum resistance, and has a highly effective antimicrobial effect on Gram-negative bacteria, Gram-positive bacteria and fungi. Therefore, the antimicrobial peptide Loong-3 of the present invention can be used to prepare drugs for antibacterial and / or fungal infections.

[0011] Furthermore, the drug comprises the antimicrobial peptide Loong-3 and also comprises one or more pharmaceutically acceptable carriers and / or additives.

[0012] Furthermore, the bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria include Staphylococcus aureus, and the Gram-positive bacteria include Escherichia coli and Salmonella.

[0013] Furthermore, the fungus includes Candida albicans.

[0014] Since the antimicrobial peptide Loong-3 of the present invention has a good application prospect in the preparation of drugs for preventing bacterial and / or fungal infections in animals and humans, according to the general characteristics of known antimicrobial peptides, the antimicrobial peptide Loong-3 will also have good applications in feed additives, disinfectants, preservatives, detergents or cosmetic additives, antimicrobial molecular materials, etc. Therefore, the present invention also provides the use of the antimicrobial peptide Loong-3 in the preparation of feed additives, disinfectants, preservatives, detergents, cosmetic additives, antimicrobial molecular materials or pesticides.

[0015] Compared with the prior art, the present invention has the following positive and beneficial effects: The antimicrobial peptide Loong-3 of the present invention is composed of 18 amino acid residues, has antimicrobial activity against Gram-positive bacteria (such as Staphylococcus aureus), Gram-negative bacteria (such as Escherichia coli, Salmonella) and fungi (such as Candida albicans), has a spiral structure, strong antimicrobial activity, and has good heat resistance, salt resistance, acid and alkali resistance, repeated freezing and thawing resistance, serum resistance, and organic solvent resistance. Therefore, the antimicrobial peptide Loong-3 of the present invention has broad application prospects in the preparation of antibacterial and / or fungal infection drugs, animal husbandry, human disease treatment and prevention, food preservation and antiseptic, environmental disinfection, pesticide preparation, antimicrobial molecular materials, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a high performance liquid chromatogram of the antimicrobial peptide Loong-3 of the present invention; Figure 2 is the mass spectrum of the antimicrobial peptide Loong-3 of the present invention; Figure 3 This is a diagram showing the isoelectric point analysis results of the antimicrobial peptide Loong-3 of the present invention; Figure 4 The helical wheel structure model diagram of the antimicrobial peptide Loong-3 of the present invention; Figure 5 This is a 3D model diagram of the antimicrobial peptide Loong-3 of the present invention; Figure 6 The minimum inhibitory concentration of the antimicrobial peptide Loong-3 of the present invention against clinical drug-resistant strains of Escherichia coli is measured; Figure 7 The minimum inhibitory concentration of the antimicrobial peptide Loong-3 of the present invention against Escherichia coli CVCC1568 was measured; Figure 8 The minimum inhibitory concentration of the antimicrobial peptide Loong-3 of the present invention against Salmonella pullorum CVCC533 is measured; Fig. 9 The minimum inhibitory concentration of the antimicrobial peptide Loong-3 of the present invention against Staphylococcus aureus ATCC25923 was determined; Fig.10 The minimum inhibitory concentration of the antimicrobial peptide Loong-3 of the present invention against Staphylococcus aureus CVCC6538 was determined; Fig.11 The minimum inhibitory concentration of the antimicrobial peptide Loong-3 of the present invention against Candida albicans ATCC10231 is measured; Fig.12 The hemolysis rate test results of the antimicrobial peptide Loong-3 of the present invention; Fig.13 The heat resistance test results of the antimicrobial peptide Loong-3 of the present invention; Fig.14 The salt tolerance test results of the antimicrobial peptide Loong-3 of the present invention are as follows; Fig.15 The results of the acid and alkali resistance test of the antimicrobial peptide Loong-3 of the present invention are shown in FIG. Fig.16 The results of repeated freeze-thaw stability test of the antimicrobial peptide Loong-3 of the present invention are shown in FIG. Fig.17 The results of serum stability test of the antimicrobial peptide Loong-3 of the present invention are shown in FIG. Fig.18 The figure shows the organic solvent stability test result of the antimicrobial peptide Loong-3 of the present invention. DETAILED DESCRIPTION

[0017] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] In the examples, Fmoc-Ile-Wang resinin, Fmoc-Leu, Fmoc-Arg, Fmoc-Leu, Fmoc-Ile, Fmoc-Lys, Fmoc-Arg, Fmoc-Ile, Fmoc-Ala, Fmoc-Lys, Fmoc-Leu, Fmoc-Ala, Fmoc-Leu, Fmoc-Lys, Fmoc-Ala, Fmoc-Ile, Fmoc-Arg and Fmoc-Lys were purchased from Gill Biochemical (Shanghai) Co., Ltd.

[0019] Example 1 An antimicrobial peptide Loong-3, whose amino acid sequence is as follows: Lys-Arg-Is-Ala-Lys-Leu-Ala-Leu-Lys-Ala-Is-Arg-Lys-Is-Leu-Arg-Leu-Is-Arg (SEQ ID NO: 1).

[0020] The antimicrobial peptide Loong-3 contains 18 amino acid residues, has a molecular weight of 2117.75 Da, and an isoelectric point of 12.4. The antimicrobial peptide Loong-3 is positively charged and can attract negative charges on the surface of bacteria.

[0021] The synthesis of the antimicrobial peptide Loong-3 is as follows: The antimicrobial peptide Loong-3 was synthesized using an automatic peptide synthesizer according to conventional peptide solid phase chemical synthesis, and the synthesis direction was from the C-terminus to the N-terminus. The detailed steps of its synthesis were as follows: Step a, swelling resin: Add Fmoc-Ile-Wang resin into the reactor of the automatic peptide synthesizer, and add dimethylformamide (DMF) for swelling. The amount of DMF added to 1g of Fmoc-Ile-Wang resin is about 12mL (DMF should completely immerse the resin), the swelling time is 10 to 30min, and the swelling can be repeated 1 to 2 times.

[0022] Step b, deprotection: after the resin swells, 20% piperidine in a storage tank (20% piperidine is prepared by dissolving piperidine in DMF) is added to the reactor, and the resin is immersed (1g Fmoc-Ile-Wang resinin resin needs to add about 10mL20% piperidine), and the resin deprotection after the swelling is about 30min, and then DMF is added to wash.

[0023] Step c, condensation reaction: then, according to the order of the amino acid sequence of the antimicrobial peptide Loong-3, the penultimate amino acid, i.e., leucine Fmoc-Leu, is added, and a condensation agent HCTU (6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate) and a catalyst NMM (N-methylmorpholine) are added for condensation reaction; after the reaction is completed, DMF is added for repeated washing to remove unreacted amino acids; the molar ratio of the leucine to the Fmoc-Ile-Wang resinin is 5:1; the molar ratio of the condensation agent HCTU to the leucine Fmoc-Leu is 1:1; the molar ratio of the catalyst NMM to the leucine is 4:1; The product obtained after washing is deprotected and washed according to the method of step b, and Fmoc-Arg, Fmoc-Leu, Fmoc-Ile, Fmoc-Lys, Fmoc-Arg, Fmoc-Ile, Fmoc-Ala, Fmoc-Lys, Fmoc-Leu, Fmoc-Ala, Fmoc-Leu, Fmoc-Lys, Fmoc-Ala, Fmoc-Ile, Fmoc-Arg and Fmoc-Lys are added in sequence according to the method of step c for condensation reaction and washing. After the last condensation reaction and washing, a polypeptide product is obtained; the product obtained after each condensation reaction and washing is deprotected and washed according to the method of step b, and then the next amino acid is added for condensation reaction and washing (in each condensation reaction, the added amino acid and Fmoc-Ile-Wang are added in sequence for condensation reaction and washing). The molar ratio of the resinin was 5:1; the molar ratio of the condensing agent HCTU to each added amino acid was 1:1; the molar ratio of the catalyst NMM to each added amino acid was 4:1).

[0024] Step d, deprotection and cleavage of the polypeptide product: 20% piperidine is added to the polypeptide product obtained in step c in the reactor, and the polypeptide product is immersed (the amount of 20% piperidine used for deprotection of 1g of resin is about 10mL), so that the polypeptide product is deprotected; after deprotection, DMF and dichloromethane (DCM) are added in sequence for repeated washing; after washing, trifluoroacetic acid (TFA) is added for cleavage (the volume of TFA used for cleavage of 1g of resin is about 20mL), so that the polypeptide is cleaved from the resin; the cleavage product is filtered (filtered with a sand core funnel) to obtain a filtrate.

[0025] Step e, precipitating the synthetic polypeptide: washing the obtained filtrate with cold ether and centrifuging it to precipitate the synthetic polypeptide, the process is as follows: adding 3 times the volume of cold ether (pre-stored at 4°C) to the filtrate obtained in step d, sealing and inverting to mix, ice bathing for 10 minutes, centrifuging at 4000r / m for 10 minutes, discarding the supernatant liquid, and recovering the precipitate; resuspending the precipitate with cold ether, repeating the above steps three times, and then drying the obtained precipitate for 12 to 16 hours to obtain a crude synthetic peptide.

[0026] Step f, purification of crude synthetic peptide: The crude synthetic peptide was purified by column purification using a semi-preparative HPLC P2000, and fractions with a purity of ≥95% were collected to obtain the purified antimicrobial peptide Loong-3 with a purity of ≥95%. The semi-preparative high performance liquid chromatograph was P2000 provided by Beijing Wohua Innovation Technology Co., Ltd. The chromatographic column was a C18 reverse phase column (4.6*250mm), the mobile phase A was a 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid dissolved in 100mL acetonitrile solution), and the mobile phase B was a 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid dissolved in 100mL deionized water). Using the above-mentioned treatment column, within 25 minutes after loading, the proportion of mobile phase A was gradually adjusted from 27% to 52%, and the proportion of mobile phase B was gradually adjusted from 73% to 48%. At 25.1 minutes, the proportion of mobile phase A became 100%, and the proportion of mobile phase B became 0%. The separation was carried out at a flow rate of 1mL / min for 30 minutes, and the detection wavelength was 220nm. The fractions with a purity ≥95% were collected (such as attached Figure 1 as shown).

[0027] Identification of the obtained antimicrobial peptide Loong-3 product: (1) Determination of molecular weight: The molecular weight of the antimicrobial peptide Loong-3 was determined by analytical liquid chromatography-mass spectrometry (Waters micromass ZQ-2000). The molecular weight was 2117.75 Da. Figure 2 shown.

[0028] The specific detection conditions are as follows: ① Chromatographic conditions: the chromatographic column is a C18 reverse phase column (4.6*250mm), the mobile phase A is a 0.1% trifluoroacetic acid acetonitrile solution (0.1mL trifluoroacetic acid is dissolved in 100mL acetonitrile solution), and the mobile phase B is a 0.1% trifluoroacetic acid aqueous solution (0.1mL trifluoroacetic acid is dissolved in 100mL deionized water). Within 25 minutes after loading, the proportion of mobile phase A is gradually adjusted from 25% to 50%, and the proportion of mobile phase B is gradually adjusted from 75% to 50%, at 25.1min, the proportion of mobile phase A became 100%, and the proportion of mobile phase B became 0%, and the separation was carried out at a flow rate of 1mL / min for 30min, and the detection wavelength was 220nm; ②Mass spectrometry conditions: positive ionization mode was used, with a capillary voltage of 3.00KV, a capillary outlet voltage of 50V, a fragmentation voltage of 5V, a drying gas flow rate of 1.5L / min, a drying gas temperature of 350℃, and a scanning range of 400-1900m / z.

[0029] (2) Amino acid sequence determination: The amino acid sequence of the antimicrobial peptide Loong-3 was determined using an automatic amino acid sequencer. It was determined that the antimicrobial peptide Loong-3 contains 18 amino acid residues, and its amino acid sequence is: lysine-arginine-isoleucine-alanine-lysine-leucine-alanine-leucine-lysine-alanine-isoleucine-arginine-lysine-isoleucine-leucine-arginine-leucine-isoleucine.

[0030] (3) Isoelectric point analysis: Open EditSeq in DNAStar software, open file, click "newprotein" in "new", input the amino acid sequence of antimicrobial peptide Loong-3, and save it as a pro format document. Then open Protean in DNAStar software, open file, click "open", select the document just saved in pro format, click "open", select "titration curve" in "analysis", and you can get the isoelectric point data. The results are shown in Figure 3 The isoelectric point of the antimicrobial peptide Loong-3 of the present invention was determined to be 12.4.

[0031] (4) Helical wheel structure model analysis: Open EditSeq in DNAStar software, open file, click "new protein" in "new", input the amino acid sequence of antimicrobial peptide Loong-3, and save it as a pro format document. Then open Protean in DNAStar software, open file, click "open", select the document just saved in pro format, click "open", select "Model Structure" in "Analysis", click "Helical Wheel", and you can get the helical wheel structure model diagram. The results are shown in Figure 4 .Depend on Figure 4 It can be seen that the positively charged amino acids are all located on one side of the helical structure, and the other uncharged amino acids are all located on the other side of the helical structure, indicating that the antimicrobial peptide Loong-3 has a better amphipathic structure.

[0032] (5) 3D model analysis: The online tool PEP-FOLD 3 was used for analysis. Log in to the online website of this tool (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ), click “Run PEP-FOLD3” on the page, enter the amino acid sequence of the antimicrobial peptide Loong-3 in the “Input Data” area, select “PEPFOLD” in “Run label” in “Options”, select “100” in “Number of simulations”, select “sOPEP” in “Sort model by”, and then click the “RUN” button. After waiting for a while, the 3D model image can be obtained. The results are shown in the figure. Figure 5 .Depend on Figure 5 It can be seen that the antimicrobial peptide Loong-3 has a helical structure.

[0033] Example 2 Performance analysis of antimicrobial peptide Loong-3: 2.1 Analysis of antibacterial activity of antimicrobial peptide Loong-3 The antibacterial activity of the antimicrobial peptide Loong-3 was determined by the agar plate diffusion method. The tested microbial strains included Escherichia coli, Salmonella, Staphylococcus aureus and Candida albicans. Staphylococcus aureus CVCC6538, Escherichia coli CVCC1568 and Salmonella pullorum CVCC533 were purchased from the China Veterinary Microbiological Culture Collection Center, Staphylococcus aureus ATCC25923 and Candida albicans ATCC10231 were purchased from Nanjing Beneficial Biotechnology Co., Ltd. The clinical resistant strains of Escherichia coli were isolated and identified by the applicant (Li Jie, Hang Bolin, Dong Mengmeng, et al. Isolation and identification of resistant Escherichia coli from pigs [J]. China Swine Industry, 2018, 13(12): 42-46, 50).

[0034] The above-mentioned test microorganisms (1×10 6CFU / mL) were diluted in a 50°C bottom culture medium (TSB 0.45 g, ultrapure agarose 0.15 g, ultrapure water to 15 mL, pH 7.4), poured into a sterilized plate, solidified, punched with a hole puncher (aperture of about 4 mm), slightly heated with an alcohol lamp to seal the bottom, and 15 μL of antimicrobial peptide (1 mg / mL) was added to each well with a pipette. Each plate included a positive control and a negative control. Positive control: gentamicin (for Gram-negative and Gram-positive bacteria), antimicrobial peptide BSN-37 (for clinical resistant strains of Escherichia coli, the antimicrobial peptide reference patent (a Bac5-like antimicrobial peptide, patent application number: 201710643182.8, application publication number: CN107298707A), copper sulfate (for fungi); negative control: PBS buffer. The plate was left to stand for 1 hour to allow the test solution to diffuse into the agarose. Then, the upper covering culture medium (TSB 0.3g, ultrapure agarose 0.1g, ultrapure water to 10ml, pH 7.4, about 50°C) was added. The culture plate was inverted and cultured overnight at 37°C, and then the diameter of the transparent circle around the well was recorded. Each strain was measured three times and the average value was calculated.

[0035] Table 1. Antibacterial activity analysis results of antimicrobial peptide Loong-3 The antibacterial activity test results in Table 1 show that the antimicrobial peptide Loong-3 of the present invention has antibacterial activity against Escherichia coli, Salmonella, Staphylococcus aureus and Candida albicans, and also has antibacterial activity against clinically resistant Escherichia coli (Li Jie, Hang Bolin, Dong Mengmeng, et al. Isolation and identification of drug-resistant Escherichia coli from pigs [J]. China Swine Industry, 2018, 13 (12): 42-46, 50.). Therefore, the antimicrobial peptide Loong-3 of the present invention can have a good application prospect in the preparation of drugs for antibacterial and / or fungal infections in animals and humans. At the same time, according to the general characteristics of known antimicrobial peptides, the antimicrobial peptide Loong-3 will also have good applications in feed additives, disinfectants, preservatives, detergents or cosmetic additives, antibacterial molecular materials, etc.

[0036] 2.2 Minimum inhibitory concentration (MIC) determination of antimicrobial peptide Loong-3 The tested strains included Escherichia coli, Salmonella, Staphylococcus aureus and Candida albicans. Among them, Staphylococcus aureus CVCC6538, Escherichia coli CVCC1568 and Salmonella pullorum CVCC533 were purchased from the China Veterinary Microbiological Culture Collection Center, Staphylococcus aureus ATCC25923 and Candida albicans ATCC10231 were purchased from Nanjing Bianzhen Biotechnology Co., Ltd., and the clinical resistant strains of Escherichia coli were isolated and identified by the applicant (Li Jie, Hang Bolin, Dong Mengmeng, et al. Isolation and identification of resistant Escherichia coli from pigs [J]. China Swine Industry, 2018, 13(12): 42-46, 50).

[0037] The tested strain was cultured in TSB liquid medium and the OD of the bacterial solution was measured. 600 When the OD value is between 0.6 and 0.8, centrifuge the bacterial solution at 6000 r / min for 10 min, discard the supernatant, collect the bacterial precipitate, and resuspend the bacterial precipitate with an equal volume of PBS buffer (0.01 mol / L); then dilute the bacterial suspension to 2×10 6 CFU / mL (Escherichia coli, Salmonella, Staphylococcus aureus), fungal suspension diluted to 2×10 4 CFU / mL (Candida albicans). The antimicrobial peptide Loong-3 sample was diluted with TSB liquid culture medium using the two-fold dilution method to a concentration of 0.244-500μg / mL. In a 96-well bacterial culture plate, 50μL of different concentrations of antimicrobial peptide Loong-3 and 50μL of diluted bacterial solution were added to each well. Each concentration was repeated three times and cultured at 37℃ for 16-18h. Then the solution in each well was mixed and its OD was measured. 600 The value, the concentration of antimicrobial peptide Loong-3 corresponding to the sudden change in OD value is its minimum inhibitory concentration (MIC), the results are shown in Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 and Fig.11 .

[0038] Depend on Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 and Fig.11It can be seen that the minimum inhibitory concentration (MIC) of the antimicrobial peptide Loong-3 against Escherichia coli CVCC1568 is 15.625 μg / mL, the minimum inhibitory concentration (MIC) against clinical drug-resistant strains of Escherichia coli is 7.8125 μg / mL, the minimum inhibitory concentration (MIC) against Salmonella pullorum CVCC533 is 0.976 μg / mL, the minimum inhibitory concentration (MIC) against Staphylococcus aureus ATCC25923 is 31.25 μg / mL, the minimum inhibitory concentration (MIC) against Staphylococcus aureus CVCC6538 is 31.25 μg / mL, and the minimum inhibitory concentration (MIC) against Candida albicans ATCC10231 is 125 μg / mL. The results show that the minimum inhibitory concentration of the antimicrobial peptide Loong-3 against common bacteria reaches the microgram level. This shows that the antimicrobial peptide Loong-3 of the present invention has extremely strong antibacterial activity.

[0039] 2.3 Hemolytic analysis of antimicrobial peptide Loong-3 Take fresh chicken blood, anticoagulate with 3.8% sodium citrate, centrifuge the anticoagulated blood at 3000r / min for 10min, wash the precipitate with sterile saline for 3 times until the supernatant is colorless and transparent, and then prepare 1% red blood cells with sterile saline. Use the two-fold dilution method to adjust the concentration of the antimicrobial peptide Loong-3 to 0.244-125μg / mL with sterile saline, and add an equal volume of red blood cell suspension. Use sterile saline as the negative control and 1% Triton X-100 as the positive control, act at 37℃ for 1h, centrifuge at 2000r / min for 5min, add the supernatant to a 96-well plate in turn, and measure the OD value at 540nm with an enzyme marker. Then calculate the hemolysis rate of the antimicrobial peptide Loong-3 according to the calculation formula [hemolysis rate (%) = (OD value of the detection well - OD value of the negative well) / (OD value of the positive well - OD value of the negative well) × 100%], and the results are shown in Fig.12 .

[0040] Depend on Fig.12 It can be seen that the hemolysis rate gradually increased with the increase of the concentration of antimicrobial peptide Loong-3. The hemolysis rate was 67.5% at 125 μg / mL and 5.78% at 0.98 μg / mL.

[0041] 2.4 Analysis of the heat resistance of the antimicrobial peptide Loong-3 The antimicrobial peptide Loong-3 solution with a concentration of 0.5 mg / mL was placed at 0℃, 20℃, 40℃, 60℃, 80℃, and 100℃ for 15 minutes respectively; then the antibacterial activity of the antimicrobial peptide Loong-3 solution after treatment was tested according to the minimum inhibitory concentration determination method in 2.2. The tested microbial strain was Escherichia coli CVCC1568. The results are shown in Fig.13 .

[0042] Depend on Fig.13 It can be seen that with the increase of temperature, the antibacterial activity of antimicrobial peptide Loong-3 against Escherichia coli first increases and then decreases. The antibacterial activity is lowest after treatment at 40°C and highest after treatment at 100°C. This shows that the antimicrobial peptide Loong-3 has good heat resistance.

[0043] 2.5 Analysis of salt-tolerance stability of antimicrobial peptide Loong-3 The antimicrobial peptide Loong-3 solution with a concentration of 0.5 mg / mL was mixed with equal amounts of physiological concentrations of sodium chloride (NaCl, 150 mM), potassium chloride (KCl, 5 mM), magnesium chloride (MgCl2, 1 mM), calcium chloride (CaCl2, 5 mM), ferric chloride (FeCl3, 4 μM) and copper sulfate (CuSO4, 25 μM), respectively, and placed at 37°C for 15 minutes. The antibacterial activity of the antimicrobial peptide Loong-3 solution after treatment was then tested according to the minimum inhibitory concentration determination method in 2.2. The test microorganism was Escherichia coli CVCC1568.

[0044] Depend on Fig.14 It can be seen that after being treated with the above-mentioned physiological concentrations of salt solutions, except for copper ions, other salt ions have almost no effect on the antibacterial activity of the antimicrobial peptide Loong-3 against Escherichia coli. This shows that the antimicrobial peptide Loong-3 has good salt tolerance to most salt ions.

[0045] 2.6 Analysis of acid and alkali stability of antimicrobial peptide Loong-3 The antimicrobial peptide Loong-3 solution with a concentration of 0.5 mg / mL was mixed with an equal amount of sterile aqueous solution with a pH value of 4, 5, 6, 7, 8, 9, and 10 (adjusted with 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide), placed at 37°C for 15 minutes, and then the antibacterial activity of the antimicrobial peptide Loong-3 solution after treatment was tested according to the minimum inhibitory concentration determination method in 2.2. The test microorganism was Escherichia coli CVCC1568.

[0046] Depend on Fig.15 It can be seen that after being treated with the above-mentioned different pH solutions, the antibacterial activity of the antimicrobial peptide Loong-3 against Escherichia coli is higher in the acidic and alkaline regions than in the neutral region, but both have good antibacterial activity. This shows that the antimicrobial peptide Loong-3 has good acid and alkali resistance.

[0047] 2.7 Analysis of the repeated freeze-thaw stability of the antimicrobial peptide Loong-3 The antimicrobial peptide Loong-3 solution with a concentration of 0.5 mg / mL was repeatedly frozen and thawed at room temperature for 4, 6, 8, 10, and 12 times, and then the antibacterial activity of the antimicrobial peptide Loong-3 solution after treatment was tested according to the minimum inhibitory concentration determination method in 2.2. The test microorganism was Escherichia coli CVCC1568.

[0048] Depend on Fig.16 It can be seen that the antibacterial activity of the antimicrobial peptide Loong-3 against Escherichia coli changes slightly with the increase of freeze-thaw cycles, but it still retains good antibacterial activity. This shows that the antimicrobial peptide Loong-3 has good resistance to repeated freeze-thaw cycles.

[0049] 2.8 Analysis of serum stability of antimicrobial peptide Loong-3 The antimicrobial peptide Loong-3 solution with a concentration of 0.5 mg / mL was mixed with an equal amount of calf serum (0%, 5%, 10%, 15%, 20%, 25%), placed at 37°C for 15 minutes, and then the antibacterial activity of the antimicrobial peptide Loong-3 solution after treatment was tested according to the minimum inhibitory concentration determination method in 2.2. The test microorganism was Escherichia coli CVCC1568.

[0050] Depend on Fig.17 It can be seen that the antibacterial activity of the antimicrobial peptide Loong-3 against Escherichia coli changes slightly with the increase of serum concentration, but it still retains good antibacterial activity. This shows that the antimicrobial peptide Loong-3 has good serum resistance.

[0051] 2.9 Analysis of the organic solvent stability of the antimicrobial peptide Loong-3 The antimicrobial peptide Loong-3 solution with a concentration of 0.5 mg / mL was mixed with an equal amount of organic solvents (10% ethanol, 10% ethyl acetate, 10% toluene), placed at 37°C for 15 minutes, and then the antibacterial activity of the antimicrobial peptide Loong-3 solution after treatment was tested according to the minimum inhibitory concentration determination method in 2.2. The test microorganism was Escherichia coli CVCC1568.

[0052] Depend on Fig.18 It can be seen that after being treated with organic solvents, the antibacterial activity of the antimicrobial peptide Loong-3 against Escherichia coli changed slightly, but it still retained good antibacterial activity. This shows that the antimicrobial peptide Loong-3 has good resistance to organic solvents.

[0053] In summary, the antimicrobial peptide Loong-3 product of the present invention has a certain hemolytic property, a broad antimicrobial spectrum, good heat resistance, salt resistance, acid and alkali resistance, repeated freezing and thawing resistance, organic solvent resistance and serum resistance, and has a highly effective antimicrobial effect on Gram-negative bacteria, Gram-positive bacteria and fungi. Therefore, the antimicrobial peptide Loong-3 product of the present invention can be well applied in the preparation of drugs for resisting Gram-positive bacteria, Gram-negative bacteria and / or fungal infections, and can also be used to prepare feed additives, disinfectants, preservatives, detergents, cosmetic additives, antimicrobial molecular materials or pesticides.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antimicrobial peptide, characterized in that: The amino acid sequence of the antimicrobial peptide is as follows: lysine-arginine-isoleucine-alanine-lysine-leucine-alanine-leucine-lysine-alanine-isoleucine-arginine-lysine-isoleucine-leucine-arginine-leucine-isoleucine, as shown in SEQ ID NO:

1.

2. Use of the antimicrobial peptide according to claim 1 in the preparation of drugs for resisting bacterial and / or fungal infections.

3. The use according to claim 2, characterized in that: The drug comprises the antimicrobial peptide according to claim 1, and further comprises one or more pharmaceutically acceptable carriers and / or additives.

4. The use according to claim 2, characterized in that: The bacteria include Gram-positive bacteria and Gram-negative bacteria. The Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Escherichia coli and Salmonella.

5. The use according to claim 2, characterized in that: The fungi include Candida albicans.

6. Use of the antimicrobial peptide according to claim 1 in the preparation of feed additives, disinfectants, preservatives, detergents, cosmetic additives, antimicrobial molecular materials or pesticides.

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