A transdermal recombinant keratin and its preparation method and application

Abstract

The present invention provides a transdermal recombinant keratin, its preparation method, and application, relating to the field of genetic engineering technology. The transdermal recombinant keratin comprises recombinant keratin and a cell-penetrating peptide; the transdermal recombinant keratin exhibits cell adhesion activity and transdermal permeability. 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 transdermal recombinant keratin significantly enhances keratin's transdermal performance and cell adhesion activity, addressing the poor skin permeability of conventional recombinant keratin.

Description

Technical Field

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

[0002] Cell-penetrating peptides (CPPs) are a class of peptides with unique bioactivities, typically composed of 5 to 30 amino acids. Their core function is to penetrate cell membranes, thereby intervening in the intracellular environment. The penetrating ability of CPPs stems primarily from their abundance of basic amino acid residues, which enable them to interact with negatively charged glycosaminoglycan chains on the cell membrane, leading to cellular uptake through mechanisms such as endocytosis or micropinocytosis. Leveraging this property, CPPs can serve as delivery vehicles for a variety of molecules, including non-cell-penetrating peptides, proteins, nanoparticles, quantum dots, and nucleic acids, finding widespread application in biomedicine and biotechnology. Currently, a vast array of CPPs has been reported, numbering approximately 1,000. Based on their physicochemical properties, they are primarily classified into three categories: cationic peptides, amphipathic peptides, and hydrophobic peptides. Common CPPs include DPV3 / 6, VP22, TP10, TD, Pep-1, TAT, R7-9, cTAT, and MAP.

[0003] In the field of biomaterials, recombinant keratin, a key material, has been widely used in a variety of fields, including drug delivery, wound healing, tissue engineering, and cosmetics and skincare, due to its numerous excellent properties, including biocompatibility, degradability, hemostasis, non-immunogenicity, antimicrobial activity, and anti-inflammatory and antioxidant properties. However, currently commercialized recombinant keratin has certain limitations. Its large molecular weight results in poor skin permeability. This characteristic makes it difficult for recombinant keratin to effectively penetrate the skin and exert its intended efficacy in practical applications, especially in lotions and cleansing products, limiting its application in related products.

[0004] While CPPs have considerable potential for delivering recombinant proteins, their compatibility with different proteins varies significantly. This means that in practical applications, CPPs may be incompatible with recombinant keratin, impacting delivery efficiency and effectiveness. Furthermore, the inherent skin permeability of recombinant keratin makes it difficult to effectively penetrate deep into the skin without external intervention (such as microneedles or injections). This limitation not only complicates drug delivery and increases patient discomfort, but also restricts the effectiveness of recombinant keratin in topical and shampoo products, hindering its full biocompatibility and multifunctionality.

[0005] In summary, existing recombinant keratin applications face the challenge of poor skin permeability. While CPPs can serve as delivery vehicles, their compatibility with recombinant keratin varies significantly, making it difficult to effectively address the permeability challenge. These challenges collectively limit the widespread application of recombinant keratin in topical and skincare products. A technical solution that effectively improves the skin permeability of recombinant keratin is urgently needed to fully realize its potential in the biomedical and cosmetic fields.

[0006] In view of this, the present invention is 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, solving the problem of poor skin permeability of traditional recombinant keratin.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0009] In a first aspect, the present invention provides a transdermal recombinant keratin protein, wherein the transdermal recombinant keratin protein comprises recombinant keratin and a cell penetrating peptide;

[0010] The transdermal recombinant keratin has cell adhesion activity and transdermal properties;

[0011] 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.

[0012] In a second aspect, the present invention provides a polynucleotide encoding the transdermal recombinant keratin as described in the above embodiments.

[0013] In an optional embodiment, the nucleotide sequence of the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

[0014] In a third aspect, the present invention provides an expression vector comprising the polynucleotide as described in the aforementioned embodiment.

[0015] In a fourth aspect, the present invention provides a host cell, comprising the expression vector described in the above embodiment;

[0016] In an alternative embodiment, the host cell is Escherichia coli.

[0017] In a fifth aspect, the present invention provides a method for preparing transdermal recombinant keratin as described in any one of the aforementioned embodiments, comprising:

[0018] The host cells are cultured in a production medium; and the transdermal recombinant keratin is isolated from the host cells.

[0019] In a sixth aspect, the present invention provides a composition comprising the transdermal recombinant keratin as described in the above embodiments.

[0020] In a seventh aspect, the present invention provides a product comprising the transdermal recombinant keratin according to the aforementioned embodiment; or comprising the composition according to the aforementioned embodiment;

[0021] The product is at least one of a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic, and a health product;

[0022] The pharmaceutical composition is an external preparation; and / or, the pharmaceutical composition is an external preparation; and the external preparation is an external smear preparation; and / or, the pharmaceutical composition is an external preparation; and the external preparation is an external smear preparation; the external smear preparation is selected from any one of an external microneedle preparation, an external hydrogel and an external infiltration preparation.

[0023] In an eighth aspect, the present invention provides a use of the transdermal recombinant keratin, the polynucleotide, the expression vector, the host cell, or the composition in the aforementioned embodiments in preparing a product.

[0024] The product is selected from at least one of medical devices, tissue engineering products, cosmetics and skin care products.

[0025] The transdermal recombinant keratin provided in this application significantly enhances its transdermal effectiveness by combining recombinant keratin with a cell-penetrating peptide. This transdermal recombinant keratin not only effectively penetrates the skin barrier but also maintains its biological activity after penetration, thereby exerting its cell adhesion activity within the skin, promoting cell proliferation and tissue repair. This innovative production method and application offer broad application prospects for transdermal recombinant keratin in the pharmaceutical, cosmetic, and skincare industries, demonstrating significant beneficial effects in promoting wound healing and enhancing the efficacy of skin care products. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a representative diagram of the plasmid construction using the vector pPicZalpha-K1 as an example in Example 1 of the present application;

[0028] Figure 2 This is the protein electrophoresis diagram of the four transdermal recombinant keratin proteins expressed in Pichia pastoris in Example 2 of this application (the electrophoresis detected molecular weight of the proteins of the recombinant proteins K1, K2, K3, and K4 genes is approximately 19KDa);

[0029] Figure 3 This is the protein electrophoresis diagram obtained after the expression and purification of K1 to K5 proteins in Example 2 of the present application (the electrophoresis detected molecular weight of the K1 to K5 proteins is approximately 19 kDa);

[0030] Figure 4 These are the transdermal test results of the recombinant transdermal keratin and recombinant keratin 1h in Example 3 of this application;

[0031] Figure 5 These are the transdermal test results of the recombinant transdermal keratin and recombinant keratin 6h in Example 3 of this application;

[0032] Figure 6 These are the transdermal test results of the recombinant transdermal keratin and recombinant keratin 12h in Example 3 of this application;

[0033] Figure 7 This is a diagram of the cell proliferation activity detection results in Example 4 of this application. DETAILED DESCRIPTION

[0034] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all commercially available conventional products.

[0035] The present invention provides a transdermal recombinant keratin protein in an embodiment, which comprises recombinant keratin protein and a cell-penetrating peptide. The transdermal recombinant keratin protein has cell adhesion activity and transdermal properties.

[0036] The transdermal recombinant keratin comprises a fusion of recombinant keratin and cell-penetrating peptides, and is capable of effectively penetrating the skin and promoting intercellular adhesion.

[0037] Recombinant keratin is a keratin fragment or derivative produced through genetic engineering. Keratin is a biomaterial with numerous advantages, including biocompatibility, biodegradability, hemostasis, non-immunogenicity, antimicrobial activity, and anti-inflammatory and antioxidant properties. Recombinant keratin is typically derived from specific functional regions of keratin and optimized and expressed through genetic engineering to meet specific application requirements.

[0038] Cell-penetrating peptides are a class of small peptides capable of penetrating cell membranes. They can be composed of 5-30 amino acids and are rich in basic amino acid residues (such as lysine and arginine). These peptides can interact with glycosaminoglycan chains on the cell membrane and enter the cell through mechanisms such as endocytosis or micropinocytosis. Cell-penetrating peptides can serve as carriers to deliver molecules such as non-cell-penetrating peptides, proteins, nanoparticles, and nucleic acids into cells.

[0039] Cell adhesion activity refers to the ability of a substance to promote cell-to-cell or cell-to-matrix adhesion. This activity is crucial for processes such as cell growth, differentiation, migration, and tissue repair. The cell adhesion activity of transdermal recombinant keratin enables it to form strong adhesion to the surface of skin cells, thereby enhancing its effectiveness in the skin.

[0040] Transdermal permeability refers to a substance's ability to penetrate the surface layer of the skin and reach deeper into the skin's tissues. Transdermal permeability is a key indicator of the effectiveness of topical preparations for skin application, such as medications and cosmetics. For transdermal recombinant keratin, its transdermal permeability means it can pass through the skin barrier and reach deeper into the skin's tissues, where it can exert its biological activities, such as promoting cell proliferation and repairing damaged skin.

[0041] By fusing the recombinant keratin with a cell-penetrating peptide, the recombinant keratin can effectively penetrate the skin barrier and reach deeper into the skin's tissues. This overcomes the problem of traditional recombinant keratin's difficulty penetrating the skin due to its large molecular weight, expanding its application in skin care and treatment. The recombinant keratin exhibits cell adhesion, promoting cell-to-cell and cell-to-matrix adhesion. This property aids cell growth, differentiation, and migration, thereby accelerating wound healing and tissue repair. Recombinant keratin itself possesses multiple biological activities, such as antibacterial, anti-inflammatory, and antioxidant properties. Combined with the cell-penetrating peptide, these activities can be more effectively exerted in the skin, enhancing the overall performance of the product. This recombinant keratin has applications not only in the medical field (such as topical medications and wound dressings), but also in cosmetics and skincare products, such as anti-wrinkle serums and repair masks, enhancing product efficacy and market competitiveness. Due to its excellent transdermal properties, the recombinant keratin can be administered via simple topical application, eliminating the need for complex procedures like microneedling or injections, improving ease of use and patient compliance.

[0042] In summary, the transdermal recombinant keratin significantly improves the transdermal performance and cell adhesion activity of keratin by fusing it with cell-penetrating peptides, making it more advantageous in skin care and treatment, while also expanding its application range in the fields of medicine and cosmetics.

[0043] 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.

[0044] In some embodiments, linker may not be added. The cell penetrating peptide sequence is shown in Table 1.

[0045] Table 1. Cell-penetrating peptides

[0046]

[0047] The sequence of the human keratin type 81 described above 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:

[0048] MTCGSGFGGRAFSCISACGPRPGRCCITAAPYRGISCYRGLTGGFGSHSVCGGFRAGSCGRSFGYRSGGVCGPSPPCITSVNESLLTPLNLEIDPNAQCVKQEEKEQIKSLNSRFAAFIDKVRFLEQQNKLLETKLQFYQNRECCQS NLEPLFEGYIETLRREAECVEADSGRLASELNHVQEVLEGYKKKYEEEVSLRATAENEFVALKKDVDCAYLRKSDLEANVEALIQEIDFLRRLYEEEILILQSHISDTSVVVKLDNSRDLNMDCIIAEIKAQYDDIVTRSRAEAESWYRS KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK LEAAVAQSEQQGEAALSDARCKLAELEGALQKAKQDMACLIREYQEVMNSKLGLDIEIATYRRLLEGEEQRLCEGIGAVNVCVSSSRGGVVCGDLCVSGSRPVTGSVCSAPCNGNVAVSTGLCAPCGQLNTTCGGGSCGVGSCGISSLGVGSCGSSCRKC (SEQ ID NO. 9).

[0049] The bold underlined portion in the above sequence is the target amino acid sequence selected in this example.

[0050] In this example, after extensive research, it was found that the selected sequence has strong water solubility, high recombinant expression yield, simple purification process, and better skin penetration effect than keratin without added cell-penetrating peptide, and has multiple excellent biomaterial properties.

[0051] It should be noted that the transdermal recombinant keratin in the examples of the present application is not the full-length sequence of SEQ ID NO.9.

[0052] This example is based in part on the following discovery: transdermal recombinant keratin comprising at least 36 consecutive amino acid residues of SEQ ID NO. 10 can have better biomaterial properties than commercial transdermal keratin, as demonstrated in the examples.

[0053] Those skilled in the art can appropriately select the consecutive amino acid residues constituting the recombinant transdermal keratin. In this embodiment, the following K5:

[0054] The sequence of the amino acid region of KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK (SEQ ID NO. 10) was tested:

[0055] In this embodiment, the transdermal recombinant keratin can be a sequence of recombinant human transdermal keratin K5 repeated three times, comprising 138 amino acids, and the basic repeating unit is:

[0056] KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK (SEQ ID NO. 10) is a human transdermal keratin 81 type peptide.

[0057] The amino acid sequence of K5-3 is:

[0058] KCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSKKCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSKKCEEMKATVIRHGETLRRTKEEINELNRMIQRLTAEVENAKCQNSK (SEQ ID NO. 11).

[0059] The DNA sequence of K5-3 is as follows:

[0060] AAGTGTGAAGAAATGAAGGCTACTGTTATTAGACATGGTGAAACTTTGAGAAGAACTAAGGAAGAAATTAACGAATTGAACAGAATGATCCAAAGATTGACTGCTGAAGTTGAAAACGCTAAATGTCAAAATTCTAAGAAGTGTGAAGAGATGAAGGCTACCGTTATTAGACACGGTGAAACTCTTAGAAGAACTAAAGAAGAAATCAA CGAATTGAATAGAATGATCCAGAGATTGACTGCCGAAGTTGAAAATGCTAAATGTCAGAACTCTAAGAAGTGCGAAGAAATGAAAGCTACTGTTATCAGACATGGTGAGACTTTGAGAAGAACCAAGGAAGAAATCAATGAATTGAACAGAATGATTCAGAGATTGACCGCTGAAGTTGAGAACGCTAAATGCCAAAATTCTAAA (SEQ ID NO.12).

[0061] 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.

[0062] 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.

[0063] 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:

[0064] Table 2. Amino acid sequence of transdermal recombinant keratin

[0065]

[0066] 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.

[0067] 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 set forth in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4. Based on the aforementioned amino acid sequences, appropriate parameters for aligning sequences can be determined, including any algorithms required to achieve maximal alignment over the full length of the compared sequences.

[0068] 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, as long as the sequence has keratin characteristics and a high transdermal rate.

[0069] Amino acid substitution refers to the replacement of an amino acid residue at a certain position in any of the three transdermal keratin amino acid sequences by other amino acid residues, as long as the protein has keratin characteristics and a high transdermal rate.

[0070] Amino acid insertion refers to inserting an amino acid residue at an appropriate position in the sequence of any one of the three transdermal keratin amino acid sequences. The inserted amino acid residues may be adjacent to each other in whole or in part, or the inserted amino acids may not be adjacent to each other, as long as the protein has keratin characteristics and a high transdermal rate.

[0071] Amino acid deletion refers to deleting 1, 2 or 3 or more amino acids in the sequence of any one of the three transdermal keratin amino acid sequences, as long as the protein has keratin characteristics and a high transdermal rate.

[0072] In the present invention, substitutions may be conservative amino acid substitutions, meaning that compared to the amino acid sequence of any of the three transdermal keratin amino acid sequences, three, preferably two, or one amino acid is replaced with an amino acid having similar or similar properties to form a peptide. These conservative variant peptides can be generated by amino acid substitution according to Table 1.

[0073] The present application provides a polynucleotide in an embodiment, which encodes the transdermal recombinant keratin as described in the above embodiment.

[0074] In some embodiments, the nucleotide sequence of the polynucleotide is shown in any one of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

[0075] An expression vector is provided in an embodiment of the present application, and the expression vector comprises the polynucleotide described in the aforementioned embodiment.

[0076] A host cell is provided in an embodiment of the present application, wherein the host cell comprises the expression vector described in the above embodiment;

[0077] In some embodiments, the host cell is Escherichia coli.

[0078] The present application provides a method for preparing transdermal recombinant keratin as described in any of the aforementioned embodiments, comprising:

[0079] The host cells are cultured in a production medium; and the transdermal recombinant keratin is isolated from the host cells.

[0080] Recombinant human transdermal keratin can be obtained by the following preparation method, which may include:

[0081] (1) Construction of genetically engineered Pichia pastoris;

[0082] (2) Fermentation culture of genetically engineered Pichia pastoris;

[0083] (3) Induction and expression of transdermal recombinant keratin;

[0084] (4) Purification of transdermal recombinant keratin.

[0085] It should be understood that those skilled in the art can replace the above-mentioned Pichia pastoris with other expression strains as host cells. The host cell can be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Enterobacteriaceae, such as Escherichia coli.

[0086] The present application provides a composition in an embodiment, comprising the transdermal recombinant keratin as described in the above embodiment.

[0087] In the embodiments of the present application, a product is provided, comprising the transdermal recombinant keratin as described in the aforementioned embodiment; or comprising the composition as described in the aforementioned embodiment;

[0088] The product is at least one of a pharmaceutical composition, a medical device, a tissue engineering product, a cosmetic, and a health product;

[0089] In some embodiments, the pharmaceutical composition is a topical preparation;

[0090] In some embodiments, the topical preparation is a topical smear preparation;

[0091] In some embodiments, the topical application preparation is selected from any one of a topical microneedle preparation, a topical hydrogel, and a topical infiltration preparation.

[0092] The present application provides, in an embodiment, a use of the transdermal recombinant keratin described in the aforementioned embodiment, the polynucleotide described in the aforementioned embodiment, the expression vector described in the aforementioned embodiment, the host cell described in the aforementioned embodiment, or the composition described in the aforementioned embodiment in preparing a product;

[0093] The product is selected from at least one of medical devices, tissue engineering products, cosmetics and skin care products.

[0094] The present invention is further described below by way of specific examples. However, it should be understood that these examples are merely provided for more detailed description and are not to be construed as limiting the present invention in any form.

[0095] The test materials and reagents used in the following examples are as follows:

[0096] (1) Strains, cells and vectors: Strains containing target genes and expression plasmids, Escherichia coli DH5a, TOP10 and other strains were purchased from Biyuntian and Pichia pastoris X33 (Miaoling Biotechnology), and the vector pPICzalpha and the antibiotic Zeocin were purchased from Invitrogen.

[0097] (2) Kits and enzymes: LDH detection kit (Roche 04744926001), modified Bradford protein content assay kit (Sangon Biotechnology), seamless cloning kit In-Fusion Snap Assembly Master Mix (TaKaRa), plasmid extraction kit (Universal Biotechnology), universal gel recovery kit (Universal Biotechnology), restriction endonuclease, Q5 ultra-fidelity 2x Master Mix PCR polymerase, etc. were purchased from NEB.

[0098] (3) Culture medium:

[0099] Yeast culture media include YPG medium (1% yeast extract, 2% peptone, 1% glycerol) and YPD medium (1% yeast extract, 2% peptone, 2% glucose). YPG+Zeo medium is YPG medium supplemented with 100 μg / mL Zeocin.

[0100] YPD medium was YPD medium with 100 μg / mL Zeocin added;

[0101] yeast induction medium 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));

[0102] Recombinant yeast fermentation medium was BSM (0.5% potassium dihydrogen phosphate, 5% diammonium phosphate, 1.5% magnesium sulfate heptahydrate, 0.1% calcium sulfate, 2% potassium sulfate, and 0.03% antifoaming agent). After autoclaving, 4.35 mL of trace salt solution PTM1 was added. PTM1 consists of: 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, and 0.02% biotin.

[0103] (4) Protein purification materials: Ni-NTA filler and nickel column were purchased from Sanji Biotechnology.

[0104] Example 1: Gene synthesis of transdermal recombinant keratin

[0105] In this example, the gene design, optimization and synthesis of transdermal recombinant keratin were performed.

[0106] Experimental methods:

[0107] (1) Gene analysis: A truncated amino acid sequence of the helical region of human keratin type 81 was selected and connected with the cell-penetrating peptides listed in Table 1 through a flexible linker to obtain recombinant transdermal keratin for fusion expression. The recombinant sequence was codon-optimized for Pichia pastoris codons. The recombinant new genes obtained are genes K1, K2, K3, and K4 of the present invention, with amino acid sequences as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4.

[0108] (2) Gene synthesis: The full lengths of K1, K2, K3, and K4 genes are 480 bp, 468 bp, 480 bp, and 498 bp, respectively. The sequences after codon optimization are SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

[0109] The optimized sequence was subjected to gene synthesis. The gene fragments were provided by Universal Gene Biotechnology Co., Ltd., and the synthesized K1, K2, K3 and K4 gene fragments were inserted into the picZalpha expression vector through EcoRI and SalI, respectively. The vector was then transformed into Escherichia coli TOP10 for preservation.

[0110] Experimental results:

[0111] The vector maps constructed above, represented by A3 gene, are shown in Figure 1 .

[0112] Example 2: Construction and induced expression of transdermal recombinant keratin

[0113] In this example, an experiment on the construction and induced expression of transdermal recombinant keratin was conducted.

[0114] Experimental methods:

[0115] (1) Plasmid extraction: Take different strains containing the target gene and streak the corresponding resistance on LB+Z plates, and culture them at 37℃ overnight. Pick single clones from the overnight culture and inoculate them into 10 mL LB+Z liquid medium, and culture them at 37℃ overnight. Take the overnight cultured bacterial liquid, centrifuge it at 5000 rpm for 5 minutes, discard the supernatant and take the bacterial cells. Use the Tiangen Plasmid Extraction Kit to extract the plasmid. The extracted plasmid is measured by NanoDrop and then frozen at -20℃.

[0116] (2) Plasmid linearization and recovery: Take 20 μg of the extracted plasmid and digest it with PmeI at 37°C for 3-6 hours. Take 5 μL of the linearized gene fragment and detect it on 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 after BL equilibration. Centrifuge at 12000 rpm for 1 min, rinse twice with PW solution, and centrifuge at 12000 rpm for 2 min. Transfer the collection column to a clean 1.5 mL centrifuge tube, dry it at room temperature for 5 min, add ddH2O preheated at 55°C, let it stand at room temperature for 2 min, and centrifuge it at 12000 rpm for 2 min to obtain the linearized gene fragment.

[0117] (3) Strain construction: Take 10 μL of the linearized fragment and add 100 μL of Pichia pastoris competent medium, mix well on ice, transfer to an electrode cup, and electroporate at 150 V and 200 mA for 10 ms. Immediately after electroporation, add 500 μL of recovery medium, mix well, transfer to a 1.5 mL centrifuge tube, and let it recover in a 30°C incubator for 3 h. Take 100 μL and spread on a YPG+Z plate, and incubate inverted in a 30°C incubator for 2-3 days.

[0118] (4) Induction culture: Add BMGY to a 24-well plate, pick yeast from the cultured plate and transfer it to the 24-well plate. Incubate in a shaker at 30°C for 1 day. Add 100 μL of BMMY medium for induction. Continue induction culture for two days. At the end of the culture, centrifuge at 4000 rpm for 5 minutes. Collect the supernatant and save it or directly perform SDS-PAGE.

[0119] (5) Protein expression detection: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, place in boiling water at 100℃ for 10 min, then add 20 μL per well to SDS-PAGE protein gel, first run at 80 V for 1 h, then turn to 120 V and run until the bands are completely separated. Use Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for protein staining for 30 min, and then use protein destaining solution (10% acetic acid, 5% ethanol) for overnight destaining.

[0120] (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 calculate the volume. Use (50mM PB, 0.5M NaCl, pH 7.0) to wash 5 column volumes to balance the nickel column: Take the pretreated protein for sample loading. Use low concentration imidazole (10-50 mM) buffer to wash 3-5 column volumes to remove impurities, and then perform gradient elution with 50-500 mM imidazole. Collect protein samples eluted with different concentrations of imidazole and detect purity by SDS-PAGE. After collection, rinse the nickel column with 20% ethanol, fill it with ethanol and store it. The nickel column needs to be regenerated after repeated use. The regeneration steps are as follows: rinse with 5 column volumes with ddH2O, rinse with 5 column volumes with EDTA solution, rinse with 5 column volumes with NaOH solution, fill the affinity column with NiSO4 solution, and rinse with 5 column volumes. Take the protein purification solution of different receiving amounts and perform SDS-PAGE detection to determine the purity and size of the target protein.

[0121] Experimental results:

[0122] SDS-PAGE electrophoresis results are shown in Figure 2 The molecular weight of K1, K2, K3 and K4 is about 19KDa; the results after purification are shown in Figure 3 The molecular weight of the proteins of genes such as K1, K2, K3, K4 and K5 detected by electrophoresis was approximately 19KDa.

[0123] Example 3: Transdermal effect test

[0124] In this example, a transdermal effect test was performed. The transdermal test method was described in He et al. J Cosmet Dermatol. 2019;00:1–10. DOI: 10.1111 / jocd.13041.

[0125] Experimental methods:

[0126] Collect skin from male Kunming mice (20 ± 2 g):

[0127] After anesthesia, abdominal hair was trimmed, the abdominal epidermis and dermis were excised, and subcutaneous fat was removed. The transdermal protein concentrations and transdermal rates of K1 to K5 were measured using a LOAGN SY STEM918 dry-heated, fully automated transdermal diffusion sampling system. The treated mouse skin was mounted between a receiving cell and a diffusion cell, with an effective diffusion area of ​​1.77 cm. 2 , the diffusion cell volume is 12 mL.

[0128] The receiving cell was filled with physiological saline, and 2 mL of recombinant keratin solution (5 mg / mL) was added to the diffusion cell and stirred at 37 ± 2°C and 300 rpm.

[0129] Samples were taken after 1, 6, and 12 hours and replaced with PBS.

[0130] 2 mL PBS was used as the control group. The protein concentration in the sample solution at each time point was determined by the Lowry method, and the permeability was calculated.

[0131] The percutaneous rate (S, %) of reconstituted keratin in penetrating mouse skin was calculated by the following formula:

[0132] .

[0133] Among them 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 of the recipient cell (mL), and V i is the sample volume (mL), m is the weight of 2 mL of recombinant keratin solution (mg), and S is the transdermal permeability (%). The relative transdermal permeability is calculated by dividing the transdermal permeability of recombinant keratin by the transdermal permeability of recombinant keratin K5.

[0134] Experimental results:

[0135] The results are as follows Figures 4 to 6 As shown in the Figure 3, K1, K2, K3, and K4 containing cell-penetrating peptides have better transdermal performance than K5 without cell-penetrating peptides.

[0136] Example 4: Cell proliferation promoting activity test of recombinant transdermal keratin

[0137] In this example, a cell proliferation promoting activity test experiment was conducted on the recombinant transdermal keratin.

[0138] Experimental methods:

[0139] (1) L929 cell culture: L929 cells were placed in DMEM medium containing 10% fetal bovine serum and double antibodies, and cultured in a carbon dioxide incubator with a constant temperature of 37°C and a concentration of 5%.

[0140] The culture medium was replaced every two days, and the cells were passaged after they were essentially confluent on the fifth day.

[0141] First, remove the old culture medium and wash twice with PBS. Then, add 0.5 mL of 0.25% trypsin to digest the cells. Observe under an inverted microscope. Once the cells shrink and become round, add 10 mL of DMEM to stop the digestion.

[0142] Gently pipette the cells to suspend them, centrifuge at 1000 rpm for 5 min, remove the supernatant, add an appropriate amount of serum-containing culture medium, and gently pipette again to make a cell suspension. Take 50 μL for counting.

[0143] For L929 cells in the logarithmic growth phase, remove the upper culture medium, wash twice with PBS solution, use 0.5 mL of 5% DMEM-0.25% trypsin to digest into a single-cell suspension, wash again twice with PBS solution, and directly collect the suspended cells.

[0144] MTT assay: Select a 96-well plate and inoculate 2×10 4 Cells were plated and plated in a controlled experiment group, a blank control group (containing only PBS solution), and a calibration group (containing no cells). Each group was plated in duplicate. After 24 hours, microscopic observation revealed that most cells had adhered and expanded. Unattached cells and liquid were removed from the wells.

[0145] The experimental group was adjusted to a concentration of 0.5 mg / mL and 20 μL was added. The blank control group received 20 μL of PBS buffer, and the calibration group received only DMEM medium. When adding the sample, ensure the pipette tip is firmly against the well wall and slowly inject. Gently pipette and mix thoroughly after each well.

[0146] After 30 minutes of stabilization, the plates were placed in a CO2 incubator. Cell proliferation was observed after 72 hours of culture. The absorbance (OD) value was determined using the MTT assay. 20 μL of MTT solution was added to each well of the 96-well plate and incubated for another 4 hours to complete the color development reaction.

[0147] After that, the culture was terminated, the culture medium in the wells was aspirated or a layer of filter paper was covered on the culture plate, the culture plate was quickly turned over, 150 μL of DMSO was added to each well, and the plate was shaken for 10 minutes to fully dissolve the formazan. Finally, the absorbance of each well was measured at a wavelength of A490 nm using a microplate reader.

[0148] Experimental results:

[0149] See the results Figure 7 Compared with blank and recombinant keratin K5 without added cell-penetrating peptide, recombinant transdermal keratin 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 that promote cell proliferation.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to 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 protein consists of recombinant keratin protein and cell penetrating peptide; The transdermal recombinant keratin has cell adhesion activity and transdermal properties; The transdermal recombinant keratin is 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.

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

3. The polynucleotide according to claim 2, wherein The nucleotide sequence of the polynucleotide is 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 according to claim 2.

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

6. A method for preparing transdermal recombinant keratin according to claim 1, characterized in that: include: culturing the host cells in a production medium; And, the transdermal recombinant keratin is isolated from the host cell.

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

8. A product, characterized in that Comprising the transdermal recombinant keratin according to claim 1; or, comprising the composition according to claim 7; The product is at least one of a pharmaceutical composition, a medical device, and a cosmetic; The pharmaceutical composition is an external preparation.

9. The product according to claim 8, wherein The external preparation is an external smear preparation.

10. The product according to claim 9, wherein The external application preparation is selected from any one of an external microneedle preparation, an external hydrogel and an external infiltration preparation.

11. Use of the transdermal recombinant keratin according to claim 1, the polynucleotide according to claim 2, the expression vector according to claim 4, the host cell according to claim 5, or the composition according to claim 7 in preparing a product; The product is selected from at least one of medical devices, cosmetics and skin care products.

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