Recombinant human-derived collagen xvii fragment based on natural collagen sequence, and preparation method and application thereof

Abstract

The application provides a recombinant collagen XVII fragment, and the protein fragment sequence is shown as SEQ ID No. 1. The recombinant collagen XVII fragment is designed based on a natural collagen sequence, has a triple helix structure, and has good biological activity, and can effectively play a function, and has a good promoting effect on cell migration, cell proliferation and cell adhesion. The method for preparing the recombinant collagen XVII fragment has simplified process steps, improves the expression efficiency of the recombinant protein, and reduces the production cost. The recombinant collagen XVII fragment has good mechanical properties and biological activity, and can be used for preparing a therapeutic drug and a biological material.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for screening, optimizing and preparing recombinant human type XVII collagen fragments based on natural collagen sequences, the obtained recombinant human type XVII collagen fragments and their applications in the biomedical field. Background Technology

[0002] Type XVII collagen is a transmembrane protein mainly distributed in the junctional regions between epithelial and dermal cells, participating in maintaining the integrity of the extracellular matrix and cell signaling. Type XVII collagen plays a crucial role in dermatology, wound healing, and inflammation-related diseases. However, current research on this protein primarily focuses on the functional analysis of the full-length protein, lacking screening and optimization of its functional fragments. Therefore, developing a bioactively enhanced recombinant human type XVII collagen by screening natural collagen sequences has significant scientific and practical value.

[0003] Bovine type I collagen is currently the most widely used natural collagen material. Due to its stable source, low cost, and structural similarity to human collagen, it is widely used in tissue repair, wound dressings, injectable fillers, cell culture scaffolds, and skincare products. Multiple studies have shown that bovine type I collagen can promote cell adhesion, proliferation, and tissue regeneration to a certain extent, particularly in promoting wound closure and cell adhesion. Because it provides good extracellular matrix structural support, bovine type I collagen is one of the most mature collagen raw materials in current clinical and cosmetic applications. However, bovine collagen has relatively weak cell migration and proliferation-promoting activity, which is a limiting factor in scenarios requiring rapid cellular response and tissue regeneration (such as chronic trauma and dermal repair). Furthermore, bovine collagen is a foreign protein, posing certain risks of immunogenicity and potential animal-derived contamination, thus limiting its use in the development of novel medical or cosmetic products with higher safety requirements.

[0004] In recent years, recombinant human type XVII collagen has gradually entered the research and development stage. According to patent publication data (such as CN116640205A, CN119462893A, CN119504982A), various type XVII fragment molecules have been screened for use in tissue repair, wound dressings, injectable fillers, cell culture scaffolds, and skin care products. CJ-XVII type collagen (hereinafter referred to as Competitor 1) developed by Chuangjian Medical Technology Co., Ltd. is currently the mainstream type XVII collagen molecule on the market (disclosed in CN113185604B). Multiple studies and product data show that Competitor 1 has good cell adhesion and cell migration promotion capabilities, but its cell proliferation promotion effect has not been reported. Although it has characteristic infrared spectra of amide I, II, and III, its stability and structure have not yet been confirmed.

[0005] The XVII fragment, which possesses high biological activity, high thermal stability, and a complete triple helix structure, has not yet been developed, thus failing to provide a foundation for further enhancing the application of collagen.

[0006] Based on the current state of technological development in this field, there is an urgent need to provide human type XVII collagen with high thermal stability, a complete triple helix structure, and good biological activity. Summary of the Invention

[0007] The purpose of this application is to provide a method for preparing recombinant human type XVII collagen fragments based on natural collagen sequence screening and optimization, to obtain functional protein fragments with high activity, high stability, and easy expression. Furthermore, this application also relates to the potential applications of this protein in the biomedical field, including but not limited to therapeutic proteins, diagnostic reagents, and drug screening platforms.

[0008] Based on this, a first aspect of this application is to provide a recombinant type XVII collagen fragment, the protein fragment sequence of which is shown in SEQ ID No. 1.

[0009] Type XVII collagen (also known as COL17 / BP180 / BPAG2) is a transmembrane protein located between the epidermis and dermis with a unique key structure. It plays an important role in basement membrane repair and epidermal anti-aging. It is a key factor in skin aging and wound repair, helping to maintain "youthful" skin and maintain hair follicle stem cells (for hair loss, gray hair, etc.). In the process of skin wound repair, type XVII collagen plays an important role by influencing the migration, proliferation and differentiation of stem cells.

[0010] The recombinant type XVII collagen fragment of this application can have a protein sequence with ≥75% homology to the protein sequence shown in SEQ ID No. 1, for example, it can be 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology.

[0011] Homology refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules. Sequence analysis is the ultimate method for determining the degree of homology, and DNA-DNA hybridization or DNA-RNA hybridization are also useful estimation methods.

[0012] A second aspect of this application is to provide a nucleic acid gene encoding the recombinant type XVII collagen fragment.

[0013] Due to the degeneracy of codons, the nucleic acid encoding the recombinant type XVII collagen fragment described in this application can have multiple codons.

[0014] A third aspect of this application is to provide an expression vector thereon recombinantly encoded with nucleic acid encoding the recombinant type XVII collagen fragment.

[0015] Expression vectors are vectors that add expression elements (such as promoters, RBSs, terminators, etc.) to the basic backbone of cloning vectors, enabling the expression of target genes. In this application, the target gene to be expressed is the XVII type collagen gene. Expression vectors can be adapted to specific host cells, such as expression vectors used for expression in yeast cells.

[0016] As some embodiments of this application, the expression vector includes pCM01-COL17-1.

[0017] A fourth aspect of this application is to provide an expression cell comprising a nucleic acid encoding the recombinant type XVII collagen fragment or comprising the expression vector.

[0018] As some embodiments of this application, the expression cells include Pichia pastoris strains.

[0019] Pichia pastoris is a type of methanol-nutritional yeast that can utilize methanol as its sole carbon and energy source. Commonly used Pichia pastoris host strains include GS115 and KM71. Because the fermentation system used in this application employs a yeast expression system, the recombinant type XVII collagen obtained through fermentation exhibits good biological activity.

[0020] As some embodiments of this application, the expression cells include Pichia pastoris strain GS115.

[0021] A fifth aspect of this application is to provide a method for preparing a recombinant type XVII collagen fragment, the method comprising seeding the expression cells in a culture medium to express the recombinant type XVII collagen fragment.

[0022] As some embodiments of this application, the culture medium contains 1-9 g / L potassium dihydrogen phosphate, 0.1-5 g / L calcium sulfate dihydrate, 5-15 g / L magnesium sulfate heptahydrate, 5-15 g / L potassium sulfate, 10-80 g / L ammonium dihydrogen phosphate, 0.1-5 g / L potassium hydroxide, and 5-25 g / L glycerol.

[0023] The potassium dihydrogen phosphate content in the culture medium described in this application can be selected from 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6. 9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9g / L.

[0024] The content of calcium sulfate dihydrate can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 g / L.

[0025] The content of magnesium sulfate heptahydrate can be selected from 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8 4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9 1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 1 2.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13. 6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15g / L.

[0026] The potassium sulfate content can be selected from 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, and 8. 4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9. 1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12 .2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6 , 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15g / L.

[0027] The content of diammonium phosphate can be selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, and 80 g / L.

[0028] The potassium hydroxide content can be selected from 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5 g / L.

[0029] The glycerol content can be selected from 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8 1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11 3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 1 3.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4, 16 5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19. 1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 20.1, 20.2, 20.3, 20.4, 20.5, 20.6, 20.7, 20.8, 20.9, 21, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21 7, 21.8, 21.9, 22, 22.1, 22.2, 22.3, 22.4, 22.5, 22.6, 22.7, 22.8, 22.9, 23, 23.1, 23.2, 23.3, 23.4, 23.5, 23.6, 23.7, 23.8, 23.9, 24, 24.1, 24.2, 24.3. 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25g / L. .

[0030] As some embodiments of this application, the culture medium contains 4-5 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium sulfate dihydrate, 8-10 g / L magnesium sulfate heptahydrate, 8-10 g / L potassium sulfate, 30-40 g / L ammonium dihydrogen phosphate, 1-2 g / L potassium hydroxide, and 10-15 g / L glycerol.

[0031] As some embodiments of this application, the culture medium contains 5 g / L potassium dihydrogen phosphate, 1 g / L calcium sulfate dihydrate, 10 g / L magnesium sulfate heptahydrate, 10 g / L potassium sulfate, 40 g / L ammonium dihydrogen phosphate, 2 g / L potassium hydroxide, and 15 g / L glycerol.

[0032] As some embodiments of this application, the expressed cells are seeded in a culture medium, and glycerol is used as a feed during the bacterial growth phase.

[0033] As some embodiments of this application, after the expression cells have grown, methanol is used to induce the expression of recombinant type XVII collagen fragments.

[0034] In some embodiments of this application, the seeding amount of the expressed cells is 10% v / v.

[0035] As some embodiments of this application, the culture medium further comprises the following components at final concentrations: 24 mg / L CuSO4·5H2O, 12 mg / L MnSO4·H2O, 260 mg / L FeSO4·7H2O, 80 mg / L ZnSO4·7H2O, 0.8 mg / L CoCl2, 0.08 mg / L H3BO3, 0.8 mg / L NaMoO4·2H2O, 0.4 mg / L KI, 0.8 mg / L biotin, and 20 mg / L H2SO4.

[0036] As some embodiments of this application, the fermentation conditions for the expression cell growth stage are a temperature of 30°C and a pH of 4.5.

[0037] As some embodiments of this application, the fermentation conditions for the induction expression stage of the recombinant type XVII collagen fragment are a temperature of 28°C and a pH of 5.0.

[0038] As some embodiments of this application, methanol induction is initiated when the wet weight of the expressed cells reaches 180 g / L-220 g / L.

[0039] As some embodiments of this application, the method further includes a purification step, which separates and purifies the supernatant after the fermentation reaction.

[0040] As some embodiments of this application, the purification step includes first purifying using weak cation exchange chromatography, and then further purifying using hydrophobic chromatography to obtain the recombinant type XVII collagen fragment.

[0041] As some embodiments of this application, a concentration step is added after the purification step.

[0042] A sixth aspect of this application is to provide a composition or article comprising the aforementioned type XVII collagen fragment.

[0043] As some embodiments of this application, the composition is a skin care product or pharmaceutical composition, and the product is one or more of medical devices, biomaterials, tissue engineering products, cosmetics, and health products.

[0044] The seventh aspect of this application is to provide the use of the recombinant type XVII collagen fragment or the recombinant type XVII collagen fragment prepared by the method in the preparation of a drug or article, said drug or article having any of the following functions: a. promoting cell migration; b. promoting cell adhesion; and c. promoting cell proliferation.

[0045] As described above, the recombinant human type XVII collagen based on natural collagen sequences, its preparation method, and its applications of this application have the following beneficial effects:

[0046] 1. The recombinant human type XVII collagen fragment containing the selected sequence exhibits a distinct triple helix structure, ensuring the excellent mechanical properties of the recombinant human type XVII collagen fragment of this application. The recombinant human type XVII collagen fragment of this application possesses typical amide A, I, II, and III bands, exhibiting good thermal stability. Through stability optimization, the recombinant human type XVII collagen fragment of this application demonstrates even higher stability, is less prone to activity loss, and enhances the protein's application potential.

[0047] 2. The recombinant human type XVII collagen fragment of this application has significantly higher bioactivity than the control and can effectively perform its function. It surpasses the mainstream products in the market that have good effects on both cell migration and cell proliferation. It also surpasses bovine collagen, which has good cell adhesion promotion effects in the field, in promoting cell adhesion.

[0048] 3. The method for preparing recombinant human type XVII collagen fragments in this application is mild and preserves the biological activity of the protein.

[0049] 4. The process for preparing recombinant human type XVII collagen fragments in this application is simplified, which improves the expression efficiency of recombinant proteins and reduces production costs.

[0050] 5. This application provides novel molecular tools for type XVII collagen, expanding its application in the biomedical field. Attached Figure Description

[0051] Figure 1 This is a map of the recombinant XVII collagen fragment expression plasmid of this application;

[0052] Figure 2 These are the SDS-PAGE electrophoresis results of samples taken at different induction times for the recombinant XVII collagen fragment expression induced in this application;

[0053] Figure 3 These are the SDS-PAGE electrophoresis results of the recombinant XVII collagen fragment expressed in this application at different concentrations after purification;

[0054] Figure 4 This is a circular dichroism chromatogram of the recombinant type XVII collagen fragment of this application;

[0055] Figure 5 These are the Fourier transform infrared spectra of the recombinant collagen fragment C17-Y5 of this application, the competing product CJ-XVII type collagen, and the standard sigmaIII type collagen.

[0056] Figure 6 , Figure 7 This application compares the effects of the recombinant type XVII collagen fragment of this application with those of the competing product CJ-XVII collagen on the migration of HaCaT and 3T3 cells.

[0057] Figure 8 The effects of the recombinant type XVII collagen fragment and natural bovine type I collagen of this application on 3T3 cell adhesion;

[0058] Figure 9 This study compares the effects of the recombinant type XVII collagen fragment of this application with those of the competing product CJ-XVII collagen on HSF cell proliferation. Detailed Implementation

[0059] To make the technical means, creative features, achieved objectives, and effects of this application readily understandable, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0060] Collagen sequence screening

[0061] The collagen fragment sequences involved in this invention were systematically screened from natural collagen databases using a self-developed thermal stability (Tm) prediction algorithm. This algorithm, constructed based on protein sequence characteristics and known Tm data, is capable of high-throughput stability assessment of collagen tripeptide repeat (Gly–X–Y) regions.

[0062] In the course of this invention, we obtained the primary structure sequence of natural human type 17 collagen from public databases (such as UniProt). COL17A1-Collagen,type XVII,alpha 1,isoform CRA_b-Homo sapiens(Human)|UniProtKB|UniProt The sequence is then scanned using a sliding window to extract continuous fragments of 24 or 30 amino acids in length. Subsequently, the Tm prediction model of this invention is used to score and rank all fragments, and candidate sequences with predicted Tm values ​​significantly higher than the natural average level are selected based on their thermal stability scores.

[0063] The aforementioned candidate sequences are the core sequences described in the claims of this invention. Their stability has been evaluated by a predictive model and preliminarily verified by experiments, demonstrating good thermal stability and potential application value. All fragments can be located in natural collagen and have a clear biological origin.

[0064] [Experimental Materials]

[0065] The nucleic acid sequence encoding type XVII collagen in this application was synthesized by Anhui General Biotechnology Co., Ltd.

[0066] Pichiapastoris GS115 strain, Beyotime Biotechnology D0412

[0067] pCM01-COL17-1 plasmid Shenzhen Chengmei Biotechnology Co., Ltd.

[0068] Potassium dihydrogen phosphate Shanghai Stock Exchange 10017618

[0069] Glycerol Shanghai Test 10010618

[0070] Methanol Shanghai Trial 10014118

[0071] Glucose Shanghai Test 10010518

[0072] Yeast Nitrogen Base(YNB)Coolaber PM2070-500G

[0073] Yeast extract OXOID LP0021

[0074] Peptone OXOID LP0042B

[0075] Agar Biofroxx 8211GR500

[0076] Sorbitol BBI A610491-0500

[0077] LiAc Sigma L6883-250G

[0078] DTT Sangon Biotech A620058-0100

[0079] Tris Vetec V900483-5KG

[0080] QuickCut Sal Ⅰ Takara 1080S

[0081] QuickCut Xba ITakara1634

[0082] QuickCut BamH I Takara 1605

[0083] Not I Takara 1166S

[0084] Cpo I(Rsr II)Takara 1035A

[0085] T4 DNA ligase Takara 2011A

[0086] 2×Hieff Ultra-Rapid HotStart PCR Master Mix (with Dye)Hieff 10157ES03

[0087] Spe I Yisheng 15028ES50

[0088] BamH I Yisheng 15003ES76

[0089] FastAP TM Thermosensitive Alkaline Phosphatase Thermo Scientific EF0654

[0090] Mili-Q Ultrapure Water System (Mili-Q China Co., Ltd.)

[0091] Haier DW-86L626 Ultra-low Temperature Freezer for Biomedical Equipment

[0092] Clean bench SCB-1360, manufactured by Beijing Donglian Haer Instrument Manufacturing Co., Ltd.

[0093] C-MAG HS10 magnetic stirrer from IKA GmbH, Germany

[0094] Vertical electrophoresis system JY600C, Beijing Junyi Oriental Electrophoresis Equipment Co., Ltd.

[0095] Nanodrop micro-volume spectrophotometer, Imlen Photometer N60 (Germany)

[0096] Bio-Ray Life Sciences T100 PCR Instrument

[0097] pH meter FE20 from METTLER-TOLEDO, Switzerland

[0098] BioRad, an electro-polarization company

[0099] MicroPulser 10 L Fermenter, Shanghai Baoxing Bio-Equipment Engineering Co., Ltd., BIOTECH-10JS

[0100] JASCO J-1500 Circular Dichroism Spectrometer

[0101] Thermo Scientific Nicolet iS50 Fourier Transform Infrared Spectrometer

[0102] Weak cation exchange chromatography packing material, Jiaxing Qianchun Biotechnology Co., Ltd., CM Purose 6Fast Flow

[0103] Phenyl Purose 6Fast Flow (HighSub) hydrophobic chromatography packing material, manufactured by Jiaxing Qianchun Biotechnology Co., Ltd.

[0104] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0105] Example 1 Construction of recombinant expression plasmid

[0106] The coding nucleic acid sequence of the XVII type collagen fragment (sequence shown in SEQ ID No. 1) was selected and designed, and Anhui General Biotechnology Co., Ltd. was commissioned to synthesize the gene fragment. The XVII type collagen sequence of this application is shown in SEQ ID No. 1. The synthesized gene fragment was inserted into the pCM01-COL17-1 vector through Not I and Cop I restriction sites to obtain the XVII type collagen recombinant plasmid ( Figure 1 ).

[0107] Example 2: Screening of recombinant expression plasmid-positive strains and high-density fermentation

[0108] 2.1 Preparation of Pichia pastoris competent cells

[0109] (1) The Pichia pastoris strain stored in the low temperature freezer was activated by YPD plates and cultured until it reached a suitable size.

[0110] (2) Pick a single colony and inoculate it into a medium containing 15 mL of YPD medium, and incubate overnight until OD600 = 3.

[0111] (3) Take about 10 mL of yeast culture and transfer it to a 1 L Erlenmeyer flask containing 100 mL of YPD. Incubate for about 5 hours.

[0112] (4) When OD 600 When the viscosity reaches approximately 2.0, stop the culture. Place the two 50mL centrifuge tubes and the Erlenmeyer flask containing the bacterial culture on ice for a few minutes to pre-cool. Centrifuge at 4℃ and 4000g for 5 minutes. After discarding the supernatant, resuspend the cells twice with approximately 30mL of pre-cooled ultrapure water under the same conditions.

[0113] (5) Then resuspend the cells in 8 mL of SB solution and culture them in a shaker at 30°C and 220 rpm for 30 min.

[0114] (6) Centrifuge at low temperature, then pour out the supernatant in the centrifuge tube, and add 15 mL of pre-cooled 1M Sorbitol to each of the two centrifuge tubes. Resuspend the cells twice under the same operation and conditions, and finally add 1 mL of pre-cooled 1M Sorbitol to each of the two centrifuge tubes to resuspend the cells.

[0115] (7) Use a pipette to add 100 μL of the resuspended cells to a pre-cooled EP tube. After the cells are dispensed, freeze the competent cells in a -20°C freezer for a few hours, and then store them in an ultra-low temperature freezer.

[0116] 2.2 Linearization of Recombinant Plasmids

[0117] The recombinant plasmid was linearized by enzyme digestion using QuickCut SalⅠ at 37℃ for 2 hours. The enzyme digestion system is shown in Table 1.

[0118] Table 1. Linearization system of Sal I

[0119] Element Volume (μL) QuickCut Sal Ⅰ 2 Plasmid 30 (at least 7500ng) 10×QuickCutGreen Buffer 5 ddH2O 13

[0120] 2.3 Electroconversion of Pichia pastoris

[0121] (1) Take the appropriate number of electric transfer cups and clean them thoroughly with ddH2O, then clean them thoroughly with 75% alcohol, place them in an oven to dry, open the lid after drying, and sterilize the 1mL pipette, 1mL pipette tip, 200μL pipette and 200μL pipette tip by UV irradiation for 30min. After completion, cover the electric transfer cup lid and place it on ice to pre-cool for later use.

[0122] (2) Take out the competent cells and place them on ice to thaw naturally. Mix 100 μL of competent cells with 10 μL of linearized plasmid. Select the pic option on the electroporator and then electroporate.

[0123] (3) Immediately after completion, add 600 μL of premixed 1M Sorbital and YPD culture medium (ratio 1:1) to the ultra-clean workbench, mix gently, transfer to EP tube, and revive at 28℃ and 220 rpm for 2 h.

[0124] (4) After centrifuging at 3000 rpm for 3 min, remove 500 μL of supernatant. Gently aspirate and mix the remaining bacterial solution, then spread it all onto MD screening plates. Place the plates in an incubator at 28℃ and invert them for about 36 to 48 h to grow more single colonies.

[0125] 2.4 Screening and Identification of Positive Transformants

[0126] 2.4.1 Transfer board

[0127] After 36-48 hours of growth, single colonies from MD plates are streaked sequentially onto another MD or YPD plate with a pre-drawn grid, and marked. This facilitates the preservation of the bacterial strain after successful screening and identification of positive clones.

[0128] 2.4.2 Methods for disrupting Pichia pastoris cell walls

[0129] (1) After 12 hours of plate transfer, pick a small number of single colonies from the YPD plate in the clean bench and transfer them into an eight-pack PCR tube, then cover it.

[0130] (2) Place the selected bacteria in a microwave oven and heat on medium for 3-5 minutes. Immediately freeze with liquid nitrogen. After freezing, heat in a microwave oven on medium for 3-5 minutes.

[0131] (3) Add 10-15 μl of double-distilled water to the bacterial cells for resuspending and use as a template.

[0132] 2.4.3 Pichia pastoris colony PCR

[0133] Using the cell wall-broken Pichia pastoris culture as a template, the original plasmid of the gene (20 ng / μL) as a positive control template, and ddH2O as a negative control template, PCR amplification was performed according to the system in Table 2.2 and the procedure in Table 2.3. If the recombinant plasmid was successfully integrated into the genome, the expected size band could be obtained by PCR amplification.

[0134] Table 2. Pichia pastoris colony PCR reaction system

[0135]

[0136]

[0137] Table 3. PCR program for Pichia pastoris colonies

[0138] step temperature time 1 95℃ 10min 2 95℃ 30s 3 55℃ 30s 4 72℃ 2kb / min 5 Back to step 2, 33× - 6 72℃ 5min 7 25℃ 10s

[0139] The sequences of primers F and R are shown in SEQ ID No. 2 and 3, respectively.

[0140] 2.4.4 24-well plate screening of recombinant proteins

[0141] (1) Prepare sterilized 24-well plates in advance, and fill each well with 3 mL of BMGY culture medium in a clean bench.

[0142] (2) Pick a single colony that has been successfully amplified by PCR and produced the expected size band, transfer it to a single well, label it, and incubate it at 28℃ and 220rpm for 36-48h.

[0143] (3) After centrifugation at 1500 rpm for 15 min, remove the supernatant of the culture medium, add 1.5 mL of BMMY culture medium to each well, and add 100% methanol to a final concentration of 1% (v / v) to begin methanol induction. Take samples every 24 h and add 100% methanol to a final concentration of 1% (depending on the situation, the initial detection of protein expression induction time is generally 1-3 days). Perform SDS-PAGE electrophoresis on samples induced for 24 h to detect whether there is protein expression of the expected size, and preserve the strains that express the target protein.

[0144] 2.4.5 Preservation of positive strains

[0145] Different recombinant expression strains were named CM001-C17-YXX-XX#. A certain amount of fresh positive single colonies from the plates were selected and placed into 1.5 mL EP tubes containing a 1:1 mixture of 50% glycerol and YPD. The tubes were thoroughly mixed by pipetting and stored at -80℃.

[0146] 2.5 High-density fermentation of recombinant proteins

[0147] The selected strain GS115 / pCM01-COL17-1-C17-Y5-17# was subjected to high-density fermentation.

[0148] 2.5.1 Culture medium formulation

[0149] Seed culture medium (YPG): yeast extract 10 g / L, peptone 20 g / L, glycerol 20 g / L.

[0150] Fermentation medium: potassium dihydrogen phosphate 5g / L, calcium sulfate dihydrate 1g / L, magnesium sulfate heptahydrate 10g / L, potassium sulfate 10g / L, ammonium dihydrogen phosphate 40g / L, potassium hydroxide 2g / L, glycerol (glycerol) 15g / L.

[0151] Feeding medium: 50% (w / v) glycerol

[0152] Induction medium: 100% methanol (with 10 mL of PTM1 trace element solution added per liter)

[0153] PTM1 trace element solution: CuSO4·5H2O 6g / L, MnSO4·H2O 3g / L, FeSO4·7H2O 65g / L, ZnSO4·7H2O 20g / L, CoCl2 0.2g / L, H3BO3 0.02g / L, NaMoO4·2H2O 0.2g / L, KI 0.1g / L, biotin 0.2g / L, H2SO4 5mL. Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0154] 2.5.2 Inoculation and Culture

[0155] (1) The fermenter was sterilized by high temperature and high pressure steam sterilization. First, air sterilization was performed at 121℃ and pressure for 10 minutes. Before actual sterilization, the pH electrode and dissolved oxygen (DO) electrode were calibrated. Then, 5L of the prepared fermentation medium (with 5mL of defoamer added during preparation) was added to the 10L fermenter, and actual sterilization was performed at 121℃ and pressure for 30 minutes. After sterilization, the temperature was lowered to 30℃. The air flow meter was turned on to maintain a positive pressure of 0.05Mpa inside the tank.

[0156] (2) The engineered Pichia pastoris strain (GS115 / pCM01-COL17-1-C17-Y5-17#) stored in an ultra-low temperature freezer was inoculated into a medium containing 50 mL of liquid YPG and cultured overnight at 30 °C and 220 rpm in a constant temperature shaker to activate the cells. The primary seed culture was then transferred at an inoculation rate of 5% (V / V) to a medium containing 500 mL of YPG for scale-up culture until the cell OD reached its maximum. 600 The inoculum was 6-8, yielding a secondary seed culture. The secondary seed culture was added to the sterilized fermenter at an inoculum rate of 10% (v / v), along with 20 mL of LPT™ trace element solution, for batch fermentation.

[0157] (3) The temperature for the glycerol growth and glycerol feeding stages was set at 30℃, and the pH was automatically adjusted to 4.5 using ammonia. The temperature for the methanol induction stage was set at 28℃, and the pH was automatically adjusted to 5.0 using ammonia. Dissolved organic matter (DO) was maintained between 20% and 50% by controlling the stirring speed (200-500 rpm), the aeration rate (2-6 L / min), and the feeding rate. During the glycerol growth stage, when the DO continuously increased, it indicated that the glycerol in the culture medium had been consumed. At this point, 1 L of 50% (w / v) glycerol was added. Once the DO continued to rise and the cell wet weight reached 180 g / L-220 g / L, methanol induction was initiated. The feeding rate was periodically adjusted to maintain the DO within a cyclical fluctuation range of 10%-50%. After induction began, samples were taken at 12-hour intervals, and protein expression was detected by SDS-PAGE. The SDS-PAGE results are shown below. Figure 2 In this designation, lane M is the protein marker, lane 1 is the sample induced by C17-Y5 expression for 0 h, and lanes 2-6 are samples with different C17-Y5 expression durations, respectively.

[0158] Example 3: Purification of Recombinant Collagen

[0159] The culture medium supernatant was collected by centrifugation. The first step of purification was performed using weak cation exchange chromatography with Qianchun CM Purose 6Fast Flow, loaded into a GE AKTA chromatography system. The column was equilibrated with pure water, and the fermentation broth supernatant was diluted to a conductivity of 3 mS / cm for loading. After loading, impurities were washed with pure water, followed by elution with phosphate buffer (25 mM, pH 7.40, hereinafter the same) containing 1 M NaCl, and the eluted proteins were collected. The eluent collected by ion exchange chromatography was then subjected to hydrophobic chromatography for the second step of purification with Qianchun Phenyl Purose 6Fast Flow (High Sub), loaded into a GE AKTA chromatography system. The column was equilibrated with phosphate buffer containing 1 M ammonium sulfate, and ammonium sulfate was added to the CM eluent to a final concentration of 1 M / L for loading. After loading, impurities were washed with phosphate buffer containing 1 M ammonium sulfate, followed by elution with phosphate buffer, and the eluted proteins were collected. After concentration and fluid exchange via membrane encapsulation, recombinant type XVII collagen stock solution was obtained. The molecular weight and purity were determined using SDS-PAGE. (See attached table). Figure 3 In this context, lane M is the protein marker, lane 1 is the purified C17-Y5 sample of this application with a concentration of 0.1 mg / mL, lane 2 is the purified C17-Y5 sample of this application with a concentration of 0.25 mg / mL, and lane 3 is the purified C17-Y5 sample of this application with a concentration of 0.5 mg / mL.

[0160] Protein content was determined using the BCA protein content assay. For detailed operating procedures, please refer to the Beyotime BCA protein content assay instruction manual.

[0161] The culture medium used in the screening stage is shown in Table 4, and the composition of the buffer solution is shown in Table 5.

[0162] Table 4. Culture medium preparation

[0163]

[0164] Table 5 Buffer Preparation

[0165]

[0166] Example 4 Structural Characterization

[0167] 4.1 Circular dichroism characterization of protein structure

[0168] This study employed circular dichroism (CD) chromatography to characterize the structural features of collagen. CD is a spectroscopic method based on the difference in absorption of light by chiral molecules, and is widely used to analyze the asymmetry and conformational changes in the structures of biological macromolecules (such as proteins). Collagen possesses a typical triple helix structure, and its CD spectrum exhibits a positive absorption peak near 225 nm and a negative absorption peak near 195 nm. These characteristic peak positions can serve as indicators of triple helix formation. The position and intensity of the absorption peaks vary depending on the amino acid sequence and length of collagen. To further evaluate the thermal stability of collagen, this application also incorporated CD to determine its thermal denaturation behavior. The thermal stability of collagen can be measured by the thermal denaturation temperature (Td), which is the temperature at which the triple helix structure unwinds to 50%. Real-time monitoring of collagen conformational changes at different temperatures using CD spectroscopy can effectively assess the stability of its triple helix structure and its thermal response characteristics.

[0169] The lyophilized powder Y5 of type XVII collagen fragment prepared in Example 3 was dissolved in 25 mM PB buffer (pH 7.4) to a concentration of 0.1 mg / ml and a volume of 2 ml. The sample was transferred to a 10 mm × 10 mm sample cell of a circular dichroism chromatograph. The scanning wavelength range was set to 200 nm-250 nm, the scanning speed to 100 nm / min, and the scanning temperature to room temperature. The CD spectra were the average of three scans. The results are shown in the figure. The recombinant type XVII collagen fragment of this application has a maximum characteristic positive peak at 225 nm and a negative peak at 210 nm. Based on the known circular dichroism chromatographic characteristics of the triple helix structure of collagen, it was determined that the collagen fragment sample has a triple helix structure.

[0170] The recombinant type XVII collagen fragment sample of this application was diluted according to the method described in Example 3, and the thermal stability of the protein was detected by real-time temperature increase at a rate of 1 °C / min. The CD spectra were all averaged from three scans, and the data were analyzed and calculated on the software. The relationship between the molar ellipticity of the recombinant collagen fragment and temperature was determined at 225 nm.

[0171] See results Figure 4 , Figure 4 A circular dichroism chromatogram for identifying type XVII collagen fragments designed for this application; wherein Figure 4 A represents the full wavelength spectrum; Figure 4 B represents the thermal curve; Figure 4 C is the first derivative of the thermal curve.

[0172] like Figure 4 As shown in Figure A, Y5 exhibits a characteristic positive absorption peak at 225 nm, indicating that collagen is correctly folded to form a triple helix structure.

[0173] The thermal change temperature of the collagen domain was further determined using circular dichroism spectroscopy. The thermal change curves from 40℃ to 60℃ were detected at a wavelength of 225 nm, and the curves were fitted (see...). Figure 4 C). The results showed that the Tm value was 53.6℃.

[0174] 4.2 Fourier Transform Infrared Spectroscopy Characterization of Collagen

[0175] The recombinant collagen fragments were structurally characterized using Fourier Transform Infrared Spectroscopy (FTIR).

[0176] Fourier transform infrared spectroscopy is a spectroscopic analysis technique based on molecular vibrational energy level transitions. It analyzes the chemical bond vibrational modes and molecular structure information of a sample by detecting its absorption characteristics of infrared light. The purified recombinant collagen fragment solution was freeze-dried to obtain a solid sample. The sample was then placed directly on the surface of a diamond or ZnSe crystal, and uniform pressure was applied to ensure contact.

[0177] Spectrometer: Fourier transform infrared spectrometer (such as Thermo Scientific Nicolet iS50).

[0178] Scanning range: 4000-500cm -1 4cm resolution -1 The number of scans was 64.

[0179] Environmental control: The humidity in the laboratory should be below 30%, and water vapor interference should be avoided (especially at 3300cm). -1 (near OH peak).

[0180] Baseline correction: Use instrument software (such as OMNIC) to perform baseline correction on the original spectrum to eliminate background interference.

[0181] Characteristic peak identification: Mark the characteristic absorption peaks of collagen (such as amide I, II, and III bands) and record the peak positions (cm). -1 Peak strength and half-peak width.

[0182] Collagen characteristic peaks:

[0183] 1. Amide A band: typically located at ~3300 cm⁻¹ 1 The peaks are broad and strong, reflecting its highly ordered hydrogen bond network.

[0184] 2. CH stretching vibration (2800-3000cm- 1 ): Symmetric / antisymmetric vibration of aliphatic CH, peak position approximately 2960 cm⁻¹ 1 and 2870cm- 1 .

[0185] 3. Amide I band (1600-1700cm- 1 ): Reflects secondary structure (such as α-helix, β-sheet). The triple helix structure of collagen is typically 1650-1660 cm⁻¹. 1 Strong peaks are observed in the vicinity.

[0186] 4. Amide II band (1480-1580cm- 1 Peak position is approximately 1550cm- 1 It is often combined with the amide I band to analyze structural changes.

[0187] 5. Amide III band (1200-1300cm- 1 Approximately 1240cm 1 It has relatively weak strength and serves as an auxiliary verification structure.

[0188] The freeze-dried Y5 solid sample was placed directly on the diamond surface of a Fourier transform infrared spectrometer, and uniform pressure was applied to ensure contact. Detection and calibration were performed under the above conditions. Simultaneously, solid samples of competitor CJ-XVII type collagen and the recombinant XVII type collagen fragment C17-Y5 of this application were detected, and the results are as follows: Figure 5 As shown.

[0189] Depend on Figure 5It can be seen that the recombinant collagen fragment C17-Y5 has similar characteristic peaks and wavenumbers to the competing product CJ-XVII type collagen, indicating that the two have similar structures. Furthermore, comparing the infrared spectra of both with those of the existing standard SigmaIII type collagen reveals that both also exhibit similar characteristic peaks, indicating that they have similar structures to the standard.

[0190] Example 5: Cell efficacy evaluation of the collagen fragment prepared in this application.

[0191] 5.1 Effect of collagen fragments prepared in Example 3 on cell migration

[0192] Experimental method: 0.35 × 10⁶ cells were seeded per well in a 12-well plate. 6 After culturing cells for 16-18 hours, a monolayer of cells with a density of 100%-110% is formed in the well. The cells in each well are scratched with a pipette tip, the scratched cells are washed away, and culture medium for each group is added. A photo is taken at 0 hours and the scratch area S0 is calculated. A photo is taken at the same location at 12 hours and the scratch area S1 is calculated.

[0193] Data processing: Cell migration rate P = (S0 - S1) / S0.

[0194] Figure 6 , Figure 7 The effect of the collagen fragment designed for Example 1 of this application on the migration of HaCaT and 3T3 cells is shown in the figure. The collagen fragment Y5 promotes the migration of HaCaT and 3T3 cells significantly better than the same concentration of BSA, and is also better than competitor 1.

[0195] Figure 6 , Figure 7 In the diagram, * indicates that the P-value is significant at the 5% significance level, i.e., P < 0.05.

[0196] **: Indicates that the P-value is significant at the 1% significance level, i.e., P < 0.01.

[0197] ***: Indicates that the P-value is significant at the 0.1% significance level, i.e., P < 0.001.

[0198] 5.2 Effect of collagen fragments prepared in Example 3 on cell adhesion

[0199] Experimental Methods: Protein solution, bovine type I collagen, and blank solution (PBS) were added to 96-well plates and incubated overnight at 4°C. The coating solution was discarded, and non-specific binding sites were blocked with 100 μL of 1% BSA. The plates were incubated at 37°C for 1 h, followed by washing twice with PBS. 3T3 cells were diluted to 10⁻¹⁰ with 1% Hoechst 33342 medium. 5Add 100 μL / ml to each well, incubate at 37°C for 50 minutes, then wash four times with PBS. Use an EVOS M7000 to scan the entire well. Count the cells in each well using Fiji Image J.

[0200] Data processing: Cell adhesion rate = Number of cells in the experimental group / Total number of cells inoculated

[0201] Figure 8 The effect of the designed collagen fragment on 3T3 cell adhesion is shown in the figure. Collagen fragment Y5 has a significant promoting effect on 3T3 cell adhesion, which is superior to that of natural bovine type I collagen.

[0202] Figure 8 In the diagram, * indicates that the P-value is significant at the 5% significance level, i.e., P < 0.05.

[0203] **: Indicates that the P-value is significant at the 1% significance level, i.e., P < 0.01.

[0204] ***: Indicates that the P-value is significant at the 0.1% significance level, i.e., P < 0.001.

[0205] 5.3 Effect of collagen fragments prepared in Example 3 on cell proliferation

[0206] Experimental Methods: 3000 HSF cells were seeded into each well of a 96-well plate. After 16-18 hours of culture, a monolayer of cells with a density of 25%-35% was formed in the wells. The original culture medium was discarded, and protein solutions from the experimental and control groups were added. The cells were then incubated at 37°C for 72 hours. The protein solution was discarded, and the cells were washed twice with PBS. Serum-free culture medium containing 10% CCK8 was added, and the CCK8 solution was also added to a cell-free blank well. The reaction was carried out at 37°C for 3 hours, and the absorbance at 450 nm was measured using a microplate reader.

[0207] Data processing: Relative cell proliferation rate = (Experimental group A450 - Blank well A450) / (Negative control A450 - Blank well A450)

[0208] Figure 9 The effect of collagen on HSF cell proliferation was designed, as shown in the figure. Collagen fragment Y5 had a significantly better effect on HSF cell proliferation than competitor 1.

[0209] Figure 9 In the diagram, * indicates that the P-value is significant at the 5% significance level, i.e., P < 0.05.

[0210] **: Indicates that the P-value is significant at the 1% significance level, i.e., P < 0.01.

[0211] ***: Indicates that the P-value is significant at the 0.1% significance level, i.e., P < 0.001.

[0212] In summary, this application provides a novel and efficient method for preparing recombinant human type XVII collagen fragments by screening and optimizing natural collagen sequences, and verifies their excellent biological properties through both structural and functional analysis. This collagen fragment is suitable for various biomedical applications and has broad industrialization prospects.

[0213] The above description is merely a preferred embodiment of this application and is not intended to limit this application in any way. Although this application has disclosed the preferred embodiment as above, it is not intended to limit this application. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above to create equivalent embodiments without departing from the scope of the technical solution of this application. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of this application without departing from the content of the technical solution of this application shall still fall within the scope of the technical solution of this application.

[0214] Sequence List:

[0215] The C17-Y5 sequence of type XVII collagen fragment (SEQ ID No. 1) of this application is as follows:

[0216] GDRGPYGTDGPPGQKGEMGTPPGKGDRGPAGPPGHPGPPGPRGHKGEKGDKGPPGDRGPYGTDGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPPGPRGHKGEKGDKGPPGDRGPYGTDGPPGQKGEMGTPPGKGDRGPAGPPGHPGPPGPRGHKGEKGDKGPP

[0217] Primer F (SEQ ID No. 2):

[0218] CGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGAA

[0219] Primer R (SEQ ID No. 3):

[0220] CAGGCAAATGGCATTCTGACATCCTCTTGA.

Claims

1. A recombinant type XVII collagen fragment, the protein fragment sequence of which is shown in SEQ ID No.

1.

2. The nucleic acid encoding the recombinant type XVII collagen fragment of claim 1.

3. An expression vector wherein the nucleic acid of claim 2 is recombined.

4. The carrier according to claim 3, characterized in that, The expression vector includes pCM01-COL17-1.

5. An expression cell comprising the nucleic acid of claim 2 or the expression vector of claim 3.

6. The expression cell according to claim 5, characterized in that, The expression cells include Pichia pastoris strains.

7. A method for preparing recombinant type XVII collagen fragments, characterized in that, The method includes seeding the expression cells of claim 5 in a culture medium to express the recombinant type XVII collagen fragment.

8. The method according to claim 7, characterized in that, The culture medium contains 1-9 g / L potassium dihydrogen phosphate, 0.1-5 g / L calcium sulfate dihydrate, 5-15 g / L magnesium sulfate heptahydrate, 5-15 g / L potassium sulfate, 10-80 g / L ammonium dihydrogen phosphate, 0.1-5 g / L potassium hydroxide, and 5-25 g / L glycerol.

9. The method according to claim 7, characterized in that, The culture medium contains 4-5 g / L potassium dihydrogen phosphate, 0.5-1 g / L calcium sulfate dihydrate, 8-10 g / L magnesium sulfate heptahydrate, 8-10 g / L potassium sulfate, 30-40 g / L ammonium dihydrogen phosphate, 1-2 g / L potassium hydroxide, and 10-15 g / L glycerol.

10. The method according to claim 7, characterized in that, The culture medium contains 5 g / L potassium dihydrogen phosphate, 1 g / L calcium sulfate dihydrate, 10 g / L magnesium sulfate heptahydrate, 10 g / L potassium sulfate, 40 g / L ammonium dihydrogen phosphate, 2 g / L potassium hydroxide, and 15 g / L glycerol.

11. The method according to claim 7, characterized in that, The expression cells were inoculated into the culture medium, and glycerol was used as a feed during the growth phase of the strain.

12. The method according to claim 7, characterized in that, After the expression cells had grown, methanol was used to induce the expression of a recombinant type XVII collagen fragment.

13. The method according to claim 7, characterized in that, The seeding amount of the expression cells was 10% v / v; Alternatively, the culture medium may further contain the following components at final concentrations: 24 mg / L CuSO4·5H2O, 12 mg / L MnSO4·H2O, 260 mg / L FeSO4·7H2O, 80 mg / L ZnSO4·7H2O, 0.8 mg / L CoCl2, 0.08 mg / L H3BO3, 0.8 mg / L NaMoO4·2H2O, 0.4 mg / L KI, 0.8 mg / L biotin, and 20 mg / L H2SO4. Alternatively, the fermentation conditions for the expression cell growth stage are a temperature of 30°C and a pH of 4.5; Alternatively, the fermentation conditions for the induction expression stage of the recombinant type XVII collagen fragment are a temperature of 28°C and a pH of 5.

0. Alternatively, methanol induction can begin when the wet weight of the expressed cells reaches 180 g / L-220 g / L.

14. The method according to claim 7, characterized in that, The method further includes a purification step, in which the supernatant after the fermentation reaction is separated and purified.

15. The method according to claim 14, characterized in that, The purification steps include first purifying with weak cation exchange chromatography, and then further purifying with hydrophobic chromatography to obtain the recombinant type XVII collagen fragment.

16. The method according to claim 14, characterized in that, A concentration step follows the purification step.

17. A composition or article, characterized in that, It contains the recombinant type XVII collagen fragment as described in claim 1.

18. The composition or article according to claim 17, characterized in that, The composition is a skin care product or pharmaceutical composition, and the product is one or more of the following: medical device, biomaterial, tissue engineering product, cosmetic, and health product.

19. The use of the recombinant type XVII collagen fragment according to claim 1 or the recombinant type XVII collagen fragment prepared by the method according to any one of claims 7 to 16 in the preparation of drugs or products for skin basement membrane repair, epidermal anti-aging, and skin wound repair.

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