Recombinant protein pPIC9K-17, preparation method and application

Abstract

The application discloses a recombinant protein pPIC9K-17, a preparation method and application, relates to the technical field of gene recombination, and provides the recombinant protein pPIC9K-17, a core structural unit does not completely follow the strict (Gly-X-Y) n triple helix repeat mode of traditional collagen, the sequence of the repeat unit contains a "GAIQ" sequence and a segment containing a collagen characteristic sequence, has a certain rigid structure basis and contains a specific growth factor receptor binding or signal activation domain. Direct concatenation of multiple such units can theoretically achieve multivalent binding, enhance the affinity and signal activation efficiency with the cell surface receptor, thereby overcoming the biological function limitation of a single component (pure collagen or pure growth factor) or a simple repeat structure. The recombinant protein can not only effectively promote the proliferation and migration of skin fibroblasts, accelerate collagen synthesis, but also has clear efficacy in promoting hair regeneration (hair growth), and can be used for preparing skin repair and / or hair regeneration promoting products.

Description

Technical Field

[0001] This invention relates to the field of gene recombination technology, and in particular to a recombinant protein pPIC9K-17, its preparation method, and its applications. Background Technology

[0002] Skin repair and hair regeneration are important applications in tissue engineering and regenerative medicine. Collagen, as a major structural protein of the extracellular matrix, plays a crucial role in maintaining skin homeostasis and promoting wound healing. In recent years, the production of humanized collagen using recombinant technology has become a research hotspot. For example, patent CN120574312A discloses a highly stable recombinant type XVII collagen, which is constructed by repeating specific collagen-derived peptide segments (2-5 times) and effectively expressed in the Pichia pastoris system. This protein can self-assemble into a triple helix structure, possesses excellent thermal stability, and exhibits activity in promoting cell adhesion, proliferation, and migration, showing potential for application in skin repair. Another patent, CN119798413A, obtained recombinant proteins with expression levels exceeding 10 g / L in a yeast system by screening and repeating different collagen short peptide sequences (e.g., repeating 6 times or more), and confirmed that its cell adhesion activity is superior to that of natural human collagen. These technological advancements signify significant progress in the large-scale production and realization of basic biological functions of recombinant collagen.

[0003] In the field of hair regeneration (hair growth), common interventions currently include the use of chemically synthesized drugs (such as minoxidil and finasteride), single growth factors (such as VEGF, FGF, and EGF), and plant extracts. However, chemical drugs are often accompanied by significant side effects and are prone to relapse after discontinuation; single growth factors, although highly bioactive, have limitations such as poor stability, short half-life in vivo, and high production costs; and plant extracts generally suffer from slow onset of action, complex composition, and unclear efficacy.

[0004] The normal hair follicle cycle depends on the precise and stable interaction between dermal papilla cells and the surrounding matrix, with cell adhesion and signal transduction being the core elements initiating and maintaining hair growth. Therefore, constructing a microenvironment that mimics the natural extracellular matrix and provides physical and biological signal support for hair follicles is an important direction for the development of hair growth technology. Recombinant collagen, especially types with strong cell adhesion activity, can theoretically provide an ideal anchoring interface for dermal papilla cells, stabilizing hair follicle structure. However, existing recombinant collagen mainly considers expression levels, structural stability, and basic adhesion, resulting in limited functionality. There is an urgent need to develop novel recombinant proteins capable of achieving both skin repair and hair follicle regeneration. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a novel protein that can achieve skin repair and hair follicle regeneration.

[0006] To address the above problems, the present invention proposes the following technical solution: A recombinant protein pPIC9K-17 has an amino acid sequence comprising an N-terminal KR residue and m repeating units downstream thereof; wherein the sequence of each repeating unit is as shown in SEQ ID NO: 1; m is an integer from 2 to 8; SEQ ID NO: 1: GAIQGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ.

[0007] Preferably, the C-terminus of the recombinant protein further comprises a membrane-penetrating peptide sequence, as shown in SEQ ID NO: 2, SEQ ID NO: 2: YGRKKRRQRRR.

[0008] Preferably, m is 2, 3, 4 or 5; and each repeating unit is directly connected to the other.

[0009] The present invention also provides a nucleic acid molecule characterized in that it encodes the recombinant protein pPIC9K-17 as described in any one of claims 1-3.

[0010] Preferably, the nucleic acid molecule has a codon-optimized nucleotide sequence suitable for expression in Pichia pastoris, Escherichia coli, Bacillus subtilis, or Chinese hamster ovary cells (CHO-K1).

[0011] The present invention also provides a recombinant expression vector comprising the aforementioned nucleic acid molecule.

[0012] The present invention also provides a recombinant host cell comprising the recombinant expression vector described above, or having the nucleic acid molecule described above integrated into its genome; the host cell is Pichia pastoris, Escherichia coli, Bacillus subtilis, or CHO cells.

[0013] The present invention also provides a method for producing the recombinant protein pPIC9K-17, comprising the following steps: (a) Constructing a recombinant expression vector containing a nucleic acid sequence encoding the recombinant protein; (b) The recombinant expression vector is introduced into host cells to obtain recombinant host cells; (c) The recombinant host cells are cultured under conditions suitable for expression, thereby expressing and obtaining the recombinant protein.

[0014] Preferably, the host cell is Pichia pastoris, Escherichia coli, Bacillus subtilis, or Chinese hamster ovary cells.

[0015] Preferably, when the host cell is Pichia pastoris, the recombinant expression vector contains an AOX1 promoter and an α-factor signal peptide sequence; preferably, the Pichia pastoris strain is GS115.

[0016] Preferably, when the host cell is Escherichia coli, the recombinant expression vector contains the T7 promoter; preferably, the Escherichia coli is BL21(DE3) strain.

[0017] The present invention also provides the use of the recombinant protein pPIC9K-17, the nucleic acid molecule, the recombinant expression vector, the recombinant host cell, or the recombinant protein pPIC9K-17 prepared by the method in the preparation of products for skin repair and / or hair regeneration.

[0018] Compared with the prior art, the technical effects achieved by the present invention include: The recombinant protein pPIC9K-17 provided by this invention does not strictly follow the (Gly-XY)n triple helix repeat pattern of traditional collagen in its core structural unit. The repeating unit sequence includes a "GAIQ" sequence and a chimeric peptide segment containing collagen characteristic sequences. This chimeric peptide segment contains multiple charged and polar residues. The direct tandem arrangement of multiple such units (e.g., 2-8 repetitions) can spontaneously assemble into a rigid structural backbone dominated by triple helices. The repetition of charged and polar residues significantly enhances the affinity and binding specificity with cell surface receptors (such as integrins, growth factor receptors, etc.), thereby more effectively activating downstream signaling pathways related to cell proliferation, migration, adhesion, and hair follicle regeneration.

[0019] Based on the structural design of the aforementioned repeating units, the recombinant protein of this invention not only retains the cell adhesion and support capabilities derived from the collagen domain in terms of biological activity, but also gains the ability to directly regulate cell behavior due to its integrated functional sequence. Experiments show that it can effectively promote the proliferation and migration of skin fibroblasts and accelerate collagen synthesis, thus making it suitable for skin wound healing and anti-aging repair; at the same time, it also shows a clear effect in promoting hair regeneration (hair growth). Therefore, the recombinant protein of this invention can simultaneously address the complex physiological needs of skin repair and hair regeneration.

[0020] In terms of expression and production, despite its complex sequence, through targeted codon optimization (e.g., targeting preferences for Pichia pastoris, Escherichia coli, etc.), this protein can achieve soluble secretory expression in highly efficient expression systems such as Pichia pastoris, with considerable yield and good stability, overcoming the difficulties of soluble expression or easy degradation of full-length natural proteins or some complex recombinant proteins. This invention fundamentally solves the technical problem of existing recombinant collagen proteins having a single function (focusing only on adhesion and support), providing a novel protein that is structurally stable, has diverse activities, and is easy to scale up for production.

[0021] Furthermore, this invention utilizes a Pichia pastoris expression system (such as using the pPIC9K vector, AOX1 promoter, and α-factor signal peptide) to express recombinant proteins in *Escherichia coli*, *Bacillus*, and CHO-K1 cells. These proteins exhibit correct folding and post-translational modifications, maintaining activity for over 7 days at room temperature. This invention successfully achieves efficient and stable secretory expression of this uniquely structured recombinant protein, laying a reliable technological foundation for its large-scale application in cosmetics, pharmaceuticals, and medical devices. Detailed Implementation

[0022] The technical solutions in the embodiments will be clearly and completely described below. Obviously, the embodiments described below are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0023] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0024] It should also be understood that the terminology used in this specification of embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to limit the embodiments of the invention. As used in this specification of embodiments of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0025] Example 1: Gene design and vector construction of recombinant protein pPIC9K-17 Gene design: The gene sequence of the recombinant protein pPIC9K-17 in this embodiment contains an N-terminal KR residue and four repeat units located downstream therefrom; wherein, the sequence of each repeat unit is shown in SEQ ID NO: 1; SEQ ID NO: 1: GAIQGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGDQ.

[0026] The constructed gene sequences were commissioned to a professional company for whole-genome synthesis. To improve expression efficiency in different hosts, codon optimization was performed for the following four expression systems: a) Pichia pastoris; b) Escherichia coli BL21(DE3) strain; c) Bacillus subtilis; d) Chinese hamster ovary cells (CHO-K1). The optimization principle was to adjust the GC content of the gene to 40-45% according to the codon usage frequency table for each host, and to avoid the formation of unfavorable mRNA secondary structures. Double stop codons (TAA TAA) were added to the 3' end of all optimized sequences. To facilitate cloning, EcoRI and XhoI restriction sites were introduced at the 5' and 3' ends, respectively. For the Pichia pastoris and CHO-K1 systems, the coding sequence for the Saccharomyces cerevisiae α-factor signal peptide was also added after the start codon ATG to guide protein secretion.

[0027] It should be noted that this embodiment uses a repeat unit number m=4 as an example for illustration; however, the scope of protection of this invention is not limited to this specific number of repeats. Based on the same sequence design principle (i.e., each repeat unit is SEQ ID NO: 1), those skilled in the art can easily construct similar recombinant proteins with repeat unit numbers m of 2, 3, 5, 6, 7, or 8, or even more. These variants with different repeat numbers are also covered within the scope of this invention, and their construction methods (including codon optimization for different host systems, vector assembly, etc.) are substantially the same as the process described in this embodiment, requiring only the design and synthesis of coding sequences with the corresponding number of repeats during the gene synthesis stage. The activity data of the protein with m=4 in subsequent embodiments are merely examples to demonstrate the core concept of this invention (i.e., the repeat unit structure possesses dual biological activity). Protein variants with different repeat numbers are expected to exhibit different degrees of activity intensity or stability, which are all within the reasonable scope of the technical solution of this invention.

[0028] Vector construction (taking Pichia pastoris system as an example): a. Enzyme digestion: 5 μg of pPIC9K expression vector (purchased from Invitrogen) was double-digested with restriction endonucleases EcoR I and Xho I (purchased from Thermo Fisher) at 37°C for 3 hours. The approximately 9.3 kb linearized vector fragment was recovered by 1% agarose gel electrophoresis.

[0029] b. Ligation: The recovered vector fragment was mixed with the pPIC9K-17 gene fragment, which had been digested and purified with the same EcoRI and XhoI enzymes and optimized for Pichia pastoris, at a molar ratio of 1:3. T4 DNA ligase (purchased from NEB) was added, and ligation was carried out overnight at 16°C.

[0030] c. Transformation and Identification: The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing 100 μg / mL ampicillin. The plates were incubated upside down at 37°C for 16 hours. Single colonies were picked and inoculated into LB liquid medium (containing ampicillin) for culture, and the plasmid was extracted. The correct recombinant plasmid was obtained by EcoRI / XhoI double enzyme digestion and Sanger sequencing verification, and named pPIC9K-17.

[0031] (Note: For the construction of vectors for Escherichia coli, Bacillus subtilis, and the CHO-K1 system, corresponding commercial expression vectors, such as the pET series, pHT series, or pcDNA3.1, are used. The cloning strategy is similar, that is, the optimized gene fragment is inserted into the multiple cloning site of the vector through the corresponding restriction enzyme sites.) Example 2: Expression and purification in Pichia pastoris GS115 Yeast transformation and screening: a. Linearization: Take 20 μg of the pPIC9K-17 plasmid constructed in Example 1 and linearize it completely at 37℃ using Sac I restriction endonuclease.

[0032] b. Electroporation: Mix 10 μg of linearized DNA with 80 μL of pre-chilled Pichia pastoris GS115 competent cells (see Invitrogen manual for preparation method) and transfer the mixture into a pre-chilled 0.2 cm electroporation cuvette. Electroporate using a Bio-Rad electroporator at 1.5 kV, 200 Ω resistance, and 25 μF capacitance.

[0033] c. Screening: Immediately after electroporation, add 1 mL of pre-cooled 1 M sorbitol solution, mix well, and spread onto MD (Minimal Dextrose) plates. Incubate at 28°C for 3-5 days until single colonies appear. Pick His+ transformants and sequentially spot them onto YPD plates containing G418 at concentrations of 0.25, 0.5, 1.0, 2.0, and 4.0 mg / mL, and incubate at 28°C for 2-3 days. Select colonies that can grow normally on plates with high concentrations of G418 (such as 2.0 or 4.0 mg / mL) as candidate engineered strains for multi-copy integration.

[0034] Fermentation expression: a. Seed culture: Select a single clone of high-copy engineered bacteria, inoculate it into 25 mL of BMGY medium, and culture at 30℃ and 250 rpm for about 20 hours until the OD600 reaches 2-6.

[0035] b. Induction of expression: The bacterial cells were collected by centrifugation and resuspended in 200 mL of BMMY medium to achieve an initial OD600 of approximately 1.0. Expression was induced at 28°C and 250 rpm. Every 24 hours, 100% methanol was added to the medium until a final concentration of 0.5% (v / v) was reached to maintain induction. Induction lasted for a total of 96 hours.

[0036] Protein purification: a. Harvesting: After fermentation, centrifuge at 12000 g for 20 minutes at 4℃ and collect the supernatant.

[0037] b. Concentration and Purification: The supernatant was filtered through a 0.45 μm filter membrane and then concentrated using an ultrafiltration tube with a molecular weight cutoff of 10 kDa. The concentrated sample was equilibrated with Binding Buffer (20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, pH 7.4) and loaded onto a pre-equilibrated Ni-NTA affinity chromatography column. Gradient elution was performed using wash buffer and elution buffer containing 40 mM and 250 mM imidazole, respectively, and the 250 mM imidazole elution peak was collected.

[0038] c. Desalting and Storage: The eluent was replaced with PBS buffer using a PD-10 desalting column. Purity was analyzed by SDS-PAGE, and protein concentration was determined using the BCA method. After aliquoting, the protein was stored at -80°C. This protein was named pPIC9K-17_Y.

[0039] Example 3: Expression and purification in Escherichia coli BL21(DE3) Construction and transformation: The recombinant plasmid (construction method similar to Example 1) that was optimized for the codon of Escherichia coli BL21(DE3) and cloned into the pET-28a(+) vector was transformed into E. coli BL21(DE3) chemical competent cells to obtain engineered bacteria.

[0040] Expression: Single colonies were picked and inoculated into LB liquid medium (containing kanamycin) and incubated overnight at 37°C and 220 rpm. The colonies were then transferred 1:100 to fresh LB medium and incubated at 37°C until the OD600 reached approximately 0.6. IPTG was added to a final concentration of 0.5 mM, and expression was induced at 25°C and 180 rpm for 16 hours.

[0041] Purification: Since this fusion protein mainly forms inclusion bodies in *E. coli*, bacterial cells were collected by centrifugation, sonicated, and the inclusion body precipitate was collected by centrifugation. The precipitate was washed with washing buffer (2 M urea, 20 mM Tris-HCl, pH 8.0) and then dissolved in dissolution buffer (8 M urea, 20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0). The solution was filtered through a 0.45 μm filter and subjected to Ni-NTA affinity chromatography (same procedure as in Example 2). The eluent containing the target protein was dialyzed at 4°C in a gradient of refolding buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, pH 8.0), gradually decreasing the urea concentration to 0 M. The precipitate was removed by centrifugation, and the supernatant was the refolded protein, named pPIC9K-17_E.

[0042] Example 4: Expression and purification in Chinese hamster ovary cells (CHO-K1) Construction and transfection: The recombinant plasmid, which was codon-optimized for CHO cells and cloned into the expression vector pcDNA3.1(+) and contained its own signal peptide coding sequence, was transfected into CHO-K1 cells in the logarithmic growth phase using a liposome transfection reagent (such as Lipofectamine 3000).

[0043] Screening and Expression: Forty-eight hours after transfection, cells were selected under pressure using medium containing an appropriate concentration of G418 to obtain stably transfected cell pools. Single-clonal cell lines were obtained using the limiting dilution method. High-expression clones were screened and cultured in shake flasks or bioreactors using CD-CHO medium with a defined chemical composition. After cells grew to a suitable density, or after expression was induced by a specific method (such as using a tetracycline induction system), the cell culture supernatant was collected.

[0044] Purification: The culture supernatant was centrifuged and filtered through a 0.45 μm filter, and then purified by affinity chromatography according to the protein characteristics. The purification procedure was similar to that in Example 2. The obtained protein was named pPIC9K-17_C.

[0045] Example 5: Expression and purification of recombinant protein pPIC9K-17 containing membrane-penetrating peptide in Pichia pastoris GS115 The recombinant protein pPIC9K-17 constructed in this embodiment has a nucleotide sequence encoding the membrane-penetrating peptide SEQ ID NO: 2: YGRKKRRQRRR inserted before the stop codon of the pPIC9K-17 gene (optimized for Pichia pastoris) used in Example 2, followed by a double stop codon. Vector construction was performed as in Example 1, and yeast transformation, screening, and fermentation expression were the same as in Example 2. The finally obtained purified protein was named pPIC9K-17YGR.

[0046] Example 6: Validation of the bioefficacy of recombinant protein pPIC9K-17 To verify the dual efficacy of the proteins of this invention, the proteins (pPIC9K-17_Y, pPIC9K-17_E, pPIC9K-17_C, pPIC9K-17_YGR) expressed and purified in Examples 2, 3, 4, and 5 were subjected to the following tests.

[0047] (1) Skin repair activity test: a. Cell proliferation assay: Human skin fibroblasts (HSF) were used. Cells were seeded at a density of 5 × 10^3 cells per well in 96-well plates and cultured for 24 hours to allow cell adhesion. The medium was then replaced with serum-free medium containing different concentrations (0.01, 0.03, 0.05 μM) of the target protein, with 6 replicates for each concentration. A group supplemented with an equal volume of PBS served as a negative control, and a group supplemented with medium containing 10% FBS served as a positive control. After culturing for another 48 hours, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader. The relative cell proliferation rate was calculated, and the results are shown in Table 1.

[0048] b. Collagen Synthesis Detection: HSF cells were seeded in 6-well plates. When the cell confluence reached 70%, the medium was replaced with serum-free medium containing 0.05 μM of the target protein. After 72 hours of culture, the cell supernatant was collected. The content of type I collagen in the cell supernatant was detected using a human type I collagen ELISA kit (purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd.), strictly following the instructions. The results are shown in Table 1.

[0049] Table 1 Results of Skin Repair Activity Test Table 1 shows that all four recombinant proteins exhibited concentration-dependent promotion of HSF cell proliferation and collagen I synthesis within the tested concentration range. Among them, the proteins produced in eukaryotic expression systems (Pichia pastoris and CHO cells) generally showed higher activity than those refolded in *E. coli*. In particular, pPIC9K-17YGR, fused with a membrane-penetrating peptide, showed the best effect in promoting proliferation, especially at a concentration of 0.5 μM, where its cell proliferation-promoting activity was further enhanced compared to pPIC9K-17_Y from the same system. (2) Hair growth activity test: Experimental animals: Forty healthy male C57BL / 6 mice, aged 7 weeks and weighing 18-22g, were randomly divided into 5 groups of 8 mice each. Negative control group: Blank emulsion base was applied daily.

[0050] pPIC9K-17_Y group: Apply lotion containing 0.5 mg / mL pPIC9K-17_Y daily.

[0051] pPIC9K-17_E group: Apply lotion containing 0.5 mg / mL pPIC9K-17_E daily.

[0052] pPIC9K-17_C group: Apply lotion containing 0.5 mg / mL pPIC9K-17_C daily.

[0053] pPIC9K-17YGR group: Apply lotion containing 0.5 mg / mL pPIC9K-17YGR daily.

[0054] Sample preparation: Each recombinant protein lyophilized powder was mixed uniformly with a blank emulsion matrix (composed of water, glycerol, emulsifier, etc.) to prepare a test emulsion containing 0.5 mg / mL of the target protein.

[0055] Hair removal and treatment: The hair on the back of the mice was removed with an electric shaver, followed by the application of hair removal cream. After thorough cleaning, a bare skin area of ​​approximately 2 cm × 2 cm was created. 24 hours after hair removal (when the hair follicles simultaneously enter the growth phase), the corresponding lotion (50 μL) was applied evenly to the hair removal area once daily for 21 consecutive days.

[0056] Observation and evaluation indicators: Hair growth score: On days 7, 14 and 21 of treatment, the hair removal area was photographed. Three observers who were unaware of the group assignments independently scored the hair coverage according to the predetermined hair coverage scoring criteria (0 points: no hair growth; 1 point: sparse vellus hair; 2 points: moderate vellus hair coverage; 3 points: dense vellus hair; 4 points: complete coverage, coarse and dark hair). The average score was taken.

[0057] Hair length measurement: At the end of treatment (e.g., day 21), approximately 20 new hairs were randomly plucked or shaved from the center and periphery of the bald area on the back of each group of mice using fine tweezers.

[0058] Arrange the hairs parallel to each other on a glass slide and measure the length of each hair from root to tip using an optical microscope with a scale or professional image analysis software (Image J).

[0059] Calculate the average length of hair in each group. The difference in average hair length directly reflects the cumulative growth rate throughout the treatment period.

[0060] The test results are shown in Table 2.

[0061] Table 2 Results of hair growth activity test The results in Table 2 show that all emulsion treatment groups containing recombinant proteins were significantly better than the negative control group in terms of hair growth score and average new hair length, confirming that the pPIC9K-17 series proteins have in vivo activity to promote hair regeneration.

[0062] The pPIC9K-17YGR group, which incorporates membrane-penetrating peptides, showed the best performance across all evaluation metrics. Its hair growth score and average new hair length were significantly higher than the pPIC9K-17_Y group in the same system but without membrane-penetrating peptides. This further validates that membrane-penetrating peptides can significantly enhance the hair growth efficacy of core proteins by increasing transdermal or cellular uptake efficiency.

[0063] (3) Stability test Each purified protein was uniformly diluted to a concentration of 1.0 mg / mL using PBS buffer. Each protein sample was aliquoted into multiple sterile centrifuge tubes, 500 μL per tube. The following experimental groups were set up: T0 group (0-day control): Samples tested immediately.

[0064] 4℃ storage group: Store in a refrigerator at 4℃.

[0065] 25℃ storage group: Store in a 25℃ constant temperature incubator.

[0066] -80℃ storage group (long-term stability control): stored in a -80℃ refrigerator as a benchmark for evaluating the activity retention rate after 7 days.

[0067] All samples were protected from repeated freeze-thaw cycles and light exposure during storage. After 7 days of storage, samples from the 4°C and 25°C groups were retrieved and analyzed for human skin fibroblast (HSF) proliferation activity along with freshly thawed samples from the -80°C group (T7 baseline).

[0068] The cell viability retention rate was calculated, and the results are shown in Table 3. The relative cell proliferation rate (%) = (OD sample group - OD blank group) / (OD untreated cell group - OD blank group) × 100%; viability retention rate (%) = (relative proliferation rate of the sample group after storage / relative proliferation rate of the -80℃ storage group (T7 baseline)) × 100%.

[0069] Table 3: Stability assessment of recombinant protein after 7 days of storage at different temperatures According to the results in Table 3, the four recombinant proteins provided by this invention maintained their physical integrity and core biological activity well after being stored in PBS solution at 4°C and 25°C for 7 days, with an activity retention rate of over 85%. This indicates that the series of proteins have excellent stability and meet the basic storage requirements for subsequent formulation development and application.

[0070] (4) Transdermal absorption test Take a healthy Bama miniature pig and collect abdominal skin, carefully removing subcutaneous fat and hair. Using a scalpel, create shallow, crisscrossing incisions approximately 5 mm long on the surface of the stratum corneum, deep enough to break the stratum corneum but not the dermis. Immerse the prepared skin sample in physiological saline at 2-8°C for later use. All skin samples should be used within 24 hours of preparation.

[0071] Four recombinant protein lyophilized powders were dissolved and diluted with physiological saline to prepare test solutions of uniform concentration (1.0 mg / mL). The solutions were sterilized by filtration through a 0.22 μm filter membrane before the experiment.

[0072] A vertical static Franz transdermal absorption system was used. The receiving cell had a volume of 10.0 mL and was made of glass; the effective diffusion area (S) of the supply chamber was 0.78 cm². 2 The prepared pigskin is placed between the receiving pool and the supply chamber, with the dermis side facing the receiving pool and the stratum corneum (with the wound surface) facing upwards towards the supply chamber. Ensure the skin is in close contact with the pool opening, free of air bubbles. The receiving pool is filled with 37°C constant-temperature circulating physiological saline as the receiving medium, and a magnetic stirrer is used to continuously stir the solution to ensure uniform concentration.

[0073] Add 1.0 mL of the test solution to the supply chamber. At 0 (initial), 2, 4, 6, and 8 hours after sample addition, accurately pipette 1.0 mL of sample solution from the receiving chamber and immediately replenish with an equal volume of fresh physiological saline at the same temperature. The collected sample solution is then appropriately diluted (if necessary) for testing. The concentration (C, mg / L) of recombinant protein in the receiving solution at each time point is determined using the BCA protein quantification method. The results are shown in Table 4.

[0074] Table 4: Results of transdermal absorption of recombinant proteins This test used the Franz diffusion cell method to simulate the transdermal behavior of recombinant proteins in a superficial wound skin model. Table 4 shows that all tested proteins achieved effective penetration within 8 hours, with the penetration amount increasing over time. In particular, the pPIC9K-17YGR protein, fused with a membrane-penetrating peptide, exhibited the highest cumulative penetration amount and steady-state penetration rate, indicating that the introduction of the membrane-penetrating peptide (YGRKKRRQRRR) significantly enhanced the transdermal absorption efficiency of the protein. The transdermal performance of other protein variants was, in descending order: pPIC9K-17_C > pPIC9K-17_Y > pPIC9K-17_E.

[0075] (5) Cytotoxicity test pPIC9K-17_Y, pPIC9K-17_E, pPIC9K-17_C, and pPIC9K-17_YGR were dissolved in serum-containing cell culture medium at high concentrations (10 mg / mL) and extracted at 37°C for 24 ± 2 hours. After centrifugation, the supernatant was used as the 100% test sample extract. Serial dilutions were performed using fresh culture medium.

[0076] Cell culture and treatment: L929 cells were seeded into 96-well plates and cultured for 24 hours. The culture medium was then replaced with different concentrations of extract (100%, 50%, 25%).

[0077] Morphological observation: After culturing for 24 and 48 hours, cell morphology, adhesion, and lysis status were observed under an inverted microscope and graded according to the following criteria: Grade 0: Cells have normal morphology and are growing well.

[0078] Grade 1: Slight changes in cell morphology, growth area <20%.

[0079] Grade 2: Moderate changes in cell morphology, with 20-50% of the growth area affected.

[0080] Grade 3: Severe changes in cell morphology, affecting 50-70% of the growth area.

[0081] Grade 4: Cells are almost completely dissolved or detached, with growth area >70%. Grades greater than 2 are considered to have cytotoxic effects.

[0082] Cell viability: After 24 hours of culture, cell viability was assessed using the MTT / CCK-8 assay, and the relative cell viability compared to the blank culture control group was calculated. Cell viability < 70% was considered potentially cytotoxic.

[0083] Results: At all tested concentrations, L929 cells in all groups exhibited normal morphology and good adhesion, with no obvious shrinkage, lysis, or detachment observed. Cytotoxicity was grade 0 in all groups. The relative cell viability in the 100% extract-treated groups was >96%, indicating no potential toxicity.

[0084] The recombinant protein pPIC9K-17 of the present invention did not show potential cytotoxicity under the test conditions and has high biocompatibility and safety.

[0085] In the above embodiments, the descriptions of each embodiment have different focuses. For parts that are not described in detail in a certain embodiment, please refer to the relevant descriptions in other embodiments.

[0086] The above description describes specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A recombinant protein pPIC9K-17, characterized in that, Its amino acid sequence consists of an N-terminal KR residue and four repeating units located downstream of it; wherein the sequence of each repeating unit is as shown in SEQ ID NO:

1.

2. The recombinant protein pPIC9K-17 according to claim 1, characterized in that, The recombinant protein further includes a membrane-penetrating peptide sequence at the C-terminus of its amino acid sequence, as shown in SEQ ID NO: 2, SEQ ID NO: 2: YGRKKRRQRRR.

3. The recombinant protein pPIC9K-17 according to claim 1 or 2, characterized in that, Each repeating unit is directly connected to the others.

4. A nucleic acid molecule, characterized in that, Its encoding is the recombinant protein pPIC9K-17 as described in any one of claims 1-3.

5. The nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid molecule is codon-optimized to be suitable for expression in Pichia pastoris, Escherichia coli, Bacillus subtilis, or CHO cells.

6. A recombinant expression vector, characterized in that, It contains the nucleic acid molecules as described in claim 4 or 5.

7. A recombinant host cell, characterized in that, It comprises the recombinant expression vector as described in claim 6, or has integrated the nucleic acid molecule as described in claim 4 or 5 into its genome; the host cell is Pichia pastoris, Escherichia coli, Bacillus subtilis or CHO cells.

8. A method for producing the recombinant protein pPIC9K-17 as described in any one of claims 1-3, characterized in that, Includes the following steps: (a) Constructing a recombinant expression vector containing a nucleic acid sequence encoding the recombinant protein; (b) The recombinant expression vector is introduced into host cells to obtain recombinant host cells; (c) The recombinant host cells are cultured under conditions suitable for expression, thereby expressing and obtaining the recombinant protein.

9. The method according to claim 8, characterized in that, The host cells are Pichia pastoris, Escherichia coli, Bacillus subtilis, or Chinese hamster ovary cells.

10. The use of the recombinant protein pPIC9K-17 as described in any one of claims 1-3, the nucleic acid molecule as described in claim 4 or 5, or the recombinant protein pPIC9K-17 prepared by the method as described in claim 8 or 9 in the preparation of products for skin repair and / or promoting hair regeneration.

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