Recombinant fibronectin and its preparation, purification method and application

Abstract

The application discloses a kind of recombinant fibronectin and its preparation, purification method and application.The application first intercepts natural fibronectin core active region (amino acid sequence is shown as SEQ ID NO.2), after codon optimization (its nucleotide sequence is shown as SEQ ID NO.1), constructs a high expression recombinant fibronectin strain GS115 / pPIC9K-FN, then finds a kind of operation simple, relatively low cost preparation recombinant fibronectin fermentation and purification technology, suitable for industrialization large-scale production, and the fibronectin prepared has good effect in the adhesion effect and elastase inhibition to Hacat cell simultaneously, to have the efficacy of promoting cell repair and tightening.

Description

Technical Field

[0001] This invention belongs to the fields of protein engineering and genetic engineering technology, specifically relating to a recombinant fibronectin and its preparation, purification methods and applications. Background Technology

[0002] Fibronectin was first discovered by Morrison in 1948. Due to its property of precipitating when cold, it was initially called cold-insoluble globulin, but is now generally known as fibronectin, abbreviated as FN. Fibronectin (FN) is a large glycoprotein with a molecular weight of approximately 440 kDa, consisting of a dimer formed by two 220 kDa subunits linked by disulfide bonds. The entire molecule has a V-shaped shape composed of two similar A and B chains. There are more than 20 types of fibronectin in the human body, widely distributed in blood, body fluids, and various tissues. It is an important component of the extracellular matrix (ECM), serving as a regulator of cellular activities and a crucial scaffold protein for maintaining and guiding 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, and the quality of the prepared products varies greatly from batch to batch, with complex compositions. Furthermore, there is a risk of carrying pathogens, thus limiting the widespread application of fibronectin in medicine and many scientific research fields. Recombinant DNA technology can solve the problems in fibronectin preparation, but the preparation of recombinant fibronectin still involves complex purification issues. Currently, compared with collagen, the expression level of recombinant fibronectin is not high, and the purification process is inefficient and costly, affecting the application range of recombinant fibronectin.

[0004] Shan Yufei et al. proposed a method for obtaining recombinant fibronectin efficiently and cheaply through bio-fermentation (patent number CN202010438531.4). This process requires introducing a His tag onto the target protein, using metal affinity chromatography packing material in the purification process, and adding a specific protease to completely remove the tag. The steps are cumbersome, and the production cost of recombinant fibronectin is greatly increased. CN201810718044.6 requires passing the protein through an SP column first, without the protein adhering to the column. The flow-through liquid is then passed through a Q column (to remove endotoxins), and then through a Ni column to complete the purification process. The process is relatively complex.

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

[0006] To address the shortcomings of existing technologies, this invention provides a recombinant fibronectin, its preparation, purification methods, and applications. First, the core active region of natural fibronectin is extracted and, after codon optimization, a strain highly expressing recombinant fibronectin is constructed. Then, a simple and low-cost fermentation and purification technique for preparing recombinant fibronectin is found, suitable for large-scale industrial production. Furthermore, the prepared fibronectin exhibits good effects on Hacat cell adhesion and elastase inhibition, thus promoting cell repair and firming.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a recombinant fibronectin, 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 that has more than 90% sequence identity with the amino acid sequence shown in SEQ ID NO.2.

[0011] A second aspect of the present invention provides a nucleic acid molecule capable of encoding the aforementioned 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 that has more than 90% sequence identity with the nucleotide sequence shown in SEQ ID NO.1.

[0014] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect, preferably the recombinant expression vector pPIC9K-FN.

[0015] In a fourth aspect, the present invention provides a host bacterium containing the recombinant expression vector described in the third aspect of the present invention or a chromosome integrated with the nucleic acid molecule described in the second aspect of the present invention or 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, comprising fermentation and purification.

[0017] The fermentation method is as follows: First, the genetically engineered bacteria GS115 / pPIC9K-FN are cultured sequentially in YPD solid medium, YPD liquid medium, and BMGY medium for primary, secondary, and tertiary seed culture stages. Then, fermentation is carried out using BSM medium. Specifically, at an inoculum size of 15%-20%, the cultured BMGY seed culture is transferred to a fermenter to begin fermentation. The fermentation speed is controlled at 200-800 rpm, and the dissolved oxygen is ≤30%. When the pH reaches 5.05 and the dissolved oxygen rises to above 60%, it indicates that the carbon source in the medium has been consumed, and continuous feeding mode is started. When the bacterial cell concentration (OD) reaches a certain level... 600 When the pH reaches 320-350, feeding is stopped to allow the remaining carbon source to be completely consumed. After the pH and dissolved oxygen rise again, induction medium is added to express the target protein. Induction expression is carried out for more than 72 hours, then fermentation is stopped, and the fermentation broth is collected. The purification method is as follows: the fermentation broth obtained above is centrifuged, the supernatant is collected, the supernatant is filtered through a hollow fiber membrane, the permeate is collected, the permeate is salted out to remove impurities, and then purified using cation exchange chromatography (SPChromstar FF). After ultrafiltration and desalting, the fibronectin product is freeze-dried.

[0018] A seventh aspect of the present invention provides the application 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 promoting cell repair and firming.

[0019] The beneficial effects of this invention are:

[0020] 1. This application addresses the difficulty in preparing and purifying recombinant fibronectin in the prior art. Through steps such as sequence design, recombinant strain construction, fermentation, and purification, a recombinant fibronectin with high purity is obtained. Compared with commercially available fibronectin, 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 this application is relatively simple. The cationic chromatography method used is easy to operate, low in cost, and suitable for large-scale production. Attached Figure Description

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

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

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

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

[0026] Figure 5 This is a graph showing the results of an elastase inhibition test conducted on the recombinant fibronectin (FN-M) provided in Example 4 of this invention. Detailed Implementation

[0027] The effects are illustrated below with reference to the embodiments and accompanying drawings.

[0028] Example 1: Sequence design of recombinant fibronectin

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

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

[0031] The recombinant human fibronectin sequence was optimized based on 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 synthesized in its entirety by Shanghai Sangon Biotech Co., Ltd., 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 confirmed the correct recombinant sequence. The recombinant expression plasmid pPIC9K-FN was linearized with SalI rapid digestion enzyme and then electroporated into P. pastoris GS115 expression host cells. Recombinant transformants were screened with genimycin G418 to obtain high-copy recombinant Pichia pastoris GS115 / pPIC9K-FN.

[0032] Example 3: Fermentation of recombinant fibronectin

[0033] The fermentation method for recombinant fibronectin includes the following steps:

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

[0035] Primary seed culture: The constructed bacterial strain GS115 / pPIC 9K-FN, preserved in glycerol tubes, was streaked onto YPD solid medium (1% yeast extract, 2% peptone, 2% glucose monohydrate, and 1.5% agar powder) in a Petri dish and incubated at 30°C for 48 hours to obtain single colonies.

[0036] Secondary seed culture: Select single colonies obtained above and place them in 50 mL of YPD liquid medium (1% yeast extract, 2% peptone, 2% glucose monohydrate), incubate at 30°C and 180 rpm for 20-24 h, controlling the inoculum concentration (OD). 600 The concentration of the solution is 12-16 (preferably 12-14), which is considered a secondary seed solution.

[0037] Tertiary seed culture: Tertiary seed culture was conducted in 2L Erlenmeyer flasks. Each flask was filled with 500mL of BMGY medium (2% peptone, 1% glycerol, 1% yeast extract, 10% 1% potassium dihydrogen phosphate buffer, 1% ammonium sulfate, and biotin). Two bottles were cultured in total. The OD values ​​were determined according to the initial tertiary seed culture medium. 600 Inoculate with a certain volume of secondary seed culture at 0.2°C, incubate at 30°C and 200 rpm for 12-15 hours, and control OD. 600 The concentration is 8.0-12.0 (preferably 8.0-10.0), which is equivalent to a third-grade seed solution.

[0038] Fermentation culture: The third-stage seed was inoculated into a 20L fermenter. The fermentation medium was BSM medium (glycerol 4.0%, potassium phosphate 1.8%, potassium hydroxide 0.42%, 85% phosphate 2.67% (v:v), calcium sulfate dihydrate 0.01%, magnesium sulfate heptahydrate 1.5%, and PTM1 trace element 0.44% (v:v) was 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 dissolving, filter through a 0.22μm filter membrane and store under cold.

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

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

[0042] Fermentation conditions: 200 rpm, pH 5.0, aeration rate of 15 L / min. Transfer the cultured BMGY seed liquid into the fermenter at an inoculum rate of 15%-20% to start fermentation. Control the fermentation speed at 200-800 rpm and the dissolved oxygen at ≤30%. When the pH reaches 5.05 and the dissolved oxygen rises to above 60%, it indicates that the carbon source in the culture medium has been consumed and the continuous feeding mode is started. The feeding flow rate is referred to Table 1 below.

[0043] Table 1: Replenishment Time and Replenishment Speed

[0044]

[0045]

[0046] bacterial cell concentration (OD) 600 When the pH reaches 320-350, stop feeding to allow the remaining carbon source to be completely consumed. After the pH and dissolved oxygen rise again, add induction medium to express the target protein. Keep the rotation speed at 600 rpm and the aeration rate at 15 L / min. Adjust the feeding rate to maintain dissolved oxygen in the range of 10-30%. Induce expression for 120 h, then end the fermentation and collect the fermentation broth.

[0047] After collecting the supernatant, the content of the target protein in the supernatant was detected by agarose gel electrophoresis. Figure 1The SDS-PAGE protein gel images show that the expression level of recombinant fibronectin increased continuously with the extension of induction time from 0 to 72 h. However, the expression level of recombinant fibronectin basically stopped increasing after 72 h to 120 h of induction. Therefore, fermentation was stopped after 72 h in subsequent experiments.

[0048] Example 4: Purification of fibronectin

[0049] The fermentation broth obtained above was centrifuged, and the supernatant was collected. The supernatant was then filtered through a hollow fiber membrane, and the permeate was collected. The permeate was subjected to salting out to remove impurities, followed by purification using cation exchange chromatography (SP Chromstar FF, Bailinke Pharmaceutical Technology (Shanghai) Co., Ltd.). After ultrafiltration and desalting, the product was lyophilized to obtain the fibronectin final product. The specific purification process of the above recombinant fibronectin is as follows:

[0050] (1) Centrifugation: Centrifuge the fermentation broth at 9000 rpm for 20 min and collect the supernatant.

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

[0052] (3) Crude purification: Add 20% ammonium sulfate to the supernatant collected in (2) for salting out. Stir slowly at room temperature for 10-20 min, centrifuge at 9000 rpm for 20 min to remove flocculent proteins from the solution, and collect the centrifuged supernatant. Add 10% ammonium sulfate according to the volume of the supernatant, stir slowly at room temperature for 10-20 min, centrifuge at 9000 rpm for 20 min to remove flocculent proteins from the solution, and collect the centrifuged precipitate.

[0053] (4) Purification: The precipitate collected in (3) was reconstituted with 20 mM Tris buffer, loaded onto an 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) Desalting: The eluent collected in (4) is subjected to ultrafiltration desalting with a membrane pore size of 10 kDa. Purified water is added to reduce the conductivity of the solution to <200 uS / cm.

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

[0056] The lyophilized recombinant fibronectin was dissolved in 0.9% physiological saline to a concentration of 1 mg / ml, and then subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 2As shown, the product prepared by this purification process has a purity of 99.1% when determined by the non-reducing form of the "SDS-polyacrylamide gel electrophoresis method" in the "Electrophoresis Method" of the Pharmacopoeia of the People's Republic of China.

[0057] Example 5: Purification of fibronectin

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

[0059] (1) Centrifugation: Centrifuge the fermentation broth at 9000 rpm for 20 min and collect the supernatant.

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

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

[0062] (4) Purification: The precipitate collected in (3) was reconstituted with 20 mM Tris buffer, loaded onto an 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) Desalting: The eluent collected in (4) is subjected to ultrafiltration desalting with a membrane pore size of 10 kDa. Purified water is added to reduce the conductivity of the solution to <200 uS / cm.

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

[0065] The lyophilized recombinant fibronectin was dissolved in 0.9% physiological saline to a concentration of 1 mg / ml, and then subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 3 As shown, the product prepared by this purification process has a purity of 95.7% when determined by the non-reducing form of the "SDS-polyacrylamide gel electrophoresis method" in the "Electrophoresis Method" of the Pharmacopoeia of the People's Republic of China.

[0066] Example 6: Recombinant fibronectin cell adhesion experiment

[0067] (1) Human immortalized epidermal cells (Hacat cells, purchased from the China Center for Type Culture Collection) were processed, digested with trypsin, washed twice with PBS, collected in centrifuge tubes, 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 × 10⁻⁶ cells / mL. 4 per mL.

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

[0069] (3) Then, according to 5×10 per hole 4 Cells were seeded in 96-well plates with 3 replicate wells.

[0070] (4) Incubate the cells in a 37°C incubator for 24 hours. Add 100 μL of calcein working solution (final concentration of 10 μM) to each well for fluorescence staining. Observe the fluorescence intensity of different groups using a fluorescence microplate reader. The maximum emission wavelength is 520 nm.

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

[0072] Subtract the fluorescence intensity value of the blank well (well without cells) from the fluorescence intensity value of each test well. Take the average of the fluorescence intensity values ​​of each replicate well.

[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 : The fluorescence intensity values ​​detected in the test sample group;

[0075] F 对照细胞 : Fluorescence intensity values ​​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, within the concentration range of 3–50 nM, the higher the concentration of FN-M, the higher the adhesion rate to cells. At 200 nM, FN-M exhibits the best adhesion effect on Hacat cells, which is higher than that of commercially available FN.

[0078] Table 2: Results of Recombinant Fibronectin Cell Adhesion Rate Experiment

[0079]

[0080] Example 7: Recombinant fibronectin protease inhibition

[0081] Recombinant fibronectin protease inhibition assay:

[0082] (1) The test samples were divided into three types: recombinant fibronectin (FN-M) prepared in Example 4, recombinant fibronectin prepared in Example 5, and commercially available fibronectin (Merck Life Sciences, product number 10838039001). Two concentration groups (A and B) were set for each group. 1.6 ml of 20 μg / ml fibronectin solution was added to the reaction tube of group A, and 1.6 ml of 200 μg / ml fibronectin solution was 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 borate buffer to the blank control reaction tube.

[0084] (3) Add 5 mL of AAAPAN solution (N-succinyl-Ala-Ala-Ala-p-nitroanilide, Shanghai Aladdin Biochemical Technology Co., Ltd.) and 1.0 mL of elastase to each of the sample reaction tube, positive reaction tube, and blank control reaction tube. Add pH 8.8 borate buffer to each reaction tube to bring the volume to 10 mL. Set up three replicates for each reaction tube during the experiment.

[0085] (4) Mix the mixture thoroughly and place it in a 37°C constant temperature 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 the reaction;

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

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

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

[0091] Table 3: Data on elastase inhibitory activity

[0092]

[0093]

[0094] The results are as follows Figure 5 As shown in Table 3, elastase has the ability to degrade various proteins such as collagen and elastin. Changes in the structure of elastin during skin aging are one of the important causes of wrinkles and skin sagging, and inhibiting elastase from breaking down elastin can have an anti-wrinkle and firming effect. The experimental results show that recombinant fibronectin (FN-M) has a better inhibitory effect on elastase, and its inhibitory effect on elastase is stronger than that of commercially available FN.

Claims

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

2.

2. A nucleic acid molecule capable of encoding the recombinant fibronectin of claim 1, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.

1.

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

4. The recombinant expression vector as described in claim 3, characterized in that, The recombinant expression vector is the recombinant expression vector pPIC9K-FN.

5. A host bacterium, said host bacterium containing the recombinant expression vector of claim 3 or a chromosome integrated with the nucleic acid molecule of claim 2.

6. The host bacterium as described in claim 5, characterized in that, It is a 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 is as follows: First, the genetically engineered bacteria GS115 / pPIC9K-FN are cultured sequentially in YPD solid medium, YPD liquid medium, and BMGY medium for primary, secondary, and tertiary seed culture stages. Then, fermentation is carried out using BSM medium. The specific fermentation steps are as follows: At an inoculum size of 15%-20%, the cultured BMGY seed culture is transferred to a fermenter to begin fermentation. The fermentation speed is controlled at 200-800 rpm, and dissolved oxygen is ≤30%. When the pH reaches 5.05 and the dissolved oxygen rises above 60%, it indicates that the carbon source in the culture medium has been consumed, and continuous feeding mode is initiated. The fermentation continues until the bacterial cell concentration reaches OD... 600 When the pH reaches 320-350, stop feeding to allow the remaining carbon source to be completely consumed. After the pH and dissolved oxygen rise again, add induction medium to express the target protein. Induce expression for more than 72 hours, then end the fermentation and collect the fermentation broth. The purification method is as follows: the fermentation broth obtained above is centrifuged and the supernatant is collected. The supernatant is filtered through a hollow fiber membrane and the permeate is collected. The permeate is salted out to remove impurities and then purified using a cation exchange chromatography packing material SP Chromstar FF. After ultrafiltration and desalting, the recombinant fibronectin product is obtained by freeze drying.

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

9. The application as described in claim 8, characterized in that, It promotes cell repair and firming by simultaneously inhibiting Hacat cell adhesion and elastase activity.

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