Fusion protein containing fibroblast growth factor and fibronectin and application thereof

Abstract

The invention discloses a fusion protein containing a fibroblast growth factor and fibronectin and application of the fusion protein. The fusion protein comprises a fibronectin truncated fragment (such as FNIIIC or FNIIICFC) and a basic fibroblast growth factor (bFGF). The invention also provides a polynucleotide for coding the fusion protein, a recombinant expression vector and a host cell containing the polynucleotide, and a method for purifying the fusion protein through fermentation culture and affinity chromatography. Experiments show that compared with a single component or FN-full, the fusion protein, especially FNIIICFC-b, disclosed by the invention can remarkably promote proliferation and migration of mesenchymal stem cells (MSC) and up-regulate expression of beneficial factors such as HGF (Hepatocyte Growth Factor) and VEGF (Vascular Endothelial Growth Factor). The fusion protein, the pharmaceutical composition containing the fusion protein and the cell culture coating material containing the fusion protein have wide application prospects in the fields of tissue engineering, regenerative medicine and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a fusion protein containing fibroblast growth factor and fibronectin, and also to the application of the fusion protein. Background Technology

[0002] Currently, the culture of primary mesenchymal stem cells (MSCs) faces numerous limitations, such as gradual aging, decreased stemness, and reduced self-renewal capacity over time. This phenomenon highlights the necessity of developing supportive microenvironments to maintain the functional properties and pluripotency of MSCs, thereby enhancing their therapeutic efficacy in regenerative medicine applications.

[0003] Fibronectin (FN), an extracellular matrix glycoprotein, is often used as a culture medium additive or as a coating protein to enhance cell adhesion, proliferation, survival, and migration, playing an important role in primary cell culture and tissue engineering. In plasma, FN often exists in dimer form, with its monomers consisting of three homologous repeat modules tandemly forming type I, II, and III modules. FN has complex receptors, including heparin, collagen, and integrins, exhibiting diverse biological functions. In our previous work, we designed and produced a truncated FN fragment, FNIIIC, by linking the heparin-binding I (HepI) and FNIII8,10 (binding integrin αVβ3) domains using a linker (GGSGGSGGS); and a truncated FN fragment, FNIIICFC, by linking the heparin-binding I (HepI) and the three domains of FNIII8,10 (binding integrin αVβ3) and heparin-binding II (HepII) domains using a linker (GGSGGSGGS). Given that basic fibroblast growth factor (bFGF) is a member of the FGF family and is often used as a key supplement in cell culture media, and considering the potential crosstalk between bFGF and the integrin αvβ3-mediated signaling pathway, we are the first to fuse FNIIIC, FNIIICFC, and bFGF to obtain a functional protein with recombinant activity. Summary of the Invention

[0004] In view of this, one objective of the present invention is to provide a fusion protein containing fibroblast growth factor and fibronectin; preferably, a second objective of the present invention is to provide a nucleotide encoding the fusion protein; a third objective of the present invention is to provide a recombinant expression vector containing the nucleotide; a fourth objective of the present invention is to provide a host containing the nucleotide or the recombinant expression vector; a fifth objective of the present invention is to provide the use of the fusion protein in the preparation of a formulation that promotes the proliferation of mesenchymal stem cells; a sixth objective of the present invention is to provide the use of the fusion recombinant protein in the preparation of a formulation that promotes the migration of mesenchymal stem cells; and a seventh objective of the present invention is to provide the use of the fusion recombinant protein in the preparation of a formulation for upregulating the expression of HGF, VEGF, SDF-1, or EGF in mesenchymal stem cells.

[0005] To achieve the above objectives, the present invention provides the following technical solution: 1. A fusion protein containing fibroblast growth factor and fibronectin, wherein the fusion protein comprises a truncated fragment of fibrin and basic fibroblast growth factor bFGF, wherein the truncated fragment is selected from FNIIIC or FNIIICFC, wherein the amino acid sequence of FNIIICFC is shown in SEQ ID NO.1, and the amino acid sequence of FNIIIC is shown in SEQ ID NO.2.

[0006] Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0007] 2. A nucleotide encoding the fusion protein.

[0008] Preferably, the nucleotide sequence is as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0009] 3. A recombinant expression vector containing the said nucleotides.

[0010] 4. A host containing the nucleotide or the recombinant expression vector.

[0011] 5. Application of the fusion protein in the preparation of formulations that promote the proliferation of mesenchymal stem cells.

[0012] Application of the fusion recombinant protein in the preparation of formulations that promote the migration of mesenchymal stem cells.

[0013] 7. The use of the fusion recombinant protein in the preparation of formulations for upregulating the expression of HGF, VEGF, SDF-1 or EGF in mesenchymal stem cells.

[0014] The beneficial effects of this invention are as follows: This invention provides a fusion protein containing fibroblast growth factor and fibronectin. By designing a fusion protein of FNIIIC, FNIIICFC and bFGF, and linking them through a linker (GGGSGGGSGGGS), the recombinant proteins FNIIIC-b and FNIIICFC-b produced can improve MSC expansion efficiency while changing the paracrine spectrum and secreting more factors that help angiogenesis, providing new ideas for the treatment of wound repair and tissue regeneration, ischemic diseases, cardiovascular diseases and other diseases. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Select molecular weight and protein purity for SDSPAGE assay; Figure 2 The results are for the cell activity assay of the recombinant protein; Figure 3 Absorbance at 450 nm was measured for MSC cell proliferation assay. Figure 4 This refers to the results of cell viability testing; Figure 5 To detect the expression of each marker molecule; Figure 6 The results are from a cell cycle analysis. Figure 7 Results of cell migration analysis; Figure 8 For ELSA and quantitative analysis results; Figure 9 This is the result of the osteogenic capacity test; Figure 10 This is the result of the lipogenic capacity test. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0017] Example 1: Sequence Design The Full-FN gene sequence (Gene ID: 2335) and the bFGF gene sequence (Gene ID: 2247) can both be found on the NCBI website. The amino acid sequence of FNIIICFC is shown in SEQ ID NO.1, the amino acid sequence of FNIIIC is shown in SEQ ID NO.2, and the amino acid sequences of the designed FNIIIC-b and FNIIICFC-bFGF (abbreviated as FNIIICFC-b) fusion protein are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0018] Based on the codon preference of *E. coli*, the nucleotide sequences of FNIIIC-b and FNIIICFC-b were optimized, as shown in SEQ ID NO. 5 and SEQ ID NO. 6, and the nucleotide sequences of FNIIICFC and FNIIIC are shown in SEQ ID NO. 7 and SEQ ID NO. 8. An enterokinase cleavage site (DYKDDDDK) was introduced at the N-terminus of the FNIIIC-b protein sequence, and a 6*HIS tag (HHHHHH) was introduced at the C-terminus of FNIIIC-b. The optimized gene sequences FNIIIC-b and FNIIICFC-b were subcloned into the vectors PGEX-6P-1 (BamHI / NotI) and Pcold TF (NdeI / XbaI), respectively, to obtain plasmids GST-FNIIIC-b and TF-FNIIICFC-b.

[0019] Example 2: Transformation of recombinant expression plasmids into competent host cells The recombinant expression plasmid obtained in Example 1 was transformed into BL21 competent cells. The specific process was as follows: the competent cells were taken out of the -80℃ freezer, thawed on ice, mixed with the fresh plasmid, and incubated on ice for 30 min. After heat shock at 42℃ for 90 s, the cells were incubated on ice for 3 min. 500 µL of LB liquid medium was added, and the cells were shaken at 37℃ and 220 rpm / min for 30 min to recover.

[0020] Take an appropriate volume of the bacterial culture according to its OD value and spread it on an ampicillin-resistant (70 μg / mL) plate. Incubate upside down overnight.

[0021] Example 3: Induction of recombinant protein expression and protein purification The induction of FNIIIC-b and FNIIICFC-b recombinant proteins was performed as follows: Fresh single colonies were picked from plates and inoculated into liquid medium containing 100 μg / mL Amp LB. The culture was incubated overnight at 37°C and 220 rpm. Then, 1% of the colony was inoculated into 100 μg / mL Amp TB. The culture was shaken at 37°C and 200 rpm until the OD value of the bacterial culture reached 0.6–0.8. IPTG was added to a final concentration of 0.1 mM / L, and the culture was induced at 15°C and 200 rpm for 20 h. The bacterial culture was then aliquoted into centrifuge bottles and centrifuged at 8500 rpm for 10 min. The bacterial cells were collected and their mass recorded. FNIIIC-b was resuspended in Tris-HCl, and FNIIICFC-b was resuspended in PB buffer containing 20 mM imidazole (20 mM PB, pH 7.4). After high-pressure disruption, the cells were subjected to 12000... Centrifuge at rpm and 4℃ for 30 min, and take the supernatant for electrophoresis detection.

[0022] The recombinant proteins FNIIIC, FNIIICF, and FNIIICFC were obtained by homologous recombination of the nucleotides encoding FNIIIC and FNIIICF into the vector PGEX-6P-1. The vector backbone before homologous recombination was digested with BamHI / NotI to obtain the recombinant plasmids GST-FNIIIC and GST-FNIIICF. The FNIIICFC gene sequence was subcloned into the space between NdeI and XbaI of Pcold TF, thus obtaining the recombinant plasmid TF-FNIIICFC. This recombinant plasmid was transformed into BL21 competent cells and induced for expression according to the method described above. The nucleotide sequences of FNIIIC, FNIIICF, and FNIIICFC are shown in SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID NO. 7.

[0023] Single-clonal bacterial strains with high cell quality and high recombinant protein expression levels were preserved to obtain genetically engineered bacteria expressing recombinant proteins. The supernatants of GST-FNIIIC-b and TF-FNIIICFC-b cells were obtained after cell lysis according to the above method and filtered through a 0.45 μm filter membrane for column loading.

[0024] GST-FNIIIC-b was purified using a GST column. First, the GST column was washed with 5 column volumes of water, and then equilibrated with 5 column volumes of Tris-HCl buffer. The sample was incubated with the GST column for 2 h. After washing away contaminating proteins with 5 column volumes, the sample was digested with HRV 3C enzyme overnight at 4°C. The target protein was then eluted with 5 column volumes of Tris-HCl buffer, and the GST tag was thoroughly washed away with 20 mM glutathione. The GST column was stored in 20% ethanol for future use.

[0025] Purification of FNIIICFC-b was performed using a His column. First, the nickel column was equilibrated with 3 volumes of 20 mM imidazole. After incubating the sample with the column substrate for 10 min, it was washed with 5 column volumes of 20 mM imidazole, 10 column volumes of 50 mM imidazole, 3 column volumes of 100 mM imidazole, and finally eluted with 5 column volumes of 250 mM imidazole. The protein obtained in this step carried a TF tag. After removing the imidazole using a desalting column or ultrafiltration, it was digested with enterokinase overnight and then re-coated onto the column. After digestion, the sample was incubated with the column substrate for 1 h, and finally eluted with 5 column volumes of 20 mM imidazole.

[0026] The proteins were concentrated by ultrafiltration and then stored in 50 mM PBS (pH 8.0). The purification methods for FNIIIC-GST, FNIIICF-GST, and FNIIICFC-TF were the same. Finally, molecular weight and protein purity were determined by SDSPAGE, and protein concentration was determined by the BCA reagent method for subsequent experiments. The results are as follows. Figure 1 As shown. Based on the sequences, the molecular weights were calculated as follows: FNIIIC-GST and FNIIIC had molecular weights of 48.4 kD and 19.1 kD, respectively; FNIIIC-b-GST and FNIIIC-b had molecular weights of 65.8 kD and 37.9 kD, respectively; FNIIICFC-TF and FNIIICFC had molecular weights of 102.6 kD and 52.7 kD, respectively; and FNIIICFC-b-TF and FNIIICFC-b had molecular weights of 120.6 kD and 70.6 kD, respectively. These were then verified using SDS-PAGE. Figure 1 As can be seen, the test results are in line with expectations.

[0027] Example 4: Cell morphology observation Treatment groups: divided into FNIIIC-b and FNIIICFC-b; Positive control group: Corning® Fibronectin, product number 356008 (commercially available); Negative control group: sterile DPBS.

[0028] To investigate the effects of different treatments on MSC morphology, after 3 days of culture, observations were performed under a microscope. Figure 2 As shown, cells adhered well in all treatment groups, but differences were observed in cell morphology. However, the FN-full and FNIIICFC-b treatments exhibited more uniform growth and a typical "vortex growth" morphology.

[0029] Example 5: Detection of the Adsorption Properties of Recombinant Proteins For solid-phase enzyme-linked immunosorbent assay (ELISA), the concentrations of FNIIIC-b and FNIIICFC-b were set at 50 nM, 300 nM, 550 nM, 950 nM, 1200 nM, 1450 nM, and 1700 nM, respectively.

[0030] Proteins from the treatment group, positive group, and negative control group were added to 6-well plates (2 mL), with three replicates per group, and incubated overnight at 4°C. The plates were washed twice with sterile DPBS to remove unbound proteins, and then blocked with 2.5% BSA. Takara Anti-Human Fibronectin monoclonal antibody (M002) was used to detect bound proteins. The plates were incubated at room temperature for 1 h, followed by the addition of HRP-conjugated goat anti-mouse secondary antibody. After washing twice, TMB was used for color development, and absorbance was measured at 450 nm.

[0031] To eliminate the influence of varying adsorption amounts of different proteins on cell culture plates, a specific monoclonal antibody was used to calculate the coating efficiency of full-length FN and recombinant truncated protein, obtaining an equal amount of adsorbed protein coated on the surface. Figure 3 We found that as the concentration of the coating protein increased, the adsorption capacity of the cell culture plate gradually reached saturation. When FN-full was used at a coating concentration of 0.2 μM, FN IIIC, FN IIICFC, FNIIIC-b, and FNIIICFC-b achieved equivalent protein adsorption at concentrations of 645 nM, 910 nM, 955 nM, and 1150 nM, respectively. Therefore, in subsequent experiments, we selected corresponding working concentrations for different proteins to ensure the same protein adsorption capacity and make the experiments comparable.

[0032] Example 6: MSC Cell Proliferation Assay The 96-well culture plates were coated using the method described above. Logarithmic-phase MSCs were seeded at a rate of 5000 cells / well into the prepared 96-well plates and incubated in a 5% CO2 incubator at 37°C for 40 h. The culture medium was then removed, the plates were washed three times with PBS, and incubated for another 2 h. The absorbance was measured at 450 nm. The results are as follows: Figure 4 As shown.

[0033] The results showed that the proliferative activity was optimal when bFGF was fused with FNIIICFC, significantly higher than that of the FNIIIC-b group. This indicates that the heparin-binding domain II is necessary to enhance the activity of the fusion protein, surpassing the proliferative activity of FN-full. Therefore, the fusion protein FNIIICFC-b produced in this invention exhibits superior proliferative activity. Subsequent investigations in this invention will focus solely on the effects of FNIIICFC and FNIIICFC-b on MSCs.

[0034] Example 7: Cell Phenotype Analysis Normally growing MSCs were seeded onto T25 cell culture flasks coated with three recombinant proteins, in addition to negative and positive control groups. After confluence and passage to passage 15, cells from each group were collected to form nine samples. CD11b, CD90, CD73, CD44, CD34, CD45, and isotype control antibodies were added to each sample, respectively. After incubation on ice for 1 hour, the samples were washed twice with DPBS, and then incubated on ice for 1 hour with the corresponding secondary antibody. The expression of each marker molecule was immediately detected.

[0035] Flow cytometry analysis was performed on the expression of MSC surface markers CD11b(A), CD14(B), CD19(C), CD106(D), CD29(E), CD44(F), CD73(G), and CD90(H). The results are as follows: Figure 5 As shown in the figure. The results indicate that the recombinant protein does not affect the expression of MSC surface markers.

[0036] Example 8: Cell Cycle Analysis The TF-FNIIICFC treatment group with the best proliferation effect was selected for flow cytometry periodic analysis. Six-well culture plates were coated using the method described above, 2*10n 5 Cells were densely seeded, and logarithmically growing cells were collected by trypsin digestion. Cells were washed twice with pre-chilled DPBS. The collected cells were slowly added dropwise to 100% ethanol to a final concentration of 70%, and fixed overnight at 4°C. Cells were collected by centrifugation at 300 g for 5 min, washed once with pre-chilled DPBS, and incubated with RNase and PI at 37°C in the dark for 30 min before analysis. Results were analyzed using the cell cycle fitting software FlowJ. The results are shown below. Figure 6 As shown in the figure. The results showed that the G0 / G1 phase cell ratio of MSCs without recombinant protein treatment was 82.3%, with some cells in the dividing state. In the FNIIICFC-b group, the G0 / G1 phase cell ratio was only 29.8%, with most cells in the dividing state.

[0037] Example 9: Cell Migration Analysis MSCs were seeded evenly in 6-well plates at a density of approximately 5 × 10⁵ cells per well. When confluence reached 90%, the cells were starved with 2% serum for 24 hours. Then, MSCs were streaked using a 200 µL pipette tip. Microscopic images of the streaks were taken at 0 h, 5 h, and 10 h to observe the healing process. Results are shown below. Figure 7As shown in the figure. The results showed that in the DPBS group, cells also migrated to the scratch area at 10 h, but the migration speed was slower. In the FNIIICFC and FNIIICFC-b groups, the migration was almost completely completed at 10 h, and the migration speed was significantly faster than that of the Control group, showing the promoting effect of recombinant protein on cell migration.

[0038] Example 10: ELSA and Quantitative Analysis The levels of HGF and VEGF in MSC cell culture medium were detected by ELISA to assess the angiogenesis-promoting effect of MSC paracrine signaling. The transcriptional levels of HGF, VEGF, SDF-1, and EGF in MSCs were detected by qPCR. The results are shown below. Figure 8 As shown in the figure. The results showed that, compared with the DPBS group, stimulation of MSCs with FNIIICFC and FNIIICFC-b significantly increased the gene expression of HGF, VEGF, SDF-1, and EGF. The ELISA results were consistent with the RT-qPCR results, showing that, compared with the PBS group, the levels of HGF and VEGF in the cell culture medium of the FNIIICFC and FNIIICFC-b groups were significantly upregulated.

[0039] Example 11: Differentiation Capacity Detection Osteogenic capacity: The osteogenic differentiation capacity of MSCs was identified by selecting 5th generation MSCs, according to 1 * 10 5 Cells were seeded at a density suitable for osteogenic induction in 12-well plates. Osteogenic induction began when cell confluence reached 80%, with medium changes every 3 days. After 15 days of culture, cells were washed three times with PBS, fixed with 4% PFA at room temperature for 30 minutes, and after washing, 1.5 mL of 0.1% Alizarin Red staining solution was added to each well. The cells were incubated at room temperature in the dark for 20 minutes. After removing excess staining solution, cells were repeatedly washed with PBS, and images were taken under a microscope. The results are shown below. Figure 9 As shown.

[0040] The results showed that after 15 days of osteogenic differentiation induction, Alizarin Red staining of MSCs revealed large areas of calcium ion deposition in all treatments, and the recombinant protein did not affect the osteogenic capacity of MSCs.

[0041] Adipogenic capacity: 5th generation MSCs were selected, according to 1*10 5Cells were seeded at the appropriate density in 12-well plates and cultured until 100% confluence. Adipogenic induction was then initiated. During induction, a decline in cell condition was observed, so the induction medium was changed to regular medium. After 20 days of culture, cells were washed three times with PBS, fixed with 4% PFA at room temperature for 20 minutes, rinsed thoroughly with 60% isopropanol for 30 seconds, and 1.5 mL of Oil Red O staining solution was added to each well for staining. The cells were incubated at room temperature for 15 minutes. After discarding the staining solution, non-specific staining was removed by rinsing with 60% isopropanol, followed by three PBS washes. The staining was observed and images were taken using a microscope. Results are as follows: Figure 10 As shown in the figure. The results showed that Oil Red-O staining revealed obvious lipid droplet formation in all treatment groups, indicating that adipogenesis induction was successful and that the recombinant protein did not affect the adipogenic ability of MSCs.

[0042] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. A fusion protein containing fibroblast growth factor and fibronectin, characterized in that: The fusion protein comprises a truncated fragment of fibrin and basic fibroblast growth factor bFGF, wherein the truncated fragment is selected from FNIIIC or FNIIICFC, the amino acid sequence of FNIIICFC is shown in SEQ ID NO.1, and the amino acid sequence of FNIIIC is shown in SEQ ID NO.

2.

2. The fusion protein according to claim 1, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO.3 or SEQ ID NO.

4.

3. A nucleotide encoding the fusion protein of claim 1 or 2.

4. The nucleotide according to claim 3, characterized in that: The nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.

6.

5. A recombinant expression vector containing the nucleotides of claim 3 or 6.

6. A host containing the nucleotide of claim 3 or 6 or the recombinant expression vector of claim 4.

7. The use of the fusion protein according to any one of claims 1 to 2 in the preparation of a formulation that promotes the proliferation of mesenchymal stem cells.

8. The use of the fusion recombinant protein according to any one of claims 1 to 2 in the preparation of a formulation that promotes the migration of mesenchymal stem cells.

9. The use of the fusion recombinant protein according to any one of claims 1 to 2 in the preparation of a formulation for upregulating the expression of HGF, VEGF, SDF-1 or EGF in mesenchymal stem cells.

Images