A recombinant type xvii collagen chimeric protein and a preparation method and application thereof

Abstract

The application discloses a recombinant type XVII collagen chimeric protein and a preparation method and application thereof, and the recombinant type XVII collagen chimeric protein sequentially comprises a truncated alpha-factor signal peptide, a 6xHis label+TEV protease recognition region, a mutant NC16A domain and a COL15 collagen domain in sequence from an N terminal to a C terminal. The sensitization epitope and the enzyme cutting site of the natural NC16A domain are precisely and non-conservatively mutated, the sensitization and enzyme cutting risks are eliminated while the function of guiding triple helix assembly is retained, a function silent chimeric body is formed, the anti-inflammatory activity of the protein is significantly improved, and the secretion of inflammatory factors is synergistically inhibited, the fibroblast proliferation and COL1A1 expression are promoted in cooperation with pogostone. The application breaks through the technical bottleneck of the existing recombinant type XVII collagen protein, and provides a functional raw material with high safety, high stability and high activity for the fields of cosmetics and medical dressings.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant type XVII collagen chimeric protein, its preparation method, and its application. Background Technology

[0002] COL17 collagen is a transmembrane protein encoded by the COL17a1 gene, containing 1497 amino acids. Its N-terminus is located in the cytoplasm, and its C-terminus is located in the extracellular matrix. It is mainly distributed in the hemidesmosomes of basal cells in the epidermis, acting like "rivets" to tightly connect the epidermis and dermis, ensuring skin firmness and structural integrity. In addition, COL17 is also expressed in tissues such as hair follicles, colon, esophagus, placenta, small intestine, and stomach, but its highest concentration is found in the skin. Although COL17 collagen has significant functions, its traditional production method involves extraction from animal tissues and skin. This method is not only low-yield and costly, but also suffers from significant batch-to-batch variability, poor water solubility, and, more seriously, the risk of transmission of potential pathogens (such as viruses). Furthermore, due to differences in amino acid sequences between different species, direct injection of animal-derived collagen can easily trigger immune rejection or allergic reactions in humans. Moreover, natural COL17 collagen has a very large molecular weight (approximately 180 kDa), theoretically making it difficult to effectively secrete into the extracellular space and highly susceptible to degradation. Therefore, the current approach is to use synthetic recombinant type 17 collagen to avoid the obvious defects of extraction, reduce immune rejection, and overcome the molecular weight bottleneck in preparation.

[0003] Chinese patent CN119219762A discloses a recombinant human type XVII collagen, its preparation method, and its applications. The recombinant human type XVII collagen is any one of COL17A 3a, COL17A 3b, COL17A 3c, COL17A 3d, or COL17A 3e. This invention reduces the amino acid sequence of collagen to an extracellular functional region of less than 200 AA, lowers the molecular weight, and exhibits good biological activity.

[0004] Chinese patent CN121914251A discloses a recombinant human type 17 collagen, its preparation method, and its applications. The recombinant human type 17 collagen uses the original gene sequence of human skin type 17 collagen and contains an amino acid sequence consisting of n tandemly linked monomers A, arranged from the N-terminus to the C-terminus. This recombinant human type 17 collagen, even with a truncated amino acid sequence, retains the original functions of collagen and exhibits comparable or superior cell adhesion and cell migration activities compared to commercially available natural human collagen.

[0005] Type XVII collagen is a core structural protein of hemidesmosomes. Its extracellular domain contains 15 collagen domains (COL1-COL15) and 16 non-collagenous domains (NC1-NC16). The NC16A region (with 490-567 AAs) is a "core switch" for maintaining triple-helix assembly, but it is also a major sensitizing epitope in bullous pemphigoid and is rich in cleavage sites for ADAM family metalloproteinases. However, the design of recombinant type XVII collagen in current technologies faces the following dilemmas: (1) Although collagen activity can be obtained by simply extracting the COL15 collagen domain, the protein cannot efficiently self-assemble into a stable triple helix higher structure due to the lack of the NC16A region. Its half-life on the skin surface is extremely short and its bioavailability is low. (2) If the natural full-length NC16A region is retained, although it can guide the correct folding, it introduces two major safety hazards: ① The NC16A region contains the immune dominant epitope of bullous pemphigoid (AGADLDKIGL), which may induce autoimmune recognition and inflammatory response in sensitive skin; ② The HSDSQEELWMF flexible region of the NC16A region is a highly efficient substrate of surface metalloproteinases (Sheddases) such as ADAM9 / 10 / 17. The protein will be rapidly cleaved into pro-inflammatory free fragments on the body surface, which not only loses the anchoring function, but its degradation products will also act as chemokines to aggravate local inflammation.

[0006] Therefore, it is necessary to design a recombinant type 17 collagen that can completely eliminate the risks of sensitization and enzymatic cleavage, retain the NC16A-mediated higher structure assembly function, and obtain a functional protein that simultaneously possesses "anti-inflammatory" and "repair-promoting" activities. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a recombinant type XVII collagen chimeric protein, its preparation method, and its applications. By precisely and non-conservatively mutating the natural NC16A domain and combining it with the COL15 collagen domain, a recombinant protein with significantly enhanced anti-inflammatory activity is obtained. This breaks through the technical bottleneck of existing recombinant type XVII collagen proteins and provides a new generation of functional raw materials with high safety, high stability, and high activity for the cosmetics and medical dressing fields.

[0008] To achieve the above objectives, the present invention provides a recombinant type 17 collagen chimeric protein, wherein the recombinant type 17 collagen chimeric protein comprises, in order from N-terminus to C-terminus, a truncated α-factor signal peptide, a 6×His tag + TEV protease recognition region, a mutant NC16A domain, and a COL15 collagen domain; the complete sequence of the recombinant type 17 collagen chimeric protein is SEQ ID NO: 1.

[0009] Preferably, the sequence of the truncated α-factor signal peptide is SEQ ID NO: 2; the sequence of the 6×His tag + TEV protease recognition region is SEQ ID NO: 3; the mutant NC16A domain includes a mutant sensitizing epitope and a mutant enzyme cleavage region; and the sequence of the COL15 collagen domain is SEQ ID NO: 4.

[0010] More preferably, the sequence of the mutated sensitized epitope is SEQ ID NO: 6; the sequence of the mutated enzyme cleavage region is SEQ ID NO: 8; and the sequence of the mutated NC16A domain is SEQ ID NO: 9.

[0011] Preferably, a flexible connector is provided between the mutant NC16A domain and the COL15 collagen domain; the sequence of the flexible connector is SEQ ID NO: 10.

[0012] Preferably, the N and C segments of the 6×His tag + TEV protease recognition region each contain 2-4 protective amino acids.

[0013] More preferably, the N segment contains 4 protected amino acids and the C segment contains 2 protected amino acids.

[0014] Preferably, the C segment of the 6×His tag + TEV protease recognition region may also include an enzymatically cleaved amino acid.

[0015] More preferably, the enzymatically cleaved amino acid is any one of G, S or A.

[0016] The present invention also provides a gene for recombinant type 17 collagen chimeric protein, the nucleotide sequence of which is SEQ ID NO: 11; the gene is a gene for Pichia pastoris after codon optimization.

[0017] This invention also provides a method for preparing recombinant type 17 collagen chimeric protein, comprising the following steps: (1) Genes that synthesize recombinant type XVII collagen chimeric proteins; (2) Construct and ferment a recombinant engineered strain containing the gene for recombinant type 17 collagen chimeric protein to obtain a fermentation broth containing recombinant protein; (3) After separating the fermentation supernatant, the target protein was obtained by chromatography enrichment; (4) After purifying the target protein, a second chromatography enrichment was performed to obtain recombinant type 17 collagen chimeric protein.

[0018] Preferably, the original strain of the recombinant engineered strain in step (2) is Pichia pastoris.

[0019] Preferably, the chromatographic enrichment method described in steps (3)-(4) is to use a Ni-NTA affinity chromatography column.

[0020] The present invention also provides an application of recombinant type 17 collagen chimeric protein, wherein the application is in the preparation of formulations with anti-inflammatory, soothing, skin barrier repair promoting or anti-aging functions.

[0021] Preferably, the formulation contains recombinant type XVII collagen chimeric protein and patchouli active ingredient.

[0022] More preferably, the active ingredient of patchouli is any one of patchouli oil, patchouli alcohol, and patchouli extract.

[0023] More preferably, the preparation is a cosmetic or a topical medical excipient.

[0024] More preferably, the mass ratio of the recombinant type 17 collagen chimeric protein to the patchouli active ingredient is (1:500)-(500:1).

[0025] The beneficial effects of this invention are as follows: 1. By precisely and non-conservedly mutating the sensitizing epitope and cleavage site of natural NC16A, the sensitizing epitope and cleavage site are simultaneously eliminated while preserving the Coiled-coil backbone that guides triple helix assembly. The mutation of the cleavage site effectively blocks the enzymatic degradation by ADAM family metalloproteinases, thereby eliminating the risk of degradation products exacerbating local inflammation as pro-inflammatory chemokines. This fundamentally severs the endogenous pro-inflammatory pathway of type XVII collagen degradation products, endowing the protein with active anti-inflammatory biological functions. The mutation of the sensitizing epitope can destroy the original epitope's space charge structure, avoiding the risk of triggering autoimmune recognition and inflammatory responses in sensitive skin, and endowing the protein with safe application characteristics of active desensitization. This invention retains the coiled-coil backbone structure of natural NC16A and can efficiently guide the COL15 domain to assemble into a fully biologically active triple helix higher-order structure, forming a "functionally silent chimera". Therefore, it can efficiently bind to integrins on the surface of keratinocytes, activate the FAK / MAPK repair signaling pathway, significantly accelerate epithelialization and tight junction reconstruction, and prevent recombinant proteins from losing their ability to fold correctly, thereby preserving their biological activity as much as possible.

[0026] 2. The recombinant protein constructed in this invention exhibits several times greater resistance to ADAM9 / 10 / 17 enzyme cleavage compared to the natural protein, and its half-life on the skin surface is significantly prolonged. Furthermore, through a "dual-site" design involving Kex2 (yeast self-cleavage signal peptide) self-cleavage and TEV enzyme cleavage, the mature protein's N-terminus contains no carrier residue, fully meeting the stringent compliance requirements for cosmetic new ingredient applications regarding "no non-human fusion label residue."

[0027] 3. Patchouli can reduce oxidative stress levels in the inflammatory microenvironment by inhibiting the NF-κB pathway, providing more stable conditions for the chimeric protein to act. Meanwhile, the recombinant protein activates the FAK / MAPK repair pathway through integrin α6β4 binding, and the cells enter an active proliferation and synthesis state, enhancing the responsiveness to the anti-inflammatory signal of patchouli. Therefore, the combined use of recombinant protein and patchouli, in the same cellular microenvironment, mutually amplifies each other, thus showing a synergistic effect that is significantly better than the sum of the effects of each component used alone in three dimensions: anti-inflammation, proliferation promotion, and collagen synthesis promotion, and four core indicators (promoting fibroblast proliferation, inhibiting TNF-α secretion, inhibiting IL-6 secretion, and promoting COL1A1 mRNA expression), and the direction of the effect is highly consistent.

[0028] 4. This invention, through the innovative concept of "partial destruction and overall preservation," simultaneously resolves the long-standing technical contradictions of "efficient folding" and "safety," and breaks through the technical bottleneck of existing recombinant type XVII collagen, providing a new generation of functional raw materials with high safety, high stability, and high activity for the fields of cosmetics and medical dressings. Attached Figure Description

[0029] Figure 1 This is a gel electrophoresis image of the purified recombinant protein in Example 2, obtained by SDS-PAGE detection.

[0030] Figure 2 The diagram shows the spatial structure of the purified recombinant protein in Example 2. In the diagram, blue represents NC16A, orange represents the flexible linker, and green represents the COL15 triple helix structure.

[0031] Figure 3 The bar chart shows the content of TNF-α and IL-6 in Example 5. In the figure, A represents TNF-α and B represents IL-6.

[0032] Figure 4 The figure shows the synergistic effect of patchouli alcohol and purified protein on the inflammatory model in Example 6. In the figure, A is a bar chart of TNF-α content and B is a bar chart of IL-6 content.

[0033] Figure 5 This is a bar chart showing the cell proliferation rate in Example 7.

[0034] Figure 6 This is a bar chart showing the relative expression levels of COL1A1 mRNA in Example 8.

[0035] Figure 7 This is a bar chart showing the relative mRNA expression levels of PTK2, MMP9, and ACTA2 in Example 8.

[0036] Figure 8 This is a line graph showing the percentage of intact protein remaining at different time points in Example 9 for the purified recombinant protein. Detailed Implementation

[0037] The technical solution of the present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be construed as limiting the present invention. The scope of protection of the present invention should be determined by the contents of the claims. Modifications and substitutions made by those skilled in the art to the technical solution of the present invention without creative effort all fall within the scope of protection of the present invention.

[0038] In the following embodiments: Pichia pastoris strain GS115: provided by Yang Xiao's research group at Three Gorges University; pPIC9K: Provided by Professor Guo Jian's research group at Three Gorges University; G418: Purchased from Solarbio; Ni-NTA affinity chromatography column: GE Ni Sepharose High Performance, 71-5027-67 AF, purchased from Cytiva; Human immortalized keratinocytes (HaCaT): provided by Professor Qiu Kuncheng's research group at Three Gorges University; HDF cells: provided by Professor Qiu Kuncheng's research group at Three Gorges University; Dexamethasone: Purchased from Beyotime International Co., Ltd. TNF-α detection kit: purchased from Beyotime Biotechnology Co., Ltd. IL-6 ELISA kit: purchased from Beyotime Biotechnology Co., Ltd. KR116 Reverse Transcription Kit: Purchased from Tiangen Pharmaceuticals; Patchouli: Purchased from Shanghai Maclean Company; Recombinant human ADAM9: Purchased from Mce Company, batch number HY-P77867A; Recombinant human ADAM10: Purchased from Wuhan Sanying Biotechnology Co., Ltd. (proteintech), batch number Ag23062; Recombinant human ADAM17 protease: purchased from Wuhan Sanying Biotechnology Co., Ltd., batch number Ag32418; BMGY medium: 20.0g peptone, 10.0g yeast extract, 13.4g amino-free yeast nitrogen source (YNB), 0.4mg D-biotin and 10mL glycerol, add 100mL of 100mM potassium phosphate buffer with pH 6.0, and bring the volume to 1L with distilled water. ADAM17 reaction buffer: 25 mM Tris-HCl, pH 9.0, 2.5 μM ZnCl2, 0.005% Brij-35.

[0039] Example 1 (1) The sequence of the natural NC16A domain and the natural COL15 collagen domain (SEQ ID NO: 4) were obtained from the original human col17a1 sequence (SEQ ID NO: 12). (2) Analyze the sequence of the natural NC16A domain and find the sequence of its sensitizing epitope SEQ ID NO: 5 and the sequence of its enzyme cleavage region SEQ ID NO: 7. Mutate them respectively to obtain the mutant sensitizing epitope (SEQ ID NO: 6) and the mutant enzyme cleavage region (SEQ ID NO: 8), thereby obtaining the sequence of the mutant NC16A domain SEQ ID NO: 9. (3) Obtaining the α-factor signal peptide from the genome of Saccharomyces cerevisiae (Pablo Aza, et al. Design of an improved universal signal peptide based on the α-factor signal peptide). (factor mating secretion signal for enzyme production in yeast. 2021, 78:3691-3707.), and truncated to obtain a truncated α-factor signal peptide, the sequence of which is SEQ ID NO: 2; (4) Obtain the TEV protease recognition sequence from the literature and connect a 6×His tag to its N segment to obtain the 6×His tag + TEV protease recognition region, the sequence of which is SEQ ID NO: 3; (5) Assembly sequence: Following the order from N-terminus to C-terminus, the truncated α-factor signal peptide, four protective amino acids, 6×His tag + TEV protease recognition region, two protective amino acids, mutant NC16A domain, flexible linker (sequence of SEQ ID NO: 10) and COL15 collagen domain are sequentially connected to obtain recombinant type 17 collagen chimeric protein, sequence of SEQ ID NO: 1; (6) Codon optimization: Based on the codon preference in the Pichia pastoris genome, the codons of the recombinant type 17 collagen chimeric protein were optimized throughout the process to obtain the gene of the recombinant type 17 collagen chimeric protein, the sequence of which is SEQ ID NO: 11, and sent to Nanjing Genscript Biotech Co., Ltd. for synthesis.

[0040] Example 2 (1) The gene of the recombinant type 17 collagen chimeric protein in Example 1 was cloned into the multiple cloning site of the expression vector pPIC9K by gene synthesis to obtain the recombinant pPIC9K-col17 expression vector; wherein the synthetic primers were col17F and col17R, and the sequences were SEQ ID NO: 23 and SEQ ID NO: 24, respectively; (2) The recombinant pPIC9K-col17 expression vector was transformed into competent cells of Pichia pastoris GS115 by electroporation, and high-copy recombinant strains were obtained by G418 screening. The recombinant vector was linearized with SacI and introduced into Pichia pastoris GS115 by electroporation (1.5 kV, 25 μF, 200 Ω). It was plated on MD plates and cultured at 30℃ for 4 days. Single colonies were picked and inoculated into YPD plates containing 2.0 mg / mL G418, and strains with copy numbers of 5-8 were screened as high-copy strains. (3) The high-copy strain was inoculated into BMGY medium and cultured at 28°C and 250 rpm until OD. 600 =4, centrifuge to collect the bacterial cells, resuspend them in BMMY medium, add anhydrous methanol to a final concentration of 0.8%, induce at 30℃ for 96 h, add methanol every 24 h; after 96 h, centrifuge at 10000 rpm for 15 min to obtain the fermentation supernatant. (4) The fermentation supernatant was concentrated to 1 / 10 by ultrafiltration and then loaded into a Ni-NTA affinity chromatography column. It was eluted with a gradient of 7 column volumes of PBS buffer containing 20 mM imidazole, and then eluted with PBS buffer containing 250 mM. The eluent was collected to obtain the recombinant protein solution. (5) Add His-tagged TEV protease to the recombinant protein solution and digest at 4°C for 16 h to obtain digestion solution; wherein the ratio of TEV protease to recombinant protein is 1:50, w / w; (6) Load the digest solution back into the Ni-NTA affinity chromatography column and collect the flow-through to obtain the purified recombinant protein.

[0041] SDS-PAGE analysis revealed that the purified recombinant protein had a molecular weight of approximately 21 kDa, and its triple-helix structure, after folding, had a molecular weight of approximately 63 kDa. Figure 1The three-dimensional structure of the purified recombinant protein was predicted using AlphaFold2. The results showed that the mutant NC16A domain formed a typical coiled-coil structure with three tightly wrapped α-helices, connected to the downstream flexible linker and the COL15 triple-helical collagen domain. Figure 2 This forms a coherent, rigid rod-like structure.

[0042] Example 3 (1) Human immortalized keratinocytes (HaCaT) were cultured at 37°C and 5% CO2 in DMEM medium containing 10% FBS. Cells that had grown to the logarithmic growth phase were then harvested at 2×10⁻⁶ ppm. 5 Seeds were inoculated per well in 6-well plates and cultured for 24 hours until confluence reached approximately 80%. The medium was then replaced with DMEM containing 0.5% FBS, starved for 12 hours, and then TNF-α (20 ng / mL) was added and cultured for 48 hours to obtain the inflammation model. (2) The inflammation model obtained in step (1) was used for anti-inflammatory activity verification experiments and compared with normal cells. The grouping and processing methods of the verification experiments are as follows: Experimental group: The purified recombinant protein prepared in Example 2 was added to the inflammation model to a final concentration of 20 μg / mL; Control group 1: An equal volume of physiological saline was added to the inflammation model; Control group 2: The purified protein was added to the inflammation model to a final concentration of 20 μg / mL; the sequence of the purified protein was the same as in Example 1, except that the mutant NC16A domain was replaced with the unmutated NC16A domain, and then it was prepared according to the method in Example 2. Control group 3: The purified protein was added to the inflammation model to a final concentration of 20 μg / mL; the sequence of the purified protein was the same as in Example 1, except that the mutant NC16A domain was replaced with the NC16A domain with only the sensitized epitope mutated, and then it was prepared according to the method in Example 2. Control group 4: The purified protein was added to the inflammation model to a final concentration of 20 μg / mL; the sequence of the purified protein was the same as in Example 1, except that the mutant NC16A domain was replaced with an NC16A domain that was mutated only in the enzyme cleavage region, and then it was prepared according to the method in Example 2. Control group 5: The purified protein was added to the inflammation model to a final concentration of 20 μg / mL; the sequence of the purified protein was the same as in Example 1, except that it did not contain the NC16A domain, and it was prepared according to the method in Example 2. Control group 6: Immortalized keratinocytes from normal individuals, with an equal volume of physiological saline added; Control group 7: Dexamethasone was added to the inflammation model to a final concentration of 10 μg / mL; (3) The above 8 groups of cells were cultured at 37℃ for 48h, and then the cell culture supernatant (300g, centrifuged for 10min) was collected; then the contents of TNF-α and IL-6 were detected by human TNF-α and IL-6 ELISA kit. The results are shown in Table 1.

[0043] Table 1. Results of inflammatory factor levels and inhibition rates in different treatment groups

[0044] Note: Inhibition rate (%) = [(factor content of model control group - factor content of experimental group) / (factor content of model control group - factor content of blank control group)] × 100%, where the model control group is control group 6 and the blank control group is control group 1.

[0045] The results showed that the purified recombinant protein in the experimental group, at a concentration of 20 μg / mL, had an inhibition rate of 58.0% for TNF-α and 53.8% for IL-6, which were significantly higher than those in the control group 2-5 (P<0.01), indicating that the purified recombinant protein prepared in this invention has a good anti-inflammatory effect.

[0046] Example 4 Following the processing method in Example 3, the final concentrations of purified recombinant protein in the experimental group were adjusted to 0, 5, 10, 20, 30 and 50 μg / mL, respectively, and the contents of TNF-α and IL-6 were detected. The results are shown in Table 2.

[0047] Table 2. Effects of purified protein concentration on inflammatory factor content and inhibition rate.

[0048] The results showed that as the concentration of the purified recombinant protein increased, its inhibition rate gradually increased, indicating that its anti-inflammatory effect also gradually increased. However, when the final concentration exceeded 20 μg / mL, the anti-inflammatory effect reached saturation and the inhibition rate entered a plateau phase. As the concentration further increased, the inhibition rate slowly decreased. Therefore, the optimal concentration of the purified recombinant protein was 20 μg / mL.

[0049] Example 5 (1) Take the inflammation model from step (1) of Example 3, divide it into 6 groups, and add the following agents to each group, as follows: Experimental group: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 20 μM and the final concentration of purified recombinant protein 20 μg / mL. Control group 1: Patchouli alcohol was added to achieve a final concentration of 20 μM; Control group 2: The purified recombinant protein prepared in Example 2 was added to achieve a final concentration of 20 μg / mL; Control group 3: Add an equal amount of solvent DMSO; Control group 4: Add asiaticoside and the purified recombinant protein prepared in Example 2 to make the final concentration of asiaticoside 20 μM and the final concentration of purified recombinant protein 20 μg / mL. Control group 5: Vitamin B5 and the purified recombinant protein prepared in Example 2 were added to make the final concentration of vitamin B5 20 μM and the final concentration of purified recombinant protein 20 μg / mL. (2) Each treatment group in step (1) was cultured at 37°C for 48 hours, and then the cell culture supernatant (300g, centrifuged for 10min) was collected; then the contents of TNF-α and IL-6 were detected by TNF-α and IL-6 ELISA kit.

[0050] The results showed that the TNF-α content in the experimental group was only 64.7 pg / mL, significantly lower than that in the three control groups, and increased by about 24.1% compared with the expected value (expected value = control group 1 + control group 2 - control group 3), proving that the purified recombinant protein and patchouli alcohol can synergistically enhance the inhibitory effect on TNF-α. Figure 3 A); The IL-6 content in the experimental group was only 82.1 pg / mL, significantly lower than that in the three control groups, and increased by about 18.6% compared with the expected value (expected value = control group 1 + control group 2 - control group 3), proving that the purified recombinant protein and patchouli alcohol can synergistically enhance the inhibitory effect on IL-6. Figure 3 B).

[0051] Example 6 (1) Take the inflammation model from step (1) of Example 3, divide it into 6 groups, and adjust the concentrations of patchouli alcohol and purified recombinant protein for experiments, as follows: Experimental group 1: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 0 μM and the final concentration of purified recombinant protein 20 μg / mL. Experimental group 2: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 5 μM and the final concentration of purified recombinant protein 20 μg / mL. Experimental group 3: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 10 μM and the final concentration of purified recombinant protein 20 μg / mL. Experimental group 4: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 20 μM and the final concentration of purified recombinant protein 20 μg / mL. Experimental group 5: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 50 μM and the final concentration of purified recombinant protein 20 μg / mL. Experimental group 6: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 10 μM and the final concentration of purified recombinant protein 50 μg / mL. (2) Each treatment group in step (1) was cultured at 37°C for 48 hours, and then the cell culture supernatant (300g, centrifuged for 10min) was collected; then the contents of TNF-α and IL-6 were detected by TNF-α and IL-6 ELISA kit.

[0052] The results are as follows Figure 4 As shown: Patchouli 10uM and purified recombinant protein 20ug / ml have the best effect; at the same time, when patchouli is combined with purified recombinant protein, 10uM basically reaches the plateau phase, and further increases do not have a significant effect enhancement.

[0053] Example 7 (1) Take HDF cells in the logarithmic growth phase and use 5×10 3 Each cell / well was seeded into a 96-well plate and cultured for 24 hours until adherence. Then, the plate was replaced with DMEM containing 0.5% FBS and starved for 12 hours. The cells were then divided into the following 4 groups: Experimental group: Patchouli and the purified recombinant protein prepared in Example 2 were added to make the final concentration of patchouli 10 μM and the final concentration of purified recombinant protein 20 μg / mL. Control group 1: Patchouli alcohol was added to bring the final concentration to 10 μM; Control group 2: The purified recombinant protein prepared in Example 2 was added to achieve a final concentration of 50 μg / mL; Control group 3: Add an equal amount of solvent DMSO; Blank group: Add an equal volume of DMEM medium containing 0.5% FBS and no cells to a 96-well plate, and an equal volume of CCK-8 solution containing patchouli alcohol. (2) Each treatment group in step (1) was placed in a 37℃, 5% CO2 incubator for 48h. Then, 10 μL of CCK-8 solution (5 mM WST-8) was added to each well. After incubation for 2h, the absorbance at 450 nm was measured and the proliferation rate was calculated. The proliferation rate was calculated as follows: proliferation rate = [(OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. The control group was control group 3.

[0054] The results are as follows Figure 5As shown, the proliferation rate of the experimental group was 152.8%, which was significantly higher than that of the three control groups and increased by about 11.5% compared with the expected value (expected value = control group 1 + control group 2 - control group 3), indicating that patchouli alcohol and purified recombinant protein can synergistically promote fibroblast proliferation.

[0055] Example 8 (1) Take cells from each treatment group in Example 7, extract total RNA from the cells using the kit method, and then use the KR116 reverse transcription kit to reverse transcribe cDNA; (2) Using cDNA as a template, the relative mRNA expression levels of COL1A1 (type I collagen α1 chain), PTK2, MMP9 and ACTA2 were detected by real-time quantitative PCR (RT-qPCR); the primer sequences of GAPDH as the internal reference gene are shown in Table 1.

[0056] The results showed that the relative expression level of COL1A1 in the experimental group was upregulated by 3.12-fold, significantly higher than that in the three control groups. Bliss independent model analysis predicted an expression level of approximately 1.95-fold, while the measured expression level of 3.12-fold far exceeded this expectation, achieving 1.6 times the expected effect. Figure 6 This indicates that patchouli alcohol and purified recombinant protein significantly promoted type I collagen synthesis. Simultaneously, the relative expression levels of PTK2, MMP9, and ACTA2 were also significantly upregulated in the experimental group. Figure 7 This indicates that patchouli alcohol and purified recombinant protein can activate the FAK / MAPK repair signaling pathway, thereby significantly accelerating epithelialization and tight junction reconstruction.

[0057] Table 3 Primer sequences for RT-qPCR

[0058] Example 9 (1) Take 10 μg each of the purified recombinant protein prepared in Example 2 and the unmutated purified protein described in Control Group 2 of Example 3; dissolve them in 200 μL ADAM17 reaction buffer respectively; (2) Add 0.2 μg of recombinant human ADAM9, ADAM10 and ADAM17 proteases respectively, and incubate at 37℃. Take samples at 0, 1, 2, 4, 8, 12 and 24 h of incubation. Separate by SDS-PAGE electrophoresis, stain with Coomassie brilliant blue and use ImageJ software to quantify the gray value of intact protein bands. The amount of intact protein at 0 h of incubation is taken as 100% and the residual rate of intact protein at each time point is calculated.

[0059] The results are as follows Figure 8As shown: The purified recombinant protein prepared in Example 2 had a complete protein residue rate of over 50% at 8 hours, indicating that it can significantly prolong the half-life on the skin surface.

Claims

1. A recombinant type XVII collagen chimeric protein, characterized in that: The recombinant type XVII collagen chimeric protein, in order from N-terminus to C-terminus, includes a truncated α-factor signal peptide, a 6×His tag + TEV protease recognition region, a mutant NC16A domain, and a COL15 collagen domain; the complete sequence of the recombinant type XVII collagen chimeric protein is SEQ ID NO:

1.

2. The recombinant type 17 collagen chimeric protein according to claim 1, characterized in that: The sequence of the truncated α-factor signal peptide is SEQ ID NO: 2; the sequence of the 6×His tag + TEV protease recognition region is SEQ ID NO: 3; the mutant NC16A domain includes a mutant sensitizing epitope and a mutant enzyme cleavage region; the sequence of the COL15 collagen domain is SEQ ID NO:

4.

3. A recombinant type 17 collagen chimeric protein according to claim 2, characterized in that: The sequence of the mutated sensitized epitope is SEQ ID NO: 6; the sequence of the mutated enzyme cleavage region is SEQ ID NO: 8; and the sequence of the mutated NC16A domain is SEQ ID NO:

9.

4. A recombinant type 17 collagen chimeric protein according to claim 1, characterized in that: A flexible connector is provided between the mutant NC16A domain and the COL15 collagen domain; the sequence of the flexible connector is SEQ ID NO:

10.

5. A gene for a recombinant type 17 collagen chimeric protein as described in any one of claims 1-4, characterized in that: The nucleotide sequence of the gene is SEQ ID NO: 11; the gene is a codon-optimized gene for Pichia pastoris.

6. A method for preparing recombinant type 17 collagen chimeric protein as described in any one of claims 1-4, characterized in that: Includes the following steps: (1) Genes that synthesize recombinant type XVII collagen chimeric proteins; (2) Construct and ferment a recombinant engineered strain containing the gene for recombinant type 17 collagen chimeric protein to obtain a fermentation broth containing recombinant protein; (3) After separating the fermentation supernatant, the target protein was obtained by chromatography enrichment; (4) After purifying the target protein, a second chromatography enrichment was performed to obtain recombinant type 17 collagen chimeric protein.

7. The preparation method according to claim 6, characterized in that: The original strain of the recombinant engineered strain mentioned in step (2) is Pichia pastoris.

8. The preparation method according to claim 6, characterized in that: The chromatographic enrichment method described in steps (3)-(4) is to use a Ni-NTA affinity chromatography column.

9. The application of a recombinant type 17 collagen chimeric protein as described in any one of claims 1-4, characterized in that: The application is in the preparation of formulations with anti-inflammatory, soothing, skin barrier repair promoting, or anti-aging functions.

10. The application according to claim 9, characterized in that: The formulation contains recombinant type XVII collagen chimeric protein and patchouli active ingredient.

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