A camel blood serum polypeptide and its application in preparing a product for promoting osteoblast proliferation and / or differentiation

Abstract

The application discloses a camel blood serum polypeptide and application thereof in preparation of a product for promoting proliferation and / or differentiation of osteoblasts, and the amino acid sequence of the camel blood serum polypeptide is SEPSGTAFGGR, DEFF or EFL. Based on a camel blood serum peptide segment identified by LC-MS / MS, the target polypeptide is obtained from camel blood serum related polypeptides through computer virtual screening, and the MC3T3E1 cell is used for standardization of the proliferation activity of the osteoblasts. It is found through experiment verification that the camel blood serum polypeptide has significant proliferation and differentiation activity of the osteoblasts, and can be used as an active substance for promoting osteogenesis and used for bone tissue repair.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide technology, specifically relating to a camel blood serum polypeptide that promotes osteoblast proliferation and / or differentiation and its applications. Background Technology

[0002] Bone defects, osteoporosis, and other bone-related diseases seriously affect human health, and there is an urgent clinical need for safe and efficient osteogenic substances for bone tissue repair. Peptides, due to their small molecular weight, high bioactivity, low toxicity, and ease of synthesis, have significant advantages in regulating osteoblast proliferation and differentiation, making them a research hotspot in the field of osteogenic development. However, existing research largely focuses on artificially synthesized peptides or peptide mimics with known sequences. Artificially synthesized peptides face two major bottlenecks: first, poor biocompatibility, as their chemical synthesis process may introduce toxic solvents or fail to perfectly mimic natural conformations, leading to reduced in vivo activity or toxic side effects; second, limited sequence sources, often based on fragments of known functional proteins, making it difficult to break through existing cognitive frameworks and discover bioactive peptides with novel mechanisms of action.

[0003] As mammals surviving in extreme environments, Bactrian camels have developed a unique bioactive molecular system in their serum through long-term evolution. Camel blood serum is not only rich in albumin and immunoglobulins, but also contains a large number of low-molecular-weight natural bioactive peptides. These peptides are naturally derived, have excellent biocompatibility, and are safer than synthetic peptides, showing broad application prospects in the biomedical field. However, despite the abundance of camel blood resources, the complexity of isolation and identification techniques means that systematic screening and functional studies of specific osteogenic peptides in camel blood serum still need further development.

[0004] MC3T3-E1 mouse pre-osteoblasts are a recognized classic in vitro evaluation model in the field of bone metabolism and bone repair. Their proliferation activity, osteogenic differentiation process, and matrix mineralization capacity are highly correlated with in vivo bone formation, bone density maintenance, bone defect repair, and prevention and treatment of osteoporosis. Existing studies have fully demonstrated that regulating the proliferation and osteogenic differentiation of MC3T3-E1 cells can directly increase alkaline phosphatase (ALP) activity, promote type I collagen synthesis and calcium nodule deposition. This in vitro effect has a clear and stable positive correlation with the improvement of bone trabecular structure, bone density, osteoporosis relief, and accelerated bone defect healing in animals. For example, active substances that promote osteogenic differentiation of MC3T3-E1 cells can significantly improve bone microstructure and increase bone mass in an ovariectomy osteoporosis model; in a bone defect model, the osteogenic activity of MC3T3-E1 cells can be directly converted into in vivo new bone formation and defect repair capacity. Therefore, using MC3T3-E1 cell proliferation and osteogenic differentiation activity as evaluation indicators can reliably reflect the potential functions of active substances in promoting bone growth, accelerating bone repair, and preventing osteoporosis (Reference: Gibon, Emmanuel, et al. "MC3T3-E1 osteoprogenitor cells systemically migrate to a bone defect and enhance bone healing"). Tissue Engineering Part A 18.9-10(2012): 968-973; Yu, Wei, et al. "LGR5 enhances the osteoblastic differentiation of MC3T3-E1 cells through the Wnt / β-catenin pathway." Experimental and Therapeutic Medicine 22.2 (2021): 889.). Summary of the Invention

[0005] This application utilizes camel blood serum peptides identified by LC-MS / MS to directionally obtain target peptides from camel blood serum-related peptides through computer-based virtual screening. High-purity peptides are prepared by optimizing solid-phase synthesis parameters. Standardized osteoblast proliferation-promoting activity is verified using MC3T3E1 cells. Verification revealed that some peptides exhibit significant osteoblast proliferation and differentiation-promoting activity, providing novel natural candidate peptides and technical support for the treatment of bone-related diseases. Based on this, this application provides a camel blood serum peptide and its application in the preparation of products promoting osteoblast proliferation and / or differentiation.

[0006] A camel blood serum polypeptide, wherein the amino acid sequence of the camel blood serum polypeptide is SEPSGTAFGGR (SEQ ID NO:1), DEFF (SEQ ID NO:2), or EFL.

[0007] More preferably, the amino acid sequence of the camel blood serum polypeptide is SEPSGTAFGGR.

[0008] This application also provides the use of the camel blood serum polypeptide in the preparation of an agent that promotes osteoblast proliferation and / or differentiation.

[0009] Optionally, the osteoblasts are MC3T3E1 cells.

[0010] Optionally, the formulation is used to prepare a bone tissue repair drug.

[0011] Optionally, the bone tissue repair drug includes bone repair gel or bone-promoting agent. The bone-promoting agent may be an injectable bone-promoting agent.

[0012] This application also provides an agent that promotes osteocyte proliferation and / or differentiation, comprising a therapeutically effective amount of the camel blood serum polypeptide and a pharmaceutically acceptable carrier or excipient.

[0013] This application also provides the use of the camel blood serum polypeptide in the preparation of health products that improve bone density.

[0014] This application also provides the application of the camel blood serum polypeptide in the preparation of functional foods that improve bone density.

[0015] This application also provides a health product or functional food for improving bone density, comprising an effective dose of the camel blood serum polypeptide and a food-grade acceptable carrier or excipient.

[0016] Optionally, the screening, synthesis, verification, and application of the camel blood serum peptides include the following steps: (1) After deduplication and content screening of the camel blood serum peptides identified by LC-MS / MS, potential bioactive peptides were obtained by sequentially screening using PeptideRanker scoring, ToxinPred toxicity screening, AllerTop 2.1 sensitization screening, and Innovagentool water solubility screening. DS software was used to perform molecular docking screening of the potential bioactive peptides with EGFR (PDB ID: 1 IVO, resolution 3.3 Å) as the receptor. Finally, three target peptides from camel blood serum—DEFF, SEPSGTAFGGR, and EFL—were identified as non-cytotoxic and possessing high potential for promoting osteoblast proliferation. Targeted screening of osteogenic peptides from camel blood serum reduced screening costs and randomness, improved the hit rate of target peptides, and realized the high-value utilization of camel blood resources.

[0017] (2) For the three target peptides (DEFF, SEPSGTAFGGR, and EFL) obtained from camel blood serum screening, an Fmoc / tBu solid-phase synthesis strategy was used for synthesis and purification, with a required peptide purity of ≥95%. After synthesis and purification, the obtained peptides were identified by RP-HPLC and mass spectrometry, confirming that the molecular weight deviation from the theoretical value of the three peptides was ≤0.5 Da and the purity was ≥96%, meeting the experimental and application requirements. The standardized Fmoc / tBu solid-phase synthesis strategy, combined with subsequent rigorous testing, ensured that the peptide purity was ≥95%, meeting the needs of subsequent applications.

[0018] (3) MC3T3E1 cells were resuscitated and cultured into groups, and treated with conventional culture medium, osteogenic induction solution, and osteogenic induction solution + different concentrations of camel blood serum target peptides, respectively, for 5 days. The cell proliferation capacity and alkaline phosphatase (ALP) staining of each group were detected, with a focus on verifying the promoting effect of the peptides on osteoblast proliferation and differentiation. Based on the test results, the osteoblast proliferation-promoting activity and optimal concentration of the target peptides were determined. Using MC3T3E1 cells as a model, a standardized verification process was established to objectively and accurately evaluate the osteoblast proliferation and differentiation-promoting activity of the peptides.

[0019] (4) The camel blood serum polypeptide that was verified in step 3 and has a significant effect on promoting osteoblast proliferation was used to prepare bone tissue repair drugs or health products. The entire screening, synthesis and verification process is complete and highly practical, and has important clinical and industrialization value. Attached Figure Description

[0020] Figure 1 The total ion chromatogram (TIC) of the sample in Example 1; Figure 2 A schematic diagram of the molecular docking of the target peptide DEFF in camel blood serum with EGFR; Figure 3 A schematic diagram of the molecular docking of the target peptide SEPSGTAFGGR in camel blood serum with EGFR. Figure 4 A schematic diagram of the molecular docking of the target peptide EFL in camel blood serum with EGFR; Figure 5 The mass spectrum of DEFF is shown in Figure 1. Figure 6 The mass spectrum of SEPSGTAFGGR is shown. Figure 7 This is the first-order mass spectrum of EFL; Figure 8The following is a graph showing the effect of different concentrations of camel blood serum target peptides on the proliferation rate of MC3T3E1 cells as detected by CCK-8 assay in Example 3 (where a is the relative cell viability of the DEFF treatment group at different concentrations, b is the relative cell viability of the SEPSGTAFGGR treatment group at different concentrations, and c is the relative cell viability of the EFL treatment group at different concentrations). Figure 9 The image shows the ALP staining results of MC3T3E1 cells in each group in Example 3. Detailed Implementation

[0021] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0023] The following is a description using specific embodiments: Example 1 1. Peptide sequence analysis based on LC-MS / MS, the specific process includes: (1) Protein extraction: The camel blood sample was centrifuged for 10 min at 4℃ and 12000 rcf in a 10 kD ultrafiltration tube; then the collected material was taken and the detection head was washed twice with 2 μL of pure water; the sample was tested, with a sample volume of 2 μL, and the data at 280 nm were measured.

[0024] (2) Reductive alkylation: Take 50 μL of small peptide sample into a 1.5 mL EP tube, add 50 μL of 50 mM ammonium bicarbonate buffer (pH 8.0), dilute the small peptide sample to 100 μL, add 1 µL of 1 mol / L DTT (dithiothreitol) solution to make the final DTT concentration 10 mmol / L, and reduce in a 56℃ water bath for 1 h; then add 2 μL of 1 mol / L IAA (iodoacetamide) solution to make the final IAA concentration 20 mmol / L, and react in the dark at room temperature for 40 min; finally add 1 µL of 1 mol / L DTT solution to make the final DTT concentration 10 mmol / L to neutralize the excess IAA; and desalt with C18 stage-tip and dry under vacuum at 45℃.

[0025] (3) Liquid chromatography conditions: µPAC NEO HPLC column, high throughput analytical column; mobile phase A is 0.1% FA (formic acid), mobile phase B is 0.1% FA and 80% ACN (acetonitrile); flow rate is 2.5 μL / min; analysis time for each component: 6.9 min.

[0026] The specific chromatographic conditions are as follows: Table 1. Liquid Chromatography Analysis Conditions for Camel Blood Peptide Sequences

[0027] (4) Mass spectrometry conditions The full scan range of mass spectrometry was 100-1500 m / z. The primary mass spectrometry resolution was set to 240,000, AGC was set to Custom, and Maximum IT was set to Custom. Progenitor ions with an ion intensity of 20 in the full scan were fragmented using high-energy collisional fragmentation (HCD) and detected by secondary mass spectrometry. AGC was set to Custom, Maximum IT was set to Custom, and the peptide fragmentation collision energy was set to 28. Raw mass spectrometry data (.raw) was generated, and the results are as follows. Figure 1 As shown.

[0028] 2. Computer-aided virtual screening of target peptides in camel blood serum that promote osteoblast proliferation and differentiation, the specific steps of which are as follows: 2.1 Preliminary peptide screening: The camel blood serum peptides identified by LC-MS / MS in the previous step were summed after deduplication. Peptides modified by phosphorylation or dephosphorylation at different amino acid positions were considered as the same peptide, and their relative content was calculated based on their area. In order to study the main functional parts in the enzymatic hydrolysate, peptides with a content percentage of 0.01% or higher were screened, and 271 peptides were obtained from 503 peptides. The PeptideRanker tool was used to calculate the score of these 271 peptides, and 42 peptides with a score >0.5 were selected.

[0029] 2.2 Screening for peptide toxicity and sensitization: Peptide toxicity is a key concern in the development of functional food ingredients. The ToxinPred tool was used to predict the toxicity of the 42 peptides. An SVM (Support Vector Machine) prediction method with a threshold of 0.0 was used to separate toxic and non-toxic peptides, resulting in 41 non-toxic peptides. The AllerTop 2.1 tool was then used to predict the sensitization potential of these 41 non-toxic peptides. "NON-ALLERGEN" indicates no allergenicity, meaning the peptide can be used for further experiments (requiring a sequence length of at least 6 amino acids). Thirteen probable ALLERGEN sensitizing peptides were removed, leaving 28. Finally, Innovagentool was used to screen for 15 potentially bioactive peptides with good water solubility.

[0030] 2.3 Molecular docking screening: The DS software was used to perform molecular docking on the above 15 water-soluble potential bioactive peptides. The specific process is as follows: ① Peptide structure processing (15 peptides): Input the sequence of each peptide, use the CHARMm force field to minimize energy, then perform Prepare Ligands processing, adjust the pH to 7.5-8.5 for ionization, and select an appropriate ionization state; ② EGFR crystal structure processing: Obtain the crystal structure of EGFR (PDB ID: 1IVO, resolution 3.3 Å), perform Prepare Protein processing, retaining only one chain; perform Define Receptor processing, using the From Receptor Cavities method, select the sphere, right-click on attributes of SBD sites, and select to move the radius to encompass the entire receptor active pocket; perform Dock Ligands processing using the CDCKER method, with the following parameters: input 15 ligand peptides, Top Hits-PoseCluster Radius 0.5, Parallel Processing True, Server-Processes 10.

[0031] 3.4 Screening result verification and target peptide identification: The docking results showed that 8 out of 15 potential bioactive peptides were successfully docked, namely DFF, DEFF (SEQ ID NO:2), SEPSGTAFGGR (SEQ ID NO:1), EFL, FKI, PIPEPLNR (SEQ ID NO:3), CKL, and MVDEAESEEDLDFKGAHASKRVL (SEQ ID NO:4). Based on the physicochemical properties, water solubility, and osteogenic potential of the peptides, DEFF, SEPSGTAFGGR, and EFL were finally identified as the target osteogenic peptides from camel blood serum that are non-cytotoxic and have high potential for promoting osteoblast proliferation (Table 2).

[0032] The molecular docking results of DEFF, SEPSGTAFGGR, and EFL are as follows: Figures 2-4 As shown.

[0033] Table 2 Physicochemical properties and affinity of peptides in camel blood serum

[0034] Example 2 Solid-phase synthesis of target peptides from camel blood serum This embodiment employs the Fmoc / tBu solid-phase synthesis strategy to achieve the solid-phase synthesis, purification, and identification of the camel blood serum target peptides screened in Example 1. The specific steps are as follows: Synthesis: Peptides DEFF, SEPSGTAFGGR, and EFL were prepared using the Fmoc solid-phase synthesis method by Shenzhen Borunsida Biotechnology Co., Ltd., China. The first amino acid was anchored to Wang resin and swollen with dichloromethane; subsequently, it was coupled with Fmoc-protected amino acids, DIC, HBTU, and DMAP. The Fmoc protecting groups were removed using a 20% piperidine / DMF solution, and stepwise condensation was performed sequentially according to the above peptide sequence using a semi-automatic peptide synthesizer. At each step, a 3-molar excess of amino acid was added. The condensation reaction was carried out at room temperature for 20 min, and the deprotection reaction for 15 min. Finally, the peptide resin was cleaved and deprotected using a TFA-TIS-H2O system, and the crude peptide was obtained by centrifugation.

[0035] Purification: The crude peptides were purified by high-performance liquid chromatography (HPLC) using a Kromasil C18 column (5 μm, 250 × 4.6 mm). Mobile phase A was 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was 0.1% trifluoroacetic acid acetonitrile solution. A gradient elution program was used: at 0.01 min, the A / B ratio was 83% / 17%, which became linear at 25 min, changing to 58% / 42%; at 25.1 min, the B phase ratio increased to 100% and remained there until 30 min. The elution flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the injection volume was 10 μL. The purified peptides were identified using an Agilent 6125B mass spectrometer in ESI positive ion mode, and the purity of all target peptides was greater than 95%.

[0036] After synthesis and purification, the samples were identified and analyzed using RP-HPLC and mass spectrometry. For RP-HPLC, a C18 column was used with acetonitrile / water (containing 0.1% TFA) as the mobile phase. Gradient elution conditions were 0-30 min, with the acetonitrile concentration increasing from 10% to 40%. Mass spectrometry was used to detect molecular weight deviation, and HPLC was used to determine purity. The primary mass spectra of DEFF, SEPSGTAFGGR, and EFL are shown below. Figures 5-7 As shown, the results indicate that the molecular weight of the three peptides deviates from the theoretical value by ≤0.5 Da, and the purity is ≥96%, which meets the requirements for experiments and subsequent applications.

[0037] Example 3 Verification of the effect of target peptides in camel blood serum on promoting osteoblast proliferation and differentiation This embodiment uses MC3T3E1 cells to verify the osteocyte proliferation and differentiation-promoting effect of the camel blood serum target polypeptide synthesized in Example 2. The specific steps are as follows: 3.1 Cell Culture and Grouping: After MC3T3E1 cells were revived, they were cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin and antibiotics, and placed in a 37°C, 5% CO2 incubator. Logarithmic growth phase cells were seeded into 6-well plates and cultured until the confluence was appropriate. The cells were then divided into a control group, a positive control group, and an experimental group (3 camel blood serum target peptides, 5 concentration groups in each group).

[0038] 3.2 Experimental grouping: Control group: standard α-MEM medium (M1370044, purchased from Aladdin); Positive control group: α-MEM medium containing 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid, and 100 nmol / L dexamethasone; Experimental group: α-MEM medium containing 10 mmol / L sodium β-glycerophosphate, 50 μg / mL ascorbic acid, 100 nmol / L dexamethasone (osteogenic induction solution), and target peptides from camel blood serum at concentrations of 1 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL was added; each group had 3 replicates, with the medium changed every 2 days, and cultured for 5 days.

[0039] 3.3 Detection of indicators to promote osteoblast proliferation and activity: 3.3.1 Osteoblast proliferation detection: Cell proliferation rates were assessed using the CCK-8 assay at 24 and 48 hours of culture. The effect of different concentrations of camel blood serum target peptides on MC3T3E1 cell proliferation rates was measured using the CCK-8 assay at 24 hours. Figure 8 As shown, a represents the relative cell viability of different concentrations of DEFF treatment groups, b represents the relative cell viability of different concentrations of SEPSGTAFGGR treatment groups, and c represents the relative cell viability of different concentrations of EFL treatment groups.

[0040] The results showed that after 24 hours of culture, the second polypeptide (SEPSGTAFGGR) at a concentration of 1 μg / mL was significantly different from the control group (P<0.01). Although the other concentration groups and the other two polypeptides did not show significant differences from the control group, they all showed obvious trends of proliferation, especially in the concentration range of 1~50 μg / mL, all of which showed a certain degree of osteocyte proliferation promotion. However, under high concentration conditions, especially at a concentration of 200 μg / mL, all polypeptides showed cell proliferation inhibition effects. This result is consistent with the conclusion in existing literature that "high concentrations of polypeptides may have certain toxic effects on cells".

[0041] 3.3.2 ALP staining detection: After culturing for 5 days, cells in each group were stained using an ALP staining kit. After staining, the cells were observed and photographed under a microscope. The results are as follows: Figure 9As shown in the figure, Control is the control group, ODM is the positive control group, P1 (DEFF) 50 μg / mL is the 50 μg / mL DEFF addition group, P1 (DEFF) 200 μg / mL is the 200 μg / mL DEFF addition group, P2 (SEPSGTAFGGR) 50 μg / mL is the 50 μg / mL SEPSGTAFGGR addition group, P2 (SEPSGTAFGGR) 200 μg / mL is the 200 μg / mL SEPSGTAFGGR addition group, P3 (EFL) 50 μg / mL is the 50 μg / mL EFL addition group, P3 (EFL) 200 μg / mL is the 200 μg / mL EFL addition group.

[0042] The staining images show that the ALP staining effect in each experimental group was better than that in the control group, indicating that the target peptide has a certain promoting effect on the ALP activity of osteoblasts.

[0043] 3.4 Result Determination: Tests showed that the target peptides (DEFF, SEPSGTAFGGR, and EFL) in the serum of the three camel blood samples all had certain effects on promoting osteoblast proliferation and osteoblast differentiation. Among them, the SEPSGTAFGGR peptide showed the best effect at a concentration of 1 μg / mL and can be used as a novel candidate peptide for promoting osteoblast development.

[0044] Example 4 Application of target peptides in camel blood serum This embodiment is used to implement the application of the camel blood serum target peptide that passed the verification in Example 3. The specific steps are as follows: The camel blood serum target peptides (DEFF, SEPSGTAFGGR, EFL) that were verified as qualified in Example 3 and have the effect of promoting osteoblast proliferation were used to prepare bone tissue repair drugs (such as bone repair gels, injectable osteoproliferating preparations) or health products (such as calcium-supplemented bone-promoting dietary supplements) to realize their application in the field of bone tissue repair.

[0045] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

1. A camel blood serum polypeptide, characterized in that, The amino acid sequence of the camel blood serum polypeptide is SEPSGTAFGGR, DEFF, or EFL.

2. The camel blood serum polypeptide according to claim 1, characterized in that, The amino acid sequence of the camel blood serum polypeptide is SEPSGTAFGGR.

3. The use of the camel blood serum polypeptide as described in claim 1 or 2 in the preparation of formulations that promote osteoblast proliferation and / or differentiation.

4. The application according to claim 3, characterized in that, The osteoblasts were MC3T3E1 cells.

5. The application according to claim 3, characterized in that, The formulation is used to prepare a bone tissue repair drug.

6. The application according to claim 5, characterized in that, The bone tissue repair drugs include bone repair gels or bone-promoting preparations.

7. An agent that promotes osteoblast proliferation and / or differentiation, characterized in that, It includes a therapeutically effective amount of the camel blood serum polypeptide as described in claim 1 or 2, as well as a pharmaceutically acceptable carrier or excipient.

8. The use of the camel blood serum polypeptide as described in claim 1 or 2 in the preparation of health products or functional foods that improve bone density.

9. A health product or functional food for improving bone density, characterized in that, It includes an effective amount of the camel blood serum polypeptide as described in claim 1 or 2, as well as a food-grade acceptable carrier or excipient.

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