Recombinant human fibronectin, method of preparation and use thereof in cosmetics

Abstract

The application belongs to the technical field of recombinant proteins, and more particularly discloses a recombinant human fibronectin, a preparation method and application thereof in cosmetics, aiming to solve the technical problem of low expression amount and low activity of the existing recombinant fibronectin. The recombinant human fibronectin is obtained by recombination after cutting the active region of natural fibronectin, introduction into a vector after codon optimization, and fermentation and purification after using Escherichia coli as an expression host cell. The recombinant human fibronectin disclosed by the application has high expression amount and good biological activity, can promote cell adhesion, proliferation or migration, and has a wide application prospect in the field of skin care, beauty and other cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein technology, specifically relating to a recombinant human fibronectin, its preparation method, and its use in cosmetics. 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 (FN). It is a multifunctional glycoprotein, abundant in plasma and widely distributed in the extracellular matrix. As a cell culture matrix, fibronectin can improve the adhesion and confluence rates of various cell types, enhance cell metabolism, and significantly increase the synthesis rates of DNA, RNA, and proteins. Coating fibronectin onto microsphere carriers as a medium for mass cell production can save space and raw materials, becoming a fundamental substance for the application of large-scale cell culture technology to produce new drugs.

[0003] Fibronectin plays a vital role in cell migration, adhesion, proliferation, hemostasis, tissue repair, and embryonic development. In recent years, research on the application value and fields of fibronectin has been increasing. In skin wound repair and healing, fibronectin can shorten wound healing time and reduce scarring. In skin care, especially in anti-aging and skin repair, fibronectin, along with other structural proteins such as humanized fibronectin and elastin, provides a support network for the skin, helping to improve skin texture and appearance.

[0004] Natural fibronectin, like collagen, can be extracted from animal tissues, but its production is limited, costly, and the quality of the prepared products varies greatly from batch to batch, with complex compositions. It also carries the risk of carrying pathogens, thus limiting its widespread application in medicine and many scientific research fields. Recombinant protein 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 low, and the purification process is inefficient and costly, affecting its application range. Designing recombinant fibronectin fragments with specific biological functions using genetic engineering technology and achieving their efficient expression in microbial expression systems has become a major trend in fibronectin production. Summary of the Invention

[0005] To address the shortcomings of existing technologies in the extraction of fibronectin, which results in low yields and low bioactivity, this invention selects specific functional fragments of human fibronectin and uses an E. coli expression system to optimize the amino acid coding sequence of recombinant human fibronectin shown in SEQ ID NO.1 based on E. coli codon preference, obtaining the coding gene nucleotide sequence SEQ ID NO.3. After cloning it into an expression vector and transforming it into host cells, recombinant human fibronectin is obtained after fermentation and purification.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] The present invention provides a recombinant human fibronectin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, the recombinant human fibronectin includes a protein tag.

[0009] Furthermore, the protein tag is selected from one or more of the following: 6×His tag, Flag tag, GST tag, MBP tag, and Myc tag.

[0010] Furthermore, the amino acid sequence of the recombinant human fibronectin has at least 80% sequence identity with SEQ ID NO.1, specifically it can be any one or any two of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, and 100%.

[0011] Furthermore, the amino acid sequence of the recombinant human fibronectin may have one or more amino acid substitutions, deletions, insertions and / or additions based on the sequence shown in SEQ ID NO.1.

[0012] Optionally, the substitution can be a conserved amino acid substitution, which means that compared with the amino acid sequence of SEQ ID NO.1, 1, 2 or 3 amino acids are replaced by amino acids with similar or close properties to form a peptide, and the function of the fibronectin is not changed after the substitution.

[0013] The present invention also provides a nucleic acid molecule that encodes the recombinant human fibronectin.

[0014] Furthermore, the nucleic acid molecule can be optimized based on codon preferences.

[0015] Furthermore, the sequence of the nucleic acid molecule is shown in SEQ ID NO.2 or SEQ ID NO.3.

[0016] The present invention also provides a recombinant plasmid carrying the aforementioned nucleic acid molecule.

[0017] The present invention also provides a host cell expressing the recombinant human fibronectin, the host cell comprising the recombinant plasmid.

[0018] It should be noted that "host cell" refers to any cell type susceptible to transformation, transfection, transduction, etc., of nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. "Host cell" encompasses any progeny of the parent cell that is not entirely identical to the parent cell due to mutations during replication. The host cell can be any cell useful in the production of recombinant human fibronectin according to the present invention. To produce recombinant fibronectin, the nucleic acid encoding the recombinant fibronectin can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. This nucleic acid can be easily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of specifically binding to the gene encoding the recombinant fibronectin). The host cell refers to a cell in which exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content, but may contain mutations. Methods for introducing vectors into host cells are well-known, such as electroporation, transfection, microinjection, gene gun technology, and liposome-mediated methods. The host cell is a prokaryotic or eukaryotic cell. The host cell is selected from any one of Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis. Further, the host cell includes Escherichia coli.

[0019] Furthermore, the host cell is Escherichia coli BL21(DE3).

[0020] The present invention also provides a method for preparing the recombinant human fibronectin, comprising the following steps: taking the host cells and inducing expression to obtain the recombinant human fibronectin of the present invention.

[0021] The present invention also provides the use of recombinant human fibronectin in the preparation of cosmetics, the cosmetics being used to promote cell adhesion, promote cell proliferation and / or promote wound healing.

[0022] The present invention also provides a cosmetic product comprising recombinant human fibronectin.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The gene sequence of recombinant human fibronectin in this invention is optimized based on the preferred codons of *E. coli* BL21(DE3), and the optimized codons contain more translation pause sites. After constructing the recombinant plasmid, expression was performed using an *E. coli* expression system, resulting in high expression levels and high purity. The preparation method of this invention is convenient, the purification process is simple, the cost is low, and the production cycle is short. The recombinant human fibronectin prepared by this invention exhibits biological activity, effectively promoting cell proliferation and improving cell adhesion, and is safe and reliable, making it suitable for wide application in cosmetics and other fields. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The translation curve of the recombinant human fibronectin described in this invention is shown (the wavy line is red, and the horizontal line in the middle is blue); where A is SEQ ID NO.2 before codon optimization, and B is SEQ ID NO.3 after codon optimization.

[0027] Figure 2 This is a spatial structure diagram of the recombinant human fibronectin of the present invention;

[0028] Figure 3 This is a graph showing the SDS-PAGE analysis results of the expression of recombinant human fibronectin multicopy transformants involved in this invention.

[0029] Figure 4 These are the results of the cell proliferation assay of recombinant human fibronectin according to the present invention;

[0030] Figure 5 These are the results of the cell adhesion promotion experiment of recombinant human fibronectin in this invention;

[0031] Figure 6 The results of the recombinant human fibronectin cytotoxicity assay of this invention are shown. Detailed Implementation

[0032] The following detailed description of embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0034] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0035] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0036] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0037] The term “and / or” as used herein includes any and all combinations of one or more of the related listed items.

[0038] In this application, "preferred" is merely a description of a more effective implementation method or embodiment, and should be understood as not constituting a limitation on the scope of protection of this application.

[0039] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0040] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0041] The culture medium formula used in this invention is as follows:

[0042] Example 1: Design of recombinant human fibronectin and its encoding gene

[0043] In this embodiment, based on the amino acid sequence of natural human fibronectin (GenBank: AAA52462.1) in the GenBank database, the spatial structure of human fibronectin was analyzed using bioinformatics methods. A new amino acid sequence of recombinant human fibronectin was extracted and redesigned, and a His-Tag purification tag was added to the C-terminus, resulting in the amino acid sequence shown in SEQ ID NO.1 (328 amino acids, theoretical molecular weight 35.99 kDa):

[0044] MKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQTHPGYD TGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPTTATPIRHRPRPYPPNVGEEIQIGHIPREDVDYHLYPHGPGLNPNASTGQEALMMSCTCLGNGKGEFKCDPHEATCYDDGKTYHVGEQWQKEYLGAICSCTCFGGQRGWRCDNCRRPGGEPSPHHHHH (SEQ ID NO.1).

[0045] The nucleotide sequence encoding recombinant human fibronectin (rFN) of SEQ ID NO.1 is shown in SEQ ID NO.2:

[0046] ATGAAAGAAATTAACCTGGCGCCGGATAGCAGCAGCGTGGTGGTGAGCGGCCTGATGGTGGCGACCAAATATGAAGTGAGCGTGTATGCGCTGAAAGATACCCTGACCAGCCGCCCGGCGCAGGGCGTGGTGACCACCCTGGAAAACGTGAGCCCGCCGCGCCGCGCGCGCGTGACCGATGCGACCGAAACCACCATTACCATTAGCTGGCGCACCAAAACCGAAACCATTACCGGCTTTCAGGTGGATGCGGTGCCGGCGAACGGCCAGACCCCGATTCAGCGCACCATTAAACCGGATGTGCGCAGCTATACCATTACCGGCCTGCAGCCGGGCACCGATTATAAAATTTATCTGTATACCCTGAACGATAACGCGCGCAGCAGCCCGGTGGTGATTGATGCGAGCACCGCGATTGATGCGCCGAGCAACCTGCGCTTTCTGGCGACCACCCCGAACAGCCTGCTGGTGAGCTGGCAGACCCATCCGGGCTATGATACCGGCAACGGCATTCAGCTGCCGGGCACCAGCGGCCAGCAGCCGAGCGTGGGCCAGCAGATGATTTTTGAAGAACATGGCTTTCGCCGCACCACCCCGCCGACCACCGCGACCCCGATTCGCCATCGCCCGCGCCCGTATCCGCCGAACGTGGGCGAAGAAATTCAGATTGGCCATATTCCGCGCGAAGATGTGGATTATCATCTGTATCCGCATGGCCCGGGCCTGAACCCGAACGCGAGCACCGGCCAGGAAGCGCTGATGATGAGCTGCACCTGCCTGGGCAACGGCAAAGGCGAATTTAAATGCGATCCGCATGAAGCGACCTGCTATGATGATGGCAAAACCTATCATGTGGGCGAACAGTGGCAGAAAGAATATCTGGGCGCGATTTGCAGCTGCACCTGCTTTGGCGGCCAGCGCGGCTGGCGCTGCGATAACTGCCGCCGCCCGGGCGGCGAACCGAGCCCGCATCATCATCATCAT(SEQ ID NO.2);

[0047] Using the online codon optimization tool ExpOptimizer (https: / / www.novopro.cn / tools / codon-optimization.html), without altering the amino acid sequence of fibronectin shown in SEQ ID NO.1, and based on the codon preference of *E. coli* BL21(DE3), NcoI and KpnI restriction sites were removed during the design process. The gene sequence was optimized by simplifying the secondary structure of mRNA, optimizing repetitive sequences, and adjusting GC content. The optimized GC content decreased from 60.57% to 46.65%, which is more conducive to the efficient expression of this gene by the host bacterium *E. coli* BL21(DE3). The nucleotide sequence of fibronectin after codon optimization is shown in SEQ ID NO.3.

[0048] ATGAAAGAAATTAACCTTGCACCCGATTCCTCATCAGTTGTCGTCTCTGGACTAATGGTGGCAACAAAGTACGAGGTCTCAGTCTACGCTCTTAAGGATACTTTGACTAGTAGACCAGCACAGGGTGTTGTCACGACTCTTGAGAATGTTTCCCCCCCCAGAAGAGCAAGAGTAACAGACGCCACTGAAACCACTATCACTATTTCCTGGCGTACTAAGACTGAAACCATTACCGGATTCCAAGTTGATGCTGTACCAGCCAATGGTCAGACGCCTATACAGAGAACCATCAAGCCTGACGTGAGGTCTTACACTATTACCGGCTTACAACCAGGCACCGACTACAAGATTTATCTTTATACTTTGAATGACAACGCTCGAAGTAGTCCCGTAGTTATAGATGCTTCAACTGCTATTGATGCCCCATCTAACCTTAGATTCTTGGCAACCACCCCAAACTCTTTGCTTGTCTCATGGCAAACACACCCAGGTTACGATACTGGTAACGGAATTCAATTGCCTGGTACTTCCGGTCAACAGCCCTCAGTTGGTCAACAAATGATTTTCGAAGAGCACGGTTTCAGAAGAACCACGCCCCCTACTACAGCCACCCCAATTAGACATAGACCACGTCCATACCCTCCAAATGTAGGTGAAGAAATTCAGATTGGTCACATTCCTAGAGAGGATGTCGATTACCATCTATACCCTCACGGACCAGGCTTGAACCCAAATGCTTCCACCGGTCAGGAGGCATTGATGATGTCTTGCACATGTTTGGGAAATGGTAAGGGAGAGTTTAAGTGTGATCCTCATGAGGCAACATGCTACGATGATGGTAAAACATATCATGTGGGAGAGCAATGGCAAAAAGAGTACCTGGGTGCCATTTGTTCATGCACATGTTTCGGAGGACAAAGAGGTTGGAGATGTGATAATTGCAGAAGACCTGGAGGTGAGCCTTCCCCTCATCACCACCACCAC(SEQ ID NO.3)。

[0049] Translation pausing curves of recombinant human fibronectin before and after codon optimization, calculated using RiboTempo software, are shown below. Figure 1 As shown in A (before optimization, SEQ ID NO.2) and B (after optimization, SEQ ID NO.3), the red lines of the translation pausing curves form translation pausing sites in the predetermined regions (the red lines are lower than the blue lines). The translation rate of the unoptimized nucleotide bases in SEQ ID NO.2 on the ribosome is relatively constant, without any decrease in translation rate. However, the optimized SEQ ID NO.3 shows a significant decrease in translation rate throughout the translation process on the ribosome, with more translation pausing sites appearing (the red lines are lower than the blue lines). This allows the translated fibronectin sufficient time to fold, thus yielding a protein product with higher biological activity and expression levels.

[0050] The gene sequences shown in SEQ ID NO.2 and SEQ ID NO.3, as well as the amino acid sequence shown in SEQ ID NO.1, were all synthesized by Shanghai Qingke Biotechnology Co., Ltd.

[0051] Example 2 Expression and purification of recombinant human fibronectin

[0052] (1) Construction of recombinant human fibronectin expression vector

[0053] An NcoI restriction site and a Kex2 nucleotide sequence (AAAAGAGAGGCTGAAGCT) were added to the 5' end of the recombinant human fibronectin gene sequence (SEQ ID NO.2 and SEQ ID NO.3), and a KpnI restriction site was added to the 3' end of the sequence. The gene fragment was synthesized by Shanghai Qingke Biotechnology Co., Ltd. The synthesized gene fragment was inserted into pET32a(+) (purchased from Changsha Aibiwei Biotechnology Co., Ltd., HG-VYN0176) through the NcoI and KpnI restriction sites to obtain plasmids pET32a(+)-rFn-2 (SEQ ID NO.2) and pET32a(+)-rFn-3 (SEQ ID NO.3) expressing recombinant human fibronectin.

[0054] (2) Preparation of Escherichia coli BL21(DE3) competent cells

[0055] Escherichia coli BL21(DE3) strain (purchased from Changsha Aibiwei Biotechnology Co., Ltd., HG-VDN0199), stored at -80℃, was streaked onto LB solid medium for activation. Five single clones were picked from the newly activated E. coli BL21(DE3) agar plates and inoculated into 250mL Erlenmeyer flasks containing 40mL LB liquid medium. The culture was incubated overnight at 37℃ with shaking at 250rpm. OD was monitored.600 Value, waiting for its OD 600 When the value reaches 1.2-1.3, remove the culture flask containing the bacterial solution and place it on ice to pre-cool for later use.

[0056] Transfer the pre-chilled bacterial culture to a 50 mL centrifuge tube and centrifuge at 4°C, 4000 rpm for 5 min. Discard the supernatant and collect the cells. Resuspend the cells in 40 mL of pre-chilled sterile water and centrifuge at 4°C, 4000 rpm for 5 min. Discard the supernatant and collect the cells. Resuspend the cells again in 40 mL of pre-chilled sterile water and centrifuge at 4°C, 4000 rpm for 5 min. Discard the supernatant and collect the cells. Next, resuspend the cells in 5 mL of pre-chilled 1M D-sorbitol and centrifuge at 4°C, 4000 rpm for 5 min. Discard the supernatant and collect the cells. Finally, resuspend the cells in 1 mL of pre-chilled 1M D-sorbitol, gently vortex to mix, and place on ice for later use.

[0057] (3) Transformed into Escherichia coli BL21(DE3) competent cells

[0058] After draining the electroporation cuvette from 75% ethanol in a clean bench, add 1 mL of 1M D-sorbitol to rinse. After rinsing, aspirate the sorbitol solution. Invert the cuvette to drain the sorbitol solution and pre-cool it on ice. Take 5-10 μg (10 μL) of recombinant expression plasmids pET32a(+)-rFn-2 and pET32a(+)-rFn-3, and 80 μL of the above competent cells, respectively, and add them to the pre-cooled electroporation cuvette at 0.2 cm (add DNA first, then add BL21(DE3) competent cells). Mix gently and let stand for 5 min. Wipe the condensate off the outside of the cuvette and place it on the electroporator. Electroporate at 2000 V, 200 Ω, and 25 μF. Immediately after electroporation, add 1 mL of pre-cooled 1M D-sorbitol to the electroporation vessel. Transfer the electroporation product to a sterile 10 mL centrifuge tube and incubate at 30°C for 1 hour. Then, place the 10 mL centrifuge tube in a clean bench, add 1 mL of LB liquid medium, and incubate at 250 rpm and 28°C for 2 hours in a constant temperature shaker to restore the strain.

[0059] (4) Screening of positive transformants

[0060] Spread 100-200 μL of the above bacterial suspension onto LB agar plates containing 100 μg / ml ampicillin sodium. Incubate at room temperature for 10 min, then incubate upside down at 37°C for approximately 2-5 days until single colonies appear. Pick a single colony from a YPD plate and transfer it to a 96-well plate containing 200 μL of LB liquid medium (containing 100 μg / ml ampicillin sodium). Continue incubation at 37°C. After 48 h, rehydrate the bacterial suspension and transfer 10 μL from each well to a new 96-well plate (containing 190 μL of LB). Continue incubation for 24 h, then repeat the above steps. After 24 h, rehydrate the bacterial suspension from a third 96-well plate and spot 1 μL onto LB agar plates containing 200 μg / ml, 300 μg / ml, and 500 μg / ml ampicillin sodium, respectively, and continue incubation. If the transformant can grow on a plate containing a high concentration of ampicillin sodium (500 μg / ml), it indicates that the transformant contains a multi-copy expression cassette. This screening step yields a highly efficient recombinant human fibronectin-expressing *Escherichia coli* strain BL21(DE3).

[0061] (5) Expression and purification of recombinant human fibronectin

[0062] The *E. coli* BL21(DE3) strains containing the recombinant expression plasmids pET32a(+)-rFn-2 and pET32a(+)-rFn-3 constructed above were streaked onto LB solid medium (with 100 μg / ml ampicillin sodium) and incubated at 37°C for 12 h. Single colonies were picked and inoculated into LB liquid medium and incubated at 37°C and 220 rpm for 24 h until OD600 reached. nm =4. Adjust the volume of LB culture medium according to the measured OD value, centrifuge at 3000 rpm for 10 min to collect the cells, and then resuspend the cells in LB liquid medium to make the initial OD value 0.5. Continue to culture at 37℃ and 220 rpm, adding 0.5% methanol to the medium every 24 h. After 72 h of induction culture, stop the fermentation and collect the fermentation broth.

[0063] The fermentation broth was centrifuged at 3000g for 20 minutes at 4°C, and the supernatant 1 and the bacterial precipitate were collected. The bacterial precipitate was resuspended in 50mM PBS (pH 8.0) and then sonicated (45 min, 3 s, 2 s interval, 500 W) to release recombinant human fibronectin from the bacterial cells. The lysed bacterial cells were then centrifuged (3000g for 20 minutes at 4°C) to separate the supernatant 2 from the precipitate. The supernatant 1 and supernatant 2 were combined to obtain the protein supernatant.

[0064] Because recombinant proteins possess a His-Tag purification tag, they can bind strongly to nickel columns under suitable conditions; therefore, affinity chromatography was used for impurity separation. Before chromatography, the protein supernatant was filtered through a 0.45 μm filter. The equilibration buffer used for chromatography was 20 mM imidazole and 50 mM PBS (pH 8.0), and the eluent was 500 mM imidazole and 50 mM PBS (pH 8.0). Linear elution was performed, and peaks with appropriate molecular weights were selected for subsequent purification by SDS-PAGE. After desalting, 0.1-1% EK enzyme was added to the system, and the reaction was carried out overnight at 4°C. The enzyme-digested protein solution was then subjected to affinity chromatography again, and the flow-through peak was collected. The flow-through peak was concentrated by ultrafiltration and stored in 50 mM PBS (pH 8.0). The obtained protein was analyzed by SDS-PAGE electrophoresis to determine its molecular weight and expression. The results showed a clear protein band at 35 kDa, consistent with the theoretical molecular weight of recombinant human fibronectin (approximately 36 kDa), indicating successful initial expression. Furthermore, the expression level of the multi-copy transformant pET32a(+)-rFn-3 was higher than that of pET32a(+)-rFn-2 (e.g., ...). Figure 3 As shown in the figure, the protein bands are single, with no degradation bands, and the purity is greater than 95%.

[0065] Example 3: Cell proliferation assay using recombinant human fibronectin

[0066] BALB / c 3T3 cells (purchased from China Center for Type Culture Collection, catalog number GDC0224) were seeded in 96-well cell culture plates (5000 cells / well) and cultured at 37°C in a 5% CO2 incubator for 24 h. The culture medium was then changed to DMEM (purchased from Prologis, PM150210) and cultured for another 12 h. Recombinant human fibronectin was added at concentrations of 10 nM, 50 nM, and 100 nM, respectively. A positive control group (100 nM commercially available fibronectin, Merck Life Sciences, 10838039001) and a negative control group (containing the same volume of purified water) were also set up. Cultures were continued for 48–72 h. 10 μL of the solution was added to each well. CCK-8 reagent, incubated at 37℃ in a 5% CO2 cell culture incubator for 2 hours, then removed; the absorbance values ​​of the 96-well plate were read at 450nm and 630nm using an ELISA reader, with 630nm as the reference wavelength, and the absorbance was measured at 450nm, and the results were recorded.

[0067] Cell proliferation rate (%) = (OD450 of experimental group - OD450 of negative control group) / OD450 of negative control group × 100%.

[0068] The results of the cell proliferation assay are as follows Figure 4As shown, the results indicate that the recombinant human fibronectin of the present invention has significant cell proliferation-promoting activity, and the proliferation effect at concentrations of 10-100 nM is positively correlated with concentration. At 100 nM, the cell proliferation-promoting effect of the recombinant human fibronectin of the present invention is superior to that of commercially available fibronectin.

[0069] Example 4: Cell adhesion promotion assay using recombinant human fibronectin

[0070] Immortalized human epidermal cells (Hacat cells, purchased from the China Center for Type Culture Collection, catalog number GDC0106) were processed, digested with trypsin, washed twice with PBS, collected in centrifuge tubes, centrifuged at 1000 rpm for 5 min, and resuspended in DMEM medium containing 10% FBS to prepare a cell suspension. The cell density was controlled at 6.5 × 10⁻⁶ cells / mL. 4 Cells / mL. Different concentrations (10 nM, 50 nM, 100 nM) of recombinant human fibronectin were added to 96-well plates. A positive control group (100 nM of commercially available fibronectin, Merck Life Sciences, 10838039001) and a negative control group (with the same volume of purified water) were also set up. Then, 5 × 10⁻⁶ cells / mL were added to each well. 4 Cells were seeded in 96-well plates with three replicate wells. Cells were incubated at 37°C for 24 hours. 100 μL of calcein working solution (final concentration 10 μM) was added to each well for fluorescence staining. Fluorescence intensity was observed using a fluorescence microplate reader at a maximum emission wavelength of 520 nm. The fluorescence intensity of the blank wells (wells without cells) was subtracted from the fluorescence intensity of each test well, and the average fluorescence intensity of each replicate well was calculated. Cell adhesion promotion rate (%) = ((F...) 处理细胞 —F 空白 ) / (F 对照细胞 —F 空白 ))×100%.

[0071] F 处理细胞 : The fluorescence intensity values ​​detected in the test sample group;

[0072] F 对照细胞 : Fluorescence intensity values ​​detected in the control group;

[0073] F 空白 : Fluorescence intensity value detected in the blank group.

[0074] Results of cell adhesion promotion assay as follows Figure 5 As shown, the results indicate that the recombinant human fibronectin of the present invention has significant cell adhesion-promoting activity, and the proliferation effect at concentrations of 10-100 nM is positively correlated with concentration. At 100 nM, the cell proliferation-promoting effect of the recombinant human fibronectin of the present invention is comparable to that of commercially available fibronectin.

[0075] Example 5: Cytotoxicity Evaluation Assay of Recombinant Human Fibronectin

[0076] HeLa cells (purchased from China Center for Type Culture Collection, GDC0009) that had grown to 70%-80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin and cultured in complete medium (purchased from Beyotime Biotechnology, C7505C) to a cell density of 1×10⁻⁶ cells / year. 5 Cell suspension at 100 μL / mL was seeded into 96-well plates and cultured at 37°C with 5% CO2 saturated humidity. After 24 h of cell culture, the complete culture medium was aspirated. Recombinant human fibronectin solutions diluted to 10 nM, 50 nM, and 100 nM in DMEM medium were added to the experimental groups. The positive control group received 100 nM commercially available fibronectin (purchased from Merck Life Sciences, 10838039001), the control group received cells cultured in DMEM medium, and the blank group received cell-free DMEM medium. The cells were cultured for another 24 h at 37°C with 5% CO2 saturated humidity. 10 μL of CCK-8 reagent was added to each group, and the cells were incubated for 4 h. The absorbance (OD) of each well was measured using an ELISA reader at 450 nm. Cell viability was calculated based on the mean absorbance of each group using the following formula: Cell viability (%) = ((Experimental group OD450 - Blank group OD450) / (Control group OD450 - Blank group OD450)) × 100%.

[0077] Cytotoxicity test results as follows Figure 6 As shown, the results indicate that the cell viability of both the recombinant human fibronectin of the present invention and the commercially available fibronectin group is above 100%, indicating that the recombinant human fibronectin prepared by the present invention has no cytotoxicity and good safety.

[0078] Based on the foregoing description, those skilled in the art will understand that this disclosure can be implemented in different specific forms without altering its technical spirit and essential features. Therefore, it should be understood that the above embodiments are not restrictive in any respect but rather illustrative. The scope of this disclosure is limited by the appended claims, not by the preceding description, and thus all changes and modifications fall within the boundaries and scope of the claims, or equivalents of such boundaries and scope are therefore intended to be covered by the claims.

Claims

1. A recombinant human fibronectin, characterized in that, The amino acid sequence of the recombinant human fibronectin is shown in SEQ ID No.

1.

2. The recombinant human fibronectin according to claim 1, characterized in that, The recombinant human fibronectin includes a protein tag selected from one or more of the following: 6×His tag, Flag tag, GST tag, MBP tag, and Myc tag.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant human fibronectin as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID No. 2 or SEQ ID No.

3.

5. A recombinant plasmid, characterized in that, The recombinant plasmid carries the nucleic acid molecule according to any one of claims 3-4.

6. A host cell expressing the recombinant human fibronectin of claim 1, characterized in that, The host cell contains the recombinant plasmid as described in claim 5.

7. The host cell according to claim 6, characterized in that, The host cell was Escherichia coli BL21(DE3).

8. A method for preparing the recombinant human fibronectin of claim 1, characterized in that, The method includes the following steps: taking the host cells described in any one of claims 6-7 and inducing expression to obtain the recombinant human fibronectin described in claim 1.

9. The use of the recombinant human fibronectin according to claim 1 in the preparation of cosmetics, wherein the cosmetics are used to promote cell adhesion, promote cell proliferation and / or promote wound healing.

10. A cosmetic product, characterized in that, The cosmetic product contains the recombinant human fibronectin as described in claim 1.

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