High-activity and high-expression recombinant collagen type xvi and application thereof

Abstract

The application provides a high-activity and high-expression recombinant collagen type XVII and application thereof, and belongs to the technical field of protein engineering, wherein the amino acid sequence of the recombinant collagen type XVII is formed by repeating and connecting n core units, wherein n is an integer of 1 or greater than 1, and the amino acid sequence of the core unit is shown as SEQ ID NO:1; the application further provides a construction method of various expression systems and a preparation method of the recombinant collagen type XVII; and the application further provides actual industrial applications according to the characteristics of the protein. The recombinant collagen type XVII provided by the application has high purity, high yield and high biological activity, and the molecular weight is much smaller than that of natural collagen protein, so that the collagen protein can reduce the barrier to biological function through the skin barrier, and experiments show that the collagen protein has multiple biological activities such as promoting cell migration.

Description

Technical Field

[0001] This invention belongs to the field of protein engineering technology, and in particular relates to a highly active, highly expressed recombinant type XVII collagen and its applications. Background Technology

[0002] Type XVII collagen is present in extremely small amounts in the body and is very difficult to obtain directly through extraction, which is also costly. Therefore, it is currently mainly produced in vitro using recombinant methods. Commonly used in vitro recombinant expression systems include Escherichia coli, yeast, and mammalian cells.

[0003] Type XVII collagen has a total length of 1497 amino acids, including intracellular, transmembrane, and extracellular domains. It is further divided into 16 non-triple-helix regions and 15 triple-helix regions, which are arranged alternately. It is difficult to recombinantly express the full-length protein in vitro, resulting in low yield and easy degradation.

[0004] When used as an ingredient in skincare products, collagen's natural micellar structure and large molecular weight make it difficult for it to penetrate the skin barrier and reach the dermis to exert its effects.

[0005] In summary, the existing technology has the following problems: 1) it is limited by traditional prokaryotic expression systems, and the recombinant collagen is not modified after translation; 2) it has low biological activity; 3) natural collagen has a large molecular weight and is not easy to penetrate the skin. Summary of the Invention

[0006] In view of this, the primary objective of the present invention is to provide a recombinant type XVII collagen. The recombinant collagen provided by the present invention has high purity, high expression level, high biological activity, and a molecular weight significantly smaller than that of natural full-length collagen. Experiments have shown that it has a variety of biological activities, such as promoting cell migration.

[0007] The present invention discloses a recombinant type XVII collagen, the amino acid sequence of which is formed by repeated tandem of n core units, where n is an integer of 1 or greater than 1, and the amino acid sequence of the core unit is shown in SEQ ID NO: 1.

[0008] Preferably, n is selected from any value among 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. Most preferably, n is selected from 8.

[0009] Through previous research, the inventors discovered that recombinant collagen composed of repeating core units can form a structure similar to that of preferred proteins and achieve similar biological functions while ensuring that its molecular weight is within the range of 50 kDa. Therefore, the recombinant amino acid sequence of the present invention can contain 1-16 core units.

[0010] The repeated tandem connection can be a series connection of core units head to tail, or it can be a series connection of core units by a linker. The linker preferably contains glycine and / or serine, and the linker preferably contains 1-5 amino acids.

[0011] This invention provides a gene encoding the recombinant type XVII collagen.

[0012] The encoding includes proteins produced by transcription of DNA molecules to form an RNA product, followed by translation; or proteins produced by transcription of DNA molecules to provide an RNA product, processing to provide a processed RNA product, followed by translation.

[0013] The present invention provides a recombinant vector for expressing the recombinant type XVII collagen, comprising the aforementioned gene.

[0014] Vectors include any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), comprising nucleic acid molecules operatively linked with one or more nucleic acid molecules. Vectors may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), at least one multiple cloning site, and / or elements that facilitate stable integration of the construct into the host cell genome. Preferably, the vectors include, but are not limited to, pET28a, pCDFDuet-1, pPICZαA, and pPIC9k.

[0015] In this invention, the vector can be cloned with the recombinant human type III collagen gene alone, or it can be cloned with the proline hydroxylase gene and / or the lysine hydroxylase gene and the recombinant human type III collagen gene on the same vector.

[0016] The present invention provides recombinant cells expressing the recombinant type XVII collagen, wherein the recombinant cells are transfected with the recombinant vector.

[0017] The recombinant cells include, but are not limited to, Escherichia coli and Pichia pastoris.

[0018] A second objective of the present invention is to provide a method for expressing the recombinant type XVII collagen.

[0019] In a preferred embodiment, the recombinant type XVII collagen is expressed in *Escherichia coli*, preferably *Escherichia coli* BL21(DE3), comprising the following steps:

[0020] 1) The gene was ligated into the vector pET28a to obtain a recombinant vector;

[0021] 2) The recombinant vector was transfected into Escherichia coli BL21(DE3) to obtain recombinant cells;

[0022] 3) After culturing the recombinant cells, centrifuge and collect the supernatant;

[0023] 4) The recombinant type XVII collagen was obtained by separating and purifying it from the supernatant.

[0024] More preferably, the method further includes constructing gene fragments encoding hydroxylases L593 and L230 into the expression vector pCDFDuet-1 to obtain the recombinant vector pCDFDuet-1-L593-L230, and transfecting it into Escherichia coli BL21(DE3).

[0025] Proline hydroxylation is the most common post-translational modification in collagen, and the resulting trans-4-hydroxyproline (Hyp) enhances the stability and function of collagen. Studies have used the proline hydroxylase L593 from a small virus (Shi, J.. et al. Hydroxylation of Human Type III Collagen Alpha Chain by Recombinant Coexpression with a Viral Prolyl 4-Hydroxylase in Escherichiacoli. Protein J 36, 322–331 (2017)) to achieve in vivo hydroxylation of collagen. In the examples, L593 can be expressed alone or co-expressed with the viral lysine hydroxylase L230.

[0026] More preferably, the culture in step 3) includes adding ascorbic acid at a final concentration of 10 mg / ml, 1 mM FeSO4 and 1 mM IPTG to induce expression of the target protein, which can further improve the thermal stability of the target protein.

[0027] In another preferred embodiment, the recombinant type XVII collagen is expressed in *Escherichia coli*, preferably *Pichia pastoris* GS115, comprising the following steps:

[0028] 1) The gene was ligated into the vector pPICZαA to obtain a recombinant vector;

[0029] 2) The recombinant vector was transfected into Pichia pastoris GS115 to obtain recombinant cells;

[0030] 3) After culturing the recombinant cells, centrifuge and collect the supernatant;

[0031] 4) The recombinant type XVII collagen was separated from the supernatant.

[0032] More preferably, the method further includes constructing a gene fragment encoding the hydroxylase BaP4H into the expression vector pPIC9k to obtain the recombinant vector pPIC9k-BaP4H, and then transfecting it into Pichia pastoris GS115.

[0033] 4-Prolyl hydroxylases (P4Hs) are a class of ferrous and 2-ketoglutarate-dependent oxidases that catalyze the formation of hydryps. P4H (BaP4H) from Bacillus anthracis can modify collagen-like proline-rich peptides.

[0034] Type XVII collagen is not only a key factor in skin aging and wound repair, but also plays an important role in maintaining hair follicle stem cells and preventing hair loss and graying. Col17A1 is highly expressed in hair follicle stem cells (HFSCs), and Col17A1 is crucial for maintaining hair follicle stem cells and melanocyte stem cells (MSCs) (Shintaro Tanimura, 2011). Collagen is widely studied and applied in the field of biomaterials due to its good biocompatibility and biodegradability, such as tissue engineering scaffolds and absorbable sutures.

[0035] Therefore, a third object of the present invention is to provide the recombinant XVII type collagen, wherein the recombinant XVII type collagen prepared by the preparation method includes any of the following non-therapeutic applications:

[0036] i) Promotes cell proliferation;

[0037] ii) Promotes cell migration;

[0038] iii) Preparation of cosmetics;

[0039] iv) Preparation of medical materials.

[0040] The cell proliferation promoter mentioned above can be used as a basic component of the culture medium to promote cell proliferation, for example, in the in vitro culture and preservation of hair follicle stem cells or hair follicle tissue.

[0041] The cell migration promoter mentioned above can be used as a basic component of the culture medium to promote cell migration, for example, in the in vitro culture and preservation of hair follicle stem cells or hair follicle tissue.

[0042] The cosmetics mentioned include, but are not limited to, skin lotion, skin cream, essence water, face mask, scalp essence, and shampoo.

[0043] The medical materials mentioned include, but are not limited to, filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, and drug delivery carriers, wherein the repair materials include, but are not limited to, bone repair materials, wound dressings, and sutures.

[0044] This invention also provides a composition comprising at least one cosmetically or pharmaceutically effective amount of the aforementioned protein and at least one excipient or cosmetically or pharmaceutically acceptable adjuvant. In some embodiments, the dosage form of the composition includes, but is not limited to, creams, lotions, aqueous solutions, gels, oils, powders, muds, patches, films, or freeze-dried products; furthermore, to promote transdermal absorption of collagen molecules, a patch product can be prepared using a solid carrier such as nonwoven fabric and applied to the face to prolong the interaction time between the collagen solution and the skin surface. The recombinant type XVII collagen of this invention can be prepared and used in tandem with recombinant or natural type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, fibronectin, human epidermal growth factor, and other bioactive ingredients, or can be mixed with the above-mentioned bioactive ingredients after separate preparation.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] 1) The theoretical molecular weight of the recombinant type XVII collagen preferred in this invention is about 22.79 kDa, which is significantly smaller than that of natural full-length collagen (150 kDa). This reduces obstacles for collagen to cross the skin barrier and perform its biological functions, and also promotes cell migration.

[0047] 2) The recombinant type XVII collagen sequence of this invention is based on the original amino acid sequence of human type XVII collagen and has the characteristics of good biocompatibility;

[0048] 3) The recombinant type XVII collagen of this invention has high expression level, stable structure, and is not easily degraded, making it more suitable for industrial application;

[0049] 4) The recombinant type XVII collagen of this invention can promote cell proliferation and migration, and regulate the expression of type I and III collagen genes. Experimental results show that its biological activity is significantly higher than that of the positive control group EGF and commercially available recombinant collagen products. Attached Figure Description

[0050] Figure 1 The image shows an SDS-PAGE image of the purified recombinant type XVII collagen LY1708 Ni-NTA in the Escherichia coli expression system.

[0051] Figure 2 The image shows an SDS-PAGE image of the recombinant type XVII collagen LY1708 Q column purified in the Escherichia coli expression system.

[0052] Figure 3 This is an SDS-PAGE image of the recombinant type XVII collagen LY1708 of the present invention expressed in the Pichia pastoris expression system.

[0053] Figure 4The graph shows the wet weight curve of the recombinant XVII type collagen LY1708 of this invention as a function of induction time in a fermenter.

[0054] Figure 5 The image shows SDS-PAGE images of the recombinant type XVII collagen LY1708 of this invention at different induction times in a fermenter.

[0055] Figure 6 This is a diagram showing the cell proliferation results of the recombinant type XVII collagen LY1708 of this invention.

[0056] Figure 7 This is a graph showing the relative cell proliferation rate of the recombinant type XVII collagen LY1708 of this invention.

[0057] Figure 8 This is a diagram showing the cell scratch assay results of the recombinant type XVII collagen LY1708 of this invention.

[0058] Figure 9 This is a graph showing the cell scratch recovery area analysis of the recombinant type XVII collagen LY1708 of this invention.

[0059] Figure 10 This diagram illustrates how the recombinant type XVII collagen LY1708 of this invention promotes the synthesis of type I collagen in human fibroblasts.

[0060] Figure 11 This diagram illustrates how the recombinant type XVII collagen LY1708 of this invention promotes the synthesis of type III collagen in human fibroblasts. Detailed Implementation

[0061] In this invention, the gene can be synthesized by a biotechnology company. This invention does not impose any particular limitation on the preparation method of the recombinant vector and recombinant cells; conventional methods for preparing recombinant vectors and recombinant cells in the art can be used.

[0062] The present invention does not specifically limit the separation and purification method, and conventional protein separation and purification methods in the art can be used; preferred technical solutions are described in the embodiments.

[0063] In the following embodiments, a protein formed by repeating the core unit (SEQ ID NO: 1) eight times in tandem is named LY1708.

[0064] The specific amino acid sequence of LY1708 is shown in SEQ ID NO: 2.

[0065] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1: Expression (E. coli expression system) and purification of recombinant type XVII collagen LY1708 of the present invention.

[0067] 1. Synthesis of LY1708 sequence

[0068] The recombinant type XVII collagen LY1708 gene fragment (SEQ ID NO: 3) of this invention was synthesized by GenScript Biotech Co., Ltd., and constructed into the pET28a E. coli expression vector. Hydroxylases L593 and L230 gene fragments were also synthesized by GenScript Biotech Co., Ltd., and constructed into the two promoters of the expression vector pCDFDuet-1.

[0069] 2. Construction of Escherichia coli expression system

[0070] 2.1 Transformation

[0071] The plasmid pET28a-LY1708 (kanamycin resistance) and the hydroxylase plasmid pCDFDuet-1-L593-L230 (streptomycin resistance) were co-transformed into competent BL21(DE3) cells (Shenzhen Kangti Life). The cells were incubated on ice for 30 min, then heat-shocked at 42°C for 45 s, and immediately cooled on ice for 2-3 min. 900 μl of SOC or LB medium (without antibiotics) was added, and the cells were incubated at 37°C for 1 h (200-250 rpm). Simultaneously, LB agar plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / ml kanamycin antibiotic) were preheated at 37°C. After incubation, the cells were centrifuged at 5000 rpm (2400 × g) for 5 min, and 900 μl of the supernatant was discarded. Resuspend the bacterial cells in the remaining culture medium and gently spread them evenly on a preheated plate with double antibiotics using a sterile spreader. Incubate upside down in a 37°C incubator for 12-16 hours until clearly visible colonies appear.

[0072] 2.2 Remove the plates containing colonies, pick single colonies, and inoculate them separately into 10 ml of LB liquid medium (containing kanamycin and streptomycin antibiotics). Incubate overnight at 37°C and 220 rpm. Then, add the kanamycin to 1 L of LB medium (containing kanamycin and streptomycin antibiotics) at a ratio of 1:100, and incubate at 37°C and 220 rpm for 3-5 hours. Monitoring of OD values ​​then begins. 600 When the pH reaches 1.0-1.2, add ascorbic acid (Shanghai Sangon Biotech), 1mM FeSO4 (Shanghai Sangon Biotech), and 1mM IPTG (Biosharp) to a final concentration of 10mg / ml to induce expression. Induce at 16℃ and 180rpm for 18-24h. Centrifuge to collect the bacteria for further purification or freeze the cells at -20℃ or below.

[0073] 3. Protein purification

[0074] 3.1 After induction, the bacterial culture was sampled and run on a gel for later use. The bacterial cells were then collected by centrifugation at 8000 rpm for 20 min.

[0075] 3.2 Preparation of Ni-NTA (Sangon Biotech Co., Ltd.) purification buffer:

[0076] Lysis buffer: 50 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, 5% glycerol, pH 7.0;

[0077] Wash buffer: 50mM Tris-HCl, 500mM NaCl, 20mM imidazole, 5% glycerol, pH 7.0;

[0078] Elution buffer: 50mM Tris-HCl, 500mM NaCl, 300mM imidazole, 5% glycerol, pH 7.0.

[0079] 3.3 Preparation of pyrolysis solution

[0080] Lysis buffer components: PMSF (MIKX) at a final concentration of 1 mM, protease inhibitor (MCE), super nuclease (Beyotime), and MgCl2 (Sangon Biotech) at a final concentration of 1 mM.

[0081] Add the appropriate volume of lysis buffer at a ratio of 1:10 according to the weight of the bacteria, suspend the bacteria in a mixer, and mix thoroughly by pipetting.

[0082] 3.4 Affinity Purification

[0083] Pre-cool the homogenizer (Duoning Biotechnology) beforehand. Pour in the bacterial culture, circulate at 700-800 bar for 3 cycles, and collect the disrupted bacterial culture. Centrifuge at 4°C, 20000 rpm for 1 hour using a high-speed refrigerated centrifuge (Beckman). After centrifugation, transfer the supernatant to a new 50 ml centrifuge tube and filter through a 0.45 μm filter. Remove the Ni-NTA gravity column (1 ml Ni-NTA), wash with 5 column volumes of filtered pure water, rinse with 3 column volumes of lysis buffer, and load the sample using a peristaltic pump. First, thoroughly clean the peristaltic pump tubing with pure water, then rinse with Lysis buffer. Then, use the peristaltic pump to load the filtered supernatant into the Ni-NTA gravity column and collect the flow-through. Wash the Ni-NTA with 100 ml of washbuffer and collect the wash buffer. Elute the target protein with 1 ml of lysis buffer each time, incubate for 5 minutes, and collect the eluent. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the protein. The protein sample was mixed with protein loading buffer (DTT), placed in a metal bath, boiled at 95°C for 10 minutes, and then the sample was loaded onto the gel.

[0084] 3.5 Ion exchange purification

[0085] The target protein was dialyzed into buffer A (20 mM PB, pH 8.0). The protein sample was filtered through a 0.22 μm, 33 mm filter membrane and subjected to anion exchange using an AKTApure column (5 ml Q column). Linear elution with buffer B (20 mM PB and 1 M NaCl, pH 8.0) yielded a highly pure target protein. SDS-PAGE electrophoresis was used to verify the protein's molecular weight and purity. The protein sample was mixed with protein loading buffer (DTT), placed in a metal bath, and boiled at 95°C for 10 min before gel loading. BCA method was used to determine protein concentration and estimate protein yield. Depending on the protein purity, it was concentrated or lyophilized for storage.

[0086] 4. Results

[0087] Figure 1 , 2 The images show SDS-PAGE electrophoresis results of the recombinant type XVII collagen of this invention after induction expression in Escherichia coli, purified using Ni and Q columns.

[0088] Example 2: Expression (Pichia pastoris expression system) and purification of recombinant type XVII collagen LY1708 of the present invention.

[0089] 1. Synthesis of LY1708 sequence

[0090] The recombinant type XVII collagen LY1708 gene fragment (SEQ ID NO: 3) of this invention was synthesized by GenScript Biotech Co., Ltd., and constructed into the pPICZalphaA Pichia pastoris expression vector. The hydroxylase BaP4H gene fragment was also synthesized by GenScript Biotech Co., Ltd., and constructed into the pPIC9k vector.

[0091] 2. Construction of Pichia pastoris expression system

[0092] 2.1 Electro-rotation

[0093] Plasmid pPICZalphaA-LY1708 was amplified by PCR and linearized at the SacI site, followed by PCR fragment recovery (Thermo GeneJET PCR Purification Kit). Competent GS115 cells were thawed on ice, and 5-10 μg of the linearized plasmid was added. The cells were incubated on ice for 15 min, and then the plasmid and competent cells were added together to a pre-chilled electroporation cuvette. Electroporation was performed at 2000 V, 200 Ω, 25 μF, with a cuvette diameter of 0.2 mm and a breakdown time of 5 ms. Immediately after electroporation, 1 ml of pre-chilled sorbitol was added, and the mixture was transferred to a sterile 1.5 ml centrifuge tube and activated at 30°C and 230 rpm for 2 h. Centrifuge at 5000g for 1 min, discard 300 μl of supernatant, resuspend the cells and spread on YPDSZ solid plates (tryptone 20 g / L, yeast extract 10 g / L, D-sorbitol 182.1 g / L, 20% glucose, 100 μg / ml bleomycin), and incubate at 30℃ and 230 rpm for 48 h.

[0094] 2.2 Remove the plate containing colonies, pick out single colonies, and copy them onto YPD solid medium with high concentration (400 μg / ml or 800 μg / ml) bleomycin for screening.

[0095] 2.3 Remove the plates containing colonies, pick single colonies, and inoculate them into 50 ml of YPD liquid medium (containing 100 μg / ml bleomycin) for overnight seed culture at 30°C and 230 rpm. The overnight seed culture is then inoculated at a 1:50 ratio into 1 L BMGY medium (20 g / L tryptone, 10 g / L yeast extract, 11.73 g / L potassium dihydrogen phosphate (KH₂PO₄), 2.4 g / L dipotassium hydrogen phosphate (K₂HPO₄), 10 g / L glycerol, 134 g / L YNB, and 1× biotin) for scale-up. Incubate at 30°C and 230 rpm for 48 h. The seed culture of BMGY was centrifuged at 3000 rpm for 5 min, the supernatant was discarded, and the culture was resuspended in sterile water. It was then centrifuged again at 3000 rpm for 5 min, the supernatant was discarded, and 1 L BMMY medium (without methanol) was added for resuspending. 1% methanol was added for induction, with 1% methanol added every 24 h. After 72 h of induction, the supernatant was collected by centrifugation for purification.

[0096] 2.4 Ion exchange purification

[0097] The target protein was dialyzed into buffer A (20 mM PB, pH 4.0). The protein sample was filtered through a 0.22 μm pore size and 33 mm diameter filter membrane, followed by cation exchange using an AKTApure column (5 ml SP column). Linear elution with buffer B (20 mM PB and 1 M NaCl, pH 4.0) yielded a highly pure target protein. SDS-PAGE was used to verify the protein's molecular weight and purity. Protein loading buffer (with added DTT) was added to the protein sample, mixed, and placed in a metal bath. The mixture was heated at 95°C for 10 min, centrifuged at 1500 rpm for 1 min, and the supernatant was analyzed by SDS-PAGE. BCA was used to determine the protein concentration and estimate the protein yield. Based on the protein purity, the protein was concentrated or lyophilized for storage.

[0098] 2.5 Preparation of yeast competent cells containing LY1708 expression plasmid

[0099] Single colonies of LY1708 with high expression levels were selected and cultured overnight at 30°C and 230 rpm in 5 ml of YPD medium (bleomycin resistant). Then, they were inoculated at a ratio of 1:200 into 500 ml of fresh YPD medium (bleomycin resistant) and cultured overnight until OD (outcome limit). 600= Between 1.3 and 1.5. Centrifuge at 1500g for 5 min at 4℃ to collect bacterial cells. Wash and resuspend the bacterial cells in 500ml of pre-cooled sterile water. Centrifuge again at 1500g for 5 min at 4℃ to collect bacterial cells. Wash 3 times with pre-cooled sterile water. Wash and resuspend the bacterial cells in 20ml of 1M sorbitol. Discard the supernatant. Repeat the washing 2-3 times. Centrifuge at 1500g for 5 min at 4℃ to collect bacterial cells. Add 500μl of 1M sorbitol to resuspend the bacterial cells. Store in 100μl tubes at -80℃.

[0100] 2.6BaP4H-pPIC9k linearization and electrotransfer

[0101] Primers were designed for PCR linearization at the SalI site of pPIC9k, and the PCR fragment was recovered (Thermo GeneJET PCR Rupture Kit). Yeast competent cells containing the LY1708 expression plasmid obtained in Example 2.4 were thawed on ice, and 5-10 μg of the linearized plasmid was added. The cells were incubated on ice for 15 min, and the plasmid and competent cells were added together to a pre-chilled electroporation cuvette. Electroporation: voltage 2000 V, resistance 200 Ω, capacitance 25 μF, cuvette diameter 0.2 mm, breakdown time 5 ms. Immediately after electroporation, 400 μl of pre-chilled sorbitol was added, and the mixture was transferred to a sterile 1.5 ml centrifuge tube and activated at 30°C and 230 rpm for 2 h. Centrifuge at 5000g for 1 min, discard 300 μl of supernatant, resuspend the cells and spread on YPDSZ solid plates (tryptone 20 g / L, yeast extract 10 g / L, 20% glucose, 4 mg / ml G418), and incubate at 30℃ and 230 rpm for 48 h.

[0102] 2.7 Induced Expression

[0103] Select a single colony that has grown well on a resistant plate and inoculate it into 1 L B MMY medium. Incubate at 30°C and 230 rpm for 48 h. Centrifuge at 3000 rpm for 5 min and discard the supernatant. Resuspend the colony in 500 ml of sterile water, centrifuge again at 3000 rpm for 5 min, and discard the supernatant. Resuspend the colony in 1 L B MMY medium (without methanol), transfer it to an Erlenmeyer flask, and add 1% methanol. Add 1% methanol every 24 h. After induction for 72 h, centrifuge and collect the supernatant for purification.

[0104] 2.8 Ion Exchange Purification

[0105] The target protein was dialyzed into buffer A (20 mM PB, pH 4.0). The protein sample was filtered through a 0.22 μm pore size and 33 mm diameter filter membrane, followed by cation exchange using an AKTApure column (5 ml SP column). Linear elution with buffer B (20 mM PB and 1 M NaCl, pH 4.0) yielded a highly pure target protein. SDS-PAGE was used to verify the protein's molecular weight and purity. Protein loading buffer (with added DTT) was added to the protein sample, mixed, and placed in a metal bath. The mixture was heated at 95°C for 10 min, centrifuged at 1500 rpm for 1 min, and the supernatant was analyzed by SDS-PAGE. BCA was used to determine the protein concentration and estimate the protein yield. Based on the protein purity, the protein was concentrated or lyophilized for storage.

[0106] 3. Results

[0107] Figure 3 This is an SDS-PAGE electrophoresis image of the recombinant type XVII collagen LY1708 expressed and purified in Pichia pastoris according to the present invention. Its molecular weight is approximately 41 kDa (theoretical value 22.79 kDa).

[0108] Example 3: Scale-up, induced expression, and purification of the recombinant type XVII collagen LY1708 of the present invention.

[0109] 1. Preparation of Fermentation Seed Liquid

[0110] Pick a single colony from the plate and transfer it to a 50ml centrifuge tube containing 5ml of liquid YPD medium. Incubate overnight at 30°C. Scale up the culture to 200ml of YPG in an Erlenmeyer flask at a 1:100 inoculation ratio and incubate overnight at 30°C.

[0111] 2. Prepare the fermentation medium:

[0112] ①Basic salt medium (BSM): calcium sulfate 0.93g / L, potassium sulfate 18.2g / L, magnesium sulfate 7.28g / L, potassium hydroxide 4.13g / L, phosphate 26.7ml / L, glycerol 40g / L, adjust the pH to 5.0 with ammonia, and add an additional 4ml / L of PTM1.

[0113] ②PTM1: CuSO4-5H2O 6.0g, NaI 0.08g, MnSO4-H2O 3.0g, Na2MoO4-2H2O 0.2g, Boric acid 0.02g, CoCl2 0.5g, ZnCl2 20.0g, FeSO4-7H2O 65.0g, Biotin 0.2g, 5ml concentrated sulfuric acid.

[0114] 3. Fermentation stage

[0115] Sterilize the fermenter and culture medium. After sterilization and cooling, when the temperature drops to 30°C, turn on the agitator and aeration to the operating environment (usually maximum speed and 0.1-1.0 vvm). Adjust the pH of the culture medium to 5.0 with 28% ammonia water. Add 4.35 ml of sterile PTM1 basic salts per liter of culture medium. Inoculate 5-10% of the initial fermentation volume of the seed culture from the seed culture shake flask into the fermenter. Before the start of culture, the DO value is close to 100%. After the start of culture, oxygen consumption will cause the DO value to drop. Add the required oxygen to ensure that the DO value is above 20%. Perform batch fermentation until glycerol is completely consumed, marked by a significant rebound in the DO value. The cell yield reached at this stage is 90-150 g / L wet cells. Recombinant proteins will not be produced due to the lack of methanol induction.

[0116] After the DO rebound, the glycerol feeding phase begins, adding 50% w / v glycerol containing 12 ml of PTM1 per liter. The feeding rate is set to 18.15 ml / h per liter of initial fermentation broth volume, and the glycerol feeding will continue for approximately 4 hours or longer. After this phase, the cell yield reaches 180-220 g / L wet cells, but no recombinant proteins are produced. After stopping the glycerol feeding, wait for the DO value to rebound to ensure complete glycerol consumption.

[0117] During the induction phase, induction was initiated using 100% methanol containing 12 ml of PTM1 basic salts per liter. The feed rate was set at 3.6 ml / h per liter of initial fermentation broth volume. During methanol feeding, dissolved oxygen (DO) was maintained above 20% by increasing agitation, aeration, pressure, or oxygen supplementation. Once the culture was fully adapted to methanol (2-4 h) and methanol became its limiting growth factor, this low feed rate was maintained for at least 1 h under limiting conditions before doubling the feed rate. The feed rate was then doubled to approximately 7.3 m / h per liter of initial fermentation broth volume.

[0118] 4. Fermentation broth sample processing

[0119] Centrifuge at 8000 rpm for 40 min to separate the bacterial precipitate and fermentation broth supernatant. Filter through a Pall 0.22 μm membrane, monitoring pressure changes before and after filtration. Use a turbidity reagent to detect turbidity changes before and after filtration.

[0120] 5. Ion exchange purification

[0121] The fermentation broth after centrifugation and filtration was diluted 5-fold with buffer A (20 mM PB, pH 4.0), and cation exchange was performed using AKTA pure (SP column). Gradient elution with buffer B (20 mM PB and 1 M NaCl, pH 4.0) yielded the target protein with high purity. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the protein. Protein loading buffer (with added DTT) was added to the protein sample, mixed well, and placed in a metal bath. The mixture was heated at 95°C for 10 min, centrifuged at 1500 rpm for 1 min, and the supernatant was collected for SDS-PAGE analysis. BCA method was used to determine protein concentration and estimate protein yield. Based on protein purity, the protein was concentrated and the buffer was changed, or it was lyophilized for storage.

[0122] 6. Results

[0123] like Figure 4 As shown, the bacterial weight of LY1708 in a 20L fermenter changes with fermentation time. After 120 hours, the bacterial weight reaches approximately 500g / L.

[0124] like Figure 5 The figure shows the expression of the target protein LY1708 in a 20L fermenter over fermentation time. 1X represents the fermentation supernatant after 91 hours of fermentation, 2X represents the fermentation supernatant diluted 2 times after 91 hours of fermentation, and 4X represents the fermentation supernatant diluted 4 times after 91 hours of fermentation. Based on the grayscale values, the expression level of LY1708 was approximately 5.3 g / L.

[0125] Example 4: Cell proliferation assay to detect the cell proliferation-promoting activity of type XVII collagen.

[0126] Cell proliferation assays are a commonly used method to detect the cell proliferation activity of collagen; a higher proliferation rate reflects better biological activity of collagen. The specific steps are as follows:

[0127] 1. Cell Seeding and Culture: 96-well plates are typically used for experiments. Mouse 3T3 cells (from Haixing Biotechnology) in logarithmic growth phase are digested and resuspended, seeded at a density of 2000 cells / well. The cell suspension is diluted, and 100 μl of cell suspension is added to each well. For the culture medium group, 100 μl of complete culture medium is added to each well, with 3-6 replicates per group. Note that the wells around the perimeter should not be used as sample wells; 100 μl of PBS is added to each well.

[0128] 2. Cell Culture and Treatment: Place the 96-well plate in a cell culture incubator and continue culturing for 24 hours until the cells adhere well. Aspirate the original culture medium and add culture medium containing 5 μg / ml BSA, competitor 1, and LY1708, respectively, and continue culturing for 24 hours.

[0129] 3. Adding CCK8 solution: Directly add 10 μL of CCK8 (Beyotime) solution, or prepare a medium containing 10% CCK8 solution using complete medium. Replace the original medium with medium containing CCK8 and continue culturing for about 1-4 hours until a distinct orange-yellow color appears.

[0130] 4. Absorbance measurement: Use an ELISA reader to measure at two wavelengths: detection wavelength 450-490nm and reference wavelength 600-650nm, and record the data.

[0131] 5. Data Analysis and Calculation: Calculate the cell proliferation rate based on the absorbance values. Cell proliferation rate = (Experimental group absorbance - Blank control absorbance) / (Control group absorbance - Blank control absorbance) × 100%.

[0132] The results are as follows Figure 6 , 7 As shown, the recombinant type XVII collagen LY1708 of the present invention has significant cell proliferation-promoting activity.

[0133] Example 5: Cell Scratch Assay for Detecting the Cell Migration Activity of Type XVII Collagen

[0134] The cell scratch assay is a commonly used method to detect the cell migration activity of collagen; a higher migration rate reflects better biological activity of the collagen. The specific steps are as follows:

[0135] 1. Resuscitate and passage mouse 3T3 cells (Haixing Biotechnology) 3 days in advance to maintain good condition and viability. Once the cell viability reaches 98% or higher, seed them into 6-well plates at a density of 5 × 10⁶ cells / well. 5 Cells per ml, plated in 2 ml of water, and incubated statically at 37°C in a 5% CO2 incubator for 24 h. When the cells have covered more than 90% of the 6-well plate, proceed to the next step of the scratch assay.

[0136] 2. Experimental groups: control group PBS, positive control group EGF, and experimental group LY1708. The type XVII collagen lyophilized powder LY1708 to be tested in the experimental group was dissolved in 20mM PB buffer (pH 7.4), the protein concentration after dissolution was detected and diluted to a concentration of 0.5mg / ml.

[0137] 3. After irradiating the plate with ultraviolet light for 30 minutes, use a ruler and a black marker to draw parallel horizontal lines 0.5-1 cm apart on the bottom of the 6-well plate. Draw at least 3 lines for each well. Mark the wells after drawing the lines for easy observation later.

[0138] 4. Using a cell scraper or a 300µl pipette tip, draw a line perpendicular to the bottom of the 6-well plate on the surface of the superimposed monolayer of cells. Try to keep the width of the line consistent. Wash three times with PBS to remove free cells and cell debris caused by the line. Add 2ml of serum-free DMEM medium and the prepared protein solution to the wells, ensuring a final protein concentration of 5µg / ml. Continue to incubate at 37°C in a 5% CO2 incubator.

[0139] 5. Observe and photograph the cell scratches under a microscope at 0h and 24h. Process the cell migration images using ImageJ software to obtain the initial scratch area and the area of ​​the blank area after cell migration. Calculate the migration rate: Migration rate = (Initial scratch area - Area of ​​blank area after cell migration / Initial scratch area) * 100%.

[0140] The results are as follows Figure 8 As shown, after 24 hours of treatment, the scratch recovery area of ​​the recombinant type XVII collagen LY1708 cells of this invention was significantly reduced compared with the control group, and the cell migration promotion effect reached a level comparable to that of EGF.

[0141] Data analysis such as Figure 9 As shown, the BSA-treated group had a scratch recovery area of ​​25.55%, while the recombinant XVII type collagen LY1708 cell scratch recovery area of ​​the present invention was 66.26%, and that of the EGF-treated group was 57.16%.

[0142] It is evident that the recombinant type XVII collagen LY1708 of this invention has an excellent effect on promoting cell migration.

[0143] Example 6: Quantitative Analysis of the Effects of Recombinant Type XVII Collagen LY1708 on Other Collagen-Related Proteins

[0144] HacaT cells (human fibroblasts, CAS Cell Bank) were prematurely revived and passaged in DMEM medium containing 10% FBS when cell confluence reached 90%. Cells in logarithmic growth phase were cultured at 4 × 10⁶ cells per well. 5A certain number of HacaT cells were seeded into 6-well plates and then cultured in a static incubator at 37°C and 5% CO2 for 12 hours (at which point the cell confluence reached over 80%). After culture, the culture medium was removed, and the cells were washed twice with PBS to remove residual medium. Next, 2 mL of serum-free medium diluted with BSA, competitor-1, and LY1708 samples (final concentration 1 μg / mL) was added to each well, with PBS used as a control. The cells were then returned to the 37°C, 5% CO2 incubator and sampled after 24 hours. Before sampling, the wells were washed 2-3 times with PBS to remove excess liquid. Then, 0.5 mL of Trizol was added to each well, and the cells were repeatedly aspirated to collect the adherent cells. Finally, all samples were transferred to 1.5 mL RNase-free centrifuge tubes. RNA was extracted using a kit; detailed instructions were provided in the kit's manual (Novizan). Reverse transcription (with an equal volume of RNA) was performed to convert the RNA to cDNA, followed by qPCR; specific procedures were provided in the kit's manual (Aikerui). 2^(-ΔΔCt) is a method for calculating relative expression levels, where ΔΔCt represents the change in the cycle threshold (Ct) after baseline correction. The following are the steps for calculating 2^(-ΔΔCt):

[0145] Calculate ΔCt for a single sample: For each sample, first calculate the Ct difference between its target gene and reference gene.

[0146] The formula is: ΔCt=Ct_Target-Ct_Reference

[0147] Here, Ct_Target is the cycle threshold of the target gene, and Ct_Reference is the cycle threshold of the internal reference gene.

[0148] Calculate the average ΔCt of the control samples: Select a set of control samples (usually calibration samples or blank controls) and calculate the average ΔCt.

[0149] The formula is: ΔCt_mean = mean(ΔCt_controls)

[0150] Calculate ΔCt: For each test sample, calculate the difference between its ΔCt and the average ΔCt of the control samples.

[0151] The formula is: ΔCt=ΔCt-ΔCt_mean

[0152] Calculate the relative expression level (2^(-ΔΔCt)): Finally, use the formula 2^(-ΔΔCt) to calculate the relative expression level of the target gene relative to the control sample. This formula is based on the assumption that the cycle threshold (Ct) is inversely proportional to the initial template amount, and the 2^x function reflects this inverse relationship. This method quantifies the relative expression level of the target gene in different samples. It is important to note that this calculation method assumes that the expression of the internal reference gene is stable in all samples and that the amplification efficiency is the same. In practical applications, these assumptions should be ensured to hold or appropriate corrections should be made.

[0153] The results are as follows Figure 10 , 11 As shown, the recombinant type XVII collagen LY1708 of the present invention can significantly promote the synthesis of type I and type III collagen even at a concentration of only 1 μg / ml (1 ppm).

[0154] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant type XVII collagen, characterized in that, Its amino acid sequence is formed by repeated tandem of n core units, where n is an integer of 1 or greater than 1, and the amino acid sequence of the core unit is shown in SEQ ID NO:

1.

2. The recombinant type XVII collagen according to claim 1, characterized in that, The value n is selected from any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

3. The gene encoding the recombinant type XVII collagen as described in claim 1 or 2.

4. A recombinant vector expressing the recombinant type XVII collagen as described in claim 1 or 2, characterized in that, It contains the gene as described in claim 3.

5. Recombinant cells expressing the recombinant type XVII collagen of claim 1, characterized in that, The recombinant cells are transfected with the recombinant vector of claim 4.

6. The method for preparing recombinant type XVII collagen according to claim 1, characterized in that, Includes the following steps: 1) The gene described in claim 3 is recombined into the vector pET28a to obtain a recombinant vector; 2) The recombinant vector was transfected into Escherichia coli BL21(DE3) to obtain recombinant cells; 3) After culturing the recombinant cells, centrifuge and collect the supernatant; 4) The recombinant type XVII collagen was separated from the supernatant.

7. The preparation method according to claim 6, characterized in that, It also includes constructing gene fragments encoding hydroxylases L593 and L230 into the expression vector pCDFDuet-1 to obtain the recombinant vector pCDFDuet-1-L593-L230, which is then transfected into Escherichia coli BL21(DE3).

8. The method for preparing recombinant type XVII collagen according to claim 1, characterized in that, Includes the following steps: 1) The gene described in claim 3 is recombined into the vector pPICZαA to obtain a recombinant vector; 2) The recombinant vector was transfected into Pichia pastoris GS115 to obtain recombinant cells; 3) After culturing the recombinant cells, centrifuge and collect the supernatant; 4) The recombinant type XVII collagen was separated from the supernatant.

9. The preparation method according to claim 8, characterized in that, It also includes constructing the gene fragment encoding the hydroxylase BaP4H into the expression vector pPIC9k to obtain the recombinant vector pPIC9k-BaP4H, which is then transfected into Pichia pastoris GS115.

10. The recombinant type XVII collagen of claim 1, and the recombinant type XVII collagen prepared by the preparation method of claim 6 or 8, include any of the following applications for non-therapeutic purposes: i) Promotes cell proliferation; ii) Promotes cell migration; iii) Preparation of cosmetics; iv) Preparation of medical materials.

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