A bovine bone collagen peptide for promoting osteoblast mineralization and its application

Abstract

The present invention discloses a bovine bone collagen peptide that promotes osteoblast mineralization and its application. The amino acid sequence of the bovine bone collagen peptide of the present invention is shown in SEQ ID NO: 1 - SEQ ID NO: 6. The bovine bone collagen peptide provided by the present invention can effectively promote osteoblast mineralization, prevent and / or treat osteoporosis, relieve the pain of osteoporosis patients, and these polypeptides have the characteristics of high efficiency and low toxicity and can be developed into drugs.

Description

[0001] This divisional application is based on an invention application filed by the applicant on March 23, 2021, with the application number 202110308414.0 and the invention title "A Bovine Collagen Peptide for Promoting Osteoblast Mineralization and Its Application". Technical Field

[0002] The present invention relates to the technical field of bioactive peptides. More specifically, it relates to a bovine collagen peptide for promoting osteoblast mineralization and its application. Background Art

[0003] Bone remodeling occurs throughout a person's life. Bone remodeling mainly includes two processes: bone formation and bone resorption. Once the rate of bone formation is less than the rate of bone resorption, osteoporosis may occur. Osteoporosis is a systemic disease characterized by reduced bone density. The bone mineralization process can directly affect the amount of bone density. Therefore, finding highly efficient polypeptides that are beneficial to the bone mineralization process is of great significance for the development of anti-osteoporosis drugs. Summary of the Invention

[0004] The first object of the present invention is to provide a bovine collagen peptide with high activity and low toxicity for promoting osteoblast mineralization.

[0005] The second object of the present invention is to provide the application of the above bovine collagen peptide in promoting osteoblast mineralization, preventing and / or treating osteoporosis.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] According to the first object of the present invention, the present invention provides a bovine collagen peptide, and the amino acid sequence of the bovine collagen peptide includes one or more of the following sequences:

[0008] LPGIDGRPGPIGPAG, wherein the proline at position 2 is hydroxylated to form hydroxyproline, as shown in SEQ ID NO:1;

[0009] GLTGPIGPPGPAG, wherein the proline at position 9 is hydroxylated to form hydroxyproline, as shown in SEQ ID NO:2;

[0010] LPGIDGRPGPIGPAG, wherein the prolines at positions 2 and 8 are hydroxylated to form hydroxyproline, as shown in SEQ ID NO:3;

[0011] APGPVGPVG, wherein the proline at position 2 is hydroxylated to form hydroxyproline, as shown in SEQ ID NO:4;

[0012] GPAGPSGPAG, as shown in SEQ ID NO:5;

[0013] GAPGSPGPAG, as shown in SEQ ID NO:6.

[0014] Optionally, the bovine bone collagen peptide may also be an active peptide having 80% or more homology with the amino acid sequence of any of the above bovine bone collagen peptides, and the functions of this bovine bone collagen peptide and the above bovine bone collagen peptides are the same or similar. For example, the amino acid sequence of the bovine bone collagen peptide may be a sequence having 85%, 90%, 95% or 97% homology with the amino acid sequence of any of the above bovine bone collagen peptides.

[0015] The present invention also provides a polynucleotide encoding the above bovine bone collagen peptide.

[0016] In the case of knowing the amino acid sequence of the bovine bone collagen peptide, those skilled in the art can design a coding gene for the bovine bone collagen peptide with different nucleotide sequences based on the degeneracy principle of codons and the codon usage preferences of different species according to the need for the expression of the bovine bone collagen peptide.

[0017] In the present invention, by way of example, the nucleotide sequences of the above bovine bone collagen peptides are as shown in SEQ ID NO:7 - SEQ ID NO:12 in the sequence listing.

[0018] Furthermore, the present invention provides a fusion polypeptide, which comprises one of the above bovine bone collagen peptides. The fusion polypeptide can be obtained by chemical binding, enzymatic cleavage or physical fragmentation methods. For example, a protein complex formed by a polypeptide and ovalbumin or bovine serum albumin.

[0019] The bovine bone collagen peptide of the present invention can be obtained by separation and purification from the enzymatic hydrolysis product of bovine bone collagen, or can also be prepared by other methods known in the art, such as solid-phase synthesis methods, bioengineering methods, and the like.

[0020] According to the second object of the present invention, the present invention provides the application of the bovine bone collagen peptide in a drug for promoting osteoblast mineralization. Furthermore, the application of the bovine bone collagen peptide in the preparation of a drug for preventing and / or treating osteoporosis is provided.

[0021] The present invention further relates to a drug containing the bovine bone collagen peptide of the present invention, such as LPGIDGRPGPIGPAG (proline at position 2 is hydroxylated to form hydroxyproline), GLTGPIGPPGPAG (proline at position 9 is hydroxylated to form hydroxyproline), LPGIDGRPGPIGPAG (prolines at positions 2 and 8 are hydroxylated to form hydroxyproline), APGPVGPVG (proline at position 2 is hydroxylated to form hydroxyproline), GPAGPSGPAG, GAPGSPGPAG, or one or more of them.

[0022] The drug of the present invention can be made into various common forms in the art, including but not limited to powders suitable for oral administration, tablets (including various coated tablets, sustained-release or controlled-release tablets), lozenges, capsules (including soft capsules and hard capsules), granules, pills, dispersible powders, aqueous or oily suspensions, aqueous or oily solutions, emulsions, elixirs, syrups, etc.; creams, ointments, gels, aqueous or oily solutions, aqueous or oily suspensions, etc. suitable for topical use; powder or liquid aerosols suitable for inhalation; sterile aqueous or oily injections or lyophilized powder injections, suppositories, etc. suitable for parenteral administration.

[0023] The drug of the present invention can further contain various conventional excipients and / or other active ingredients. Suitable excipients include but are not limited to excipients, lubricants, binders, disintegrants, water-soluble polymers, inorganic salts, solvents, solubilizing agents, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, sweeteners, acidulants, foaming agents, and flavoring agents, etc.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention uses the promotion of osteoblast mineralization as the active screening index, and successively adopts reverse-phase silica gel column chromatography, ion-exchange column chromatography, and gel column chromatography, and finally screens out the component with the strongest activity, and uses a high-performance liquid chromatography-mass spectrometry instrument to identify the amino acid sequence of the component with the strongest activity. The bovine bone collagen peptide obtained by the present invention can effectively promote osteoblast mineralization, prevent and / or treat osteoporosis, and relieve the pain of osteoporosis patients, and these polypeptides have the characteristics of high efficiency and low toxicity. Description of the Drawings

[0026] The following further describes in detail the specific embodiments of the present invention with reference to the drawings.

[0027] Figure 1 Shows the fast reverse-phase column chromatography chromatogram of the ultrafiltration component CP1.

[0028] Figure 2The osteoblast mineralization activity test results of 6 components obtained by subjecting the ultrafiltration component to fast reversed-phase silica gel column chromatography are shown.

[0029] Figure 3 The ion exchange chromatogram of the fast reversed-phase silica gel column chromatography components CP1-5 is shown.

[0030] Figure 4 The gel column chromatogram of the ion exchange column chromatography components CP1-5-1 is shown.

[0031] Figure 5 The gel column chromatogram of the ion exchange column chromatography components CP1-5-2 is shown.

[0032] Figure 6 The osteoblast mineralization activity test results of 4 components obtained by subjecting the CP1-5-1 and CP1-5-2 components to gel column chromatography are shown.

[0033] Figure 7 The osteoblast mineralization deposition test results of 6 kinds of bovine bone collagen peptides of the present invention are shown.

[0034] Figure 8 The osteocalcin content test results in the supernatant of osteoblasts of 6 kinds of bovine bone collagen peptides of the present invention are shown. Detailed implementation manners

[0035] To more clearly illustrate the present invention, the present invention will be further described below in conjunction with preferred embodiments and the accompanying drawings. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0036] Example 1 Preparation of bovine bone collagen peptide

[0037] According to the content disclosed in Chinese Patent Application CN 105586379 A "A preparation method of a collagen active peptide with the effect of inhibiting cancer cell proliferation", the ultrafiltration component CP1 is obtained, that is, steps (1)-(8) of the preparation method disclosed in this patent application are completed.

[0038] RP-HPLC: The ultrafiltration fraction CP1 sample was dissolved in 1 mL of ultrapure water to prepare a 10 mg / mL solution. The solution was centrifuged and filtered through a 0.22 μm disposable filter membrane. An ODS-AQ RP-HPLC column (50 μm, 2.5×100 mm) was used to separate and purify the bioactive peptides. The sample loading volume was 1 mL, and the eluents were ultrapure water (solution A) and chromatographic ethanol (solution B). The flow rate was 5 mL / min. The elution gradient was as follows: 0 - 10 min, 100% A; 10 - 30 min, 96% A; 30 - 50 min, 90% A; 50 - 70 min, 87% A; 70 - 75 min, 83% A; 75 - 85 min, 78% A; 85 - 97 min, 0% A. The detection wavelength was 220 nm. A total of 6 fractions (CP1-1 - CP1-6) were obtained, lyophilized, and stored at -80 °C in a low-temperature refrigerator for further use. The results are shown in Figure 1 as follows. The osteoblast mineralization activity of the 6 fractions was tested. The results are shown in Figure 2 as follows. CP1-5 could significantly promote osteoblast mineralization. Therefore, this fraction was selected for further purification.

[0039] Ion exchange column chromatography: 5 mg of the fraction with the strongest activity (CP1-5) obtained from RP-HPLC was dissolved in 100 μL of ultrapure water, centrifuged, and filtered through a 0.22 μm disposable filter membrane. An ion exchange column was used to separate and purify the bioactive peptides. The sample loading volume was 100 μL, and the eluents were ultrapure water (solution A) and 1 M sodium chloride solution (solution B). The flow rate was 1 mL / min. The elution gradient was as follows: 0 - 100% B solution (0 - 1 M) for 60 min. The detection wavelength was 220 nm. A total of 2 fractions (CP1-5-1 and CP1-5-2) were obtained, lyophilized, and stored at -80 °C in a low-temperature refrigerator for further use. The results are shown in Figure 3 as follows.

[0040] Gel column chromatography: Approximately 2 mg of the fractions CP1-5-1 and CP1-5-2 obtained from ion exchange column chromatography were separately dissolved in 100 μL of ultrapure water, centrifuged, and filtered through a 0.22 μm disposable filter membrane. A Sephadex G-15 gel column was used to separate and purify the bioactive peptides. The sample loading volume was 100 μL. The eluent was ultrapure water. The flow rate was 0.8 mL / min. The chromatographic conditions were as follows: elution with 100% ultrapure water for 50 min. The detection wavelength was 220 nm. A total of 4 fractions (CP1-5-1-A and CP1-5-1-B; CP1-5-2-A and CP1-5-2-B) were obtained, lyophilized, and stored at -80 °C in a low-temperature refrigerator for further use. The results are shown in Figure 4 and Figure 5 as follows. The osteoblast mineralization activity of the 4 fractions was tested. The results are shown in Figure 6The shown CP1-5-2-B can significantly promote osteoblast mineralization.

[0041] Identification of bioactive amino acid sequences

[0042] Component CP1-5-2-B has the strongest biological activity. Therefore, LC-MS-MS liquid chromatography-mass spectrometry analysis was performed on this component, and data analysis was carried out using the proteomics software Peaks Studio. A total of 6 active peptides (bovine bone collagen peptides) were identified, and the results are shown in Table 1.

[0043] Table 1 Sequences of bovine bone collagen peptides identified from bovine bone collagen hydrolysate

[0044] Number Bull stock collagen peptide sequence Mass-to-charge ratio Theoretical molecular weight Length (amino acids) Parent protein 1 LPGIDGRPGPIGPAG 695.3779 1388.7411 15 Collagen alpha-2(I)chain 2 GLTGPIGPPGPAG 553.796 1105.5768 13 Collagen alpha-1(I)chain 3 LPGIDGRPGPIGPAG 703.3752 1404.7361 15 Collagen alpha-2(I)chain 4 APGPVGPVG 738.3782 737.3708 9 Collagen alpha-1(I)chain 5 GPAGPSGPAG 767.3674 766.3607 10 Collagen alpha-2(I)chain 6 GAPGSPGPAG 767.3674 766.3607 10 Collagen alpha-1(Ill)chain

[0045] P : indicates hydroxylation modification

[0046] Example 2

[0047] Based on the amino acid sequences of the 6 bovine bone collagen peptides identified in Example 1, experimental samples were obtained by solid-phase synthesis method to verify the osteoblast mineralization activity. As Figure 7 、 Figure 8 shown, the 6 bovine bone collagen peptides (numbered 1-6) can all promote the formation of osteoblast mineralization deposits and can increase the content of osteocalcin in osteoblasts, indicating that the 6 collagen peptides can all promote osteoblast mineralization. Solid-phase synthesis peptide company: Gil Biochemical (Shanghai) Co., Ltd.

[0048] Detection of cell mineralization deposition:

[0049] First, 50-100 mouse MT3T3-E1 cells were seeded in each well of a 24-well plate. After 24 hours when the cells adhered to the wall, the culture medium was aspirated, and 6 polypeptides (concentration 0.05 mg / mL) and three osteogenic inducers (dexamethasone 10 nmol, β-glycerophosphate 10 mmol, ascorbic acid 5 μg / ml) were added. A blank control group was set, with three replicates in each group. Then, it was placed in a constant temperature and humidity cell culture incubator at 37 °C and 5% CO2 concentration for 21 days. After 21 days, the medium was discarded and washed twice with PBS. Then, paraformaldehyde fixative was added to fix for 30 min, rinsed once with deionized water, 1 mL of 0.2% alizarin red S staining solution was added, and stained for 3 min. Then, it was rinsed twice with deionized water to terminate the staining, photographed with a 4x and 10x microscope, and after photographing, statistical analysis was carried out using image pro plus 6.0.

[0050] Detection of osteocalcin in cell supernatant:

[0051] Mouse embryonic osteoblast precursor cells MC3T3-E1 were seeded in 24-well cell culture plates and incubated overnight. Then, the culture medium was discarded, and a polypeptide solution with a concentration of 0.05 mg / mL was added for 21 days, with the medium changed every three days. After the culture ended, the culture medium was collected, and its osteocalcin content was detected using a radioimmunoassay kit. At 4 °C, the osteocalcin in the sample or the standard and the iodine isotope-labeled osteocalcin were incubated with the same antibody. Then, a separation reagent was added to separate the free and bound osteocalcin. Subsequently, the radioactivity of the bound state was measured and the binding rate was calculated. A standard curve was made with the standard, and based on the standard curve, the osteocalcin content in the sample was obtained.

[0052] Obviously, the above-mentioned embodiments of the present invention are merely examples for clearly explaining the present invention, and are not intended to limit the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to list all the implementation manners here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention. SEQUENCE LISTING <110> Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Baotou Dongbao Biotechnology Co., Ltd. <120> A Bovine Bone Collagen Peptide Promoting Osteoblast Mineralization and Its Application <130> JLP21I0347D1 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> modified_base <222> (2)..(2) <223> Hyp <400> 1 Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile Gly Pro Ala Gly 1 5 10 15 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> modified_base <222> (9)..(9) <223> Hyp <400> 2 Gly Leu Thr Gly Pro Ile Gly Pro Pro Gly Pro Ala Gly 1 5 10 <210> 3 <211> 15 <212> PRT <213> Artificial Sequence <220> <221> modified_base <222> (2)..(2) <223> Hyp <220> <221> modified_base <222> (9)..(9) <223> Hyp <400> 3 Leu Pro Gly Ile Asp Gly Arg Pro Gly Pro Ile Gly Pro Ala Gly 1 5 10 15 <210> 4 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> modified_base <222> (2)..(2) <223> Hyp <400> 4 Ala Pro Gly Pro Val Gly Pro Val Gly 1 5 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <400> 5 Gly Pro Ala Gly Pro Ser Gly Pro Ala Gly 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <400> 6 Gly Ala Pro Gly Ser Pro Gly Pro Ala Gly 1 5 10 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <400> 7 ctgcccggca tcgacggcag gcccggcccc atcggccccg ccggc 45 <210> 8 <211> 39 <212> DNA <213> Artificial Sequence <400> 8 ggcctgaccg gccccatcgg cccccccggc cccgccggc 39 <210> 9 <211> 45 <212> DNA <213> Artificial Sequence <400> 9 ctgcccggca tcgacggcag gcccggcccc atcggccccg ccggc 45 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <400> 10 gcccccggcc ccgtgggccc cgtgggc 27 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <400> 11 ggccccgccg gccccagcgg ccccgccggc 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <400> 12 ggcgcccccg gcagccccgg ccccgccggc 30

Claims

1. A bovine bone collagen peptide, characterized in that, The amino acid sequence of the bovine bone collagen peptide is APGPVGPVG; wherein, the proline at position 2 is hydroxylated to form hydroxyproline.

2. A polynucleotide, characterized in that, Encoding the bovine bone collagen peptide described in claim 1.

3. A fusion polypeptide, characterized in that, It contains the bovine bone collagen peptide described in claim 1.

4. The fusion polypeptide according to claim 3, wherein The fusion polypeptide is a protein complex formed by linking the bovine bone collagen peptide described in claim 1 with a carrier protein.

5. The fusion polypeptide according to claim 4, wherein, The carrier protein is ovalbumin or bovine serum albumin.

6. A drug, characterized in that, Containing the bovine bone collagen peptide described in claim 1.

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