Application of a polypeptide in preparing a product for preventing or treating oral diseases

Abstract

The invention belongs to the field of biotechnology, and specifically relates to an application of a polypeptide in preparing a product for preventing or treating oral diseases. The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, and the polypeptide has strong bactericidal ability against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, and Lactobacillus casei, with an MIC range of 8 μg / mL-16 μg / mL and an MBC range of 8 μg / mL-16 μg / mL.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of a polypeptide in preparing a product for preventing or treating oral diseases. Background Art

[0002] Dental caries is a chronic infectious disease caused by acid production in the hard tissues of the teeth by microbial metabolism under the influence of multiple factors, mainly bacteria. It is a major oral disease that aggravates or induces systemic diseases, and is also one of the non-communicable diseases that humans focus on preventing and treating. About 2.4 billion people in the world are affected by dental caries to varying degrees, and the proportion of people with dental caries in permanent teeth is as high as 35%. Therefore, the prevention and treatment of dental caries is of vital importance.

[0003] Antimicrobial peptides are a class of short polypeptides that are usually associated with the host's innate immune system. They are widely found in plants, amphibians, fish and humans. These peptides are usually composed of 30-100 amino acids, most of which are cationic. In addition to the net positive charge, antimicrobial peptides are usually amphipathic and contain both hydrophobic and hydrophilic domains. This property allows increased interaction and insertion with negatively charged cell walls and microbial membranes, perforating the cell membrane to form ion channels, causing a large amount of intracellular water-soluble substances to leak out, and ultimately leading to bacterial death. Chrysophsin-1 (FFGWLIKGAIHAGKAIHGLIHRRRH) is an alpha-helical cationic antimicrobial peptide from fish. Our previous studies have shown that it has an inhibitory effect on caries-related bacteria and can inhibit the formation of biofilms of Streptococcus mutans, the main oral cariogenic bacteria. However, naturally occurring antimicrobial peptides are often long and unstable, which increases the cost of synthesis and limits their application. Although the applicant has also studied polypeptides with shorter sequences than Chrysophsin-1, the sequence of this polypeptide is relatively long. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides an application of a polypeptide in the preparation of a product for preventing or treating oral diseases.

[0005] A polypeptide is used in preparing a product for preventing or treating oral diseases, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1, specifically: FFGWLIKGAIKAGKAIIG, denoted as Chm3.

[0006] Preferably, the oral disease is a disease caused by oral pathogenic bacteria.

[0007] Preferably, the pathogenic bacteria is any one of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Streptococcus sanguinis, Lactobacillus acidophilus, Lactobacillus casei, Actinomyces naeslundii, and Actinomyces viscosus.

[0008] Preferably, the pathogenic bacteria is Streptococcus gordonii.

[0009] Preferably, the oral disease is caries.

[0010] Preferably, the product is an oral product, an additive, a preparation for inhibiting the formation of dental plaque biofilm, or a preparation for removing mature dental plaque biofilm.

[0011] Preferably, the oral products include toothpaste, mouthwash, tooth paste, oral dressing, and oral spray.

[0012] Preferably, the additive is a food additive, a feed additive, a cosmetic additive or a sanitary product additive.

[0013] Preferably, the concentration of the polypeptide is 8-512 μg / mL.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention develops a polypeptide with shorter sequence, stable properties and better antibacterial / antibacterial effect by deleting the inactive central amino acids, replacing weak hydrophobic amino acids with strong hydrophobic amino acids, and maintaining the amino acid balance between the hydrophobic end and the hydrophilic end, which has good application prospects.

[0016] The amino acids of the natural cationic antimicrobial peptide Chrysophsin-1 (FFGWLIKGAIHAGKAIHGLIHRRRH) were deleted from the back end to form a new cationic antimicrobial peptide with antibacterial and bactericidal abilities; it has strong bactericidal abilities against Streptococcus mutans, Streptococcus sobrinus, Lactobacillus acidophilus, and Lactobacillus casei, with MIC ranging from 8μg / mL to 16μg / mL and MBC ranging from 8μg / mL to 16μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the structure of a polypeptide;

[0018] Figure 2 is the time-kill curve of the peptide against Streptococcus mutans;

[0019] Figure 3Figure 3 is the morphological effect of different concentrations of peptides on Streptococcus mutans, where AD is the observation result of Streptococcus mutans at 10k times magnification; EH is the observation result of Streptococcus mutans at 50k times magnification, where A and E are blank control groups without peptide treatment, and the bacterial surface is relatively smooth and the structure is intact; B and F are the observation results of Chm 3 treating Streptococcus mutans at 1×MBC for 4 hours, at which time the bacterial surface becomes swollen, wrinkled and has pores of different sizes; C and G are the observation results of Chm 3 treating Streptococcus mutans at 4×MBC for 4 hours, and the bacterial surface is wrinkled and cracked; D and H are the observation results of Chm 3 treating Streptococcus mutans at 8×MBC for 4 hours, at which time a large number of bacterial fragments can be clearly observed;

[0020] Figure 4 The treatment effects of different concentrations of peptides on mutans streptococcus biofilms. A is the negative control, which is a biofilm formed after 24 hours of growth of mutans streptococci without any treatment. At this time, the membrane is mainly green fluorescence; B is the observation result of Chm3 treating the biofilm at 2×MBC for 1 hour, and red and green fluorescence appeared in the membrane, with green fluorescence as the main fluorescence; C is the observation result of Chm 3 treating the biofilm at 8×MBC for 1 hour. Compared with Figure B, the green fluorescence is further weakened, and red fluorescence is dominant; D is the positive control group, which is the observation result of the biofilm treated with 0.12% CHX for 1 hour, with red fluorescence as the main fluorescence. Note: Green fluorescence represents live bacteria; red fluorescence represents dead bacteria;

[0021] Figure 5 The effects of different concentrations of peptides on the proliferation of human fibroblasts. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.

[0023] Example 1

[0024] Synthesis of peptides

[0025] The cationic antimicrobial peptide was synthesized by Fmoc solid phase synthesis method. 2-Chlorotrityl Chloride Resin was placed in a reaction tube, DMF (N, N-dimethylformamide) (15 mL / g) was added, and the mixture was shaken for 60 min. The amino acids were connected in the order of phenylalanine-phenylalanine-glycine-tryptophan-leucine-isoleucine-lysine-glycine-alanine-isoleucine-lysine-alanine-glycine-lysine-alanine-isoleucine-isoleucine-glycine (FFGWLIKGAIKAGKAIIG).

[0026] Connect the first amino acid F (phenylalanine), add 20% piperidine DMF solution for deprotection, remove the piperidine solution, wash three times with ethanol, add one drop each of ninhydrin, KCN (potassium cyanide), and phenol solution, heat for 5 minutes, and turn dark blue for a positive reaction, rinse, condense, and detect. Repeat the above steps until the last amino acid G (glycine) is connected, drain and cut, and the synthetic product is purified by HPLC. The purity of the antimicrobial peptide is greater than> 90%, and a polypeptide is obtained, which is recorded as chm3.

[0027] Chm3: FFGWLIKGAIKAGKAIIG, recorded as SEQ ID NO.1, with a hydrophobicity of 0.705H, a hydrophobic moment of 0.395μH, and a net charge z of 3.

[0028] Example 2

[0029] Microbial strains and culture conditions

[0030] ATCC standard strains were used to determine the in vitro killing activity of Chm3 against oral cariogenic microorganisms. The results are shown in Table 1.

[0031] Strains: Streptococcus mutans ATCC 700610; Streptococcus sanguinis ATCC 10556; Streptococcus sobrinus ACTT6715; Streptococcus gordonii ATCC 10558; Lactobacillus acidophilus ATCC4356; Lactobacillus acidophilus ATCC 393; Actinomyces viscosus ATCC 15987; Actinomyces naeslundii ATCC12104.

[0032] Culture conditions: The culture medium used in the experiment was brain heart infusion medium (BHI). Streptococcus mutans, Streptococcus sanguinis, Streptococcus sobrinus and Streptococcus gordonii were cultured in an incubator at 37°C and 5% CO2 for 24 to 48 hours; Lactobacillus acidophilus, Lactobacillus casei, Actinomyces viscosus and Actinomyces naeslundii were cultured at 37°C and anaerobic conditions (80% N2, 10% H2, 10% CO2) for 24 to 48 hours.

[0033] Example 3

[0034] Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

[0035] According to the US CLSI standard, the two-fold dilution method was used to determine the MIC and MBC of Chm3 against multiple bacteria. After the bacteria were cultured overnight in BHI medium, they were diluted to 10 6 CFU / mL for later use. Add the polypeptide to the bacterial suspension to a final concentration of 512μg / mL to 1μg / mL. Add 200μL / well to a 96-well plate, and use sterile water as a negative control. Depending on the bacterial growth, place the 96-well plate in static culture under the corresponding conditions for 24 to 48 hours. After the culture is completed, the MIC value of the bacteria is determined based on the turbidity of the culture medium and a spectrophotometer (600nm). Take out 100μL of bacterial culture solution from the clarified wells, spread on the plate, and culture overnight at 37°C. The polypeptide concentration at which no colony growth can be observed is the minimum bactericidal concentration (MBC) of the bacteria. Each experiment should be repeated at least 3 times.

[0036] Table 1 In vitro antibacterial test of Chm3 against oral cariogenic bacteria

[0037] Experimental strains Strain number MIC (μg / mL) MBC (μg / mL) Streptococcus mutans ATCC700610 16 16 Streptococcus sobrinus ATCC6715 16 16 Streptococcus gordonii ATCC10558 8 8 Streptococcus sanguinis ATCC10556 16 16 Lactobacillus acidophilus ATCC4356 16 32 Lactobacillus casei ATCC393 8 8 Actinomyces naeslundii ATCC12104 32 32 Actinomycetes viscosus ATCC15987 32 64

[0038] As shown in Table 1, the MIC of Chm3 ranges from 8 μg / mL to 32 μg / mL, and the MBC ranges from 8 μg / mL to 64 μg / mL.

[0039] Example 4

[0040] Time-kill curve of Chm3 against Streptococcus mutans

[0041] In order to determine the bactericidal ability of the peptides, we performed time-kill curves to determine the effect of Chm3 at different concentrations on the growth of S. mutans.

[0042] The specific experimental steps are as follows: prepare 10 mL of polypeptide BHI solution containing different concentrations of 1×MBC, 2×MBC, 4×MBC and 8×MBC, i.e. 8 μg / mL, 16 μg / mL, 32 μg / mL and 64 μg / mL, add Streptococcus mutans in the logarithmic phase to the above solution to control the final concentration to 10 5CFU / mL, take 0.5mL BHI solution and add it to 4.5mL diluent containing BHI for a specific time, so that the concentration of the polypeptide drops below the bactericidal concentration, and place it on ice to stop growth. After shaking and mixing, dilute it a certain number of times, take 1mL of bacterial solution and add it to an empty plate, pour 15-20mL of BHI solid culture medium cooled to 45-55℃, shake it well, and put it in a 37℃, 5% CO2 incubator for 24-48h after it is completely solidified, and count the bacteria. The experiment used BHI solution without polypeptide as the control group. The number of live bacteria at the action time of Chm3 determination was 0, 5min, 10min, 15min, 30min, 60min, 120min, 240min and 480min respectively.

[0043] like Figure 2 As shown, Chm3 can completely kill bacteria within 1 h under 8× MBC and can kill bacteria within 6 h under MBC.

[0044] Example 5

[0045] Scanning electron microscope observation of the damaging effect of peptides on Streptococcus mutans

[0046] In order to more intuitively observe the effects of Chm1, Chm2 and Chm3 on bacteria, we took Streptococcus mutans as the research object and used scanning electron microscopy to observe the damage of peptides to Streptococcus mutans at different concentrations (1×MBC, 4×MBC and 8×MBC).

[0047] The specific steps are as follows: 1mL10 6 cfu / mL bacterial suspension was added to a 24-well plate containing cell slides (Solebo, China), cultured at 37°C, 5% CO2 for 24h, washed twice with PBS, and then 500μl of different concentrations of Chm3 were treated for 4h, washed twice with PBS, and then 500μL of 2.5% glutaraldehyde was added, fixed overnight at 4°C, washed twice with PBS, dehydrated in gradient ethanol (30%, 50%, 70%, 80%, 90%, 100%, 100%) for 10 minutes, and soaked in hexamethyldisilane for 45min, dried, sprayed with gold (IonSputterE-1045, Hitachi, Japan), and the bacterial morphology on the surface of the slide was observed using a cold field emission electron microscope (S-4800, Hitachi, Japan). Untreated bacteria were used as negative controls, and the other steps were the same as above.

[0048] The scanning electron microscopy results are as follows Figure 3As shown in the figure, the surface of Streptococcus mutans without polypeptide treatment was relatively smooth and intact, with no obvious bacterial fragments or cell lysis. After treatment with polypeptide, its cell membrane and overall structure changed significantly, and the higher the polypeptide concentration, the stronger the effect. After Streptococcus mutans was treated with 1×MBC, 4×MBC and 8×MBC polypeptides for 4 hours, the bacterial surface became swollen, rough and wrinkled, with pores of varying sizes, and a large number of bacterial fragments appeared as the polypeptide concentration increased.

[0049] Example 6

[0050] Laser confocal microscopy to observe the effect of peptides on biofilm of Streptococcus mutans

[0051] The mutans Streptococcus was used as the research object to evaluate the killing effect of different concentrations (2×MBC and 8×MBC) of peptides on the biofilm formed by bacteria. First, the mutans Streptococcus in the logarithmic phase was diluted to 10 with 0.1% sucrose in BHI-S solution. 6 cfu / mL, add 1mL of bacterial solution to the 24-well plate with cell slides, and culture in a 37℃, 5% carbon dioxide incubator for 24h; secondly, aspirate the liquid in the plate, slowly wash twice with PBS, add 500μl of drug solution to the wells, and continue to culture at 37℃ for 1h; again, aspirate the liquid in the wells, rinse twice with PBS, add 350μl of LIVE / DEAD stain (InvitrogenL-7012, PI and SYTO9 volume ratio is 1:1) to the wells, and incubate in the dark for 15min; finally, take the slides out of the well plate and place them on a glass slide, and use a laser confocal microscope (Olympus, FV3000) to scan the slides at 40x and 100x respectively at a thickness of 1μm. The experiment set up a negative control group (BHI solution without polypeptide) and a positive control group (BHI solution containing 0.12% CHX). The images after layer scanning were processed with the software provided by the equipment, and the results are shown in the figure. Figure 4 .

[0052] Biofilm susceptibility test results Figure 4 : CLSM results showed that compared with the mutans Streptococcus biofilm not treated with polypeptide, the number of live bacteria (green fluorescence) in the biofilm treated with polypeptide was significantly reduced, and the number of dead bacteria (red fluorescence) decreased significantly with the increase of polypeptide concentration.

[0053] Example 7

[0054] Peptide cytotoxicity assay

[0055] The sixth generation of gingival fibroblasts were used for experimental studies. Gingival fibroblasts were inoculated in 96-well plates, with 1×105 cells per well, and cultured for 24 hours. Then, the cells were treated with 1×MBC, 2×MBC, and 4×MBC for 5 minutes, 30 minutes, and 240 minutes, respectively. The supernatant in the 96-well plate was removed, and 200 μl of culture medium was added to each well. After the peptide-treated cells were cultured for another 24 hours, the CCK8 kit (Beyotime, China) was used for quantitative evaluation according to the manufacturer's operating manual; untreated cells were used as negative controls and quantitatively evaluated in the same way. The values ​​were read at 450 nm using an ELISA reader.

[0056] like Figure 5 As shown, 64 μg / mL (4×MBC group) Chm3 treatment of cells for 30 min and 240 min can significantly affect bacterial proliferation, which is significantly different from the control group (P < 0.05).

[0057] Comparative Example 1

[0058] The sequence of the polypeptide in Comparative Example 1 is GWLIKGAIHAGKAIHGLIHRRRH, recorded as SEQ ID NO.2.

[0059] The polypeptide of Comparative Example 1 was subjected to an in vitro antibacterial experiment on oral cariogenic bacteria. The results are shown in Table 3.

[0060] Comparative Example 2

[0061] The sequence of the polypeptide in Comparative Example 2 is GWLIKGAIHAGKAIHGLIH, recorded as SEQ ID NO.3.

[0062] Comparative Example 3

[0063] The sequence of the polypeptide in Comparative Example 3 is GWLIKGAIHAGKAIHGLI, recorded as SEQ ID NO.4.

[0064] Comparative Example 4

[0065] The polypeptide sequence in Comparative Example 4 is FFGWLIKGAIKAGKAIHG, recorded as SEQ ID NO.5.

[0066] Streptococcus mutans is the core microorganism that causes dental caries, so the antibacterial activity of the polypeptides in Comparative Examples 1-4 against Streptococcus mutans was evaluated, and the results are shown in Table 2.

[0067] Table 2 Evaluation of the antibacterial activity of the peptides of Comparative Examples 1-4 and Example 1 against Streptococcus mutans

[0068] Peptides Amino acid sequence of peptide MIC (μg / mL) MBC (μg / mL) Comparative Example 1 GWLIKGAIHAGKAIHGLIHRRRH 8 8 Comparative Example 2 GWLIKGAIHAGKAIHGLIH 256 >256 Comparative Example 3 GWLIKGAIHAGKAIHGLI >256 ND Comparative Example 4 FFGWLIKGAIKAGKAIHG 128 256 Example 1 FFGWLIKGAIKAGKAIIG 16 16

[0069] Table 3 In vitro antibacterial test of the polypeptide of comparative example 1 against oral cariogenic bacteria

[0070]

[0071]

[0072] As can be seen from Table 2, after the polypeptide of Comparative Example 1 was truncated, the antibacterial properties of Comparative Examples 2 to 4 were significantly reduced, while the polypeptide of Example 1 of the present invention had a shorter sequence than that of Comparative Example 1, and the degree of reduction in antibacterial activity was very small. As can be seen from Tables 1 and 3, compared with the polypeptide of Comparative Example 1, after the polypeptide was truncated, the polypeptide of Example 1 of the present invention still maintained excellent antibacterial activity.

[0073] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0074] 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.

[0075] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a polypeptide in preparing a product for preventing or treating oral diseases, characterized in that: The amino acid sequence of the polypeptide is FFGWLIKGAIKAGKAIIG; The oral disease is a disease caused by oral pathogenic bacteria; The pathogenic bacteria is any one of Streptococcus mutans, Streptococcus sobrinus, Streptococcus gordonii, Streptococcus sanguinis, Lactobacillus acidophilus, Lactobacillus casei, Actinomyces naeslundii, and Actinomyces viscosus; The oral disease is caries.

2. The use according to claim 1, characterized in that: The pathogenic bacteria is Streptococcus gordonii.

3. The use according to claim 1, characterized in that: The product is an oral product, a hygiene product additive, a preparation for inhibiting the formation of dental plaque biofilm, or a preparation for removing mature dental plaque biofilm.

4. The use according to claim 3, characterized in that: The oral products include toothpaste, mouthwash, tooth paste, oral dressing and oral spray.

5. The use according to claim 1, characterized in that: The concentration of the polypeptide is 8-512 µg / mL.

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