Narrow-spectrum antibacterial peptide XMK-8 and application thereof

Abstract

The invention belongs to the technical field of biology, and relates to a narrow-spectrum antibacterial peptide XMK-8 and application thereof. The amino acid sequence of the narrow-spectrum antibacterial peptide XMK-8 is as shown in SEQ ID NO: 1, and the narrow-spectrum antibacterial peptide XMK-8 has no bacteriostatic activity on escherichia coli, salmonella, staphylococcus aureus and aeromonas hydrophila and only has bacteriostatic activity on pasteurella multocida and haemophilus parasuis. The antibacterial activity of the narrow-spectrum antibacterial peptide XMK-8 can still be detected after treatment with salt ions (sodium ions and potassium ions, 50-200 mmol / L), acid-base (pH 4-10) and protease K (20-100 [mu] g / mL) at the temperature of 0-100 DEG C, and the narrow-spectrum antibacterial peptide XMK-8 has low hemolytic activity on red blood cells of mice. The antibacterial peptide XMK-8 disclosed by the invention is expected to become a novel antibiotic substitute and has a good application prospect in prevention, treatment, diagnosis and identification of pasteurella multocida, haemophilus parasuis and infection thereof.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a narrow-spectrum antimicrobial peptide XMK-8 and an application thereof. Background Art

[0002] At present, bacterial resistance is a serious threat to the health of humans and animals. The iteration speed of new antibiotics is far behind the pace of bacterial resistance. Therefore, finding antibiotic substitutes has become an inevitable choice. Antimicrobial peptides (AMPs) have become the "leader" in the family of antibiotic substitutes due to their low resistance to drug resistance, simple structure, small molecular weight, diverse biological activities, and green and no residue.

[0003] Antimicrobial peptides mainly exert their bactericidal effects through unique membrane damage mechanisms and intracellular bactericidal mechanisms. This bactericidal mechanism is different from that of antibiotics, making antimicrobial peptides a highly regarded alternative to antibiotics. Natural antimicrobial peptides are one of the important components of the innate immune system of biological organisms and play an important role in resisting pathogenic infections. However, many natural antimicrobial peptides have some defects, such as low antimicrobial activity, low safety, poor stability, and immunogenicity. New antimicrobial peptides can be obtained by artificially modifying natural antimicrobial peptides. Artificially modified antimicrobial peptides are modified by modifying the amino acid sequence of existing antimicrobial peptides based on the structure-activity relationship of antimicrobial peptides, and then functional verification is performed. Based on this, antimicrobial peptides can also be artificially designed based on the structure-activity relationship of antimicrobial peptides. Modified or designed antimicrobial peptides are all non-natural antimicrobial peptides. These antimicrobial peptides often have a common feature, that is, they have broad-spectrum antimicrobial activity. This property makes antimicrobial peptides have bactericidal activity against both beneficial and harmful bacteria. However, bacteria that are beneficial to the host do not want to be killed. To this end, it is necessary to design antimicrobial peptides with a narrow antimicrobial spectrum, namely narrow-spectrum antimicrobial peptides.

[0004] Narrow-spectrum antimicrobial peptides should have antimicrobial activity against only a few bacteria or only one target bacterium. Therefore, the application of narrow-spectrum antimicrobial peptides in clinical practice will be very targeted or specific. They can be used as specific drugs for the prevention and treatment of infections caused by corresponding pathogens, as special reagents for the diagnosis and identification of corresponding pathogens, and as specific sterilization reagents or disinfectants in the preparation of inactivated vaccines. Summary of the invention

[0005] In order to solve the problem that existing broad-spectrum antimicrobial peptides have bactericidal activity against both beneficial bacteria and harmful bacteria, the present invention provides a narrow-spectrum antimicrobial peptide XMK-8, which has antimicrobial activity against Pasteurella multocida and Haemophilus parasuis, but has no antimicrobial activity against Escherichia coli, Salmonella, Aeromonas hydrophila and Staphylococcus aureus.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions: The present invention is based on the amino acid sequence of positions 1919 to 1936 of goose MyHC1 protein (NCBI database, accession number: KM675469.1), and adopts an amino acid replacement method to obtain an antimicrobial peptide XMK-8, whose amino acid sequence is arginine-leucine-leucine-proline-lysine-leucine-proline-arginine-lysine-valine-proline-arginine-phenylalanine-proline-arginine-lysine-isoleucine-proline, which is expressed as RLLPKLPRKVPRFPRKIP in single letters, as shown in SEQ ID NO: 1.

[0007] The antimicrobial peptide XMK-8 contains 18 amino acid residues, has a molecular weight of 2211.78 Da, an isoelectric point of 12.6, and an electrostatic charge of +7, and is a basic antimicrobial peptide.

[0008] The antibacterial performance analysis of the antimicrobial peptide XMK-8 showed that the antimicrobial peptide XMK-8 had good antimicrobial activity against Pasteurella multocida and Haemophilus parasuis, but had no antimicrobial activity against Escherichia coli, Salmonella, Aeromonas hydrophila and Staphylococcus aureus, which indicated that the antimicrobial peptide was a narrow-spectrum antimicrobial peptide. The minimum inhibitory concentration of the narrow-spectrum antimicrobial peptide XMK-8 against Pasteurella multocida was 0.25 mg / mL, and the minimum inhibitory concentration against Haemophilus parasuis was 1 mg / mL.

[0009] The above-mentioned narrow-spectrum antimicrobial peptide XMK-8 was subjected to hemolytic analysis, thermal stability analysis, acid-base stability analysis, enzyme stability analysis, and salt ion stability analysis. The results showed that the hemolytic activity of the narrow-spectrum antimicrobial peptide XMK-8 was low, and at a high concentration of 500 μg / mL, the hemolytic rate was still lower than 20%; the narrow-spectrum antimicrobial peptide XMK-8 had good thermal stability, and after high-temperature treatment at 75°C-100°C, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity; after treatment with different acid-base solutions, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity; after treatment with different concentrations of proteinase K, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity; after treatment with different concentrations of sodium ion and potassium ion solutions, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity.

[0010] Based on the above content, the narrow-spectrum antimicrobial peptide XMK-8 can be used to prepare products for preventing, inhibiting, diagnosing and / or identifying bacterial infections, including but not limited to inactivated vaccines, anti-infective drugs, and disinfectant products, and the bacteria are Pasteurella multocida and / or Haemophilus parasuis.

[0011] The present invention also provides a bacterial disinfection product, which uses the narrow-spectrum antimicrobial peptide XMK-8 as an effective ingredient and has antibacterial activity against Pasteurella multocida and / or Haemophilus parasuis.

[0012] The present invention also provides a product for diagnosing and / or identifying bacterial infection, the product comprising the narrow-spectrum antimicrobial peptide XMK-8, physiological saline and bacterial culture medium, and the product is used for diagnosing and / or identifying the bacteria Pasteurella multocida and / or Haemophilus parasuis. If the bacteria to be identified can be killed by the product, the bacteria to be identified are one or both of the two bacteria Pasteurella multocida and Haemophilus parasuis, and if the bacteria to be identified cannot be killed by the product, the bacteria to be identified are other bacteria other than these two bacteria. The bacterial culture medium can meet the growth of bacteria, such as the commercially available Tryptic Soy Broth (TSB).

[0013] The beneficial effects of the present invention are: According to the structure-activity relationship of antimicrobial peptides, the present invention designs an antimicrobial peptide XMK-8 with the aid of bioinformatics tools. Bioinformatics prediction shows that the molecular weight of the antimicrobial peptide XMK-8 is 2211.78 Da, the isoelectric point is 12.6, and the electrostatic charge is +7.

[0014] The present invention has found through antibacterial activity determination that the antimicrobial peptide XMK-8 has antibacterial activity against Pasteurella multocida and Haemophilus parasuis, but has no antibacterial activity against Escherichia coli, Salmonella, Aeromonas hydrophila and Staphylococcus aureus, indicating that the antimicrobial peptide XMK-8 is a narrow-spectrum antimicrobial peptide.

[0015] The narrow-spectrum antimicrobial peptide XMK-8 provided by the present invention has low hemolytic activity. Specifically, within the test concentration range, the hemolytic rate of the antimicrobial peptide XMK-8 on red blood cells is less than 20%.

[0016] Therefore, the narrow-spectrum antimicrobial peptide XMK-8 can be used as a green and safe antimicrobial substance for the treatment, prevention, diagnosis and identification of Pasteurella multocida infection and Haemophilus parasuis infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the result of reverse phase high performance liquid chromatography purification of narrow spectrum antimicrobial peptide XMK-8.

[0018] Figure 2 This is the mass spectrometry identification result of the narrow-spectrum antimicrobial peptide XMK-8.

[0019] Figure 3 This is the isoelectric point analysis result of the narrow-spectrum antimicrobial peptide XMK-8.

[0020] Figure 4 This is the helical wheel structure diagram of the narrow-spectrum antimicrobial peptide XMK-8.

[0021] Figure 5 This is a 3D model diagram of the narrow-spectrum antimicrobial peptide XMK-8.

[0022] Figure 6 It is the minimum inhibitory concentration of the narrow-spectrum antimicrobial peptide XMK-8 against Pasteurella multocida strain EO630.

[0023] Figure 7 It is the minimum inhibitory concentration of narrow-spectrum antimicrobial peptide XMK-8 against Escherichia coli ATCC25922.

[0024] Figure 8 It is the minimum inhibitory concentration of the narrow-spectrum antimicrobial peptide XMK-8 against Salmonella typhimurium CVCC541.

[0025] Fig. 9 It is the minimum inhibitory concentration of narrow-spectrum antimicrobial peptide XMK-8 against Staphylococcus aureus ATCC49525.

[0026] Fig.10 It is the minimum inhibitory concentration of the narrow-spectrum antimicrobial peptide XMK-8 against Aeromonas hydrophila strains.

[0027] Fig.11 It is the minimum inhibitory concentration of the narrow-spectrum antimicrobial peptide XMK-8 against Haemophilus parasuis strains.

[0028] FIG. 12 shows the hemolysis rate of the narrow-spectrum antimicrobial peptide XMK-8 on mouse erythrocytes.

[0029] FIG. 13 shows the results of thermal stability test of narrow-spectrum antimicrobial peptide XMK-8.

[0030] FIG. 14 shows the results of the acid-base stability test of the narrow-spectrum antimicrobial peptide XMK-8.

[0031] FIG. 15 shows the results of the proteinase K stability test of the narrow-spectrum antimicrobial peptide XMK-8.

[0032] FIG. 16 shows the results of the sodium ion stability test of the narrow-spectrum antimicrobial peptide XMK-8.

[0033] FIG. 17 shows the potassium ion stability test results of the narrow-spectrum antimicrobial peptide XMK-8. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 The amino acid sequence of the narrow-spectrum antimicrobial peptide XMK-8 of the present invention is as follows: Arginine-Leucine-Leucine-Proline-Lysine-Leucine-Proline-Arginine-Lysine-Valine-Proline-Arginine-Phe-Proline-Arginine-Lysine-Isoleucine-Proline (SEQ ID NO: 1).

[0036] The synthesis method of the narrow-spectrum antimicrobial peptide XMK-8 of the present invention is as follows: The narrow-spectrum antimicrobial peptide XMK-8 of the present invention is synthesized by an automatic peptide synthesizer according to conventional peptide solid phase chemical synthesis method, and the synthesis direction is from C-terminus to N-terminus. The detailed steps of its synthesis are as follows: Step a, swelling resin: add Fmoc-Pro-Wang resin into the reactor of the automatic peptide synthesizer, and add dimethylformamide (DMF) for swelling. The amount of DMF added to 1g Fmoc-Pro-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; Step b, deprotection: After the resin is swollen, 20% piperidine (20% piperidine is prepared by dissolving piperidine in DMF) in a storage tank is added to the reactor, and the resin is immersed (1g Fmoc-Pro-Wang resinin resin needs to add about 10mL20% piperidine), and the swollen resin is deprotected for about 30min, and then DMF is added for washing; Step c, condensation reaction: then, according to the sequence of the amino acid sequence of the narrow-spectrum antimicrobial peptide XMK-8, the penultimate amino acid, i.e., isoleucine Fmoc-Ile, 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 isoleucine to Fmoc-Pro-Wang resinin is 5:1; the molar ratio of the condensation agent HCTU to isoleucine Fmoc-Ile is 1:1; and the molar ratio of the catalyst NMM to isoleucine is 4:1.

[0037] The product obtained after washing is deprotected and washed according to the method of step b, and Fmoc-Lys, Fmoc-Arg, Fmoc-Pro, Fmoc-Phe, Fmoc-Arg, Fmoc-Pro, Fmoc-Val, Fmoc-Lys, Fmoc-Arg, Fmoc-Pro, Fmoc-Leu, Fmoc-Lys, Fmoc-Pro, Fmoc-Leu, Fmoc-Leu and Fmoc-Arg 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-Pro-Wang 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).

[0038] 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.

[0039] 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.

[0040] 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 narrow-spectrum antimicrobial peptide XMK-8 with a purity of ≥95%.

[0041] 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).

[0042] Identification of the narrow-spectrum antimicrobial peptide XMK-8 product obtained by the present invention: (1) Determination of molecular weight: The molecular weight of the obtained narrow-spectrum antimicrobial peptide XMK-8 was determined by analytical liquid chromatography-mass spectrometry (Waters micromass ZQ-2000). The molecular weight was 2211.78 Da. The results are shown in the attached figure. Figure 2 shown.

[0043] 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.

[0044] (2) Amino acid sequence determination: The amino acid sequence of the narrow-spectrum antimicrobial peptide XMK-8 was determined using an automatic amino acid sequencer. It was determined that the narrow-spectrum antimicrobial peptide XMK-8 contains 18 amino acid residues, and its amino acid sequence is: arginine-leucine-leucine-proline-lysine-leucine-proline-arginine-lysine-valine-proline-arginine-phenylalanine-proline-arginine-lysine-isoleucine-proline.

[0045] Isoelectric point analysis of the narrow-spectrum antimicrobial peptide XMK-8 obtained in the present invention: Open EditSeq in DNAStar software, open file, click "new protein" in "new", input the amino acid sequence of narrow-spectrum antimicrobial peptide XMK-8, 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 It was determined that the isoelectric point of the narrow-spectrum antimicrobial peptide XMK-8 of the present invention is 12.6.

[0046] Structural analysis of the narrow-spectrum antimicrobial peptide XMK-8 obtained in the present invention: (1) Analysis of the helical wheel structure model: Open EditSeq in the DNAStar software, open the file, click "new protein" in "new", enter the amino acid sequence of the narrow-spectrum antimicrobial peptide XMK-8 (single-letter format), and save it as a pro format document. Then open Protean in the DNAStar software, open the file, click "open", select the document just saved in pro format, click "open", select "Model Structure" in "Analysis", and click "HelicalWheel" to obtain 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 narrow-spectrum antimicrobial peptide XMK-8 has a good amphipathic structure.

[0047] (2) 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 narrow-spectrum antimicrobial peptide XMK-8 (single-letter format) 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 Figure 2. Figure 5 .Depend on Figure 5 It can be seen that the narrow-spectrum antimicrobial peptide XMK-8 is a partially helical structure.

[0048] Example 2 This example is a performance analysis of the narrow-spectrum antimicrobial peptide XMK-8.

[0049] 2.1 Analysis of antibacterial properties of narrow-spectrum antimicrobial peptide XMK-8 The tested strains were Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Pasteurella multocida, Aeromonas hydrophila, and Haemophilus parasuis. Staphylococcus aureus ATCC49525 was purchased from Hangzhou Baosai Biotechnology Co., Ltd., Escherichia coli ATCC25922 was purchased from Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd., Salmonella typhimurium CVCC541 was purchased from the China Veterinary Drug Administration, Pasteurella multocida EO630 was isolated and identified from the commercially available triple live vaccine of swine fever-swine erysipelas-swine Pasteurella multocida in the laboratory, Aeromonas hydrophila was donated by Associate Professor Wang Li of Henan Institute of Science and Technology, and Haemophilus parasuis was isolated and identified from diseased pigs by the research team (Wang Lingcai, Wang Qing, Wang Xiaofei, et al. Isolation and identification of Haemophilus parasuis type 14 and its pathogenicity detection [J]. Chinese Journal of Preventive Veterinary Medicine, 2020, 42(8): 766-771.). Other commercially available products may also be used for Pasteurella multocida, Aeromonas hydrophila, and Haemophilus parasuis.

[0050] The antibacterial activity of the narrow-spectrum antimicrobial peptide XMK-8 was tested by the microbroth dilution method. A single colony of the test strain was picked and placed in 5 mL of TSB liquid culture medium. The culture was shaken at 37 °C and 180 r / min for about 4-5 h until the bacterial solution concentration was about 1×10 8 CFU / mL, dilute the bacterial solution with TSB liquid medium to 1×10 5 CFU / mL. Add 50μL of TSB liquid culture medium to a row of wells in a 96-well bacterial culture plate, add 50μL of 1mg / mL narrow-spectrum antimicrobial peptide XMK-8 to the first well of the 96-well bacterial culture plate, pipette to mix, take 50μL of the mixed liquid and place it in the second well, pipette to mix, and so on. Take out 50μL of the mixed liquid from the 10th well and discard it. The 11th well is a control with bacteria and no drug (narrow-spectrum antimicrobial peptide XMK-8), and the 12th well is a control without bacteria and no drug. Add 50 μL of diluted bacterial solution to wells 1-11. Gently rotate the culture plate to mix the liquid. Place the culture plate in a 37°C constant temperature incubator, culture it for 18-24 hours, and then read the OD 600 Value, in OD 600 The concentration of the narrow-spectrum antimicrobial peptide XMK-8 corresponding to the wells with sudden changes in the value is the minimum inhibitory concentration (MIC) for the tested strain.

[0051] The results showed that the minimum inhibitory concentration of the narrow-spectrum antimicrobial peptide XMK-8 against porcine Pasteurella multocida was 250 μg / mL ( Figure 6 coli (as shown in Figure 7 as shown), Salmonella typhimurium (as shown Figure 8 as shown), Staphylococcus aureus (as Fig. 9 ) and Aeromonas hydrophila (as Fig.10 The minimum inhibitory concentration against Haemophilus parasuis was 1 mg / mL (as shown in Fig.11 as shown).

[0052] 2.2 Hemolytic analysis of narrow-spectrum antimicrobial peptide XMK-8 The mouse blood anticoagulated with 3.8% sodium citrate was centrifuged at 3000 rpm for 10 min, the precipitate was taken, the red blood cells were washed with 0.9% saline, and then centrifuged at 3000 rpm for 10 min, the precipitate was taken, and the washing was repeated 3 times. Finally, a mouse red blood cell suspension with a concentration of 2% was prepared with saline. 100 μL of saline was added to a row of wells in a 96-well bacterial culture plate, 100 μL of narrow-spectrum antimicrobial peptide XMK-8 (2 mg / mL) was added to the first well, and then the antimicrobial peptide was double-diluted with saline according to the method in 2.1. After the dilution in the 10th well, 100 μL of the mixed liquid was discarded, no antimicrobial peptide was added to the 11th well as a negative control, 100 μL of 1% Triton X-100 was added to the 12th well as a positive control, and then 100 μL of each well was added 2% red blood cell suspension, gently shake the culture plate to make all the objects in the wells fully contact, then put it in a 37℃ constant temperature incubator for 1h, take out the culture plate, gently mix the liquid in the wells, transfer the liquid to 1.5mL centrifuge tubes, centrifuge at 3000 rpm for 5min, take 150μL of the upper layer liquid and transfer it to another row of wells in the 96-well bacterial culture plate, use a microplate reader to measure the absorbance (OD) value of the liquid in the well at a wavelength of 540nm, and finally calculate the hemolysis rate of the antimicrobial peptide according to the formula [hemolysis rate = (OD sample-OD negative) / (OD positive-OD negative) × 100%]. The results are as follows: Fig.12 The results showed that the narrow-spectrum antimicrobial peptide XMK-8 had low hemolytic activity, and at a high concentration of 500 μg / mL, the hemolytic rate was still less than 20%.

[0053] 2.3 Thermal stability analysis of narrow-spectrum antimicrobial peptide XMK-8 A 1 mg / mL narrow-spectrum antimicrobial peptide XMK-8 solution was divided into 5 equal portions, 50 μL / portion, and treated at 0°C, 25°C, 50°C, 75°C, and 100°C for 30 min, respectively. Then, the minimum inhibitory concentration of the antimicrobial peptide after treatment at different temperatures was determined according to the method described in 2.1. The test bacteria were porcine Pasteurella multocida strains. The results are as follows: Fig.13 The results showed that the narrow-spectrum antimicrobial peptide XMK-8 had good thermal stability and after high-temperature treatment at 75℃-100℃, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity.

[0054] 2.4 Analysis of acid-base stability of narrow-spectrum antimicrobial peptide XMK-8 Use 1mol / L hydrochloric acid solution and 1mol / L sodium hydroxide solution to prepare solutions with pH values ​​of 4, 5, 6, 7, 8, 9, and 10. Divide the 2mg / mL narrow-spectrum antimicrobial peptide XMK-8 solution into 7 equal parts, 50μL / part, and mix them with equal volumes of solutions with different pH values, and act at 37℃ for 30min. Then, the minimum inhibitory concentration of the antimicrobial peptide after treatment with different pH acid and alkali solutions is determined according to the method described in 2.1. The test bacteria are porcine Pasteurella multocida strains. The results are as follows Fig.14 The results showed that after being treated with different acid and alkali solutions, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity.

[0055] 2.5 Analysis of enzyme stability of narrow-spectrum antimicrobial peptide XMK-8 Prepare 0μg / mL, 20μg / mL, 40μg / mL, 60μg / mL, 80μg / mL and 100μg / mL proteinase K solutions with sterile water. Divide the 2mg / mL narrow-spectrum antimicrobial peptide XMK-8 solution into 6 equal portions, 50μL / portion, and mix with equal volumes of proteinase K solutions of different concentrations, incubate at 37℃ for 30min, incubate in a boiling water bath for 5min to terminate the activity of the enzyme, and then determine the minimum inhibitory concentration of the antimicrobial peptides after treatment with different concentrations of proteinase K according to the method described in 2.1. The test bacteria are Pasteurella multocida strains. The results are as follows: Fig.15 The results showed that after being treated with different concentrations of proteinase K, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity.

[0056] 2.6 Analysis of salt ion stability of narrow-spectrum antimicrobial peptide XMK-8 Sodium chloride solution and potassium chloride solution with concentrations of 50mmol / L, 100mmol / L, 150mmol / L, and 200mmol / L were prepared respectively. 2mg / mL narrow-spectrum antimicrobial peptide XMK-8 solution was divided into 8 equal portions, 50μL / portion, and mixed with equal volumes of salt ion solutions of different concentrations, and reacted at 37℃ for 30min. Then, the minimum inhibitory concentration of the antimicrobial peptide after treatment with salt ion solutions of different concentrations was determined according to the method described in 2.1. The test bacteria were porcine Pasteurella multocida strains. The results are as follows: Fig.16 and Fig.17 The results showed that after being treated with sodium ion and potassium ion solutions of different concentrations, the narrow-spectrum antimicrobial peptide XMK-8 still had good antibacterial activity.

[0057] In summary, the present invention provides a narrow-spectrum antimicrobial peptide XMK-8, which has no antibacterial activity against Escherichia coli, Salmonella, Staphylococcus aureus, and Aeromonas hydrophila, and only has antibacterial activity against Pasteurella multocida and Haemophilus parasuis. The antibacterial activity of the narrow-spectrum antimicrobial peptide XMK-8 can still be detected after treatment with temperature (0-100°C), salt ions (50-200mmol / L, sodium ions and potassium ions), acid and alkali (pH4-10) and proteinase K (20-100μg / mL), and has low hemolytic activity against mouse erythrocytes.

[0058] 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. A narrow-spectrum antimicrobial peptide, characterized in that: The amino acid sequence of the narrow-spectrum antimicrobial peptide is arginine-leucine-leucine-proline-lysine-leucine-proline-arginine-lysine-valine-proline-arginine-phenylalanine-proline-arginine-lysine-isoleucine-proline, as shown in SEQ ID NO:

1.

2. Use of the narrow-spectrum antimicrobial peptide according to claim 1 in the preparation of products for preventing, inhibiting, diagnosing and / or identifying bacterial infections, characterized in that: The bacteria are Pasteurella multocida and / or Haemophilus parasuis.

3. The use according to claim 2, characterized in that: The products include inactivated bacterial vaccines, anti-bacterial infection drugs, and bacterial disinfection products.

4. The use according to claim 2, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide to Pasteurella multocida is 250 μg / mL.

5. The use according to claim 2, characterized in that: The minimum inhibitory concentration of the antimicrobial peptide to Haemophilus parasuis is 1 mg / mL.

6. A bacterial disinfection product, characterized in that: The bacterial disinfection product uses the narrow-spectrum antimicrobial peptide XMK-8 as an effective component and has antibacterial activity against Pasteurella multocida and / or Haemophilus parasuis.

7. A product for diagnosing and / or identifying bacterial infections, characterized in that The product comprises the narrow-spectrum antimicrobial peptide according to claim 1, physiological saline and bacterial culture medium, and is used for diagnosing and / or identifying bacteria Pasteurella multocida and / or Haemophilus parasuis.

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