A dual domain recombinant humanized fibronectin and preparation and application thereof

Abstract

The application discloses a double-domain recombinant humanized fibronectin, the sequence of which is shown as SEQ ID NO. 3, which is composed of the collagen binding domain and the cell binding domain of human fibronectin. The application also provides a recombinant expression vector and a host cell containing the gene coding the protein. The protein is efficiently expressed by eukaryotic cells and engineered Escherichia coli, and a fermentation and purification process suitable for industrial scale is established. Functional experiments prove that, compared with the single-domain commercial fibronectin product, the double-domain recombinant humanized fibronectin of the application can significantly promote the proliferation, migration, adhesion and differentiation activity of human skin fibroblasts, and has wide application potential in the fields of skin repair, beauty and skin care and biomedical materials.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and protein engineering, specifically relating to a dual-domain recombinant humanized fibronectin and its preparation and application. Background Technology

[0002] Fibronectin (FN) was discovered in 1948 by Morrison and was initially named "cold-insoluble globulin" based on its property of precipitation at low temperatures. It is a large glycoprotein widely found in plasma and on cell surfaces, playing a crucial role in cell adhesion, migration, proliferation, and tissue repair. FN usually exists in dimer form, consisting of two nearly identical subunits of about 250 kDa covalently linked near the C-terminus by a pair of disulfide bonds. Each monomer consists of three types of repeating units: type I, type II, and type III, and contains multiple independent functional domains, such as a collagen-binding domain that binds to extracellular matrix components (e.g., collagen) and a cell-binding domain that interacts with cell surface integrins.

[0003] Fibronectin has applications spanning multiple fields, including medical repair, aesthetic medicine, and biomaterials. In medical repair, fibronectin promotes fibroblast migration and collagen deposition, accelerating wound healing. A team at Shanghai Jiao Tong University developed a click chemistry-based fibronectin-modified cell layer (FACS), which significantly improves wound healing rates and reduces inflammatory responses. In aesthetic medicine, fibronectin enhances skin barrier function, reduces inflammation, and is used for sensitive skin repair. It stimulates collagen production, reduces wrinkles, and improves skin elasticity for anti-wrinkle and firming effects. It can also bind moisture to form a moisturizing film, improving dryness and promoting keratinocyte renewal. In biomaterials, fibronectin, as a cell adhesion protein, is widely used in the in vitro culture of stem cells and primary cells.

[0004] Currently, fibronectin raw materials produced using recombinant technology mostly focus on expressing a single functional domain. For example, existing technologies contain only collagen-binding domains or, although peptides, incorporate non-fibronectin-derived functional fragments (such as transdermal peptides). These single-domain products have functional limitations and cannot fully mimic the synergistic biological effects of the multi-domain synergistic effects of natural fibronectin. This invention overcomes the limitations of single-domain expression by rationally designing and fusing multiple key functional domains of human fibronectin to develop a novel dual-domain recombinant humanized fibronectin. This protein possesses both collagen-binding and cell-binding functions, and through the establishment of a corresponding efficient preparation process, it provides a superior product option for applications in the medical, cosmetic, and biomaterials fields.

[0005] To systematically evaluate its functional advantages, we used human skin fibroblasts as a model and set up multiple experimental groups, including a blank group, two commercially available fibronectin control groups, and the sample group of this invention. The commercially available fibronectins used in the control groups were recombinant human type III fibronectin from MELLPRO (Shenzhen) (trade name: MELLPRO-fibronectin solution, catalog number: 232407040102) and recombinant fibronectin from Shenzhen Baiyin Biotechnology Co., Ltd. (trade name: HythermFN-01, batch number: FN(S)-2.0-240402). The concentrations used in all experiments were consistent with the sample group of this invention, both being 1 mg / mL, to ensure comparability of results. A comprehensive evaluation was conducted from multiple dimensions, including cell proliferation, migration, adhesion, and differentiation. The experimental results showed that compared to commercially available products containing only a single domain, the dual-domain recombinant humanized fibronectin constructed in this invention exhibited significant improvements in multiple functional indicators, laying a solid foundation for its specific efficacy in applications. Summary of the Invention

[0006] One of the technical solutions provided by this invention is to provide a dual-domain recombinant human fibronectin, wherein the protein is composed of a collagen-binding domain and a cell-binding domain of human fibronectin connected by a flexible linker peptide (G4S). n The amino acid sequences of the collagen-binding domains are arranged in any order, as shown in SEQ ID NO.1, the amino acid sequences of the cell-binding domains are shown in SEQ ID NO.2, and the amino acid sequences of the complete protein are selected from the following group:

[0007] a) The amino acid sequence as shown in SEQ ID NO: 3;

[0008] b) Has at least 90%, 95%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO: 3, and retains the derived sequence that promotes cell adhesion and collagen binding.

[0009] The present invention also provides a nucleic acid molecule encoding the dual-domain recombinant human fibronectin of claim 1, wherein the sequence comprises a sequence encoding a collagen-binding domain as shown in SEQ ID NO.4 and a sequence encoding a cell-binding domain as shown in SEQ ID NO.5, and the complete nucleotide sequence thereof is selected from the following group:

[0010] a) The nucleotide sequence shown in SEQ ID NO: 6;

[0011] b) The nucleotide sequence having at least 90%, 95%, 98% or 99% identity with the nucleotide sequence shown in SEQ ID NO: 6, and encoding the dual-domain recombinant human fibronectin sequence;

[0012] The present invention also provides a recombinant vector or recombinant strain containing the above-described encoding gene;

[0013] Furthermore, the recombinant vector uses prokaryotic expression plasmids and eukaryotic expression plasmids as expression vectors;

[0014] Furthermore, the host system used for protein expression is a prokaryotic expression system or a eukaryotic expression system;

[0015] The present invention also provides a method for preparing the above-mentioned dual-domain recombinant humanized fibronectin, including fermentation, separation, purification and drying;

[0016] The present invention also provides applications of the above-mentioned dual-domain recombinant human fibronectin, particularly in the proliferation, migration, adhesion and differentiation of human skin fibroblasts.

[0017] Beneficial effects of the present invention

[0018] This invention uses human-derived sequences, which have low immunogenicity and high biosafety, making them suitable for widespread clinical application. By fusing the collagen-binding domain with the cell-binding domain, synergistic and enhanced functions are achieved, resulting in superior biological activity compared to products with single domains in cell experiments. An integrated process has been established, from fermentation in a 100L fermenter to affinity chromatography, ion exchange chromatography purification, and internal drying, achieving a target protein purity of up to 92.4%, with stable process and easy scale-up. Attached Figure Description

[0019] Figure 1 Electrophoresis results during E. coli expression: The target protein was detected in the supernatant after bacterial cell lysis. Lane 1: 8-180 kDa protein marker; Lane 2: Homogenized supernatant of bacteria induced by dual-domain recombinant human fibronectin for 4 h; Lane 3: Uninduced dual-domain recombinant human fibronectin; Lane 4: Homogenized supernatant of bacteria induced by dual-domain recombinant human fibronectin for 4 h.

[0020] Figure 2 Electrophoretic detection results during CHO cell expression: The target protein was in the fermentation supernatant, with lane 1: 8-180 kDa protein marker; lane 2: fermentation supernatant of dual-domain recombinant human fibronectin.

[0021] Figure 3Electrophoretic analysis results of the purification process of dual-domain recombinant humanized fibronectin: Lane 1: Supernatant after bacterial cell separation; Lane 2: Affinity chromatography flow-through; Lane 3: Affinity chromatography washing; Lane 4: Affinity chromatography elution; Lane 5: Desalting; Lane 6: Anion exchange flow-through; Lane 7: Lyophilized dual-domain recombinant humanized fibronectin; Lane 8: 8-180 kDa protein marker; Lane 9: Dual-domain recombinant humanized fibronectin 1 mg / mL.

[0022] Figure 4 The control group and sample group were based on the proliferation rate of human skin fibroblasts (HSF);

[0023] Figure 5 The blank group, control group, and sample group were based on the 24-hour scratch map and migration rate quantification results of human skin fibroblasts (HSF);

[0024] Figure 6 Adhesion fluorescence staining patterns and quantitative results of human skin fibroblasts (HSF) in the blank group, control group, and sample group;

[0025] Figure 7 Results of the expression levels of Mus α-SMA and Mus Vimentin genes in the blank group, control group, and sample group. Detailed Implementation

[0026] The present invention is further illustrated below through specific embodiments. Unless otherwise specified, the technical means and materials involved in the following embodiments are all known to those skilled in the art, and suitable means and materials that can solve the corresponding technical problems can be selected. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention; the essence and scope of the invention are defined only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments without departing from the essence and scope of the invention also fall within the protection scope of the invention.

[0027] The present invention relates to a dual-domain recombinant humanized fibronectin derived from human fibronectin, comprising the collagen-binding domain amino acid sequence shown in SEQ ID NO. 1:

[0028] SEQ ID NO.1

[0029] MSSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRDFDFTTTSTSTP

[0030] Contains the amino acid sequence of the cell-binding domain shown in SEQ ID NO.2:

[0031] SEQ ID NO.2

[0032] PTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVP HSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS

[0033] The complete amino acid sequence is shown in SEQ ID NO.3:

[0034] SEQ ID NO.3

[0035] MSSSGPVEVFITETPSQPNSHPIQWNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPPTDLRFTNIGPDTMRVTWAPPPSIDLTNFLVRYSPVKNEEDVAELSISPSDNAVVLTNLLPGTEYVVSVSSVYEQHESTPLRGRQKTG LDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS

[0036] The recombinant humanized fibronectin gene with dual domains involved in this invention is obtained by sequencing analysis of human fibronectin gene fragments and is heterologously expressed by Escherichia coli BL21(DE3) and CHO cells.

[0037] The gene encoding a dual-domain recombinant human fibronectin involved in this invention comprises the collagen-binding domain nucleotide sequence shown in SEQ ID NO.4:

[0038] SEQ ID NO.4

[0039] ATGTCAAGTTCTGGACCGGTTGAGGTGTTCATCACCGAAACCCCTAGCCAGCCGAACTCCCATCCGATTCAGTGGAACGCTCCGCAACCAAGCCATATTAGCAAATACATTCTGCGCTGGCGTCCGAAGAACTCTGTTGGCCGTTGGAAA GAAGCGACTATTCCGGGTCACTTGAATAGCTATACCATCAAGGGTCTGAAGCCGGGTGTCGTTTATGAGGGCCAGCTGATCTCCATCCAACAATACGGCCACCAGGAGGTGACCCGTTTTGACTTCACCACGACCAGCACGTCGACCCCG

[0040] Contains the cell-binding domain nucleotide sequence shown in SEQ ID NO.5:

[0041] SEQ ID NO.5

[0042] CCCACTGACCTGCGATTCACCAACATTGGTCCAGACACCATGCGTGTCACCTGGGCTCCACCCCCATCCATTGATTTAACCAACTTCCTGGTGCGTTACTCACCTGTGAAAAATGAGGAAGATGTTGCAGAGTTGTCAATTTCTCCTTCAGACAATGCAGTGGTCTTAACAAATCTCCTGCCTGGTACAGAATATGTAGTGAGTGTCTCCAGTGTCTACGAACAACATGAGAGCACACCTCTTAGAGGAAGACAGAAAACAGGTCTTGATTCCCCAACTGGCATTGACTTTTCTGATATTACTGCCAACTCTTTTACTGTGCACTGGATTGCTCCTCGTGCCACCATCACTGGCTACAGGATTCGCCATCATCCCGAGCACTTCAGTGGGAGACCTCGTGAAGATCGGGTGCCCCACTCTCGCAATTCCATCACCCTCACCAACCTCACTCCAGGCACAGAGTATGTGGTCAGCATCGTTGCTCTTAATGGCAGAGAGGAAAGTCCCTTATTGATTGGCCAACAATCAACAGTTTCTGATGTTCCGAGGGACCTGGAAGTTGTTGCTGCGACCCCCACCAGCCTACTGATCAGCTGGGATGCTCCTGCTGTCACAGTGAGATATTACAGGATCACTTACGGAGAGACAGGAGGAAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGGAGCAAGTCTACAGCTACCATCAGCGGCCTTAAACCTGGAGTTGATTATACCATCACTGTGTATGCTGTCACTGGCCGTGGAGACAGCCCCGCAAGCAGCAAGCCAATTTCCATTAATTACCGAACAGAAATTGACAAACCATCC

[0043] The complete nucleotide sequence is shown in SEQ ID NO.6:

[0044] SEQ ID NO.6

[0045]

[0046] The present invention will be further explained and described below with reference to specific embodiments.

[0047] Example 1. Preparation of recombinant humanized fibronectin with two domains

[0048] Gene construction: The amino acid sequence of the dual-domain recombinant human fibronectin was obtained using bioinformatics methods. The nucleotide sequence of the dual-domain recombinant human fibronectin was then entrusted to Beijing Qingke Biotechnology Co., Ltd. for whole-gene synthesis and cloned into an expression vector.

[0049] Expression System 1: *E. coli* was selected as the prokaryotic expression system. The constructed expression vector was transformed into competent *E. coli* cells. The plasmid contained the kanamycin resistance gene. High-expression clones were selected for shake-flask culture. After the cell density reached the required level, the cells were transferred to a 100 L fermenter. Cell density was maintained in the 100 L fermenter by adding culture medium. Once the target cell density was reached, the temperature was lowered for protein expression. The protein expression level was determined using SDS-PAGE. Electrophoresis results are shown below. Figure 1 As shown, the expressed target protein is in the supernatant after homogenization, which is suitable for subsequent purification.

[0050] Expression System 2: CHO cells were selected as the eukaryotic expression system. The constructed expression vector was transfected into CHO cells via electroporation. Cell line selection was then performed, and clones with the highest expression levels in each batch were selected for shake-flask culture. Once the cell density reached the required level, the cells were transferred to a 5 L fermenter. Cell density was maintained in the 5 L fermenter by supplementing with culture medium. After the cell density reached the target level, the temperature was lowered for protein expression. The protein expression level in the fermentation supernatant was measured using SDS-PAGE. Electrophoresis results are shown below. Figure 2 As shown, the expressed target protein was in the fermentation supernatant, which is suitable for subsequent purification.

[0051] Protein purification: *E. coli* culture cells and CHO cell fermentation supernatant were collected, and the expressed dual-domain recombinant human fibronectin was purified using affinity chromatography and ion exchange chromatography. After purification, the protein was diluted to 1 mg / mL, and its purity was verified using non-reducing SDS-PAGE. Figure 3 The electrophoretic bands of the two-domain recombinant human fibronectin obtained by SDS-PAGE electrophoresis are shown. The bands in the lanes are clear. Grayscale analysis using ImageJ software shows that the purity of the protein reaches 92.4%.

[0052] Example 2. Lyophilization process of dual-domain recombinant human fibronectin

[0053] The purified dual-domain recombinant humanized fibronectin was dialyzed into water for injection, the fibronectin solution was concentrated using ultrafiltration, and the fibronectin solution was then lyophilized. The specific steps are as follows:

[0054] Pre-freezing: Pre-freeze at -42 ℃ for 4 hours;

[0055] Sublimation drying: Sublimation drying was carried out at 0 ℃, at a pressure of 20 Pa, and for 30 hours;

[0056] Desorption drying: Desorption drying was carried out at 35 ℃, pressure of 5 Pa, and time of 20 hours.

[0057] The resulting dual-domain recombinant human fibronectin was then freeze-dried and stored in the dark at 2-8 ℃.

[0058] Example 3. Cell proliferation experiment of recombinant human fibronectin with two domains

[0059] HSF is 5*10 3 Cells were seeded at a density of 3:1 in 96-well plates (with blank, control, and sample groups, each with 3 replicates) and incubated at 37 ℃ and 5% CO2 for 24 h. After cell attachment, the control and sample groups were replaced with culture medium containing the sample, while the control group was replaced with regular culture medium. Incubation continued for 48 h. After incubation, 10 μL of CCK-8 reagent was added to each well, mixed well, and incubated in the dark for 1-2 h. The absorbance at 450 nm was measured using a microplate reader, and the cell proliferation rate was calculated using the formula: (proliferation rate = (…)). OD 实验 - OD 空白 )-( OD 对照 - OD 空白 )*100%).

[0060] The results are as follows Figure 4 As shown, in the cell proliferation experiment, by comparing the cell proliferation rate of the sample group and the control group, the cell proliferation fold of the sample group was significantly higher than that of the control group within the same culture time, and the cell activity was better maintained, indicating that it has a more superior cell proliferation-promoting effect and can significantly enhance the cell proliferation capacity.

[0061] Example 4. Cell scratch assay of recombinant human fibronectin with two domains

[0062] HSF was seeded into 96-well plates. After confluence and the formation of a monolayer, uniform scratches were made along the same direction on the bottom of the plate using a sterile pipette tip. The plates were then washed with PBS to remove detached cells, followed by replacement with serum-free medium (to avoid interference from cell proliferation with migration results). Three control groups (blank, control, and sample) were established and cultured at 37 °C with 5% CO2. At 24 h, images of the scratches were taken in the same field of view using a microscope. The area of ​​the scratched region was measured using ImageJ software, and the scratch healing rate was calculated (healing rate = (initial scratch area - 24 h scratch area) / initial scratch area × 100%) to assess cell migration ability.

[0063] The results are as follows Figure 5 As shown, within the same culture time, the healing rate of the scratch area in the sample group was significantly faster than that in the control group, the reduction in scratch width was greater, and at the observation endpoint, the area covered by migrating cells in the scratch area of ​​the sample group was significantly wider, with more cells migrating to the center of the scratch. The results indicate that, compared with the control group, the sample group has a superior effect in promoting cell migration and can more effectively drive cells to migrate to the damaged area to complete the repair process.

[0064] Example 5. Cell adhesion assay of recombinant human fibronectin with two domains

[0065] Cells were digested with trypsin, counted, and seeded into 96-well plates, then cultured in a 37 ℃, 5% CO2 incubator. After cell attachment, cells were treated into blank, control, and sample groups, respectively. Matrigel was prepared at a concentration of 0.4 μg / μL (1:500) using serum-free DMEM / F12 medium. 50 μL / well of Matrigel was used to coat 96-well plates, which were then air-dried overnight in a clean bench. The plates were washed three times with PBS, blocked with 1% BSA at 37 ℃ for 1 h, and rinsed three times with PBS to block uncoated areas. The previously treated cells were digested and prepared into single-cell suspensions. Calcein AM was loaded, cells were counted, and cell concentration was adjusted. Cells were then cultured at 4*10-1... 3 / The cells were seeded into 96-well plates containing matrix gel and incubated in an incubator for 1 hour; then the cells were washed 3 times with PBS to remove unattached and loosely attached cells by gentle washing; the cells were photographed, counted and processed using a fluorescence microscope.

[0066] The results are as follows Figure 6 As shown, under the same culture conditions and time, the number of cells successfully adhering to the matrix surface in the sample group was significantly greater than that in the control group, and the degree of cell spread was also higher. Quantitative detection revealed that the cell adhesion rate in the sample group was significantly higher than that in the control group, indicating that this sample can more effectively promote the interaction between cells and the matrix and enhance cell adhesion ability, that is, the sample group has a superior cell adhesion-promoting effect.

[0067] Example 6. Cell differentiation experiment of recombinant human fibronectin with two domains

[0068] HSF was cultured in a 37 ℃, 5% CO2 incubator; HSF was then incubated at a rate of 5000 cells / cm². 2 The cells were seeded at a specific density on well plates and allowed to adhere for 24 h. The original culture medium was removed, and filtered, sterile culture medium containing fibronectin from the control group and the sample group was added. The blank group received complete culture medium without added fibronectin. After co-culturing to the predetermined time, intracellular messenger RNA (mRNA) was extracted using a kit. This mRNA was reverse transcribed into cDNA and amplified by qRT-PCR. The fluorescence values ​​of each group after amplification were compared using a 2... -∆∆Ct The method is used for analysis.

[0069] See results Figure 7 By detecting the gene expression level of a specific differentiation marker, it was found that under the same culture conditions, the gene expression level of the marker in the sample group was significantly higher than that in the control group. The sample group was able to induce cells to differentiate in the target direction more effectively and showed a better effect in promoting cell differentiation.

[0070] The above biological evaluation results all indicate that it has great application potential in the field of tissue repair: the protein can efficiently drive the expansion of the number of key repair cells (such as fibroblasts), providing a sufficient cellular basis for wound healing and tissue regeneration. In the future, if it is integrated into high-end dressings, hydrogels or bioengineered scaffolds, it is expected to significantly shorten the healing cycle in the clinical treatment of refractory injuries such as diabetic foot ulcers, burns, and surgical wounds by accelerating cell renewal and tissue reconstruction. Furthermore, it can improve the quality of repair by maintaining better cell activity, promoting functional regeneration rather than simple scar healing.

Claims

1. A recombinant human fibronectin with two domains, wherein, The protein is composed of a collagen-binding domain and a cell-binding domain of human fibronectin linked together in sequence, with a protein purification tag attached to the C-terminus. The amino acid sequence of the collagen-binding domain is shown in SEQ ID NO.1, and the amino acid sequence of the cell-binding domain is shown in SEQ ID NO.

2.

2. The dual-domain recombinant human fibronectin according to claim 1, characterized in that, The amino acid sequence of the complete protein is selected from the group consisting of the amino acid sequence shown in SEQ ID NO:3 or its functional fragments or derivatives.

3. The dual-domain recombinant human fibronectin according to claim 1, characterized in that, The collagen-binding domain and the cell-binding domain are linked by a linker peptide, which is (G4S). n , where n = 0, 1, 2 or 3.

4. The dual-domain recombinant human fibronectin according to claim 1, characterized in that, The protein has a His-tag protein purification tag attached to its C-terminus.

5. A nucleic acid molecule encoding the dual-domain recombinant human fibronectin of claim 1, wherein, The sequence includes a sequence encoding a collagen-binding domain as shown in SEQ ID NO.4 and a sequence encoding a cell-binding domain as shown in SEQ ID NO.

5.

6. The nucleic acid molecule of recombinant human fibronectin with dual domains according to claim 5, characterized in that... Its complete nucleotide sequence is selected from the following group: nucleotide sequence as shown in SEQ ID NO: 6 or nucleotide sequence encoding its functional fragment or derivative.

7. An expression vector comprising the nucleic acid molecule according to claim 5 SEQ ID NO.

6.

8. The expression vector according to claim 7, wherein, The expression vector is a prokaryotic expression plasmid or a eukaryotic expression plasmid; the prokaryotic expression plasmid is pET-28a; the eukaryotic expression plasmid is pEGFP-N1.

9. A host cell, characterized in that, It comprises the recombinant expression vector as described in claim 7.

10. The host cell according to claim 9, wherein, The host cell is a prokaryotic expression system or a eukaryotic expression system; the prokaryotic expression system is Escherichia coli, and the eukaryotic expression system is CHO cells.

11. A method for preparing the dual-domain recombinant human fibronectin according to claim 1, characterized in that, It includes fermentation, separation, purification and drying steps.

12. The method according to claim 11, wherein, The separation and purification method is selected from one or more of affinity chromatography, ion exchange chromatography, molecular sieve and hydrophobic chromatography.

13. The use of the dual-domain recombinant humanized fibronectin according to claim 1 in the preparation of cosmetics or biomaterials for promoting cell proliferation, migration, adhesion and differentiation.

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