Improved osteogenic protein with high biological activity based on BMP9 (bone morphogenetic protein 9) and preparation method of improved osteogenic protein

Abstract

The invention relates to an improved high-biological-activity osteogenic protein based on BMP9 (bone morphogenetic protein 9). The improved high-biological-activity osteogenic protein is formed by inserting a heparin binding site structure from a human fibroblast growth factor 2 into a protein structure of the BMP9. According to the invention, a recombinant adenovirus plasmid pAd-HB-hBMP9 is constructed, an SA (AGCGCC) is reserved at the C terminal of an HB structural domain so as to ensure the function of a wild type hBMP9 mature peptide, and before the HB structural domain is respectively inserted into the wild type hBMP9 mature peptide, the main functional structure of the wild type hBMP9 is not changed and influenced. The improved osteogenic protein with high biological activity has osteogenic activity higher than that of WT-hBMP9, can significantly induce bone marrow mesenchymal stem cell osteogenesis, and promotes healing of mouse skull defects. The method has a wide application prospect in the research fields of basis and clinical transformation of adenovirus-mediated osteogenic factor expression, gene therapy and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a high-bioactive osteogenic protein and a preparation method thereof. Background Art

[0002] The process of bone formation is regulated by a variety of growth factors, signaling pathways, and even non-coding RNA. Among them, bone morphogenetic proteins (BMP) have long been proven to be the most effective osteogenic induction factor and are widely used in clinical practice. The actual application method is mainly to combine BMP recombinant protein with biological delivery materials to achieve the purpose of releasing BMP to induce osteogenesis at the target site. There are many delivery materials, such as hydrogel, collagen sponge, hydroxy (Hydroxyapatite), polylactic acid scaffold, etc. In order to increase the adsorption capacity of BMP on the delivery material and control the sustained release efficiency, the delivery material will be modified to a certain extent. Among them, research related to heparin has gradually become a hot spot in BMP research. Heparin is a naturally occurring mucopolysaccharide that is widely present in collagen, animal cell capsules, and extracellular matrix. It interacts with various heparin-binding proteins and plays complex functions such as information transmission and biological regulation. Studies have found that BMP2 protein has a clear heparin binding site (HB), which can regulate its biological activity after binding with heparin, giving it better osteogenic ability.

[0003] Recent studies have confirmed that BMP9 (also known as Growth differentiation factor 2, GDF2) has the strongest osteogenic potential among many BMPs, and is superior to the currently recognized and widely used BMP2 and BMP7. However, there are few heparin-related studies on BMP9. The main reason is that when simulating protein structure in computer, BMP9 itself lacks the so-called heparin binding site compared to BMP2. Second, BMP2 has been widely used in clinical practice, so people pay more attention to BMP2 rather than BMP9. Summary of the invention

[0004] In view of this, the object of the present invention is to provide an improved high bioactive osteogenic protein based on BMP9, and also to provide the application of the protein in bone tissue engineering.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A modified high bioactive osteogenic protein based on BMP9, which is composed of a protein structure derived from human fibroblast growth factor 2 inserted into BMP9.

[0007] Furthermore, the amino acid sequence of the improved high bioactive osteogenic protein is shown in SEQ ID No.8.

[0008] 2. A gene encoding an improved high bioactive osteogenic protein based on BMP9, wherein the gene contains a DNA fragment of at least one of the following nucleotide sequences:

[0009] 1) The nucleotide sequence of SEQ ID NO.3 in the sequence listing;

[0010] 2) A nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence shown in SEQ ID NO. 3 in the sequence listing and encoding a protein with the same biological function.

[0011] 3. The recombinant plasmid, recombinant virus, expression cassette, transgenic cell line or recombinant bacteria containing the above gene are all within the scope of protection of the present invention.

[0012] Furthermore, the invention also provides the use of a recombinant plasmid, a recombinant virus, an expression cassette, a transgenic cell line or a recombinant bacterium containing the above gene in bone defect repair.

[0013] The bone defect includes but is not limited to bone tissue defects such as skull, long bones, and teeth.

[0014] 4. A method for preparing a recombinant adenovirus plasmid, comprising:

[0015] 1) The coding region of BMP9 was connected to the pBR322 plasmid with modified restriction sites to construct a recombinant adenovirus vector plasmid pBR322-hBMP9 expressing WT-hBMP9;

[0016] 2) Inserting the HB fragment into the Furin restriction site of the recombinant adenovirus vector plasmid pBR322-hBMP9 CDS mature peptide to obtain the recombinant adenovirus vector pBR322-hBMP9-HB;

[0017] 3) The HB2-hBMP9 fragment in pBR322-hBMP9-HB was amplified by PCR and inserted into the adenovirus shuttle plasmid pAdTrack-TO4 to form the shuttle plasmid pAdTrack-TO4-hBMP9-HB, which was homologously recombined with the adenovirus backbone plasmid pAdEasy1 to form the recombinant adenovirus plasmid pAd-HB-hBMP9.

[0018] Furthermore, the specific sequence of the coding region of BMP9 is shown in SEQ ID No.2.

[0019] Furthermore, the nucleotide sequence of the HB fragment is shown in SEQ ID No.1.

[0020] 5. A method for preparing a recombinant adenovirus, comprising packaging the recombinant adenovirus plasmid pAd-HB-hBMP9 obtained by any of the above preparation methods through 293pTP or 293GP cells and amplifying the virus through HEK-293 cells to obtain the recombinant adenovirus Ad-HB-hBMP9.

[0021] 6. Application of improved highly bioactive osteogenic protein in bone defect repair.

[0022] The bone defect includes but is not limited to bone tissue defects such as skull, long bones, and teeth.

[0023] The beneficial effect of the present invention is that after multiple experimental studies and layer-by-layer screening, the present invention constructs the five HB domains of hBMP2, hFGF2, hVEGFA, hHBEGV, and hTGF-β1 into the BMP9 coding region, and the active protein with the hFGF2 HB domain and the BMP9 functional coding region finally obtained has higher osteogenic activity than WT-hBMP9, and is also better than BMP9 with HB of BMP2. The recombinant adenovirus plasmid pAd-HB-hBMP9 constructed by the present invention retains a "SA (AGCGCC)" at the C-terminus of the HB domain to ensure the function of the wild-type hBMP9 mature peptide, and the HB domain is respectively inserted before the mature peptide of the wild-type hBMP9 to ensure that the addition of the HB domain will not change and affect the main functional structure of the wild-type hBMP9. The recombinant adenovirus finally obtained from the plasmid can overexpress the gene shown in SEQ ID NO.3 in immortalized mouse adipose-derived mesenchymal stem cells (iMAD) to obtain the HB-hBMP9 active protein. In addition, the in vivo and in vitro related biological activity assays and comparisons prove that the improved HB-hBMP9 protein of the present invention can significantly induce bone marrow mesenchymal stem cells to form bones and promote the healing of skull defects in mice. At the same time, there is a certain synergistic effect with WT-hBMP9, and no obvious immunogenicity is observed. It has high medical value in the application of preparing bone injury treatment products. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0025] Figure 1a This is the commercialized PBR322 plasmid map.

[0026] Figure 1b The following are the recombinant plasmid images of pAdTrack-TO4 (A) and pAdTrack-TO4-HB-BMP9 (B).

[0027] Figure 2This is the PBR322-Linker plasmid map.

[0028] Figure 3 Schematic diagram of the construction of HB-BMP9 recombinant adenovirus plasmid, where A: the HB-BMP9 fragment was inserted into the pBR322-Linker plasmid, named pBR322N2HM-hBMP9; B: the HB-BMP9 fragment cloned from the pBR322N2HM-hBMP9 plasmid was inserted into the adenovirus shuttle plasmid pAdTrack-TO4, named pAdTrack-TO4-HB-BMP9; C: the pAdTrack-TO4-HB-BMP9 plasmid and the recombinant adenovirus backbone plasmid underwent homologous recombination to form a complete recombinant adenovirus plasmid, named pAd-HB-hBMP9.

[0029] Figure 4 Schematic diagram of the HB domain insertion site and amino acid sequence in pAd-HB-hBMP9 (A) and the protein structure predicted by AlphaFold2 (B).

[0030] Figure 5 Overexpression and function test of hFGF2 HB site after adenovirus infection, where A: fluorescence image of iMAD cells 24 hours after infection with Ad-HB-hBMP9; B: HB-specific TqPCR results; C: schematic diagram of collagen sponge scaffold protein adsorption test; D: remaining cell alkaline phosphatase (ALP) activity ratio (activity after adsorption / activity before adsorption*100%).

[0031] Figure 6 To determine the precise titer of each recombinant adenovirus.

[0032] Figure 7 Comparison of the in vitro basal osteogenic activity of each recombinant adenovirus, where A: ALP qualitative staining of iMAD cells after infection; B: ALP quantitative detection of iMAD cells after infection; C: ALP quantitative detection results of other cell lines after infection.

[0033] Figure 8 These are the results of the matrix mineralization test, where A is the Alizarin red staining image and B is the reading result.

[0034] Fig. 9 Micro-CT three-dimensional reconstruction and quantitative analysis of subcutaneous injection masses, where A: representative three-dimensional reconstruction image and the ratio of bone volume to total volume at week 3; B: representative three-dimensional reconstruction image and the ratio of bone volume to total volume at week 6.

[0035] Fig.10 This is the histological analysis of the subcutaneous mass at 3 weeks, including HE staining, Masson's trichrome staining, and safranin & fast green staining.

[0036] Fig.11 The results of the skull defect repair test in 8-week-old C57 mice are shown in Figure 1. A: Schematic diagram of the skull defect repair test; B: 3D reconstruction of the mouse skull by Micro-CT scanning; C: H&E staining and Masson trichrome staining (20X and 200X) of the skull defect area; NB refers to new bone; LB refers to lamellar bone. DETAILED DESCRIPTION

[0037] The preferred embodiment of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The experimental methods for which specific conditions are not specified in the embodiments are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] Immortalized mouse adipose-derived mesenchymal stem cells (iMAD) are extracted from mouse embryos and immortalized.

[0040] The animal experiments in this invention were approved by the Ethics Committee for the Use of Research and Experimental Animals of Chongqing Medical University, and all experimental procedures were performed in accordance with the guidelines.

[0041] Other reagents were from Thermo Fisher Scientific (Waltham, MA) or Millipore Sigma (St. Louis, MO).

[0042] Example 1

[0043] Construction of recombinant adenovirus-mediated Ad-HB-hBMP9 containing HB domain and hBMP9 functional coding region

[0044] The HB domain was successfully introduced into hBMP9 through gene editing technology and stably expressed by recombinant adenovirus.

[0045] Five heparin binding (HB) domains were finally screened from the PubMed gene library and combined with some preliminary experimental studies. The sequences are as follows:

[0046] HB1 is derived from human bone morphogenetic protein 2 (hBMP2), and its amino acid sequence is QAKHKQRKRLKSSCKRH;

[0047] HB2 is derived from human fibroblast growth factor 2 (hFGF2), and its amino acid sequence is KRTGQYKLGSKTGPGQK;

[0048] HB3 is derived from human epithelial growth factor (hEGF), and its amino acid sequence is KRKKKGKGLGKKRDPCLRKYK;

[0049] HB4 is derived from human transforming growth factor β1 (hTGF-β1), and its amino acid sequence is DFRKDLGWKWIHEPKGYHA;

[0050] HB5 comes from human vascular endothelial growth factor A (hVEGFA), and the amino acid sequence is PCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR.

[0051] The HB-hFGF2 fragment sequence was designed, and the specific nucleotide sequence was shown in SEQ ID No. 1. HB-hFGF2 is the heparin binding site domain (HB) of human fibroblast growth factor 2 (hFGF2).

[0052] SEQ ID No.1:

[0053] AAACGAACTGGGCAGTATAAACTTGGATCCAAACAGGACCTGGGCAGAAA

[0054] WT-hBMP9 is the functional coding region of wild-type human bone morphogenetic protein 9 (WT-BMP9, HumanBMP9CDS, NM_016204.4), and the specific nucleotide sequence is shown in SEQ ID No.2.

[0055] SEQ ID No.2:

[0056]

[0057] hBMP9-HB2 HB from hFGF2,SEQ ID No.3:

[0058]

[0059] hBMP9-HB1 HB from hBMP2,SEQ ID No.4:

[0060]

[0061] hBMP9-HB3 HB from hHBEGF,SEQ ID No.5:

[0062]

[0063] hBMP9-HB4 HB from hTGF-β1,SEQ ID No.6:

[0064]

[0065] hBMP9-HB5 HB from hVEGF-A,SEQ ID No.7:

[0066]

[0067] hBMP9-HB2 HB from hFGF2 amino acid sequence,SEQ ID No.8:

[0068] MCPGALWVALPLLSLLAGSLQGKPLQSWGRGSAGGNAHSPLGVPGGGLPEHTFNLKMFLENVKVDFLRSLNLSGVPSQDKTRVEPPQYMIDLYNRYTSDKSTTPASNIVRSFSMEDAISITATEDFPFQKHILLFNISIPRHEQITRAELRLYVSCQNHVDPSHDLKGSVVIYDVLDGTDAWDSATETKTFLVSQDIQDEGWETLEVSSAVKRWVRSDSTKSKN KLEVTVESHRKGCDTLDISVPPGSRNLPFFVVFSNDHSSGTKETRLELREMISHEQESVLKKLSKDGSTEAGESSHEEDTDGHVAAGSTLARRKRSAKRTGQYKLGSKTGPGQKSAGAGSHCQKTSLRVNFEDIGWDSWIIAPKEYEAYECKGGCFFPLADDPTKHAIVQTLVHLKFPTKVGKACCVPTKLSPISVLYKDDMGVPTLKYHYEGMSVAECGCR

[0069] Linker SEQ ID No.9:

[0070]

[0071] Figure 1- Figure 3 Schematic diagram of the construction of HB-BMP9 recombinant adenovirus plasmid. Figure 1a This is the commercialized PBR322 plasmid map; Figure 2 This is the PBR322-Linker plasmid map. The PBR322 plasmid was modified by introducing a restriction site sequence (SEQ ID No. 9) at the EcoRI site, starting with EcoRI and ending with BsaBI, and named PBR322-Linker. Figure 3 Middle A: The HB-BMP9 fragment was inserted into the pBR322-Linker plasmid, named pBR322N2HM-hBMP9, with HindⅢ site at the beginning, the hBMP9 coding region indicated by lines at the bottom, and HB fragments at the end (HB here applies to HB1, HB2, HB3, HB4 and HB5), BseRI at the beginning and StuⅠ at the end; Figure 3 Middle B: The HB-BMP9 fragment was cloned from the pBR322N2HM-hBMP9 plasmid and inserted into the adenovirus shuttle plasmid pAdTrack-TO4, named pAdTrack-TO4-HB-BMP9; Figure 3 Middle C: pAdTrack-TO4-HB-BMP9 plasmid undergoes homologous recombination with the recombinant adenovirus backbone plasmid to form a complete recombinant adenovirus plasmid, named pAd-HB-hBMP9.

[0072] First, the coding region (Coding sequence, CDS, specific sequence such as SEQ ID No. 2) of wild-type human BMP9 was connected to the pBR322N2HM plasmid with modified restriction sites (pBR322N2HM is a tool plasmid modified based on the commercial plasmid pBR322, and a nucleotide sequence containing multiple restriction sites was introduced into the TcR sequence segment) to form a recombinant adenovirus vector plasmid pBR322-hBMP9 expressing WT-hBMP9. Subsequently, the designed and synthesized HB fragment was inserted into the Furin restriction site (between the BseRI and StuI restriction sites, as shown in the above sequence SEQ ID No.3-SEQ ID No.7) of the CDS mature peptide of the pBR322-hBMP9 recombinant adenovirus vector plasmid to obtain the same gene vector pBR322-hBMP9-HB that simultaneously overexpresses the HB domain and the BMP9 functional coding region. When designing the gene editing, a "SA (AGCGCC)" was retained at the C-terminus to ensure the function of the wild-type hBMP9 mature peptide, and the HB domain was inserted before the mature peptide of the wild-type hBMP9 (the other HB insertion methods were the same). Figure 4The "SA" in A is marked in red, representing the first two amino acids of the mature peptide after the protein is cleaved by Furin, and is also the starting amino acid of the mature peptide. To ensure successful Furin cleavage, the HB domain is inserted after the "SA" sequence of WT, and another "SA" sequence is left to maintain the original structure of the mature peptide.

[0073] Secondly, the HB2-hBMP9 fragment in pBR322-hBMP9-HB was amplified by PCR and inserted into the adenovirus shuttle plasmid pAdTrack-TO4 to form the shuttle plasmid pAdTrack-TO4-hBMP9-HB, and finally homologously recombined with the adenovirus backbone plasmid pAdEasy1. The recombination method is to cut the front shuttle plasmid through the PmeI restriction site to expose the left arm and the right arm, and electrotransform the BJ5183 competent cells containing the backbone plasmid pAdEasy1 to homologously recombine the shuttle plasmid and the left arm and the right arm on the backbone plasmid. After electrotransformation, the bacterial solution was incubated at 37°C for 20 minutes, applied to an LB plate containing kanamycin and cultured for 18 hours, and then a smaller single colony was selected for shaking, and the plasmid was extracted for enzyme cutting verification. The single plasmids that were correctly selected and verified were mixed together to form the recombinant adenovirus plasmid pAd-HB-hBMP9. The virus was then packaged using 293pTP or 293GP cells and amplified using HEK-293 cells to obtain high titer recombinant adenovirus Ad-HB-hBMP9. When adenovirus was used to infect target cells, Polybrene (final concentration of 6 μg / ml) was added to improve infection efficiency.

[0074] Similarly, only the WT-hBMP9 fragment in the recombinant adenovirus vector plasmid pBR322-hBMP9 expressing WT-hBMP9 was amplified and inserted into the shuttle plasmid and then recombined with the backbone plasmid pAdEasy1 to obtain the recombinant adenovirus plasmid pAd-WT-hBMP9. At the same time, the empty shuttle plasmid pAdTrack-TO4 containing green fluorescent protein (GFP) was recombined with the backbone plasmid pAdEasy1 to obtain the control recombinant adenovirus plasmid pAd-GFP. The virus was then packaged and amplified by 293pTP or HEK-293 cells to obtain high-titer recombinant adenovirus Ad-WT-hBMP9 and Ad-GFP, respectively. Figure 4B in the figure is the protein structure prediction result of Alphafold2. The red area indicates the area with lower prediction accuracy, and the blue area indicates the area with higher prediction accuracy. The circled part is the structure of the HB domain, and the boxed part is the mature peptide of WT-hBMP9. The results show that adding the HB domain will not change the main functional structure of wild-type hBMP9. In the figure, WT refers to Ad-WT-hBMP9, and HB1-HB5 refer to Ad-HB-hBMP9 with the corresponding numbered HB domains added respectively. In the present invention, HB represents the general description of HB1-HB5, and HBX, X is any natural number from 1 to 5, referring to the corresponding HB domain in the aforementioned specific HB1-HB5.

[0075] The above results suggest that the HB domain was successfully introduced into WT-hBMP9 through gene editing technology and stably expressed by recombinant adenovirus. After 24 hours of infection of cells by recombinant adenovirus Ad-HB2-hBMP9, highly bioactive More Osteogenic Protein-1 (MOP-1) was synthesized.

[0076] Example 2

[0077] HB locus overexpression and functional assay

[0078] After adenovirus packaging and amplification, the infection efficiency was tested. Equal volumes of Ad-WT-hBMP9 and Ad-HB-hBMP9 were co-infected with polybrene (final concentration of 1.7 mg / mL) in iMAD cells for 24 hours. It was found that both adenoviruses expressing the constructed constructs could produce strong fluorescent signals. The results are as follows Figure 5 As shown in Figure A, the constructed adenovirus can efficiently infect iMAD cells. Similar infection efficiencies were observed in other cell lines used in this study. Figure 5 As shown in B. Cells infected with each Ad-HB-hBMP9 only expressed the corresponding primers, without cross-expression with other primers. In addition, Ad-GFP and Ad-WT-hBMP9 did not express any expression under any pair of primers because the forward primer used was specific to the HB sequence. These results preliminarily indicate that the designed HB-hBMP9 gene was successfully overexpressed by adenovirus, that is, each Ad-HB-hBMP9 cell only specifically expressed its own HB sequence.

[0079] Collagen sponge scaffold protein adsorption test example:

[0080] Conditioned medium preparation: Take 5 × 10 6HCT116 cells in the logarithmic growth phase were infected with Ad-WT-hBMP9 and Ad-HB-hBMP9, and an appropriate amount of polybrene (final concentration of 1.7 mg / mL) was added to the culture medium to improve the efficiency of viral infection. The infection rate was 30% at 24 hours. After 48 hours, 20 mL of protein-containing culture medium supernatant was collected and centrifuged at 800 RPM for 3 minutes to remove cell debris. Then, 1 cm 3 (L group), 2cm 3 (M group), 3cm 3 The collected culture medium was evenly divided into centrifuge tubes containing type I collagen sponges (group H), and placed in a 4°C refrigerator for 24 hours for adsorption to obtain each conditioned culture medium, such as Figure 5 As shown in the schematic diagram C.

[0081] An appropriate amount of iMAD cells were plated in a 48-well plate, and after adherence, they were cultured with the above-mentioned conditioned medium. After 3 days, the quantitative readings of ALP (alkaline phosphatase) were tested for comparison. The results showed that after different volumes of type I collagen sponge were absorbed, the WT group did not show a significant decrease in ALP activity, while the HB group showed different gradient decreases, especially the HB2 group showed a significant gradient decrease ( Figure 5 D), suggesting that HB2 can bind to collagen, thereby reducing the remaining protein in the culture medium and thus reducing ALP activity.

[0082] The above results indicate that the recombinant adenovirus Ad-HB2-hBMP9 was successfully constructed and expressed the highly bioactive osteogenic protein 1 (MOP-1), and the HB site was successfully expressed and had the ability to bind to the extracellular matrix.

[0083] Example 3

[0084] In vitro osteogenic differentiation potential analysis example:

[0085] Since BMP9 itself exhibits strong osteogenic activity, in order to ensure the accuracy of subsequent experiments, it is necessary to accurately determine the adenovirus titer. All in vitro and in vivo experiments of the present invention were performed at a cell infection rate of about 30%. Maintaining a low infection rate allows reasonable and observable comparisons between different groups. The results of accurate titer determination are shown in Figure 1. Figure 6 The group with about 30% cell infection rate was selected under the microscope and determined as the relative titer of the virus.

[0086] The in vitro basal osteogenic capacity of each modified BMP9 was determined: an appropriate amount of primary bone marrow mesenchymal stem cells were plated in a 24-well plate, and after they adhered to the plate, Ad-GFP (GFP group), Ad-WT-hBMP9 (WT group) and Ad-HB-hBMP9 (HB1-HB5 group) were mixed with polybrene (final concentration of 1.7 mg / mL) and infected with 30% infection efficiency. ALP qualitative staining was then performed on each group of cells, and the results are shown in Figure 2. Figure 7 As shown. The results showed that HB2 showed higher ALP activity than WT on days 3 and 5. The ALP activity of HB4 was lower than that of WT, while the activity of HB5 was lower than that of HB4. In contrast, although HB1 and HB3 were slightly higher than GFP, their osteogenic activity was very limited. The main cell line in this study was iMAD, which was selected because of its sensitivity to BMP9 stimulation and low background. In addition, in order to confirm its universality in inducing osteogenesis, we also tested other cell lines, including imBMSCs, iMEFs, iCALs, and iHAFCs, and the results are shown in Figure 7 As shown in C. In brief, all four cell lines responded to our constructed adenovirus, among which HB2 still showed the highest osteogenic activity, followed by WT, HB4 and HB5, while HB1 and HB3 had limited activity, which was consistent with the iMADs results. These findings indicate that the modified HB2-hBMP9 can not only induce osteogenesis in mesenchymal cells, but also play a role in other osteoblasts.

[0087] Similarly, the three groups of cells were cultured in mineralization medium (containing 0.1 μM dexamethasone, 50 μM vitamin C, and 10 mM sodium β-glycerophosphate) for 21 days and then stained with Alizarin red, as shown in Figure 5. Figure 8 As shown in the figure, the results showed that after 21 days of culture of mesenchymal stem cells infected with different adenoviruses, the HB2 group showed the strongest mineralization ability, exceeding WT, indicating that the HB2 domain can enhance the interaction with ECM. The results of the other HBs were basically the same as the ALP results, weaker than WT. It is worth mentioning that the WT+HB2 group was stronger than the simple WT group and the simple HB2 group, suggesting that there may be a synergistic effect between the two. The GFP group was the lowest.

[0088] Example 4

[0089] In vivo osteogenic differentiation potential analysis example:

[0090] To further explore whether the modified BMP9 is indeed superior to WT-hBMP9, primary bone marrow mesenchymal stem cells in the logarithmic growth phase were infected with Ad-GFP (GFP group), Ad-WT-hBMP9 (WT group), Ad-HB-hBMP9 (HB1-HB5 group), and mixed group (HB+WT group) with an infection efficiency of 30%. After 36 hours of infection, the cells were digested and resuspended in PPCNg (0.2% gelatin / PBS dilution, PPCN final concentration of 50 mg / mL, polyethyleneglycolcitrate-co-N-isopropylacrylamidegelatin, PPCNg) to approximately 5×10 6 pcs / 80μL.

[0091] Athymic nude mice (6 weeks old, weighing 15-20 g, regardless of gender, randomly divided into GFP group, WT group and MOP-1 group, n = 5 in each group) were subcutaneously injected with 80 μL of cell suspension (corresponding to the cell suspension of each infection group mentioned above) on both sides of the abdomen. The tumors at the subcutaneous injection site were removed 21 days and 42 days after injection for Micro-CT imaging analysis and histological analysis. The results are shown in Fig. 9 As shown, whether from 3 weeks ( Fig. 9 A) or 6 weeks ( Fig. 9 In the results of the mass in (B), HB2 formed larger and stronger bone tissue compared to WT, while HB4 and HB5 had relatively small bone formation. HB1 and HB3 had limited bone formation. The bar graph of the ratio of bone volume to total volume (BV / TV) showed similar results. At the same time, the HB2+WT group showed the highest osteogenesis, suggesting that there may be a synergistic effect, which is consistent with the results of the in vitro test. Comparing the results of 6 weeks and 3 weeks, it can be seen that after 6 weeks of subcutaneous osteogenesis, the bone tissue is significantly more mature and the bone mass is larger.

[0092] Fig.10 This is the histological analysis of the subcutaneous mass at 3 weeks, including HE staining, Masson trichrome staining, and safranin & fast green staining. From the histological staining, at 3 weeks, it can be seen from the left column of the figure that the subcutaneous osteogenesis has not yet fully matured after 3 weeks. The most mature one is the HB2 group, with obvious trabeculae and less cartilage-like matrix. The WT is mostly cartilage-like matrix that is not fully mature.

[0093] The above results suggest that the MOP-1 produced by Ad-HB2-hBMP9 introduced with hFGF2HB has a significantly better ability to induce osteogenic differentiation of bone marrow mesenchymal stem cells than WT-hBMP9.

[0094] Example 5.

[0095] In addition to the above-mentioned in vitro marker determination and in vivo subcutaneous osteogenesis assay, we also conducted a skull defect repair assay in C57 black mice to explore the potential of different BMP9s in tissue repair and whether there is an obvious immune response.

[0096] C57 mice (4-6 weeks old, regardless of sex) were randomly divided into Ad-GFP (GFP group), Ad-WT-BMP (WT group), Ad-HB-hBMP9 (HB group), and Ad-WT-BMP and Ad-HB2-hBMP9 mixed group (HB+WT group), with 5 mice in each group. A 3 mm diameter skull defect was created on the parietal bones on both sides of the mice, and a type I collagen sponge with a diameter of 3 mm and a height of 1 mm was placed in the defect.

[0097] Mouse primary bone marrow mesenchymal stem cells were infected with a mixture of 30% adenovirus Ad-GFP, Ad-WT-BMP, and Ad-HB-hBMP9 and polybrene (final concentration of 1.7 mg / mL) for 24 hours, then collected and suspended to 2×10 with PPCNg (0.2% gelatin / PBS dilution, final PPCN concentration of 50 mg / mL). 6 The cells were then loaded onto a type I collagen scaffold and placed into the skull defect. Eight weeks after implantation, the mice were killed and skull samples were retrieved and fixed for Micro-CT imaging and histological analysis. Fig.11 As shown in the figure, HB2 and WT+HB2 showed superior repair effects compared to WT alone. The figure is marked as GFP WT. The GFP group and WT group were placed on the same mouse skull to make models, which is convenient for comparison with other groups. They can all be regarded as control groups. It is worth mentioning that WT+HB2 formed a structure similar to a cover, which may be due to the rapid formation of bone tissue before the collagen scaffold degrades. In the histological sections, it can be seen that the trabeculae formed by the HB2 group are significantly more mature than those of the WT group. Because the blood supply conditions of skull defects and subcutaneous osteogenesis are very different, the blood supply of skull defects is significantly better than that of subcutaneous osteogenesis. Therefore, the interaction between HB and ECM is stronger, which supports the better osteogenesis of HB2. Since this part of the experiment used C57 black mice instead of thymic nude mice, it was proved that there was no obvious rejection reaction after the addition of HB2.

[0098] The above results suggest that after the recombinant adenovirus Ad-HB2-hBMP9 expressing MOP-1 infects mesenchymal stem cells and is co-placed with a collagen sponge scaffold, more mature trabeculae are formed at the bone defect site, which has better skull repair ability than WT-hBMP9.

[0099] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. An improved high bioactive osteogenic protein based on BMP9, characterized in that: It consists of a protein structure in which the heparin binding site from human fibroblast growth factor 2 is inserted into the BMP9 structure.

2. The improved high bioactive osteogenic protein according to claim 1, characterized in that: The amino acid sequence of the improved high bioactivity osteogenic protein is shown in SEQ ID No.

8.

3. A gene encoding the improved high bioactive osteogenic protein according to claim 1 or claim 2, characterized in that: The gene contains a DNA fragment of at least one of the following nucleotide sequences: 1) The nucleotide sequence of SEQ ID NO.3 in the sequence listing; 2) A nucleotide sequence having at least 90%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence shown in SEQ ID NO. 3 in the sequence listing and encoding a protein with the same biological function.

4. A recombinant plasmid, recombinant virus, expression cassette, transgenic cell line or recombinant bacteria comprising the gene of claim 3.

5. A method for preparing a recombinant adenovirus plasmid, characterized in that: include: 1) The coding region of BMP9 was connected to the pBR322 plasmid with modified restriction sites to construct a recombinant adenovirus vector plasmid pBR322-hBMP9 expressing WT-hBMP9; 2) Inserting the HB fragment into the Furin restriction site of the recombinant adenovirus vector plasmid pBR322-hBMP9 CDS mature peptide to obtain the recombinant adenovirus vector pBR322-hBMP9-HB; 3) The HB2-hBMP9 fragment in pBR322-hBMP9-HB was amplified by PCR and inserted into the adenovirus shuttle plasmid pAdTrack-TO4 to form the shuttle plasmid pAdTrack-TO4-hBMP9-HB, which was homologously recombined with the adenovirus backbone plasmid pAdEasy1 to form the recombinant adenovirus plasmid pAd-HB-hBMP9.

6. The method for preparing a recombinant adenovirus plasmid according to claim 5, characterized in that: The wild-type human bone morphogenetic protein 9 coding region, the specific sequence is shown in SEQ ID No.

2.

7. The method for preparing a recombinant adenovirus plasmid according to claim 5, characterized in that: The nucleotide sequence of the HB2-BMP9 fragment is shown in SEQ ID No.

3.

8. A method for preparing a recombinant adenovirus, characterized in that: The recombinant adenovirus plasmid pAd-HB-hBMP9 obtained by any preparation method of claims 5-7 is packaged by 293pTP or 293GP cells and amplified by HEK-293 cells to obtain the recombinant adenovirus Ad-HB-hBMP9.

9. Use of the improved high bioactive osteogenic protein according to claim 1 or claim 2 in repairing bone defects.

10. Use of the recombinant plasmid, recombinant virus, expression cassette, transgenic cell line or recombinant bacteria according to claim 4 in bone defect repair.

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