Recombinant human fibronectin, preparation method and application of recombinant human fibronectin in cosmetics

Abstract

The invention belongs to the technical field of recombinant proteins, particularly discloses recombinant human fibronectin, a preparation method and application of the recombinant human fibronectin in cosmetics, and aims to solve the technical problems that existing recombinant fibronectin is small in expression quantity and relatively low in activity. The recombinant human fibronectin disclosed by the invention is recombined after an active region of natural fibronectin is intercepted, the recombinant human fibronectin is introduced into a vector after codon optimization, escherichia coli is used as an expression host cell, and the recombinant human fibronectin disclosed by the invention is obtained after fermentation and purification. The recombinant human fibronectin disclosed by the invention is high in expression quantity and relatively good in biological activity, can promote cell adhesion, proliferation or migration, and has a wide application prospect in the field of cosmetics for skin care, beauty and the like.

Description

Technical Field

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

[0002] Fibronectin was originally discovered by Morrison in 1948. Due to its tendency to precipitate when exposed to cold, it was initially called cold-insoluble globulin. It is now generally referred to 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 attachment and confluence rates of various cells, enhance cellular metabolism, and significantly increase the rates of DNA, RNA, and protein synthesis. Coating fibronectin onto microsphere carriers as a medium for mass cell production saves space and raw materials, making it a foundational substance for the production of new pharmaceuticals using large-scale cell culture technology.

[0003] In the life activities of the body, fibronectin is involved in cell migration, adhesion, proliferation, hemostasis, tissue repair and embryonic development. In recent years, there has been more and more research on the application value and field of fibronectin. In skin wound repair and healing, fibronectin can shorten the wound healing time and reduce wound scars; in skin care, especially in anti-aging and skin repair, fibronectin and other structural proteins such as humanized fibronectin and elastin provide a support network for the skin, which helps to improve skin texture and appearance.

[0004] Natural fibronectin, like collagen, can be extracted from animal tissues, but its production is limited, the cost is high, the quality of the prepared products varies greatly from batch to batch, and the composition is complex. There is also the risk of pathogens being carried, which limits the widespread application of fibronectin in medicine and many scientific research fields. Protein recombinant technology can solve the difficulties in fibronectin preparation, but the preparation of recombinant fibronectin also involves the more complex purification problem. Currently, compared with collagen, the expression level of recombinant fibronectin is low, and the purification process is inefficient and costly, which limits the scope of application of recombinant fibronectin. The use of genetic engineering technology to design recombinant fibronectin fragments with specific biological functions and achieve their efficient expression in microbial expression systems has become a major trend in fibronectin production. Summary of the Invention

[0005] In order to overcome the technical defects of the existing technology of artificially extracted fibronectin in small amounts and low biological activity, the present invention selects a specific functional fragment of human fibronectin, utilizes an Escherichia coli expression system, and performs codon optimization on the amino acid coding sequence of recombinant human fibronectin shown in SEQ ID NO.1 according to the codon preference of E. coli to obtain the coding gene nucleotide sequence SEQ ID NO.3. The nucleotide sequence is cloned into an expression vector and then transferred into host cells. After fermentation and purification, the recombinant human fibronectin is obtained.

[0006] To achieve the above objectives, this application provides the following technical solutions: The present invention provides a recombinant human fibronectin, the amino acid sequence of the recombinant human fibronectin is shown as SEQ ID NO.1.

[0007] Furthermore, the recombinant human fibronectin includes a protein tag.

[0008] Furthermore, the protein tag is selected from one or more of a 6×His tag, a Flag tag, a GST tag, an MBP tag and a Myc tag.

[0009] Furthermore, the amino acid sequence of the recombinant human fibronectin has at least 80% sequence identity with SEQ ID NO.1, specifically can be any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100%, or a range between any two of them.

[0010] 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.

[0011] Alternatively, the substitution may be a conservative 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 similar properties to form a peptide, and the replacement does not change the function of the fibronectin.

[0012] The present invention also provides a nucleic acid molecule encoding the recombinant human fibronectin.

[0013] Furthermore, the nucleic acid molecule can be optimized based on codon preference.

[0014] Furthermore, the sequence of the nucleic acid molecule is shown as SEQ ID NO.2 or SEQ ID NO.3.

[0015] The present invention also provides a recombinant plasmid, which carries the nucleic acid molecule.

[0016] The present invention also provides a host cell expressing the recombinant human fibronectin, wherein the host cell comprises the recombinant plasmid.

[0017] It should be noted that host cell refers to any cell type, which is susceptible to transformation, transfection, transduction, etc. of the nucleic acid construct or expression vector comprising the polynucleotide of the present invention." host cell" encompasses any offspring of a parental cell, which is not identical to the parental cell due to mutation during the replication process. The host cell can be any cell useful in the production of recombinant human fibronectin of the present invention. In order to produce recombinant fibronectin, the nucleic acid encoding recombinant fibronectin can be separated and inserted into one or more vectors to further clone and / or express in the host cell. Conventional techniques (for example, by using oligonucleotide probes that can specifically bind to the gene encoding recombinant fibronectin) can be easily separated and sequenced. The host cell refers to a cell into which exogenous nucleic acid has been introduced, including the offspring of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be identical to the parental cell in nucleic acid content, but may contain mutations. Methods for introducing vectors into host cells are well known, such as electroporation. Alternatively, transfection, microinjection, gene gun technology, liposome-mediated methods, and the like may be employed. 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. Furthermore, the host cell includes Escherichia coli.

[0018] Furthermore, the host cell is Escherichia coli BL21 (DE3).

[0019] The present invention also provides a method for preparing the recombinant human fibronectin, comprising the following steps: taking the host cell and inducing expression to obtain the recombinant human fibronectin of the present invention.

[0020] The present invention also provides use of recombinant human fibronectin in preparing cosmetics, wherein the cosmetics are used to promote cell adhesion, promote cell proliferation and / or promote wound healing.

[0021] The present invention also provides a cosmetic comprising recombinant human fibronectin.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The gene sequence of the recombinant human fibronectin of the present invention is codon-optimized according to the preferred codons of Escherichia coli BL21 (DE3), and the optimized codons contain more translation pause sites. After constructing the recombinant plasmid, it is expressed in an E. coli expression system, resulting in high expression levels and high purity. The preparation method of the present invention is convenient to prepare, has a simple purification process, is low in cost, and has a short production cycle. The recombinant human fibronectin prepared by the present invention has biological activity, can effectively promote cell proliferation activity, enhance cell adhesion activity, and is safe and reliable, and can be widely used in fields such as cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 This is a translation curve of the recombinant human fibronectin of the present invention (the wavy line is the red line, and the middle horizontal line is the blue line); wherein A is SEQ ID NO. 2 before codon optimization, and B is SEQ ID NO. 3 after codon optimization; Figure 2 This is a diagram showing the spatial structure of the recombinant human fibronectin of the present invention; Figure 3 This is a diagram showing the results of SDS-PAGE analysis of the expression of multiple copies of the recombinant human fibronectin transformant involved in the present invention; Figure 4 The results of the cell proliferation promotion test of the recombinant human fibronectin of the present invention are as follows; Figure 5 The results of the cell adhesion promotion test of the recombinant human fibronectin of the present invention are as follows; Figure 6 The figure shows the cytotoxicity test results of the recombinant human fibronectin of the present invention. DETAILED DESCRIPTION

[0025] The following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention as claimed, but rather merely represents selected embodiments of the invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort shall fall within the scope of protection of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.

[0027] Recombination: In a broad sense, any gene exchange process that causes genotype changes is called recombination.

[0028] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0029] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not completely identical to the parent cell due to mutations that occur during replication.

[0030] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0031] In this application, "preferred" is only used to describe an implementation method or example with better effects. It should be understood that it does not constitute a limitation on the scope of protection of this application.

[0032] Unless otherwise specified, in the following embodiments, all reagents or instruments used without manufacturer indication are conventional products that can be purchased commercially. If specific conditions are not indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0033] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art.

[0034] The culture medium formula used in the present invention is as follows: Example 1 Design of recombinant human fibronectin and its encoding gene In this example, based on the amino acid sequence of natural human fibronectin (GenBank: AAA52462.1) in the GenBank database, bioinformatics methods were used to analyze the spatial structure of human fibronectin. The amino acid sequence of a new recombinant human fibronectin was extracted and redesigned, and a His-Tag purification tag was added to the C-terminus to obtain the amino acid sequence shown in SEQ ID NO. 1 (328 amino acids, theoretical molecular weight 35.99 kDa): MKEINLAPDSSSVVVSGLMVATKYEVSVYALKDTLTSRPAQGVVTTLENVSPPRRARVTDATETTITISWRTKTETITGFQVDAVPANGQTPIQRTIKPDVRSYTITGLQPGTDYKIYLYTLNDNARSSPVVIDASTAIDAPSNLRFLATTPNSLLVSWQTHPGYD TGNGIQLPGTSGQQPSVGQQMIFEEHGFRRTTPPTTATPIRHRPRPYPPNVGEEIQIGHIPREDVDYHLYPHGPGLNPNASTGQEALMMSCTCLGNGKGEFKCDPHEATCYDDGKTYHVGEQWQKEYLGAICSCTCFGGQRGWRCDNCRRPGGEPSPHHHHH (SEQ ID NO.1).

[0035] The nucleotide sequence encoding SEQ ID NO.1 recombinant human fibronectin (rFN) is shown in SEQ ID NO.2: ATGAAAGAAATTAACCTGGCGCCGGATAGCAGCAGCGTGGTGGTGAGCGGCCTGATGGTGGCGACCAAATATGAAGTGAGCGTGTATGCGCTGAAAGATACCCTGACCAGCCGCCCGGCGCAGGGCGTGGTGACCACCCTGGAAAACGTGAGCCCGCCGCGCCGCGCGCGCGTGACCGATGCGACCGAAACCACCATTACCATTAGCTGGCGCACCAAAACCGAAACCATTACCGGCTTTCAGGTGGATGCGGTGCCGGCGAACGGCCAGACCCCGATTCAGCGCACCATTAAACCGGATGTGCGCAGCTATACCATTACCGGCCTGCAGCCGGGCACCGATTATAAAATTTATCTGTATACCCTGAACGATAACGCGCGCAGCAGCCCGGTGGTGATTGATGCGAGCACCGCGATTGATGCGCCGAGCAACCTGCGCTTTCTGGCGACCACCCCGAACAGCCTGCTGGTGAGCTGGCAGACCCATCCGGGCTATGATACCGGCAACGGCATTCAGCTGCCGGGCACCAGCGGCCAGCAGCCGAGCGTGGGCCAGCAGATGATTTTTGAAGAACATGGCTTTCGCCGCACCACCCCGCCGACCACCGCGACCCCGATTCGCCATCGCCCGCGCCCGTATCCGCCGAACGTGGGCGAAGAAATTCAGATTGGCCATATTCCGCGCGAAGATGTGGATTATCATCTGTATCCGCATGGCCCGGGCCTGAACCCGAACGCGAGCACCGGCCAGGAAGCGCTGATGATGAGCTGCACCTGCCTGGGCAACGGCAAAGGCGAATTTAAATGCGATCCGCATGAAGCGACCTGCTATGATGATGGCAAAACCTATCATGTGGGCGAACAGTGGCAGAAAGAATATCTGGGCGCGATTTGCAGCTGCACCTGCTTTGGCGGCCAGCGCGGCTGGCGCTGCGATAACTGCCGCCGCCCGGGCGGCGAACCGAGCCCGCATCATCATCATCAT(SEQ ID NO.2); Using the online codon optimization tool (ExpOptimizer) (https: / / www.novopro.cn / tools / codon-optimization.html), the fibronectin amino acid sequence shown in SEQ ID NO. 1 was not altered. Based on the codon preference of E. coli BL21 (DE3), the Nco I and Kpn I restriction sites were removed during the design process. The gene sequence was optimized by simplifying the secondary structure of the mRNA, optimizing the repetitive sequences, and adjusting the GC content. The optimized GC content increased from 60.57% to 46.65%, which is more conducive to efficient expression of the gene in the host strain E. coli BL21 (DE3). The codon-optimized fibronectin nucleotide sequence is shown in SEQ ID NO. 3: ATGAAAGAAATTAACCTTGCACCCGATTCCTCATCAGTTGTCGTCTCTGGACTAATGGTGGCAACAAAGTACGAGGTCTCAGTCTACGCTCTTAAGGATACTTTGACTAGTAGACCAGCACAGGGTGTTGTCACGACTCTTGAGAATGTTTCCCCCCCCAGAAGAGCAAGAGTAACAGACGCCACTGAAACCACTATCACTATTTCCTGGCGTACTAAGACTGAAACCATTACCGGATTCCAAGTTGATGCTGTACCAGCCAATGGTCAGACGCCTATACAGAGAACCATCAAGCCTGACGTGAGGTCTTACACTATTACCGGCTTACAACCAGGCACCGACTACAAGATTTATCTTTATACTTTGAATGACAACGCTCGAAGTAGTCCCGTAGTTATAGATGCTTCAACTGCTATTGATGCCCCATCTAACCTTAGATTCTTGGCAACCACCCCAAACTCTTTGCTTGTCTCATGGCAAACACACCCAGGTTACGATACTGGTAACGGAATTCAATTGCCTGGTACTTCCGGTCAACAGCCCTCAGTTGGTCAACAAATGATTTTCGAAGAGCACGGTTTCAGAAGAACCACGCCCCCTACTACAGCCACCCCAATTAGACATAGACCACGTCCATACCCTCCAAATGTAGGTGAAGAAATTCAGATTGGTCACATTCCTAGAGAGGATGTCGATTACCATCTATACCCTCACGGACCAGGCTTGAACCCAAATGCTTCCACCGGTCAGGAGGCATTGATGATGTCTTGCACATGTTTGGGAAATGGTAAGGGAGAGTTTAAGTGTGATCCTCATGAGGCAACATGCTACGATGATGGTAAAACATATCATGTGGGAGAGCAATGGCAAAAAGAGTACCTGGGTGCCATTTGTTCATGCACATGTTTCGGAGGACAAAGAGGTTGGAGATGTGATAATTGCAGAAGACCTGGAGGTGAGCCTTCCCCTCATCACCACCACCAC(SEQ ID NO.3)。

[0036] The translation pause curves of recombinant human fibronectin before and after codon optimization calculated by RiboTempo software are as follows: Figure 1 As shown in Figures A (before optimization, SEQ ID NO. 2) and B (after optimization, SEQ ID NO. 3), the red lines of the translation pause curves form translation pause sites in the predetermined region (the red line is lower than the blue line). The unoptimized nucleotide bases of SEQ ID NO. 2 are translated at a relatively constant rate on the ribosome, without any reduction in translation speed. However, the optimized SEQ ID NO. 3 experiences a significant reduction in translation speed throughout the ribosome translation process, with a significant number of translation pause sites (the red line is lower than the blue line). This allows the translated fibronectin ample time to fold, resulting in a more biologically active and highly expressed protein product.

[0037] The gene sequences shown in SEQ ID NO.2 and SEQ ID NO.3 and the amino acid sequence shown in SEQ ID NO.1 were commissioned to Shanghai Qingke Biotechnology Co., Ltd. for synthesis.

[0038] Example 2 Expression and purification of recombinant human fibronectin (1) Construction of recombinant human fibronectin expression vector An Nco I 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 Kpn I restriction site was added to the 3' end of the sequence. Gene fragment synthesis was commissioned to Shanghai Qingke Biotechnology Co., Ltd. The synthesized gene fragments were inserted into pET32a(+) (purchased from Changsha Abiwei Biotechnology Co., Ltd., HG-VYN0176) via the Nco I and Kpn I restriction sites to obtain the recombinant human fibronectin-expressing plasmids pET32a(+)-rFn-2 (SEQ ID NO. 2) and pET32a(+)-rFn-3 (SEQ ID NO. 3).

[0039] (2) Preparation of Escherichia coli BL21(DE3) competent cells The original E. coli BL21 (DE3) strain (purchased from Changsha Abiwei Biotechnology Co., Ltd., HG-VDN0199) stored at -80°C was streaked onto LB solid medium for activation. Five monoclonal strains were picked from the newly activated E. coli BL21 (DE3) plate and inoculated into a 250 mL conical flask containing 40 mL LB liquid medium. The culture was shaken at 250 rpm and 37°C overnight; the OD was monitored. 600 Value, wait for its OD 600When the value reaches 1.2-1.3, take out the culture bottle containing the bacterial solution and place it on ice for pre-cooling.

[0040] Transfer the ice-cold bacterial suspension to a 50 mL centrifuge tube and centrifuge at 4°C, 4000 rpm, for 5 minutes. Discard the supernatant and collect the cells. Resuspend the cells in 40 mL of pre-cold sterile water and centrifuge at 4°C, 4000 rpm, for 5 minutes. Discard the supernatant and collect the cells. Resuspend the cells again in 40 mL of pre-cold sterile water and centrifuge at 4°C, 4000 rpm, for 5 minutes. Discard the supernatant and collect the cells. Then, resuspend the cells in 5 mL of pre-cold 1 M D-sorbitol and centrifuge at 4°C, 4000 rpm, for 5 minutes. Discard the supernatant and collect the cells. Finally, resuspend the cells in 1 mL of pre-cold 1 M D-sorbitol, gently swirl to mix, and place on ice until ready to use.

[0041] (3) Transform into Escherichia coli BL21 (DE3) competent cells Drain 75% ethanol from the cuvette in a clean bench and rinse with 1 mL of 1M D-sorbitol. Aspirate the sorbitol solution after rinsing. Invert the cuvette to drain the sorbitol solution and place on ice for later use. Add 5-10 μg (10 μL) of the recombinant expression plasmids pET32a(+)-rFn-2 and pET32a(+)-rFn-3, along with 80 μL of the competent cells, to a 0.2 cm pre-chilled cuvette (add DNA first, then BL21(DE3) competent cells). Gently mix and let stand for 5 minutes. Wipe away any condensation on the outside of the cuvette, place it on an electroporator, and electroporate at 2000 V, 200 Ω, and 25 μF. Immediately after electroporation, add 1 mL of pre-cooled 1 M D-sorbitol to the electroporation cup, transfer the electroporation product to a sterile 10 mL centrifuge tube, and place it in a 30°C incubator for 1 hour. Then, place the 10 mL centrifuge tube in a clean bench, add 1 mL of LB liquid medium, and shake in a constant temperature shaker at 250 rpm and 28°C for 2 hours to recover the strain.

[0042] (4) Screening of positive transformants Spread 100-200 μL of the above bacterial solution onto an LB plate containing 100 μg / mL ampicillin sodium. Let stand at room temperature for 10 minutes. Incubate inverted in a 37°C incubator for approximately 2-5 days until a single colony appears. Pick a single colony from the YPD plate and transfer it to a 96-well plate containing 200 μL of LB liquid medium (containing 100 μg / mL ampicillin sodium). Continue incubating at 37°C. After 48 hours, homogenize the bacterial solution by airflow and transfer 10 μL per well to a new 96-well plate containing 190 μL of LB. Continue incubating for 24 hours and repeat the above steps. After 24 hours, homogenize the bacterial solution from a third 96-well plate by airflow and apply 1 μL of the solution to LB plates containing 200 μg / mL, 300 μg / mL, and 500 μg / mL ampicillin sodium for further incubation. If the transformant can grow on a plate containing 500 μg / ml of high-concentration ampicillin sodium, it indicates that the transformant contains multiple copies of the expression cassette. After this step of screening, an E. coli BL21 (DE3) strain that efficiently expresses recombinant human fibronectin can be obtained.

[0043] (5) Expression and purification of recombinant human fibronectin 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 (added with 100 μg / ml ampicillin sodium) and cultured at 37°C for 12 h. A single colony was picked and inoculated into LB liquid medium and cultured at 37°C, 220 rpm for 24 h until the OD600 was 0. nm = 4. Adjust the LB culture volume based on the measured OD value. Collect the cells by centrifugation at 3000 rpm for 10 min. Resuspend the cells in LB liquid medium to an initial OD value of 0.5. Continue culturing at 37°C and 220 rpm. Add 0.5% methanol to the culture medium every 24 h. End the fermentation after 72 h of induction culture and collect the fermentation broth.

[0044] The fermentation broth was centrifuged at 3000 g for 20 minutes at 4°C to collect supernatant 1 and bacterial pellet; the bacterial pellet was resuspended in 50 mM PBS (pH 8.0) and ultrasonically disrupted (ultrasound for 45 minutes, ultrasonication for 3 seconds, intervals of 2 seconds, power 500 watts) to release recombinant human fibronectin from the bacteria. The disrupted bacteria were centrifuged (3000 g for 20 minutes at 4°C) to separate supernatant 2 from the pellet; supernatant 1 and supernatant 2 were combined to obtain a protein supernatant.

[0045] Because the recombinant protein contains a His-Tag purification tag, it binds strongly to a nickel column under appropriate conditions. Therefore, affinity chromatography was used to separate impurities. The protein supernatant was filtered through a 0.45 μm filter before chromatography. The chromatography buffer used was 20 mM imidazole in 50 mM PBS (pH 8.0), and the elution buffer was 500 mM imidazole in 50 mM PBS (pH 8.0). Linear elution was performed. SDS-PAGE analysis was performed and peaks of appropriate molecular weight were selected for subsequent purification. After desalting the eluted peak, 0.1-1% EK enzyme was added to the system and the reaction was incubated at 4°C overnight. The enzymatically digested protein solution was subjected to affinity chromatography again as described above, 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 for the molecular weight and expression of the target protein. The results showed that a clear protein band was detected at 35 kDa, which was consistent with the theoretical molecular weight of recombinant human fibronectin (about 36 kDa), indicating that the initial expression was successful. The expression level of the multi-copy transformant pET32a(+)-rFn-3 was higher than that of pET32a(+)-rFn-2 (such as Figure 3 The protein band was single, without degradation bands, and the purity was greater than 95%.

[0046] Example 3 Cell proliferation promotion test of recombinant human fibronectin BALB / c 3T3 cells (purchased from China Center for Type Culture Collection, No. GDC0224) were seeded into 96-well cell culture plates (5000 cells / well) and cultured in a 37°C, 5% CO2 cell culture incubator for 24 hours. DMEM medium (purchased from Pronose, PM150210) was used for a further 12 hours. Recombinant human fibronectin was added at 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 (with the same volume of purified water) were also set up. The culture was continued for 48 to 72 hours. 10 μL of DMEM was added to each well. CCK-8 reagent was incubated in a cell culture incubator at 37°C and 5% CO2 for 2 h before removal. The absorbance of the 96-well plate at 450 nm and 630 nm was read using a microplate reader. The absorbance at 450 nm was measured with 630 nm as the reference wavelength, and the measurement results were recorded.

[0047] Cell proliferation rate (%) = (OD450 of experimental group - OD450 of negative control group) / OD450 of negative control group × 100%.

[0048] The results of cell proliferation test were as follows Figure 4As shown, the results show that the recombinant human fibronectin of the present invention has significant cell proliferation promoting activity, and the proliferation effect at a concentration of 10-100 nM is positively correlated with the concentration. At 100 nM, the cell proliferation promoting effect of the recombinant human fibronectin of the present invention is better than that of commercially available fibronectin.

[0049] Example 4 Cell Adhesion Promotion Assay of Recombinant Human Fibronectin Human immortalized epidermal cells (Hacat cells, purchased from China Center for Type Culture Collection, No. GDC0106) were treated, digested with trypsin, washed twice with PBS, collected in a centrifuge tube, centrifuged at 1000 rpm for 5 min, and resuspended in DMEM medium containing 10% FBS to prepare a cell suspension. The cell density was controlled at 6.5×10 4 Recombinant human fibronectin at different concentrations (10nM, 50nM, 100nM) was added to a 96-well plate. A positive control group (100nM commercial fibronectin, Merck Life Sciences, 10838039001) and a negative control group (same volume of purified water) were also set up. 5×10 4 Cells were seeded in a 96-well plate and three replicate wells were set up. The cells were incubated in a 37°C incubator for 24 hours. 100 μL of calcein working solution (final concentration of 10 μM) was added to each well for fluorescence staining. The fluorescence intensity of different groups was observed using a fluorescence microplate reader with a maximum emission wavelength of 520 nm. The fluorescence intensity value of the blank well (well without cells) was subtracted from the fluorescence intensity value of each test well, and the fluorescence intensity value of each replicate well was averaged. Cell adhesion promotion rate (%) = ((F 处理细胞 —F 空白 ) / (F 对照细胞 —F 空白 ))×100%.

[0050] F 处理细胞 : Fluorescence intensity value detected by the test sample group; F 对照细胞 : Fluorescence intensity value of the control group; F 空白 : Fluorescence intensity value of blank group detection.

[0051] The results of cell adhesion test were as follows Figure 5 As shown, the results show that the recombinant human fibronectin of the present invention has significant cell adhesion promoting activity, and the proliferation effect at a concentration of 10-100 nM is positively correlated with the 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.

[0052] Example 5 Cytotoxicity Evaluation Test of Recombinant Human Fibronectin HeLa cells (purchased from China Center for Type Culture Collection, No. GDC0009) grown to 70%-80% of the bottom area of ​​the culture flask were digested with 0.25% trypsin and cultured with complete culture medium (purchased from Beyotime Biotechnology, C7505C) to a cell density of 1×10 5 Cells were suspended at a concentration of 100 μL / mL. 100 μL of the cell suspension was inoculated into a 96-well culture plate and cultured in a 37°C, 5% CO2, saturated humidity incubator. After 24 hours of cell culture, the complete culture medium was aspirated. Recombinant human fibronectin solutions diluted in DMEM at concentrations of 10 nM, 50 nM, and 100 nM 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 consisted of cells cultured in DMEM, while the blank group received DMEM without cells. Culture was continued for 24 hours in a 37°C, 5% CO2, saturated humidity incubator. 10 μL of CCK-8 reagent was added to each group and incubated in the cell culture incubator for 4 hours. The absorbance (OD) of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA). The cell viability was calculated based on the mean absorbance of each group according to the following formula: cell viability (%) = ((OD450 of experimental group - OD450 of blank group) / (OD450 of control group - OD450 of blank group)) × 100%.

[0053] The results of cytotoxicity test were as follows Figure 6 As shown, the results show that the cell viability of 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.

[0054] Based on the above description, it will be understood by those skilled in the art that the present disclosure may be implemented in different specific forms without changing its technical spirit and essential features. Therefore, it should be understood that the above embodiments are not restrictive in all aspects, but illustrative. The scope of the present disclosure is limited by the appended claims, rather than by the description preceding them, and therefore all changes and modifications fall within the boundaries and scope of the claims, or the 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, and the protein tag is selected from one or more of a 6×His tag, a Flag tag, a GST tag, an MBP tag, and a Myc tag.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the recombinant human fibronectin according to 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 as 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 according to claim 1, characterized in that: The host cell comprises the recombinant plasmid according to claim 5.

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

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

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

10. A cosmetic, characterized in that: The cosmetic comprises the recombinant human fibronectin according to claim 1.

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