Recombinant human XVII type collagen fragment based on natural collagen sequence as well as preparation method and application of recombinant human XVII type collagen fragment

Abstract

The invention provides a recombinant XVII type collagen fragment, and the protein fragment sequence of the recombinant XVII type collagen fragment is shown as SEQ ID No.1. The recombinant XVII type collagen fragment is screened and designed based on a natural collagen sequence, has a triple helix structure, has good biological activity, can effectively exert functions, and has a good promotion effect on cell migration, cell proliferation and cell adhesion. The method for preparing the recombinant XVII type collagen fragment has the advantages that the process steps are simplified, the expression efficiency of the recombinant protein is improved, and the production cost is reduced. The recombinant XVII type collagen fragment provided by the invention has good mechanical properties and biological activity, and can be used for preparing therapeutic drugs and biological materials.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for screening, optimizing and preparing a recombinant human type XVII collagen fragment based on a natural collagen sequence, the obtained recombinant human type XVII collagen fragment and the application of the protein fragment in the biomedical field. Background Art

[0002] Type XVII collagen is a transmembrane protein primarily located in the junctional region between epithelial and dermal cells, where it participates in maintaining extracellular matrix integrity and cell signaling. Type XVII collagen plays an important role in dermatology, wound healing, and inflammatory diseases. However, current research on this protein primarily focuses on functional analysis of the full-length protein, lacking the screening and optimization of its functional fragments. Therefore, developing a recombinant human type XVII collagen with enhanced bioactivity, prepared by screening natural collagen sequences, has important scientific significance and application value.

[0003] Bovine type I collagen is currently the most widely used natural collagen material. It is widely used in tissue repair, wound dressings, injection fillings, cell culture scaffolds and skin care products because of its stable source, low cost and structure close to human collagen. Many studies have shown that bovine type I collagen can promote cell adhesion, proliferation and tissue regeneration to a certain extent, especially in promoting wound closure and cell adhesion. Because it provides good extracellular matrix structural support, bovine type I collagen is one of the more mature collagen materials in current clinical and cosmetic applications. However, the cell migration and proliferation activity of bovine collagen is relatively weak, which becomes a limiting factor in scenarios that require rapid cell response and tissue regeneration (such as chronic trauma and dermal repair). At the same time, bovine collagen is a xenogeneic protein, which has certain immunogenicity risks and potential animal-derived contamination problems, and there are restrictions on its use in the development of new 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 disclosure data (such as CN116640205A, CN119462893A, and CN119504982A), a variety of 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 XVII collagen developed by Chuangjian Medical Technology Co., Ltd. (hereinafter referred to as Competitor 1) is the mainstream type XVII collagen molecule on the market (disclosed in CN113185604B). Multiple studies and product data have shown 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 the characteristic infrared spectra of amide I, II, and III, its stability and structure have not been confirmed.

[0005] Fragments of XVII with high biological activity, high thermal stability, and a complete triple helical structure have not yet been developed, which cannot provide a basis for further improving the application of collagen.

[0006] Based on the current technological development in this field, there is an urgent need to provide human type XVII collagen with high thermal stability, a complete triple helical 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, thereby obtaining functional protein fragments with high activity, high stability, and easy expression. In addition, this application also involves 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, the first aspect of the present 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 with a unique key structure located between the epidermis and dermis. It plays an important role in basement membrane repair and epidermal anti-aging. It is a key factor in skin aging and trauma repair, keeping the skin "youthful" and maintaining hair follicle stem cells (hair loss, gray hair, etc.); in the process of skin trauma repair, type XVII collagen plays an important role by affecting the migration, proliferation and differentiation of stem cells.

[0010] The recombinant type XVII collagen fragment of the present application can have a protein sequence that is ≥75% homologous 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% homologous.

[0011] Homology refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between 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] The second aspect of the present application is to provide a nucleic acid gene encoding the recombinant type XVII collagen fragment.

[0013] Due to the degeneracy of codons, there may be multiple nucleic acids encoding the recombinant type XVII collagen fragment in the present application.

[0014] The third aspect of the present application is to provide an expression vector on which is recombined a nucleic acid encoding the recombinant type XVII collagen fragment.

[0015] Expression vectors are constructed from a basic cloning vector framework, with the addition of expression elements (such as a promoter, RBS, and terminator) to enable the expression of a target gene. In this application, the target gene is type XVII collagen. Expression vectors can be adapted to specific host cells, such as those used for expression in yeast cells.

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

[0017] The fourth aspect of the present application is to provide an expression cell, which contains the nucleic acid encoding the recombinant type XVII collagen fragment or the expression vector.

[0018] As certain embodiments of the present application, the expression cell comprises a Pichia pastoris strain.

[0019] Pichia pastoris is a type of methanol-trophic 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 employed in this application utilizes a yeast expression system, the recombinant type XVII collagen obtained through fermentation exhibits excellent biological activity.

[0020] As certain embodiments of the present application, the expression cell comprises the Pichia pastoris GS115 strain.

[0021] The fifth aspect of the present application is to provide a method for preparing a recombinant type XVII collagen fragment, the method comprising inoculating the expressing cells into a culture medium to express the recombinant type XVII collagen fragment.

[0022] As certain embodiments of the present 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 content of potassium dihydrogen phosphate in the culture medium of the present 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 calcium sulfate dihydrate 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, 5 g / L.

[0025] The magnesium sulfate heptahydrate 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, 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, 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] 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 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, 5 g / L.

[0029] The glycerol content may 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 certain embodiments of the present 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 certain embodiments of the present 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 certain embodiments of the present application, the expression cells are inoculated into a culture medium and glycerol is used as a feed during the growth phase of the strain.

[0033] As certain embodiments of the present application, after the growth of the expressing cells is completed, methanol is used to induce the expression of the recombinant type XVII collagen fragment.

[0034] As certain embodiments of the present application, the inoculation amount of the expressing cells is 10% v / v.

[0035] As certain embodiments of the present application, the culture medium further contains a final concentration of 24 mg / L CuSO4·5H2O, 12 mg / L MnSO4·H2O, 260 mg / L FeSO4·7H2O, 80 mg / L ZnSO4·7H2O, 0.8 mg / L CoC12, 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 certain embodiments of the present application, the fermentation conditions during the growth phase of the expression cells are a temperature of 30° C. and a pH of 4.5.

[0037] As certain embodiments of the present application, the fermentation conditions during 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 certain embodiments of the present application, when the wet weight of the expressing cells reaches 180 g / L-220 g / L, methanol induction is started.

[0039] As certain embodiments of the present application, the method further comprises a purification step, wherein the purification step separates and purifies the supernatant after the fermentation reaction.

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

[0041] As certain embodiments of the present application, there is a concentration step after the purification step.

[0042] The sixth aspect of the present application is to provide a composition or product comprising the type XVII collagen fragment.

[0043] As certain embodiments of the present application, the composition is a skin care product or a pharmaceutical composition, and the product is one or more of a medical device, a biomaterial, a tissue engineering product, a cosmetic, and a health care product.

[0044] The seventh aspect of the present 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 product, wherein the drug or product is a drug or product having any one 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 the natural collagen sequence of the present application, and its preparation method and application, have the following beneficial effects:

[0046] 1. The recombinant human type XVII collagen fragment containing the screened sequence has a distinct triple-helical 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 has typical amide A, I, II, and III bands and exhibits excellent thermal stability. After stability optimization, the recombinant human type XVII collagen fragment of this application is more stable and less likely to lose activity, thereby enhancing the protein's potential for application.

[0047] 2. The biological activity of the recombinant human type XVII collagen fragment of the present application is significantly higher than that of the control, and it can effectively exert its function. It exceeds the competitor 1 of the mainstream product in the existing technology that has good effects on both aspects in promoting cell migration and cell proliferation. It also promotes cell adhesion better than the bovine collagen in the field that has good cell adhesion promotion effects.

[0048] 3. The method for preparing recombinant human type XVII collagen fragments of the present application has mild conditions and retains the biological activity of the protein.

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

[0050] 5. This application provides a new molecular tool for type XVII collagen, expanding its application in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0052] Figure 2 The SDS-PAGE electrophoresis results of samples taken at different induction times of the recombinant XVII collagen fragment induced expression of the present application are shown;

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

[0054] Figure 4 This is a circular dichroism spectrum identification diagram of the recombinant type XVII collagen fragment of the present application;

[0055] Figure 5 This is the Fourier transform infrared spectra of the recombinant collagen fragment C17-Y5 of the present application, the competitor CJ-XVII type collagen, and the standard sigma III type collagen;

[0056] Figure 6 、 Figure 7 The effects of the recombinant type XVII collagen fragment of the present application and the competitor CJ-XVII collagen on the migration of HaCaT and 3T3 cells;

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

[0058] Figure 9 The effect of the recombinant type XVII collagen fragment of the present application and the competitor CJ-XVII collagen on the proliferation of HSF cells. DETAILED DESCRIPTION

[0059] In order to make the technical means, creative features, objectives and effects achieved by this application easier to understand, the following will be combined with the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall 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 a native collagen database using a proprietary thermal stability (Tm) prediction algorithm. This algorithm, based on protein sequence characteristics and known Tm data, enables high-throughput stability assessment of the collagen tripeptide repeat (Gly–X–Y) region.

[0062] In the implementation of the present invention, we obtained the primary structure sequence of natural human type 17 collagen from a public database (such as UniProt) COL17A1-Collagen,type XVII,alpha 1,isoform CRA_b-Homo sapiens(Human)|UniProtKB|UniProt ), a sliding window scan was performed to extract continuous segments of 24 or 30 amino acids in length. Subsequently, all segments were scored and ranked using the Tm prediction model of the present invention, and multiple candidate sequences with predicted Tm values ​​significantly higher than the natural average were screened based on the thermal stability scores.

[0063] These candidate sequences are the core sequences described in the claims of this invention. Their stability, as assessed by predictive models and preliminarily verified by experiments, demonstrates good thermal stability and potential application value. All fragments can be located within native collagen and have a clear biological origin.

[0064] [Experimental Materials]

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

[0066] Pichiapastoris GS115 strain Biyuntian Biotech D0412

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

[0068] Potassium dihydrogen phosphate Shanghai test 10017618

[0069] Glycerol Shanghai Test 10010618

[0070] Methanol Shanghai Test 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 Yisheng15028ES50

[0088] BamH I Yisheng15003ES76

[0089] FastAP TM Thermosensitive Alkaline Phosphatase Thermo Scientific EF0654

[0090] Ultrapure Water Instrument Millipore China Co., Ltd. Mili-Q

[0091] Ultra-low temperature refrigerator Haier Biomedical Equipment DW-86L626

[0092] Clean Bench SCB-1360 Beijing Donglian Har Instrument Manufacturing Co., Ltd.

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

[0094] Vertical electrophoresis apparatus JY600C from Beijing Junyi Dongfang Electrophoresis Equipment Co., Ltd.

[0095] Nano drop micro-spectrophotometer German Implen company Photometer N60

[0096] PCR instrument Bio-Rad Bio-Medical Products Co., Ltd. T100

[0097] pH meter Swiss METTLER-TOLEDO FE20

[0098] BioRad Electroporation System

[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 medium Jiaxing Qianchun Biotechnology Co., Ltd. CM Purose 6Fast Flow

[0103] Hydrophobic chromatography filler Jiaxing Qianchun Biotechnology Co., Ltd. Phenyl Purose 6 Fast Flow (High Sub)

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

[0105] Example 1 Construction of recombinant expression plasmid

[0106] The nucleic acid sequence encoding the type XVII collagen fragment (sequence shown in SEQ ID No. 1) was screened and designed and commissioned to Anhui General Biotechnology Co., Ltd. for gene fragment synthesis. The type XVII collagen sequence of the present application is shown in SEQ ID No. 1. The synthesized gene fragment was inserted into the pCM01-COL17-1 vector through the Not Ⅰ and Cop Ⅰ restriction sites to obtain the type XVII 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 a low-temperature refrigerator was activated on a YPD plate and cultured to a suitable size.

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

[0111] (3) Transfer about 10 mL of yeast culture solution to a 1 L Erlenmeyer flask containing 100 mL of YPD and incubate for about 5 h.

[0112] (4) When OD 600 When the pH is approximately 2.0, stop the culture and place the two 50 mL centrifuge tubes and the Erlenmeyer flask containing the bacterial suspension on ice for a few minutes. Centrifuge at 4°C, 4000 g for 5 minutes. Discard the supernatant and resuspend the cells twice in approximately 30 mL of pre-chilled ultrapure water under the same conditions.

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

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

[0115] (7) Use a pipette to take 100 μL of the resuspended cells and add them to the pre-cooled EP tube. After the packaging is completed, first freeze the competent cells in a -20℃ refrigerator for a few hours, and then store them in an ultra-low temperature refrigerator.

[0116] 2.2 Linearization of recombinant plasmid

[0117] The recombinant plasmid was linearized using QuickCut SalⅠ. The enzyme digestion conditions were 37°C for 2 h. The enzyme digestion system is shown in Table 1:

[0118] Table 1 Sal I linearization system

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

[0120] 2.3 Electrotransformation of Pichia pastoris

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

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

[0123] (3) Immediately after completion, add 600 μL of premixed 1 M Sorbital and YPD medium (ratio 1:1) in a clean bench, mix gently, transfer to an EP tube, and shake at 28°C, 220 rpm for 2 h.

[0124] (4) After centrifugation at 3000 rpm for 3 min, 500 μL of supernatant was removed. The remaining bacterial solution was gently pipetted and mixed, and then all was spread on the MD screening plate. The plate was placed in an incubator at 28°C and inverted for about 36 to 48 hours to grow a large number of single colonies.

[0125] 2.4 Screening and identification of positive transformants

[0126] 2.4.1 Transfer

[0127] After 36-48 hours of growth, single colonies on the MD plate were picked and streaked onto another MD or YPD plate with grids drawn on it. Mark them well to facilitate the preservation of the strain after successful screening and identification of positive clones.

[0128] 2.4.2 Pichia pastoris cell wall disruption method

[0129] (1) 12 hours after transfer, pick a small number of single colonies from the YPD plate in a clean bench and place them in eight rows of PCR tubes and cover them.

[0130] (2) Heat the selected bacteria in a microwave oven at medium heat for 3 to 5 minutes, then freeze them immediately with liquid nitrogen. After freezing, heat them in a microwave oven at medium heat for 3 to 5 minutes.

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

[0132] 2.4.3 Pichia pastoris colony PCR

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

[0134] Table 2 Pichia pastoris colony PCR reaction system

[0135]

[0136]

[0137] Table 3 Pichia pastoris colony PCR program

[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 a sterilized 24-well plate in advance and add 3 mL of BMGY medium to each well in a clean bench.

[0142] (2) Pick a single colony that successfully amplifies a band of the expected size by PCR and place it in a single well, mark it, and culture it in a shaker at 28°C and 220 rpm for 36 to 48 hours;

[0143] (3) After centrifugation at 1500 rpm for 15 min, the supernatant of the culture medium was removed and 1.5 mL of BMMY medium and 100% methanol were added to each well to start methanol induction. Samples were taken every 24 h and 100% methanol was added to a final concentration of 1% (depending on the situation, the initial detection of protein expression induction time is generally 1-3 days). The samples after 24 h of induction were subjected to SDS-PAGE electrophoresis to detect whether the expected size of protein was expressed. The strains expressing the target protein were preserved.

[0144] 2.4.5 Preservation of positive strains

[0145] The different recombinant expression strains were named CM001-C17-YXX-XX#. Fresh positive single colonies were selected from a certain number of plates and placed into enzyme-free, pyrogen-free 1.5 mL EP tubes containing a 1:1 mixture of 50% glycerol and YPD. Mix thoroughly with a pipette and store at -80°C.

[0146] 2.5 High-density fermentation of recombinant proteins

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

[0148] 2.5.1 Culture medium formulation

[0149] Seed culture medium (YPG): yeast powder 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 (propylene glycol) 15g / L.

[0151] Feed medium: 50% (W / V) glycerol

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

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

[0154] 2.5.2 Inoculation and culture

[0155] (1) The fermentation tank was sterilized with steam at high temperature and high pressure. First, air sterilization was performed, and the temperature and pressure were kept at 121°C for 10 minutes. The pH electrode and dissolved oxygen (DO) electrode were calibrated before sterilization. Then, 5L of the prepared fermentation medium (5mL of defoaming agent was added during preparation) was added to the 10L fermentation tank and sterilized at 121°C for 30 minutes. After sterilization, the temperature was lowered to 30°C. The air flow meter was turned on to maintain the positive pressure in the tank at 0.05 MPa.

[0156] (2) Inoculate the engineered Pichia pastoris (GS115 / pCM01-COL17-1-C17-Y5-17#) stored in an ultra-low temperature freezer into 50 mL of liquid YPG medium and culture overnight in a constant temperature shaker at 30°C and 220 rpm to activate the bacteria. Transfer the primary seed solution at a 5% (V / V) inoculum volume to 500 mL of YPG medium and culture until the cell OD reaches 0. 600 The secondary seed solution was added to the sterilized fermentation tank at an inoculum volume of 10% (v / v), and 20 mL of PTM1 trace element solution was added at the same time for batch fermentation.

[0157] (3) The temperature of the glycerol growth and glycerol feeding stages of the bacteria was set to 30°C, and the pH was automatically adjusted to 4.5 by ammonia water; the temperature of the methanol induction stage was set to 28°C, and the pH was automatically adjusted to 5.0 by ammonia water. The DO was maintained in the range of 20%-50% by controlling the stirring speed to 200-500rpm, the ventilation volume to 2-6L / min and the feeding rate. During the glycerol growth stage, when DO continued to rise, it indicated that the glycerol in the culture medium had been consumed. At this time, 1L of 50% (W / V) glycerol was added, and when DO continued to rise again and the wet weight of the bacteria reached 180g / L-220g / L, methanol induction was started. The feeding rate was adjusted regularly to maintain DO in the range of 10%-50% and to show periodic fluctuations. After the induction began, samples were taken at intervals of 12h from the start of induction, and protein expression was detected by SDS-PAGE. The SDS-PAGE results are shown in Figure 2 Among them, lane M is a protein marker, lane 1 is a sample of C17-Y5 induced expression for 0 hours, and lanes 2-6 are samples of C17-Y5 expression for different lengths.

[0158] Example 3 Purification of recombinant collagen

[0159] The culture supernatant was collected by centrifugation. The first step of purification was performed using weak cation exchange chromatography, with a chromatographic filler of 100% pure CM Purose 6Fast Flow loaded on a GE AKTA chromatography system. The column was balanced with pure water, and the fermentation broth supernatant was diluted to a conductivity of 3 mS / cm for sample loading. After sample loading, the supernatant was washed with pure water, and then eluted with a phosphate buffer containing 1 M Nacl (25 mM, pH 7.40, the same below), and the eluted protein was collected. The eluate collected by ion exchange chromatography was subjected to a second step of purification using hydrophobic chromatography, with a chromatographic filler of 100% pure Phenyl Purose 6Fast Flow (High Sub) loaded on a GE AKTA chromatography system. The column was balanced with a phosphate buffer containing 1 M ammonium sulfate, and ammonium sulfate was added to the CM eluate at a final concentration of 1 M / L for sample loading. After sample loading, the supernatant was washed with a phosphate buffer containing 1 M ammonium sulfate, and then eluted with a phosphate buffer, and the eluted protein was collected. After membrane pack concentration and liquid exchange, the recombinant type XVII collagen stock solution was obtained, and the molecular weight and purity were detected by SDS-PAGE. Figure 3 . Among them, lane M is a protein marker, lane 1 is a purified sample of C17-Y5 of the present application with a concentration of 0.1 mg / mL, lane 2 is a purified sample of C17-Y5 of the present application with a concentration of 0.25 mg / mL, and lane 3 is a purified sample of C17-Y5 of the present application with a concentration of 0.5 mg / mL.

[0160] Protein content was determined using the BCA protein assay. For detailed instructions, see the Biyuntian BCA protein assay instructions.

[0161] The culture medium in the screening stage is shown in Table 4, and the composition of the buffer 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 Characterization of protein structure by circular dichroism

[0168] This study used circular dichroism (CD) to characterize the structural characteristics of collagen. CD is a spectroscopic method based on the differential absorption of left-handed and right-handed light by chiral molecules. It is widely used to analyze the asymmetry of the structure of biological macromolecules (such as proteins) and their conformational changes. Collagen has a typical triple helical structure, and its CD spectrum shows a positive absorption peak near 225nm and a negative absorption peak near 195nm. This characteristic peak position can be used as an indication of the formation of a triple helical structure. The position and intensity of the absorption peak will vary with the amino acid sequence and length of the collagen. In order to further evaluate the thermal stability of collagen, this application also combines CD to measure its thermal denaturation behavior. The thermal stability of collagen can be measured by the thermal denaturation temperature (Td), which is the temperature corresponding to the unwinding of the triple helical structure to 50%. Real-time monitoring of collagen conformational changes at different temperatures through CD spectroscopy can effectively evaluate the stability of its triple helical structure and thermal response characteristics.

[0169] The type XVII collagen fragment freeze-dried powder Y5 prepared in Example 3 was dissolved in 25mM PB buffer (pH 7.4) to a concentration of 0.1mg / ml and a volume of 2ml. The sample to be tested was transferred to a 10mm×10mm sample cell of a circular dichroism spectrometer, and the scanning wavelength range was set to 200nm-250nm, the scanning speed was 100nm / min, the scanning temperature was room temperature, and the CD spectra were the average values ​​of three scans. As shown in the figure, the recombinant type XVII collagen fragment of the present application had a maximum characteristic positive peak at 225nm and a negative peak at 210nm. According to the circular dichroism characteristics of the known collagen triple helix structure, it was judged that the collagen fragment sample had a triple helix structure.

[0170] A sample of the recombinant type XVII collagen fragment of this application was diluted as described in Example 3, and the protein's thermal stability was monitored by real-time temperature ramping at 1°C / min. CD spectra were averaged from three scans. The data were analyzed and calculated using software, and the relationship between the molar ellipticity of the recombinant collagen fragment and temperature was measured at 225 nm.

[0171] See the results Figure 4 , Figure 4 Circular dichroism spectrum identification diagram of type XVII collagen fragment designed for this application; wherein Figure 4 A is the full wavelength spectrum; Figure 4 B is the thermal curve; Figure 4 C is the first-order derivative of the thermal curve.

[0172] like Figure 4 As shown in A, Y5 shows a characteristic positive absorption peak at 225 nm, indicating that collagen is correctly folded and forms a triple helical structure.

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

[0174] 4.2 Characterization of collagen by Fourier transform infrared spectroscopy

[0175] The structure of the recombinant collagen fragment was characterized by Fourier transform infrared spectroscopy (FTIR).

[0176] Fourier transform infrared spectroscopy is a spectroscopic analysis technique based on molecular vibrational energy level transitions. By examining a sample's absorption characteristics of infrared light, it can reveal chemical bond vibration patterns and molecular structure. A solid sample is obtained by freeze-drying the purified recombinant collagen fragment solution. The sample is placed directly on the surface of a diamond or ZnSe crystal, applying uniform pressure to ensure contact.

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

[0178] Scanning range: 4000-500cm -1 , resolution 4cm -1 , scan times 64 times.

[0179] Environmental control: The humidity in the laboratory should be lower than 30% to avoid water vapor interference (especially at 3300cm -1 OH peak nearby).

[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 intensity and half-peak width.

[0182] Collagen characteristic peaks:

[0183] 1. Amide A band: usually located at ~3300 cm- 1 , the peak is 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 at about 2960cm- 1 and 2870cm- 1 .

[0185] 3. Amide I band (1600-1700cm- 1 ): reflects the secondary structure (such as α-helix, β-sheet). The triple helix structure of collagen is usually at 1650-1660cm- 1 There is a strong peak nearby.

[0186] 4. Amide II band (1480-1580cm- 1 ): Peak position is about 1550cm- 1 , often combined with amide I band to analyze structural changes.

[0187] 5. Amide III band (1200-1300cm- 1 ): about 1240cm- 1 , weak strength, auxiliary verification structure.

[0188] The freeze-dried Y5 solid sample was placed directly on the diamond surface of the Fourier transform infrared spectrometer, and uniform pressure was applied to ensure contact. The test and calibration were performed according to the above conditions. At the same time, the solid samples of the competitor CJ-XVII collagen and the recombinant type XVII collagen fragment C17-Y5 of the present application were tested. The results are as follows: Figure 5 shown.

[0189] Depend on Figure 5The recombinant collagen fragment C17-Y5 and the competitor's CJ-XVII collagen share similar characteristic peaks and close wavenumbers, indicating a similar structure. Furthermore, a comparison of the infrared spectra of both with the prior art standard, sigma III collagen, revealed similar characteristic peaks, indicating a similar structure.

[0190] Example 5 Description of Cellular Efficacy Evaluation of Collagen Fragments Prepared in This Application

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

[0192] Experimental method: 0.35×10 cells were seeded per well of a 12-well plate. 6 After culturing for 16-18 hours, a monolayer of cells with a density of 100%-110% was formed in the wells. The cells in each well were scratched with a pipette tip, the scratched cells were washed away, and the culture medium of each group was added. At 0 hours, photos were taken to calculate the scratch area S0. At 12 hours, photos were taken at the same position to calculate the scratch area S1.

[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 the present application on the migration of HaCaT and 3T3 cells. As shown in the figure, the effect of collagen fragment Y5 in promoting the migration of HaCaT and 3T3 cells is significantly better than BSA of the same concentration, and better than competitor 1.

[0195] Figure 6 、 Figure 7 In the table, * indicates that the P value is significant at the 5% significance level, that is, P < 0.05

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

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

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

[0199] Experimental method: Protein solution, bovine type I collagen and blank solution (PBS) were added to a 96-well plate and incubated at 4°C overnight. The coating solution was discarded and nonspecific binding sites were blocked with 100 μl of 1% BSA at 37°C for 1 hour, followed by washing twice with PBS. 3T3 cells were diluted to 10 μl in medium containing 1% Hoechst 33342. 5100 μl of culture medium was added to each well and incubated at 37°C for 50 minutes. The cells were then washed four times with PBS. The entire well was scanned and photographed using an EVOS M7000. The number of cells in each well was counted using FijiImage J.

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

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

[0202] Figure 8 In the table, * indicates that the P value is significant at the 5% significance level, that is, P < 0.05

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

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

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

[0206] Experimental Methods: 3000 HSF cells were seeded per well of a 96-well plate. After 16-18 hours of culture, a monolayer of cells with a density of 25%-35% was formed within the well. The original culture medium was aspirated, and the protein solutions of the experimental and control groups were added. The cells were incubated at 37°C for 72 hours. The protein solution was aspirated, and the cells were washed twice with PBS. Serum-free culture medium containing 10% CCK8 was added. Simultaneously, the CCK8 solution was added to a blank well containing no cells. The cells were incubated at 37°C for 3 hours, and the absorbance at 450nm was measured using a microplate reader.

[0207] Data processing: relative cell proliferation rate = (A450 of experimental group - A450 of blank well) / (A450 of negative control - A450 of blank well)

[0208] Figure 9 The design is to study the effect of collagen on HSF cell proliferation. As shown in the figure, the effect of collagen fragment Y5 on HSF cell proliferation is significantly better than competitor 1.

[0209] Figure 9 In the table, * indicates that the P value is significant at the 5% significance level, that is, P < 0.05

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

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

[0212] In summary, this application provides a novel method for efficiently preparing recombinant human type XVII collagen fragments by screening and optimizing natural collagen sequences. Its excellent biological properties have been verified from both structural and functional perspectives. This collagen fragment is suitable for a variety of biomedical applications and has broad industrial prospects.

[0213] The above description is merely a preferred embodiment of the present application and does not constitute any form of limitation to the present application. Although the present application has been disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present application. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present application without departing from the content of the technical solution of the present application are still within the scope of the technical solution of the present application.

[0214] Sequence Listing:

[0215] The sequence of the type XVII collagen fragment C17-Y5 of the present application (SEQ ID No. 1):

[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. A nucleic acid encoding the recombinant type XVII collagen fragment according to claim 1.

3. An expression vector containing a nucleic acid encoding the recombinant type XVII collagen fragment according to claim 1; Preferably, the expression vector comprises pCM01-COL17-1.

4. An expression cell comprising the nucleic acid encoding the recombinant type XVII collagen fragment of claim 1 or the expression vector of claim 2; Preferably, the expression cell comprises a Pichia pastoris strain.

5. A method for preparing recombinant type XVII collagen fragment, characterized in that: The method comprises inoculating the expressing cell according to claim 3 into a culture medium to express the recombinant type XVII collagen fragment; Preferably, 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; More preferably, 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; Further preferably, 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; Preferably, when the expression cells are inoculated into the culture medium, glycerol is used as a feed during the growth phase of the strain; Preferably, after the growth of the expressing cells is completed, methanol is used to induce the expression of the recombinant type XVII collagen fragment.

6. The method according to claim 5, characterized in that The inoculation amount of the expressing cells was 10% v / v; Alternatively, the culture medium further contains a final concentration of 24 mg / L CuSO4·5H2O, 12 mg / L MnSO4·H2O, 260 mg / L FeSO4·7H2O, 80 mg / L ZnSO4·7H2O, 0.8 mg / L CoC12, 0.08 mg / L H3BO3, 0.8 mg / LNaMoO4·2H2O, 0.4 mg / L KI, 0.8 mg / L biotin, and 20 mg / L H2SO4; Alternatively, the fermentation conditions during the growth phase of the expression cells are a temperature of 30° C. and a pH of 4.5; Alternatively, the fermentation conditions during the induction expression stage of the recombinant type XVII collagen fragment are a temperature of 28° C. and a pH of 5.0; Alternatively, when the wet weight of the expressing cells reaches 180 g / L-220 g / L, methanol induction is started.

7. The method according to any one of claims 5 to 6, characterized in that The method further comprises a purification step, wherein the purification step separates and purifies the supernatant after the fermentation reaction; Preferably, the purification step comprises first purifying using weak cation exchange chromatography and then further purifying using hydrophobic chromatography to obtain the recombinant type XVII collagen fragment; More preferably, the purification step is followed by a concentration step.

8. A composition or product, characterized in that The invention comprises the recombinant type XVII collagen fragment according to claim 1.

9. The composition or product according to claim 8, characterized in that The composition is a skin care product or a pharmaceutical composition, and the product is one or more of a medical device, a biomaterial, a tissue engineering product, a cosmetic, and a health care product.

10. 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 5 to 7 in the preparation of a drug or product, wherein the drug or product has any of the following functions: a. Promote cell migration; b. Promote cell adhesion; c. Promote cell proliferation.

Images