A recombinant chimeric protein SAF2 and its preparation method and application

Abstract

The application discloses a recombinant chimeric protein SAF2 and a preparation method and application thereof. Based on an active short peptide chimeric strategy, a human FGF2 mature peptide and human amelogenin N-terminal and C-terminal active short peptides are connected in series to construct a recombinant chimeric protein SAF2, and the amino acid sequence of the recombinant chimeric protein SAF2 is shown as SEQ ID NO. 2. The recombinant chimeric protein SAF2 retains the cell proliferation and migration activity of FGF2 and the osteogenic differentiation activity of amelogenin, and has the advantages of small molecular weight, low immunogenicity, high expression in a prokaryotic system and the like, and can be used for promoting periodontal tissue regeneration and osteogenic differentiation of periodontal ligament stem cells.

Description

Technical Field

[0001] This invention relates to the field of recombinant proteins, and particularly to a recombinant chimeric protein SAF2, its preparation method, and its applications. Background Technology

[0002] Periodontal disease is a common chronic inflammatory disease of the oral cavity that can lead to alveolar bone resorption, periodontal ligament damage, and tooth loosening and loss, seriously endangering oral and systemic health. Current clinical treatments mainly rely on mechanical scaling and guided tissue regeneration, which can control inflammation and partially repair tissue, but cannot achieve complete functional regeneration of the alveolar bone and periodontal ligament. This is primarily due to the lack of active molecules that can simultaneously promote the repair of both bone and soft tissue.

[0003] Fibroblast growth factor 2 (FGF2) promotes the proliferation, migration, and angiogenesis of various cells and is widely used in tissue repair. Amelogenin (Am) is a key matrix protein in tooth germ development, capable of inducing the regeneration of cementum and alveolar bone and promoting periodontal ligament cell differentiation. However, FGF2 readily forms inclusion bodies and is unstable in the prokaryotic system; while full-length amelogenin, although solublely expressible, has a large molecular weight and high immunogenicity, limiting its direct application in periodontal tissue engineering.

[0004] To overcome the aforementioned shortcomings, existing technologies attempt to fuse different functional proteins for expression, but often the fusion method is inappropriate, leading to decreased activity or difficulty in expression of the fused protein. Therefore, developing a novel chimeric protein with a simplified structure, possessing the dual functions of FGF2 and amelogenin, and capable of efficient expression, has significant clinical value and market prospects for the treatment of periodontal disease. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a recombinant chimeric protein, SAF2.

[0006] Another object of the present invention is to provide the encoding gene of the recombinant chimeric protein SAF2.

[0007] Another object of the present invention is to provide the application of the recombinant chimeric protein SAF2.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A recombinant chimeric protein SAF2, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The nucleotide sequence of the gene encoding the recombinant chimeric protein SAF2 is shown in SEQ ID NO.1.

[0011] The method for preparing the recombinant chimeric protein SAF2 includes the following steps:

[0012] (1) The gene sequence shown in SEQ ID NO.1 is ligated into an expression vector to obtain a recombinant expression plasmid;

[0013] (2) The recombinant expression plasmid was transformed into a prokaryotic expression strain to obtain the recombinant expression strain;

[0014] (3) The recombinant expression strain was induced to express by isopropyl-β-D-thiogalactoside (IPTG), and then the bacterial precipitate was collected to obtain the recombinant chimeric protein SAF2.

[0015] The expression vector mentioned in step (1) is preferably PET-21a(+).

[0016] The expression strain mentioned in step (1) is Escherichia coli; preferably Escherichia coli BL21 (DE3).

[0017] In step (3), the final concentration of isopropyl-β-D-thiogalactoside (IPTG) in the induction expression system is 0.1 mM.

[0018] The temperature for inducing expression in step (3) is 25℃~37℃; preferably 37℃.

[0019] The induction time in step (3) is 4 h.

[0020] The method for preparing the recombinant chimeric protein SAF2 further includes, after step (3), steps such as CM ion exchange chromatography, heparin affinity chromatography purification, protein desalting, dialysis refolding and / or ultrafiltration concentration of the recombinant chimeric protein SAF2 to obtain the purified recombinant chimeric protein SAF2.

[0021] A recombinant vector, expression cassette, and recombinant bacteria containing the encoding gene of the recombinant chimeric protein SAF2.

[0022] The recombinant bacteria are derived from Escherichia coli; preferably Escherichia coli BL21(DE3).

[0023] The application of the recombinant chimeric protein SAF2 in the preparation of products for periodontal tissue regeneration and / or osteogenic differentiation of periodontal ligament stem cells.

[0024] The products mentioned include pharmaceuticals and medical materials.

[0025] This invention utilizes an active short peptide chimerism strategy to tandemly link mature human FGF2 peptides with N-terminal and C-terminal active short peptides of human amelogenin, constructing a novel recombinant chimeric protein, SAF2. This protein is also expressed in inclusion body form, but high-purity protein preparation was achieved through CM-heparin affinity tandem chromatography. This process retains the dual functional activities of FGF2 in promoting cell proliferation and migration, and amelogenin in promoting bone differentiation, while reducing the immunogenicity of the full-length amelogenin.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] 1. In this invention, amelogenin retains only the N-terminal and C-terminal active short peptides and discards the middle non-functional region. While maintaining key biological functions such as mineralization regulation and cell proliferation, it significantly reduces the protein molecular weight, which is beneficial to improving expression efficiency and protein stability.

[0028] 2. The SAF2 chimeric protein provided by this invention has been verified to have significant effects at the cellular level, promoting the proliferation and migration of fibroblasts, periodontal ligament stem cells and osteoblast progenitor cells. It also enhances ALP activity, promotes the formation of mineralized nodules and upregulates the expression of osteogenic genes in the early stage of osteogenic induction, and has a clear application prospect for periodontal tissue regeneration. Attached Figure Description

[0029] Figure 1 This is a graph showing the expression analysis results of the pET-21a(+)-SAF2 recombinant plasmid (in the graph, lane T: whole cell disruption; lane S: supernatant after disruption; lane P: precipitate after disruption; lane (-): before induction).

[0030] Figure 2 These are the CM ion exchange chromatography electrophoresis diagram and CM ion chromatography peak diagram of inclusion body SAF2; where A is the CM ion exchange chromatography electrophoresis diagram of inclusion body SAF2 (in the figure, lane 1: dissolved sample; lane 2: sample loading and exit; lane 3: washed sample; lanes 4-11: 0.15M NaCl eluted sample; lane 12: 0.3M NaCl eluted sample; lane 13: 2M NaCl eluted sample; lane 14: 0.5M NaOH eluted sample); B is the CM ion chromatography peak diagram of inclusion body SAF2.

[0031] Figure 3The first image shows the Heparin affinity chromatography electrophoresis diagram and peak chromatogram of inclusion bodies SAF2. The first image shows the Heparin affinity chromatography electrophoresis diagram of inclusion bodies SAF2 (lane 1: dissolved sample; lane 2: sample loading and exiting; lane 3: sample eluted with 0.2 M NaCl; lanes 4-5: sample eluted with 0.3 M NaCl; lane 6: sample eluted with 0.75 M NaCl; lane 7: sample eluted with 1 M NaCl); the second image shows the Heparin affinity chromatography peak chromatogram of inclusion bodies.

[0032] Figure 4 This is an electrophoresis image of SAF2 after tandem chromatography on a CM-heparin column and dialysis refolding.

[0033] Figure 5 This is a diagram of the SAF2 identification results.

[0034] Figure 6 The graph shows the effects of SAF2, FGF2, and rhAm on the activity of PDLSCs, NIH3T3, and MC3T3-E1 cells after 48 hours (compared to the control group (concentration of 0), *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, n=3).

[0035] Figure 7 This is a graph showing the effect of SAF2 on cell migration; where AC represents the effects of SAF2, FGF2, and rhAm on the migration of NIH3T3, MC3T3-E1, and PDLSCs cells, respectively; DF represents the quantitative graphs of cell migration rates of NIH3T3 (21h), MC3T3-E1 (24h), and PDLSCs (22h) (compared with the NC group, *P<0.05, **P<0.01, n=3).

[0036] Figure 8 This is a graph showing the effects of SAF2, FGF2, and rhAm on ALP activity in MC3T3-E1 cells.

[0037] Figure 9 This is a graph showing the effects of SAF2, FGF2, and rhAm on ALP activity in PDLSCs cells.

[0038] Figure 10 This is a diagram showing the influence of SAF2, FGF2, and rhAm on the formation of mineralized nodules in PDLSCs.

[0039] Figure 11 The effects of SAF2, FGF2, and rhAm on the expression of osteogenic differentiation-related genes in PDLSCs (*P<0.05, n=3 compared with the NC group). Detailed Implementation

[0040] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0041] Example 1

[0042] 1. Experimental materials and reagents

[0043] 1.1 Materials:

[0044] PET-21a(+)-SAF2 (Genscript Biotechnology); Plasmid Mini Kit I (Omega); HiTrap CM FF 5 mL weak cation exchanger (Cytiva); HiTrap HeparinHP 5 mL heparin affinity chromatography column (Cytiva); PD-10 desalting column (Cytiva); standard dialysis bag (Yuanye Company); CCK-8 kit (Synthetic Biotech); osteogenic differentiation induction kit (Oricell); alkaline phosphatase assay kit (Beyotime); rapid RNA extraction kit (Albatross); reverse transcription premixed kit (Aikerui Biotechnology).

[0045] 1.2 Reagent Preparation:

[0046] Denaturation buffer: 6 mol / L urea, 2 mmol / L EDTA (ethylenediaminetetraacetic acid), 5 mmol / L DTT (dithiothreitol), 10% (v / v) glycerol, basal buffer PBS, pH 7.0;

[0047] Refolding buffer: 10% (v / v) glycerol, basal buffer PBS, pH 7.0;

[0048] Equilibration buffer: 8 mol / L urea, 2 mmol / L EDTA, 5 mmol / L DTT, basal buffer 20 mmol / L PB, pH 6.9;

[0049] Elution buffer: 8 mol / L urea, 2 mmol / L EDTA, 5 mmol / L DTT, 2 mol / L NaCl, basal buffer 20 mmol / L PB, pH 6.9.

[0050] 2. Construction of SAF2 recombinant bacteria

[0051] The SAF2 gene sequence consists of three tandemly linked parts: its N-terminus and C-terminus correspond to the coding sequences of the N-terminal and C-terminal active short peptides of human amelogenin, respectively, while the middle region codes for the mature peptide of human FGF2. These functional fragments are linked by flexible glycine-serine linkers, and the final sequence (SEQ ID NO.1) was obtained after codon optimization. Specifically, 1–96 bp encodes the N-terminal active short peptide of human amelogenin, 97–129 bp encodes the first flexible linker peptide, 130–594 bp encodes the mature peptide of human FGF2, 595–627 bp encodes the second flexible linker peptide, and 628–705 bp encodes the C-terminal active short peptide of human amelogenin. This sequence was then ligated into an expression vector to construct the recombinant expression plasmid pET-21a(+)-SAF2, which was subsequently transformed into the host bacterium BL21(DE3). This process was outsourced to GenScript Biotech.

[0052] The SAF2 codon-optimized gene sequence (SEQ ID NO.1) is shown below:

[0053] ATGCCTCTACCACCGCACCCCGGACATCCCGGCTACATCAACTTCAGCTATGAAGTGCTGACTCCGCTGAAGTGGTACCAGAGCATTAGACCTCCAGGTGGCGGAGGCTCTGGTGGCGGTCGCGGTTCCATGGCAGCGGGTAGCATCACGACCTTACCGGCATTGCCGGAAGATGGCGGTTCCGGCGCGTTTCCGCCGGGTCACTTTAAAGATCCGAAACGTTTGTACTGCAAGAACGGTGGCTTTTTCCTGCGTATCCACCCGGATGGCCGTGTTGACGGCGTTCGTGAGAAAAGCGACCCGCATATTAAGCTGCAGCTGCAAGCGGAAGAACGTGGTGTCGTGAGCATCAAGGGTGTATGTGCTAATCGTTATTTGGCTATGAAAGAAGACGGCCGGCTGCTGGCGAGCAAGTGCGTTACCGACGAGTGCTTTTTCTTCGAGCGCTTGGAGAGCAATAACTATAACACCTATCGTAGCCGTAAATACACCAGCTGGTACGTGGCCCTGAAGCGCACCGGTCAGTACAAACTGGGTTCGAAGACGGGTCCGGGCCAAAAAGCGATTCTGTTCCTTCCGATGTCAGCCAAAAGCGGTCGTGGCGGCGGTTCTGGTGGTGGCGGGTCCCCGCTCCCACCGATGCTGCCGGACCTGACCTTGGAGGCGTGGCCGTCTACCGATAAGACCAAACGCGAGGAAGTTGAC。

[0054] The codon-optimized amino acid sequence of SAF2 (SEQ ID NO. 2) is shown below:

[0055] MPLPPHPGHPGYINFSYEVLTPLKWYQSIRPPGGGGSGGGRGSMAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIK GVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKSGRGGGSGGGGSPLPPMLPDLTLEAWPSTDKTKREEVD.

[0056] The molecular weight and isoelectric point of SAF2 were predicted to be 25.5 kDa and 9.46 kDa using the ProtParam online tool.

[0057] 3. Prokaryotic expression

[0058] (1) Activation of bacterial strain: When activating the SAF2 recombinant bacterial strain under aseptic conditions, take the glycerol bacterial tube stored at -80℃ and place it directly on ice. Use a sterile pipette tip to scrape off a small amount of frozen bacterial growth. Then transfer the obtained bacterial suspension or bacterial growth to a fresh ampicillin (Amp) resistant plate (working concentration of ampicillin 100 mg / L) for streaking. Finally, place the inoculated plate in a constant temperature incubator at 37℃ for incubation until new single colonies grow.

[0059] (2) Induction of SAF2 expression: single colonies were picked and inoculated into 5 mL of LB medium with Amp resistance (100 μg / mL) in a test tube and incubated overnight (12-14 h) at 37℃ and 220 rpm in a shaker. 1% (v / v) of the overnight bacteria was inoculated into a 250 mL shake flask of LB medium with Amp resistance and amplified at 37℃ and 220 rpm for 2 h (OD value reached 0.6-0.8). 1 mL of the amplified bacterial solution before induction was stored at -20℃. Then, 0.1 mmol / L IPTG (isopropyl-β-D-thiogalactopyranoside) inducer was added to the shake flask and induced at 150 rpm in a shaker for 4 h. The culture was incubated at 16℃, 25℃, 30℃ and 37℃ in separate shake flasks. The shake flasks were incubated overnight at 16℃.

[0060] (3) Sterilization and SDS-PAGE: After sterilization using a microfluidic homogenizer, the bacterial solution before induction, the whole bacteria after rupture, the supernatant after rupture, and the precipitate after rupture were analyzed by SDS-PAGE.

[0061] (4) Results: To determine the expression form (soluble or insoluble) of pET-21a(+)-SAF2 in Escherichia coli BL 21(DE 3), the induced expression bacterial culture was homogenized and the components were collected for SDS-PAGE. Figure 1 As shown, the uninduced bacterial culture did not express SAF2 (SAF2 was 25.5 kDa). SAF2 induced by 0.1 M IPTG was primarily expressed as inclusion bodies. Low-temperature expression of SAF2 at 16℃, 25℃, and 30℃ did not increase soluble expression levels; instead, it reduced overall SAF2 expression. The final determined expression temperature for SAF2 was 37℃. This indicates that recombinant SAF2 is highly expressed in E. coli in the form of inclusion bodies.

[0062] 4. Purification and Identification

[0063] 4.1 Sample preparation:

[0064] The bacterial culture induced by pET-21a(+)-SAF2 plasmid was centrifuged at 8000 rpm for 15 min, the culture medium was discarded, and the cells were resuspended in pre-chilled PBS buffer. The culture was then homogenized using a microfluidic autoclave. After homogenization, the bacterial culture was centrifuged at 12000 rpm for 30 min to precipitate SAF2 inclusion bodies. Soluble components in the supernatant were discarded. The inclusion bodies were then resuspended in equilibration buffer for purification chromatography, the inclusion body solution was thoroughly vortexed, and then dissolved using a microfluidic autoclave.

[0065] 4.2 CM ion exchange chromatography and heparin affinity chromatography:

[0066] (1) Sample and buffer preparation: The sample buffer needs to be filtered through a 0.45 μm filter membrane. Other solutions that need to flow through the column, including deionized water, equilibration buffer, elution buffer, alcohol, etc., also need to be filtered to prevent impurities from clogging the column.

[0067] (2) Install the chromatography column: Connect the appropriate ion exchange column to the AKTA system, ensuring the correct flow direction and avoiding air bubbles from entering.

[0068] (2) System equilibration (cleaning and equilibration): First, rinse the alcohol stored in the tubing with water until the monitored conductivity and UV baseline levels are reached, then rinse with equilibration buffer for 5 column volumes.

[0069] (3) Loading: After the baseline stabilizes, reduce the flow rate to 1-2 mL / min for loading. At this time, the target protein will bind to the ion exchange groups on the medium, while the flow-through peak records the substances that failed to bind.

[0070] (4) Washing or rebalancing: Wash away non-specific or weakly binding proteins with equilibration buffer until the UV detector signal returns to near baseline.

[0071] (5) Elution: A salt concentration (NaCl) gradient from 0% to 100% is used. As the proportion of high-salt buffer increases, competitive ions replace the target protein one by one.

[0072] (6) Collection: The separation effect can be judged based on the fluctuation of the ultraviolet absorption peak, and the components can be collected according to the changes of the ultraviolet peak.

[0073] (7) Column regeneration and preservation: After elution, thoroughly wash the column with high-salt buffer to remove all tightly bound residues. Finally, replace the salt with pure water, seal the column with 20% (V / V) ethanol to prevent microbial growth, and seal both ends of the column before removing it from the system.

[0074] (8) Detection and analysis: The collected components (SAF2 inclusion body dissolved sample, sample loading and exit, washing, elution with 0.15M NaCl, elution with 0.3M NaCl, elution with 0.5M NaOH, elution with 2M NaCl) were subjected to SDS-PAGE electrophoresis and then detected by Coomassie brilliant blue staining.

[0075] (9) Results: such as Figure 2 As shown in Figure 2 (A), recombinant SAF2 was purified by CM ion exchange chromatography, starting with elution from 0.15 M NaCl. The eluent contained other proteins. As shown in Figure 2 (B), the chromatographic peaks show three elution peaks. The first elution peak was from 0.15 M NaCl, and the eluent contained a large amount of other proteins besides the target protein SAF2. The second and third elution peaks were from 0.3 M and 2 M NaCl, respectively, and the eluent was mainly composed of SAF2. Figure 3 As shown in A, recombinant SAF2 was purified by heparin column elution with 0.3M NaCl, and the eluted protein contained contaminating proteins. Figure 3 As shown in Figure B, the chromatogram shows three elution peaks. The first elution peak is from 0.15 M NaCl elution, and the eluent contains a large amount of other proteins besides the target protein SAF2. The second and third elution peaks are from 0.3 M and 0.75 M NaCl elutions, respectively, with SAF2 being the predominant eluent. These results indicate that using CM columns and heparin columns alone yields poor purification results.

[0076] 4.3 Protein desalting:

[0077] After eluting the target protein by CM ion exchange chromatography, a desalting column is used for solution displacement to replace the target protein from the high-salt solution to the equilibration buffer for heparin affinity chromatography. The following are the steps for solution displacement using a PD-10 desalting column:

[0078] 1. Desalting column: Remove the top and bottom caps of the column and fix it vertically. Allow the stock solution in the column to drain naturally, then equilibrate the column with the desired equilibration buffer (or deionized water), adding a total of 25 mL (usually in 5 portions of 5 mL each), allowing each portion to drain completely before adding the next.

[0079] 2. Sample loading: After the final equilibration buffer has drained, carefully add the sample to the center of the gel bed. The maximum loading volume is 2.5 mL (1 / 4 of the column volume). Allow the sample to completely enter the gel bed.

[0080] 3. Elution and Collection: After the sample liquid surface has dried, add an appropriate amount of equilibration buffer for elution. Immediately begin collecting the eluent, collecting the elution peak according to the target molecular weight (usually proteins are in the first 1-3 mL of eluent, and salt ions are in subsequent eluents).

[0081] 4. Regeneration or Disposal: After use, reequilibrate the column with deionized water, and finally equilibrate and store it with 20% (V / V) ethanol to inhibit microbial growth.

[0082] 4.4 Dialysis refolding:

[0083] The target protein solution eluted by CM-heparin column tandem chromatography contains high concentrations of urea and NaCl. Dialysis with a dialysis bag can desalt, replace the solution, and refold the protein. The following is the dialysis procedure:

[0084] (1) Pretreatment of dialysis bags: The size of SAF2 protein is 25.5 kDa. Dialysis bags with a molecular weight cutoff of 10 kDa are selected, cut into appropriate lengths, boiled in 2% (w / v) sodium bicarbonate solution containing 1 mmol / L EDTA for 10 minutes, then thoroughly washed with distilled water, boiled in distilled water for 10 minutes, cooled and soaked at 4℃ for later use.

[0085] (2) Sample loading: Slowly inject the denatured protein solution containing high concentration of urea into the prepared dialysis bag, leaving 1.5-2 times the volume of space to prevent bursting. Seal both ends with dialysis clamps to ensure no leakage.

[0086] (3) Gradient dialysis equilibration: Place the dialysis bag containing the sample into pre-cooled denaturing buffer (4°C, with slow magnetic stirring), and gradually reduce the denaturant (urea) concentration by diluting with refolding buffer. Use gradient dialysis, i.e., successively reduce the denaturant concentration (from 6 mol / L, 4 mol / L, 2 mol / L to 0 mol / L), with each step dialysis lasting 4-8 hours.

[0087] (4) Post-renaturation treatment: After the denaturant is completely removed, continue dialyzing in refolding buffer overnight to stabilize the protein structure. Remove the contents of the dialysis bag, centrifuge at 2000 rpm for 30 minutes at 4°C, collect the supernatant to obtain the refolded protein, and detect the refolding effect by SDS-PAGE.

[0088] (5) Results: such as Figure 4 As shown, the SAF2 protein eluted by CM ion exchange chromatography was desalted by a PD-10 desalting column and then purified by heparin affinity chromatography. After dialysis and refolding, the target protein obtained was virtually free of impurities. This indicates that using CM and heparin columns in series can yield SAF2 protein with high purity.

[0089] 4.5 Ultrafiltration Concentration:

[0090] (1) Before ultrafiltration, add 5% (w / v) trehalose to the protein solution to protect the stability of the protein structure. Select a 10 kDa ultrafiltration tube. Before use, add ultrapure water to completely cover the membrane surface and perform a brief centrifugation rinse. Add the sample to the upper filter inner tube. The liquid level should not exceed the maximum graduation line (white line) indicated by the manufacturer. The operation should be gentle. Centrifuge at 3000-5000 × g (15 mL centrifuge tube) at 4℃. Concentrate the protein to 1-2 mL. After centrifugation, remove the filter inner tube and invert it to insert it into a new collection tube. Centrifuge at a low speed of 1000 × g for 1 minute to transfer the retained concentrate from the inner tube to the collection tube. Avoid prolonged drying of the ultrafiltration membrane during use.

[0091] (2) Protein concentration assay: After appropriate dilution of the concentrated protein, the protein concentration was detected by BCA kit. The SAF2 protein was stored at -80℃.

[0092] 4.6 Western Blot Identification of SAF2

[0093] First, perform SDS-PAGE electrophoresis. After electrophoresis, do not perform Coomassie brilliant blue staining. Follow the steps below:

[0094] (1) Transfer: The protein gel separated by SDS-PAGE electrophoresis is combined with a PVDF membrane (which needs to be activated with methanol beforehand) and filter paper to form a "sandwich" structure (in the order of negative electrode-sponge-filter paper-gel-membrane-filter paper-sponge-positive electrode), and placed in the transfer tank. The protein is transferred from the gel to the solid membrane under the action of an electric field by wet transfer.

[0095] (2) Blocking: After the transfer is completed, cut the membrane according to the molecular weight of the target protein, put the membrane into the blocking solution (5% (v / v) skim milk) and incubate at 60 rpm for 1-2 hours at room temperature (or overnight at 4°C) to block the non-specific sites of unbound proteins on the membrane and reduce background interference.

[0096] (3) Primary antibody incubation: Discard the blocking solution, add the primary antibody (YP-Ab-15832) diluted with antibody dilution solution according to the ratio, and incubate overnight at 4℃ on a shaker at 60 rpm to allow the primary antibody to specifically bind to the target protein on the membrane.

[0097] (4) Secondary antibody incubation: After recovering the primary antibody, wash the membrane 3-5 times with washing buffer (TBST) at 130 rpm on a shaker for 5-10 minutes each time. Then add the enzyme-labeled HRP secondary antibody (Fode Bio FD0060) corresponding to the species of the primary antibody and incubate at room temperature for 1-2 hours.

[0098] (5) Colorimetric detection: Discard the secondary antibody and wash the membrane 3-5 times at 130 rpm on a shaker for 5-10 minutes each time. After thorough washing, add ECL chemiluminescent substrate and acquire the signal using a chemiluminescence imaging system.

[0099] (6) Results: The identification results are as follows Figure 5 As shown.

[0100] 5. In vitro activity assay

[0101] 5.1 CCK-8 test experiment:

[0102] (1) Seeding: Periodontal ligament stem cells (PDLSCs, Shenzhen Youli Biotechnology Co., Ltd.), mouse embryonic fibroblasts (NIH3T3, Pronosai), and mouse embryonic osteoblast precursor cells (MC3T3-E1, ATCC) were seeded into 96-well plates containing 100 μL / well medium (α-MEM / DMEM + 10% (v / v) fetal bovine serum (FBS)), with 4000 cells per well. No cells were seeded around the outer edge of the 96-well plate. An equal volume of PBS buffer was added, and the plates were incubated for 6 h.

[0103] (2) Starvation: After the cells have completely adhered to the wall for 6 hours, change the medium and replace it with 100 μL of low serum medium (α-MEM / DMEM + 0.4% (v / v) FBS) per well. Then, put the cells back into the incubator for starvation overnight.

[0104] (3) Drug administration: After starvation for 14-16 h, the cells were replaced with 100 μL of low serum medium with different treatments in each well. The experimental group (SAF2 protein correctly identified in 4.6 above, with concentrations of 0.1, 0.5, 2.5 and 12.5 μg / ml, respectively), blank group (without cells) and 3 control groups (without protein treatment (concentration of 0 μg / ml), human amelogenin (purchased from Tianzhengyuan Biotechnology, catalog number TP01145, trade name Recombinant Amelogenin, X-Linked (AMELX) with the same concentration as the experimental group), and fibroblast growth factor 2 (FGF2, Yiqiao Shenzhou 10014-HNAE) with the same concentration as the experimental group), with 4 replicates in each well.

[0105] (4) CCK-8 detection: 48 h after administration, the old culture medium was discarded and 100 μL of fresh low serum culture medium containing CCK-8 solution was added to each well (CCK-8 and culture medium were 1:10). The medium was incubated for 1-2 h (OD value of blank group was 0.8-1.0). The whole process was kept in the dark. After incubation, the absorbance value at 450 nm was detected.

[0106] (5) Calculation: Cell viability is calculated according to the following formula: Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%; where, As: absorbance of experimental wells; Ac: absorbance of control wells; Ab: absorbance of blank wells.

[0107] (6) Results: such as Figure 6 As shown, the purified recombinant SAF2 protein had no toxic effect on NIH3T3, PDLSCs, and MC3T3-E1 cells and significantly promoted cell proliferation. rhAm at a concentration of 12.5 μg / mL had no significant effect on the viability of MC3T3-E1 and PDLSCs cells; however, at this concentration, both FGF2 and SAF2 promoted the proliferation of MC3T3-E1 and PDLSCs cells. These results indicate that SAF2 and FGF2 can significantly promote the proliferation of NIH3T3, PDLSCs, and MC3T3-E1 cells.

[0108] 5.2 Cell scratches:

[0109] (1) Pre-experiment preparation and marking: First, use a marker pen to draw horizontal lines evenly on the back of the six-well plate (approximately every 1 cm) as observation and positioning marks. After digesting and resuspending cells in the logarithmic growth phase (PDLSCs, NIH3T3, and MC3T3-E1), use approximately 5 × 10⁶ cells per well. 5100 cells (the specific density varies depending on the cell type, but the principle is that they can grow to full size overnight) are seeded into well plates and cultured in an incubator until the cell confluence reaches 95% to form a monolayer of cells.

[0110] (2) Starvation: Discard the old culture medium (α-MEM / DMEM + 10% (v / v) FBS), add low serum culture medium (α-MEM / DMEM + 0.4% (v / v) FBS), put it back into the incubator, and starve for 14 h.

[0111] (3) Creating a scratch: Using a 200μL sterile pipette tip, hold it vertically against the bottom of the well plate (perpendicular to the horizontal line on the back) and quickly and evenly scratch across the monolayer of cells to create a cell-free scratch area. Maintain consistent pressure during the scratching process to ensure that the scratch width is basically uniform in each well.

[0112] (4) Cleaning and removal of cell debris: After scratching, discard the old culture medium immediately. Gently rinse the cell layer 2-3 times with sterile PBS buffer to remove the cell debris generated by the scratching and avoid it interfering with the migration results.

[0113] (5) Dosing of drugs and culture:

[0114] After washing, add fresh culture medium containing SAF2 to each well. At this time, take a picture under a microscope (usually recorded as 0 hours) and record the initial scratch width. At the same time, use serum-free or low-serum culture medium as a control (NC).

[0115] (6) Observation and data collection: Return the well plate to the incubator for further incubation. Depending on the experimental needs, remove the plate at specific time points (e.g., 21 hours, 22 hours, 24 hours, 48 ​​hours) and take photos at the same marked locations. Pay attention to the consistency of contrast, brightness, and location when taking photos.

[0116] (7) Data Analysis: Use ImageJ image analysis software to measure the width or area of ​​the scratch at different time points and calculate the cell migration rate. The formula is: (0-hour width - specified time width) / 0-hour width × 100%.

[0117] (8) Results:

[0118] NIH3T3, PDLSCs, and MC3T3-E1 cells were treated with 20 μg / mL, 5 μg / mL, and 10 μg / mL SAF2, respectively, for 21 h, 24 h, and 22 h, respectively. Figure 7As shown, SAF2 significantly promoted the migration of NIH3T3, PDLSCs, and MC3T3-E1 cells, with NIH3T3 and MC3T3-E1 showing extremely significant promotion of migration (P < 0.01). These results indicate that SAF2 can significantly promote the migration of NIH3T3, PDLSCs, and MC3T3-E1 cells.

[0119] 5.3 Osteogenesis Induction

[0120] 5.3.1 The osteogenic-induced alkaline phosphatase staining experiment of MC3T3-E1 cells was performed using the following method:

[0121] (1) Pre-treatment: Cells were cultured in ordinary complete medium (α-MEM / DMEM + 10% (v / v) FBS) to ensure normal cell growth. They were then plated at low density. After 8 h of plating, the following treatments were administered: blank group, SAF2 group, FGF2 group, and rhAm group. The treatment concentrations for the SAF2 and rhAm groups were 0.1 μg / ml, and the treatment concentration for the FGF2 group was 0.01 μg / ml. No drug was added to the blank group. Three days after administration, the treatment was stopped, and the cells were cultured in commercial osteogenic differentiation-inducing medium (Oricell) (ordinary complete medium + osteogenic differentiation inducers including sodium β-glycerophosphate, ascorbic acid, dexamethasone, etc.) for 7 days. The experiment was conducted in triplicate.

[0122] 5.3.2 Related experiments on PDLSCs cells:

[0123] For the osteogenic differentiation induction experiments of PDLSCs, pre-drug administration was performed, and the method was the same as that for MC3T3-E1 cells described above. Specifically, the alkaline phosphatase staining experiment and osteogenic differentiation gene induction experiment were performed with pre-drug administration for 3 days and induction for 7 days. The Alizarin Red staining experiment for calcium nodule formation was performed with pre-drug administration for 3 days and induction for 14 days. In the alkaline phosphatase staining experiment, the treatment concentrations for the SAF2 group were 0.1 μg / ml and 5 μg / ml, the treatment concentration for the rhAm group was 0.1 μg / ml, and the treatment concentrations for the FGF2 group were 0.01 μg / ml and 0.25 μg / ml. No drug was added to the blank group.

[0124] 5.4 Alkaline phosphatase (ALP) staining experiment

[0125] (1) After osteogenic induction culture in 5.3, the culture medium was aspirated and the cells were gently rinsed once with PBS;

[0126] (2) Remove PBS, add 4% paraformaldehyde, and fix for 15-20 min;

[0127] (3) Remove paraformaldehyde, add PBS and rinse for 5 min, rinse 3-5 times;

[0128] (4) Prepare staining solution according to the instructions of alkaline phosphatase test kit (Beyotime), discard PBS, add staining solution, and stain in the dark.

[0129] (5) After the staining difference between the control group and the treatment group was observed, the staining solution was aspirated, PBS was added, the staining was terminated, and the image was taken and recorded under a microscope.

[0130] (6) Results: Alkaline phosphatase is an early marker for identifying osteogenic differentiation of cells. The effects of FGF2, SAF2, and rhAm on ALP activity in MC3T3-E1 and PDLSCs cells were detected by ALP staining. In the detection, the color intensity represented the level of enzyme activity. MC3T3-E1 cells were pre-treated for 3 days and induced for 7 days before osteogenic differentiation. Figure 8 The results showed that rhAm (0.1 μg / mL) and SAF2 (0.1 μg / mL) enhanced ALP activity, while FGF2 (0.01 μg / mL) had no significant effect on ALP activity. Pre-drug administration for 3 days prior to osteogenic differentiation induction of PDLSCs, followed by 7 days of induction, resulted in… Figure 9 The results indicate that the effects of FGF2, rhAm, and SAF2 on ALP activity in PDLSCs were largely consistent with those in MC3T3-E1 cells. Furthermore, the results showed that increasing the concentration of FGF2 (0.25 μg / mL) and SAF2 (5 μg / mL) inhibited ALP activity. These results suggest that SAF2 and rhAm can enhance ALP activity in both PDLSCs and MC3T3-E1 cells during the early stages of cell differentiation induction.

[0131] 5.5 Alizarin Red Staining Experiment

[0132] (1) After osteogenic induction culture in 5.3, the PDLSCs cells were aspirated and the culture medium was removed. The cells were then gently rinsed once with PBS.

[0133] (2) Remove PBS, add 4% paraformaldehyde, and fix for 15-20 min;

[0134] (3) Remove paraformaldehyde, add PBS and rinse for 5 min, rinse 3-5 times;

[0135] (4) Discard the PBS, add alizarin red staining solution, and stain in the dark.

[0136] (5) After the staining difference between the control group and the treatment group was observed, the staining solution was removed, deionized water was added, and the results were recorded by microscopy.

[0137] (6) Results: Mineralized nodules are a marker of osteoblast maturation and the main morphological manifestation of osteoblast-driven bone formation. Observing mineralized nodules of osteoblasts is a common method for assessing their differentiation degree. The effects of FGF2, SAF2, and rhAm on mineralized nodules of PDLSCs were detected by Alizarin Red staining. PDLSCs were pre-treated with drugs for 3 days before osteogenic induction and induced for 14 days. Figure 10 The results showed that rhAm (0.1 μg / mL) and SAF2 (0.1 μg / mL) promoted the formation of mineralized nodules, while FGF2 (0.01 μg / mL) had no significant effect on the formation of mineralized nodules. These results indicate that SAF2 and rhAm can promote the formation of mineralized nodules in PDLSCs during the early stages of cell-induced differentiation.

[0138] 5.6 qPCR

[0139] (1) RNA was extracted from PDLSCs cells after osteogenic induction culture in section 5.3 according to the instructions of the rapid RNA extraction kit. Reverse transcription was performed according to the instructions of the reverse transcription premix kit, and qPCR reaction was performed according to the instructions of the ChamQ SYBR qPCR Master Mix kit.

[0140] (2) Results: The effects of FGF2 (0.01 μg / mL), SAF2 (0.1 μg / mL), and rhAm (0.1 μg / mL) on the expression of osteogenic differentiation-related genes (POSTN, RUNX2, OCN, ALP, CAP, and BMP2) in PDLSCs were detected by real-time quantitative PCR. PDLSCs were pre-treated for 3 days and induced for 7 days before osteogenic differentiation. Figure 11 The results showed that SAF2 significantly promoted the expression of POSTN, RUNX2, ALP, and BMP2 genes, and rhAm significantly promoted the expression of RUNX2, ALP, and BMP2 genes (P < 0.05). FGF2 had no significant effect on the expression of osteogenic differentiation-related genes. These results indicate that SAF2 significantly promotes the expression of POSTN, RUNX2, ALP, and BMP2 genes in the early stages of cell differentiation induction.

[0141] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A recombinant chimeric protein SAF2, characterized in that: The amino acid sequence is shown in SEQ ID NO.

2.

2. The gene encoding the recombinant chimeric protein SAF2 according to claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

1.

3. The method for preparing the recombinant chimeric protein SAF2 according to claim 1, characterized in that, Includes the following steps: (1) The gene sequence shown in SEQ ID NO.1 is ligated into an expression vector to obtain a recombinant expression plasmid; (2) The recombinant expression plasmid was transformed into a prokaryotic expression strain to obtain the recombinant expression strain; (3) The recombinant expression strain was induced to express with isopropyl-β-D-thiogalactoside, and then the bacterial precipitate was collected to obtain the recombinant chimeric protein SAF2.

4. The method for preparing recombinant chimeric protein SAF2 according to claim 1, characterized in that: The expression vector mentioned in step (1) is PET-21a(+); The expression strain mentioned in step (1) is Escherichia coli.

5. The method for preparing recombinant chimeric protein SAF2 according to claim 1, characterized in that: The expression strain mentioned in step (1) is Escherichia coli BL21(DE3).

6. The method for preparing recombinant chimeric protein SAF2 according to claim 1, characterized in that: In step (3), the final concentration of isopropyl-β-D-thiogalactoside in the induction expression system is 0.1 mmol / L; The temperature for inducing expression in step (3) is 25℃~37℃; The induction time in step (3) is 4 h.

7. A recombinant vector, expression cassette, and recombinant bacteria containing the encoding gene of the recombinant chimeric protein SAF2 as described in claim 1.

8. The use of the recombinant chimeric protein SAF2 of claim 1 in the preparation of products for periodontal tissue regeneration and / or osteogenic differentiation of periodontal ligament stem cells.

9. The application according to claim 8, characterized in that: The products mentioned include pharmaceuticals or medical materials.

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