Recombinant fibronectin and its freeze-dried powder Recomtein WHT and its application in whitening cosmetics

Abstract

The present invention discloses recombinant fibronectin and its lyophilized powder Recomtein WHT and application in whitening cosmetics. Its amino acid sequence is shown in SEQ NO.1, and its nucleotide sequence is shown in SEQ NO.2. The recombinant fibronectin prepared by the present invention is formed by fusion of fibronectin with a peptide sequence having whitening activity, and has good biological tissue compatibility and strong whitening effect. The recombinant fibronectin of the present invention not only has the function of fibronectin, but also has whitening function, and has outstanding advantages such as safety and effectiveness, small side effects, humanization, low immunogenicity, and not prone to allergic reactions.

Description

Technical Field

[0001] The invention relates to the field of cosmetics, in particular to a recombinant fibronectin and a whitening application thereof. Background Art

[0002] Among the numerous whitening mechanism pathways, the synthesis and regulation of melanin have attracted much attention, among which MITF (Microphthalmia-associated transcription factor), TRP-1 (Tyrosinase-related protein 1), TYR (Tyrosinase) and tyrosinase play key roles. As an important transcription factor, MITF plays a central role in melanocytes, affecting the development, function and melanin production of melanocytes by regulating the expression of multiple genes including TYR and TRP-1. In particular, TYR, the rate-limiting enzyme in the melanin synthesis pathway, is responsible for converting tyrosine into dopaquinone, which is a key initial step in the melanin formation process. At the same time, TRP-1 is also involved in this process, affecting the quality, quantity and distribution of melanin. Therefore, in the whitening mechanism, the regulation of these key factors has become an important means to achieve skin color changes. Whether by directly inhibiting the activity of tyrosinase, reducing the expression level of MITF, or interfering with the synthesis and transport of melanin, the production of melanin can be reduced to a certain extent, thereby achieving the purpose of whitening the skin. Many whitening products and treatments are developed based on this mechanism, using specific whitening ingredients to regulate this pathway. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a novel recombinant fibronectin and its lyophilized powder Recomtein WHT and application. The present invention activates the whitening pathway in cells through the action of a specific amino acid sequence, thereby inhibiting melanin production. The fibronectin designed by genetic engineering means combines fibronectin with a small molecule peptide segment that enhances the phosphorylation of the extracellular signal kinase ERK and degrades MITF, giving the recombinant fibronectin a whitening effect. The addition of this small molecule peptide not only has a whitening effect, but also improves the stability and bioavailability of recombinant fibronectin. The amino acid sequence in the selected small molecule peptide segment is optimized to achieve maximum binding affinity and target selectivity.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] A recombinant fibronectin, the amino acid sequence of which is shown in SEQ NO.1.

[0006] The nucleotide sequence of the nucleic acid encoding the recombinant fibronectin is shown in SEQ NO.2.

[0007] The preparation method of the above recombinant fibronectin comprises the following steps:

[0008] (1) Construction of pET20b-rhFNL recombinant plasmid: Gene sequences related to skin whitening and melanin production inhibition were collected from the Genebank database. Fragments with potential activity were identified through screening and comparison. They were then docked with the human fibronectin gene sequence for target and fused to construct a recombinant fibronectin gene sequence. NdeI and Xhol restriction sites were introduced at the 5' and 3' ends, respectively, and a histidine tag was added before the 3' end stop codon. The fragments were then double-digested with NdeI / Xhol to be connected to the pET20b vector to obtain the pET20b-rhFNL recombinant plasmid. ; pET20b-rhFNL recombinant plasmid was double-digested with XbaI / XhoI, and the product was subjected to agarose gel electrophoresis, and the target band was cut out and recovered with a DNA gel recovery kit for later use; (2) Expression of rhFNL: The fusion gene expression vector was transformed into competent cells such as BL21, and screened and cultured in LB (Amp+) medium; positive clones were picked and inoculated in LB (Amp+) culture, and cultured at 37°C for 3-5h until OD600 was 0.5, IPTG was added to a final concentration of 1mM, and induced at 37°C, the cells were collected by centrifugation, pH6.8 PBS buffer was added, and the cells were broken by a homogenizer at 400bar;

[0009] (3) Purification of rhFNL: After centrifugation, the collected supernatant was purified by nickel column at a flow rate of 0.6 mL / min. After equilibration with pH 6.8 PBS buffer, imidazole gradient elution was used and the elution peak was collected at 300 mM. The eluted sample was desalted by G25 and freeze-dried to obtain highly active recombinant fibronectin rhFNL.

[0010] A recombinant fibronectin Recomtein WHT lyophilized powder is prepared by the following method: (1) adding 0.05 parts by weight of the rhFNL prepared above into 30 parts by weight of purified water, stirring and dissolving evenly until completely transparent, and pre-preparing as phase A;

[0011] (2) adding 0.2 parts by weight of mannitol and 0.05 parts by weight of trehalose to 35 parts by weight of purified water, stirring and dissolving them uniformly until they are completely transparent, and preparing phase B;

[0012] (3) Mix phases A and B and stir until they are homogeneous and transparent;

[0013] (4) Add 10 parts by weight of purified water, filter the material through a sterilized 0.22 μ filter, and vacuum freeze-dry to obtain recombinant fibronectin Recomtein WHT freeze-dried powder.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the recombinant fibronectin prepared by the present invention is formed by the fusion of fibronectin and whitening polypeptide, has a unique fibrous structure and good biological tissue compatibility, and has a strong whitening effect. The recombinant fibronectin of the present invention not only has the function of fibronectin, but also has a whitening function, and has the outstanding advantages of being safe and effective, having few side effects, being humanized, having low immunogenicity, and not prone to allergic reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the map of the Escherichia coli expression plasmid pET20b-rhFNL.

[0016] Figure 2 SDS-PAGE analysis of the target protein rhFNL.

[0017] Figure 3 The whitening activity of recombinant fibronectin Recomtein WHT lyophilized powder on B16 cells. Figure 4 The effect of recombinant fibronectin Recomtein WHT freeze-dried powder on the formation of melanin on the surface of zebrafish embryos.

[0018] Figure 5 The effect of recombinant fibronectin Recomtein WHT lyophilized powder on the inhibition rate of melanin production in zebrafish embryos, compared with the blank control group: *p<0.05, **p<0.01, ***p<0.001. Figure 6 The effect of recombinant fibronectin RecomteinWHT lyophilized powder on the inhibition rate of tyrosinase activity in zebrafish embryos, compared with the blank control group: *p<0.05, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0019] Embodiment 1:

[0020] The present invention first artificially synthesizes the gene sequence of fibronectin, introduces NdeI and Xhol restriction sites at the 5' end and 3' end respectively, connects it into the pET3c vector through NdeI / Xhol double restriction digestion, thereby obtaining the pET20b-rhFNL recombinant plasmid. At the same time, the Xhol restriction site is introduced at the 5' end through primer design, and the (GGGGS)3 sequence and NdeI restriction site are introduced at the 3' end. After the NdeI / Xhol double restriction digestion, it is connected into the pET20b-rhFNL vector, thereby constructing the pET20b-rhFNL recombinant plasmid, introducing it into the competent Escherichia coli BL21 (DE3) for cultivation, inducing the expression of rhFNL fusion protein with IPTG, obtaining the purified rhFNL through affinity chromatography, and identifying the molecular weight of rhFNL through SDS-PAGE. The specific steps are as follows:

[0021] (1) Construction of pET20b-rhFNL recombinant plasmid: The present invention collects gene sequences related to skin whitening and melanin production inhibition in the Genebank database, identifies fragments with potential activity through screening and comparison, and performs target docking with the human fibronectin gene sequence to fuse and construct a recombinant fibronectin gene sequence with whitening efficacy, and introduces NdeI and Xhol restriction sites at the 5' and 3' ends, respectively. To facilitate subsequent purification, a histidine tag is added before the 3' end stop codon, and it is connected to the pET20b vector through NdeI / Xhol double restriction digestion to obtain the pET20b-rhFNL recombinant plasmid, such as Figure 1 The pET20b-rhFNL recombinant plasmid was double-digested with XbaI / XhoI, and the product was subjected to agarose gel electrophoresis, the target band was cut out, and recovered with a DNA gel recovery kit for later use.

[0022] (2) Expression of rhFNL: The fusion gene expression vector was transformed into competent cells such as BL21 (DE3), and screened and cultured in LB (Amp+) medium. Positive clones were picked and inoculated into LB (Amp+) culture medium, cultured at 37°C for 3-5h until OD600 reached 0.5, IPTG was added to a final concentration of 1mM, and induced at 37°C for about 4h. The cells were collected by centrifugation, pH 6.8 PBS buffer was added, and the cells were broken by homogenizer at 400bar.

[0023] (3) Purification of rhFNL: Centrifuge at 18000rpm for 20min, and purify the collected supernatant with a nickel column at a flow rate of 0.6mL / min. After equilibration with pH 6.8 PBS buffer, use imidazole gradient elution, collect the effluent peak at 300mM, and desalt the eluted sample with G25 to obtain high-purity rhFNL. SDS-PAGE analysis was used to identify the purity and molecular weight of rhFNL. Figure 2As shown, the target protein is marked with its unique red arrow. After elution with 200mmol / L imidazole concentration, only a trace amount of impurity protein is left, with a molecular weight of 15kDa. The nucleotide sequence encoding the recombinant fibronectin rhFNL is shown in SEQ NO.2, and the amino acid of the recombinant fibronectin rhFNL is shown in SEQ NO.1.

[0024] Example 2: Preparation of recombinant fibronectin Recomtein WHT lyophilized powder:

[0025] (1) 0.05 parts by weight of rhFNL prepared in Example 1 was added to 30 parts by weight of purified water, and the mixture was stirred and dissolved evenly until completely transparent to prepare phase A.

[0026] (2) Add 0.2 parts by weight of mannitol and 0.05 parts by weight of trehalose into 35 parts by weight of purified water, stir and dissolve evenly until completely transparent, and prepare phase B.

[0027] (3) Mix phases A and B and stir for 9 minutes until they become homogeneous and transparent.

[0028] (4) Add 10 parts by weight of purified water, filter the material through a sterilized 0.22 μ filter, and freeze-dry in vacuum to obtain recombinant fibronectin Recomtein WHT lyophilized powder, referred to as Recomtein WHT.

[0029] Embodiment 3:

[0030] The whitening effect of recombinant fibronectin Recomtein WHT lyophilized powder on B16 cells was determined: Recomtein WHT solution sample was used as the test substance, kojic acid was used as the positive control, and PBS solution was used as the blank control. B16 cells in the logarithmic growth phase were routinely cultured with 1640 medium containing 10% FBS (purchased from Gibco, USA) until 80% to 90% confluence, digested with 0.25% trypsin, and 2.0×10 5 The number of cells / mL was inoculated in a 96-well plate. After culturing for 24 hours, the serum-free 1640 medium was changed for culturing. After 24 hours, the medium was aspirated and Recomtein WHT with concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 ng / mL was added respectively (all samples were diluted to 0.5 mg / mL with PBS). There were 3 replicate wells for each concentration, and 6 replicate wells for the blank group. After administration, the cells were placed in the incubator and cultured for 24 hours. When the cell fusion rate reached more than 90%, the cells were subjected to RNA extraction kit. The extracted cell RNA was reverse transcribed according to the reverse transcription kit. Fluorescence quantitative PCR was used to detect the expression of genes related to melanin synthesis and its whitening effect. The results are as follows Figure 3As shown, within the concentration range investigated, the Recomtein WHT group had a whitening effect, and both Recomtein and kojic acid could inhibit the gene expression of key factors involved in melanin synthesis.

[0031] Embodiment 3:

[0032] The whitening effect of recombinant fibronectin Recomtein WHT lyophilized powder on zebrafish embryos was determined. The specific steps are as follows:

[0033] (1) Collection of experimental fish and fertilized eggs: Sexually mature zebrafish of different sexes were kept in separate tanks in a zebrafish breeding unit. Water temperature: 26±2℃; pH: 7.2; conductivity: 520μs / cm; light / dark cycle: 14h:10h. One day before the start of the exposure experiment, sexually mature zebrafish were paired in a 1:2 ratio, mated naturally and spawned, and embryos were collected.

[0034] (2) Exposure experiment: A blank control group and sample groups with different concentrations were set up in the experiment. Normally developed wild-type AB zebrafish embryos at 6-8 hpf were selected under a stereomicroscope and placed in a 6-well cell culture plate. 30 embryos were placed in each well. Each group had 3 replicate wells in parallel, and 5 mL of the corresponding solution was added to each well. Standard dilution water was added to the blank control group, 40 μM N-phenylthiourea solution was added to the positive control group, and sample solutions of corresponding concentrations (0.23125 mg / mL, 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT) were added to the sample group. The zebrafish embryos were then placed in an incubator and incubated until 72 hpf. The melanin deposition on the surface of the zebrafish embryos was observed under a stereomicroscope at 48 hpf and 72 hpf, and photographed and recorded. The effect of recombinant fibronectin Recomtein WHT on the formation of melanin on the surface of zebrafish embryos is as follows. Figure 4 As shown, after zebrafish were treated with 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT to 72 hpf, the number of black spots produced was reduced compared with the blank control group, indicating that 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT have the effect of inhibiting the production of body surface melanin.

[0035] (3) Melanin production detection: The experiment set up a blank control group and sample groups with different concentrations. Under a stereomicroscope, wild-type AB zebrafish embryos with normal development at 6-8 hpf were selected and placed in a 6-well cell culture plate, with 30 embryos placed in each well. Each group had 3 replicates in parallel, and 5 mL of the corresponding solution was added to each well. Standard dilution water was added to the blank control group, 40 μM N-phenylthiourea solution was added to the positive control group, and sample solutions of corresponding concentrations (0.23125 mg / mL, 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT) were added to the sample group. The zebrafish embryos were then placed in an incubator and incubated until 72 hpf, and the melanin production in the zebrafish was detected at 72 hpf. After incubation to 72 hpf, the zebrafish embryos were transferred to an EP tube, lysis buffer (5 mg / mL sodium deoxycholate) was added, homogenized, centrifuged, the precipitate was taken, 1 M sodium hydroxide solution was added, and the assay was performed under an ELISA instrument. The calculation formula for the inhibition rate of melanin production is as follows:

[0036]

[0037] Effect of recombinant fibronectin Recomtein WHT freeze-dried powder on the inhibition rate of melanin production in zebrafish embryos Figure 5 As shown, from Figure 5 It can be seen that 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT can significantly inhibit the production of melanin.

[0038] (4) Tyrosinase activity detection: The experiment set up a blank control group and sample groups with different concentrations. Under a stereomicroscope, wild-type AB zebrafish embryos with normal development at 6-8 hpf were selected and placed in a 6-well cell culture plate. 30 embryos were placed in each well. Each group had 3 replicate wells in parallel, and 5 mL of the corresponding solution was added to each well. Standard dilution water was added to the blank control group, 40 μM N-phenylthiourea solution was added to the positive control group, and sample solutions of corresponding concentrations (0.23125 mg / mL, 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT) were added to the sample group. The zebrafish embryos were then placed in an incubator and incubated until 72 hpf, and the tyrosinase activity in the zebrafish was detected at 72 hpf. After incubation to 72 hpf, the zebrafish embryos were transferred to EP tubes, lysis buffer (5 mg / mL sodium deoxycholate) was added, homogenized, centrifuged, the supernatant was taken, 5 mM L-DOPA solution was added, incubated at 37 °C for 1 h, and then detected under a microplate reader. The calculation formula for the inhibition rate of tyrosinase activity is as follows:

[0039]

[0040] Effect of recombinant fibronectin Recomtein WHT freeze-dried powder on the inhibition rate of tyrosinase activity in zebrafish embryos Figure 6 As shown, from Figure 6 It can be seen that 0.4625 mg / mL and 0.925 mg / mL of Recomtein WHT can significantly inhibit tyrosinase activity.

Claims

1. A recombinant fibronectin, the amino acid sequence of which is shown in SEQ NO.

1.

2. A nucleic acid encoding the recombinant fibronectin according to claim 1, wherein the nucleotide sequence is shown in SEQ NO.

2.

3. A method for preparing the recombinant fibronectin as claimed in claim 1, characterized in that The steps include: (1) Construction of pET20b-rhFNL recombinant plasmid: Gene sequences related to skin whitening and melanin production inhibition were collected from the Genebank database, and fragments with potential activity were identified through screening and comparison. The fragments were docked with the human fibronectin gene sequence for target docking, and the gene sequence encoding the recombinant fibronectin described in claim 1 was fused and constructed; NdeI and Xhol restriction sites were introduced at the 5' and 3' ends respectively, and a histidine tag was added before the 3' end stop codon, and it was connected to the pET20b vector through NdeI / Xhol double restriction digestion to obtain the pET20b-rhFNL recombinant plasmid; the pET20b-rhFNL recombinant plasmid was double-digested with XbaI / XhoI, the product was subjected to agarose gel electrophoresis, the target band was cut out, and a DNA gel recovery kit was used to recover it for standby use; (2) Expression of rhFNL: The fusion gene expression vector was transformed into competent cells BL21 and screened and cultured in LB medium; positive clones were picked and inoculated into LB medium and cultured at 37°C for 3-5 h until OD600 reached 0.5, IPTG was added to a final concentration of 1 mM, and the cells were induced at 37°C. The cells were collected by centrifugation, pH 6.8 PBS buffer was added, and the cells were broken using a homogenizer at 400 bar; (3) Purification of rhFNL: After centrifugation, the collected supernatant was purified by nickel column with a sample loading flow rate of 0.6 mL / min. After equilibration with pH 6.8 PBS buffer, imidazole gradient elution was used, and the elution peak was collected at 300 mM. The eluted sample was desalted by G25 and freeze-dried to obtain recombinant fibronectin.

4. A recombinant fibronectin Recomtein WHT lyophilized powder, characterized in that Made by the following method: (1) Add 0.05 parts by weight of the recombinant fibronectin prepared according to claim 3 to 30 parts by weight of purified water, stir and dissolve evenly until completely transparent, and prepare phase A; (2) adding 0.2 parts by weight of mannitol and 0.05 parts by weight of trehalose to 35 parts by weight of purified water, stirring and dissolving them uniformly until they are completely transparent, and preparing phase B; (3) Mix phases A and B and stir until they are homogeneous and transparent; (4) Add 10 parts by weight of purified water, filter the material through a sterilized 0.22 μ filter, and vacuum freeze-dry to obtain recombinant fibronectin Recomtein WHT freeze-dried powder.

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

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