High-transdermal recombinant humanized 17-type collagen and application thereof

Abstract

The invention belongs to the technical field of gene engineering, and relates to a high-transdermal recombinant humanized type 17 collagen and application thereof. The cell-penetrating peptide is composed of a cell-penetrating peptide segment and a collagen segment, the collagen segment is an amino acid sequence as shown in SEQ ID NO: 1, and the cell-penetrating peptide segment is cell-penetrating peptide SPACE, TAT or ANTP. The high-transdermal recombinant humanized type 17 collagen provided by the invention not only has relatively high skin permeation efficiency, but also has various biological functions such as barrier repair, tightening and wrinkle resistance, and is beneficial to efficient application in the fields of cosmetics, medicines and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and relates to a high-transdermal recombinant humanized collagen type 17 (XVII) and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art already known to a person of ordinary skill in the art.

[0003] Collagen type 17 (COL17) is a core functional protein existing in the epidermal basement layer of the skin. COL17 is a core component of the "anchoring junction" between the epidermis and the dermis, and is essential for stabilizing the basement membrane and preventing the epidermis from falling off. At the same time, COL17 can regulate the activity of epidermal stem cells, promote the orderly replacement of epidermal cells, and maintain the continuous repair ability of the skin barrier. Therefore, COL17 is regarded as the "guardian of skin basement membrane integrity" and the "anti-aging key target". However, with the increase of age (such as the human body COL17 synthesis rate decreases by 1%-2% per year after the age of 30), ultraviolet radiation (UVB can induce COL17 degradation enzyme activity to increase by 3-5 times), environmental pollution and bad living habits, the content of COL17 in the skin will gradually decrease, and the function will significantly decline: on the one hand, the anchoring structure of the basement membrane is loose, which leads to the expansion of the gap between the epidermis and the dermis, and the decrease of skin elasticity, sagging; on the other hand, the efficiency of epidermal stem cell renewal is reduced, and the skin barrier repair ability is weakened, and dry, sensitive, wrinkles and other signs of aging appear.

[0004] In order to improve the problem of skin aging, exogenous supplementation of COL17 has become the core research direction in the field of cosmetics and skin medical repair. Compared with animal source COL17, recombinant human 17 type collagen has the advantages of low immunogenicity, high biocompatibility, and clear functional targeting, and is regarded as an ideal material to replace traditional collagen. However, the core technical bottleneck that limits the application of recombinant human 17 type collagen is the extremely low transdermal absorption efficiency. The reason is that: the molecular weight of the recombinantly expressed protein is generally high (far greater than the 500 Da penetration threshold of the skin keratin layer gap), and the molecular structure is in a linear rigid conformation, which itself is difficult to pass through the structural barrier of the skin keratin layer, and the transdermal absorption rate is less than 5%, most of the active ingredients only stay in the skin surface layer, and cannot effectively reach the target site in the epidermal basement layer to play a repair and anti-aging effect. Therefore, it is necessary to develop a high-efficiency, safe and COL17-transdermal-promoting technology, so as to promote the efficient application of COL17 in the fields of cosmetics and medicine. SUMMARY

[0005] In order to solve the problems of the prior art, the present application aims to provide a high skin permeability recombinant humanized collagen type 17 and application, the high skin permeability recombinant humanized collagen type 17 provided by the present application has higher skin penetration efficiency, and has multiple biological functions such as barrier repair, firming and anti-wrinkle, which is beneficial to efficient application in the fields of cosmetics, medicine and the like.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: In a first aspect, a high skin permeability recombinant humanized collagen type 17 is composed of a transmembrane peptide segment and a collagen segment, the collagen segment is an amino acid sequence as shown in SEQ ID NO: 1, and the transmembrane peptide segment is a transmembrane peptide SPACE, TAT or ANTP.

[0007] In the collagen provided by the present application, the amino acid sequence of the collagen segment is designed to contain multiple KGD active motifs, which can mediate the adhesion and migration of keratinocytes and is directly related to the stable and skin basement membrane repair of hair follicle stem cells. In the design of the collagen segment, the structural integrity, expression efficiency and subsequent delivery feasibility are taken into account: if the functional domain is too short, it is difficult to form a stable triple helix structure and enough active sites, resulting in weak biological function; and a too long sequence may increase the difficulty of gene synthesis and expression, and affect the delivery efficiency of the transmembrane peptide due to the crowding of the molecular conformation. Therefore, the present application can achieve the best balance between biological activity and delivery efficiency.

[0008] In a second aspect, a nucleic acid molecule for encoding the high skin permeability recombinant humanized collagen type 17 of the first aspect of the present application is provided.

[0009] In a third aspect, a recombinant vector comprising the nucleic acid molecule of the second aspect of the present application is provided.

[0010] In a fourth aspect, a recombinant cell, which is a host cell containing the nucleic acid molecule of the second aspect of the present application or the recombinant vector of the third aspect of the present application, is provided.

[0011] In a fifth aspect, a method for preparing the high skin permeability recombinant humanized collagen type 17 of the first aspect of the present application is provided, which comprises the steps of: culturing the recombinant cell of the fourth aspect of the present application to express the high skin permeability recombinant humanized collagen type 17; and isolating the high skin permeability recombinant humanized collagen type 17.

[0012] In a sixth aspect, the high skin permeability recombinant humanized collagen type 17 of the first aspect of the present application or the nucleic acid molecule of the second aspect of the present application or the recombinant vector of the third aspect of the present application or the recombinant cell of the fourth aspect of the present application is used in the preparation of a product for improving skin aging or skin repair.

[0013] The beneficial effects of the present application are: 1、The present application designs and modifies specific cell-penetrating peptides on humanized type 17 collagen, so that the obtained recombinant humanized type 17 collagen has extremely strong transdermal absorption capacity. Through efficient transdermal absorption, the collagen of the present application can effectively penetrate the structure barrier of the stratum corneum of the skin, reach the epidermal basal layer as the physiological function target site in sufficient amount, so as to realize its repair and anti-aging biological activity, greatly improve the product efficacy and application efficiency. It can be widely applied in high-end functional skin care products, medical repair dressings, wound healing materials and other biomedical material fields, and meet the urgent needs of the market for efficient, targeted anti-aging and skin repair technology.

[0014] 2、The recombinant humanized type 17 collagen provided by the present application can be stably and highly expressed in recombinant cells, especially in recombinant strains. This characteristic makes the collagen provided by the present application suitable for industrial large-scale production, effectively solving the problems of high cost, limited yield and poor batch stability caused by natural extraction or mammalian cell expression, and laying a solid raw material supply foundation for its wide application in the fields of biological materials, medical devices and skin care products. BRIEF DESCRIPTION OF DRAWINGS

[0015] The drawings accompanying the specification of the present application serve to provide a further understanding of the present application, and the illustrative embodiments of the present application and their descriptions serve to explain the present application, and do not constitute an improper limitation on the present application.

[0016] Figure 1 Figure 1 is a fermentation time sampling detection diagram of COL1502, SP1502, TA1502 and AN1502 in Example 3 of the present application; a is TA1502 and COL1502, M: Marker, lane 1 is TA1502 fermentation 0h, lane 2 is TA1502 fermentation 12h, lane 3 is TA1502 fermentation 24h, lane 4 is TA1502 fermentation 36h, lane 5 is TA1502 fermentation 48h, lane 6 is COL1502 fermentation 0h, lane 7 is COL1502 fermentation 12h, lane 8 is COL1502 fermentation 24h, lane 9 is COL1502 fermentation 36h, lane 10 is COL1502 fermentation 48h; b is AN1502 and SP1502, M: Marker, lane 1 is AN1502 fermentation 0h, lane 2 is AN1502 fermentation 12h, lane 3 is AN1502 fermentation 24h, lane 4 is AN1502 fermentation 36h, lane 5 is AN1502 fermentation 48h, lane 6 is SP1502 fermentation 0h, lane 7 is SP1502 fermentation 12h, lane 8 is SP1502 fermentation 24h, lane 9 is SP1502 fermentation 36h, lane 10 is SP1502 fermentation 48h.

[0017] Figure 2 Electrophoresis result chart after purification of COL1502, SP1502, TA1502, and AN1502 in Example 4 of the present application.

[0018] Figure 3 Transdermal experiment result chart in Example 5 of the present application.

[0019] Figure 4 Repairing efficacy result chart of recombinant collagen type XVII TA1502 in Example 6 of the present application; P<0.05 (#), P<0.01 (##), P<0.001 (###), and P<0.0001 (####) are considered to have significant difference compared with the BC group; P<0.05 (*), P<0.01 (**), P<0.001 (***), and P<0.0001 (****) are considered to have significant difference compared with the NC group.

[0020] Figure 5 Tightening and anti-wrinkle efficacy result chart of recombinant collagen type XVII TA1502 in Example 7 of the present application; a is the content of COL-I; P<0.05 (#), P<0.01 (##), P<0.001 (###), and P<0.0001 (####) are considered to have significant difference compared with the BC group; P<0.05 (*), P<0.01 (**), P<0.001 (***), and P<0.0001 (****) are considered to have significant difference compared with the NC group; b is the content of ELN; P<0.05 (#), P<0.01 (##), P<0.001 (###), and P<0.0001 (####) are considered to have significant difference compared with the BC group; P<0.05 (*), P<0.01 (**), P<0.001 (***), and P<0.0001 (****) are considered to have significant difference compared with the NC group. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0022] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, components, and / or groups thereof, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof.

[0023] In view of the fact that the transdermal absorption efficiency of human-derived collagen type 17 is extremely low, which limits its application in cosmetics and skin medical repair, the present application provides a high-transdermal recombinant humanized collagen type 17 and application thereof.

[0024] In a typical embodiment of the present application, a high-transdermal recombinant humanized collagen type 17 is provided, which is composed of a transmembrane peptide segment and a collagen segment, the collagen segment is an amino acid sequence as shown in SEQ ID NO: 1, and the transmembrane peptide segment is a transmembrane peptide SPACE, TAT or ANTP.

[0025] In some embodiments, the transmembrane peptide segment is modified at the N-terminus of the collagen segment.

[0026] In some embodiments, the amino acid sequence of the transmembrane peptide SPACE is as shown in SEQ ID NO: 2.

[0027] In some embodiments, the amino acid sequence of the transmembrane peptide TAT is as shown in SEQ ID NO: 3.

[0028] In some embodiments, the amino acid sequence of the transmembrane peptide ANTP is as shown in SEQ ID NO: 4.

[0029] In some embodiments, the amino acid sequence of the high-transdermal recombinant humanized collagen type 17 is as shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. Studies have shown that the skin penetration efficiency of the high-transdermal recombinant humanized collagen type 17 with the amino acid sequence as shown in SEQ ID NO: 5 is higher than that of the other two.

[0030] In another embodiment of the present application, a nucleic acid molecule for encoding the above-mentioned high-transdermal recombinant humanized collagen type 17 is provided.

[0031] In some embodiments, the DNA sequence of the nucleic acid molecule is as shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.

[0032] In a third embodiment of the present application, a recombinant vector comprising the above-mentioned nucleic acid molecule is provided. In a third embodiment of the present application, a recombinant vector comprising the above-mentioned nucleic acid molecule is provided.

[0033] The recombinant vector of the present application employs any one of the vectors of pPIC9, pPIC9K, pHIL-S1, pPICZ alpha A, pYAM75P, pGAPZ alpha A, pGAPZ A and the vectors modified therefrom. In some embodiments, the recombinant vector employs the vector of pPIC9K.

[0034] In a fourth embodiment of the present application, a recombinant cell is provided, which is a host cell containing the nucleic acid molecule or the recombinant vector as described above.

[0035] The recombinant strain of the present application employs any one or more of bacterial cells, fungal cells or plant cells. Specifically, the bacterial cells can be Escherichia (e.g., Escherichia coli), Bacillus (e.g., Bacillus subtilis) and the like. Specifically, the fungal cells can be yeast and the like.

[0036] In a fifth embodiment of the present application, a method for preparing a high transdermal recombinant humanized collagen XVII is provided, which comprises the steps of: culturing the recombinant cell as described above, so as to express the high transdermal recombinant humanized collagen XVII; and isolating the high transdermal recombinant humanized collagen XVII.

[0037] In some embodiments, the method comprises the following steps: (1) activating the recombinant engineering bacteria as described above; (2) stopping the feeding when the wet bacteria amount reaches 250-400 g / L, and adding methanol in stages to induce for 48-96 h to obtain a fermentation broth; (3) purifying the obtained collagen protein using a multi-mode weak cation exchange chromatography filler to obtain the high transdermal recombinant humanized collagen XVII.

[0038] Specifically, the activation method of step (1) is: picking a single colony of the recombinant engineering bacteria and inoculating it in a first-stage seed culture medium for overnight culture, and then transferring it to a second-stage seed culture medium at a volume ratio of 1.5-2.5% inoculation amount to activate the OD600 to 6.0-10.0. More specifically, the first-stage seed culture medium and the second-stage seed culture medium used in the activation process are both YPD liquid culture medium. More specifically, the culture conditions in the activation process are: temperature of 28-32 ℃, and rotation speed of 200-250 rpm.

[0039] Specifically, the fermentation process of step (2) is as follows: the activated bacteria in step (1) are inoculated into the fermentation medium at a volume ratio of 9-11% for fermentation culture, the initial culture temperature is 28-32°C, the rotation speed is 280-320 rpm, the ventilation is 0.8-1.2 vvm, the pH is adjusted to 4.8-5.2 by using 45-55% ammonia water, and the DO value in the fermentation tank is maintained above 20% by adjusting the stirring rotation speed in connection with the dissolved oxygen; when the glycerol is consumed, the stirring is associated with the dissolved oxygen feedback, when the DO value is greater than 30%, the glycerol is fed at a feeding rate of 0.8-1.2 mL / min, when the wet bacterial mass is 250-400 g / L, the feeding is stopped, the rotation speed is increased to 850-950 rpm, and when the DO value rises stably, the induction is started by feeding the methanol feed solution, the induction temperature is 20-30°C, the methanol feed solution is fed at a flow rate of 0.05-0.07 mL / min for 3.5-4.5 h before induction; after 3.5-4.5 h of induction, the feeding is stopped, and when the DO value starts to rise, the remaining methanol feed solution is fed by setting the dissolved oxygen feedback condition, and the feeding is carried out at a feeding rate of 0.11-0.13 mL / min, while the dissolved oxygen is associated, and the DO value is controlled to be above 30% to start the feeding, and the induction is continued for 48-96 h.

[0040] More specifically, the fermentation medium is BSM medium. The formula of the BSM medium is as follows: 85% phosphoric acid 26-27 mL / L, calcium sulfate 0.9-1.0 g / L, potassium sulfate 18-19 g / L, magnesium sulfate 14-15 g / L, potassium hydroxide 4.1-4.2 g / L, glycerol 35-45 g / L, and solvent water; after sterilization at 120-125°C for 10-30 min, the temperature is reduced to room temperature, and PTM1 trace element mother liquor is added at 4.3-4.4 mL / L, and the pH value is adjusted to 5.0 with 45-55% ammonia water.

[0041] More specifically, the formula of the glycerol feed solution during the feeding process is as follows: mass ratio of 45-55% glycerol, 120-125°C sterilization for 10-30 min, and then temperature is reduced to room temperature, and PTM1 trace element mother liquor is added at 11-13 mL / L, and mixed uniformly.

[0042] More specifically, the formula of the methanol feed solution is as follows: 100% methanol, PTM1 trace element mother liquor is added at 11-13 mL / L, and mixed uniformly.

[0043] The PTM1 trace element mother liquor has the following formula: copper sulfate 5.5-6.5 g / L, sodium iodide 0.07-0.09 g / L, manganese sulfate 2.5-3.5 g / L, sodium molybdate 14.5-15.0 g / L, boric acid 0.01-0.03 g / L, cobalt chloride 0.4-0.6 g / L, zinc chloride 18-22 g / L, ferrous sulfate heptahydrate 60-70 g / L, biotin 0.1-0.3 g / L, sulfuric acid 4.5-5.5 mL / L, and solvent water; the bacteria are removed by filtering with a filter membrane (the specification can be 0.22 μm), and the product is stored at 3-5 ℃ for standby.

[0044] In a sixth embodiment of the present application, the use of the above-mentioned high-transdermal recombinant humanized type 17 collagen, the above-mentioned nucleic acid molecule, the above-mentioned recombinant vector or the above-mentioned recombinant cell in the preparation of a product for improving skin aging or skin repair is provided.

[0045] The product can be a cosmetic, a medical device (such as a medical dressing), or a drug.

[0046] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0047] The contents not described in detail in the examples are all according to the existing conventional technology in the art; the experimental materials and reagents not described in detail are all ordinary commercially available products. Among them, the Pichia pastoris GS115 is purchased from Biyun Tian Biotechnology Co., Ltd., the pPIC9K vector and Escherichia coli DH5a are provided by Beijing Lihe Huada Gene Technology Co., Ltd., and are all conventional commercially available products.

[0048] Example 1: Selection and codon optimization of recombinant humanized XVII type collagen SP1502, TA1502 and AN1502 sequences (1) According to the full-length sequence of human XVII type collagen in Genbank (Genbank accession number: Q9UMD9.3), the 644-673 amino acids in the 15th collagen region were taken as a repeating unit, and the collagen segment was obtained after 10 times of repeating splicing of the repeating unit, and the total number of amino acids was 300.

[0049] The amino acid sequence of the collagen segment is: GERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSP, as shown in SEQ ID NO: 1.

[0050] (2) The N-terminus of the collagen segment obtained in step (1) is modified by adding a transmembrane peptide, the amino acid sequence of the SPACE transmembrane peptide is ACTGSTQHQCG, as shown in SEQ ID NO: 2; the amino acid sequence of the TAT transmembrane peptide is RKKKRRQRRR, as shown in SEQ ID NO: 3; the amino acid sequence of the ANTP transmembrane peptide is RQIKIYFQNRRMKWKK, as shown in SEQ ID NO: 4.

[0051] The SPACE transmembrane peptide is derived from a modified sequence of a skin penetration-related natural peptide, which contains two cysteines that can form disulfide bonds and has an amphiphilic structure, can specifically penetrate the cell membrane of skin keratinocyte cells or tumor cells, and has low cytotoxicity.

[0052] The TAT transmembrane peptide is derived from the 47-57 amino acid fragment of the transcriptional activator TAT protein of HIV-1 virus, which contains six positively charged arginines / lysines and is a typical cationic transmembrane peptide that can efficiently penetrate the cell membrane without the need for receptor mediation, with a transmembrane efficiency of more than 85% within 1 h at 37°C.

[0053] The ANTP transmembrane peptide is derived from the 43-58 amino acid fragment of the antennal control gene encoding protein of Drosophila, which contains five positively charged amino acids and can realize transmembrane by interacting with the cell membrane phospholipid bilayer, and has a better penetration rate on the nuclear membrane than the TAT transmembrane peptide.

[0054] In addition, the two ends of the collagen segment were modified respectively. A start codon sequence and a membrane-penetrating peptide sequence were added to the amino terminus, and a DNA sequence encoding a stop codon was added to the carboxyl terminus to form the amino acid sequences encoding SP1502, TA1502 and AN1502 proteins. The modified amino acid sequences are shown below, with the underlined part being the membrane-penetrating peptide sequence.

[0055] The amino acid sequence of TA1502 is as follows: M ACTGSTQHQCG GERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGE RGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSP, such as SEQ ID NO: 5 is shown.

[0056] The amino acid sequence of AN1502 is as follows: M RKKKRRQRRR GERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGE RGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSP, such as SEQ ID NO: 6.

[0057] The amino acid sequence of SP1502 is as follows: M RQIKIYFQNRRMKWKKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGE RGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGERGAAGEPGPHGPPGVPGSVGPKGSSGSP, such as SEQ ID NO: 7 is shown.

[0058] (3) GenScript was commissioned to optimize the codons of the above amino acids for the Pichia pastoris expression system and synthesize them, and then cloned them into the pPIC9K vector. EcoR I, Not Between the I restriction sites.

[0059] The DNA sequences encoding the proteins SP1502, TA1502, and AN1502 are shown below: The DNA sequence encoding the SP1502 protein is: ATGAGACAGATTAAAATCTACTTCCAAAACCGTAGGATGAAATGGAAAAAGGGAGAGAGAGGTGCCGCTGGAGAGCCTGGACCACATGGTCCACCAGGAGTCCCAGGTTCAGTTGGACCTAAAGGTTCTTCTGGTTCCCCCGGCGAGAGAGGAGCTGCTGGTGAACCTGGCCCACACGGTCCACCAGGTGTTCCTGGCTCAGTAGGACCTAAAGGTTCTTCCGGAAGTCCTGGTGAAAGAGGAGCCGCCGGTGAACCTGGCCCCCATGGCCCTCCAGGTGTCCCAGGTTCAGTAGGACCAAAGGGATCCTCTGGATCTCCTGGAGAAAGGGGAGCTGCCGGAGAGCCAGGACCACACGGACCACCTGGTGTTCCAGGATCAGTCGGACCTAAAGGATCTAGTGGAAGTCCTGGAGAAAGAGGCGCTGCTGGTGAACCTGGTCCTCACGGACCCCCTGGTGTTCCTGGAAGTGTGGGACCAAAGGGATCATCCGGTTCTCCAGGTGAGAGAGGAGCAGCTGGCGAGCCCGGACCCCATGGTCCCCCTGGAGTCCCTGGCTCCGTCGGCCCTAAGGGTTCATCAGGATCTCCTGGAGAGCGTGGAGCTGCTGGTGAGCCAGGACCTCATGGACCACCTGGCGTACCAGGCAGTGTGGGACCTAAGGGCTCCTCAGGTTCTCCAGGCGAGAGAGGTGCCGCAGGCGAACCTGGTCCACATGGTCCACCTGGAGTTCCTGGTTCAGTTGGTCCTAAGGGATCATCTGGTTCTCCTGGTGAAAGGGGTGCCGCTGGTGAGCCCGGACCACACGGACCCCCAGGAGTTCCAGGTTCAGTTGGACCAAAGGGTTCTTCAGGTTCCCCTGGCGAAAGAGGTGCTGCCGGTGAACCTGGACCACATGGACCTCCTGGTGTTCCAGGTTCTGTTGGACCTAAGGGCTCATCCGGATCTCCA, as set forth in SEQ ID NO: 8.

[0060] The DNA sequence encoding the TA1502 protein is: ATGGCTTGCACTGGTTCTACTCAGCATCAATGTGGAGGTGAAAGAGGAGCAGCTGGTGAACCTGGTCCCCACGGACCACCCGGTGTTCCTGGAAGTGTTGGACCTAAGGGTTCTTCCGGCAGTCCAGGTGAGAGAGGTGCAGCTGGTGAGCCCGGTCCTCACGGTCCCCCAGGTGTACCAGGTTCTGTCGGCCCTAAGGGTTCAAGTGGATCTCCAGGCGAGAGAGGCGCCGCTGGTGAACCCGGACCACATGGTCCTCCAGGTGTTCCTGGATCTGTTGGCCCAAAAGGTTCTTCAGGATCTCCAGGTGAGAGAGGAGCTGCTGGTGAACCTGGTCCTCATGGTCCACCCGGTGTGCCTGGTTCTGTTGGTCCCAAAGGATCATCAGGTTCCCCTGGAGAGCGTGGTGCTGCTGGTGAACCTGGACCTCACGGTCCACCCGGAGTTCCAGGTTCAGTTGGTCCAAAGGGTTCCTCAGGTAGTCCTGGAGAGAGAGGTGCCGCAGGTGAGCCAGGTCCTCATGGTCCTCCAGGTGTGCCAGGATCCGTAGGACCCAAAGGTTCTTCCGGATCTCCAGGAGAGAGGGGTGCCGCAGGTGAACCTGGTCCACACGGTCCACCTGGTGTTCCTGGCTCAGTCGGTCCAAAAGGTTCATCCGGTAGTCCAGGTGAAAGAGGTGCCGCCGGCGAACCAGGTCCTCATGGACCTCCTGGTGTGCCAGGTTCTGTTGGCCCAAAGGGTTCTAGTGGTAGTCCAGGAGAAAGGGGTGCTGCTGGAGAACCAGGTCCACACGGACCCCCAGGAGTGCCAGGTTCCGTAGGACCTAAGGGAAGTTCAGGAAGTCCTGGAGAAAGAGGAGCAGCCGGAGAGCCTGGCCCACATGGTCCTCCTGGAGTTCCAGGTTCTGTTGGACCTAAGGGATCCTCTGGTAGTCCC, as set forth in SEQ ID NO: 9.

[0061] The DNA sequence encoding the AN1502 protein is: ATGAGGAAAAAAAAGAGACGTCAAAGAAGAAGAGGTGAAAGAGGCGCTGCTGGAGAACCAGGTCCACACGGACCTCCAGGTGTACCTGGTTCCGTCGGCCCTAAAGGCAGTAGTGGAAGTCCTGGTGAAAGAGGTGCAGCAGGAGAACCTGGTCCCCATGGACCCCCAGGAGTTCCTGGTTCCGTTGGTCCAAAGGGTTCTTCTGGAAGTCCAGGAGAAAGAGGAGCTGCTGGTGAGCCTGGACCACATGGACCCCCTGGTGTTCCTGGTTCCGTTGGACCAAAGGGCTCCTCTGGTTCACCCGGTGAAAGAGGTGCTGCTGGTGAACCAGGCCCTCACGGTCCACCTGGCGTCCCCGGTTCTGTAGGACCCAAGGGATCATCTGGCAGTCCAGGCGAACGAGGTGCTGCAGGAGAACCTGGTCCTCACGGTCCTCCCGGAGTGCCTGGTTCCGTAGGTCCTAAAGGAAGTAGTGGTTCTCCTGGTGAACGTGGTGCCGCTGGAGAACCAGGACCTCATGGCCCCCCTGGTGTCCCAGGATCCGTCGGACCTAAGGGATCCTCAGGATCCCCAGGCGAGAGAGGAGCAGCTGGTGAGCCAGGTCCACATGGTCCACCCGGTGTTCCCGGAAGTGTTGGACCAAAAGGTAGTTCCGGTAGTCCAGGAGAGAGAGGTGCAGCTGGCGAGCCTGGACCACACGGACCACCTGGTGTTCCAGGTTCAGTGGGCCCAAAAGGATCCAGTGGATCTCCAGGTGAGAGAGGCGCCGCCGGTGAACCAGGTCCTCACGGTCCTCCTGGAGTCCCTGGTTCAGTAGGTCCTAAAGGTAGTTCTGGTTCTCCTGGTGAGAGGGGTGCTGCAGGTGAACCAGGACCACACGGACCTCCTGGCGTTCCAGGATCTGTAGGTCCCAAGGGCAGTTCTGGCTCTCCA, as set forth in SEQ ID NO: 10.

[0062] Example 2 Construction and screening of recombinant expression strains of high transdermal recombinant humanized collagen XVII SP1502, TA1502, AN1502 (1) The recombinant constructed vector was verified by DNA sequencing, and the recombinant expression vector pPIC9K- sp1502, pPIC9K- ta1502, pPIC9K- an1502 was constructed.

[0063] (2) Extract the correct recombinant plasmid, take 10 μg plasmid, cut with sal I fast enzyme (reagent purchased from Takara company, specific operation according to reagent box instruction book) at 37℃ for 10 min, and then recover the linearized product by using PCR product purification kit (purchased from Shengong Bioengineering (Shanghai) Co., Ltd.), and the elution volume is 10 μL.

[0064] (3) Preparation of Pichia pastoris GS115 competent cells: pick Pichia pastoris GS115 single colony into YPD liquid medium, cultivate at 30℃, 220 rpm overnight, transfer 1% by volume to YPD liquid medium, cultivate to OD 600 1.3-1.5, take 50 mL bacterial liquid, ice bath for 10 min, centrifuge at 4000 rpm, 4℃, then collect the bacterial body, add pre-cooled 1 M D-sorbitol for washing twice, add 5 mL 1M D-sorbitol, mix well, then distribute, 100 μL / branch.

[0065] The above YPD liquid medium formula is: yeast powder 10 g / L, peptone 20 g / L, glucose 20 g / L, solvent is water.

[0066] (4) Electroporation: take 1 branch of the above Pichia pastoris GS115 competent cells, add 10 μL linearized recovery product, select PIC program in electroporation instrument (purchased from Bole) for electric shock once, quickly take out and add 1 mL pre-cooled 1M D-sorbitol, mix well, then static culture for 2-3 h, centrifuge at 4000 rpm, then remove part of supernatant, mix well by blowing the remaining liquid, then spread MD plate, cultivate at 30℃ for 2-4 days, until single colony grows.

[0067] The above MD medium formula is: amino acid-free yeast nitrogen source (YNB) 13.4 g / L, glucose 10 g / L, agar 2 g / L, biotin 4×10 -4 g / L, solvent water.

[0068] (5) G418 screening: configure G418 YPD solid medium with a concentration of 4 g / L, draw a 1cm*1cm grid and number, and then transfer the single colony to the screening medium, and culture at 30°C for 2-4 days until the colony size is obviously different.

[0069] (6) Pick the single colony with the largest diameter for shake flask culture, extract the genome, and then perform PCR verification. The correct strain is preserved after verification.

[0070] (7) The correct strain verified above is inoculated into YPD medium and cultured at 30°C, 220 rpm overnight. The culture is inoculated into 50 mL BMGY medium at a volume ratio of 2%, and cultured at 30°C, 220 rpm for 24 h. The culture is centrifuged at 5000 rpm for 5 min, washed once with sterile water, resuspended in 5 mL BMMY medium, and then inoculated into 100 mL BMMY medium. The culture is induced at 25°C, and 1% methanol is added every 24 h. The sample is stored at -20°C until the induction is completed at 96 h.

[0071] The BMGY medium has the following formulation: yeast powder 10 g / L, peptone 20 g / L, 100 mM potassium phosphate buffer (pH 6.0), YNB 13.4 g / L, biotin 4×10 -4 g / L, glycerol 10 g / L, and water as solvent.

[0072] The BMMY medium has the following formulation: yeast powder 10 g / L, peptone 20 g / L, 100 mM potassium phosphate buffer (pH 6.0), YNB 13.4 g / L, biotin 4×10 -4 g / L, methanol 5 g / L, and water as solvent.

[0073] Example 3 Fermentation and analysis of high-transdermal recombinant humanized collagen XVII SP1502, TA1502 and AN1502 The recombinant engineering bacteria Pichia.pastoris GS115 sp1502 , Pichia.pastoris GS115 ta1502 , Pichia.pastoris GS115 an1502 are inoculated into YPD liquid medium and cultured at 30°C, 220 rpm overnight to obtain primary seed. The culture is inoculated into secondary seed flask at a volume ratio of 1%, and cultured at 30°C, 220 rpm until the OD 600The pH range was 8.0-12.0. Fermentation was conducted using BSM fermentation medium, with successfully activated secondary seed culture inoculated into the fermenter at a 10% (v / v) inoculum. The initial culture temperature was 30 °C, the agitator speed was 200 rpm, and aeration was 1 vvm. The pH was adjusted to 5.0 using 28% ammonia. As the cells grew, dissolved oxygen gradually decreased. When dissolved oxygen dropped to 20%, the dissolved oxygen (DO) in the fermenter was maintained above 20% by adjusting the dissolved oxygen-correlated agitation speed. After 18-20 h of fermentation, the glycerol in the medium was depleted, and the DO began to rise. Dissolved oxygen feedback agitation was used, and when the DO exceeded 30%, glycerol was fed at a rate of 1.0 mL / min. Feeding was stopped when the wet cell volume reached 250-400 g / L, and the agitator speed was increased to 900 rpm. Once the DO stabilized, methanol was added to induce induction. The induction temperature was 25 ℃. For the first 4 hours of induction, the methanol feed solution was fed at a rate of 0.06 mL / min to promote the cell adaptation to the carbon source and conversion to methanol. Feeding was stopped after 4 hours of induction. Once the dissolved oxygen (DO) value began to rise, indicating that the methanol in the fermenter had been consumed, the remaining methanol feed solution was added under dissolved oxygen feedback conditions at a rate of 0.12 mL / min. Simultaneously, dissolved oxygen was monitored, and feeding was initiated when the DO value was maintained above 30%. Induction continued for 48 hours to obtain the fermentation broth. Samples were taken every 12 hours during the induction period for subsequent analysis and detection.

[0074] SDS-PAGE analysis was performed on samples taken at each time point, and the results are as follows: Figure 1 As shown in figures a and b, after 12 hours of induced fermentation, the characteristic band corresponding to the target protein began to appear clearly. With the induction time extended to 48 hours, the grayscale value of the target protein band continued to increase, and no significant degradation bands were produced. Grayscale analysis of the gel image of the sample induced for 48 hours using ImageJ software showed that the target protein band accounted for ≥80% of the total protein bands. These results indicate that the recombinant protein described in this embodiment can be stably expressed without significant degradation during 48 hours of continuous fermentation induction, demonstrating good expression stability.

[0075] The above BSM fermentation medium formula is as follows: phosphate (85%) 26.7 mL / L, calcium sulfate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40 g / L, with water as the solvent; after sterilization at 121 ℃ for 20 min, PTM1 trace element stock solution is added at 4.35 mL / L after the temperature drops to room temperature, and the pH is adjusted to 5.0 with 50% (v / v) ammonia water. The above-mentioned glycerol feed solution formula is: 50% (mass ratio) glycerol, after sterilization at 121 ℃ for 20 min, add PTM1 trace element mother liquor at 12 mL / L after the temperature drops to room temperature, mix well; The above-mentioned methanol feed solution formula is: 100% methanol, add PTM1 trace element mother liquor at 12 mL / L, mix well; The above-mentioned trace element mother liquor PTM1 formula is: copper sulfate 6 g / L, sodium iodide 0.08 g / L, manganese sulfate 3 g / L, sodium molybdate 14.9 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate heptahydrate 65 g / L, biotin 0.2 g / L, sulfuric acid 5 mL / L, solvent is water; filter sterilization with a 0.22 μm filter membrane, store at 4 ℃ for standby.

[0076] Example 4 Purification of high transdermal recombinant humanized type XVII collagen SP1502, TA1502 and AN1502 (1) Sample treatment: collect the fermentation supernatant of Example 3, centrifuge at 8000 rpm for 10 min at 4 ℃, discard the precipitate, collect the supernatant, and filter with a 0.45 μm filter membrane to obtain a filtrate, adjust the pH of the filtrate to 5.0 with dilute acetic acid, and adjust the conductivity to below 10 mS / cm, and store on ice for standby.

[0077] (2) Equilibration of chromatography column: select a multi-mode weak cation exchange chromatography filler, the filler is Diamond MM Mustang (purchased from Bioron (Shanghai) Biotechnology Co., Ltd.), use buffer solution Buffer A as equilibration liquid, equilibrate for 5 column volumes, until the pH value and conductivity of the effluent are stable and consistent with Buffer A.

[0078] (3) Sample loading and elution: load the filtrate of step (1) into the equilibrated chromatography column, after loading is completed, rinse with buffer solution Buffer A until the baseline is flat, and then elute for 3 column volumes; to improve the purity of the target protein, use a 10% B eluent for impurity removal, rinse for 4 column volumes; then use 40% B for elution, start collecting after the OD220 peak appears, collect for 4 column volumes; (4) Regeneration and preservation of chromatography column: regenerate the chromatography column with 3 column volumes of buffer C solution, then pass through deionized water, rinse until the conductivity of the effluent is stable and the pH is 7.0±0.2, then pass through 3 column volumes of 20% ethanol to preserve the column, store at 4 ℃.

[0079] (5) Desalting and freeze-drying: the purified elution solution is concentrated and desalted by using a membrane with a molecular weight cut-off of 10 kDa, concentrated to a sample concentration of 10-20 g / L, desalted with pure water to a conductivity of ≤50 μS / cm, and then freeze-dried to obtain a freeze-dried powder.

[0080] (6) The purity of the product is detected by HPLC, the chromatographic column is BioCore SEC-150, the mobile phase is 50 mM phosphate buffer (pH 6.8) containing 300 mM sodium chloride, isocratic elution, the flow rate is 0.5 mL / min, the injection volume is 2 μL, the column temperature is 30 °C, the freeze-dried powder is diluted to 1 mg / mL for injection, and detection is performed at 220 nm, the chromatogram is as shown in Figure 2 , and the purity is 97.58%.

[0081] The formula of Buffer A is: 50 mmol / L sodium acetate, and the pH is adjusted to 5.0 by using acetic acid. The formula of Buffer B is: 50 mmol / L sodium acetate, 1 mol / L NaCl, and the pH is adjusted to 5.0 by using acetic acid. The formula of Buffer C is: 1 mol / L NaOH.

[0082] Example 5: Transdermal performance test of recombinant protein To verify the skin penetration performance of the cell-penetrating peptide modified recombinant humanized type XVII collagen SP1502, TA1502 and AN1502 according to the present application, the following experiments were performed. (1) Experimental materials Control group: recombinant humanized type XVII collagen COL1502 (fluorescently labeled, consistent with the collagen segment of the experimental group protein, purity ≥95%); Experimental group 1: recombinant humanized type XVII collagen SP1502 (fluorescently labeled, purity ≥95%); Experimental group 2: recombinant humanized type XVII collagen TA1502 (fluorescently labeled, purity ≥95%); Experimental group 3: recombinant humanized type XVII collagen AN1502 (fluorescently labeled, purity ≥95%); All samples were prepared into a concentration of 10 mg / mL with PBS buffer at pH 7.4.

[0083] Ex vivo skin: the skin on the back of a 3-5 day old piglet was removed, cut into 2 cm × 2 cm after removing the subcutaneous fat, and then sterilized with 0.2% glutaraldehyde and washed with PBS to balance before use.

[0084] (2) Experimental method Diffusion cell preparation: the receiving chamber was added with 37℃ preheated PBS buffer (containing 0.02% sodium azide), magnetic stirring at 300 rpm, maintaining the temperature at 37±0.5℃; the stratum corneum of the piglet skin was fixed between the two chambers of the diffusion cell with the stratum corneum facing up, and the effective diffusion area was 1.77 cm 2 .

[0085] Sample administration: 5 mL of fluorescently labeled test sample was added to the supply chamber, slowly injected into the supply pool, and the timer was started. At 0 h, 4 h, and 8 h, 200 μL of the collected receiving chamber buffer was taken, and an equal amount of fresh PBS buffer preheated to 37℃ was added immediately; Fluorescence quantification: the receiving chamber samples collected at each time point were placed in a centrifuge tube, centrifuged at 4℃ and 5000 rpm for 10 min, and 180 μL of the supernatant was added to a 96-well fluorescence plate for detection in the dark; Instrument and parameters: Thermo Scientific Varioskan LUX enzyme label instrument was used, with excitation wavelength set at 488 nm, emission wavelength set at 525 nm, sensitivity level set at "high", and detection temperature set at 37±0.5℃ (consistent with the temperature of the diffusion cell); Detection operation: three replicate wells were set for each sample, and a blank control (receiving chamber buffer without sample) was also set. The relative fluorescence units (RFU) of each well were read by the enzyme label instrument, and the average value of the replicate wells was taken. The blank control RFU value was subtracted to obtain the net fluorescence intensity of the receiving chamber samples at each time point; (3) Experimental results: the test results are shown in Figure 3 . Time trend: the fluorescence accumulation of the four groups of samples significantly increased with the prolongation of penetration time (P<0.01), indicating that they all had transdermal ability and the penetration amount was positively correlated with time.

[0086] Inter-group comparison: at the same time point, the fluorescence intensity of experimental group 2 (TA1502) was significantly higher than that of the control group and other experimental groups. For example, at 8 h of penetration, the fluorescence intensity of experimental group 2 was 2.2 times that of the control group, 1.5 times that of experimental group 1 (P<0.001), and 1.2 times that of experimental group 3 (P<0.001).

[0087] Example 6: Barrier repair performance test of recombinant protein TA1502 Filaggrin (FLG) is one of the important components of the keratin envelope, which ensures the integrity of the skin barrier. FLG deficiency can cause disorder of the lipid bilayer structure, delay of maturation, and decrease of cell tightness, intercellular permeability and light protection, ultimately leading to damage of the skin barrier.

[0088] HaCaT cells were collected and prepared into a cell suspension with high-sugar DMEM cell culture solution, 2 mL of the cell suspension was added to each well of a 6-well plate, and the cell number was 2.6 x 10 5 / well. Blank control (BC), bovine serum albumin BSA negative control (NC), commercially available recombinant collagen positive control (PC), and recombinant collagen TA1502 prepared by the present application (experimental group) were set up, and each group had three replicate wells. The 6-well plate was placed in a cell incubator (5% CO2, 37°C) for 24 h, and when the cell fusion rate reached 50%-60%, the culture solution was discarded, 2 mL of high-sugar DMEM cell culture solution was added to each well of the blank control group; 2 mL of high-sugar DMEM cell culture solution containing the corresponding protein was added to the other three groups, and the concentration of each protein in the high-sugar DMEM cell culture solution was 2.5 mg / mL. After incubation in the incubator (37°C, 5% CO2) for 24 h, the cells in each well were washed twice with 2 mL of PBS buffer, and the RNA extraction, reverse transcription, and fluorescence quantitative PCR detection experiments were carried out according to the instructions of the Total RNA extraction kit. The mRNA relative expression of the barrier-related protein FLG was detected, and the 2 -△△CT method was used for calculation.

[0089] The mRNA expression promoting ability of different collagens in HaCaT cells is shown in Figure 4 Compared with the BC group, the FLG expression of the NC group was significantly reduced, indicating that the stimulation conditions of this experiment were effective; compared with the NC group, the FLG expression of the PC group was significantly increased, indicating that the detection of the positive control group was effective; compared with the NC group, the FLG expression of the recombinant collagen TA1502 at a concentration of 2.5 mg / mL was significantly increased. In summary, the recombinant collagen TA1502 at a concentration of 2.5 mg / mL can significantly promote the expression of the FLG gene in UVB-induced human immortalized keratinocytes HaCaT, and has a repair effect.

[0090] Example 7: Performance test of the tightening and anti-wrinkle effect of the recombinant protein Collagen is the main component of dermal collagen fibers, mainly maintaining skin tension, among which type I collagen (COL-I) accounts for 80-85% of the collagen component of adult skin. Elastin (ELN) is the main component of skin elastic fibers, which forms a three-dimensional fiber network structure with collagen to give the skin elasticity and toughness. Both of them maintain the skin firm and smooth, and reduce wrinkles. Based on the UVA-induced human primary skin fibroblast HSF aging model, after the action of the test substance, the content changes of COL-I and ELN were detected to evaluate the firming and anti-wrinkle efficacy of the test substance.

[0091] Take out the HSF cell cryopreservation tube from the liquid nitrogen tank, quickly melt it in a 37°C constant temperature water bath (1-2 min), add 9 mL of preheated complete medium, centrifuge at 1000 rpm for 5 min, then discard the supernatant, resuspend the cells with 5 mL of complete medium and inoculate into a T25 culture flask, incubate in a 37°C, 5% CO2 incubator until the cell confluence rate reaches 80%-90% for passage; when passing, discard the old culture medium of the culture flask, wash it twice with PBS (pH 7.4), add 0.25% trypsin-EDTA, incubate at 37°C for 3-4 min, then add 5 mL of complete medium to terminate digestion, blow it into a single cell suspension, adjust the concentration to 1.3×10 5 cells / mL, add 2 mL of cell suspension to each well of a 6-well plate (2.6×10 5 cells per well), and incubate in a 37°C, 5% CO2 incubator for 24 h; observe the HSF cells (spindle-shaped and evenly distributed) under an inverted microscope when the confluence rate reaches 50%-60%, and then divide them into groups for treatment, with 3 replicate wells in each group. The blank control group (BC) discards the old culture medium and adds 2 mL of fresh complete medium, the negative control group (NC) adds 2 mL of complete medium containing 2.5 mg / mL BSA, the positive control group (PC) adds 2 mL of complete medium containing 2.5 mg / mL of commercially available recombinant collagen protein, and the experimental group (TA1502 group) adds 2 mL of complete medium containing 2.5 mg / mL of recombinant collagen protein TA1502. Then put them back into the incubator for continuous incubation for 24 h (error <15 min). After incubation, carefully aspirate the supernatant of each well into a 1.5 mL centrifuge tube, centrifuge at 4°C, 12000 r / min for 10 min, remove the cell debris, take the supernatant, and perform ELISA detection according to the instructions of the COL-I ELISA kit and the ELN ELISA kit.

[0092] For example, Figure 5As shown in a, compared with the BC group, the COL-I content in the NC group was significantly lower, indicating that the stimulation conditions in this experiment were effective; compared with the NC group, the COL-I content in the PC group was significantly higher, indicating that the positive control group was effective; compared with the NC group, the COL-I content of recombinant collagen TA1502 at a concentration of 2.5 mg / mL was significantly higher. Compared with the BC group, the ELN content in the NC group was significantly lower, as shown in a diagram. Figure 5 As shown in b, this indicates that the stimulation conditions in this experiment were effective. Compared with the NC group, the ELN content in the PC group was significantly increased. Compared with the NC group, the ELN content of recombinant collagen TA1502 at a concentration of 2.5 mg / mL was significantly increased. In conclusion, recombinant collagen TA1502 at a concentration of 2.5 mg / mL can significantly promote the increase of COL-I and ELN content in UVA-induced human primary skin fibroblasts (HSF), and has a firming and anti-wrinkle effect.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A highly transdermally permeable recombinant humanized collagen type 17, characterized by, consisting of a transmembrane peptide segment and a collagen segment, the collagen segment being an amino acid sequence as shown in SEQ ID NO: 1, the transmembrane peptide segment being a transmembrane peptide SPACE, TAT or ANTP; the amino acid sequence of the transmembrane peptide SPACE is shown in SEQ ID NO: 2; the amino acid sequence of the transmembrane peptide TAT is shown in SEQ ID NO: 3; the amino acid sequence of the transmembrane peptide ANTP is shown in SEQ ID NO:

4.

2. The high-transdermic recombinant humanized type 17 collagen according to claim 1, characterized by, The transmembrane peptide segment is modified at the N-terminus of the collagen segment; Or, the amino acid sequence of the high skin permeability recombinant humanized collagen type XVII is shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:

7.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule for encoding the high skin permeability recombinant humanized collagen type XVII as claimed in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The DNA sequence of the nucleic acid molecule is shown in SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

5. A recombinant vector, characterized in that, The nucleic acid molecule as claimed in claim 3 or 4.

6. The recombinant vector of claim 5, wherein, The recombinant vector adopted is pPIC9K.

7. A recombinant cell, characterized in that, It is a host cell containing the nucleic acid molecule as claimed in claim 3 or 4 or the recombinant vector as claimed in claim 5 or 6.

8. A method for preparing a high transdermal recombination humanized collagen type 17, characterized by, The steps include: culturing the recombinant cell as claimed in claim 7, so as to express the high skin permeability recombinant humanized collagen type XVII; and isolating the high skin permeability recombinant humanized collagen type XVII.

9. The method of claim 8, comprising The steps are as follows: (1) activating the above-mentioned recombinant engineering bacteria; (2) stopping feeding when the wet bacterial mass reaches 250-400 g / L, and adding methanol in stages to induce for 48-96 h to obtain a fermentation broth; (3) using a multi-mode weak cation exchange chromatography filler to purify the obtained collagen protein to obtain the high skin permeability recombinant humanized collagen type XVII.

10. Use of the high skin permeability recombinant humanized collagen type XVII as claimed in claim 1 or 2 or the nucleic acid molecule as claimed in claim 3 or 4 or the recombinant vector as claimed in claim 5 or 6 or the recombinant cell as claimed in claim 7 in the preparation of a product for improving skin aging or skin repair.

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