An S-layer protein-derived polypeptide and its application

Abstract

The present invention discloses an S-layer protein-derived polypeptide and its application, belonging to the technical field of biomedicine. The S-layer protein polypeptide disclosed in the present invention comprises the amino acid sequence shown in SEQ ID No.1: VTVNV. The S-layer protein-derived polypeptide disclosed in the present invention comprises the amino acid sequence shown in SEQ ID No.2: PVTVNVPNVTPAAAQETTKTV. The composition of the present invention contains the above polypeptide. The present invention discloses the application of the above polypeptide in the preparation of oral care products. The polypeptide of the present invention has good collagen-binding ability and calcium-binding ability, can effectively adsorb on the surface of collagen while aggregating calcium ions in the environment, and realizes the biomimetic mineralization of dentin with ordered mineral arrangement in collagen fibers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an S-layer protein-derived polypeptide and its application. Background Art

[0002] Dentin is the hard tissue that constitutes the main body of the tooth, located inside the enamel and cementum. It contains approximately 30% organic matter and water, and 70% inorganic matter, and has a lower hardness than enamel. Dentin caries mostly develops from enamel caries, and dentin caries will show demineralization of dentin and exposure of type I collagen fibers. In the prior art, the classical treatment method for dentin caries is to remove the affected dental tissue and then replace it with an artificial filling material.

[0003] Early intervention through non-invasive treatment methods to remineralize demineralized dentin, thereby preventing and stopping the progression of the disease, has been regarded as an important means of clinical treatment. Designing mineralization-promoting functional polypeptides by means of bionic ideas has become a new and ideal way for the prevention and treatment of dental caries. Summary of the Invention

[0004] One of the purposes of the present invention is to provide an S-layer protein-derived polypeptide, which has good collagen-binding ability and calcium-binding ability, can effectively adsorb on the surface of collagen while aggregating calcium ions in the environment, and realize the bionic mineralization of dentin with orderly arrangement of minerals in collagen fibers.

[0005] Another purpose of the present invention is to provide a composition containing the S-layer protein-derived polypeptide.

[0006] The third purpose of the present invention is to provide the application of the S-layer protein-derived polypeptide.

[0007] To achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0008] The present invention discloses an S-layer protein polypeptide, and the polypeptide contains the amino acid sequence shown in SEQ ID No.1: VTVNV.

[0009] The present invention discloses an S-layer protein-derived polypeptide, and the polypeptide contains the amino acid sequence shown in SEQ ID No.2: PVTVNVPNVTPAAAQETTKTV.

[0010] The applicant analyzed the key sequences for collagen adhesion and regulation of mineralization in the S-layer protein SlpB structural sequence of Lactobacillus crispatus K313, and obtained the self-assembled derivative polypeptide of the present invention with collagen-binding ability and calcium-binding ability.

[0011] In some embodiments of the present invention, the polypeptide is C-terminally amidated or is a pharmaceutically acceptable salt or ester thereof.

[0012] In some embodiments of the present invention, the pharmaceutically acceptable salts are hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate or organic amine salts.

[0013] A composition disclosed by the present invention contains the above-mentioned polypeptide, as well as a pharmaceutically acceptable carrier and / or excipient.

[0014] Use of the polypeptide disclosed by the present invention in the preparation of an oral care product.

[0015] In some embodiments of the present invention, the oral care product includes an oral care product having the effect of promoting dentin mineralization.

[0016] In some embodiments of the present invention, the oral care product includes an oral care product for repairing demineralized dentin or / and improving dentin sensitivity.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The polypeptide sequence of the present invention is inspired by the key sequences for bacterial S-layer protein collagen adhesion and regulation of mineralization, and is obtained by analyzing the sequences of key mineralization proteins.

[0019] The polypeptide of the present invention has strong self-assembly ability, can spontaneously self-assemble into a fiber network structure in aqueous solution, and is less affected by environmental factors (ions, pH); can adsorb calcium ions, regulate the nucleation process of hydroxyapatite, and induce the orderly growth of hydroxyapatite crystals; has strong collagen-binding ability and can evenly bind to the demineralized dentin collagen fiber network; can accelerate the biomimetic mineralization process of type I collagen, thereby accelerating the biomimetic mineralization process of demineralized dentin. Moreover, the polypeptide of the present invention has good biosafety.

[0020] Using the polypeptide of the present invention for biomimetic mineralization can enable the polypeptide to play the role of an "intermediate gripper" in the biomimetic mineralization process, effectively adsorb on the collagen surface while aggregating calcium ions in the environment, and achieve dentin biomimetic mineralization with orderly arrangement of minerals within collagen fibers. It has good application prospects in common oral clinical diseases such as dental caries and dentin hypersensitivity, such as being applied to repair dentinal tubules exposed due to caries and significantly improving dentin hypersensitivity, etc. Thus, it provides new ideas for the research on dentin biomimetic mineralization repair. Description of the Drawings

[0021] Figure 1 It is a transmission electron microscope image of the self-assembled morphology of the polypeptide.

[0022] Figure 2It is a transmission electron microscope image of the in vitro mineralization product.

[0023] Figure 3 It is a confocal microscope image of the polypeptide-adsorbed demineralized dentin collagen fiber network; where A: Image of the surface of normal dentin treated with FITC; B: Image of the surface of normal dentin treated with FITC-polypeptide; C: Image of the surface of demineralized dentin treated with FITC; D: Image of the surface of demineralized dentin treated with FITC-polypeptide; E: Magnified image of the surface of demineralized dentin treated with FITC; F: Magnified image of the surface of demineralized dentin treated with FITC-polypeptide.

[0024] Figure 4 It is the TEM and SAED images of collagen mineralization for 1 day after polypeptide treatment; where A1, A2, B1, B2 are TEM images, and A3, B3 are SAED images.

[0025] Figure 5 It is the transmission electron microscope and elemental distribution images of collagen mineralization for 1 day and 3 days after polypeptide treatment; where A is the transmission electron microscope image of collagen mineralization for 1 day in the control group; B is the transmission electron microscope image of collagen mineralization for 3 days in the control group; C is the transmission electron microscope image of collagen mineralization for 1 day in the polypeptide group; D is the transmission electron microscope image of collagen mineralization for 3 days in the polypeptide group; in each group, the second to fourth images are in turn: calcium (Ca) elemental distribution image, phosphorus (P) elemental distribution image, and mixed element image (mixture of N, O, P, Ca elements).

[0026] Figure 6 It is the SEM morphology images of the cross-section of dentin after mineralization for 3 days and 7 days, where sound represents the dentin before acid etching, that is, A1 - A2: normal dentin group; etched is the dentin after acid etching, that is, B1 - B2: demineralized dentin group; C1 - C2: control group mineralized for 3 days; D1 - D2: control group mineralized for 7 days; E1 - E2: polypeptide group mineralized for 3 days; F1 - F2: polypeptide group mineralized for 7 days. Figure 7 It is the cell survival rate image after co-culturing different concentrations of polypeptide with HDPCs. Detailed implementation manners

[0027] In order to make the objectives, technical solutions and advantages of the present invention clearer, the following further details the present invention in combination with specific examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0028] The commercial type I collagen used in the embodiments of the present invention is mouse type I collagen (Solarbio, USA).

[0029] The amino acid sequence of the polypeptide adopted in the embodiments of the present invention is as shown in SEQ ID No.2:

[0030] PVTVNVPNVTPAAAQETTKTV.

[0031] Example 1

[0032] This example provides an S-layer protein-derived polypeptide, and the amino acid sequence is as follows:

[0033] SEQ ID No.2: PVTVNVPNVTPAAAQETTKTV.

[0034] The S-layer protein-derived polypeptide in this example was synthesized by Nano-Micro Life Sciences Co., Ltd. (Xi'an, China).

[0035] The solid-phase synthesis method of the polypeptide includes the following steps:

[0036] Step 1, first Fmoc-Val-OH is coupled to the resin, and then Fmoc-Val-OH; Fmoc-Lys(Boc)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Thr(tBu)-OH; Fmoc-Glu(OtBu)-OH; Fmoc-Gln(Trt)-OH; Fmoc-Ala-OH; Fmoc-Ala-OH; Fmoc-Ala-OH: Fmoc-Pro-OH; Fmoc-Thr(tBu)-OH; Fmoc-Val-OH; Fmoc-Asn(Trt)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Val-OH; Fmoc-Asn(Trt)-OH; Fmoc-Val-OH; Fmoc-Thr(tBu)-OH; Fmoc-Val-OH; Fmoc-Pro-OH are sequentially coupled. All couplings are carried out in DMF with DIC(3eq), HOBt(3eq) as coupling reagents.

[0037] Step 2, the Fmoc protecting group is cleaved with 20% Pip / DMF solvent, washed with DMF solvent, and finally cut from the resin with TFA solvent at room temperature, and the solution is filtered.

[0038] Step 3, add ether to the filtrate, the polypeptide precipitates in ether, and then centrifuged and washed to obtain the crude peptide

[0039] H-Pro-Val-Thr-Val-Asn-Val-Asp-Asn-Val-Thr-Pro-Ala-Ala-Ala-Gln-Glu-Thr-Thr-Lys-Thr-Val-OH.

[0040] Step 4, the crude peptide is purified on a C18 preparative column by high performance liquid chromatography to a purity of 95% to obtain the purified polypeptide.

[0041] Step 5: The purified polypeptide is placed in a freeze dryer and freeze-dried into a white powder.

[0042] Example 2

[0043] This example discloses the investigation of the self-assembly ability of the polypeptide of the present invention.

[0044] After incubating the 1 mg / mL polypeptide solution at 37 °C for 24 h, 20 μL is dropped onto a copper grid, then stained with 2% phosphotungstic acid for 3 - 4 minutes, and immediately rinsed with pure water (DIW) to remove the excess dye. After drying, it is observed under a transmission electron microscope (TEM).

[0045] The results are as Figure 1 shown. Under the microscope, the polypeptide is a mixture of nanospheres and nanofibers. A large number of nanofibers can be seen intertwined and arranged in a network, with a diameter of about 4 nm.

[0046] The results indicate that the polypeptide of the present invention has strong self-assembly ability and can spontaneously self-assemble into a fibrous network structure in aqueous solution.

[0047] Example 3

[0048] This example discloses the test for investigating the ability of the polypeptide of the present invention to regulate the nucleation of hydroxyapatite (HAP).

[0049] Polypeptide group: The polypeptide is assembled according to the method of Example 2. After the polypeptide assembly is completed, a CaCl2 solution and a phosphorus stock solution are added to the peptide assembly solution to prepare a mineralization solution. The mineralization solution contains 1.67 mM CaCl2, 1 mM Na2HPO4, 0.85×PBS, and 1 mg / mL polypeptide. The mineralization solution is prepared freshly and filtered through a bacteria filter before use. After wrapping with a sealing film, it is incubated at 37 °C for 2 h. 200 μL of the mineralization samples at different time points are dropped onto a copper grid, washed with DIW, dried, and observed under a transmission electron microscope.

[0050] Another negative control group without polypeptide is set up. Incubated in the same way, 200 μL is dropped onto a copper grid, washed with DIW, dried, and observed under a transmission electron microscope.

[0051] The results are as Figure 2 shown. Amorphous calcium phosphate (ACP) appears initially in both the negative control group and the polypeptide group. However, at 1 h, the mineralization samples in the negative control group change from ACP to the typical HAP morphology, while the mineralization samples in the polypeptide group do not show the HAP structure at the same time point, indicating that the polypeptide regulates the initial nucleation process of hydroxyapatite and can stabilize ACP in a short time.

[0052] Example 4

[0053] This example discloses the test for measuring the adsorption force of the polypeptide of the present invention on the demineralized dentin collagen fiber network. The specific process is as follows:

[0054] 1. Preparation of dentin: First, the extracted teeth were made into dentin slices with a thickness of 100 μm to obtain normal dentin.

[0055] 2. Preparation of demineralized dentin: The dentin slices were polished with sandpaper, then etched with 37 wt% phosphoric acid for 30 s, rinsed with DIW, and ultrasonically cleaned for 10 min to obtain completely demineralized dentin.

[0056] 3. Grouping and experiments: In the polypeptide sample group, both normal dentin and completely demineralized dentin were treated with fluorescein isothiocyanate (FITC) and FITC-polypeptide: Approximately 100 μL of FITC-polypeptide solution and FITC solution were respectively added to the surfaces of normal dentin and demineralized dentin, allowed to contact fully for 3 min, then rinsed for 5 min, and the binding of FITC-polypeptide to dentin collagen fibers was observed with a confocal laser scanning microscope (CLSM).

[0057] Another negative control group was set up: Both normal dentin and completely demineralized dentin were only treated with FITC solution.

[0058] In the FITC-polypeptide solution, the polypeptide content was 1 mg / ml and the FITC content was 0.153 mg / ml.

[0059] In the FITC solution, the FITC content was 0.153 mg / ml. 4. Results: As Figure 3 shown, in the polypeptide sample group, after treatment with FITC-polypeptide, the fluorescence was still evenly distributed in the demineralized dentinal tubules ( Figure 3 D), while in the negative control group, only a small amount of fluorescence was scattered in the peritubular dentin after treatment with FITC ( Figure 3 C). It shows that the polypeptide can effectively adsorb the demineralized dentin collagen fiber network and can effectively bind to the demineralized dentinal tubules. This adsorption specificity is beneficial to the closure of dentinal tubules in the application of biomimetic mineralization of demineralized dentin.

[0060] Example 5

[0061] This example discloses the investigation of the in-fiber mineralization effect of the polypeptide of the present invention.

[0062] 1. Preparation of nickel mesh loaded with recombinant collagen: Dissolve commercial type I collagen in 0.1 M acetic acid solution to prepare the original collagen solution (3 mg / mL), and store it at 4 °C. Prepare a collagen assembly solution containing 50 mM glycine and 200 mM KCl (pH 9.2), and use it immediately after preparation. Add 16.7 μL of the original collagen solution to 983.3 μL of the assembly solution, mix by shaking immediately, and incubate at room temperature for 20 min to obtain a recombinant collagen solution with a final concentration of 50 μg / mL. The recombinant collagen after incubation shows slight gelation and no fiber precipitation. Subsequently, place the nickel mesh face up on the wax sheet at the bottom of a six-well plate, drop 14 μL of the recombinant collagen solution onto each nickel mesh, and add DIW to the remaining wells to maintain a humid environment. Seal the well plate with a sealing film, incubate at 37 °C for 24 hours, wash three times with DIW, fix in 0.5 wt% glutaraldehyde solution for 2 h, wash again with DIW, and air dry for the next treatment and characterization.

[0063] 2. Investigation of mineralization within fibers

[0064] Polypeptide group: Treat the nickel mesh loaded with recombinant collagen with 200 μL of a polypeptide solution with a concentration of 1 mg / ml for 24 h, then wash with DIW and air dry. Invert the nickel mesh in a six-well plate containing a PAA mineralization solution, seal it with a sealing film, and incubate at 37 °C.

[0065] Negative control group: Take another nickel mesh loaded with recombinant collagen, directly invert it in a six-well plate containing a PAA mineralization solution, seal it with a sealing film, and incubate at 37 °C as a negative control.

[0066] 3. Detection: Extract the nickel meshes mineralized for different times, rinse with DIW, air dry, observe the collagen mineralization situation under a transmission electron microscope (TEM), select representative areas for imaging, perform selected area electron diffraction (SAED) detection at the same time, and detect the elemental distribution of the mineralized collagen by energy dispersive spectrometer (EDS) surface scanning. The results are as Figure 4 and Figure 5 shown.

[0067] 4. Results

[0068] The TEM and SAED images of the samples mineralized for 1 day are as Figure 4 shown: After 1 day of mineralization, the collagen density in the control group increased, indicating ACP infiltration, and there were no scattered crystals outside the fibers. In the polypeptide group, obvious mineralization within the fibers was achieved, and the SAED images of the mineralized sites also showed characteristic crystal planes of (004), (002), and (211).

[0069] The elemental distribution map of the samples mineralized for 1 day is as Figure 5 shown: No obvious mineralization was observed in the negative control group at 1 day, so no calcium and phosphorus elements were distributed along the collagen fibers. In the polypeptide group, calcium and phosphorus elements were found to aggregate on the collagen at 1 day of mineralization ( Figure 5-3C), and its distribution is consistent with the collagen orientation.

[0070] The above results indicate that the polypeptide of the present invention can accelerate the process of type I collagen biomineralization, thereby accelerating the process of biomineralization of demineralized dentin.

[0071] Example 6

[0072] This example discloses an investigation test on the mineral deposition of the polypeptide of the present invention.

[0073] 1. Preparation of dentin samples: With the consent of the patient, sound third molars (wisdom teeth) were collected. Dentin samples with a thickness of 2 mm were cut perpendicularly to the direction of dentinal tubules from the middle region of the crown of the selected teeth using a diamond blade with continuous water cooling. The sample surfaces were polished successively using SiC papers (1000, 1500, 2000, 3000, and 5000#).

[0074] 2. Acid etching of dentin samples: A 37 wt.% phosphoric acid solution was prepared. 10 mL of the phosphoric acid solution was added to a small beaker, and the prepared dentin slice samples were clamped and immersed in the phosphoric acid solution, sealed with a sealing film, and the solution was changed once every 24 h. After acid etching at 37 °C for 48 h, the samples were taken out. After rinsing thoroughly with deionized water, they were placed in DIW and ultrasonically cleaned for 10 minutes.

[0075] 3. Mineralization: The experiment was set up with a polypeptide group and a control group. Among them, in the polypeptide group, the acid-etched dentin was pretreated with the polypeptide solution and then incubated in the mineralization solution for mineralization; in the control group, the acid-etched dentin was directly incubated in the mineralization solution for mineralization, and n = 15 for each group of samples. Specifically as follows:

[0076] The acid-etched dentin was immersed in the polypeptide solution for 24 hours, taken out, then washed with DIW and air-dried to obtain the dentin pretreated with the polypeptide solution.

[0077] Another portion of acid-etched dentin and the dentin pretreated with the polypeptide solution as described above were respectively immersed in 4 mL of PAA mineralization solution for mineralization, and the mineralization medium was changed once every 24 h. The well plates were sealed with a sealing film and incubated in an oven at 37 °C. After incubation for 3 d and 7 d, dentin samples were randomly taken from each group for further detection.

[0078] The mineralization solution in this example consisted of: 1.67 mM CaCl2, 1 mM Na2HPO4, 0.85×PBS, 100 μg / L PAA (polyacrylic acid, 450 kDa).

[0079] After ultrasonic cleaning of the samples for 20 min, they were placed in ethanol (volume fractions were successively: 30%, 50%, 70%, 90%, 100%) for gradient dehydration. After dehydration was completed, they were placed in hexamethyldisilane for continued fixation for 2 h, and then placed in a fume hood overnight. After the samples were sputter-coated with gold in a vacuum environment for 30 s, they were observed with a scanning electron microscope at an accelerating voltage of 20 keV.

[0080] The results were as Figure 6 shown. In the control group, no obvious mineral deposition was seen ( Figure 6 A1, A2). In the polypeptide group, a large amount of minerals covered the surface of peritubular dentin. Some of these minerals were dispersed on the dentin surface, some were interconnected to form a mineral layer, and a small number of dentinal tubules were also covered by minerals but not completely sealed ( Figure 6 B1, B2).

[0081] The above results indicate that the S-layer protein-derived polypeptide can promote the occurrence of three-dimensional demineralized dentin collagen biomineralization in vitro.

[0082] Example 7

[0083] In this example, the toxicity to HDPCs was evaluated by the CCK-8 method.

[0084] The cytotoxicity of the S-layer protein-derived polypeptide was evaluated using human dental pulp cells (HDPCs), and the cell viability was measured by the CCK-8 method. Solutions of S-layer protein-derived polypeptide at different concentrations (1, 3, 5 mg / mL) were prepared. First, the HDPCs cell suspension was inoculated into a 96-well plate, with 5×10 4 cells per well, and cultured at 37 °C overnight to allow the cells to adhere. Six replicate wells were set for each concentration. After adhesion, they were washed with PBS, and the cells were placed in DMEM medium containing S-layer protein-derived polypeptide at different concentrations (1, 3, 5 mg / mL) and incubated for 1, 3, 5 d. Then, 10 μL of CCK-8 reagent and 90 μL of medium were added to each well and incubated at 37 °C for 2 h. 90 μL of supernatant was aspirated from each well, and the absorbance at 450 nm was detected with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0085] After co-culturing different concentrations of polypeptide with HDPCs, the cell survival rate was as Figure 7 shown. The CCK-8 results showed that after HDPCs were co-cultured with polypeptides at different concentrations (1, 3, 5 mg / mL) for 1, 3, 5 d, the cell survival rate was higher than 95%, indicating no obvious cytotoxicity.

[0086] The above are only the preferred embodiments of the present invention, which are illustrative rather than restrictive to the present invention; those of ordinary skill in the art understand that many changes, modifications, and even equivalent changes can be made to it within the spirit and scope defined by the claims of the present invention, but all will fall within the protection scope of the present invention.

Claims

1. A polypeptide, polypeptide derivative, or pharmaceutically acceptable salt or ester of a polypeptide derived from an S-layer protein, characterized in that, The amino acid sequence of the polypeptide is as follows: PVTVNVDNVTPAAAQETTKTV; The polypeptide derivative is the polypeptide with its C-terminus amidated; The pharmaceutically acceptable salts are hydrochloride, sulfate, acetate, mesylate, succinate, fumarate, citrate, malate or organic amine salts.

2. A composition, characterized in that, Containing the polypeptide, polypeptide derivative or pharmaceutically acceptable salt, ester of the polypeptide as claimed in claim 1, and pharmaceutically acceptable excipients.

3. Use of the polypeptide, polypeptide derivative or pharmaceutically acceptable salt or ester of the polypeptide according to claim 1, characterized in that, Use in the preparation of oral care products; the oral care products are oral care products having the effect of promoting dentin mineralization or / and repairing demineralized dentin or / and improving dentin sensitivity.

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