Recombinant human fibronectin as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of optimized coding genes, and discloses recombinant human fibronectin as well as a preparation method and application thereof, and the amino acid sequence of the fibronectin is as shown in SEQ ID No.1. Experiments prove that the fibronectin freeze-dried product provided by the invention has good stability and better performance of promoting cell proliferation, has no cytotoxicity within a certain range, is high in safety, and can be widely applied to the fields of cosmetics, medicines, biological materials and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of optimized coding genes, and in particular relates to a recombinant human fibronectin and a preparation method and application thereof. Background Art

[0002] Fibronectin (FN) is a high-molecular-weight (approximately 440-500 kDa) extracellular matrix glycoprotein. It is a dimer composed of two similar subunits (each with a molecular weight of 220-250 kDa) linked by a C-terminal disulfide bond, resulting in a V-shaped molecule. Each FN subunit has multiple binding domains closely related to its function, including two heparin-binding domains, three fibrin-binding domains, and one collagen-binding domain. The collagen-binding domains on FN bind to collagen in the cellular matrix, thereby adsorbing cells to the extracellular matrix.

[0003] FN is widely distributed in plasma, body fluids, on various cell surfaces, and in the extracellular matrix. Within human tissues and body fluids, it exists in a soluble form in plasma and an insoluble form in the extracellular matrix. FN participates in a wide range of physiological processes, including cell adhesion, migration, growth, and differentiation, helping to maintain extracellular matrix homeostasis. It also mobilizes the mononuclear phagocyte system to clear harmful substances that damage tissues, acting as a growth factor. FN's primary cosmetic and skincare benefits include wrinkle reduction and whitening. Wrinkles are primarily caused by the loss of collagen, elastin, and reticular fibers that support the skin. Collagen is a key component of the extracellular matrix and, along with other extracellular matrix components, maintains skin elasticity, toughness, and radiance. FN acts as a signaling molecule, inducing the expression and secretion of collagen in the extracellular matrix. It also promotes the secretion of hyaluronic acid and other glycoproteins and influences the directional arrangement of the extracellular matrix, thereby smoothing fine lines. FN can also prevent abnormal melanocyte secretion, resulting in whitening and lightening of skin spots. In skin wound repair and healing, FN can shorten wound healing time and reduce scarring. Applying fibronectin to microsphere carriers as a medium for mass cell production saves space and raw materials, becoming a foundational material for the large-scale production of new pharmaceuticals using cell culture technology. Therefore, fibronectin has broad application prospects in medicine, beauty, and skincare.

[0004] Currently, there are two main methods for producing FN: one is the traditional method of extracting natural fibronectin from human or animal blood and tissues, and the other is the production of recombinant fibronectin through microbial fermentation. Naturally extracted fibronectin has a large molecular weight and is not uniform, with extremely limited production, high cost, and low purity, which limits its application. In addition, non-human proteins also pose the risk of carrying pathogens, and the safety issues of animal raw materials also limit the application of traditional preparation methods. DNA recombinant technology can solve the difficulties in the preparation of fibronectin. Currently, the development of recombinant human fibronectin has been carried out. For example, CN117820462A discloses a recombinant human fibronectin prepared using Pichia pastoris. However, there are still some challenges and problems. For example, the expression level of fibronectin is low and insufficient to meet the needs of large-scale production, which increases production costs; the purity is low, and large-scale production has certain safety risks; the activity is low, and the cost of large-scale production is high; the stability is poor, and the cost of large-scale production is high, which is not conducive to product use and has simple application scenarios.

[0005] Therefore, developing a method for preparing recombinant fibronectin and achieving low-cost preparation of highly active and stable recombinant fibronectin is of great significance for the application field of recombinant fibronectin. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a low-cost, high-activity, and good-stability recombinant human fibronectin, and at the same time provide a preparation method and application thereof.

[0007] In order to solve the above problems, the technical solution adopted by the present invention is: In one aspect, the present invention provides a recombinant human fibronectin, wherein the recombinant human fibronectin comprises one or more of the following characteristics: (1) Its amino acid sequence is shown in SEQ ID No. 1, which is as follows: MGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDA PAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMK EINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKI YLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTHHHHHH (2) A protein derived from (1) having fibronectin activity, wherein the amino acid sequence of the protein is replaced, deleted or added with one or more amino acids based on SEQ ID No. 1.

[0008] (3) An amino acid sequence that is more than 80% identical to the amino acid sequence shown in SEQ ID No. 1.

[0009] As some preferred embodiments of the present invention, the nucleotide sequence of the recombinant human fibronectin comprises one or more of the following features: (1) Its nucleotide sequence is shown in SEQ ID No. 2, which is as follows: (2) Any nucleotide sequence obtained by replacing synonymous codons in (1).

[0010] Another aspect of the present invention provides a vector comprising any one or more of the above-mentioned nucleotide sequences.

[0011] Another aspect of the present invention provides a host cell, wherein the host cell contains the above-mentioned vector.

[0012] In another aspect, the present invention provides a method for preparing the above-mentioned recombinant human fibronectin, which specifically comprises the following steps: (1) Protein sequence design Based on human fibronectin, the amino acid sequence shown in SEQ ID No. 1 was designed; (2) Gene design and synthesis Reversely designing a coding nucleic acid sequence according to the amino acid sequence of the recombinant human fibronectin in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human fibronectin, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human fibronectin fragment; (3) Construction of expression vector The nucleic acid fragment obtained in step (2) was ligated to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites to obtain an expression plasmid; (4) Construction and screening of expression strains The expression plasmid described in step (3) is transferred into competent host cells by heat shock method, spread on a resistance plate containing kanamycin (Kan), and cultured to obtain an expression strain; (5) Inducible expression Induce the expression strain obtained in step (4) to express, and collect the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption, centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a freeze-dried product of recombinant human fibronectin.

[0013] In some preferred embodiments of the present invention, the host cell is Escherichia coli BL21 (DE3).

[0014] Another aspect of the present invention provides a use of the recombinant human fibronectin in cosmetics, medicines, health products, medical devices or biomaterials.

[0015] The beneficial effects of adopting the above technical solution are: (1) The recombinant human fibronectin provided by the present invention can be expressed in an Escherichia coli expression system, is easy to operate, and has the potential for large-scale production.

[0016] (2) Experimental results show that the recombinant human fibronectin provided by the present invention has excellent ability to promote cell migration and cell proliferation, as well as good stability.

[0017] (3) The recombinant human fibronectin provided by the present invention has no cytotoxicity below 1 mg and no animal-derived infection source, has high biosafety, and has the prospect of being applied in the fields of medicine, medical devices, biomaterials, tissue engineering, cosmetics, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The electrophoresis diagrams are of the affinity chromatography samples of the bacteria prepared in Example 4 and the recombinant human fibronectin FN prepared in Example 5; Figure 2 This is the electrophoresis diagram of the sample of recombinant human fibronectin FN prepared in Example 5 during ion chromatography; Figure 3 This is a statistical chart of the cytotoxicity experimental data of different concentrations of recombinant human fibronectin FN in Effect Example 1; Figure 4 This is the statistical graph of the data of the recombinant human fibronectin FN-induced HaCaT cell proliferation test in effect example 2. * indicates P < 0.05 compared with the blank group, and ** indicates P < 0.01 compared with the blank group; Figure 5 This is a statistical diagram of the data of the cell migration promotion test of recombinant human fibronectin FN in effect example 3; Figure 6 This is the scratch area diagram of effect example 3: recombinant human fibronectin FN promotes cell migration; Figure 7 This is the electrophoresis diagram of the recombinant human fibronectin FN thermal stability test sample in Effect Example 4. DETAILED DESCRIPTION

[0019] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is described clearly and completely below in conjunction with specific embodiments.

[0020] Example 1 Gene Design and Synthesis (1) Genetic design: Based on the sequence characteristics of human fibronectin, a fibronectin sequence FN was designed. Its amino acid sequence is shown in SEQ ID NO. 1. Its N-terminus begins with methionine to reduce the degradation rate of the protein. A histidine tag is also added to facilitate identification and purification. The amino acid sequence of the recombinant human fibronectin fragment is shown below: SEQ ID NO.1 MGLDSPTGIDFSDITANSFTVHWIAPRATITGYRIRHHPEHFSGRPREDRVPHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDVPRDLEVVAATPTSLLISWDA PAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTAIPAPTDLKFTQVTPTSLSAQWTPPNVQLTGYRVRVTPKEKTGPMK EINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKI YLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQPPRARITGYIIKYEKPGSPPREVVPRPRPGVTEATITGLEPGTEYTIYVIALKNNQKSEPLIGRKKTHHHHHH The coding nucleic acid sequence was reverse-designed using the online design tool Jcat (http: / / www.jcat.de / ), and codon optimization was performed for expression in the host E. coli. After these optimizations, the corresponding nucleic acid fragment encoding recombinant human fibronectin was obtained. The nucleotide sequence corresponding to the above amino acid sequence is shown below: SEQ ID NO.2 (2) Gene synthesis: Based on the nucleic acid sequence shown above, a nucleic acid fragment encoding a recombinant human fibronectin fragment was synthesized by GenScript Biotech Co., Ltd.

[0021] Example 2 Construction of expression vector pET30a(+)-FN The nucleic acid fragment SEQ ID NO. 2 obtained in Example 1 was ligated into the pET30a(+) plasmid (via the NdeI and XhoI multiple cloning sites) to obtain the recombinant plasmid pET30a(+)-FN.

[0022] Example 3 Construction of expression strain BL21(DE3) / pET30a(+)-FN The construction of the expression strain was carried out according to the method described in "Molecular Cloning Laboratory Manual (3rd Edition)" (J. Sambrook et al.). The specific steps are as follows: Pick a single colony of E. coli BL21 (DE3) and inoculate it into an LB tube, then shake and culture it overnight at 37°C. Add 0.5 ml of the overnight culture to a 50 ml LB flask and shake vigorously at 37°C for about 2 hours to allow the bacteria to grow to the early logarithmic phase. Aseptically transfer the bacteria to an ice-cold 50 ml polypropylene tube and place it on ice for 10 minutes. Centrifuge at 4°C, 4000 rpm, pour off the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible. Add 6 ml of ice-cold 0.1 mol / L CaCl2 to resuspend the pellet and place it on ice for 30 minutes. Centrifuge at 4°C, 3000 rpm, pour off the supernatant, and invert the tube to allow the residual liquid to flow out as much as possible. Add 1.2 ml of ice-cold 0.1 mol / L CaCl2 to resuspend the pellet (If you want to prepare competent cells for storage at -70°C, add 0.1 mol / L containing 20% ​​glycerol). CaCl2 suspension of the cells), after incubation at 4°C for 5-24 hours, 200 μl of the competent cell suspension was aspirated, the recombinant plasmid pET30a(+)-FN prepared in Example 2 (volume <10 μl, DNA <50 ng) was added, the mixture was gently mixed, and the cells were placed on ice for 30 minutes; a static heat shock was performed in a 42°C water bath for 90 seconds, and the cells were immediately cooled on ice; 500 μl of liquid LB culture medium was added, the mixture was mixed, and the cells were shaken at low speed at 37°C for 45 minutes (LB can also be added and the cells can be directly recovered in a 37°C water bath for 1 hour, with the tube shaken in between to suspend the cells); the transformed cells were aspirated and spread on a plate containing antibiotics (kanamycin), and the plate was incubated upside down in a 37°C incubator. The colonies that grew were the expression strain BL21(DE3) / pET30a(+)-FN.

[0023] Example 4 Induced expression of expression strain BL21(DE3) / pET30a(+)-FN A single colony of the expression strain BL21(DE3) / pET30a(+)-FN prepared in Example 3 was picked and placed in LB liquid medium containing 50 μg / mL Kan. After culturing overnight at 37°C and 200 rpm, it became an activated seed. Then, it was inoculated into a 5 L fermenter containing 3 L complete medium at a 3% inoculum size. The fermentation process was controlled at 37°C, 30% dissolved oxygen, and pH 7.0. When OD 600 When the expression reached 60, IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.5 mM to induce expression, and the culture was continued for 12 hours, and the bacteria were collected by centrifugation.

[0024] Example 5 Purification of recombinant human fibronectin FN The theoretical isoelectric point of FN is 9.44, and the pH of the buffer solution is 7.5. The specific purification steps are as follows: 1. Bacteria destruction The bacterial cells prepared in Example 4 were resuspended in an appropriate amount of lysis solution (20 mM Tris, 500 mM NaCl, 20 mM imidazole, 1 M urea, pH 7.5) at a ratio (m / v) of 1 g of cells to lysis solution of 5 to 1:20 ml, with 1 g of cells to 10 ml being used in this example. The cells were disrupted using a high-pressure homogenizer. The disrupted bacterial solution was centrifuged at 12,000 g for 1 hour, and the supernatant was collected and filtered through a 0.45 μm filter.

[0025] 2. Affinity chromatography (1) Column balance: The Ni Sepharose FF column was equilibrated with Buffer A (20 mM Tris, 500 mM NaCl, 20 mM imidazole, pH 7.5) for 5 CVs until the baseline was stable.

[0026] (2) Loading: The supernatant was loaded onto the chromatography column, and the column retention time was controlled to be no less than 5 min.

[0027] (3) Column flushing: Rinse the column with Buffer A until the baseline is stable and not less than 7 CVs.

[0028] (4) Target protein elution: Elution was performed with 60% Buffer A and 40% Buffer B (20 mM Tris, 500 mM NaCl, 500 mM imidazole, pH 7.5), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human fibronectin FN.

[0029] 3. Ion exchange chromatography (1) Sample processing: The affinity chromatography eluate was diluted with ion exchange solution A (20 mM Tris, pH 7.5) or ultrafiltered to a conductivity of less than 5 mS / cm.

[0030] (2) Column balance: The SP Chrpmstar FF chromatography column was equilibrated with ion exchange solution A for 5 CV until the baseline was stable.

[0031] (3) Loading: The replacement liquid is loaded into the chromatography column, and the column retention time is controlled to be no less than 5 minutes.

[0032] (4) Column flushing: Rinse the column with ion exchange solution A until the baseline is stable and not less than 7 CV.

[0033] (5) Target protein elution: The solution was eluted with 50% ion exchange solution A and 50% ion exchange solution B (20 mM Tris, 1 M NaCl, pH 7.5), and the eluate was collected into a new centrifuge tube to obtain a solution containing recombinant human fibronectin FN.

[0034] 4. Desalination The FN solution obtained in step 3 was replaced with purified water by equal volume replacement using a 3 kDa ultrafiltration membrane device to remove salt from the solution, and then filtered with a 0.45 μm filter membrane and freeze-dried to obtain a recombinant fibronectin freeze-dried product.

[0035] Example 6 SDS-PAGE protein electrophoresis detection Sample Preparation: Collect the bacterial cells prepared in Example 4 and the samples from the affinity chromatography and ion chromatography processes in Example 5, add loading buffer, mix thoroughly, incubate in a metal bath at 100°C for 10 minutes, cool naturally, and set aside. Load the samples onto a GenScript SurePAGE™ precast gel (4-12%) and run electrophoresis at 140 V for 45-55 minutes until the bromophenol blue band reaches the bottom of the gel.

[0036] Microwave staining with Coomassie Brilliant Blue R-250: 1) Prepare the staining solution: Dissolve Coomassie Brilliant Blue R-250 to a final concentration of 0.1% (w / v) in 40% ethanol and 10% acetic acid. 2) Prepare the destaining solution: Dissolve 10% (v / v) ethanol and 7.5% (v / v) acetic acid to a final concentration. 3) After electrophoresis, remove the gel from the slides and place it in a staining container containing 100 ml of staining solution. 4) Cover the container and microwave on high for 8 minutes. To avoid any risk, be careful not to allow the solution to boil. 5) Remove the staining container from the microwave and gently shake it on a destaining shaker at room temperature for 5 minutes. 6) Discard the staining solution and carefully rinse the gel with deionized water. 7) Discard the deionized water and add 100 ml of destaining solution. 8) Cover the container and microwave on high for 8 minutes. 9) Pour off the destaining solution, add new destaining solution, and repeat step 8. 10) Remove from the microwave and place on a destaining shaker at room temperature and gently shake until the background is clear.

[0037] The results of protein electrophoresis test are shown in the attached Figure 1 and attached Figure 2 As shown, it proves that recombinant human fibronectin FN was successfully prepared and separated.

[0038] Effect Example 1 Cytotoxicity Experiment of Recombinant Human Fibronectin FN The cytotoxicity of FN was evaluated according to the method specified in GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test Appendix C MTT Cytotoxicity Test. The experiment was divided into three groups: sample group (recombinant human zonulin FN solution prepared in Example 5 dissolved in complete MEM medium), positive control group (extract of ZDEC Polyurethane Film, 0.2g / ml cell culture medium, extracted at 37℃±1℃ for 24±2h), and blank / negative control group (100μL complete MEM culture medium). L929 cells were seeded in 96-well plates, and after 24 hours, the buffer or cell culture medium described in each group was treated. After 24 hours, if the negative control group and the positive control group met the requirements, the relative viability of the cells was detected by MTT. Figure 3 As shown in the figure, FN had no obvious cytotoxicity in the concentration range of 0.1mg / ml~1mg / ml.

[0039] Effect Example 2: Recombinant human fibronectin FN promotes cell proliferation test The cell proliferation test was conducted using the recombinant human fibronectin FN prepared in Example 5. The main experimental process was as follows: human skin keratinocytes HaCaT were cultured at 1×10 4Cells were seeded in a 96-well plate and incubated in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was discarded. 100 μL of culture medium was added to each well of the blank control group, and 100 μL of FN solution was added to each well of the sample group. Five replicate wells were set up for each group. After 24 hours of incubation, the supernatant was discarded, MTT solution was added, shaken, and the cells were incubated in the incubator for 2 hours. After incubation, DMSO was added and shaken for 15 minutes, and the absorbance was measured using a microplate reader. Cell viability (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) * 100%.

[0040] The sample groups in the experiment were as follows: the lyophilized FN obtained in Example 5 was dissolved in complete MEM medium and sterilized by filtration using a 0.22 μm filter membrane. The FN solution was diluted with complete MEM medium to 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 0.9 mg / mL, and 1 mg / mL, respectively.

[0041] like Figure 4 As shown, the experimental results showed that compared with the control group, recombinant human fibronectin FN had a significant proliferation-promoting effect at a concentration of 1 mg / mL.

[0042] Effect Example 3 Recombinant human fibronectin FN promotes cell migration test The cells were digested and counted, and 1 × 10 cells were seeded per well in a six-well plate. 6 HaCaT skin keratinocytes were cultured for 24 hours at 37°C and 5% CO2. Then, a 10 μL pipette tip was used to align the well plate vertically with a ruler (a special cell culture dish for scratching can also be used). The longitudinal (horizontal) scratch line was gently pushed down to form a scratch. The cells were rinsed 3 times with PBS to remove the scratched cells. A sample group (FN sample dissolved in DMEM serum-free medium) and a control group (DMEN serum-free medium) were set up. 2 ml of serum-free medium was added to the sample group. The sample content was 0.1, 0.3, 0.5, 1, and 2 mg / mL, respectively. The control group only added serum-free medium. Continue to culture at 37°C and 5% CO2, and take pictures under a 40x microscope at 0h, 24h, 48h, and 72h with the intersection of the transverse and longitudinal scratch lines as the core. Cell migration rate (%) = (initial scratch area - scratch area at time t) / initial scratch area. The cell migration rate of each group was calculated as follows: Figure 5 、 Figure 6 As shown in Figure 2, compared with the blank control group, the FN samples at a concentration of 2 mg / mL all had a migration promoting effect. Effect Example 4: Thermal Stability Test of Recombinant Human Fibronectin FN High temperatures can break down the bonds within proteins, causing changes in their spatial structure and irreversible denaturation. The spatial structure of proteins is crucial to their function; once irreversible denaturation occurs, they lose their biological activity. High temperatures also increase the interactions between protein molecules, leading to aggregation and precipitation. Generally, when ambient temperatures exceed 60°C, proteins begin to undergo irreversible denaturation and precipitation. The recombinant human fibronectin heat resistance test verifies the thermal stability of recombinant human fibronectin by treating the protein in a high-temperature water bath and then comparing and analyzing the protein's appearance and purity before and after treatment.

[0043] Specific implementation method: Heat a water bath to 80°C, prepare the recombinant human fibronectin lyophilized product obtained in Example 6 into a 10 mg / ml stock solution, and dispense it into 8 EP tubes, 1 ml of stock solution per tube. (The samples are sealed and no protein stabilizer is added). Place them in hot water and heat them for 0, 1, 2, 3, 4, 5, 6, and 7 hours. Take out the samples and observe if there is any change in appearance. Take samples and perform SDS-PAGE electrophoresis. The recombinant human fibronectin stock solution samples show no degradation after high-temperature boiling ( Figure 7 ).

[0044] The results of this experiment showed that heating treatment at 80℃ for 7 hours had no significant effect on the appearance and purity of the recombinant human fibronectin stock solution.

[0045] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human fibronectin, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. The recombinant human fibronectin according to claim 1, characterized in that: The nucleotide sequence of the recombinant human fibronectin is shown in SEQ ID No.

2.

3. A carrier, characterized in that The vector contains the nucleotide sequence shown in SEQ ID No.

2.

4. A host cell, characterized in that The host cell contains the vector according to claim 3.

5. A method for preparing recombinant human fibronectin according to claim 1, characterized in that: The specific steps include: (1) Protein sequence design Based on human fibronectin, the amino acid sequence shown in SEQ ID No. 1 was designed; (2) Gene design and synthesis Reversely designing a coding nucleic acid sequence based on the amino acid sequence of the recombinant human fibronectin in step (1), and performing codon optimization to obtain a nucleotide sequence encoding the recombinant human fibronectin, and then performing gene synthesis to obtain a nucleic acid fragment encoding the recombinant human fibronectin fragment; (3) Construction of expression vector The nucleic acid fragment obtained in step (2) was ligated to the pET30a(+) plasmid via the NdeI and XhoI multiple cloning sites to obtain an expression plasmid; (4) Construction and screening of expression strains The expression plasmid described in step (3) is transferred into competent host cells by heat shock method, spread on a resistance plate containing kanamycin, and cultured to obtain an expression strain; (5) Inducible expression Induce the expression strain obtained in step (4) and collect the bacterial solution; (6) Purification The bacterial liquid collected in step (5) is subjected to bacterial cell disruption, centrifugation, affinity chromatography, ion exchange chromatography, desalting, and freeze-drying to obtain a freeze-dried product of recombinant human fibronectin.

6. The preparation method according to claim 5, characterized in that The host cell is Escherichia coli BL21 (DE3).

7. Use of the recombinant human fibronectin according to claim 1 in cosmetics, medicines, health products, medical devices or biomaterials.

Images