CN117777250A - Antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of oral cavity and application thereof - Google Patents
Antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of oral cavity and application thereof Download PDFInfo
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- CN117777250A CN117777250A CN202410002993.XA CN202410002993A CN117777250A CN 117777250 A CN117777250 A CN 117777250A CN 202410002993 A CN202410002993 A CN 202410002993A CN 117777250 A CN117777250 A CN 117777250A
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses an antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of an oral cavity and application thereof. The amino acid sequence of the antibacterial peptide PA9 is shown as SEQ ID NO. 1. The antibacterial peptide PA9 provided by the invention consists of 36 amino acids, the molecular weight is 4049.99, the isoelectric point is 10.4, the net charge at pH 7.0 is 12, the average hydrophilicity is 0.6, and the hydrophilic residue proportion is 47%. The antibacterial peptide shown as SEQ ID NO.1 has the advantages of simple preparation and low cytotoxicity, has antibacterial and bactericidal activity on periodontal pathogenic bacteria of the oral cavity, can target and destroy the biomembrane of the periodontal pathogenic bacteria of the oral cavity, plays a bactericidal role, can be used for preparing oral supplies, inhibiting the growth of the periodontal pathogenic bacteria of the oral cavity, regulating the balance of oral micro-ecology, and further can be used for preventing and treating oral diseases, especially caries, periodontitis, oral mucosa diseases or oral cancers and the like.
Description
Technical Field
The invention relates to an antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of an oral cavity and application thereof, belonging to the technical field of antibacterial peptides.
Background
Periodontal disease is a common oral disease, and oral pathogenic bacteria and dental plaque play a direct role in the occurrence and progression of periodontal disease. The auxiliary therapeutic medicine for periodontal disease mainly uses antibiotics, and can easily cause bacterial drug resistance and oral dysbacteriosis after long-term use. Therefore, there is an urgent need to develop new drugs to avoid the risk of bacterial resistance.
The antibacterial peptide is used as a novel antibacterial agent, has wide sources and broad-spectrum antibacterial activity, and compared with the traditional antibiotics, most antibacterial peptides kill bacteria mainly by destroying bacterial membranes, are not easy to cause the bacteria to generate drug resistance, and are considered as good antibiotic substitutes. Boman et al, 1980, succeeded in separating and purifying cecropin from the silkworm pupae for the first time, which was the first antimicrobial peptide discovered. Antibacterial peptides were subsequently found in bacteria, fungi, amphibians, higher plants, mammals, and humans. Currently, more than 40 antimicrobial peptides are isolated from human oral epithelial cells, saliva and periodontal tissue. Some antimicrobial peptides have been shown to have antimicrobial activity against periodontal pathogens, for example, LL-37 has a killing effect on actinomycetes and Proteus intermedia, beta defensins have a killing effect on actinomycetes, fusobacterium nucleatum, porphyromonas gingivalis and Proteus intermedia, and lactoferrin has a killing effect on Porphyromonas gingivalis and Proteus intermedia.
The natural antibacterial peptide has high production cost, low antibacterial activity and poor stability, and can not meet the requirements of practical application. With the continuous intensive research on the properties, structure and action mechanism of the existing natural antibacterial peptide, more and more researches focus on developing artificial synthetic antibacterial peptide, and researchers have successfully utilized various modern biotechnology to perform molecular improvement on the antibacterial peptide or synthesize novel antibacterial peptide. Therefore, the development of the antibacterial peptide with simple structure and strong antibacterial activity has important significance.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an antibacterial peptide PA9 for inhibiting oral periodontal pathogenic bacteria and application thereof. The antibacterial peptide has good antibacterial and bactericidal activity on clostridium nucleatum, porphyromonas gingivalis and Proprietaria intermedia for periodontal pathogens of oral cavity, and has the advantages of simple structure, small synthesis difficulty, low cell hemolytic activity and high safety.
The technical scheme of the invention is as follows:
the first aspect of the invention provides an antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of the oral cavity, wherein the amino acid sequence of the antibacterial peptide PA9 is shown as SEQ ID NO. 1.
The invention discovers for the first time that the antibacterial peptide shown as SEQ ID NO.1 has simple preparation and low cytotoxicity, has antibacterial and bactericidal activity on oral cavity periodontal pathogens, can target and destroy oral cavity periodontal pathogen biomembrane, and plays a bactericidal role. More specifically, the antibacterial peptide PA9 provided by the invention consists of 36 amino acids, has a molecular weight of 4049.99, an isoelectric point of 10.4, a net charge of 12 at pH 7.0, an average hydrophilicity of 0.6 and a hydrophilic residue ratio of 47%.
The antibacterial peptide PA9 can be synthesized according to specific amino acid sequences by a solid phase synthesis method, and is prepared by HPLC reversed phase column chromatography desalination and purification.
According to the invention, the oral cavity periodontal pathogenic bacteria are preferably one or more of clostridium nucleatum, porphyromonas gingivalis and praecox intermedia.
The second aspect of the invention provides application of the antibacterial peptide PA9 in preparing a bacteriostatic agent for periodontal pathogens of an oral cavity.
According to the invention, the oral cavity periodontal pathogenic bacteria are preferably one or more of clostridium nucleatum, porphyromonas gingivalis and praecox intermedia.
In a third aspect, the invention provides the use of the above antimicrobial peptide PA9 in the manufacture of an oral product for the treatment and/or prophylaxis of oral diseases.
According to the present invention, the oral disease is preferably caused by one or more of Fusobacterium nucleatum, porphyromonas gingivalis, and Proteus intermedia.
According to the invention, the oral product is preferably one or more of toothpaste, mouthwash effervescent tablets or oral care solution.
In the oral cavity, if the microecological balance is caused, the microbial population is easily converted into related populations of pathogens, and the oral health is negatively affected; meanwhile, the addition of pathogenic bacteria can easily cause bad breath (halitosis) in the oral cavity, thereby causing caries, periodontitis, oral mucosa diseases or oral cancers and other diseases, and the antibacterial peptide PA9 can effectively inhibit periodontal pathogenic bacteria in the oral cavity, thereby effectively regulating the balance of micro-ecology in the oral cavity, so that the oral products containing the antibacterial peptide PA9 are all within the protection scope of the invention.
The invention has the beneficial effects that:
1. the antibacterial peptide PA9 provided by the invention has antibacterial and bactericidal activity on oral cavity periodontal pathogens, can target and destroy oral cavity periodontal pathogens biomembrane, inhibit the formation of oral cavity periodontal pathogens biomembrane, and play a bactericidal role. In particular to the minimum antibacterial concentration of the fusobacterium nucleatum which is 500 mug/mL, and the minimum antibacterial concentration of the fusobacterium nucleatum which is 500 mug/mL; the minimum antibacterial concentration for Porphyromonas gingivalis is 250 mug/mL, and the minimum antibacterial concentration is 250 mug/mL; the minimum antibacterial concentration of the intermediate Proprietaria is 125 mug/mL, and the minimum antibacterial concentration is 500 mug/mL, so that the intermediate Proprietaria can be used for preparing oral products, inhibiting the growth of pathogenic bacteria around the oral cavity and regulating the balance of oral micro-ecology, and further can be used for preventing and treating oral diseases, especially caries, periodontitis, oral mucosa diseases or oral cancers.
2. The antibacterial peptide PA9 provided by the invention has the advantages of short synthetic sequence, small molecular weight, small chemical synthesis difficulty, low hemolytic activity, high safety, definite effect and wide application, and can be directly combined into a high-purity product.
Drawings
FIG. 1 is a high performance liquid chromatogram of the antimicrobial peptide PA9 of the present invention;
FIG. 2 is a mass spectrum of the antimicrobial peptide PA9 of the present invention;
FIG. 3 shows the experimental results of the minimum inhibitory concentration of the antibacterial peptide PA9 of the invention on periodontal pathogens of the oral cavity;
FIG. 4 shows the experimental results of the minimum bactericidal concentration of the antibacterial peptide PA9 of the present invention against periodontal pathogens;
FIG. 5 shows the experimental results of the crystal violet staining of the antibacterial peptide PA9 of the present invention on the oral periodontal pathogenic bacteria biofilm;
FIG. 6 shows the experimental results of the antibacterial peptide PA9 of the present invention on the biological membrane biomass of periodontal pathogens of the oral cavity;
FIG. 7 shows the result of experiment of hemolytic toxicity of the antibacterial peptide PA9 of the present invention to mammalian erythrocytes.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental technical methods and scientific terms used in the following examples have the same meanings as commonly understood by one of ordinary skill unless otherwise indicated. The experimental consumables and reagents involved, as without any special remarks, are commercially available in general.
EXAMPLE 1 Synthesis of antibacterial peptide PA9
The amino acid sequence of the antibacterial peptide PA9 is as follows:
LGAAVLKMLKPGLQTNTLFKKKKKKKKKKGEKFSGF (specifically SEQ ID NO. 1).
Then, the Shanghai Nuo Biotechnology Co., ltd is entrusted to the solid phase synthesis method, the specific amino acid sequence is synthesized artificially, and then the antibacterial peptide PA9 is prepared by HPLC reversed phase column chromatography desalination and purification.
The high performance liquid chromatogram of the antibacterial peptide PA9 prepared in the embodiment is shown in figure 1, and the mass chromatogram is shown in figure 2.
As can be seen from fig. 1 and 2, the basic physicochemical properties of the antimicrobial peptide PA9 prepared in this example are: the molecular weight was 4049.99, the isoelectric point was 10.4, the net charge at pH 7.0 was 12, the average hydrophilicity was 0.6, and the hydrophilic residue ratio was 47%.
Example 2 determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of antibacterial peptide PA9
1. The periodontal disease-causing strains of the oral cavity involved in this example were: fusobacterium nucleatum (Fusobacterium nucleatum, F.n), porphyromonas gingivalis (Porphyromonas gingivalis, P.g) are commercially available from American type culture Collection, and Proprietaria intermedia (Prevotella intermedia, P.i) are commercially available from Beijing Bai Bo Wei Biotechnology Co.
2. The method for determining the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of the antibacterial peptide PA9 is specifically as follows:
(1) Activating strains, streaking the periodontal disease-causing strains of the oral cavity on a BHI blood agar solid culture medium, and performing stationary culture in an anaerobic incubator at 37 ℃; after the colony grows to a proper size, randomly picking a single colony into a BHI liquid culture medium, and culturing in an anaerobic incubator at 37 ℃ until the colony is in a logarithmic phase;
(2) Centrifuging the bacterial liquid cultured in the step (1) to logarithmic phase at 5000rpm for 5min to collect bacterial cells, and diluting with BHI liquid culture medium to give bacterial cell concentration of 1×10 8 CFU/mL；
(3) Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the antimicrobial peptide PA9 were determined using standard micro broth dilution: preparing double serial dilutions of the antimicrobial peptide PA9 in 96-well plates at a volume of 100 μl/well in culture medium, the concentration of the antimicrobial peptide ranging from 1 μg/mL to 500 μg/mL; positive controls (periodontal disease strains and medium) and negative controls (medium only) were set simultaneously, three in parallel per group;
(4) Culturing the 96-well plate in the step (3) in an anaerobic incubator at 37 ℃ for 24 hours, and visually observing the turbidity degree of the culture medium in each well, wherein the concentration corresponding to the clarified culture medium well is an MIC value; 10. Mu.L of each bacterial liquid was streaked from each well corresponding to MIC and above, and the concentration corresponding to colony-free growth on the surface of the plate was observed to be MBC value, and the results are shown in Table 1, FIG. 3 and FIG. 4.
Table 1 Minimum Inhibitory Concentration (MIC) and minimum inhibitory concentration (MBC) of antibacterial peptide PA9
As can be seen from Table 1, FIG. 3 and FIG. 4, the minimum inhibitory concentration of the antibacterial peptide PA9 on Fusobacterium nucleatum is 500. Mu.g/mL, and the minimum inhibitory concentration is 500. Mu.g/mL; the minimum antibacterial concentration for Porphyromonas gingivalis is 250 mug/mL, and the minimum antibacterial concentration is 250 mug/mL; the minimum antibacterial concentration of the antibacterial peptide PA9 for the intermediate Propionibacterium is 125 mug/mL and the minimum antibacterial concentration of the antibacterial peptide PA9 is 500 mug/mL, which fully shows that the antibacterial peptide PA9 provided by the invention has obvious antibacterial and bactericidal activity for oral periodontal pathogenic bacteria such as Fusobacterium nucleatum, porphyromonas gingivalis and intermediate Propionibacterium.
Example 3 influence of the antibacterial peptide PA9 on the bacterial biofilm
The influence of the antibacterial peptide PA9 on the formation of the oral periodontal pathogenic bacteria biomembrane is evaluated by a crystal violet staining method, and the specific method is as follows:
(1) Preparing bacterial liquid of periodontal pathogenic bacteria of the oral cavity according to the method of the example 2;
(2) Taking a 96-well plate, and respectively adding 50 mu L of bacterial liquid of oral periodontal pathogenic bacteria and 50 mu L of antibacterial peptide PA9 solution with different concentrations into each well to ensure that the final concentration of the antibacterial peptide PA9 in the well plate is MIC, 1/2MIC, 1/4MIC and 1/8MIC. Positive controls (oral periodontal pathogen and medium) and negative controls (medium only) were set simultaneously, three in parallel per group;
(3) Placing the 96-well plate in an anaerobic incubator at 37 ℃ for anaerobic incubation for 24 hours;
(4) Discarding the supernatant, slowly flushing with 200 μl PBS buffer solution for 3 times to remove planktonic oral periodontal pathogenic bacteria, and drying at room temperature;
(5) Adding 100 μl of methanol into each hole, fixing for 15min, sucking methanol out, and air drying;
(6) Adding 100 mu L of 1% crystal violet coloring agent into each hole to dye the biological film, and dyeing for 20min at room temperature;
(7) Sucking out crystal violet, washing out excessive coloring agent with PBS, and air drying;
(8) Then 100. Mu.L of 30% acetic acid was added to each well, and the mixture was decolorized at room temperature by gentle shaking for about 10 minutes, and the OD at 595nm was recorded using a microplate reader, and the results are shown in FIGS. 5 and 6.
As can be seen from fig. 5, the control groups F.n, P.g, P.i all formed complete biofilms; when the concentration of the antimicrobial peptide PA9 is MIC, the biological film can hardly be colored; at concentrations of 1/2MIC and 1/4MIC, only very thin biofilms formed; at a concentration of 1/8MIC, the antimicrobial peptide PA9 was able to form a complete biofilm with a deep coloration.
As can be seen from FIG. 6, the concentrations of the antimicrobial peptides PA9 were MIC, 1/2MIC and 1/4MIC, respectively, as compared with the control group, and the biological membrane OD of F.n and P.i 595 Reduced biofilm formation and reduced biofilm OD of P.g at concentrations of antimicrobial peptide PA9 of MIC and 1/2MIC 595 The reduction of the biofilm formation amount and the difference are statistically significant (P < 0.05). The antibacterial peptide PA9 provided by the invention can be used for targeted destruction of the biomembrane of the periodontal pathogenic bacteria of the oral cavity, inhibiting the biomembrane formation of the periodontal pathogenic bacteria of the oral cavity, and has obvious antibacterial and bactericidal activity on the periodontal pathogenic bacteria of the oral cavity.
Example 4: haemolytic toxicity of the antimicrobial peptide PA9 on mammalian erythrocytes
1mL of sterile, anticoagulated sheep blood was centrifuged at 3000rpm for 10min, sheep red blood cells were collected by washing 3 times with sterile PBS buffer, and incubated in PBS buffer containing various concentrations of the antimicrobial peptide PA9 (62.5. Mu.g/mL-500. Mu.g/mL) at 37℃for 30min while 1% Triton X-100 was used as positive control and sterile PBS buffer was used as negative control. After incubation was completed, the supernatant was centrifuged at 3000rpm for 10min, and the OD was measured at 570nm with a microplate reader, three replicates were set for each group, and the results are shown in table 2 and fig. 7.
The calculation formula of the hemolysis rate is as follows: the hemolysis ratio= (AT-AO)/(AC-AO) ×100%.
In the formula: AT is the absorbance of the experimental group, AC is the absorbance of the positive control group, and AO is the absorbance of the negative control group.
TABLE 2 haemolytic Activity of antibacterial peptide PA9
As shown in Table 2 and FIG. 7, under the condition of inhibiting the Minimum Inhibitory Concentration (MIC) of periodontal pathogens in the oral cavity, the hemolysis rate of the antibacterial peptide PA9 is lower than 1%, even if the hemolysis rate is only 0.27% at the maximum bactericidal concentration of 500 mug/mL, the antibacterial peptide PA9 has better safety and larger application prospect, and can be further studied and developed for use.
In conclusion, the antibacterial peptide PA9 provided by the invention has antibacterial and bactericidal activity on periodontal pathogenic bacteria of the oral cavity, can target and destroy the biomembrane of the periodontal pathogenic bacteria of the oral cavity, inhibit the biomembrane formation of the periodontal pathogenic bacteria of the oral cavity, play a role in sterilization, can be used for preparing oral supplies, inhibit the growth of the periodontal pathogenic bacteria of the oral cavity, regulate the balance of oral micro-ecology, and further prevent and treat oral diseases, especially caries, periodontitis, oral mucosa diseases or oral cancers and other diseases.
Claims (8)
1. An antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of the oral cavity, which is characterized in that the amino acid sequence of the antibacterial peptide PA9 is shown as SEQ ID NO. 1.
2. The antibacterial peptide PA9 for inhibiting oral periodontal pathogenic bacteria according to claim 1, wherein the antibacterial peptide PA9 is composed of 36 amino acids, has a molecular weight of 4049.99, an isoelectric point of 10.4, a net charge at ph 7.0 of 12, an average hydrophilicity of 0.6, and a hydrophilic residue ratio of 47%.
3. The antibacterial peptide PA9 for inhibiting oral periodontal pathogenic bacteria according to claim 1, wherein the oral periodontal pathogenic bacteria is one or more of fusobacterium nucleatum, porphyromonas gingivalis, and praecox intermedia.
4. The use of the antibacterial peptide PA9 for inhibiting oral periodontal pathogenic bacteria according to claim 1 for preparing antibacterial agent for oral periodontal pathogenic bacteria.
5. The use according to claim 4, wherein the oral periodontal pathogen is one or more of fusobacterium nucleatum, porphyromonas gingivalis, and praecox intermedia.
6. Use of the antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria in the oral cavity according to claim 1 for the preparation of an oral product for the treatment and/or prevention of oral diseases.
7. The use according to claim 6, wherein the oral disease is caused by one or more of fusobacterium nucleatum, porphyromonas gingivalis, and praecox intermedia.
8. The use according to claim 6, wherein the oral product is one or more of a toothpaste, a mouthwash effervescent tablet or an oral care solution.
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| CN202410002993.XA CN117777250A (en) | 2024-01-02 | 2024-01-02 | Antibacterial peptide PA9 for inhibiting periodontal pathogenic bacteria of oral cavity and application thereof |
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2024
- 2024-01-02 CN CN202410002993.XA patent/CN117777250A/en active Pending
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