A polypeptide and its application in preparing mutans streptococcus inhibitor

Abstract

The present invention provides a polypeptide and its application in preparing a mutans streptococcus inhibitor. The amino acid sequence of the polypeptide of the present invention is shown in SEQ ID NO: 1, which can not only effectively inhibit mutans streptococcus, but also is non-toxic to cells, and is suitable for preparing oral products with excellent antibacterial effect and long-term use, thereby preventing and treating oral diseases, especially preventing and treating caries. Moreover, the polypeptide of the present invention is short in length, has low difficulty in chemical synthesis, can be directly synthesized into a high-purity product, and has clear effects and wide applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptides, and more specifically, relates to a polypeptide and its application in the preparation of a mutans streptococcus inhibitor. Background Art

[0002] Streptococcus mutans ( Streptococcus mutans ), is a Gram-positive bacterium and one of the most abundant streptococci in the natural oral flora. Infection with mutans streptococci may cause a series of problems in the oral cavity, such as: it can decompose sugars and produce acidic substances, which dissolve calcium and phosphorus on the surface of teeth, causing enamel demineralization and the formation of tooth cavities (i.e., caries). Therefore, it is urgent to find a method that can effectively inhibit mutans streptococci, which is very important for maintaining oral health.

[0003] Peptides are important components of the innate immune system of biological organisms and may have antibacterial activity, such as C16G2, which has strong inhibitory activity against Streptococcus mutans. However, C16G2 is synthesized by connecting the C-terminal 16 amino acid sequence of the Streptococcus mutans receptor stimulating peptide (CSPC16) with the cationic antimicrobial peptide Novinspirin G2 through a linker (-GGG-). The Novinspirin G2 in its structure is derived from the broad-spectrum antimicrobial peptide novispirin G10, which has certain cytotoxicity and is not suitable for long-term use. Summary of the invention

[0004] The present invention aims to provide a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1, which can not only effectively inhibit Streptococcus mutans but also is non-toxic to cells and is suitable for preparing oral products with excellent antibacterial effect and long-term use.

[0005] The first object of the present invention is to provide a polypeptide.

[0006] The second object of the present invention is to provide a polypeptide having more than 85% homology with the above polypeptide.

[0007] The third object of the present invention is to provide the use of the above polypeptide in the preparation of an antibacterial agent.

[0008] The fourth object of the present invention is to provide the use of the above polypeptide in the preparation of mutans streptococcus inhibitors.

[0009] The fifth object of the present invention is to provide the use of the above polypeptide in the preparation of oral products.

[0010] A sixth object of the present invention is to provide an oral product.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention first discovered that a polypeptide having an amino acid sequence as shown in SEQ ID NO:1 can not only effectively inhibit Streptococcus mutans, but also is non-toxic to cells, and is suitable for preparing oral products with excellent antibacterial effect and long-term use. Therefore, the present invention provides a polypeptide having an amino acid sequence as shown in SEQ ID NO:1, a polypeptide having more than 85% homology with the above polypeptide, the use of the above polypeptide in preparing an antibacterial agent, the use of the above polypeptide in preparing a Streptococcus mutans inhibitor, and the use of the above polypeptide in preparing oral products.

[0013] Preferably, the oral product has the function of treating and / or preventing oral diseases.

[0014] Further preferably, the oral disease is an oral disease caused by oral pathogenic bacteria, such as dental caries.

[0015] More preferably, the oral pathogen is Streptococcus mutans.

[0016] Based on this, the present invention also provides an oral product containing the above polypeptide.

[0017] Preferably, the oral product is one or more of toothpaste, mouthwash, effervescent mouthwash tablets, oral lozenges or oral care liquid.

[0018] The present invention has the following beneficial effects:

[0019] The polypeptide of the present invention can not only effectively inhibit Streptococcus mutans, but also is non-toxic to cells, and is suitable for preparing oral products with excellent antibacterial effect and long-term use, thereby preventing and treating oral diseases, especially dental caries. Moreover, the polypeptide of the present invention is short in length, has low difficulty in chemical synthesis, can be directly synthesized into a high-purity product, and has clear effects and wide applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the peptide toxicity test on cells. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0022] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0023] Example 1 Synthesis of Peptide 1

[0024] The peptide 1 (amino acid sequence as shown in SEQ ID NO: 1: GLDWWQL) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0025] Comparative Example 1 Synthesis of Comparative Peptide 1

[0026] The comparative polypeptide 1 (amino acid sequence as shown in SEQ ID NO: 2: GLQWWTL) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0027] Comparative Example 2 Synthesis of Comparative Peptide 2

[0028] The comparative polypeptide 2 (amino acid sequence as shown in SEQ ID NO: 3: GLRWWGL) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0029] Comparative Example 3 Synthesis of Comparative Peptide 3

[0030] The comparative polypeptide 3 (amino acid sequence as shown in SEQ ID NO: 4: GLSWWML) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0031] Comparative Example 4 Synthesis of Comparative Peptide 4

[0032] The comparative polypeptide 4 (amino acid sequence as shown in SEQ ID NO: 5: GLAWWTL) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0033] Comparative Example 5 Synthesis of Comparative Peptide 5

[0034] The comparative polypeptide 5 (amino acid sequence as shown in SEQ ID NO: 6: GLHWWSL) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0035] Comparative Example 6 Synthesis of Comparative Peptide 6

[0036] The comparative peptide 6 (amino acid sequence as shown in SEQ ID NO: 7: GLKWWSL) was synthesized by commissioning Sangon Biotechnology Co., Ltd.

[0037] The amino acid sequences of polypeptide 1 obtained in Example 1 and comparative polypeptides 1 to 6 obtained in Comparative Examples 1 to 6 are shown in Table 1.

[0038] Table 1 Amino acid sequences of peptides

[0039]

[0040] Test Example 1 Test of the inhibitory activity of peptides against Streptococcus mutans

[0041] Use BHI medium to revive and culture Streptococcus mutans (No. ATCC 700610, purchased from the American Type Culture Collection ATCC), collect Streptococcus mutans in the logarithmic phase, wash with PBS, and resuspend in CDM medium. In a 96-well plate, place Streptococcus mutans at 1×10 7CFU / mL was inoculated in 200 μL CDM medium. Then, the polypeptide 1 obtained in Example 1 and the comparative polypeptides 1 to 6 obtained in Comparative Examples 1 to 6 were added to the medium to make their final concentrations 0.78, 1.56, 3.13, 6.25, and 12.5 μM, respectively (the experiment without adding polypeptide was used as the blank control group). 2 The culture was carried out under anaerobically conditions for 24 h, and the OD of the bacterial solution was detected by an ELISA instrument. 595 The results are shown in Table 2.

[0042] Table 2 Activity of Streptococcus mutans after peptide action (OD 595 )

[0043]

[0044] It can be seen that the activity of Streptococcus mutans treated with polypeptide 1 obtained in Example 1 is significantly lower than that of the blank control group, and its inhibitory activity against Streptococcus mutans is significantly better than that of comparative polypeptides 1 to 6 obtained in comparative examples 1 to 6, indicating that the polypeptide of the present invention can more effectively inhibit Streptococcus mutans.

[0045] Test Example 2 Test of the inhibitory activity of peptides of different concentrations against Streptococcus mutans at different action times

[0046] Use BHI medium to revive and culture Streptococcus mutans (No. ATCC 700610, purchased from the American Type Culture Collection ATCC), collect Streptococcus mutans in the logarithmic phase, wash with PBS, and resuspend in CDM medium. In a 96-well plate, place Streptococcus mutans at 1×10 7 The initial concentration of CFU / mL was inoculated in 200 μL CDM medium. Subsequently, the polypeptide 1 obtained in Example 1 and the comparative polypeptides 1 to 6 obtained in Comparative Examples 1 to 6 were added to the culture medium to make their final concentrations 3.13, 6.25, 12.5, and 25 μM, respectively (the experiment without adding polypeptide was used as the blank control group), and 50 μL mineral oil was added to each well to isolate oxygen. Using a LogPhase 600 growth curve instrument, anaerobically cultured at 37 ° C and 500 rpm for 48 h, and the OD of the bacterial solution was detected at 0, 12, 24, 36, and 48 h. 600 The results are shown in Tables 3 to 6.

[0047] Table 3 Inhibitory activity of peptides at a concentration of 3.13 μM against Streptococcus mutans at different action times (OD 600 )

[0048]

[0049] Table 4 Inhibitory activity of peptides at a concentration of 6.25 μM against Streptococcus mutans at different action times (OD 600 )

[0050]

[0051] Table 5 Inhibitory activity of peptides at a concentration of 12.5 μM against Streptococcus mutans at different action times (OD 600 )

[0052]

[0053] Table 6 Inhibitory activity of peptides at 25 μM against Streptococcus mutans at different action times (OD 600 )

[0054]

[0055] It can be seen that the polypeptide 1 obtained in Example 1 treats Streptococcus mutans at different concentrations (3.13-25 μM) and different action times (12-48 h), so that the activity of Streptococcus mutans is significantly lower than that of the blank control group, and its inhibitory activity against Streptococcus mutans is significantly better than that of the comparative polypeptides 1-6 obtained in comparative examples 1-6, indicating that the polypeptide of the present invention can more effectively inhibit Streptococcus mutans.

[0056] Test Example 3: Toxicity test of peptides to cells

[0057] Experimental group: Gingival epithelial cells were inoculated in a medium containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) in DMEM medium containing penicillin / streptomycin at 37°C and 5% CO 2 After reviving and subculturing under the conditions of 5 The cells were seeded at a density of 100 / mL in a 96-well plate and incubated at 37°C and 5% CO 2 When the gingival epithelial cells reached 70% confluence, fresh medium containing 10% ( v / v ) fetal bovine serum and 1% ( v / v ) penicillin / streptomycin DMEM medium, and then added the polypeptide 1 obtained in Example 1 to make the final concentrations of 3.13, 6.25, 12.5, and 25 μM, respectively, at 37°C and 5% CO 2 Then, the gingival epithelial cells were washed with PBS, and 100 μL of CCK-8 working solution (10 μL of CCK8 solution + 90 μL of DMEM medium) was added to each well and incubated at 37 °C in the dark for 0.5 h.

[0058] Control (Con) group: Same as the experimental group, except that no peptide was added.

[0059] Blank group: CCK-8 working solution.

[0060] The OD of each group was detected by microplate reader 450 The cell viability (%) was calculated according to the formula “Cell viability (%) = (OD 实验组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%” to calculate the survival rate of gingival epithelial cells to reflect the toxicity of the peptide to gingival epithelial cells. Figure 1 shown.

[0061] It can be seen that after co-culture with different concentrations of polypeptide 1 for 24 h, the activity of gingival epithelial cells had no significant difference compared with the control group, indicating that the polypeptide of the present invention is non-toxic to cells and has good cell compatibility. The oral products prepared as raw materials can be used for a long time.

[0062] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the polypeptide according to claim 1 in the preparation of a mutans streptococcus inhibitor.

3. Use of the polypeptide according to claim 1 in the preparation of oral products for treating and / or preventing oral diseases caused by Streptococcus mutans.

4. An oral product, characterized in that: Contains the polypeptide according to claim 1.

5. The oral product according to claim 4, characterized in that: The oral product is one or more of toothpaste, mouthwash, oral lozenges or oral care liquid.

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