CN117586379B - Preparation method and application of recombinant fibronectin - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of recombinant fibronectin. The invention discloses a preparation technology of a recombinant small fragment fibronectin mutant. The recombinant fibronectin has small molecular weight but comprises a main functional region of FN, can be efficiently expressed in engineering bacteria, has simple preparation process and low cost, and is suitable for large-scale production. The recombinant protein has high activity in the aspect of promoting proliferation of skin fibroblasts or keratinocytes, has remarkable effects of relieving, repairing, moisturizing, enhancing skin elasticity and resisting wrinkles, and has great potential as a raw material and a finished product in the aspects of cosmeceutical, beauty, skin care and the like.

Description

A preparation method and application of recombinant fibronectin

Technical Field

The invention belongs to the field of biotechnology, and specifically relates to a preparation method and application of recombinant fibronectin.

Background technique

Fibronectin (FN) is a multifunctional glycoprotein widely present in vertebrates. It not only participates in the formation of ECM (extracellular matrix), but also regulates cell adhesion, migration and differentiation, thereby providing tissue development and regeneration. The FN molecule consists of two subunits of 220kDa and 250kDa in size, connected by two disulfide bonds at the C-termini of the two chains. The molecule consists of three repeating module structures - types I, II and III (FNI, FNⅡ and FNⅢ). Each subunit includes 12 FNI modules (each about 40 amino acid residues), 2 FNⅡ modules (each about 50 residues) and 15-17 FNⅢ modules (each about 90-100 residues). FN's Ⅲ8-10 is the central cell domain, among which Ⅲ-10 contains the RGD sequence, which can interact with integrin receptors, thereby affecting cell adhesion, migration, etc. Ⅲ-9 contains the synergistic motif Pro-His-Ser-Arg-Asn (PHSRN) that can bind to integrins, and Ⅲ-8 can promote the spatial directional stability of Ⅲ-9.

Traditional natural fibronectin is often extracted from human or animals, with complex processes and high costs. Recombinant fibronectin is a protein with high purity and high activity obtained by combining recombinant DNA or recombinant RNA technology with advanced separation and purification technology, which solves the cost and safety issues of natural fibronectin extraction. Since natural FN has a large molecular weight and low feasibility of full sequence expression, different functional domains of FN can be selected for expression to obtain recombinant FN with different functions.

FN has been used in tumors, biomaterials and other fields, and has also begun to be used in the field of beauty and skin care. Although patents CN111548410B, CN113717290A, CN114702571A, CN110204608B, CN113186109A and CN113527523A state that recombinant FN has anti-wrinkle, anti-aging, skin permeation and repair effects, soothing and moisturizing effects are very important in beauty and skin care, and there is currently no report on recombinant FN with such effects; NHDF is a human skin fibroblast, which is more suitable for the activity determination of recombinant FN in beauty and skin care applications, but there is currently no relevant report on the use of NHDF to determine the activity.

In order to overcome the technical problems such as the complex process and high cost of natural FN purification; the large molecular weight of natural FN and the low feasibility of full sequence expression; the low solubility, complex separation and purification, and low activity of existing recombinant proteins, the present invention provides a preparation technology for efficiently expressing recombinant fibronectin in Escherichia coli and a method for detecting its activity and function.

Summary of the invention

In order to solve the above problems, the present invention provides a recombinant fibronectin expressed in E. coli, which overcomes the technical problems of complex and high cost of natural FN purification process; large molecular weight of natural FN and low feasibility of full sequence expression; low solubility of existing recombinant protein FN and complex separation and purification.

In one aspect, the present invention provides a recombinant fibronectin, including a functional fragment of FN and/or a tag sequence; the amino acid sequence of the functional fragment of FN is SEQ ID NO.1.

Specifically, the tag sequence includes, but is not limited to: His tag, FLAG tag, c-Myc tag, HA tag, GST tag, MBP tag and/or SUMO tag.

Preferably, the tag sequence is a His tag, and the amino acid sequence is SEQ ID NO.2.

Specifically, the nucleotide sequence of the recombinant fibronectin is SEQ ID NO.3.

In another aspect, the present invention provides a method for preparing the aforementioned recombinant fibronectin, the method comprising the following steps:

S1. Construct a vector and clone SEQ ID NO.3 into an expression vector to obtain a recombinant vector;

S2, constructing an engineered bacterium, transforming the recombinant vector of step S1 into a host cell, screening positive clones, and obtaining an engineered bacterium;

S3, high-density fermentation of engineered bacteria, using pseudo-exponential feeding for high-density induction culture to obtain bacterial sludge;

S4, purification of recombinant fibronectin, crushing the bacterial sludge in step S3, centrifuging, passing through a membrane, and purifying by a column to obtain a crude extract; desalting the crude extract, purifying by ion exchange chromatography, and obtaining a recombinant fibronectin working solution.

Specifically, the expression vector in step S1 is a prokaryotic expression vector or a eukaryotic expression vector.

More specifically, the eukaryotic expression vector can be selected from an Escherichia coli expression vector, a yeast expression vector, an insect expression vector or a mammalian expression vector.

Preferably, the expression vector is pET-22b(+) vector.

Specifically, the host cell in step S2 may be a prokaryotic cell or a eukaryotic cell.

Preferably, the host cell is a prokaryotic cell.

More preferably, the cells are Escherichia coli BL21 (DE3) competent cells.

Specifically, the pseudo-exponential feeding in step S3 is divided into a first stage and a second stage; the first stage is to start feeding when the glucose concentration of the culture solution drops to 0, or the pH and dissolved oxygen increase significantly.

Specifically, the induction in step S3 is to add the inducer when _OD600 is not less than 20.

Specifically, the second stage is to carry out constant pH feeding at the beginning of induction.

Specifically, the column purification in step S4 uses a nickel column; the desalting uses a gel filtration chromatography column; and the ion exchange chromatography purification uses a DEAE anion exchange chromatography column.

In yet another aspect, the present invention provides a method for detecting the activity of the aforementioned recombinant fibronectin in promoting cell proliferation and adhesion, the method comprising measuring the growth activity of NHDF cells.

In yet another aspect, the present invention provides use of the aforementioned recombinant fibronectin in in vitro adherent cell culture.

In yet another aspect, the present invention provides the use of the aforementioned recombinant fibronectin in the preparation of skin damage repair, soothing, anti-wrinkle and moisturizing products.

The technical effects achieved by the present invention are as follows: the total expression level of the recombinant fibronectin of the present invention is 10%, and the soluble expression level is 87%; the high-density fermentation OD ₆₀₀ can reach 86; and the recombinant fibronectin with a purity of more than 95% can be obtained by three purification methods. The recombinant fibronectin of the present invention has a better cell growth promoting effect than the prior art. The anti-wrinkle cream prepared by using the recombinant fibronectin of the present invention has significant moisturizing and anti-wrinkle effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a map of the pET-22b(+)-rFN vector framework of the present invention.

FIG2 is a flow chart of the preparation of recombinant fibronectin.

Figure 3 is a graph showing the agarose gel electrophoresis results of PCR amplification of the target gene and the linearized vector, wherein channel 1 in the figure is the maker, channel 2 in the figure is the agarose gel electrophoresis result of the target gene, and channel 3 is the agarose gel electrophoresis result of the linearized vector.

FIG. 4 is a protein electrophoresis diagram of recombinant fibronectin expressed by shake flask fermentation.

Figure 5 is a nickel column electrophoresis diagram of recombinant fibronectin purification, wherein channel 1 is the supernatant of the crushing; channel 2 is the flow-through; channels 3-5 are the washing solution; channels 6-9 are 50mM imidazole eluent; channel 10 is 100mM imidazole eluent.

Figure 6 is an electrophoresis diagram of the recombinant fibronectin purification, wherein channels 1, 2, and 3 are the electrophoresis results before, during, and after the target protein peak purified by gel filtration chromatography, respectively, and channel 4 is the electrophoresis result of the target protein peak purified by DEAE ion exchange chromatography.

FIG. 7 shows the OD ₄₅₀ value and fitting curve of Example 1.

FIG8 shows the EC ₅₀ values of Example 1 and Comparative Examples 1-10 acting on NHDF.

FIG. 9 shows the OD ₄₅₀ value and fitting curve of Comparative Example 1.

FIG. 10 shows the OD ₄₅₀ value and fitting curve of Comparative Example 2.

FIG. 11 shows the OD ₄₅₀ value and fitting curve of Comparative Example 3.

FIG. 12 shows the OD ₄₅₀ value and fitting curve of Comparative Example 4.

FIG. 13 shows the OD ₄₅₀ value and fitting curve of Comparative Example 5.

FIG. 14 shows the OD ₄₅₀ value and fitting curve of Comparative Example 6.

FIG. 15 shows the OD ₄₅₀ value and fitting curve of Comparative Example 7.

FIG. 16 shows the OD ₄₅₀ value and fitting curve of Comparative Example 8.

FIG. 17 shows the OD ₄₅₀ value and fitting curve of Comparative Example 9.

FIG. 18 shows the OD ₄₅₀ value and fitting curve of Comparative Example 10.

Figure 19 shows the changing trend of moisture content in the stratum corneum of the test subjects' skin.

Figure 20 shows the changing trend of skin elasticity R2 in the test area of the subjects.

Figure 21 shows the changing trend of skin elasticity R5 in the test area of the subjects.

Figure 22 shows the trend of wrinkle length changes in the test area of the subjects.

Detailed ways

The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, especially in the claims, may be replaced by the term "consisting of".

The terms "optionally", "any", "arbitrarily" or "any" as used herein mean that the event or circumstance described subsequently may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specific sequence may but need not be present.

As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

The term "and/or" used herein should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

The recombinant FNIII8-10 described in the present invention is recombinant fibronectin.

Example 1

Based on the function of the natural fibronectin domain, the central cell domain FNⅢ8-10 of natural fibronectin was selected. The amino acid sequence of the FNⅢ8-10 domain is shown in SEQ ID NO.1. The amino acid sequence of SEQ ID NO.1 contains three consecutive amino acid residue sequences of Arg-Gly-Asp and a Pro-His-Ser-Arg-Asn (PHSRN) synergistic sequence, which is used to bind to the integrin receptors of specific cell types.

The preparation method of the recombinant fibronectin comprises the following steps:

1.1 Construction of recombinant vector

The FNⅢ8-10 domain of natural fibronectin was intercepted, and the coding DNA of FNⅢ8-10 was obtained by whole gene optimization and synthesis. The gene sequence before optimization is SEQ ID NO.4; the gene sequence after optimization is SEQ ID NO.5: In this embodiment, the target gene is His-FNⅢ8-10 gene, and the target gene includes FN functional fragment FNⅢ8-10 and tag sequence.

In this embodiment, first, amplification primers are designed according to the His-FNⅢ8-10 sequence. The amplification primers include a forward primer and a reverse primer, the nucleic acid sequence of the forward primer is shown in SEQ ID NO.6, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.7; the bases 5208-5354 of the pET-22b(+) vector are deleted to obtain a linearized vector, and amplification primers are designed according to the sequence of the linearized vector, and the linearized vector is amplified by PCR, and the amplification primers include a forward primer and a reverse primer, the nucleic acid sequence of the forward primer is shown in SEQ ID NO.8, and the nucleic acid sequence of the reverse primer is shown in SEQ ID NO.9.

Secondly, PCR amplification of the target gene and the linearized vector was performed. The PCR conditions for PCR amplification of the target gene and the linearized vector were: 98°C denaturation for 10s, 55°C annealing for 5s, and 68°C extension for 30s, for a total of 30 cycles, to obtain PCR amplification products; the PCR amplification products were separated, and the results are shown in Figure 3. The target bands were cut off, and the target bands were the PCR amplification products of the target gene and the linearized vector, respectively; and the PCR amplification products were recovered.

In this example, the PCR amplification products were separated by agarose gel electrophoresis, the target band was quickly cut out under ultraviolet light, and the target gene PCR amplification products were recovered using a DNA gel recovery kit.

After that, the recombinant vector was constructed. The target gene PCR amplification recovery product, the linearized vector PCR amplification recovery product and the seamless cloning enzyme (purchased from YEASEN) were mixed at a ratio of 2.5 μL: 2.5 μL: 5 μL, and reacted at 50°C for 30 minutes to obtain the recombinant vector. The map of the recombinant vector is shown in Figure 1.

The calculation formulas for the optimal linearized vector dosage and insert fragment dosage of the seamless cloning reaction system in this embodiment are as follows: the optimal vector dosage = 0.02 × vector base pair number] ng, the optimal insert fragment dosage = [0.04 × insert fragment base pair number] ng.

1.2 Construction of engineered bacteria

In this example, the recombinant vector was transformed into competent Escherichia coli BL21 (DE3) by heat shock method (the preparation of competent cells is a conventional experimental method in the art).

Add all the recombinant vectors obtained by seamless cloning to 100 μL of E. coli BL21 (DE3) competent cells, mix well, let stand on ice for 30 minutes, heat shock in a 42°C water bath for 90 seconds, and then quickly transfer to ice and let stand for 2 minutes. Add 200 μL of non-antibiotic LB medium to the competent cells in the clean bench, culture at 37°C and 200rpm for 1 hour, pour it onto ampicillin-resistant LB solid medium, and invert it in a 37°C constant temperature incubator for overnight culture. After ampicillin resistance screening, select transformants for culture, and verify the correct transformation through colony PCR and sequencing to obtain positive engineering bacteria.

1.3 Induced expression of engineered bacteria

A single colony of positive engineering bacteria was picked and inoculated into LB medium, cultured at 37°C and 200rpm for 8h to obtain seed solution, and then the seed solution was inoculated into the fermentation medium at 1%, cultured at 37°C and 200rpm for 3h, and then 0.5mM IPTG was added and induced at 30°C overnight. The cells were collected by centrifugation at 4°C and 8000rpm for 10min, and the cells were resuspended at a ratio of 1:10 of cell volume (g): PBS (mL), ultrasonically broken in an ice bath, centrifuged at 12000rpm for 10min, and the whole cell lysate and supernatant were collected. The expression of the recombinant protein was detected by SDS-PAGE analysis, and the results are shown in Figure 4.

1.4 High-density fermentation of engineered bacteria

The frozen glycerol tube strain was inoculated into LB medium at 1%, and cultured at 37°C and 200rpm for 8h to obtain seed liquid. The seed liquid was inoculated into a 3L fermenter at 2.5%, and the fermenter was filled with 2L. The pH was controlled to 7.0 by adding ammonia water, and the DO was controlled to be above 20% by ventilation and stirring speed. When the glucose concentration in the fermenter dropped to 0, or the pH and dissolved oxygen increased significantly, feeding began, using a pseudo-exponential feeding strategy, and setting the specific growth rate to 0.2h ^-1 . When _OD600 reached 20, 0.3mM IPTG was added for induction, and the temperature was lowered to 20°C. At the beginning of induction, a constant pH strategy was used for feeding. The fermentation was terminated after 8h of induction, when _OD600 reached 86.

Among them, the recombinant expression vector pET-22b(+) itself has ampicillin resistance and a T7 promoter. After being transferred into the Escherichia coli BL21(DE3) expression strain, the T7 RNA polymerase contained in the strain can initiate the transcription of the recombinant protein gene. Under IPTG induction conditions, the protein translation system of Escherichia coli itself can be used to express the recombinant protein pET-22b(+)-rFN. The recombinant protein has a His-tag label and can be used for metal affinity purification.

The shake flask fermentation medium formula in this embodiment is 10 g/L peptone, 20 g/L yeast powder, 10 g/L glucose, 2 g/L potassium dihydrogen phosphate, 4 g/L dipotassium hydrogen phosphate, 7 g/L disodium hydrogen phosphate, 1.2 g/L ammonium sulfate, and 1 g/L magnesium sulfate.

In this embodiment, the fermentation medium formula of the fermentation tank is 10g/L peptone, 20g/L yeast powder, 2g/L glucose, 2g/L potassium dihydrogen phosphate, 4g/L dipotassium hydrogen phosphate, 7g/L disodium hydrogen phosphate, 1.2g/L ammonium sulfate, 1g/L magnesium sulfate, and 0.3mL/L bubble damping fluid; the feed medium is: C source-500g/L glucose, N source-60g/L peptone, 20g/L yeast powder, and ammonia water.

The PBS buffer in this embodiment is prepared as follows: 10 mM disodium hydrogen phosphate-sodium dihydrogen phosphate, 50 mM sodium chloride, pH 7.4.

1.4 Purification of recombinant fibronectin

The homogenate supernatant is collected by centrifugation and filtered with a filter membrane for use as the sample; a nickel ion chelate affinity chromatography column is used for crude purification to obtain a crude pure liquid, and then a gel filtration chromatography is used to remove sodium chloride and imidazole from the crude pure liquid, and finally an ion exchange chromatography is used for fine purification to obtain a recombinant fibronectin working solution, which can be further lyophilized into a certain amount of fibronectin lyophilized powder by adding excipients such as mannitol, mannan, dextran, erythrose, and trehalose.

In the present embodiment, the broken supernatant was collected by centrifugation, and a 0.22 μm filter membrane was used for filtration. The Ni Focurose FF (IMAC) nickel ion chelate affinity chromatography column of Huiyan Biology was used to perform the crude purification of recombinant fibronectin. First, 10 CVs were rinsed with purified water at a flow rate of 5 mL/min, and then 10 CVs were rinsed with PBS at a flow rate of 5 mL/min. After the conductivity value and the 280 nm wavelength absorption value were stable, the sample was loaded at a flow rate of 2 mL/min. After the loading was completed, the nickel column was rinsed with PBS at a flow rate of 5 mL/min until the ultraviolet absorption value was stable. Finally, the following three eluents were used to elute in the order of imidazole concentration from low to high, and the protein corresponding to the elution peak was collected to obtain crude pure recombinant fibronectin, and the SDS-PAGE electrophoresis of the eluted sample was detected, and the results were shown in Figure 5.

Among them, the equilibrium solution is PBS buffer (10mM PB, 50mM NaCl, pH7.4);

Washing solution: PBS buffer containing 20 mM imidazole;

Elution buffer 1: PBS buffer containing 50 mM imidazole;

Eluent 2: PBS buffer containing 100 mM imidazole.

In this example, the recombinant fibronectin was eluted in a buffer solution containing 50 mM imidazole to obtain a crude pure solution.

In this embodiment, the gel filtration chromatography column is rinsed with 10mM PB (pH 7.4) buffer at a flow rate of 8mL/min until the conductivity of the solution after passing through the gel filtration chromatography is close to that of the initial PB, that is, the balance of the gel filtration chromatography column is completed. The crude recombinant fibronectin solution is loaded onto the gel filtration chromatography column at a flow rate of 2mL/min, and after loading, the gel filtration chromatography column is continuously rinsed with 10mM PB buffer at a flow rate of 8mL/min, and the elution peak is collected according to the conductivity and the absorption value of 280nm wavelength to obtain a desalted solution, and the desalted solution is subjected to SDS-PAGE electrophoresis detection, and the results are shown in Figure 6.

In the present embodiment, the desalted solution is combined and ion exchange chromatography is performed. The DEAE Focurose FF anion exchange chromatography column of Huiyan Biology is used to purify recombinant fibronectin. First, the DEAE column is rinsed with 10mM PB (pH7.4) buffer at a flow rate of 5mL/min, until the conductivity and pH of the outflowing solution are close to the initial 10mM PB buffer, and the balance of the ion exchange chromatography column is completed. The desalted solution is loaded at a flow rate of 2mL/min, and after loading, 10 CVs are rinsed with buffer at a flow rate of 5mL/min, and finally eluted with eluent in the order of sodium chloride concentration from low to high, and the protein corresponding to the elution peak is collected to obtain pure recombinant fibronectin, and the SDS-PAGE electrophoresis test of the eluted sample is performed, and the test results are shown in Figure 6.

Among them, the equilibrium solution is 10mM PB (pH 7.4);

Elution buffer 1: equilibration buffer containing 50 mM NaCl;

Elution buffer 2: Equilibration buffer containing 200 mM NaCl.

The E. coli expression system has low cost and simple operation. The present invention utilizes the E. coli expression system to obtain a total expression level of 10% for recombinant fibronectin and a soluble expression level of 87%; the high-density fermentation OD ₆₀₀ can reach 86; and the three purification methods can obtain recombinant fibronectin with a purity of more than 95%.

Comparative Example 1

According to the patent "Recombinant fibronectin with anti-wrinkle repair function and its preparation method and application" (patent number: CN111548410B), a fusion protein with RGD domain + conopeptide was prepared.

Comparative Example 2

FNⅢ1-C was prepared according to the patent "A recombinant human fibronectin Ⅲ1-C and its preparation method and application" (patent number: CN111217903B).

Comparative Example 3

According to the patent "A composite transdermal recombinant fibronectin and its application" (patent number: CN113717290A), a fusion protein of transdermal short peptide + FN sequence + collagen was prepared.

Comparative Example 4

According to the patent "A recombinant fibronectin and its application" (patent number: CN115976031B), a fusion protein of integrin + laminin was prepared.

Comparative Example 5

Recombinant human fibronectin was prepared according to the patent "Recombinant human fibronectin and its preparation, activity determination and stability experimental methods" (patent number: CN113480636A).

Comparative Example 6

Fibronectin was prepared according to the patent "Coding sequence of fibronectin mutant with high expression level and strong activity and its application" (patent number: CN112941081A).

Comparative Example 7

Fibronectin was prepared according to the patent "A fibronectin having the function of promoting stem cell colonization and a preparation method thereof" (patent number: CN114702571A).

Comparative Example 8

According to the patent "A yeast fermentation small molecule recombinant fibronectin peptide and its preparation method and application" (patent number: CN110204608B), a fusion protein of fibrin + collagen + heparin domain + integrin was prepared.

Comparative Example 9

According to the patent "A brewer's yeast expressing long-acting recombinant fibronectin and its application in cosmetics" (patent number: CN113186109A), the FNⅢ1-C+HAS fusion protein was prepared.

Comparative Example 10

According to the patent "A recombinant protein and its construction method and use" (patent number: CN113527523A), a fusion protein of transdermal peptide+FN sequence+elastase was prepared.

Application Examples

500 ppm fibronectin freeze-dried powder prepared with mannan as an excipient in the example was added to the formula. The specific formula and process are shown in Table 1:

Table 1: Fibronectin Anti-wrinkle Cream C3 Formulation Process

Effect Example 1

This example discloses a method for detecting the activity of recombinant fibronectin expressed by Escherichia coli in promoting cell proliferation and adhesion, which is used to verify the activity of recombinant fibronectin in Example 1 and Comparative Examples 1-10 in promoting human dermal fibroblasts (NHDF).

The reagents include: DMEM complete medium, DMEM serum-free medium, 0.25% trypsin, PBS buffer, 15 g/L BSA solution, and CCK-8 kit.

Specific steps are as follows:

The cells were revived and subcultured in complete cell culture medium. After the cells grew to 80% confluence, they were digested with trypsin and resuspended in serum-free culture medium to obtain a cell suspension.

Pre-dilute the recombinant fibronectin to 10 μg/mL with PBS, and then dilute it 2-fold after pre-dilution, making a total of 9 dilutions. Add 50 μL of protein dilution solution to the 96-well plate, set up 5 parallels for each concentration, and set up a negative control (PBS only), and incubate at 37°C for 3 hours.

After incubation, discard the liquid in the plate, add 100 μL 15% BSA to each well for blocking, and incubate in a 37°C incubator for 1 hour; after incubation, discard the liquid in the plate, add 100 μL PBS to each well for washing once; then add cell suspension with an inoculation density of 1×10 ⁵ cells/mL, inoculate 100 μL in each well, that is, each well contains 1×10 ⁴ cells, and incubate at 37°C for 3 hours.

After the incubation is completed, the medium in the plate is discarded, and the plate is washed twice with serum-free medium. A serum-free medium containing 10% CCK-8 solution (prepared immediately before use) is prepared and added to the 96-well plate in the form of liquid replacement. After gently shaking the 96-well plate, the plate is placed in a 37°C incubator for incubation for 1 hour.

The absorbance at a wavelength of 450 nm was detected using an enzyme-labeled instrument, and the values were analyzed and the activity of the recombinant fibronectin was determined by fitting the curve.

According to the OD ₄₅₀ value and the fitting curve, the EC ₅₀ value of recombinant FNⅢ8-10 acting on NHDF is about 872 ng/mL, as shown in Figure 7, while the EC ₅₀ of comparative examples 1-10 (Figures 9-18) is greater than that of recombinant FNⅢ8-10, as shown in Figure 8. The experimental results show that recombinant FNⅢ8-10 has a good effect of promoting cell growth, which means that the recombinant protein is active.

This example uses NHDF cells for the first time to determine the activity of recombinant FN. NHDF cells are derived from human skin, and FN is an important chemokine for NHDF, which can promote the migration and growth of NHDF cells, thereby accelerating wound healing. The proliferation and adhesion promoting effect of recombinant FNⅢ8-10 on NHDF confirms that the recombinant protein is suitable for use as a cosmetic skin care raw material in the human body.

Effect Example 2

1. Materials and instruments

1.1 Test samples: fibronectin anti-wrinkle cream of application example, and blank control anti-wrinkle cream C0 in which fibronectin freeze-dried powder was replaced with mannan.

1.2 Usage: Half-face test, subjects used the product once in the morning and once in the evening every day, with blank cream on the left and fibronectin anti-wrinkle cream on the right. The skin stratum corneum moisture content, skin elasticity, wrinkle length and other indicators were measured before using the product and 4 weeks after using the product.

1.3 Instruments: 3D skin imaging system (Antera 3D, Miravex, Ireland); skin stratum corneum moisture content tester (Corneometer, C&K, Germany); skin elasticity tester (Cutometer, C&K, Germany).

1.4 Test indicators: moisture content of skin stratum corneum, skin elasticity R2, skin elasticity R5, wrinkle length.

1.5 Test location: The measurement location for the moisture content of the skin stratum corneum and skin elasticity is the cheek, and the measurement location for wrinkle length is the area with a diameter of 15mm at the outer corner of the eye.

1.6 Testing Process

Day 0: After cleaning the face and equilibrating in the test environment for 20 minutes, measure the various test indicators of the test site.

Day 28 (±1): After cleaning the face and equilibrating in the test environment for 20 minutes, the subjects were asked to evaluate the product through a questionnaire and the various test indicators of the test area were measured.

1.7 Data Analysis

Calculate the mean and standard deviation of a skin physiological parameter for all subjects at the same time point. Calculate the change value (△ value) and change rate relative to before using the product through the mean value of the skin physiological parameter at each time point. The calculation formula is as follows:

Change value (Δ value) = T _n - _{T 0}

Where, T ₀ is the skin background value before using cosmetics in the test area.

_Tn ——Skin parameter measurement value after using cosmetics on the test area

n——revisit time

SPSS22.0 software was used for statistical testing to analyze whether there were significant differences in the changes in skin physiological parameters at each time point after using the sample compared with before using the product. If the data were normally distributed, a paired t-test, two-tailed test, and test level α = 0.05 were used; if the data were non-parametric or not normally distributed, a rank sum test of two related samples, two-tailed test, and test level α = 0.05 were used.

1.8 Test Results

1.8.1 Skin Moisture Content

The descriptive statistical results of the moisture content of the skin stratum corneum during the test period are shown in Table 2:

Table 2 Descriptive statistics of skin stratum corneum moisture content

Note: The significance analysis was performed compared with that before using the product, n.s.p≥0.05, *p<0.05, **p<0.01.

The trend of moisture content in the stratum corneum of the test area of the subjects is shown in Figure 19:

Result description:

Compared with before using the test sample, after using the product for 4 weeks, the average moisture content of the stratum corneum of the skin of anti-wrinkle cream C0 increased by about 8.6%, and the difference was extremely significant (p<0.01). The average moisture content of the stratum corneum of the skin of anti-wrinkle cream C3 increased by about 15.2%, and the difference was extremely significant (p<0.01). The increase in the moisture content of the stratum corneum of the skin using anti-wrinkle cream C3 was higher than that using anti-wrinkle cream C0. Fibronectin can significantly increase the moisture content of the skin.

1.8.2 Skin elasticity R2

The descriptive statistical results of skin elasticity R2 during the test period are shown in Table 3:

Table 3 Descriptive statistics of skin elasticity R2

Note: The significance analysis was performed before using the product, n.s.p≥0.05, *p<0.05, **p<0.01, ***p<0.001,

****p<0.0001.

The change trend of skin elasticity R2 in the test area of the subjects is shown in Figure 20:

Result description:

Compared with before using the test sample, after using the product for 4 weeks, the average skin elasticity R2 of anti-wrinkle cream C0 increased by about 1.16%, and the difference was not significant (p>0.05); the average skin elasticity R2 of anti-wrinkle cream C3 increased by about 6.5%, which was a very significant difference (p<0.0001). Fibronectin can significantly improve skin elasticity R2.

1.8.3 Skin elasticity R5

The descriptive statistical results of skin elasticity R5 during the test period are shown in Table 4:

Table 4 Descriptive statistics of skin elasticity R5

Note: The significance analysis was performed compared with that before using the product, n.s.p≥0.05, *p<0.05, **p<0.01.

The change trend of skin elasticity R5 in the test area of the subjects is shown in Figure 21:

Result description:

Compared with before using the test sample, after using the product for 4 weeks, the average skin elasticity R5 of anti-wrinkle cream C0 increased by about 1.31%, and the difference was not significant (p>0.05); the average skin elasticity R5 of anti-wrinkle cream C3 increased by about 9.8%, which was a very significant difference (p<0.01). Fibronectin can significantly improve skin elasticity R5.

1.8.4 Wrinkle length

The descriptive statistics of wrinkle length during the test period are shown in Table 5:

Table 5 Descriptive statistics of wrinkle length

Note: The significance analysis was performed compared with that before using the product, n.s.p≥0.05, *p<0.05, **p<0.01.

The trend of wrinkle length changes in the test area of the subjects is shown in Figure 22:

Result description:

Compared with before using the test sample, after using the product for 4 weeks, the average wrinkle length of anti-wrinkle cream C0 decreased by about 3.8%, with no significant difference (p>0.05), and the average wrinkle length of anti-wrinkle cream C3 decreased by about 23.4%, with a very significant difference (p<0.01), indicating that the fibronectin of the present invention has the effect of significantly shortening the length of wrinkles.

1.9 Conclusion

Fibronectin has excellent moisturizing and anti-wrinkle effects.

Claims (13)

1. A recombinant fibronectin comprising a functional fragment of FN and a tag sequence; the amino acid sequence of the FN functional fragment is SEQ ID NO.1.

2. The recombinant fibronectin according to claim 1, wherein the tag sequence is His tag, FLAG tag, c-Myc tag, HA tag, GST tag, MBP tag and/or SUMO tag.

3. The recombinant fibronectin according to claim 2, wherein the tag sequence is His-tag and the amino acid sequence is SEQ ID No.2.

4. A recombinant fibronectin according to any of claims 1-3, wherein the nucleotide sequence of the recombinant fibronectin is SEQ ID No.3.

5. The method for producing a recombinant fibronectin according to any one of claims 1 to 4, comprising the steps of:

S1, constructing a vector, and cloning SEQ ID NO.3 onto an expression vector to obtain a recombinant vector;

s2, constructing engineering bacteria, transforming the recombinant vector in the step S1 into host cells, and screening positive clones to obtain the engineering bacteria;

S3, high-density fermentation of engineering bacteria, namely performing high-density induction culture by adopting a quasi-exponential feed supplement and a constant pH feed supplement to obtain bacterial sludge;

s4, purifying recombinant fibronectin, namely crushing the bacterial sludge obtained in the step S4, centrifuging, passing through a membrane, and purifying by a column to obtain a crude extract; desalting the crude extract, and purifying by ion exchange chromatography to obtain recombinant fibronectin working solution.

6. The method according to claim 5, wherein the expression vector of step S1 is a prokaryotic expression vector or a eukaryotic expression vector.

7. The method according to claim 5, wherein the host cell in step S2 is a prokaryotic cell or a eukaryotic cell.

8. The method according to claim 5, wherein the pseudo-exponential feeding in step S3 is started when the glucose concentration of the culture solution decreases to 0 or the pH and dissolved oxygen significantly increase.

9. The method according to claim 5, wherein the induction culture of step S3 is performed by adding an inducer at an OD ₆₀₀ of 20 or more.

10. The method according to claim 5, wherein the constant pH feeding in step S3 is performed at the beginning of induction.

11. The method according to claim 5, wherein the column purification of step S4 uses a nickel column; the desalination uses a gel filtration chromatographic column; the ion exchange chromatography purification uses a DEAE anion exchange chromatography column.

12. The method for detecting the activity of recombinant fibronectin in promoting cell proliferation adhesion according to any one of claims 1 to 4, comprising the measurement of the activity of NHDF cell growth.

13. Use of the recombinant fibronectin according to any of claims 1-4 for the preparation of an anti-wrinkle and moisturizing product.