Recombinant coral protein and preparation method thereof

Abstract

The invention relates to the technical field of biology, in particular to a recombinant coral protein and a preparation method thereof. The preparation method of the recombinant coral protein comprises the following steps: S1, constructing an expression vector: cloning a coding sequence of a galaxin gene into a pcDNA3.1 expression vector to obtain the expression vector; s2, cell culture and transfection: culturing the cells in a culture medium containing fetal calf serum until the cell density reaches 80-90%, and transfecting the expression vector into the cells to obtain transfected cells; s3, protein expression and culture: continuously culturing the transfected cells; and S4, cell lysis and protein extraction: collecting, washing and lysing the transfected cells continuously cultured in the step S3, and centrifuging the obtained lysate to prepare the recombinant coral protein. The recombinant coral protein shows excellent bone regeneration capacity, can promote osteogenesis after defects, and can be used for bone tissue engineering.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically relates to a recombinant coral protein and a preparation method thereof. Background Art

[0002] Coral bone meal is a natural biomaterial that is widely used in the field of bone defect repair. Currently, the existing technologies for coral bone meal in bone defect repair mainly include the following: coral bone meal filling, which fills the coral bone meal into the bone defect site to fill and repair the missing part of the bone tissue. This method can promote the growth of bone cells and blood vessels and provide a scaffold structure for new bone tissue, helping the healing and regeneration of bone defects; coral bone meal composite materials, which combine coral bone meal with other biomaterials or growth factors to form composite materials with better mechanical properties or biological activities. This technology can enhance the repair effect of coral bone meal and improve the success rate of bone defect repair; coral bone meal scaffold implantation, which processes coral bone meal into a scaffold and implants it into the bone defect site as a support framework and a structure to promote healing. The scaffold can provide sufficient mechanical support and is conducive to the growth of bone cells and the regeneration of bone tissue.

[0003] Although coral materials have good biocompatibility, their performance in inducing bone regeneration in surrounding tissues is limited. The rapid dissolution of CaCO 3 in coral scaffolds limits their clinical application. Determining the ideal combination of coral species with appropriate mechanical strength and cell compatibility is crucial for the application effect. Directly applying coral skeletons to the field of regenerative biomedicine has poor environmental sustainability. Since using corals will affect the coral reef ecosystem, the sustainability of the material source and environmental friendliness need to be considered. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the present invention provides a recombinant coral protein and a preparation method thereof to solve the problems that the performance of the above-mentioned coral materials in inducing bone regeneration in surrounding tissues is limited, the rapid dissolution of CaCO 3 in coral scaffolds limits their clinical application, determining the ideal combination of coral species with appropriate mechanical strength and cell compatibility is crucial for the application effect, directly applying coral skeletons to the field of regenerative biomedicine has poor environmental sustainability, and since using corals will affect the coral reef ecosystem, the sustainability of the material source and environmental friendliness need to be considered, and other technical problems.

[0005] To achieve the above object, the solution of this application is as follows: In the first aspect, the present invention provides a preparation method of a recombinant coral protein, including the following steps: S1. Construct an expression vector: Clone the coding sequence of the galaxin gene into the pcDNA3.1 expression vector to obtain the expression vector; S2. Cell culture and transfection: The cells are cultured in a medium containing fetal bovine serum until the cell density reaches 80 - 90%, and the expression vector is transfected into the cells to obtain transfected cells. S3. Protein expression and culture: The transfected cells are continuously cultured. S4. Cell lysis and protein extraction: The transfected cells continuously cultured in step S3 are collected, washed and lysed, and the lysate is centrifuged to obtain the recombinant coral protein.

[0006] Optionally, in step S1, cloning the coding sequence of the galaxin gene into the pcDNA3.1 expression vector includes: respectively digesting the coding sequence of the galaxin gene and the pcDNA3.1 expression vector with enzymes, and ligating the digested products.

[0007] Optionally, in step S1, restriction endonucleases are used for digestion.

[0008] Optionally, in the medium of step S2, the concentration of the fetal bovine serum is 8% - 12%.

[0009] Optionally, in step S2, the culture temperature is 35 - 40 °C.

[0010] Optionally, in step S4, cell lysate is used to lyse the transfected cells continuously cultured in step S3.

[0011] Optionally, after centrifugation, the following step is further included: purifying the supernatant obtained by centrifugation using an affinity column.

[0012] In a second aspect, the present application also provides a recombinant coral protein prepared according to the method described above.

[0013] Advantages of the present invention: The recombinant coral protein of the present application exhibits excellent bone regeneration ability and can be used in bone tissue engineering.

[0014] The recombinant coral protein of the present application can promote osteogenesis after defect. Brief description of the drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the structures shown in these drawings.

[0016] Figure 1Results graphs of in vivo and in vitro osteogenesis assays. 1A shows Galaxea fascicularis in an aquarium. 1B shows the organic part of the exoskeleton of Galaxea fascicularis visible when it is in water. 1C shows the predicted structure of the recombinant coral protein. 1D shows the His-tagged fusion construct transfected into HEK293 cells and the results of analyzing and detecting the His-tagged fusion protein by Western blotting. 1E shows the results of QRT-PCR analysis of osteogenesis-related gene expression in MC3T3-E1 cells incubated with different concentrations of galaxin. 1F shows the results graph of alkaline phosphatase staining of MC3T3-E1 cells treated with different concentrations of galaxin. 1G shows the results graph of alizarin red staining of MC3T3-E1 cells treated with different concentrations of galaxin. relative mRNA expression level represents the relative mRNA expression level; Figure 2 Results graphs of the osteogenesis assay for promoting the osteogenesis of mouse mandibular defects by the recombinant coral protein. Control represents the control group. Detailed implementation manners

[0017] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0018] In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the fact that those of ordinary skill in the art can implement it. When the combination of technical solutions results in contradictions or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the protection scope required by the present invention.

[0019] In the following description, a large number of details are explored to provide a more thorough explanation of the embodiments of the present invention. However, it is obvious to those skilled in the art that the embodiments of the present invention can be implemented without these specific details.

[0020] (I) Preparation of recombinant coral protein Prepare the recombinant coral protein, which is entrusted to a biological company ( Apak Biology ) for implementation. The specific steps are as follows: S1. Construct an expression vector: Clone the coding sequence of the galaxin gene into the pcDNA3.1 expression vector. Specifically: Digest the coding sequence of the galaxin gene with a restriction endonuclease: Mix the coding sequence of the galaxin gene and the restriction endonuclease at a molar ratio of 1:10 to 1:50 to obtain a DNA fragment containing the coding sequence of the galaxin gene; Synthesize the his-tag linker and add restriction endonuclease sites compatible with the target gene at both ends of the linker.

[0021] Digest the pcDNA3.1 expression vector with a restriction endonuclease: Mix the same restriction endonuclease as the coding sequence of the galaxin gene and the pcDNA3.1 expression vector (commercially available) at a molar ratio of 1:10 to 1:50 to obtain the digestion product of the pcDNA3.1 expression vector; Ligate the digestion products to obtain the expression vector: Mix the coding sequence of the galaxin gene, the digestion product of the pcDNA3.1 expression vector, and the his-tag linker at a ratio of 3:10:1 (unit: 10 μg) to obtain the expression vector; S2. Cell culture and transfection: Place HEK293 cells in DMEM medium containing 10% fetal bovine serum and incubate them in an incubator at 37°C and 5% CO 2 2. When cultured to the logarithmic growth phase and the cell density reaches 80 - 90%, transfect the HEK293 cells with the expression vector obtained in step S1 using Lipofectamine 2000. The specific steps are as follows: Dilute the expression vector obtained in step S1 at 0.1 to 1 μg / μL in serum-free medium, mix the diluted Lipofectamine 2000 and the expression vector at a ratio of 1:2 to 1:4, and incubate at room temperature for 20 min. Add the transfection complex to the HEK293 cell culture dish, gently shake the culture dish, and place the HEK293 cells in an incubator at 37°C and 5% CO 2 2 for 48 h, and confirm the transfection efficiency by fluorescence labeling. Observe the transfection efficiency using a fluorescence microscope or a flow cytometer to obtain the transfected cells; S3. Protein expression and culture: Place the transfected cells in an incubator at 37°C and 5% CO 2 2 and continue to culture for 3 days, and then induce the HEK293 cells to express and produce galaxin protein (i.e., coral protein) through cell culture; S4. Cell lysis and protein extraction: After 3 days, collect the HEK293 cells and wash them 3 times with PBS buffer; Place the washed HEK293 cells in cell lysis buffer to dissolve the cell membrane with the lysis buffer and release the internal galaxin protein (i.e., coral protein); The centrifuged lysate was collected to obtain the supernatant. The His affinity column was used for purifying the galaxin protein (the principle is: specific binding of six histidines (His-tag) to nickel ions for protein purification, and His-tag is usually fused to the N-terminus or C-terminus of the recombinant protein to facilitate purification using the His affinity column). Specifically: The His affinity column was connected to the chromatography column. After equilibration of the column with 8 column volumes of equilibration buffer, the recombinant protein sample containing His-tag (i.e., the supernatant) was loaded onto the equilibrated His affinity column. The column was washed with 8 column volumes of wash buffer to remove unbound proteins and other impurities, and then the His-tag protein was eluted with elution buffer to obtain the purified galaxin protein (coral protein). The BCA method was used to determine the concentration of the purified galaxin protein. After detection, the concentration of the purified galaxin protein was 0.5 mg / mL.

[0022] SDS-PAGE and Western blot were used to analyze the purity and quality of the purified galaxin protein. After detection, the molecular weight of the purified galaxin protein was consistent with the literature report (53 kda).

[0023] (II) In vitro osteogenesis assay of recombinant coral protein The in vitro osteogenesis assay was performed on the recombinant coral protein (i.e., the purified coral protein) prepared in (I). The specific steps were as follows: Cell culture: MC3T3-E1 cells were seeded in 6-well plates and placed in an incubator at 37°C and 5% CO 2 to continue culturing until 80% confluence was reached. Coral protein treatment: MC3T3-E1 cells at 80% confluence were divided into a control group and coral protein treatment groups with different concentrations. Each group had 3 replicates. The control group was not supplemented with coral protein, and the final concentrations of coral protein in the treatment groups were 0.1 ng / mL, 1 ng / mL, 10 ng / mL, and 100 ng / mL, respectively. They were placed in an incubator at 37°C and 5% CO 2 to continue culturing for 4 days. QRT-PCR reaction: Total cellular RNA was extracted using commercially available Trizol reagent according to the instructions. RNA was reverse transcribed into cDNA using a commercially available reverse transcription kit. QRT-PCR reaction was performed using SYBR Green PCR kit (TAKARA), and the target genes (runx2, alpl, sp7, bglap) and the internal reference gene (β-actin) were amplified using the corresponding specific primers. Data analysis: The 2^-ΔΔCt method was used to analyze the QRT-PCR data, and the relative expression levels of the target genes among different treatment groups were compared. The results are shown in Figure 1 Figure E.

[0024] Alkaline phosphatase (ALP) staining: The medium was removed, and the cells were fixed with 4% paraformaldehyde solution for 15 min. The cells were stained with a commercially available ALP staining reagent, and the ALP activity of the cells was observed under a microscope. The appearance of blue-black precipitates indicated ALP positivity. The results are shown in Figure 1 Figure F.

[0025] Alizarin red staining: The medium was removed, and the cells were fixed with 4% paraformaldehyde solution for 15 min. The cells were stained with alizarin red staining solution for 1 h, and the calcification of the cells was observed under a microscope. The appearance of red or orange precipitates indicated calcification positivity. The results are shown in Figure 1 Figure G.

[0026] As can be seen from Figure 1 Figures E to G, the expression levels of osteogenic genes (Runx2, Alpl, Sp7, Bglap) treated with 0.1 ng / mL galaxin were significantly higher than those of the control group. As the concentration of galaxin increased, the expression levels of osteogenic genes showed a bell-shaped curve pattern. Qualitative analysis of ALP and alizarin red staining showed that in the experimental group, 0.1 ng / mL galaxin mediated the highest levels of ALP staining and alizarin red staining of the cells. The results indicate that the recombinant coral protein of the present application can significantly promote the expression of osteogenic genes in MC3T3-E1 osteoblasts, as well as the expression of alkaline phosphatase and alizarin red.

[0027] And AlphaFold3 was used to predict the structure of the recombinant coral protein. The results are shown in Figure 1 Figure C.

[0028] In summary Figure 1 it can be seen that in in vitro tests, the recombinant coral protein of the present application all showed excellent bone regeneration ability and can be used in bone tissue engineering.

[0029] (III) Osteogenic test of recombinant coral protein in promoting mandibular bone defect in mice An in vivo osteogenic test was performed on the recombinant coral protein (i.e., purified coral protein) prepared in (I). The specific steps were as follows: 20 mL of PBS buffer was added to a brown bottle containing the initiator LAP (i.e., lithium phenyl-2,4,6-trimethylbenzoylphosphinate) (containing 0.05 g LAP), and it was heated and dissolved at a water bath temperature of 45 °C for 15 min to obtain a standard solution of LAP initiator with a concentration of 0.25% (w / v); The initiator LAP (i.e., lithium phenyl-2,4,6-trimethylbenzoylphosphinate) was added to GelMa (i.e., methacrylated gelatin), and GelMA was dissolved by heating in a water bath at 65 °C in the dark for 25 min with several oscillations during this period to obtain a 10% GelMA solution. The GelMA solution was immediately sterilized using a 0.22 μm sterile needle filter; The recombinant coral protein was dispersed in the GelMA solution at final concentrations of 0 ng / mL (control group), 100 ng / mL (galaxin-100 group), and 1000 ng / mL (galaxin-1000 group) to form an injectable coral protein-GelMA complex; A 5-mm incision was made in the skin of the mouse mandible, the masseter muscle was bluntly dissected, and the periosteum was incised. On the mandible, a full-thickness penetrating defect with a diameter of 2.3 mm was created using a dental drill under continuous irrigation with 0.9% saline; the injectable coral protein-GelMA complex was filled into the mouse mandibular defect and irradiated with a 405-nm light source for 30 seconds to cause gelation.

[0030] The coral protein of different groups was injected at the defect site. Immediately after the surgery, the coral protein-GelMA complex was implanted into the penetrating defect area, and then the wound was sutured layer by layer. After 8 weeks, micro-CT was used to measure the BMD, BV / TV, etc. in the bone defect area, and the results are as Figure 2 shown.

[0031] It can be seen from Figure 2 that compared with the control group, the relative bone mineral density (i.e., BMD) of the galaxin-100 group and the galaxin-1000 group was significantly increased, and the bone volume / tissue volume ratio (BV / TV) was significantly increased. It can be seen from Figure 2 Figures B and 2C that the recombinant coral protein of this application exhibits excellent in vivo bone regeneration performance, indicating that the recombinant coral protein can be used in bone tissue engineering. The results show that the recombinant coral protein of this application can promote osteogenic repair of the mouse mandible, indicating that the recombinant coral protein of this application can promote osteogenesis after defect.

[0032] The above embodiments only exemplarily illustrate the principles and effects of the present invention, rather than limiting the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A method for preparing a recombinant coral protein, characterized in that: The following steps are involved: S1. Construction of expression vector: clone the coding sequence of galaxin gene into pcDNA3.1 expression vector to obtain expression vector; S2. Cell culture and transfection: The cells are cultured in a culture medium containing fetal bovine serum until the cell density reaches 80-90%, and the expression vector is transfected into the cells to obtain transfected cells; S3. Protein expression and culture: continuing to culture the transfected cells; S4. Cell lysis and protein extraction: Collect, wash and lyse the transfected cells that continue to be cultured in step S3, centrifuge the obtained lysate, and obtain the recombinant coral protein.

2. The method for preparing the recombinant coral protein according to claim 1, characterized in that: In step S1, cloning the coding sequence of the galaxin gene into the pcDNA3.1 expression vector includes: performing enzyme digestion on the coding sequence of the galaxin gene and the pcDNA3.1 expression vector respectively, and connecting the enzyme digestion products.

3. The method for preparing the recombinant coral protein according to claim 2, characterized in that: In step S1, restriction endonucleases are used for enzyme digestion.

4. The method for preparing the recombinant coral protein according to claim 1, characterized in that: In the culture medium of step S2, the concentration of fetal bovine serum is 8%-12%.

5. The method for preparing the recombinant coral protein according to claim 1, characterized in that: In step S2, the culture temperature is 35-40°C.

6. The method for preparing the recombinant coral protein according to claim 1, characterized in that: In step S4, the transfected cells that were cultured in step S3 are lysed using a cell lysis solution.

7. The method for preparing the recombinant coral protein according to claim 1, characterized in that: After centrifugation, the method further comprises the following steps: purifying the supernatant obtained by centrifugation using an affinity column.

8. The recombinant coral protein obtained according to the method according to any one of claims 1 to 7.

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