A recombinant human K14 keratin and its preparation method

Abstract

This invention provides a recombinant human K14 keratin and its preparation method, relating to genetic engineering technology. The recombinant human K14 keratin exhibits cell proliferation-promoting activity; it is composed of the amino acid sequence shown in SEQ ID NO. 6. The recombinant human K14 keratin possesses higher cell proliferation activity and skin repair function, with low production cost, making it suitable for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically, to a recombinant human K14 keratin and its preparation method. Background Technology

[0002] As the outermost protective barrier of the human body, the skin plays a crucial role in resisting physical, chemical, and microbial attacks. The epidermis, primarily composed of keratinocytes, directly determines the integrity and functionality of the skin barrier through their differentiation. Epidermal renewal depends on the continuous proliferation of basal cells and their upward differentiation into the stratum spinosum, stratum granulosum, and stratum corneum. During this process, intracellular intermediate filament structural proteins, especially members of the keratin family, construct the cytoskeleton system, providing essential mechanical support and biological stability for the skin.

[0003] Intermediate keratin is widely distributed in epithelial cells, and its types and expression patterns vary with the cell differentiation stage, forming a distinct expression profile. These proteins not only play a role in maintaining cell morphology and structural stability but also participate in regulating cell proliferation, migration, and adhesion. Some keratins can mediate cell-to-cell or cell-to-basement membrane adhesion through interactions with desmosomes and hemidesmosomes. Changes in keratin expression are often used to identify different types of epithelial tissues or disease states, and are important biomarkers for cell differentiation and disease.

[0004] For skin barrier damage, current interventions primarily rely on exogenous skincare ingredients, such as ceramides, peptides, amino acids, hydrolyzed protein products, plant extracts, and their complex formulations. These products often improve skin condition by enhancing hydration, alleviating dryness, or regulating the skin's microenvironment. However, the effectiveness of these strategies is limited by the stability, penetration ability, and targeting compatibility of the active ingredients with the tissue structure. Clinical observations frequently reveal short-term improvements but poor long-term effects, and some products exhibit irritation or significant individual variability.

[0005] Current skincare technologies lack targeted approaches to address abnormalities or functional impairments in epidermal structural proteins. While some studies indicate that certain intermediate filament proteins have potential regulatory roles in epidermal barrier function, a systematic technical approach has not yet been developed regarding their expression structure, key functional fragments, bioactivity characteristics, and engineering expression strategies. The lack of mature methods for the stable expression, segmented optimization, and functional validation of these structural proteins hinders the effective application of their potential value in areas such as skin repair, barrier reconstruction, and materials development. Therefore, there is an urgent need to establish more precise, efficient, and standardized solutions to address the clinical and application needs arising from skin barrier dysfunction.

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

[0007] The purpose of this invention is to provide a recombinant human K14 keratin and its preparation method. The recombinant human K14 keratin exhibits higher cell proliferation activity and skin repair function, has low production cost, and is suitable for large-scale production.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides a recombinant human K14 keratin, wherein the recombinant human K14 keratin has cell adhesion activity and comprises at least x consecutive amino acid residues as in SEQ ID NO.10; wherein 472 > x ≥ 36.

[0009] Secondly, the present invention provides a recombinant human K14 keratin, wherein the recombinant human K14 keratin has cell adhesion activity, comprising: (A) n The sequence shown; or, by (A) n The sequence shown is composed of; Each A is selected from any one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; Alternatively, each A may be selected from any one of the amino acid sequences in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in which one or more amino acid residues have been substituted, added, or deleted; Alternatively, each A may be selected from a sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity with the amino acid sequence shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3; Where n is an integer between 1 and 16; each A is the same or different, and two adjacent A are directly connected by peptide bonds; or, two adjacent A are connected by more than one amino acid residue.

[0010] In an optional embodiment, the recombinant human K14 keratin comprises or is composed of the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0011] Thirdly, the present invention provides a polynucleotide that encodes recombinant human K14 keratin as described in the foregoing embodiments.

[0012] Fourthly, the present invention provides an expression vector comprising the polynucleotides described in the foregoing embodiments.

[0013] Fifthly, the present invention provides a host cell comprising the expression vector as described in the foregoing embodiments, or expressing recombinant human K14 keratin as described in the foregoing embodiments; In an optional implementation, the host cell is Escherichia coli.

[0014] Sixthly, the present invention provides a method for preparing recombinant human K14 keratin as described in the foregoing embodiments, comprising: The host cells were cultured in a culture medium; and the recombinant human K14 keratin was isolated from the host cells.

[0015] In a seventh aspect, the present invention provides a composition comprising recombinant human K14 keratin as described in the foregoing embodiments.

[0016] Eighthly, the present invention provides an article comprising recombinant human K14 keratin as described in the foregoing embodiments, or a composition as described in the foregoing embodiments; The product is a pharmaceutical composition, medical device, tissue-engineered product, cosmetic or health product.

[0017] In optional embodiments, the recombinant human K14 keratin, polynucleotide, expression vector, host cell, or composition described in the foregoing embodiments are used in the preparation of medical devices, tissue-engineered products, cosmetics, or skin care products.

[0018] The recombinant human K14 keratin provided in this application exhibits higher cell proliferation activity compared to full-length expressed K14 keratin, more effectively promoting cell growth and division, and accelerating tissue repair and regeneration. It possesses excellent skin stratum corneum repair function, significantly reducing transepidermal water loss, enhancing skin's moisturizing ability and barrier function, and fundamentally improving the problem of a weak skin barrier. This recombinant has high expression yield in host cells such as Escherichia coli, and its purification process is simple, reducing production costs and improving production efficiency, making it more suitable for large-scale industrial production. Furthermore, these characteristics endow recombinant human K14 keratin with broad application prospects in pharmaceuticals, cosmetics, and skincare products, especially in tissue engineering materials, skincare serums, and topical formulations, providing strong support for technological advancements and product innovation in related industries. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 This is a representative diagram of plasmid construction using the vector pET32a-K14A as an example in the embodiments of this application; Figure 2 The following are electrophoretic images of the three recombinant keratins expressed in Escherichia coli in the embodiments of this application. The molecular weights of the recombinant proteins K14A3, K14B2, K14C2 and K14, as determined by electrophoresis, are approximately 29 kDa, 34 kDa, 50 kDa and 68 kDa, respectively. Figure 3 This is an electrophoresis image of the K14B2 protein obtained after expression and purification in the embodiments of this application; the molecular weight of the K14B2 protein detected by electrophoresis is approximately 34 kDa; Figure 4 The results show the cell proliferation activity detection results of recombinant keratin and full-length K14 expressed protein in the embodiments of this application. Detailed Implementation

[0021] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0022] This application provides a recombinant human K14 keratin, based on the screening and optimization of the human type 14 keratin K14 sequence. The sequence of human type 14 keratin can be found at https: / / www.ncbi.nlm.nih.gov / protein / AAP35850.1; it is the NCBI reference sequence: AAP35850.1 (SEQ ID NO.10). The specific sequence is as follows: MTTCSRQFTSSSSMKGSCGIGGGIGGGSSRISSVLAGGSCRAPSTYGGGLSVSSSSRFSSGGAYGLGGGYGGGFSSSSSSFGSGFGGGYGGGLGTGLGGGFGGGFAGGDGLLVGS EKVTMQNLNDRLASYLDKVRALEEANADLEVK IRDWYQRQRPAEIKDYSPYFKTIEDLRNKILTATVDNANVLL QIDNARLAADDFRTKYETELNLRMSVEADINGLRR VLDELTLARADLEMQIESLKEELAYLKKNHEEEMNAL RGQVGGDVNVEMDAAPGVDLSRI LNEMRDQYEKMAEKNRK DAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLEETKGRYCMQLAQIQEMI GSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGE DAHLSSSQFSSGSQSSRDVTSSSRQIRTKVMDVHDGKVVSTHEQVLRTKN The bold underlined portion of the above sequence is the amino acid sequence selected in this invention. In this embodiment, extensive research has shown that the selected sequence exhibits high water solubility, high recombinant expression yield, and simple purification process. Furthermore, it demonstrates better cell adhesion and stratum corneum repair effects than commercially available keratin or other sequences in SEQ ID NO. 10, exhibiting a variety of excellent biomaterial properties.

[0023] The recombinant human K14 keratin provided in this application embodiment is not the full-length sequence of SEQ ID NO.10, but has cell adhesion activity and contains at least 36 consecutive amino acid residues as in SEQ ID NO.10, where the full-length sequence is 472. Therefore, the number of consecutive amino acid residues x needs to meet the condition: 472 > x ≥ 36.

[0024] The embodiments of this application are partly based on the following finding: Keratin containing n consecutive amino acid residues as specified in SEQ ID NO. 10 exhibits better biomaterial properties compared to commercially available keratin, as demonstrated by the examples. Those skilled in the art can appropriately select the consecutive amino acid residues constituting the recombinant keratin. For example, the length x of the consecutive amino acid residues can be 36~72, 72~139, etc., such as x=36, 40, 60, 80, 100, 150, 200, 300, 400, or 471, etc.

[0025] In the embodiments of this application, the sequences of several specific amino acid regions were tested: (1) K14A: EKVTMQNLNDRLASYLDKVRALEEANADLEVKIRDW (SEQ ID NO. 1).

[0026] (2) K14B: QIDNARLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLARADLEMQIESLKEELAYLKKNHEEEMNAL (SEQ ID NO.2) (3) K14C: LNEMRDQYEKMAEKNRKDAEEWFFTKTEELNREVATSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLEETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGE (SEQID NO.3) In this article, it can be recombinant human keratin K14A3, which is a K14A sequence repeated three times, including 108 amino acids. The basic repeat unit is: EKVTMQNLNDRLASYLDKVRALEEANADLEVKIRDW (SEQ ID NO.1) is a human keratin type 14 peptide.

[0027] The amino acid sequence of K14A3 is as follows: EKVTMQNLNDRLASYLDKVRALEEANADLEVKIRDWEKVTMQNLNDRLASYLDKVRALEEANADLEVKIRDWEKVTMQNLNDRLASYLDKVRALEEANADLEVKIRDW (SEQ ID NO. 4).

[0028] The DNA sequence of K14A3 is as follows: GAAAAAGTAACAATGCAAAACCTAAATGATCGCTTGGCGAGCTACCTGGACAAGGTTAGAGCGTTGGAAGAGGCCAACGCCGACCTGGAGGTTAAGATCCGCGACTGGGAAAAAGTGACGATGCAGAATCTGAATGATCGTCTGGCTTCCTACCTGGATAAAGT ACGTGCGTTAGAGGAAGCGAACGCAGATCTTGAGGTGAAAATCCGTGATTGGGAAAAAGTCACCATGCAAAACCTGAACGACCGTCTGGCTAGCTATCTGGATAAGGTGCGCGCTGGAGGAAGCGAATGCAGACTTGGAGGTTAAGATTCGTGACTGG (SEQ ID NO.5).

[0029] In this article, it can be recombinant human keratin K14B2, which is a repeat of the K14B sequence twice, including 144 amino acids. The basic repeating unit is: QIDNARLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLARADLEMQIESLKEELAYLKKNHEEEMNAL (SEQ ID NO.2), which is a human keratin type 14 peptide.

[0030] The amino acid sequence of K14B2 is as follows: QIDNARLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLARADLEMQIESLKEELAYLKKNHEEEMNALQIDNARLAADDFRTKYETELNLRMSVEADINGLRRVLDELTLARADLEMQIESLKEELAYLKKNHEEEMNAL (SEQ ID NO.6) The DNA sequence of K14B2 is as follows: CAAATAGACAATGCTAGGCTAGCGGCAGATGACTTCCGCACCAAGTATTGAAACCGAATTGAATCTGCGCATGTCCGTGGAAGCTGATATTAACGGTTTGAGACGTGTTCTTGATGAGCTGACCCTGGCGCGTGCTGACCTGGAAATGCAAATCGAAAGCCTGAAAGAGGAGTTGGCGTATCTGAAGAAAAACCATGAAGAGGAGATGAACGCCCTGCA GATTGATAATGCGCGTCTGGCGGCAGATGATTTTCGTACCAAATACGAAACTGAGTTGAATCTGCGTATGAGCGTTGAAGCGGACATCAACGGCCTGCGTCGCGTGCTGGACGAATTGACGCTCGCTCGTGCAGACCTGGAAATGCAGATCGAGTCTCTGAAAGAGGAGTTAGCCTACTTAAAGAAGAACCACGAAGAGGAGATGAACGCGCTG (SEQ ID NO.7) In this article, it can be recombinant human keratin K14C2, which is a repeat of the K14C sequence twice, including 278 amino acids, the basic repeat unit: LNEMRDQYEKMAEKNRKDAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLEETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGE (SEQ ID NO.3) is a human keratin type 14 peptide.

[0031] The amino acid sequence of K14C2 is as follows: LNEMRDQYEKMAEKNRKDAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLEETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGELN EMRDQYEKMAEKNRKDAEEWFFTKTEELNREVATNSELVQSGKSEISELRRTMQNLEIELQSQLSMKASLENSLEETKGRYCMQLAQIQEMIGSVEEQLAQLRCEMEQQNQEYKILLDVKTRLEQEIATYRRLLEGE (SEQ ID NO.8).

[0032] The DNA sequence of K14C2 is as follows: (SEQ ID NO.9).

[0033] The recombinant human K14 keratin provided in this embodiment of the application, which has cell adhesion activity, may include: (A) n The sequence shown; or, by (A) nThe sequence shown is composed of; each A can satisfy any of the following conditions: (1) Each A is selected from any one of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3; (2) Each A is selected from any one of the amino acid sequences in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in which one or more amino acid residues have been substituted, added or deleted; (3) Each A is selected from a sequence having 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or 97% sequence identity with any of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3; Where n is an integer between 1 and 16. Each A is the same or different, and two adjacent A are directly connected by a peptide bond; or two adjacent A are connected by one or more amino acid residues. For example, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, etc.

[0034] Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared.

[0035] In the above-mentioned modification methods of keratin amino acid sequences, amino acid addition refers to adding an amino acid to the C-terminus or N-terminus of any one of the three keratin amino acid sequences. The added amino acid must have keratin characteristics and cell proliferation-promoting activity.

[0036] The aforementioned amino acid substitution refers to replacing a certain amino acid residue with another amino acid residue at any position in the three keratin amino acid sequences; the substituted residue must possess keratin characteristics and cell proliferation-promoting activity.

[0037] The above-mentioned amino acid insertion refers to inserting an amino acid residue at an appropriate position in any of the three keratin amino acid sequences. The inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other. The inserted amino acid residues must have keratin characteristics and cell proliferation-promoting activity.

[0038] The aforementioned amino acid deletion refers to the deletion of 1, 2, or 3 or more amino acids from any of the three types of keratin amino acid sequences; the deleted amino acid sequence must still possess keratin characteristics and cell proliferation-promoting activity.

[0039] All four modification methods mentioned above are based on the premise of having keratin characteristics and cell proliferation-promoting activity.

[0040] In the embodiments of this application, substitution can be conservative amino acid substitution, which means that compared with the amino acid sequence of any of the three keratin amino acid sequences, three, more preferably two or one amino acid is replaced by amino acids with similar or related properties to form a peptide.

[0041] In some embodiments, the recombinant human K14 keratin comprises or is composed of the amino acid sequences shown in or described above as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0042] This application provides a polynucleotide that encodes recombinant human K14 keratin as described in the foregoing embodiments.

[0043] This application provides an expression vector comprising the polynucleotides described in the foregoing embodiments.

[0044] This application provides a host cell that includes the expression vector as described in the foregoing embodiments, or that expresses the recombinant human K14 keratin as described in the foregoing embodiments.

[0045] In some embodiments, the host cell is Escherichia coli.

[0046] This application provides a method for preparing recombinant human K14 keratin as described in the foregoing embodiments, comprising: Step S1: Culture host cells in culture medium; Step S2, and based on the host cell, the recombinant human K14 keratin is isolated.

[0047] It should be noted that the above-mentioned recombinant human keratin can be prepared using conventional methods in the art. For example, it can be prepared by: (1) constructing a genetically engineered *Escherichia coli* strain; (2) fermenting and culturing the genetically engineered *E. coli* strain; (3) inducing and expressing the recombinant keratin; and (4) purifying the recombinant keratin. It should be understood that those skilled in the art can replace the above-mentioned *E. coli* strain with other expression strains as the host cell. The host cell can be a eukaryotic cell, such as fungi and yeast, or a prokaryotic cell, such as Enterobacteriaceae bacteria, such as *Escherichia coli*.

[0048] This application provides a composition comprising recombinant human K14 keratin as described in the foregoing embodiments.

[0049] This application provides an article comprising recombinant human K14 keratin as described in the foregoing embodiments, or a composition as described in the foregoing embodiments.

[0050] The core ingredient of the product is recombinant human K14 keratin. This has a specific amino acid sequence and can be obtained through corresponding preparation methods. The product can also be a composition containing this ingredient, such as pharmaceutical compositions, cosmetic formulations, etc.

[0051] In some embodiments, the article is a pharmaceutical composition, a medical device, a tissue-engineered product, a cosmetic, or a health product.

[0052] In some embodiments, the pharmaceutical composition is a topical preparation; In some embodiments, the topical formulation includes a smear formulation or a transdermal delivery formulation; In some embodiments, the topical formulation includes at least one of a topical ointment, a hydrogel formulation, and a topical wetting formulation; In some embodiments, the transdermal drug delivery formulation includes at least one of microneedle formulations, patches, and transdermal gel formulations.

[0053] In some embodiments, the recombinant human K14 keratin, polynucleotide, expression vector, host cell, or composition described in the foregoing embodiments are used in the preparation of medical devices, tissue-engineered products, cosmetics, or skin care products.

[0054] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0055] Unless otherwise specified, all biological materials, reagents, and devices used in the following examples are available from conventional commercial sources or obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0056] Example 1: Gene Synthesis of Recombinant Keratin Experimental methods: 1. Gene Analysis: The amino acid sequence of natural human hair keratin K14 (Genebank: AAP35850.1) was selected for analysis of its hydrophilicity, isoelectric point, and other physicochemical properties. The helical region and hydrophilic conserved structural regions in human keratin K14 were selected for recombination. The recombinant sequences were codon-optimized against the *E. coli* codons. The resulting new genes are K14A3, K14B2, and K14C2, as described in this application, with amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.6, or SEQ ID NO.8.

[0057] 2. Gene synthesis: The full lengths of the K14A3, K14B2, K14C2 and K14 genes are 324 bp, 432 bp, 834 bp and 1419 bp, respectively. The optimized sequences based on the codons are SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.9, respectively.

[0058] The optimized sequences were used for gene synthesis. The gene fragments were provided by Universal Gene Biotechnology Co., Ltd. The synthesized K14A3, K14B2, K14C2 and K14 gene fragments were inserted into the expression vector PET32a through NcoI and XhoI, respectively. The vectors were then transformed into E. coli TOP10 for preservation.

[0059] The vector maps constructed using the K14A3 gene as an example are shown below. Figure 1 .

[0060] Example 2: Construction and Induced Expression of Recombinant Keratin Experimental methods: (1) Construction of genetically engineered Escherichia coli: Plasmid extraction: Referring to Example 1, different bacterial strains containing the target gene were streaked onto LB+A or LB+K plates containing the corresponding antibiotic resistance and incubated overnight at 37°C. Subsequently, single colonies were selected from the overnight culture plates and inoculated into 10 mL of LB+A or LB+K liquid medium, and incubated overnight at 37°C. An appropriate amount of the overnight culture was centrifuged at 5000 rpm for 5 min, and the supernatant was removed to collect the bacterial pellet. Plasmid extraction was performed using the Tiangen Plasmid Extraction Kit. The concentration of the extracted plasmids was determined by NanoDrop, and the plasmids were either directly used for transformation of the expression host or stored at -20°C as needed.

[0061] Strain construction: Add 2 μL of the target gene plasmid to 100 μL of competent *E. coli* cells and incubate on ice for 15-30 min. Heat shock the mixture in a 42°C water bath for 45 s, incubate on ice for 2-3 min, add 500 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 190 rpm for 20 min. Spread 100 μL of the bacterial culture evenly onto LB+K or LB+Amp plates. Incubate overnight at 37°C until clearly visible colonies appear.

[0062] Induction of target protein expression: Single clones were picked from overnight culture plates and cultured overnight in 10 mL LB+A or LB+K liquid medium. Then, they were transferred to TB medium at a ratio of 1% for expansion culture. The culture was carried out at 37℃ and 190 rpm until the OD600 of the bacterial culture reached 0.5 to 0.8. IPTG was then added to a final concentration of 0.1 to 0.5 mM for induction expression. Induction conditions were 16 to 26℃ and 190 rpm for 16-20 h. The bacterial cells were collected by centrifugation and washed three times repeatedly with phosphate buffer. The washed bacterial cells could be frozen at -20℃ or directly lysed using an autoclave, followed by centrifugation at 10,000 rpm for 30 min. The supernatant was then collected and stored.

[0063] SDS-PAGE detection of the target protein: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, and boil in 100℃ water for 10 min. Then add 20 μL to each well of the SDS-PAGE protein gel, run at 80 V for 1 h, and then at 120 V until the bands are completely separated. Stain with Coomassie Brilliant Blue solution (0.1% Coomassie Brilliant Blue R). Stain the protein with 250, 25% isopropanol, and 10% glacial acetic acid for 30 min, then decolorize overnight with protein decolorizing solution (10% acetic acid and 5% ethanol).

[0064] Experimental results: See results Figure 2 The recombinant proteins K14A3, K14B2, K14C2, and K14 had molecular weights of approximately 29 kDa, 34 kDa, 50 kDa, and 68 kDa, respectively, as determined by electrophoresis.

[0065] Example 3: Purification of recombinant keratin Experimental methods: (1) Crude protein treatment: The frozen bacterial cells washed with phosphate buffer were weighed and resuspended in 8 times the volume of lysis buffer (50 mM PB, 10 mM imidazole, 0.5 M NaCl, pH 7.0). The cells were then homogenized using a high-pressure homogenizer at low temperature. The homogenized mixture was centrifuged at 10,000 rpm for 25 min at 4°C. The precipitate was discarded, and the supernatant was collected and its volume was recorded.

[0066] (2) Ni-NTA nickel column purification of proteins: Wash the column for 5 column volumes with (50 mM PB, 0.5 M NaCl, pH 7.0) to equilibrate the nickel column. Load the pretreated protein onto the column. Wash the column for 3-5 column volumes with a low concentration of imidazole (10-50 mM) buffer to remove impurities, and then perform gradient elution with 50 mM to 500 mM imidazole. Collect protein samples eluted with different concentrations of imidazole and determine the purity by SDS-PAGE. After collection, wash the nickel column with 20% ethanol, fill it with ethanol, and store it. For nickel columns that have been reused multiple times, nickel column regeneration is required. The regeneration steps are as follows: Wash the column for 5 column volumes with ddH2O, wash the column for 5 column volumes with EDTA solution, wash the column for 5 column volumes with NaOH solution, fill the affinity column with NiSO4 solution, and wash the column for 5 column volumes. Take different received protein purification solutions and perform SDS-PAGE to determine the purity and size of the target protein.

[0067] Experimental results: See results Figure 3 The molecular weight of K14B2, as determined by protein electrophoresis, is approximately 29 kDa.

[0068] Example 4: Cell proliferation activity test of recombinant keratin Experimental methods: (1) L929 cell culture: L929 cells were placed in DMEM medium containing 10% fetal bovine serum and antibiotics and cultured in a CO2 incubator at 5% concentration, 95% relative humidity, and 37℃. The medium was changed every two days, and the cells were passaged after they were basically confluent on the fifth day. First, the old medium was removed, and the cells were washed twice with PBS solution. Then, 0.5 mL of 0.25% trypsin was added for digestion. Under an inverted microscope, after the cells shrank and became round, 10 mL of DMEM medium was added to stop the digestion. The cells were gently pipetted to suspend them, centrifuged at 1000 r / min for 5 min, the supernatant was removed, an appropriate amount of medium containing serum was added, and the cells were gently pipetted again to make a cell suspension. 50 μL was taken for counting. For L929 cells in the logarithmic growth phase, the upper medium was removed, the cells were washed twice with PBS solution, and digested into a single-cell suspension with 0.5 mL of 5% DMEM-0.25% trypsin. The cells were washed twice with PBS solution again, and the suspended cells were collected directly.

[0069] (2) MTT assay: 96-well plates were selected, with 2 × 10⁴ cells seeded in each well. Experimental groups, a blank control group (containing only PBS solution), and a calibration / zeroing group (containing no cells) were set up. Two replicate wells were set up for each group. After 24 h, microscopic observation revealed that most cells had adhered and spread. At this point, unattached cells and liquid were removed from the wells. The concentration in the experimental group was adjusted to 0.5 mg / mL, and 20 μL was added. The blank control group received 20 μL of PBS buffer, and the calibration / zeroing group received only DMEM medium. When adding the sample, ensure the pipette tip was close to the well wall and inject slowly. After each well, gently pipette to mix. After standing for 30 min, the plate was placed in a CO₂ incubator. Cell proliferation was observed after 72 h of culture. The absorbance (OD) value was measured using the MTT assay. 20 μL of MTT solution was added to each well of the 96-well plate, and the plate was cultured for another 4 h to complete the colorimetric reaction. Afterwards, terminate the culture, aspirate the culture medium from the wells or cover the culture plate with a layer of filter paper, quickly invert the culture plate, add 150 μL DMSO to each well, shake for 10 min to fully dissolve the formazan, and finally use an ELISA reader to measure the absorbance of each well at an A490 nm wavelength.

[0070] Experimental results: See results Figure 4 Compared with blank and full-length expressed K14, recombinant keratinogens K14A3, K14B2, and K14C2 exhibit better effects in promoting the proliferation and differentiation of L929 cells and enhancing cell activity. The human recombinant keratinogens of this invention have the function of promoting cell proliferation and can be used to prepare biomaterials or drugs that promote cell proliferation.

[0071] Example 5: Transdermal Moisture Loss Test Experimental methods: (1) Preparation of keratin essence: K14 keratin and recombinant K14A3, K14B2 and K14C2 were respectively combined with glycerin, propylene glycol, xanthan gum and other ingredients to prepare keratin essence with a content of 0.5% and recombinant keratin essence, and were divided into 4 groups.

[0072] (2) Recruit volunteer subjects according to requirements and obtain written informed consent. Before enrollment, ask subjects a series of questions about their medical history and health status according to the inclusion and exclusion criteria. At the same time, conduct conformity assessment and skin color test screening for skin conditions such as pigmentation at the test site, and record the results. For qualified subjects, conduct pre-product use skin baseline value assessment and testing, including visual assessment, instrument testing, and standard image shooting, and record the results. The same assessment and testing are conducted again 2 weeks and 4 weeks after product use. On the day the trial begins, distribute test samples and control products to the subjects. The test sites are randomly divided into left and right sides and two groups of products are used. Use begins after the researcher's guidance. Follow up weekly to confirm the usage status. After 4 weeks of continuous use, weigh and record the usage, and collect the samples. 4 weeks of continuous use (allowing for a 2-day error). Inner arm skin. The experimental schedule is as follows.

[0073] Table 1. Experiment Schedule

[0074] Experimental results: Table 2. Transdermal water loss experiment (K14A3 serum)

[0075] Table 3. Transdermal water loss experiment (K14B2 serum)

[0076] Table 4. Transdermal water loss experiment (K14C2 serum)

[0077] Table 5. Transdermal moisture loss experiment (K14 serum)

[0078] Table 6. Transdermal water loss experiment (control)

[0079] In this embodiment, the effect of recombinant human K14 keratin serum on skin barrier function was evaluated using a transepidermal water loss (TEWL) assay. Four keratin serums (K14A3, K14B2, K14C2, and K14) were used in the experiment, along with a control group for comparison. The results showed that subjects using the three recombinant keratin serums (K14A3, K14B2, and K14C2) experienced a significant reduction in transepidermal water loss after two weeks, indicating that these serums effectively enhance skin's moisturizing ability and barrier function.

[0080] Specifically, the average TEWL value of the K14A3 serum group decreased from 9.15 to 5.78, the average TEWL value of the K14B2 serum group decreased from 8.51 to 6.2, and the average TEWL value of the K14C2 serum group decreased from 9.03 to 6.1, all showing significant improvement (P values ​​less than 0.05).

[0081] In contrast, although the control group using K14 protein serum also showed some improvement, its average TEWL value decreased from 8.77 to 6.97, which was a relatively small improvement. Moreover, the P value was 0.024, which was less significant than that of the recombinant keratin serum group.

[0082] The results are shown in Tables 3-6. Compared with the control serum containing K14 protein, the three serums containing recombinant keratin significantly reduced transepidermal water loss after two weeks of use, contributing to the repair of the skin's stratum corneum barrier. This finding indicates that recombinant human K14 keratin has significant application potential in the field of skin care, effectively improving skin barrier function and providing strong scientific evidence for the development of novel skin care products.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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 recombinant human K14 keratin, characterized in that, The recombinant human K14 keratin has cell proliferation-promoting activity; the recombinant human K14 keratin consists of the amino acid sequence shown in SEQ ID NO.

6.

2. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant human K14 keratin as described in claim 1.

3. An expression carrier, characterized in that, Includes the polynucleotides as described in claim 2.

4. A host cell, characterized in that, Includes the expression vector as described in claim 3, or expresses the recombinant human K14 keratin as described in claim 1; The host cell is Escherichia coli.

5. A method for preparing recombinant human K14 keratin as described in claim 1, characterized in that, include: The host cells were cultured in a culture medium; and the recombinant human K14 keratin was isolated from the host cells.

6. A composition, characterized in that, Includes the recombinant human K14 keratin as described in claim 1.

7. An article characterized in that, Includes the recombinant human K14 keratin as described in claim 1, or the composition as described in claim 6; The product is a pharmaceutical composition or a medical device.

8. The article of claim 7, characterized in that, The product in question is a tissue-engineered product.

9. The use of the recombinant human K14 keratin as described in claim 1 in the preparation of medical devices.

10. The application as described in claim 9, characterized in that, The medical device in question is a tissue-engineered product.

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