Transdermal recombinant keratin as well as preparation method and application thereof

Abstract

The invention provides transdermal recombinant keratin as well as a preparation method and application thereof, and relates to the technical field of genetic engineering. The transdermal recombinant keratin comprises a recombinant keratin and a cell penetrating peptide; the transdermal recombinant keratin has cell adhesion activity and transdermal property. The transdermal recombinant keratin comprises any one amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 or consists of the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4. According to the transdermal recombinant keratin, the transdermal performance and the cell adhesion activity of keratin are remarkably improved, and the problem that traditional recombinant keratin is poor in skin permeability is solved.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular, to a transdermal recombinant keratin and its preparation method and application. Background Technique

[0002] Cell-penetrating peptides (CPPs) are a class of peptides with unique biological activities, usually consisting of 5 - 30 amino acids. Their core function is to penetrate cell membranes, thereby enabling intervention in the intracellular environment. The penetration ability of CPPs mainly stems from their property of being rich in basic amino acid residues, which allows them to interact with negatively charged glycosaminoglycan chains on cell membranes and then be taken up by cells through endocytosis or micropinocytosis mechanisms. Relying on this property, CPPs can be used as carriers to mediate the delivery of various molecules such as non-cell-penetrating peptides, proteins, nanoparticles, quantum dots, and nucleic acids, and are widely used in the fields of biomedicine and biotechnology. Currently, there are about 1,000 reported types of CPPs, which are mainly divided into three categories according to their physical and chemical properties: cationic peptides, amphiphilic peptides, and hydrophobic peptides. Common CPPs include DPV3 / 6, VP22, TP10, TD, Pep-1, TAT, R7-9, cTAT, MAP, etc.

[0003] In the field of biomaterials, recombinant keratin, as an important material, has been widely used in multiple fields such as drug delivery, wound healing, tissue engineering, and cosmetic skin care products due to its many excellent properties such as biocompatibility, biodegradability, hemostasis, non-immunogenicity, antibacterial activity, and anti-inflammatory and antioxidant properties. However, currently commercially available recombinant keratin has certain limitations. Its large molecular weight results in poor skin permeability. This property makes it difficult for recombinant keratin to effectively penetrate the skin in practical applications, especially for topical and wash-and-care products, to exert its due effects, restricting its application scope in related products.

[0004] Although CPPs have certain potential in delivering recombinant proteins, there are significant differences in their compatibility with different proteins. This means that in practical applications, the combination of CPPs and recombinant keratin may be incompatible, affecting the delivery efficiency and effect. In addition, the skin permeability problem of recombinant keratin itself also makes it difficult to effectively enter the deep skin tissues without external forces (such as microneedles or injections). This limitation not only increases the complexity of drug administration and the discomfort of patients, but also restricts the application effect of recombinant keratin in topical and wash-and-care products, and cannot fully exert its biocompatibility and multifunctional advantages.

[0005] In summary, the existing recombinant keratin faces the problem of poor skin permeability in skin applications. Although CPPs can be used as delivery tools, there are significant differences in their compatibility with recombinant keratin, making it difficult to effectively solve the penetration problem of recombinant keratin. These problems together limit the wide application of recombinant keratin in topical and wash-care products. There is an urgent need to develop a technical solution that can effectively improve the skin permeability of recombinant keratin to fully exert its potential in the fields of biomedicine and beauty skin care.

[0006] In view of this, the present invention is specifically proposed. Summary of the Invention

[0007] The purpose of the present invention is to provide a transdermal recombinant keratin and its preparation method and application. The transdermal recombinant keratin significantly improves the transdermal performance and cell adhesion activity of keratin, and solves the problem of poor skin permeability of traditional recombinant keratin.

[0008] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: In the first aspect, the present invention provides a transdermal recombinant keratin, which comprises recombinant keratin and a cell-penetrating peptide; The transdermal recombinant keratin has cell adhesion activity and transdermal permeability; The transdermal recombinant keratin contains any one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 or is composed of them.

[0009] In the second aspect, the present invention provides a polynucleotide, which encodes the transdermal recombinant keratin as described in the foregoing embodiment.

[0010] In an optional embodiment, the nucleotide sequence of the polynucleotide is as shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

[0011] In the third aspect, the present invention provides an expression vector, which contains the polynucleotide as described in the foregoing embodiment.

[0012] In the fourth aspect, the present invention provides a host cell, which contains the expression vector as described in the foregoing embodiment; In an optional embodiment, the host cell is Escherichia coli.

[0013] In the fifth aspect, the present invention provides a preparation method of the transdermal recombinant keratin as described in any one of the foregoing embodiments, comprising: Culturing host cells in a production medium; and isolating the transdermal recombinant keratin from the host cells.

[0014] In a sixth aspect, the present invention provides a composition comprising the transdermal recombinant keratin as described in the foregoing embodiments.

[0015] In a seventh aspect, the present invention provides an article comprising the transdermal recombinant keratin as described in the foregoing embodiments; or comprising the composition as described in the foregoing embodiments; The article is at least one of a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic, and a health product; The pharmaceutical composition is a topical preparation; and / or, the pharmaceutical composition is a topical preparation; and the topical preparation is a topical application preparation; and / or, the pharmaceutical composition is a topical preparation; and the topical preparation is a topical application preparation; the topical application preparation is any one selected from topical microneedle preparations, topical hydrogel agents, and topical infiltration preparations.

[0016] In an eighth aspect, the present invention provides an application of the transdermal recombinant keratin as described in the foregoing embodiments, the polynucleotide as described in the foregoing embodiments, the expression vector as described in the foregoing embodiments, the host cell as described in the foregoing embodiments, or the composition as described in the foregoing embodiments in the preparation of a product; The product is at least one selected from medical devices, tissue engineering products, cosmetics, and skin care products.

[0017] The transdermal recombinant keratin provided in the present application significantly improves the transdermal effect of keratin by binding recombinant keratin to a cell-penetrating peptide. This transdermal recombinant keratin can not only effectively penetrate the skin barrier, but also maintain its biological activity after penetration, thereby exerting its cell adhesion activity inside the skin and promoting cell proliferation and tissue repair. This innovative production method and application enable the transdermal recombinant keratin to have broad application prospects in industries such as medicine, cosmetics, and skin care products, especially showing significant beneficial effects in promoting skin wound healing and improving the efficacy of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required to be used in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is a representative diagram of plasmid construction taking the vector pPicZalpha-K1 in Example 1 of the present application as an example; Figure 2 This is the protein electrophoresis pattern of 4 kinds of transdermal recombinant keratins expressed in Pichia pastoris in Example 2 of this application (the electrophoretic detection molecular weights of the proteins of recombinant proteins K1, K2, K3, K4, etc. genes are all about 19 kDa); Figure 3 This is the protein electrophoresis pattern obtained after the protein expression and purification of K1-K5 proteins in Example 2 of this application (the electrophoretic detection molecular weights of the proteins of K1-K5 are all about 19 kDa); Figure 4 This is the transdermal test result of recombinant transdermal keratin and recombinant keratin for 1 h in Example 3 of this application; Figure 5 This is the transdermal test result of recombinant transdermal keratin and recombinant keratin for 6 h in Example 3 of this application; Figure 6 This is the transdermal test result of recombinant transdermal keratin and recombinant keratin for 12 h in Example 3 of this application; Figure 7 This is the cell proliferation activity detection result diagram in Example 4 of this application. Detailed implementation manners

[0020] The following will describe the implementation schemes of the present invention in detail in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0021] A transdermal recombinant keratin is provided in the examples of this application, which includes recombinant keratin and a cell-penetrating peptide. The transdermal recombinant keratin has cell adhesion activity and transdermal permeability.

[0022] The above transdermal recombinant keratin includes a fusion of recombinant keratin and a cell-penetrating peptide, and this can effectively penetrate the skin and promote adhesion between cells.

[0023] Recombinant keratin is a keratin fragment or derivative produced by genetic engineering technology. Keratin is a biomaterial with various advantages such as biocompatibility, biodegradability, hemostasis, non-immunogenicity, antibacterial activity, and anti-inflammatory and antioxidant properties. Recombinant keratin usually comes from specific functional regions of keratin and is optimized and expressed by genetic engineering means to meet specific application requirements.

[0024] Cell-penetrating peptides are a class of small peptides that can penetrate cell membranes. They can consist of 5 - 30 amino acids and are rich in basic amino acid residues (such as lysine and arginine). These peptides can interact with the glycosaminoglycan chains on the cell membrane and enter the cell interior through mechanisms such as endocytosis or micropinocytosis. Cell-penetrating peptides can act as carriers to deliver molecules such as non-cell-penetrating peptides, proteins, nanoparticles, and nucleic acids into cells.

[0025] Cell adhesion activity refers to the ability of a substance to promote adhesion between cells or between cells and the matrix. This activity is crucial for processes such as cell growth, differentiation, migration, and tissue repair. In transdermal recombinant keratin, cell adhesion activity enables it to form good adhesion on the surface of skin cells, thereby enhancing its effect in the skin.

[0026] Transdermal permeability refers to the ability of a substance to penetrate the skin surface and enter the deep skin tissues. Transdermal permeability is an important indicator for measuring the effectiveness of topical skin preparations (such as drugs, cosmetics, etc.). For transdermal recombinant keratin, its transdermal permeability means that it can pass through the skin barrier and reach the deep skin tissues to exert its biological activities, such as promoting cell proliferation and repairing damaged skin.

[0027] The described transdermal recombinant keratin can effectively penetrate the skin barrier and enter the deep skin tissues by fusing with cell-penetrating peptides. This solves the problem that traditional recombinant keratin is difficult to penetrate the skin due to its large molecular weight, making its application in skin care and treatment more extensive; Transdermal recombinant keratin has cell adhesion activity, which can promote adhesion between cells or between cells and the matrix. This property helps cell growth, differentiation, and migration, thus accelerating wound healing and tissue repair; Recombinant keratin itself has various biological activities, such as antibacterial, anti-inflammatory, and antioxidant. After combining with cell-penetrating peptides, these activities can be more effectively exerted in the skin, enhancing the comprehensive performance of the product; This transdermal recombinant keratin can be used not only in the medical field (such as topical drugs, wound dressings, etc.), but also widely in cosmetics and skin care products, such as anti-wrinkle essence, repair masks, etc., to improve the efficacy and market competitiveness of the products; Due to its good transdermal performance, transdermal recombinant keratin can be administered by simple topical application without the need for complex means such as microneedles or injections, improving the convenience of use and patient compliance.

[0028] In summary, the described transdermal recombinant keratin significantly improves the transdermal performance and cell adhesion activity of keratin by fusing with cell-penetrating peptides, making it more advantageous in skin care and treatment. At the same time, it also expands its application scope in the medical and cosmetic fields.

[0029] In this embodiment, a truncated amino acid sequence of human keratin type 81 helix region and the cell-penetrating peptide in Table 1 are connected through a flexible Linker to obtain a recombinantly expressed transdermal keratin.

[0030] In some embodiments, the Linker may not be added. The cell-penetrating peptide sequences are shown in Table 1.

[0031] Table 1. Cell-penetrating peptides

[0032] The sequence of the above-mentioned human keratin type 81 is the NCBI reference sequence: Q14533 (SEQ ID NO.9), see "https: / / www.ncbi.nlm.nih.gov / protein / Q14533 / ". The amino acid sequence is as follows: MTCGSGFGGRAFSCISACGPRPGRCCITAAPYRGISCYRGLTGGFGSHSVCGGFRAGSCGRSFGYRSGGVCGPSPPCITTVSVNESLLTPLNLEIDPNAQCVKQEEKEQIKSLNSRFAAFIDKVRFLEQQNKLLETKLQFYQNRECCQSNLEPLFEGYIETLRREAECVEADSGRLASELNHVQEVLEGYKKKYEEEVSLRATAENEFVALKKDVDCAYLRKSDLEANVEALIQEIDFLRRLYEEEILILQSHISDTSVVVKLDNSRDLNMDCIIAEIKAQYDDIVTRSRAEAESWYRS KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK LEAAVAQSEQQGEAALSDARCKLAELEGALQKAKQDMACLIREYQEVMNSKLGLDIEIATYRRLLEGEEQRLCEGIGAVNVCVSSSRGGVVCGDLCVSGSRPVTGSVCSAPCNGNVAVSTGLCAPCGQLNTTCGGGSCGVGSCGISSLGVGSCGSSCRKC (SEQ ID NO.9).

[0033] The bold and underlined part in the above sequence is the target amino acid sequence selected in this embodiment.

[0034] In this embodiment, through a large amount of research, it is found that the selected above sequence has strong water solubility, high recombinant expression yield, simple purification process, and better transdermal effect than keratin without adding cell-penetrating peptide, and has a variety of excellent biomaterial properties.

[0035] It should be noted that the transdermal recombinant keratin in the embodiments of the present application is not the full-length sequence of SEQ ID NO.9.

[0036] This embodiment is partly based on the following discovery: The transdermal recombinant keratin containing at least 36 consecutive amino acid residues in SEQ ID NO.10 has better biomaterial properties than the commercial transdermal keratin, as demonstrated in the examples.

[0037] Those skilled in the art can appropriately select the consecutive amino acid residues constituting the recombinant transdermal keratin. In this embodiment, for the following K5: The sequence of the amino acid region of KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK (SEQ ID NO.10) was tested: The transdermal recombinant keratin in this embodiment can be the sequence of recombinant human transdermal keratin K5 repeated three times, including 138 amino acids, and the basic repeating unit is: KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK (SEQ ID NO.10), which is a peptide segment of human transdermal keratin type 81.

[0038] The amino acid sequence of K5-3 is: KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSKKCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSKKCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK (SEQ ID NO.11).

[0039] The DNA sequence of K5-3 is as follows: AAGTGTGAAGAAATGAAGGCTACTGTTATTAGACATGGTGAAACTTTGAGAAGAACTAAGGAAGAAATTAACGAATTGAACAGAATGATCCAAAGATTGACTGCTGAAGTTGAAAACGCTAAATGTCAAAATTCTAAGAAGTGTGAAGAGATGAAGGCTACCGTTATTAGACACGGTGAAACTCTTAGAAGAACTAAAGAAGAAATCAA CGAATTGAATAGAATGATCCAGAGATTGACTGCCGAAGTTGAAAATGCTAAATGTCAGAACTCTAAGAAGTGCGAAGAAATGAAAGCTACTGTTATCAGACATGGTGAGACTTTGAGAAGAACCAAGGAAGAAATCAATGAATTGAACAGAATGATTCAGAGATTGACCGCTGAAGTTGAGAACGCTAAATGCCAAAATTCTAAA (SEQ ID NO.12).

[0040] In this example, a truncated amino acid sequence of the helical region of human keratin type 81 and the cell-penetrating peptides listed in Table 1 were selected and connected through a flexible linker to obtain a fusion-expressed recombinant transdermal keratin.

[0041] The flexible linker may be GGGGS(n), where n may be 1 to 6. The cell penetrating peptide may be linked to the N-terminus or C-terminus of the recombinant keratin.

[0042] In some embodiments, the transdermal recombinant keratin comprises or consists of any one of the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4. The specific sequences are shown in the following table: Table 2. Amino acid sequence of transdermal recombinant keratin

[0043] In the above table, NO. represents SEQ ID NO. In this embodiment, the transdermal recombinant keratin sequence may include one or more substitutions, additions, deletions or insertions in the amino acid sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4.

[0044] In some embodiments, an amino acid sequence of 2, 4, 6, or 8 amino acid residues, or an amino acid sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity to the amino acid sequence shown in any one of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4. Based on the foregoing amino acid sequences, suitable parameters for aligning the sequences can be determined, including any algorithm required to achieve maximum alignment over the entire length of the sequences being compared.

[0045] Amino acid addition refers to adding amino acids to the C-terminus or N-terminus of any one of the three transdermal keratin amino acid sequences, provided that the resulting sequence has keratin characteristics and a high transdermal rate.

[0046] Amino acid substitution refers to replacing an amino acid residue at a certain position in any one of the three transdermal keratin amino acid sequences with another amino acid residue, provided that the resulting sequence has keratin characteristics and a high transdermal rate.

[0047] Amino acid insertion refers to inserting amino acid residues at appropriate positions in the sequence of any one of the three transdermal keratin amino acid sequences. The inserted amino acid residues may all or partially be adjacent to each other, or none of the inserted amino acids are adjacent to each other, provided that the resulting sequence has keratin characteristics and a high transdermal rate.

[0048] Amino acid deletion refers to deleting one, two, or more than three amino acids from the sequence of any one of the three transdermal keratin amino acid sequences, provided that the resulting sequence has keratin characteristics and a high transdermal rate.

[0049] In the present invention, the substitution can be a conservative amino acid substitution, which means that compared with the amino acid sequence of any one of the three transdermal keratin amino acid sequences, three, preferably two or one amino acid is replaced by an amino acid with similar or close properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 1.

[0050] An embodiment of the present application provides a polynucleotide that encodes a transdermal recombinant keratin as described in the foregoing embodiments.

[0051] In some embodiments, the nucleotide sequence of the polynucleotide is as shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0052] An embodiment of the present application provides an expression vector that contains the polynucleotide as described in the foregoing embodiments.

[0053] In an embodiment of the present application, a host cell is provided, and the host cell contains the expression vector as described in the foregoing embodiment; In some embodiments, the host cell is Escherichia coli.

[0054] In an embodiment of the present application, a method for preparing a transdermal recombinant keratin as described in any one of the foregoing embodiments is provided, including: culturing the host cell in a production medium; and isolating the transdermal recombinant keratin from the host cell.

[0055] Recombinant human transdermal keratin can be obtained by the following preparation method, for example, it may include: (1) Construction of a Pichia pastoris genetic engineering bacterium; (2) Fermentation culture of the Pichia pastoris genetic engineering bacterium; (3) Induction and expression of the transdermal recombinant keratin; (4) Purification of the transdermal recombinant keratin.

[0056] It should be understood that those skilled in the art can replace the above-mentioned Pichia pastoris with other expression strains as the host cell. The host cell can be a eukaryotic cell, such as a fungus and yeast, or a prokaryotic cell, such as a bacterium of the Enterobacteriaceae family, such as Escherichia coli.

[0057] In an embodiment of the present application, a composition is provided, which contains the transdermal recombinant keratin as described in the foregoing embodiment.

[0058] In an embodiment of the present application, a product is provided, which contains the transdermal recombinant keratin as described in the foregoing embodiment; or contains the composition as described in the foregoing embodiment; The product is at least one of a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic, and a health product; In some embodiments, the pharmaceutical composition is a topical preparation; In some embodiments, the topical preparation is a topical application preparation; In some embodiments, the topical application preparation is any one selected from topical microneedle preparations, topical hydrogel agents, and topical infiltration preparations.

[0059] In an embodiment of the present application, an application of the transdermal recombinant keratin as described in the foregoing embodiment, the polynucleotide as described in the foregoing embodiment, the expression vector as described in the foregoing embodiment, the host cell as described in the foregoing embodiment, or the composition as described in the foregoing embodiment in the preparation of a product is provided; The product is at least one of a medical device, a tissue engineering product, a cosmetic, and a skin care product.

[0060] The present invention will be further described below through specific embodiments. However, it should be understood that these embodiments are only for more detailed description and should not be construed as limiting the present invention in any form.

[0061] The test materials and reagents used in the following examples are as follows: (1) Strains, cells and vectors: Strains containing the target gene and expression plasmids, such as Escherichia coli DH5α, TOP10, etc., were purchased from Beyotime; Pichia pastoris X33 (Miaoling Biology), and the vectors pPICzα and the antibiotic Zeocin were purchased from Invitrogen.

[0062] (2) Kits and enzymes: LDH detection kit (Roche 04744926001), improved Bradford protein content determination kit (Sangon Biotech), seamless cloning kit In-Fusion Snap Assembly Master Mix (TaKaRa), plasmid extraction kit (General Biosystems), general gel extraction kit (General Biosystems), restriction endonucleases, Q5 ultra-fidelity 2x Master Mix PCR polymerase, etc. were purchased from NEB.

[0063] (3) Media: The yeast media were YPG medium (1% yeast extract, 2% peptone, 1% glycerol) and YPD medium (1% yeast extract, 2% peptone, 2% glucose). YPG + Zeo medium was YPG medium supplemented with 100 μg / mL of Zeocin.

[0064] YPD medium with Zeo was YPD medium supplemented with 100 μg / mL of Zeocin; Yeast induction media BMGY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 1% glycerol (v / v)) and BMMY (1% yeast extract, 2% peptone, 1.34% YNB, 0.00004% biotin, 0.5% methanol (v / v)); The recombinant yeast fermentation medium was BSM medium (0.5% potassium dihydrogen phosphate, 5% diammonium hydrogen phosphate, 1.5% magnesium sulfate heptahydrate, 0.1% calcium sulfate, 2% potassium sulfate, 0.03% antifoaming agent). After autoclaving, 4.35 mL of trace salt solution PTM1 was added to the BSM medium, and the composition of PTM1 was: 6.5% ferric sulfate heptahydrate, 0.5% copper sulfate, 0.3% manganese sulfate, 0.05% cobalt chloride hexahydrate, 0.02% sodium molybdate dihydrate, 0.002% boric acid, 0.018% potassium iodide, 2% zinc chloride, 0.5% concentrated sulfuric acid, 0.02% biotin.

[0065] (4)Protein purification materials: Ni-NTA filler and nickel column were both purchased from Sanji Biotechnology Co., Ltd.

[0066] Example 1: Gene synthesis of transdermal recombinant keratin In this example, gene design, optimization and synthesis of transdermal recombinant keratin were carried out.

[0067] Experimental method: (1)Gene analysis: The truncated amino acid sequence of human keratin type 81 helix region and the cell-penetrating peptide in Table 1 were selected and connected by a flexible Linker to obtain a recombinant transdermal keratin with fusion expression. The recombinant sequence was codon-optimized for Pichia pastoris codons. The newly obtained genes, namely genes K1, K2, K3 and K4 of the present invention, have amino acid sequences as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4.

[0068] (2)Gene synthesis: The full lengths of genes K1, K2, K3 and K4 are 480bp, 468bp, 480bp and 498bp respectively, and the sequences after codon optimization are SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 respectively.

[0069] The optimized sequences were subjected to gene synthesis. The gene fragments were provided by General Gene Biotechnology Co., Ltd., and the synthesized gene fragments of K1, K2, K3 and K4 were inserted into the expression vector of picZalpha by EcoRI and SalI respectively. Then the vector was transformed into Escherichia coli TOP10 for preservation.

[0070] Experimental results: The vector maps constructed with the A3 gene as a representative are shown respectively in Figure 1 .

[0071] Example 2: Construction and induced expression of transdermal recombinant keratin In this example, experiments on the construction and induced expression of transdermal recombinant keratin were carried out.

[0072] Experimental method: (1)Plasmid extraction: Different strains containing the target gene were streaked on the corresponding resistant LB+Z plates and cultured overnight at 37°C. Single colonies cultured overnight were picked and inoculated into 10 mL LB+Z liquid medium respectively, and cultured overnight at 37°C. The cultured bacterial liquid was taken, centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were taken. The plasmid was extracted with the Tiangen Plasmid Extraction Kit. The extracted plasmid was measured for concentration with NanoDrop and then stored at -20°C.

[0073] (2) Plasmid linearization and recovery: Take 20 μg of the extracted plasmid, and perform single enzyme digestion with PmeI at 37°C for 3 - 6 h. Take 5 μL of the linearized gene fragment and detect it with 1% agarose gel to confirm that the fragment is completely linearized. Use the Tiangen Universal DNA Purification and Recovery Kit for liquid recovery. The specific steps are as follows: Take the completely linearized mixed solution, add an equal volume of PC solution, mix well and transfer it to the collection column balanced with BL. Centrifuge at 12000 rpm for 1 min, wash twice with PW solution, and centrifuge at 12000 rpm for 2 min. Transfer the collection column to a clean 1.5 mL centrifuge tube, air dry at room temperature for 5 min, add ddH2O preheated at 55°C, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain the linearized gene fragment.

[0074] (3) Strain construction: Take 10 μL of the linearized fragment and add 100 μL of Pichia pastoris competent cells. Mix well on ice and transfer it to an electrode cup. Electroporate at 150 V and 200 mA for 10 ms. Immediately add 500 μL of recovery medium after electroporation, mix well and transfer it to a 1.5 mL centrifuge tube, and let it stand and recover in a 30°C incubator for 3 h. Take 100 μL and spread it on the YPG + Z resistance plate, and incubate it upside down in a 30°C incubator for 2 - 3 d.

[0075] (4) Induction culture: Add BMGY to a 24-well plate. Pick the yeast cultured on the plate and transfer it to the 24-well plate. Incubate it in a 30°C shaker for 1 day, add 100 μL of BMMY medium for induction, and continuously induce for two days. After the culture is completed, centrifuge at 4000 rpm for 5 min, take the supernatant for storage or directly perform SDS-PAGE.

[0076] (5) Protein expression detection: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, boil it in boiling water at 100°C for 10 min, and then add 20 μL to each well of the SDS-PAGE protein gel. First run at 80 V for 1 h, and then transfer to 120 V until the bands are completely separated. Stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 30 min, and then decolorize it overnight with protein decolorizing solution (10% acetic acid, 5% ethanol).

[0077] (6) Protein purification: Take the fermented yeast mixture, centrifuge at 4°C, 10,000 rpm for 25 min, discard the precipitate, harvest the supernatant, and measure the volume. Rinse with (50 mM PB, 0.5 M NaCl, pH 7.0) for 5 column volumes to equilibrate the nickel column: Load the pretreated protein. Rinse with a low-concentration imidazole (10 - 50 mM) buffer for 3 - 5 column volumes to remove impurities, and then perform gradient elution with 50 - 500 mM imidazole. Collect the protein samples eluted with different concentrations of imidazole and detect the purity by SDS-PAGE. After collection, rinse the nickel column with 20% ethanol, fill it with ethanol, and store it. For nickel columns reused multiple times, nickel column regeneration is required. The regeneration steps are as follows: Rinse with ddH2O for 5 column volumes, rinse with an EDTA solution for 5 column volumes, rinse with an NaOH solution for 5 column volumes, fill the affinity column with a NiSO4 solution, and rinse for 5 column volumes. Take the protein purification solutions received at different times and perform SDS-PAGE detection to determine the purity and size of the target protein.

[0078] Experimental results: The SDS-PAGE electrophoresis results are shown in Figure 2 , and the molecular weights of K1, K2, K3, and K4 are approximately 19 kDa; the purification results are shown in Figure 3 , and the electrophoretically detected molecular weights of the proteins of genes K1, K2, K3, K4, and K5 are all approximately 19 kDa.

[0079] Example 3: Transdermal effect test In this example, a transdermal effect test was conducted. Among them, the transdermal test method refers to He et. al. J Cosmet Dermatol. 2019;00:1–10. DOI: 10.1111 / jocd.13041.

[0080] Experimental method: Collect the skin of male Kunming mice (20 ± 2 g): After anesthesia, trim the abdominal hair, cut off the abdominal epidermis and dermis, and remove the subcutaneous fat. Use the LOAGN SYSTEM918 dry heating fully automatic transdermal diffusion sampling system to detect the transdermal protein concentration and transdermal rate of K1 to K5. Install the treated mouse skin between the receiving cell and the diffusion cell, with an effective diffusion area of 1.77 cm 2 , and the volume of the diffusion cell is 12 mL.

[0081] The receiving cell is filled with normal saline, and 2 mL of recombinant keratin solution (in 5 mg / mL) is added to the diffusion cell, and it is stirred at 37 ± 2°C and 300 rpm.

[0082] Samples were taken after 1, 6, and 12 hours and replaced with PBS.

[0083] Using 2 mL of PBS as the control group, the protein concentration in the sampled solution at each time point was measured by the Lowry method, and the permeability was calculated.

[0084] The percutaneous rate (S, %) of recombinant keratin penetrating excised mouse skin was calculated by the following formula: .

[0085] Where C n is the sample protein concentration at time point "n" (mg / mL), C i is the sample protein concentration at time point "i" (mg / mL), V is the volume (mL) of the receptor cell, V i is the volume (mL) of the sample, m is the weight (mg) of 2 mL of recombinant keratin solution, and S is the percutaneous permeability rate (%). The relative percutaneous permeability rate is obtained by dividing the percutaneous permeability rate of the transdermal recombinant keratin by the percutaneous permeability rate of recombinant keratin K5.

[0086] Experimental results: The results are as Figures 4 to 6 shown. Compared with K5 without cell-penetrating peptide, K1, K2, K3, and K4 containing cell-penetrating peptide have better transdermal performance.

[0087] Example 4: Test on the cell proliferation promoting activity of recombinant transdermal keratin In this example, an experiment on the cell proliferation promoting activity was conducted for recombinant transdermal keratin.

[0088] Experimental method: (1) L929 cell culture: L929 cells were placed in DMEM medium containing 10% fetal bovine serum and double antibiotics and cultured in a carbon dioxide incubator at a concentration of 5%, relative humidity of 95%, and a constant temperature of 37 °C.

[0089] The medium was changed every two days, and subculture was carried out after the cells were basically confluent on the fifth day.

[0090] First, the old medium was aspirated, washed twice with PBS solution, and then 0.5 mL of 0.25% trypsin was added for digestion. Observation was carried out under an inverted microscope. After the cells shrank and became round, 10 mL of DMEM medium was added to stop digestion.

[0091] The cells were gently pipetted to make them suspended, centrifuged at 1000 r / min for 5 min, the supernatant was removed, an appropriate amount of serum-containing medium was added, and the cells were gently pipetted again to make a cell suspension, and 50 μL was taken for counting.

[0092] For L929 cells in the logarithmic growth phase, aspirate the upper culture medium, wash twice with PBS solution, digest into single-cell suspension with 0.5 mL of 5% DMEM-0.25% trypsin, wash twice again with PBS solution, and directly collect the suspended cells.

[0093] MTT assay: Select a 96-well plate, seed 2×10 4 cells per well, and set up an experimental group, a blank control group (containing only PBS solution), and a calibration and zeroing group (without cells). Two replicate wells are set up for each group. After 24 h, upon microscopic observation, it is found that most cells have adhered and spread. At this time, remove the non-adherent cells and liquid in the wells.

[0094] Adjust the concentration of the experimental group to 0.5 mg / mL and add 20 μL, while the blank control group adds 20 μL of PBS buffer, and the calibration and zeroing group only adds DMEM medium. When adding the samples, ensure that the pipette tip is close to the well wall and inject slowly. After adding each well, gently pipette to mix evenly.

[0095] Then let it stand for 30 min and place the plate in a CO2 incubator. Observe the cell proliferation after culturing for 72 h. Use the MTT method to measure the absorbance (OD) value. Add 20 μL of MTT solution to each well of the 96-well plate and continue to culture for 4 h to complete the color reaction.

[0096] Then terminate the culture, aspirate the culture medium in the wells or cover a layer of filter paper on the culture plate, quickly invert the culture plate, add 150 μL of DMSO to each well, shake for 10 min to fully dissolve the formazan, and finally use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 490 nm.

[0097] Experimental results: The results are shown in Figure 7 . Compared with the blank and recombinant keratin K5 without cell-penetrating peptide, recombinant transdermal keratins K1, K2, K3, and K4 have better effects in promoting the proliferation and differentiation of L929 cells and enhancing cell activity, and can be used to prepare biomaterials or drugs for promoting cell proliferation.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A transdermal recombinant keratin, characterized in that, The transdermal recombinant keratin comprises recombinant keratin and a cell-penetrating peptide; The transdermal recombinant keratin has cell adhesion activity and transdermal permeability; The transdermal recombinant keratin comprises any one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 or is composed of the same.

2. A polynucleotide, characterized in that, The polynucleotide encodes the transdermal recombinant keratin as claimed in claim 1.

3. The polynucleotide according to claim 2, wherein The nucleotide sequence of the polynucleotide is as shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.

8.

4. An expression vector, characterized in that, The expression vector comprises the polynucleotide as claimed in claim 2.

5. A host cell, characterized in that, The host cell comprises the expression vector as claimed in claim 4.

6. A method for preparing the transdermal recombinant keratin as described in claim 1, characterized in that, Comprising: Culturing the host cell in a production medium; And isolating the transdermal recombinant keratin from the host cell.

7. A composition, characterized in that, Comprising the transdermal recombinant keratin as claimed in claim 1.

8. An article, characterized in that, Comprising the transdermal recombinant keratin as claimed in claim 1; or, comprising the composition as claimed in claim 7; The article is at least one of a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic and a health product; The pharmaceutical composition is a topical preparation; and / or, the pharmaceutical composition is a topical preparation; and the topical preparation is a topical application preparation; and / or, the pharmaceutical composition is a topical preparation; and the topical preparation is a topical application preparation; the topical application preparation is selected from any one of a topical microneedle preparation, a topical hydrogel and a topical infiltration preparation.

9. Use of a transdermal recombinant keratin as claimed in claim 1, a polynucleotide as claimed in claim 2, an expression vector as claimed in claim 4, a host cell as claimed in claim 5, or a composition as claimed in claim 7 in the preparation of a product; The product is selected from at least one of a medical device, a tissue engineering product, a cosmetic and a skin care product.

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