Antimicrobial peptides and their applications

Abstract

The present invention discloses an antimicrobial peptide and its application, wherein the antimicrobial peptide comprises one or more of an antimicrobial peptide PFK having an amino acid sequence as shown in SEQ ID NO: 1, an antimicrobial peptide PVK having an amino acid sequence as shown in SEQ ID NO: 2, and an antimicrobial peptide PIK having an amino acid sequence as shown in SEQ ID NO: 3. The antimicrobial peptides PFK, PVK and PIK of the present invention have antimicrobial effects on Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus, and the antimicrobial peptides PFK, PVK and PIK have low hemolytic activity and low cytotoxicity, and can be used to prepare biological antimicrobial preparations, anti-infective drugs or oral products.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to antimicrobial peptides and applications thereof. Background Art

[0002] Periodontitis is a chronic inflammation that affects the gum tissue. In severe cases, it can lead to alveolar bone atrophy, tooth loss and loss of chewing function. Studies have also found that periodontitis is associated with diabetes, cardiovascular disease, Alzheimer's disease, rheumatoid arthritis and premature birth. Therefore, periodontitis is defined by the medical community as the third largest killer threatening human health after cancer and cardiovascular and cerebrovascular diseases. Periodontitis is caused by the imbalance of periodontal flora, and Porphyromonas gingivalis is considered to be the basic pathogen causing the imbalance of periodontal flora. Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that can destroy the host cell defense system and activate the host's immune response by secreting virulence factors. The nutrients produced by the destruction of host cells will promote the growth of other pathogens, thereby breaking the balance of oral flora and causing periodontitis.

[0003] At present, the main treatment for periodontitis is mechanical plaque removal combined with the use of antibiotics. The disadvantage is that it is difficult to completely eradicate periodontitis and it can also cause bacterial resistance. Antimicrobial peptides are considered to be a promising alternative to antibiotics. Antimicrobial peptides (AMPs) are short, positively charged, cationic, amphiphilic amino acid sequences that have a wide range of antimicrobial activities and play an important role in the host's innate immunity. The lack or dysfunction of antimicrobial peptides is associated with infectious diseases including periodontitis. Porphyromonas gingivalis is the main pathogen associated with periodontal disease and is resistant to antimicrobial peptides from human and non-human sources. There are a large number of gingipains on the cell surface of Porphyromonas gingivalis that can hydrolyze antimicrobial peptides. Studies have found that Porphyromonas gingivalis has proteolytic activity and can render the antimicrobial peptides K4-S4 (1-15) a, Dhvar4, and LL-37 ineffective (Bachrach G, Altman H, Kolenbrander PE, Chalmers NI, Gabai-Gutner M, Mor A, Friedman M, Steinberg D. Resistance of Porphyromonas gingivalisATCC 33277 to direct killing by antimicrobial peptides is proteaseindependent. Antimicrob Agents Chemother. 2008 Feb; 52(2): 638-42. doi:10.1128 / AAC.01271-07. Epub 2007 Dec 17. PMID: 18086848; PMCID: PMC2224744), because these antimicrobial peptides have low affinity for Porphyromonas gingivalis.

[0004] Cecropin B, a naturally found antimicrobial peptide sequence, has relatively low antibacterial activity, with a minimum inhibitory concentration of 95 μg / mL against Porphyromonas gingivalis (Devine DA, Marsh PD, Percival RS, Rangarajan M, Curtis MA. Modulation of antibacterial peptide activity by products of Porphyromonas gingivalis and Prevotella spp. Microbiology (Reading). 1999 Apr; 145 (Pt4):965-971. doi: 10.1099 / 13500872-145-4-965. PMID: 10220176), and its clinical value is relatively low.

[0005] China CN110305193A discloses an anti-Porphyromonas gingivalis polypeptide, the amino acid sequence of which is: Gly-Leu-Gln-Phe-Pro-Val-Gly-Arg-Ile-His-Arg-Leu-Leu-Arg-Lys. The minimum inhibitory concentration of the polypeptide against Porphyromonas gingivalis ATCC33277 is 12.5 μg / mL, but the minimum inhibitory concentration for bacteria such as Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli is greater than 100 μg / mL, and the therapeutic effect on periodontitis and dental caries is limited. Summary of the invention

[0006] In order to overcome the above-mentioned deficiencies in the prior art and improve the bactericidal activity of antimicrobial peptides, the present invention provides antimicrobial peptides and their applications. The present invention analyzes the sequence and structure of the natural antimicrobial peptide cecropin, and on this basis designs and synthesizes a group of antimicrobial peptides (antimicrobial peptide PFK, antimicrobial peptide PVK and antimicrobial peptide PIK) containing about 20 amino acids, providing ideas for the research and development of new periodontitis treatment drugs. Antimicrobial peptide PFK, antimicrobial peptide PVK and antimicrobial peptide PIK have antimicrobial effects on Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus, and antimicrobial peptides PFK, PVK and PIK have low hemolyticity and low cytotoxicity.

[0007] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0008] The first aspect of the present invention provides an antimicrobial peptide comprising one or more of the antimicrobial peptide PFK with an amino acid sequence as shown in SEQ ID NO: 1, the antimicrobial peptide PVK with an amino acid sequence as shown in SEQ ID NO: 2, and the antimicrobial peptide PIK with an amino acid sequence as shown in SEQ ID NO: 3.

[0009] The second aspect of the present invention provides the use of the aforementioned antimicrobial peptide in the preparation of biological antimicrobial preparations, anti-infection drugs or oral products.

[0010] Furthermore, the dosage forms of the biological antibacterial preparation, anti-infective drug or oral product include sprays, aerosols, gargles, ointments, films or effervescents.

[0011] Furthermore, the biological antibacterial preparation inhibits the growth of one or more bacteria among Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus; the anti-infective drug is a drug for preventing and / or reducing or treating oral diseases caused by one or more bacteria among Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus; the oral product is an oral product for preventing and / or reducing or treating oral diseases caused by one or more bacteria among Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus.

[0012] Furthermore, the oral disease includes periodontitis and / or dental caries.

[0013] Further, the biological antibacterial preparation contains 1 μg / mL-1 mg / mL of antimicrobial peptide PFK, and / or 1 μg / mL-1 mg / mL of antimicrobial peptide PVK, and / or 1 μg / mL-1 mg / mL of antimicrobial peptide PIK; the anti-infective drug contains 1 μg / mL-1 mg / mL of antimicrobial peptide PFK, and / or 1 μg / mL-1 mg / mL of antimicrobial peptide PVK, and / or 1 μg / mL-1 mg / mL of antimicrobial peptide PIK; the oral product contains 1 μg / mL-1 mg / mL of antimicrobial peptide PFK, and / or 1 μg / mL-1 mg / mL of antimicrobial peptide PVK, and / or 1 μg / mL-1 mg / mL of antimicrobial peptide PIK.

[0014] Furthermore, the biological antibacterial preparation contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK, and / or 32 μg / mL-64 μg / mL of antimicrobial peptide PVK, and / or 32 μg / mL-64 μg / mL of antimicrobial peptide PIK; the anti-infective drug contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK, and / or 32 μg / mL-64 μg / mL of antimicrobial peptide PVK, and / or 32 μg / mL-64 μg / mL of antimicrobial peptide PIK; the oral product contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK, and / or 32 μg / mL-64 μg / mL of antimicrobial peptide PVK, and / or 32 μg / mL-64 μg / mL of antimicrobial peptide PIK.

[0015] The third aspect of the present invention provides a biological antibacterial preparation, an anti-infection drug or an oral product, comprising antimicrobial peptide PFK, and / or antimicrobial peptide PVK, and / or antimicrobial peptide PIK; one or more of the carriers or excipients acceptable in pharmacology or oral products may be added thereto.

[0016] The fourth aspect of the present invention provides a method for preparing a biological antibacterial preparation, comprising the following steps:

[0017] (1) Purified water is heated to 60-70°C, hydroxypropyl methylcellulose (HPMC) is dispersed in the water, and then cooled to room temperature to obtain a dispersion of hydroxypropyl methylcellulose;

[0018] (2) adding poloxamer 407 to the dispersion of hydroxypropyl methylcellulose and refrigerating at 2-8° C. until poloxamer 407 is fully dissolved to obtain a mixed solution;

[0019] (3) Add glycerol to the mixed solution, stir to dissolve, and leave at room temperature to obtain a matrix gel;

[0020] (4) Add antimicrobial peptide PFK and / or antimicrobial peptide PVK and / or antimicrobial peptide PIK to the matrix gel and adjust the pH to 7 to obtain a biological antimicrobial preparation.

[0021] Furthermore, the raw materials of the biological antibacterial preparation include, by weight, 0.1-10 parts of antimicrobial peptide PFK and / or 0.1-10 parts of antimicrobial peptide PVK and / or 0.1-10 parts of antimicrobial peptide PIK, 18 parts of poloxamer 407, 0.5 parts of hydroxypropyl methylcellulose and 2 parts of glycerol; and / or, in step (4), the pH is adjusted using one or more of citric acid monohydrate, hydrochloric acid and sodium hydroxide.

[0022] Furthermore, the raw materials of the biological antibacterial preparation also include 100 parts of purified water.

[0023] Furthermore, the raw materials of the biological antibacterial preparation include 6.4 mg of antimicrobial peptide PFK and / or 6.4 mg of antimicrobial peptide PVK and / or 6.4 mg of antimicrobial peptide PIK, 100 mL of purified water, 18 g of poloxamer 407, 0.5 g of hydroxypropyl methylcellulose and 2 g of glycerol.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] (1) The antimicrobial peptides PFK, PVK and PIK of the present invention have antibacterial effects on Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus, and the antimicrobial peptides PFK, PVK and PIK have low hemolytic activity and low cytotoxicity, and can be used to prepare biological antibacterial preparations, anti-infection drugs or oral products.

[0026] (2) The antimicrobial peptide PFK of the present invention has the strongest antibacterial activity, with minimum inhibitory concentrations of 16 μg / mL and 32 μg / mL for Porphyromonas gingivalis and Streptococcus mutans, respectively; the minimum inhibitory concentrations for Acinetobacter baumannii and Escherichia coli are both 4 μg / mL; and the minimum inhibitory concentration for Staphylococcus aureus is 32 μg / mL. When the concentration of antimicrobial peptide PFK is 1 mg / mL, the hemolysis rate is only 1.7%, and when the concentration is less than or equal to 100 μg / mL, antimicrobial peptide PFK has no hemolytic activity. When the concentration of antimicrobial peptide PFK, PVK and PIK is less than or equal to 64 μg / mL, they all have low cytotoxicity and high safety, and have the potential to be developed as drugs for the treatment of periodontitis and dental caries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the three-dimensional structure diagram of the antimicrobial peptides PFK, PVK and PIK.

[0028] Figure 2 This is a bar graph showing the hemolytic assay results of the antimicrobial peptides PFK, PVK and PIK in Example 3.

[0029] Figure 3This is a bar graph showing the cytotoxicity assay results of the antimicrobial peptides PFK, PVK and PIK in Example 4. DETAILED DESCRIPTION

[0030] The antimicrobial peptide provided by the present invention comprises one or more of the antimicrobial peptide PFK with an amino acid sequence as shown in SEQ ID NO: 1, the antimicrobial peptide PVK with an amino acid sequence as shown in SEQ ID NO: 2, and the antimicrobial peptide PIK with an amino acid sequence as shown in SEQ ID NO: 3.

[0031] The synthetic antimicrobial peptide provided by the present invention is obtained by shortening the sequence of the naturally discovered antimicrobial peptide Cecropin B (KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL) from 35 amino acids to 20 amino acids, reducing its molecular weight of 3.8 kDa to below 3 kDa, thereby reducing the difficulty of synthesizing the antimicrobial peptide. By analyzing the polarity of amino acids and the isoelectric point of polypeptides, the proportion of positively charged amino acids is increased, thereby increasing the binding ability of the antimicrobial peptide to the bacterial cell membrane. By replacing amino acids of the same nature, the alpha helix structure of the antimicrobial peptide remains unchanged after the sequence is shortened. Finally, three new antimicrobial peptides PFK, PVK and PIK were designed, and their three-dimensional structures were displayed using the Alphafold 3 protein structure prediction software. Figure 1 The three new sequences of antimicrobial peptides designed, PFK (SEQ ID NO: 1), PVK (SEQ ID NO: 2), and PIK (SEQ ID NO: 3), have hydrophilic and lipophilic properties. Experiments have shown that the antimicrobial peptides PFK, PVK, and PIK have antibacterial effects on Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli, and Staphylococcus aureus. Among them, the antimicrobial peptide with the sequence PFK has the best antibacterial effect. The antimicrobial peptide with the sequence PFK has good antibacterial activity against Porphyromonas gingivalis, Streptococcus mutans, and other common pathogens.

[0032] In the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably in the present invention and refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones.

[0033] The sequence information of the amino acid sequence or nucleotide sequence used in the present invention is as follows:

[0034] SEQ ID NO: 1 Antimicrobial peptide PFK amino acid sequence:

[0035] FKKASHLFKKTLKKIFSKWK

[0036] SEQ ID NO: 2 Antimicrobial peptide PVK amino acid sequence:

[0037] IVAVRIRWQVTLKKIFSKWK

[0038] SEQ ID NO: 3 Antimicrobial peptide PIK amino acid sequence:

[0039] VQWRIRVAVIRKTLKKIFSKWK

[0040] SEQ ID NO: 4 Antimicrobial peptide Cecropin B amino acid sequence:

[0041] KWKVFKKIEKMGRNIRNGIVKAGPAIAVLGEAKAL

[0042] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0043] Comparative Example 1

[0044] Studies have reported that the antimicrobial peptides K4-S4 (1-15) a, Dhvar4 and LL-37 have no antibacterial effect on Porphyromonas gingivalis. The antimicrobial peptide Cecropin B has a weak antibacterial effect on Porphyromonas gingivalis, with a minimum inhibitory concentration of 95 μg / ml.

[0045] Example 1 Preparation, separation and purification of antimicrobial peptides

[0046] After the antimicrobial peptides PFK, PVK and PIK of the present invention are designed, the structures predicted by Alphafold 3 are as follows: Figure 1As shown, all three peptides form an α-helical structure. They were prepared and separated and purified by Gill Biochemical (Shanghai) Co., Ltd. according to the sequence. The preparation of antimicrobial peptides is to connect amino acids one by one from the C-terminus to the N-terminus, which is automatically controlled by the synthesizer. First, weigh 0.1mmol of the resin (Rink Amide-AM, Gill Biochemical Shanghai Co., Ltd.) that combines the first amino acid, namely Fmoc-Phe-OH, and load it into the column. Then, deprotect it with 20% piperidine dimethylformamide solution to remove Fmoc, and then wash it with dimethylformamide. The corresponding free amino acids protected with 9-Fmoc are dissolved in HATU / DIPEA, and the dissolved solutions are cyclically coupled on the column one after another, each reaction lasts for 30 minutes, and Fmoc is removed by washing with dimethylformamide. Repeat the above deprotection and coupling reaction steps until the preparation is completed (for specific operation steps, see the operation guide of Applied Biosystems pioneer peptide synthesizer of Applied Biosystems, USA). The prepared polypeptide was sheared by the following steps: removing the reacted resin, adding type B shearing solution (88% trifluoroacetic acid, 5% phenol, 5% water, 2% triisopropylsilane), reacting at room temperature for 2 hours, filtering, adding 10 times the volume of pre-cooled anhydrous ether to the filtrate, centrifuging at 4000 rpm for 10 minutes, collecting the precipitate and drying it at room temperature, and finally obtaining an antimicrobial peptide sample with a purity of more than 95%.

[0047] Example 2 Antibacterial activity detection of synthetic antimicrobial peptides

[0048] Streptococcus mutans ATCC 25175 and Porphyromonas gingivalis ATCC BAA-308 stored at -80°C were inoculated on blood agar plates, and Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were inoculated on LB plates. Porphyromonas gingivalis was cultured in an anaerobic environment at 37°C for 4 days, and other strains were cultured at 37°C for 24 h. The colonies of Streptococcus mutans, Porphyromonas gingivalis, Escherichia coli and Staphylococcus aureus were resuspended in BHI liquid medium and the bacterial solution concentration was adjusted to about 10 5 CFU / mL and reserve for use.

[0049] The antimicrobial peptides PFK, PVK and PIK were diluted 10 times to 8 × 10 15 U / mL, the above samples (antimicrobial peptides PFK, PVK and PIK) were diluted 2-fold in BHI medium, for a total of 6 gradient dilutions. 50 μL of sample dilutions and test bacterial solution were added to 96-well plates and cultured at 37°C for 24 h. Porphyromonas gingivalis was cultured in an anaerobic environment at 37°C for 72 h. 10 μL of resazurin dye was added to each well of the 96-well plate to observe the MIC results.

[0050] Table 1 Minimum inhibitory concentration (MIC) determination

[0051]

[0052] The results of the test in Table 1 show that the antimicrobial peptide PFK has stronger antibacterial activity. The minimum inhibitory concentrations of common oral pathogens Porphyromonas gingivalis and Streptococcus mutans were 16 μg / mL and 32 μg / mL, respectively. The antibacterial effect on other common pathogens Acinetobacter baumannii and Escherichia coli was the best, with the minimum inhibitory concentrations of 4 μg / mL. It also had a certain antibacterial effect on Staphylococcus aureus, with a minimum inhibitory concentration of 32 μg / mL. The antibacterial effect of antimicrobial peptide PVK on Porphyromonas gingivalis and Escherichia coli was weaker than that of antimicrobial peptide PFK, both of which were higher than 100 μg / mL. It had a certain antibacterial effect on Streptococcus mutans, Acinetobacter baumannii and Staphylococcus aureus, with the minimum inhibitory concentrations of 64 μg / mL, 64 μg / mL and 32 μg / mL, respectively. The antibacterial effect of antimicrobial peptide PIK on Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii and Escherichia coli was weaker than that of antimicrobial peptide PFK, all of which were higher than 200 μg / mL; it had a certain antibacterial effect on Staphylococcus aureus, with the minimum inhibitory concentration of 32 μg / mL.

[0053] It can be seen that the antimicrobial peptides PFK, PVK and PIK have antibacterial effects on common oral pathogens and other common pathogens, and have the potential to be developed into drugs for the treatment of periodontitis and dental caries. Among them, the antimicrobial peptide PFK has the best antibacterial effect.

[0054] Example 3 In vitro hemolytic activity detection of synthetic antimicrobial peptides

[0055] Sterile defibrinated horse blood samples (Solarbio, TX0040) were collected in centrifuge tubes and centrifuged at 3000 rpm for 5 min to remove impurities. The cells were then washed three times with 10 mM PBS (pH 7.4), and the red blood cells were collected and diluted with 10 mM PBS (pH 7.4) to a final concentration of 4% (i.e., the red blood cell volume fraction was 4%). Thereafter, 100 μL of antimicrobial peptides of different concentrations were mixed with 100 μL of 4% red blood cells, incubated in a 37°C water bath for 30 min, and finally centrifuged at 10,000 rpm for 5 min at room temperature. The absorbance of the supernatant at 540 nm was measured using a UV spectrophotometer. Red blood cells were suspended in PBS and 1% Triton X-100 alone as negative and positive controls, respectively. The experiment was repeated three times, and the standard deviation was calculated using the function STDEV.

[0056] The calculation formula of hemolysis rate is: hemolysis rate (%) = (Dt-Dnc) / (Dpc-Dnc) × 100%.

[0057] Wherein, Dt represents the absorbance of the test samples treated with different concentrations of antimicrobial peptides at 540 nm, and Dpc and Dnc refer to the absorbance of the positive control and negative control at 540 nm, respectively.

[0058] Table 2 Determination of hemolysis rate of antimicrobial peptides

[0059]

[0060] Table 2 and Figure 2 The results of the test showed that the antimicrobial peptides PFK, PVK and PIK all had low hemolysis and high safety. The hemolysis rate of the antimicrobial peptide PFK was 64.4% at a high concentration of 10 mg / mL, but it dropped to 1.7% at a concentration of 1 mg / mL. When the concentration was less than or equal to 100 μg / mL, the antimicrobial peptide PFK had no hemolysis. The antimicrobial peptide PVK had the lowest hemolysis rate at a high concentration of 10 mg / mL, which was only 18.9%; at 1 mg / mL, the hemolysis rate was 7.1%; at 100 μg / mL, the hemolysis rate was 4.2%; when the concentration was less than or equal to 10 μg / mL, the antimicrobial peptide PVK had no hemolysis. The antimicrobial peptide PIK had a hemolysis rate of 51.6% at a high concentration of 10 mg / mL. As the concentration decreased, the hemolysis gradually decreased, but it still had weak hemolysis. In summary, when the concentration is 10 mg / mL, the antimicrobial peptide PVK has the lowest hemolytic activity and is the safest; when the concentration is less than or equal to 1 mg / mL, the antimicrobial peptide PFK has the lowest hemolytic activity and is the safest.

[0061] Example 4 Cytotoxicity detection of synthetic antimicrobial peptides

[0062] Mouse lung fibroblast L929 cells stored in liquid nitrogen were subcultured. 100 μL of cell suspension (about 5000 cells per well) was added to a 96-well plate and pre-incubated in a cell culture incubator (37°C, 5% CO2) for 24 hours. 10 μL of antimicrobial peptides of different concentrations were added to the cell suspension and cultured in the incubator for 24 hours. 10 μL of CCK-8 solution was added to each well and cultured in the incubator for 1 hour. The absorbance value OD at 450 nm was measured using a microplate reader 450 The formula for calculating the survival rate of CCK-8 cells is: survival rate (100%) = (OD value of the experimental group - OD value of the blank control group) / (OD value of the control group - OD value of the blank control group) × 100%. The OD value of the experimental group is the absorbance value of the cells after antimicrobial peptide treatment, the OD value of the control group is the absorbance value of the untreated cells, and the OD value of the blank control group is the absorbance value of the culture medium.

[0063] Table 3 Cell viability determination

[0064]

[0065] Table 3 and Figure 3 The results of the test showed that the antimicrobial peptides of the present invention had low cytotoxicity and high safety when the concentration was less than or equal to 64 μg / mL. At a concentration of 256 μg / mL, the antimicrobial peptides PFK, PVK and PIK all showed high cytotoxicity. When the concentration of the antimicrobial peptides dropped to 64 μg / mL or below, the cell survival rate increased significantly, among which the cell survival rates of PFK and PVK were higher than those of PIK, indicating that PFK and PVK were safer as antimicrobial ingredients in this concentration range.

[0066] Example 5 The synthesized antimicrobial peptide can be prepared into a biological antimicrobial preparation

[0067] The antimicrobial peptides PFK, PVK and PIK prepared in Example 1 of the present invention can all be used as active ingredients to prepare biological antimicrobial preparations. Here, as an example, 100 mL of the biological antimicrobial preparation is prepared.

[0068] 1. Prescription: Poloxamer 407 (BASF, GNC22022B) 18g, HPMC (4000) 0.5g, glycerol 2g, antimicrobial peptide 0.1-10mg, purified water 100 mL (100g), citric acid monohydrate amount.

[0069] 2. Preparation steps

[0070] (1) Take 100 mL of purified water and heat it to 60-70°C. Then disperse the weighed HPMC into the water and cool it to room temperature for later use.

[0071] (2) Weigh 18 g of Poloxamer 407 and add it to the dispersion in step 1. Refrigerate overnight at 2-8°C until Poloxamer 407 is fully dissolved.

[0072] (3) Weigh 2 g of glycerol and add it to the mixed solution in step 2. Stir to dissolve and allow to stand at room temperature to form a gel.

[0073] (4) Weigh 0.1-10 mg of PFK, PVK or PIK and add it to the matrix gel in step 3.

[0074] (5) Adjust the pH to 7 with citric acid monohydrate.

[0075] After the gel is prepared, it is packaged in medical syringes and injected into the pockets formed by periodontitis. It can inhibit the growth of oral pathogens such as Porphyromonas gingivalis, thereby slowing down or eliminating periodontitis. In this biological antibacterial preparation, the antimicrobial peptide has almost no hemolysis and cytotoxicity at a suitable concentration, is safe and mild, and will not cause bacterial resistance.

[0076] The above embodiments are only preferred implementation modes of the present invention and are only used to explain the present invention rather than to limit the present invention. Any changes, substitutions, modifications, etc. made by those skilled in the art without departing from the spirit of the present invention should fall within the scope of protection of the present invention.

Claims

1. An antimicrobial peptide, characterized in that The antimicrobial peptide is an antimicrobial peptide PFK with an amino acid sequence as shown in SEQ ID NO:

1.

2. Use of the antimicrobial peptide according to claim 1 in the preparation of biological antimicrobial preparations, anti-infection drugs or oral products; The biological antibacterial preparation inhibits the growth of one or more bacteria selected from Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus; The anti-infective drug is a drug for preventing and / or reducing or treating oral diseases caused by one or more bacteria selected from Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus; The oral product is an oral product for preventing and / or reducing or treating oral diseases caused by one or more bacteria selected from Porphyromonas gingivalis, Streptococcus mutans, Acinetobacter baumannii, Escherichia coli and Staphylococcus aureus; The oral diseases include periodontitis and / or dental caries.

3. The use according to claim 2, characterized in that: The dosage forms of the biological antibacterial preparation, anti-infection medicine or oral product include spray, aerosol, gargle, paste, film or effervescent.

4. The use according to claim 2, characterized in that: The biological antibacterial preparation contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK; The anti-infective drug contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK; The oral product contains 4 μg / mL-64 μg / mL of the antimicrobial peptide PFK.

5. A biological antibacterial preparation, anti-infection drug or oral product, characterized in that: It comprises antimicrobial peptide PFK; one or more of the carriers or excipients acceptable in pharmacology and oral products may be added thereto, and the amino acid sequence of the antimicrobial peptide PFK is shown in SEQ ID NO: 1; The biological antibacterial preparation contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK; The anti-infective drug contains 4 μg / mL-64 μg / mL of antimicrobial peptide PFK; The oral product contains 4 μg / mL-64 μg / mL of the antimicrobial peptide PFK.

6. A method for preparing a biological antibacterial preparation, characterized in that: The steps include: (1) Purified water is heated to 60-70° C., hydroxypropyl methylcellulose is dispersed in the purified water, and then cooled to room temperature to obtain a dispersion of hydroxypropyl methylcellulose; (2) adding poloxamer 407 to the dispersion of hydroxypropyl methylcellulose and refrigerating at 2-8° C. until poloxamer 407 is fully dissolved to obtain a mixed solution; (3) Add glycerol to the mixed solution, stir to dissolve, and leave at room temperature to obtain a matrix gel; (4) Adding the antimicrobial peptide PFK to the matrix gel and adjusting the pH to 7 to obtain a biological antimicrobial preparation; the amino acid sequence of the antimicrobial peptide PFK is shown in SEQ ID NO: 1; the biological antimicrobial preparation contains 4 μg / mL-64 μg / mL of the antimicrobial peptide PFK.

7. The preparation method according to claim 6, characterized in that: The raw materials of the biological antibacterial preparation include, by weight, 0.1-10 parts of antimicrobial peptide PFK, 18 parts of poloxamer 407, 0.5 parts of hydroxypropyl methylcellulose and 2 parts of glycerol; And / or, in step (4), the pH is adjusted using one or more of citric acid monohydrate, hydrochloric acid and sodium hydroxide.

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