Preparation process and application of a recombinant collagen bone morphogenetic protein biomaterial

Abstract

The present invention provides a recombinant collagen-like bone morphogenetic protein (BMP2C, named as a must-complement factor, research and development code FLM4.x), encoding nucleic acid, and vector. A recombinant collagen-like bone morphogenetic protein (BMP2C) biomaterial is also provided, characterized in that the biomaterial comprises a protein, nucleic acid or vector as described in the present invention. A preparation method of the recombinant collagen-like bone morphogenetic protein is further provided, and the preparation method can effectively reduce the formation of inclusion bodies, so that the concentration of recombinant protein in the supernatant is significantly improved, and there is no need to treat the inclusion bodies, thereby reducing the complexity of subsequent treatments and reducing production costs. Therefore, the scheme of the present invention has higher expression efficiency and a simpler purification process, is a very valuable advancement in protein production and research, and has huge commercial value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a recombinant collagen bone morphogenetic protein biomaterial and a preparation process and application thereof. Background Art

[0002] Dental pulp stem cells can be induced to differentiate into odontoblasts and have osteogenic differentiation ability, which plays an important role in periodontal tissue regeneration. ALP can be used as a marker for early odontoblast differentiation.

[0003] Bone morphogenetic protein (BMP), also known as bone morphogenetic protein, is a group of highly conserved functional proteins with similar structures and belongs to the TGF-β family. BMP is the main factor that induces bone and cartilage formation in the body, and is expressed during limb growth, endochondral ossification, early fractures, and cartilage repair, playing an important role in the embryonic development and regeneration and repair of bones. Today, there are 43 members in the BMP family, which are widely present in embryos, blood cells, kidneys, spleens and other tissues of pigs, cattle, sheep, rabbits, mice and humans, and have a high degree of homology between different species. BMP is not only involved in the regulation of bones, but also plays a certain role in the development of fat, kidneys, liver, bones and nervous systems. Bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor B family, which promotes the differentiation and maturation of osteoblasts, participates in the growth and development of bones and cartilage and their reconstruction process, and thus accelerates the repair of bone defects. BMP-2 is synthesized in vivo in the form of a precursor, and the signal peptide and propeptide are removed by protease cleavage to obtain a mature peptide consisting of 114 amino acid residues. Summary of the invention

[0004] In a first aspect of the present invention, a protein is provided, characterized in that the protein comprises an amino acid sequence as shown in SEQ ID NO: 1, 3 or 5.

[0005] In some embodiments of the present invention, the protein comprises the amino acid sequence shown in SEQ ID NO:1.

[0006] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO: 1, 3 or 5.

[0007] In some embodiments of the present invention, the amino acid sequence of the protein is shown in SEQ ID NO:1.

[0008] In some embodiments of the present invention, the protein is recombinant collagen-like bone morphogenetic protein (BMP2C, named as essential factor, research and development code FLM4.x).

[0009] In the second aspect of the present invention, a nucleic acid is provided, characterized in that the nucleic acid comprises a nucleotide sequence encoding the protein as described in the first aspect of the present invention.

[0010] In some embodiments of the invention, the nucleic acid is a DNA nucleic acid. In some embodiments of the invention, the nucleic acid is an RNA nucleic acid.

[0011] In some embodiments of the present invention, the nucleotide sequence is codon optimized. In some embodiments of the present invention, the nucleotide sequence is codon optimized to improve expression in cells. Further, the cell is selected from: Escherichia coli, such as BL21 (DE3), Rosetta (DE3), TOP10 or JM109 expression strains; yeast, such as Pichia pastoris; insect cells, such as Sf9, Sf21 expression systems, High Five cell lines; mammalian cell systems, such as HEK293 cells, CHO cells, NS0 cells; plant cell systems, such as tobacco cells.

[0012] In the third aspect of the present invention, a vector is provided, characterized in that the vector comprises the nucleic acid as described in the second aspect of the present invention.

[0013] In some embodiments of the present invention, a vector is provided, characterized in that the vector comprises a nucleotide sequence encoding the protein as described in the first aspect of the present invention.

[0014] In some embodiments of the present invention, the vector is a plasmid vector:

[0015] In some embodiments of the present invention, the vector is a pET vector, a pGEX vector or a pUC vector.

[0016] In some embodiments of the invention, the vector is a viral vector:

[0017] In some embodiments of the present invention, the vector is an adenoviral vector, a lentiviral vector or an adeno-associated viral vector.

[0018] In some embodiments of the present invention, the vector is an expression vector.

[0019] In some embodiments of the invention, the vector comprises a tag sequence.

[0020] In some embodiments of the present invention, the tag sequence is a His tag or a FLAG tag.

[0021] In the fourth aspect of the present invention, a cell is provided, characterized in that the cell comprises the protein as described in the first aspect of the present invention, the nucleic acid as described in the second aspect of the present invention, or the vector as described in the third aspect of the present invention.

[0022] In the fifth aspect of the present invention, a recombinant collagen bone morphogenetic protein biomaterial is provided, characterized in that the biomaterial comprises the protein as described in the first aspect of the present invention, the nucleic acid as described in the second aspect of the present invention, or the vector as described in the third aspect of the present invention.

[0023] In some embodiments of the present invention, the biological material comprises the protein as described in the first aspect of the present invention.

[0024] In some embodiments of the present invention, the recombinant collagen-like bone morphogenetic protein is the protein as described in the first aspect of the present invention.

[0025] In a sixth aspect of the present invention, a method for preparing a recombinant collagen-like bone morphogenetic protein is provided, wherein the amino acid sequence of the recombinant collagen-like bone morphogenetic protein is as shown in SEQ ID NO: 1, and the preparation method comprises the following steps:

[0026] The expression vector plasmid of the protein is transformed into the expression strain E.coli BL21 (DE3); inoculated into LB culture medium containing kanamycin; the culture medium is cultured at 37°C overnight, and IPTG is added to 0.04-0.06mM; mixed evenly, and induced at 25-27°C for 20-22h; the bacteria are collected by centrifugation, a lysis solution is added, and the supernatant is obtained by centrifugation; the recombinant collagen-like bone morphogenic protein is obtained by purification using nickel column chromatography and then by purification using HPLC column, and the eluate is freeze-dried to obtain the recombinant collagen-like bone morphogenic protein.

[0027] In some embodiments of the present invention, the plasmid sequence is shown as SEQ ID NO:2.

[0028] In some embodiments of the present invention, the preparation method comprises the following steps:

[0029] The expression vector plasmid of the protein is transformed into the expression strain E.coli BL21 (DE3), wherein the plasmid sequence is shown in SEQ ID NO: 2; the plasmid is inoculated into LB culture medium containing kanamycin; the culture medium is cultured at 37° C. overnight, and IPTG is added to 0.05 mM; the mixture is mixed evenly, and the mixture is induced at 26° C. for 21 hours; the bacteria are collected by centrifugation, a lysis solution is added, and the supernatant is obtained by centrifugation; the supernatant is purified by nickel column chromatography after filtering with a 0.45 um filter membrane, and then purified by HPLC column chromatography, and the eluate is freeze-dried to obtain the recombinant collagen-like bone morphogenic protein.

[0030] In some embodiments of the present invention, the preparation method is as described in Example 4.

[0031] In the seventh aspect of the present invention, there is provided use of the protein according to the first aspect of the present invention, the nucleic acid according to the second aspect of the present invention, and the vector according to the third aspect of the present invention in the preparation of a drug for promoting bone formation.

[0032] In some embodiments of the present invention, there is provided use of the protein according to the first aspect of the present invention in the preparation of a drug for promoting bone formation.

[0033] In some embodiments of the present invention, there is provided use of the protein according to the first aspect of the present invention in the preparation of a medicament for promoting tooth formation.

[0034] In some embodiments of the present invention, there is provided use of the protein according to the first aspect of the present invention in the preparation of a medicament for regulating bone formation and absorption.

[0035] In some embodiments of the present invention, there is provided use of the protein according to the first aspect of the present invention in the preparation of a drug for alleviating osteolysis.

[0036] Collagen promotes bone tissue formation, and BMP2 also promotes bone tissue formation. The former participates in cytoskeleton and tissue support, and the latter participates in signal transduction of cell growth, both of which can promote the growth of mesenchymal stem cells and promote osteogenic differentiation. The inventor creatively fused collagen variants with BMP2 variants to obtain the present invention.

[0037] The fusion protein (recombinant collagen bone morphogenetic protein BMP2C) of the present invention contains human type I collagen domain variant, type III collagen domain variant, and BMP2-derived mutant peptide segments, and alleviates osteolysis by dual regulation of bone formation and absorption, as well as macrophage polarization, reduces the toxic and side effects of BMP2, and improves drugability. The fusion protein of the present invention has excellent osteogenic activity and can significantly increase the activity of dental pulp stem cells and alkaline phosphatase ALP activity.

[0038] The present invention further provides a preparation process of the fusion protein (recombinant collagen-like bone morphogenetic protein). The technical solution of the present invention can effectively reduce the formation of inclusion bodies, so that the concentration of recombinant protein in the supernatant is significantly increased, and the inclusion bodies do not need to be processed, thereby reducing the complexity of subsequent processing and reducing production costs. Therefore, the scheme of the present invention has higher expression efficiency and a simpler purification process, which is a very valuable advancement in protein production and research, and has great commercial value. DETAILED DESCRIPTION

[0039] The present disclosure is further illustrated by way of examples below, but the present disclosure is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product instructions. Unless otherwise specified, the reagents and raw materials used in the present disclosure are all commercially available.

[0040] Example 1: Fusion protein design

[0041] The inventors designed and tested the fusion protein of BMP2-derived mutant peptide and collagen. The mutant peptide derived from BMP2 replaced C with two S residues and M with T residues to enhance the molecular stability, promote the binding with BMP receptor II and improve the efficacy; two C residues were used to replace L to promote the binding with BMP receptor I. Then, after fusing with the variants of two collagen fragments, a highly active fusion protein was unexpectedly obtained. Named: Recombinant collagen bone morphogenetic protein BMP2C, essential factor (SEQID NO: 1).

[0042] MGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNPGAPGAKGAHGSAGPPGATGFPGAAGRPGPPGSGNAGPVGPPGPAGKEGGKGPHGETGPAGRPGEVGPPGPPGPAGEKGSP GADGPAGAPGTPGPQGIAGQRGPVGLPGQHGERGFPGLVGPPGEPGKQGVSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGP VGPAGKPGDRGETGPAGPAGSVGPVGARGPAGPQGSRGDKGETGEQGDRGIKGKRGFSGLQGSPGPPGSPGEQGPPGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDA GPVGGGGSLKSVKGQIESLISPDGSRKKPAHNCRDLKFCHPELNSGEYWVDPNQGCKLDAINVMCNFETETCGGGGSKIPKASSVPTELSAISTCYLDENEKVVCKNYQHHHHHH(SEQ ID NO:1).

[0043] Example 2: Preparation of protein

[0044] Preparation of fusion protein:

[0045] A DNA nucleic acid fragment containing the coding sequence of the fusion protein (SEQ ID NO: 1) (codon optimization for Escherichia coli was performed by ExpOptimizer under default parameters) was prepared at an outsourcing service company, and then digested with the pET28a (+) vector using NcoI enzyme and BlpI enzyme, respectively, and the digested products were connected using T4 DNA ligase. The reaction solution was transformed into stbl3 competent cells, cultured for 14 hours, and positive clones were selected for sequencing and identification. After the positive clones with correct sequencing were cultured, the expression vector plasmid (SEQ ID NO: 2) of the fusion protein (SEQ ID NO: 1) was extracted.

[0046] The expression vector plasmid of the fusion protein was transformed into the expression strain E. coli BL21 (DE3). Cultured overnight in LB culture medium containing kanamycin at 37°C. IPTG was added to 0.3mM and induced at 16°C for 21h. The cells were collected by centrifugation, lysate was added, and supernatant was obtained by centrifugation. After filtering with a 0.45um filter membrane, it was purified by nickel column chromatography, then purified by ion chromatography and RP-HPLC, and the eluate was freeze-dried to obtain a fusion protein with a purity of 95.1% (SEQ ID NO: 1). It showed a single band in SDS-PAGE.

[0047] Preparation of other proteins: Using the same method, the expression vector plasmids of each protein were obtained, and then BMP2 protein fragment (SEQ ID NO: 3), variant of BMP2 protein fragment (SEQ ID NO: 4), collagen fragment (SEQ ID NO: 5), variant of collagen fragment (SEQ ID NO: 6) were prepared and purified.

[0048] BMP2 protein fragment:

[0049] MKIPKACCVPTELSAISMLYLDENEKVVLKNYQ (SEQ ID NO: 3).

[0050] Variants of BMP2 protein fragments:

[0051] MKIPKASSVPTELSAISTCYLDENEKVVCKNYQ (SEQ ID NO: 4).

[0052] Collagen fragments:

[0053] MGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNVGAPGAKGARGSAGPPGATGFPGAAGRVGPPGPSGNAGPPGPPGPAGKEGGKGPRGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGVVGLPGQRGERGFPGLPGPSGEPGKQGPSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPAGPVGPVGARGPAGPQGPRGDKGETGEQGDRGIKGHRGFSGLQGPPGPPGSPGEQGPSGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPV(SEQ ID NO:5).

[0054] Variant of collagen fragment:

[0055] MGQPGAKGEPGDAGAKGDAGPPGPAGPAGPPGPIGNPGAPGAKGAHGSAGPPGATGFPGAAGRPGPPGPSGNAGPVGPPGPAGKEGGKGPHGETGPAGRPGEVGPPGPPGPAGEKGSPGADGPAGAPGTPGPQGIAGQRGPVGLPGQHGERGFPGLVGPPGEPGKQGVSGASGERGPPGPMGPPGLAGPPGESGREGAPGAEGSPGRDGSPGAKGDRGETGPAGPPGAPGAPGAPGPVGPAGKPGDRGETGPAGPAGSVGPVGARGPAGPQGSRGDKGETGEQGDRGIKGKRGFSGLQGSPGPPGSPGEQGPPGASGPAGPRGPPGSAGAPGKDGLNGLPGPIGPPGPRGRTGDAGPV(SEQ ID NO:6).

[0056] Example 3. Osteogenic activity test

[0057] Cell culture: Human dental pulp stem cells (Beijing Zeping Technology, catalog number PT-5025) were cultured in DMEM medium (containing 10% fetal bovine serum) at 37°C, 5% CO2, and saturated humidity. The medium was replaced every 2 days. When the cell confluence reached 90%, trypsin was used for digestion and passage. Then, human dental pulp stem cells of the third generation in the logarithmic growth phase were taken, resuspended in DMEM medium, and then inoculated in a 24-well plate, 3×10 cells per well. 4 Cells were cultured continuously. When the cell density reached about 80%, they were randomly divided into a blank control group, a positive control group and each experimental group. The cells in the blank control group were not treated in any way. The cells in the positive control group were cultured with DMEM medium containing 100 ng / ml BMP2 protein (abcam, ab155700). The cells in each experimental group were cultured with DMEM medium containing 100 ng / ml of the protein to be tested. The culture was continued for 15 days.

[0058] Detection of the effect on the activity of human dental pulp stem cells: The cells of each group after 15 days of culture were collected and made into a cell density of 1×10 7 Cell suspension of 100 μl / ml was inoculated into a 96-well plate, and the inoculation volume per well was 100 μl. The culture was continued for 6 hours, and then 10 μl of CCK-8 working solution was added to each well. After gently mixing, the plate was incubated at 37 degrees Celsius for 30 minutes, and the absorbance OD value was measured (the wavelength of the microplate reader was set to 450 nm). The larger the OD value, the stronger the activity of the corresponding living cells. The experiment was repeated 6 times, and the results were averaged.

[0059] Detection of the effect on alkaline phosphatase ALP activity: Collect the cells of each group after 15 days of culture, wash the cells with PBS, then add cell lysis solution, lyse on ice for 15 minutes, centrifuge at 1000 rpm for 5 minutes at 4 degrees Celsius, aspirate the supernatant, add buffer and colorimetric solution in sequence, and use a spectrophotometer to detect the absorbance A value at a wavelength of 510 nanometers. According to the instructions, draw a standard curve, and then calculate the sample concentration based on the standard curve. The ratio of the sample concentration to the standard protein (Solarbio, C9442) concentration value is the ALP activity. The experiment was repeated 6 times, and the results were averaged.

[0060] As shown in Tables 1 and 2, the activity of dental pulp stem cells in the fusion protein group (experimental group 1) was higher than that in the other groups, with statistically significant differences (P<0.01); the activity of dental pulp stem cells in experimental groups 3 and 5 was significantly higher than that in the positive control group, experimental group 2, and experimental group 4 (P<0.05). The activity of alkaline phosphatase ALP in experimental group 1 was also higher than that in the other groups, with statistically significant differences (P<0.01).

[0061] Table 1. Results of dental pulp stem cell activity test

[0062]

[0063]

[0064] Table 2. Alkaline phosphatase ALP activity test results

[0065]

[0066] Example 4. Preparation process of recombinant collagen-like bone morphogenetic protein BMP2C

[0067] The fusion protein expression vector plasmid was obtained using the method described in Example 2.

[0068] Transform the expression vector plasmid of the recombinant collagen-like bone morphogenetic protein BMP2C into the expression strain E. coli BL21 (DE3). Inoculate into LB culture medium containing kanamycin. Each experimental group is inoculated with the same and equal amount of strains and inoculated from the same colony, and the same inoculation volume is controlled by OD value. Culture 2L culture medium at 37°C overnight. Add IPTG to concentration C1. Mix well. Induce at temperature T1 for 21 hours. The OD600 value shows that the growth curves of bacteria in each group are basically the same. Take a small amount of cells for staining and observe under a microscope to see if inclusion bodies are generated. Collect the bacteria by centrifugation, add lysis buffer, and centrifuge to obtain the supernatant. After filtering with a 0.45um filter membrane, the product was purified by nickel column chromatography (GenScript High Affinity Ni-NTAResin) and then purified by Hypersil GOLD C18 selective liquid chromatography column. The eluate was freeze-dried to obtain a fusion protein (SEQ ID NO: 1) with an HPLC purity of 92.2%-98.3%. The mass of the obtained fusion protein was divided by the culture volume 2L to obtain the fusion protein yield in the supernatant. The results are shown in Table 3. The experiment was repeated 5 times, and all data in the table are the results after taking the average value.

[0069] In terms of increasing the yield of fusion protein in the supernatant: when the IPTG concentration was 0.05 mM and the induction temperature was 26°C, the yield of fusion protein in the supernatant was the highest (much higher than other groups, with statistically significant difference, P<0.01), no inclusion bodies were generated in the cells, and the purity of the final product was also the highest. Therefore, an unexpected technical effect was achieved.

[0070] Table 3. Screening and optimization of recombinant protein production conditions

[0071]

[0072] Although the embodiments of the present disclosure have been described above, it is understood that the above embodiments are exemplary and do not constitute limitations to the present disclosure. A person skilled in the art may make changes, modifications and variations to the above embodiments within the scope of the present disclosure.

Claims

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

1.

2. A nucleic acid, characterized in that The nucleic acid comprises a nucleotide sequence encoding the protein of claim 1.

3. A carrier, characterized in that The vector comprises the nucleic acid of claim 2.

4. A recombinant collagen bone morphogenetic protein biomaterial, characterized in that: The biological material comprises the protein according to claim 1 , the nucleic acid according to claim 2 , or the vector according to claim 3 .

5. A method for preparing a recombinant collagen-like bone morphogenetic protein, characterized in that: The amino acid sequence of the recombinant collagen-like bone morphogenetic protein is shown in SEQ ID NO: 1, and the preparation method comprises the following steps: The expression vector plasmid of the protein is transformed into the expression strain E.coli BL21 (DE3); inoculated into LB culture medium containing kanamycin; the culture medium is cultured at 37°C overnight, IPTG is added to 0.04-0.06mM; mixed evenly, and induced at 25-27°C for 20-22h; the cells are collected by centrifugation, lysate is added, and supernatant is obtained by centrifugation; the recombinant collagen-like bone morphogenic protein is obtained by purification using nickel column chromatography and then HPLC column purification, and the eluate is freeze-dried to obtain the recombinant collagen-like bone morphogenic protein; The plasmid sequence is shown in SEQ ID NO:

2.

6. Use of the protein according to claim 1 in the preparation of a drug for promoting bone formation or tooth formation.

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