Recombinant fibronectin as well as preparation method, purification method and application thereof

Abstract

The invention discloses recombinant fibronectin as well as a preparation method, a purification method and application thereof. A natural fibronectin core active region (the amino acid sequence is shown as SEQ ID NO.2) is firstly intercepted, after codon optimization (the nucleotide sequence of the natural fibronectin core active region is shown as SEQ ID NO.1), a strain GS115 / pPIC9K-FN for highly expressing recombinant fibronectin is constructed, and then a fermentation and purification technology which is simple and convenient to operate and low in cost and is used for preparing the recombinant fibronectin is found. And the prepared fibronectin has a relatively good effect in the aspects of adhesion effect on Hacat cells and elastase inhibition at the same time, so that the fibronectin has the effects of promoting cell repair and tightening.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering and gene engineering, and specifically relates to a recombinant fibronectin and a preparation, purification method and application thereof. Background Art

[0002] Fibronectin was first discovered by Morrison in 1948. Due to its property of precipitation when exposed to cold, it was originally called cold insoluble globulin. It is now generally called fibronectin, abbreviated as FN. Fibronectin (FN) is a large glycoprotein with a molecular weight of about 440KDa. It is a dimer formed by two subunits with a molecular weight of 220KDa connected by disulfide bonds. The entire molecule is composed of two similar A chains and B chains, forming a V shape. There are more than 20 types of fibronectin in the human body, which are widely present in blood, body fluids and various tissues. It is an important component of the extracellular matrix (ECM). It is not only a regulatory factor in the process of cell activities, but also an important scaffold protein that maintains and guides tissue structure and ECM composition.

[0003] Both natural fibronectin and collagen can be extracted from animal tissues, but the yield is limited, the cost is high, the quality of the prepared products varies greatly from batch to batch, and the composition is complex; there is also the risk of carrying pathogens, which limits the wide application of fibronectin in medicine and many scientific research fields. DNA recombinant technology can solve the problem of fibronectin preparation, but the preparation of recombinant fibronectin also involves a more complex purification problem. At present, compared with collagen, the expression level of recombinant fibronectin is not high, the purification process is inefficient and costly, which affects the application range of recombinant fibronectin.

[0004] Shan Yufei et al. proposed a method for obtaining recombinant fibronectin efficiently and inexpensively by a bio-fermentation method (patent number CN202010438531.4). This process requires the introduction of a His tag on the target protein, the use of a metal affinity chromatography filler in the purification process, and the addition of a specific protease to remove all tags. The steps are cumbersome and the production cost of recombinant fibronectin is greatly increased; CN201810718044.6 needs to pass through an SP column first, the protein does not hang on the column, and the flow-through liquid is taken and then passed through a Q column (to remove endotoxins), and then through a Ni column to complete the purification process. The process is relatively complicated.

[0005] Currently, most of the fibronectin on the market is extracted, with a large molecular weight and weak transdermal ability. The survey found that the fermentation yield of fibronectin under development by major research institutions is still at the gram level. Therefore, in the process of strain sequence construction, some optimization work is often done on the basis of the fibronectin sequence, such as adding a His tag sequence, which is called recombinant fibronectin. However, this recombinant fibronectin has low safety and is prone to adverse reactions such as allergies. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a recombinant fibronectin and its preparation, purification method and application. The present invention first intercepts the core active region of natural fibronectin, and after codon optimization, constructs a strain that highly expresses recombinant fibronectin, and then finds a fermentation and purification technology for preparing recombinant fibronectin that is simple to operate and low in cost, which is suitable for industrial large-scale production, and the prepared fibronectin has good effects on adhesion to Hacat cells and inhibition of elastase, thereby having the effect of promoting cell repair and firming.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect of the present invention, a recombinant fibronectin is provided, wherein the recombinant fibronectin is selected from:

[0009] (1) the amino acid sequence shown in SEQ ID NO.2;

[0010] (2) An amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO.2.

[0011] The second aspect of the present invention provides a nucleic acid molecule capable of encoding the above-mentioned recombinant fibronectin. Specifically, the nucleic acid molecule is selected from:

[0012] (1) the nucleotide sequence shown in SEQ ID NO.1;

[0013] (2) A nucleotide sequence having a sequence identity of more than 90% with the nucleotide sequence shown in SEQ ID NO.1.

[0014] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid molecule described in the second aspect, and is preferably a recombinant expression vector pPIC9K-FN.

[0015] The fourth aspect of the present invention provides a host bacterium, which contains the recombinant expression vector described in the third aspect of the present invention or has the nucleic acid molecule described in the second aspect of the present invention integrated into its chromosome, or is capable of expressing the recombinant fibronectin described in the first aspect of the present invention, preferably recombinant Pichia pastoris GS115 / pPIC9K-FN.

[0016] A sixth aspect of the present invention provides a method for preparing the above-mentioned recombinant fibronectin, which comprises fermentation and purification.

[0017] The fermentation method comprises the following steps: firstly, the genetically engineered bacteria GS115 / pPIC9K-FN is cultured in a YPD solid medium, a YPD liquid medium and a BMGY medium for primary, secondary and tertiary seed liquid culture in turn, and then the BSM medium is used for fermentation culture, specifically, the cultured BMGY seed liquid is transferred into a fermentation tank according to an inoculation amount of 15%-20% to start fermentation, the rotation speed is controlled at 200-800rpm, the dissolved oxygen is ≤30%, and when the pH reaches 5.05 and the dissolved oxygen rises to more than 60%, indicating that the carbon source in the culture medium has been consumed, a continuous feeding mode is started; when the bacterial cell concentration (OD 600 ) reaches 320-350, stop feeding to completely consume the residual carbon source; after the pH and dissolved oxygen rise again, add induction medium to express the target protein, induce expression for more than 72 hours, end fermentation, and collect fermentation broth. The purification method is: centrifuge the fermentation broth obtained above, collect the supernatant, filter the supernatant through a hollow fiber membrane, collect the permeate, salt out the permeate to remove impurities and proteins, and then purify it with a cationic chromatography filler (SPChromstar FF), ultrafiltration desalination and freeze-drying to obtain the fibronectin product.

[0018] The seventh aspect of the present invention provides the use of the above-mentioned recombinant fibronectin in the preparation of cosmetics, which has a good effect on the adhesion of Hacat cells and the inhibition of elastase, thereby having the effect of promoting cell repair and firming.

[0019] The beneficial effects of the present invention are:

[0020] 1. This application aims to solve the problem of difficulty in preparing and purifying recombinant fibronectin in the prior art, and obtains recombinant fibronectin with high purity through sequence design, recombinant strain construction, fermentation, purification and other steps; compared with fibronectin from commercial sources, the recombinant fibronectin obtained by this application has a higher cell adhesion rate and a higher inhibitory effect on elastase, and has good application value;

[0021] 2. The process for purifying recombinant fibronectin in the present application is relatively simple. The cationic chromatography method adopted is easy to operate, low in cost, and suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1Electrophoresis diagram of protein expression of GS115 / pPIC9K-FN-M strain at different fermentation times; Lane 1: 10-180 kDa protein marker; Lane 2: recombinant fibronectin induced for 24 h; Lane 3: recombinant fibronectin induced for 48 h; Lane 4: recombinant fibronectin induced for 72 h; Lane 5: recombinant fibronectin induced for 96 h; Lane 6: recombinant fibronectin induced for 120 h; Lane 7: recombinant fibronectin not induced;

[0023] Figure 2 This is the SDS-PAGE electrophoresis result of the recombinant fibronectin (FN-M) prepared in Example 3 of the present invention; Lane 1: 10-180 kDa protein marker; Lane 2: lyophilized recombinant fibronectin;

[0024] Figure 3 This is the SDS-PAGE electrophoresis result of the recombinant fibronectin prepared in Example 4 of the present invention; Lane 1: 10-180 kDa protein marker; Lane 2: lyophilized recombinant fibronectin;

[0025] Figure 4 is a diagram showing the results of a cell adhesion experiment conducted using the recombinant fibronectin (FN-M) provided in Example 4 of the present invention;

[0026] Figure 5 This is a graph showing the results of an elastase inhibition test conducted using the recombinant fibronectin (FN-M) provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0027] The effects are described below in conjunction with the embodiments and drawings.

[0028] Example 1: Sequence design of recombinant fibronectin

[0029] The natural human fibronectin amino acid sequence (sequence number NP_000081) was analyzed, and based on the reported active sites of the natural fibronectin amino acid sequence, a highly active amino acid sequence was screened out. The amino acid sequence is shown in SEQ ID NO.2, which is a 438 amino acid sequence including p421-p858 in the full-length sequence of natural human fibronectin (NCBI accession P02751).

[0030] Example 2: Construction of recombinant human fibronectin gene expression system

[0031] The sequence of recombinant human fibronectin was optimized according to the codon preference of Pichia pastoris, and the optimized protein nucleotide sequence is shown in SEQ ID NO.1. The codon-optimized recombinant human fibronectin nucleotide sequence was commissioned to Shanghai Sangon Biotechnology Co., Ltd. for full gene synthesis and cloned into the EcoRI and NotI restriction sites of the Pichia pastoris expression vector pPIC9K to obtain the recombinant expression vector pPIC9K-FN. DNA sequencing and alignment showed that the recombinant sequence was correct. The recombinant expression plasmid pPIC9K-FN was linearized by SalI fast cutting enzyme and then electroporated into the P. pastoris GS115 expression host cell. The recombinant transformant was screened by geneticin G418 to obtain high-copy recombinant Pichia pastoris GS115 / pPIC9K-FN.

[0032] Example 3: Fermentation of recombinant fibronectin

[0033] The fermentation method of recombinant fibronectin comprises the following steps:

[0034] (1) The genetically engineered bacteria GS115 / pPIC9K-FN was fermented to obtain a fermentation broth containing recombinant fibronectin.

[0035] Primary seed culture: Streak the constructed strain GS115 / pPIC 9K-FN stored in a glycerol tube on a YPD solid medium (1% yeast extract powder, 2% peptone, 2% monohydrate glucose, 1.5% agar powder) plate, and culture at 30°C for 48 hours to obtain a single colony.

[0036] Secondary seed culture: Pick the single colony obtained above and place it in 50 mL of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose monohydrate), culture at 30°C, 180 rpm for 20-24 h, and control the bacterial concentration (OD 600 ) is 12-16 (preferably 12-14), which is the secondary seed liquid.

[0037] Tertiary seed culture: Use 2L conical flasks to culture tertiary seeds, add 500mL BMGY medium (2% peptone, 1% glycerol, 1% yeast extract powder, 10% 1% potassium dihydrogen phosphate buffer, 1% ammonium sulfate, biotin ) and cultured two bottles. According to the OD 600 A certain volume of secondary seed solution was added to 0.2, and cultured at 30°C and 200 rpm for 12-15 h to control OD 600 It is 8.0-12.0 (preferably 8.0-10.0), which is the third-level seed liquid.

[0038] Fermentation culture: The tertiary seeds were inoculated into a 20L fermenter. The fermentation medium was BSM medium (4.0% glycerol, 1.8% potassium phosphate, 0.42% potassium hydroxide, 2.67% 85% phosphoric acid (v:v), 0.01% calcium sulfate dihydrate, 1.5% magnesium sulfate heptahydrate, and 0.44% (v:v) PTM1 trace elements were added after sterilization). The initial volume of the fermentation medium was 10L.

[0039] PTM1 trace elements: copper sulfate pentahydrate 0.6%, potassium iodide 0.01%, manganese sulfate monohydrate 0.3%, boric acid 0.002%, sodium molybdate dihydrate 0.02%, cobalt chloride hexahydrate 0.09%, zinc chloride 2.0%, ferrous sulfate heptahydrate 4.3%, biotin 0.02%, concentrated sulfuric acid 0.5% (v:v), after dissolution, filter with 0.22μm filter membrane and store in refrigerator.

[0040] Feed medium: 63% (w:v) glycerol, 1.2% (v:v) PTM1 trace elements, and the remainder water.

[0041] Induction medium: methanol 88%, PTM1 trace elements 1.2%.

[0042] Fermentation culture conditions: 200rpm, pH 5.0, ventilation 15L / min, according to the inoculation amount of 15%-20%, the cultured BMGY seed liquid was transferred to the fermentation tank to start fermentation, the rotation speed was controlled at 200-800rpm, the dissolved oxygen was ≤30%, and when the pH reached 5.05 and the dissolved oxygen rose to more than 60%, indicating that the carbon source in the culture medium was consumed, the continuous feeding mode was started, and the feeding flow rate was referred to Table 1 below.

[0043] Table 1: Feeding time and feeding rate

[0044]

[0045]

[0046] The bacterial concentration (OD 600 ) reached 320-350, the feeding was stopped to completely consume the residual carbon source. After the pH and dissolved oxygen rose again, the induction medium was added to express the target protein, the rotation speed was fixed at 600 rpm, the ventilation volume was 15 L / min, and the dissolved oxygen was controlled to be maintained in the range of 10-30% by adjusting the feeding rate. The expression was induced for 120 hours, the fermentation was terminated, and the fermentation liquid was collected.

[0047] After collecting the supernatant, agarose gel electrophoresis was used to detect the target protein content in the supernatant. Figure 1From the SDS-PAGE protein gel map, it can be seen that the expression level of recombinant fibronectin increases continuously with the extension of the induction time from 0 to 72 h. After 72 h to 120 h of induction, the expression level of recombinant fibronectin basically stops increasing. Therefore, in subsequent experiments, the fermentation was stopped at 72 h.

[0048] Example 4: Purification of fibronectin

[0049] The fermentation broth obtained above was centrifuged to collect the supernatant, the supernatant was filtered through a hollow fiber membrane and the permeate was collected, the permeate was subjected to salting out to remove impurities and proteins, and then purified using a cationic chromatography filler (SP Chromstar FF, Biolink Pharmaceutical Technology (Shanghai) Co., Ltd.), and then lyophilized after ultrafiltration and desalting to obtain the finished fibronectin. The specific implementation process of the purification of the above recombinant fibronectin is as follows:

[0050] (1) Centrifugation: The fermentation broth was centrifuged at 9000 rpm for 20 min and the supernatant was collected.

[0051] (2) Clarification: The supernatant collected in (1) was clarified using a hollow fiber membrane (membrane pore size: 300 kDa), the feed liquid temperature was controlled to <20°C, and the supernatant was collected.

[0052] (3) Crude purification: Add 20% ammonium sulfate to the supernatant collected in (2) for salting out, slowly stir at room temperature for 10-20 minutes, centrifuge at 9000 rpm for 20 minutes to remove the flocculent protein in the solution, and collect the centrifugal supernatant. According to the volume of the supernatant, continue to add 10% ammonium sulfate, slowly stir at room temperature for 10-20 minutes, centrifuge at 9000 rpm for 20 minutes to remove the flocculent protein in the solution, and collect the centrifugal precipitate.

[0053] (4) Purification: The precipitate collected in (3) was re-dissolved in 20 mM Tris buffer, loaded on a SP Chromstar FF ion chromatography column, eluted with Tris buffer containing 1 M sodium chloride, and the elution peak containing recombinant fibronectin was collected.

[0054] (5) Desalination: The eluate collected in (4) was subjected to ultrafiltration desalination, the membrane pore size was 10 kDa, and purified water was added to reduce the solution conductivity to <200 uS / cm.

[0055] (6) Freeze-drying: The desalted solution in (5) is freeze-dried to obtain recombinant fibronectin freeze-dried fibers (FN-M).

[0056] The lyophilized recombinant fibronectin was dissolved in 0.9% saline to a 1 mg / ml sample and subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 2As shown, the product prepared by the purification process has a purity of 99.1% as measured by the non-reduced "SDS-polyacrylamide gel electrophoresis method" of the "electrophoresis method" in the "Pharmacopoeia of the People's Republic of China".

[0057] Example 5: Purification of fibronectin

[0058] The specific implementation process of purification of recombinant fibronectin is as follows:

[0059] (1) Centrifugation: The fermentation broth was centrifuged at 9000 rpm for 20 min and the supernatant was collected.

[0060] (2) Clarification: The supernatant collected in (1) was clarified using a hollow fiber membrane (membrane pore size: 500 kDa), the feed liquid temperature was controlled to <20°C, and the supernatant was collected.

[0061] (3) Crude purification: Add 20% ammonium sulfate to the supernatant collected in (2) for salting out, slowly stir at room temperature for 10-20 minutes, centrifuge at 9000 rpm for 20 minutes to remove flocculent proteins in the solution, and collect the centrifugal supernatant. According to the volume of the supernatant, continue to add 20% ammonium sulfate, slowly stir at room temperature for 10-20 minutes, centrifuge at 9000 rpm for 20 minutes to remove flocculent proteins in the solution, and collect the centrifugal precipitate.

[0062] (4) Purification: The precipitate collected in (3) was re-dissolved in 20 mM Tris buffer, loaded on a SP Chromstar FF ion chromatography column, eluted with Tris buffer containing 0.5 M sodium chloride, and the elution peak containing recombinant fibronectin was collected.

[0063] (5) Desalination: The eluate collected in (4) was subjected to ultrafiltration desalination, the membrane pore size was 10 kDa, and purified water was added to reduce the solution conductivity to <200 uS / cm.

[0064] (6) Freeze-drying: The desalted solution in (5) is freeze-dried to obtain freeze-dried recombinant fibronectin fibers.

[0065] The lyophilized recombinant fibronectin was dissolved in 0.9% saline to a 1 mg / ml sample and subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 3 As shown, the product prepared by the purification process has a purity of 95.7% as measured by the non-reduced "SDS-polyacrylamide gel electrophoresis method" of the "electrophoresis method" in the "Pharmacopoeia of the People's Republic of China".

[0066] Example 6: Recombinant fibronectin cell adhesion assay

[0067] (1) Human immortalized epidermal cells (Hacat cells, purchased from China Center for Type Culture Collection) were treated, digested with trypsin, washed twice with PBS, collected in a centrifuge tube, centrifuged at 1000 rpm for 5 min, and resuspended in DMEM cell culture medium containing 10% FBS to prepare a cell suspension. The cell density was controlled at 6.5×10 4 Pieces / mL.

[0068] (2) Different concentrations of FN-M solution (fibronectin obtained in Example 4) were added to a 96-well plate. A control group (200 nM, commercially available fibronectin, Merck Life Sciences, product number 10838039001) and a blank group (adding the same volume of purified water) were also set up.

[0069] (3) Then, 5×10 4 The cells were seeded in 96-well plates and 3 replicate wells were set up.

[0070] (4) The cells were incubated in a 37°C incubator for 24 h. 100 μL of calcein working solution (final concentration of 10 μM) was added to each well for fluorescence staining. The fluorescence intensity of different groups was observed using a fluorescence microplate reader. The maximum emission wavelength was 520 nm.

[0071] (5) Calculation of cell adhesion rate of recombinant fibronectin.

[0072] The fluorescence intensity value of each test well was subtracted from the fluorescence intensity value of the blank well (well without cells), and the fluorescence intensity values ​​of each replicate well were averaged.

[0073] Cell adhesion rate = ((F 处理细胞 -F 空白 ) / (F 对照细胞 -F 空白 ))×100%

[0074] F 处理细胞 : Fluorescence intensity value detected by the test sample group;

[0075] F 对照细胞 : Fluorescence intensity value detected in the control group;

[0076] F 空白 : Fluorescence intensity value detected in the blank group.

[0077] The results are as follows Figure 4 As shown in Table 2: In the concentration range of 3 to 50 nM, the higher the concentration of FN-M, the higher the adhesion rate to cells. At 200 nM, the adhesion effect of FN-M on Hacat cells is the best and higher than that of commercially available FN.

[0078] Table 2: Experimental results of recombinant fibronectin cell adhesion rate

[0079]

[0080] Example 7: Recombinant fibronectin protease inhibition

[0081] Recombinant Fibronectin Protease Inhibition Assay:

[0082] (1) There are three types of test samples, namely, the recombinant fibronectin (FN-M) prepared in Example 4, the recombinant fibronectin prepared in Example 5, and commercially available fibronectin (Merck Life Sciences, product number 10838039001), and two groups of concentrations (A and B) are set respectively. 1.6 ml of 20 μg / ml fibronectin solution is added to the reaction tube of group A, and 1.6 ml of 200 μg / ml fibronectin solution is added to the reaction tube of group B.

[0083] (2) Add 1.6 ml of 200 μg / ml oleanolic acid solution (Shanghai Aladdin Biochemical Technology Co., Ltd., content ≥ 98%) to the positive reaction tube, and add 1.6 mL of pH 8.8 boric acid buffer to the blank control reaction tube.

[0084] (3) 5 mL of AAAPAN solution (N-succinyl-Ala-Ala-Ala-p-nitroanilide, Shanghai Aladdin Biochemical Technology Co., Ltd.) and 1.0 mL of elastase were added to the sample reaction tube, positive reaction tube, and blank control reaction tube, respectively. pH 8.8 boric acid buffer was added to each reaction tube to make the volume 10 mL. Three parallel reactions were set for each reaction tube during the experimental test.

[0085] (4) Mix the mixture thoroughly and shake it in a 37°C water bath shaker for 30 min. Measure the absorbance at 405 nm.

[0086] Elastase inhibitory activity (%) = 1-(As-As0) / (Ac-Ac0) × 100%

[0087] As: absorbance of the sample group after reaction;

[0088] As0: absorbance of the sample group before reaction;

[0089] Ac: absorbance of the control group after reaction;

[0090] Ac0: absorbance of the control group before reaction.

[0091] Table 3: Elastase inhibitory activity data

[0092]

[0093]

[0094] The results are as follows Figure 5 As shown in Table 3: Elastase has the ability to degrade collagen, elastin and other proteins. The change in elastin structure during skin aging is one of the important causes of wrinkles and skin sagging, and inhibiting elastase from decomposing elastin can play an anti-wrinkle and firming role. From the test results, it can be seen that recombinant fibronectin (FN-M) has a better inhibitory effect on elastase, and has a stronger inhibitory effect on elastase than commercially available FN.

Claims

1. A recombinant fibronectin, characterized in that it is selected from: (1) the amino acid sequence shown in SEQ ID NO.2; (2) An amino acid sequence having a sequence identity of more than 90% with the amino acid sequence shown in SEQ ID NO.

2.

2. A nucleic acid molecule capable of encoding the recombinant fibronectin according to claim 1, wherein the nucleic acid molecule is selected from: (1) the nucleotide sequence shown in SEQ ID NO.1; (2) A nucleotide sequence having a sequence identity of more than 90% with the nucleotide sequence shown in SEQ ID NO.

1.

3. A recombinant expression vector comprising the nucleic acid molecule according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The recombinant expression vector is the recombinant expression vector pPIC9K-FN.

5. A host bacterium, wherein the host bacterium contains the recombinant expression vector according to claim 3 or the nucleic acid molecule according to claim 2 or the recombinant fibronectin according to claim 1 is integrated into the chromosome.

6. The host bacteria according to claim 5, characterized in that: It is recombinant Pichia pastoris GS115 / pPIC9K-FN.

7. The method for preparing the recombinant fibronectin according to claim 1, comprising fermentation and purification, The fermentation method comprises: firstly, the genetically engineered bacteria GS115 / pPIC9K-FN is cultured in a first-level, second-level and third-level seed solution in YPD solid medium, YPD liquid medium and BMGY medium in sequence, and then fermented and cultured in BSM medium. The fermentation steps are as follows: according to an inoculation amount of 15%-20%, the cultured BMGY seed solution is transferred into a fermentation tank to start fermentation, the rotation speed is controlled at 200-800rpm, the dissolved oxygen is ≤30%, and when the pH reaches 5.05 and the dissolved oxygen rises to more than 60%, indicating that the carbon source in the culture medium is consumed, a continuous feeding mode is started; when the bacterial cell concentration OD 600 When the pH reaches 320-350, stop feeding to completely consume the remaining carbon source; after the pH and dissolved oxygen rise again, add induction medium to express the target protein, induce expression for more than 72 hours, end fermentation, and collect the fermentation broth; The purification method comprises the following steps: centrifuging the fermentation broth obtained above, collecting the supernatant, filtering the supernatant through a hollow fiber membrane, collecting the permeate, salting out the permeate to remove impurity proteins, purifying the permeate with a cationic chromatography filler SP Chromstar FF, ultrafiltration desalting, and freeze-drying to obtain a recombinant fibronectin product.

8. Use of the recombinant fibronectin according to claim 1 in the preparation of cosmetics.

9. The use according to claim 8, characterized in that: It promotes cell repair and firming through its simultaneous adhesion to Hacat cells and elastase inhibition.

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