An anti-periodontal pathogen polypeptide, a polypeptide solution and application thereof

Abstract

The application relates to the medical application field, and particularly discloses an anti-periodontal pathogenic bacteria polypeptide, a polypeptide solution and application thereof, and the amino acid sequence of the antibacterial polypeptide is Lys-Thr-Lys-Ile-Gly-Ile-Asn-Gly-Phe-Gly-Lys-Ile-Gly-Lys-Leu-Val-Leu-Lys. The polypeptide provided by the application can effectively inhibit periodontal pathogenic bacteria Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans, and has extremely small toxicity to human red blood cells. The polypeptide can be developed into a drug, a mouthwash, an antibacterial gel and a toothpaste additive for resisting Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to an anti-periodontal pathogenic polypeptide, a polypeptide solution and application thereof. BACKGROUND

[0002] Periodontal health problems are increasingly prominent, which has a great impact on people's overall health and quality of life. Periodontal infection is the main factor of periodontal health problems, which is mainly caused by infection of related pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans. Therefore, effectively inhibiting and killing the pathogenic bacteria such as Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans in the oral cavity has important significance for the prevention and treatment of periodontal infection. As an active antibacterial molecule of polypeptide, the antibacterial polypeptide not only has small antibacterial effect and low toxicity and side effects, but also has the characteristics of immune regulation and difficulty in inducing bacterial drug resistance. In recent years, the antibacterial polypeptide has become a research hotspot in the field of new antibacterial drugs in the field of medicine. SUMMARY

[0003] The present application provides an anti-periodontal pathogenic polypeptide, a polypeptide solution and application thereof. The polypeptide provided by the present application can effectively inhibit the periodontal pathogenic bacteria Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans, and has very small toxicity to human red blood cells. The polypeptide can be developed into a drug, mouthwash, antibacterial gel and toothpaste additive for the treatment of Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans infection.

[0004] The present application provides an anti-periodontal pathogenic polypeptide, and the amino acid sequence of the polypeptide is shown in SEQ ID NO. 1: Lys-Thr-Lys-Ile-Gly-Ile-Asn-Gly-Phe-Gly-Lys-Ile-Gly-Lys-Leu-Val-Leu-Lys.

[0005] The present application also provides a polypeptide solution containing the anti-periodontal pathogenic polypeptide, and the antibacterial polypeptide solution is prepared by mixing a solvent and the anti-periodontal pathogenic polypeptide.

[0006] Further, the solvent is any one of physiological saline, ultrapure water, phosphate buffer and acetic acid.

[0007] The present application also provides an application of the anti-periodontal pathogenic polypeptide or the polypeptide solution in the preparation of an antibacterial agent, and the antibacterial agent can inhibit Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans.

[0008] Further, the minimum inhibitory concentration of the antibacterial agent to Porphyromonas gingivalis is 6.25 μg / mL, the minimum inhibitory concentration of the antibacterial agent to Fusobacterium nucleatum is 12.5 μg / mL, and the minimum inhibitory concentration of the antibacterial agent to Streptococcus mutans is 12.5 μg / mL.

[0009] The application also provides the use of the anti-periodontal pathogenic bacteria polypeptide or the solution of the anti-bacterial polypeptide in the preparation of a bactericide, which can kill Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans.

[0010] The application also provides the use of the anti-periodontal pathogenic bacteria polypeptide or the solution of the polypeptide in the preparation of a medicine for preventing and / or treating periodontal infection, which takes the anti-periodontal pathogenic bacteria polypeptide as the only effective component.

[0011] The application also provides the use of the anti-periodontal pathogenic bacteria polypeptide or the solution of the anti-bacterial polypeptide in the preparation of toothpaste, anti-bacterial gel, toothpaste additive or mouthwash.

[0012] Further, the toothpaste also includes but is not limited to abrasives, thickening agents, foaming agents, preservatives and fluoride substances.

[0013] Further, the abrasives are any one of calcium carbonate, calcium hydrogen phosphate, silicon dioxide and aluminum hydroxide; the abrasives account for 50% of the toothpaste and play a role in cleaning teeth.

[0014] Further, the moisturizing agent is one or a combination of several of glycerol, sorbitol, xylitol and polyethylene glycol; and has the functions of keeping the moisture of the toothpaste body and maintaining the flow phase of the body.

[0015] Further, the foaming agent is sodium dodecanol sulfate or 2-acyloxy sodium sulfonate; and has the functions of reducing the surface tension of liquid, and good wetting, emulsifying and detergency.

[0016] Further, the preservative is sodium benzoate.

[0017] Further, the fluoride substance is sodium fluoride, sodium monofluorophosphate or stannous fluoride or other substances containing 0.1-0.4% fluorine. Fluorine can promote the remineralization of the surface enamel of teeth and inhibit the growth and reproduction of cariogenic bacteria, thereby reducing the occurrence of dental caries.

[0018] Further, the mouthwash also includes any one or a combination of several of water, glycerol, sorbitol, ethanol, tea extract and propolis extract, citric acid, sodium citrate, octanol, physiological saline, flavoring agent, sweetener, surfactant, colorant, preservative, essential oil and triclosan.

[0019] Compared with the prior art, the application has the beneficial effects that:

[0020] 1. The anti-periodontal pathogenic polypeptide provided by the application can efficiently inhibit and kill Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans, has good stability, and has very low toxicity. The polypeptide has the potential to be developed into an anti-Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans mouthwash, an antibacterial gel, and a toothpaste additive, and is used for preventing and treating periodontal infections caused by Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative effort on the basis of these drawings.

[0022] Figure 1 Hemolytic activity of the anti-periodontal pathogenic polypeptide prepared in Example 1 at different concentrations. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application will be described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort belong to the protection scope of the present application. The experimental methods described in the embodiments of the present application are all conventional methods, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0024] Example 1: Preparation of an anti-periodontal pathogenic polypeptide.

[0025] The amino acid sequence of the anti-periodontal pathogenic polypeptide provided by the present application is shown in SEQ ID NO. 1. According to the shown amino acid sequence, the polypeptide was synthesized by Gil Biochemical (Shanghai) Co., Ltd. using solid-phase chemical synthesis method, and was purified by reverse-phase high-performance liquid chromatography and identified by electrospray mass spectrometry, to obtain a polypeptide pure product with a purity of more than 95%.

[0026] SEQ ID NO. 1:

[0027] Lys-Thr-Lys-Ile-Gly-Ile-Asn-Gly-Phe-Gly-Lys-Ile-Gly-Lys-Leu-Val-Leu-Lys.

[0028] Example 2: Study on the antibacterial activity of the anti-periodontal pathogenic polypeptide obtained in Example 1.

[0029] I. Experimental method

[0030] 1. Study on antibacterial activity of anti-periodontal pathogenic bacteria polypeptide

[0031] (1) The culture medium for Porphyromonas gingivalis and Fusobacterium nucleatum is ATCC Medium 2722, and the culture medium for Streptococcus mutans is BHI. The bacteria in the logarithmic growth phase are adjusted to 10 5 -10 6 cfu / mL with the corresponding culture medium, and the polypeptide is dissolved in physiological saline and diluted by two-fold gradient. 80 μL of the above diluted bacteria solution and 20 μL of the above diluted polypeptide solution are added to a sterile 96-well sterile cell culture plate, and then the culture plate is placed in a 37°C constant temperature anaerobic biochemical incubator for incubation for 24 / 48 h. After the incubation is completed, the minimum polypeptide concentration for sterile growth is the minimum inhibitory concentration of the polypeptide on the tested bacteria. The results are shown in Table 1.

[0032] Table 1: Antibacterial activity of anti-periodontal pathogenic bacteria polypeptide obtained in Example 1

[0033] Strains Minimum inhibitory concentration (pg / mL) P. gingivalis ATCC 33277 6.25 F. nucleatum ATCC 10953 12.5 S. mutans ATCC 25175 12.5

[0034] The antibacterial activity of the anti-periodontal pathogenic bacteria polypeptide of the present application is shown in Table 1. The minimum inhibitory concentration of the polypeptide on Porphyromonas gingivalis is 6.25 μg / mL, the minimum inhibitory concentration on Fusobacterium nucleatum is 12.5 μg / mL, and the minimum inhibitory concentration on Streptococcus mutans is 12.5 μg / mL. It can be seen that the polypeptide can effectively inhibit Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans.

[0035] Example 3: Study on bactericidal effect of anti-periodontal pathogenic bacteria polypeptide obtained in Example 1.

[0036] The bacteria in the logarithmic growth phase are adjusted to 10 6 -10 7 cfu / mL with the culture medium, and the polypeptide is added so that the final concentration of the polypeptide is 4 times the minimum inhibitory concentration. After incubation in a 37°C constant temperature anaerobic biochemical incubator for 15 min, the sample is diluted by 10-fold gradient and plated on solid nutrient plates. The plates are placed in a 37°C constant temperature anaerobic biochemical incubator for incubation for 24 / 48 h. After the incubation is completed, the number of bacteria is counted, compared with the initial amount of bacteria, and the clearance rate is calculated. The results are shown in Table 2.

[0037] Table 2: Bactericidal activity of anti-periodontal pathogenic bacteria polypeptide

[0038] Strains Clearance (%) P. gingivalis ATCC 33277 99.9 F. nucleatum ATCC 10953 99.1 S. mutans ATCC 25175 99.9

[0039] The bactericidal effects of the antimicrobial peptides are shown in Table 2. As can be seen from Table 2, after 15 minutes of treatment with a 4-fold minimum inhibitory concentration of the antimicrobial peptide, 99.7% of P. gingivalis, 98.9% of F. nucleatum, and 99.2% of S. mutans were killed, respectively. This demonstrates that the peptides are highly effective in rapidly and effectively killing P. gingivalis, F. nucleatum, and S. mutans.

[0040] Example 4: Study on the hemolytic activity of the anti-periodontal pathogenic bacteria polypeptide prepared in Example 1.

[0041] 1. Experimental Methods

[0042] 1. Study on the hemolytic activity of antimicrobial peptides

[0043] (1) The antimicrobial peptide was diluted twice with normal saline to obtain antimicrobial peptide solutions with concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively.

[0044] (2) Anticoagulated blood from healthy individuals was centrifuged at 1000 rpm / min for 5 min to collect red blood cells. The collected red blood cells were washed three times with physiological saline and resuspended into a 2% (V / V) red blood cell suspension. 100 μL of red blood cell suspension and 100 μL of anti-periodontal pathogen peptide solution were added to 96-well cell culture plates, with final peptide concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The plates were then incubated at 37°C for 1 hour. After incubation, the 96-well cell culture plates were centrifuged at 3000 rpm / min for 10 minutes. Equal volumes of supernatant were taken to new 96-well cell culture plates, and the absorbance of the supernatant at 570 nm was detected using an enzyme-linked microplate reader to calculate the hemolysis rate of the peptide.

[0045] 2. Experimental Results

[0046] Hemolytic activity of peptides against periodontal pathogens Figure 1 As shown, the hemolysis rate of the polypeptide at a concentration of 25 μg / mL was 0.3%, at a concentration of 50 μg / mL was 0.5%, and at a concentration of 100 μg / mL was 5.9%. This shows that the cytotoxicity of the anti-periodontal pathogen polypeptide is very low.

[0047] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0048] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. A polypeptide for resisting periodontal pathogens, characterized in that: The amino acid sequence of the polypeptide is: Lys-Thr-Lys–Ile–Gly–Ile–Asn–Gly–Phe–Gly–Lys–Ile–Gly–Lys–Leu–Val–Leu–Lys.

2. A polypeptide solution containing the anti-periodontal pathogen polypeptide according to claim 1, characterized in that: The polypeptide solution is prepared by mixing a solvent with the anti-periodontal pathogen polypeptide.

3. The polypeptide solution according to claim 2, characterized in that The solvent is any one of physiological saline, ultrapure water, phosphate buffer and acetic acid.

4. Use of the anti-periodontal pathogen polypeptide according to claim 1 or the polypeptide solution according to any one of claims 2 to 3 in the preparation of an antibacterial agent, characterized in that: The antibacterial agent can inhibit Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans.

5. Use of the anti-periodontal pathogen polypeptide according to claim 1 or the polypeptide solution according to any one of claims 2 to 3 in the preparation of a bactericide, characterized in that: The bactericide can kill Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mutans.

6. Use of the anti-periodontal pathogen polypeptide according to claim 1 or the polypeptide solution according to any one of claims 2 to 3 in the preparation of a drug for preventing and / or treating periodontal infection, characterized in that: The drug has an anti-periodontal pathogenic bacteria polypeptide as the only effective ingredient, and the periodontal infection is caused by Porphyromonas gingivalis, Fusobacterium nucleatum or Streptococcus mutans.

7. Use of the anti-periodontal pathogen polypeptide according to claim 1 or the polypeptide solution according to any one of claims 2 to 3 in the preparation of toothpaste, antibacterial gel, toothpaste additive or mouthwash, characterized in that: The antibacterial gel is used for resisting Porphyromonas gingivalis, Fusobacterium nucleatum or Streptococcus mutans.

8. The use according to claim 7, characterized in that The toothpaste further comprises an abrasive, a thickener, a foaming agent, an antiseptic and a fluorine-containing substance.

9. The use according to claim 7, characterized in that The mouthwash further comprises any one or a combination of water, glycerin, sorbitol, ethanol, tea extract and propolis extract, citric acid, sodium citrate, caprylyl glycol, normal saline, flavoring agent, sweetener, surfactant, colorant, preservative, essential oil and triclosan.

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