Recombinant type XVII collagen with triple helical structure and its application

Abstract

The present invention provides a recombinant type XVII collagen with a triple helix structure and its application, belonging to the field of protein engineering technology. The amino acid sequence of the recombinant type XVII collagen is formed by repeated series connection of n core units, where n is an integer of 1 or greater, and the amino acid sequence of the core unit is shown in SEQ ID NO: 1. The present invention also provides methods for constructing various expression systems and preparing recombinant type XVII collagen. The present invention also provides practical industrial applications based on the characteristics of the protein. The recombinant type XVII collagen provided by the present invention has high purity, good thermal stability, and high biological activity. Its molecular weight is much smaller than that of natural collagen, reducing obstacles for collagen to cross the skin barrier and exert its biological function. Experiments have shown that it has multiple biological activities such as promoting cell migration.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein engineering, and in particular relates to recombinant type XVII collagen with a triple helical structure and its application. Background Art

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

[0003] Natural collagen has a unique triple-helical structure, which is closely related to its stability and various biological activities, such as promoting cell proliferation, adhesion, and migration (Marion, 2010). Type XVII collagen is 1497 amino acids long and comprises an intracellular domain, a transmembrane domain, and an extracellular domain. These domains are further divided into 16 non-triple-helical regions and 15 triple-helical regions arranged in an alternating pattern. This makes in vitro recombinant expression difficult, resulting in low yields and easy degradation. Assembly into its native conformation, where non-triple-helical and triple-helical regions intersect, is even more challenging.

[0004] Patent publication number CN110845603A uses a truncated sequence of the C15 helical region and approximately 130 amino acids from the C-terminus of type XVII collagen for expression verification, achieving good yields and lacking post-translational modification activity using an E. coli expression system. Patent publication number CN116640205A uses a Pichia pastoris expression system to splice truncated sequences of the non-helical region NC16 and C15 helical region with the C-terminus of type XVII collagen. Patent publication number CN116751282A uses a Saccharomyces cerevisiae expression system to splice truncated sequences of the C15 helical region with the C1 helical region. However, none of these technologies have been shown to form the natural triple helical structure of collagen.

[0005] When used as an ingredient in skin care products, collagen's natural micelle structure and large molecular weight make it difficult for it to penetrate the skin barrier and reach the dermis to exert its effects.

[0006] In summary, the existing technologies have the following problems: 1) Due to the limitations of the traditional prokaryotic expression system, the recombinant collagen is not modified after translation; 2) an advanced triple helical conformation is not formed; 3) biological activity is low; 4) stability is poor; 5) natural collagen has a large molecular weight and is not easy to penetrate the skin, etc. Summary of the Invention

[0007] In view of this, the first purpose of the present invention is to provide a recombinant type XVII collagen with a triple helix structure. The recombinant collagen provided by the present invention has high purity, good thermal stability, high biological activity, and a molecular weight significantly smaller than that of natural full-length collagen. Experiments have shown that it has multiple biological activities such as promoting cell migration.

[0008] The amino acid sequence of the recombinant type XVII collagen of the present invention is formed by repeated series connection of 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 in SEQ ID NO: 1.

[0009] Preferably, n is selected from any one of 3, 4, 5, 6, 7 and 8. Most preferably, n is selected from 8.

[0010] The repeated series can be a head-to-tail series of core units, or a series of core units connected by a linker, wherein the linker preferably contains glycine (Gly) and / or serine (Ser), and the linker preferably contains 1-5 amino acids.

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

[0012] The encoding includes a protein produced by transcribing a DNA molecule to form an RNA product, which is then translated; or a protein produced by transcribing a DNA molecule to provide an RNA product, which is processed to provide a processed RNA product, which is then translated.

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

[0014] Vectors include any nucleic acid molecule derived from any source and capable of genome integration or autonomous replication (e.g., plasmids, cosmids, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecules), comprising a nucleic acid molecule to which one or more nucleic acid molecules are operably linked. 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 promote 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 the present invention, the recombinant human type III collagen gene may be cloned into the vector alone, or the proline hydroxylase gene and / or lysine hydroxylase gene and the recombinant human type III collagen gene may be cloned into the same vector.

[0016] The present invention provides a recombinant cell expressing the recombinant type XVII collagen, wherein the recombinant cell is transfected with the recombinant vector.

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

[0018] The second object 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 in Escherichia coli BL21 (DE3), comprising the following steps:

[0020] 1) Connecting the gene to the vector pET28a to obtain a recombinant vector;

[0021] 2) Transfecting the recombinant vector into Escherichia coli BL21 (DE3) to obtain recombinant cells;

[0022] 3) culturing the recombinant cells, centrifuging, and collecting the supernatant;

[0023] 4) Separating and purifying the recombinant type XVII collagen from the supernatant.

[0024] More preferably, the method further comprises 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 the recombinant vector into Escherichia coli BL21 (DE3).

[0025] More preferably, the culturing in step 3) includes adding ascorbic acid with a final concentration of 10 mg / ml, 1 mM FeSO4 and 1 mM IPTG to induce the expression of the target protein, which can further improve the thermal stability of the target protein.

[0026] In another preferred embodiment, the recombinant type XVII collagen is expressed in Escherichia coli, preferably Pichia pastoris GS115, comprising the following steps:

[0027] 1) Connecting the gene to the vector pPICZαA to obtain a recombinant vector;

[0028] 2) transfecting the recombinant vector into Pichia pastoris GS115 to obtain recombinant cells;

[0029] 3) culturing the recombinant cells, centrifuging, and collecting the supernatant;

[0030] 4) Separating and obtaining the recombinant type XVII collagen from the supernatant.

[0031] More preferably, the method further comprises constructing a gene fragment encoding hydroxylase BaP4H into an expression vector pPIC9k to obtain a recombinant vector pPIC9k-BaP4H, and transfecting the recombinant vector into Pichia pastoris GS115.

[0032] Type XVII collagen is not only a key factor in skin aging and wound repair, but also plays a crucial role in maintaining hair follicle stem cells, preventing hair loss and graying. Col17A1 is highly expressed in hair follicle stem cells (HFSCs) and is crucial for maintaining both HFSCs and melanocyte stem cells (MSCs) (Shintaro Tanimura, 2011). Collagen, due to its excellent biocompatibility and biodegradability, is widely studied and applied in biomaterials such as tissue engineering scaffolds and absorbable sutures.

[0033] Therefore, the third object of the present invention is to provide the recombinant type XVII collagen and the recombinant type XVII collagen prepared by the preparation method, including any of the following uses:

[0034] i) promote cell proliferation;

[0035] ii) promote cell migration;

[0036] iii) preparing cosmetics;

[0037] iv) Preparation of medical materials.

[0038] The cell proliferation promoting agent can be used as a basic component of a culture medium to promote cell proliferation, including application in the culture and preservation of hair follicle stem cells or hair follicle tissue in vitro.

[0039] The cell migration promoting agent can be used as a basic component of a culture medium to promote cell migration, including application in the culture and preservation of hair follicle stem cells or hair follicle tissue in vitro.

[0040] The cosmetics include but are not limited to skin care lotion, skin care cream, essence water, facial mask, scalp essence, and shampoo.

[0041] The medical materials include but are not limited to filling materials, repair materials, implants, tissue engineering scaffolds, hemostatic agents, and drug release carriers, wherein the repair materials include but are not limited to bone repair materials, wound dressings, and sutures.

[0042] The present invention also provides a composition comprising a cosmetically or pharmaceutically effective amount of at least one protein according to the aforementioned method 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; further, in order to promote transdermal absorption of collagen molecules, a solid carrier such as a non-woven fabric can be used to prepare a patch product, which is applied to the face to prolong the interaction time between the collagen solution and the skin surface. The recombinant type XVII collagen of the present invention can be prepared and used in series 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, and can also be mixed with the above-mentioned bioactive ingredients after being prepared separately.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1) The recombinant type XVII collagen of the present invention has a theoretical molecular weight of approximately 33 kDa, which is significantly smaller than the natural full-length collagen (150 kDa). This reduces obstacles for collagen to cross the skin barrier and exert its biological function, and has the effect of promoting cell migration;

[0045] 2) The recombinant type XVII collagen of the present invention has an advanced triple helical structure and a stable molecular structure, and its thermal stability can be maintained at 40°C.

[0046] 3) The recombinant type XVII collagen sequence of the present invention is based on the original amino acid sequence of human type XVII collagen. By optimizing and selecting the parts with high biological activity and stable higher-order structure, a new recombinant type XVII collagen sequence is obtained, which has the characteristics of good biocompatibility;

[0047] 4) The recombinant type XVII collagen of the present invention has very good cell migration promoting activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=3) of the present invention expressed in an E. coli system.

[0049] Figure 2 This is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=4) of the present invention expressed in an E. coli system.

[0050] Figure 3 This is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=5) of the present invention expressed in an E. coli system.

[0051] Figure 4This is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=6) of the present invention expressed in an E. coli system.

[0052] Figure 5 This is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=7) of the present invention expressed in an E. coli system.

[0053] Figure 6 This is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=8) of the present invention expressed in an E. coli system.

[0054] Figure 7 The figure is an SDS-PAGE diagram of the recombinant type XVII collagen LY1701 (n=8) of the present invention in the Pichia pastoris expression system.

[0055] Figure 8 This is the circular dichroism spectrum of the recombinant type XVII collagen LY1701 (n=3, 4, 5, 6, 7, 8) of the present invention at room temperature.

[0056] Figure 9 Circular dichroism spectra of the recombinant type XVII collagen LY1701 (n=3, 4, 5, 6, 7, 8) of the present invention at different temperatures.

[0057] Figure 10 Graph showing the results of a cell scratch test using the recombinant type XVII collagen LY1701 (n=8) of the present invention.

[0058] Figure 11 This is a graph showing the cell scratch recovery area analysis of the recombinant type XVII collagen LY1701 (n=8) of the present invention. DETAILED DESCRIPTION

[0059] In the present invention, the gene can be synthesized by a biotechnology company. The present invention has no particular limitation on the preparation method of the recombinant vector and recombinant cell, and conventional preparation methods of recombinant vectors and recombinant cells in the art can be used.

[0060] The present invention has no particular limitation on the separation and purification method, and conventional protein separation and purification methods in the art may be used; for preferred technical solutions, please refer to the examples.

[0061] The present invention characterizes the structure of collagen using circular dichroism (CD), a spectroscopic method commonly used in the art. CD is used to determine the structure of compounds with chiral structures that produce differential absorption of left- and right-handed optical rotation, and is primarily used to measure molecular structural asymmetry. Biomacromolecules generally contain chiral groups and structures, and therefore CD is often used to measure and observe changes in the structure and conformation of biomacromolecules. The CD characteristic of the collagen triple helix structure is generally characterized by a positive absorption peak near 221nm and a negative absorption peak near 195nm (industry standard YY / T1849-2022). The positions of the absorption peaks shift with changes in amino acid sequence and length. The thermal stability of collagen includes the thermal shrinkage temperature (Ts) of collagen fibers and the thermal denaturation temperature (Td) of collagen. The thermal shrinkage temperature of collagen fibers refers to the temperature at which, when heated, collagen fibers undergo axial contraction, shortening to approximately 5% of their original length. The thermal denaturation temperature of collagen refers to the temperature at which, when heated in a medium, the triple helix unwinds to form single strands, reaching 50% of its original size. Therefore, CD spectroscopy can be used to study the helical structure of collagen and its thermal denaturation process.

[0062] In the following examples, LY1701 represents a series of proteins formed by repeating a core unit (SEQ ID NO: 1) in series, wherein the core unit is repeated 3, 4, 5, 6, 7, and 8 times.

[0063] The technical solutions provided by the present invention are 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.

[0064] Example 1 Expression (E. coli expression system) and purification of recombinant type XVII collagen LY1701 of the present invention

[0065] 1. LY1701 sequence synthesis

[0066] GenScript Biotech Co., Ltd. was commissioned to synthesize gene fragments of the amino acid sequence of recombinant type XVII collagen LY1701 (SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, and SEQ ID NO. 7) and to construct them into the pET28a E. coli expression vector. GenScript Biotech Co., Ltd. was commissioned to synthesize gene fragments of hydroxylases L593 and L230 and to construct them into the expression vector pCDFDuet-1 behind two promoters.

[0067] 2. Construction of E. coli Expression System

[0068] 2.1 Conversion

[0069] Competent BL21(DE3) cells (Shenzhen Kangti Life Science) were co-transformed with the type XVII collagen plasmid pET28a-LY1701 (kanamycin resistance) and the hydroxylase plasmid pCDFDuet-1-L593-L230 (streptomycin resistance). The cells were incubated on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, and immediately cooled on ice for 2-3 minutes. 900 μl of SOC or LB medium (without antibiotics) was added and incubated at 37°C for 1 hour (200-250 rpm). Simultaneously, a kanamycin- and streptomycin-resistant LB solid medium plate (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / ml kanamycin antibiotic) was preheated in a 37°C incubator. After incubation, the cells were centrifuged at 5000 rpm (2400 × g) for 5 minutes, and the 900 μl supernatant was discarded. Resuspend the cells in the remaining culture medium and spread evenly on a preheated double-resistant plate using a sterile spreader. Incubate inverted at 37°C for 12-16 hours until visible colonies form.

[0070] 2.2 Take out the plate with colonies, pick out the monoclonal colonies, inoculate them into 10 ml LB liquid medium (containing kanamycin and streptomycin antibiotics), culture them at 37 ° C, 220 rpm overnight, then add them into 1 L LB medium (containing kanamycin and streptomycin antibiotics) at a ratio of 1:100, culture them at 37 ° C, 220 rpm for 3-5 hours, and then monitor the OD value. 600 When the pH value reaches 1.0-1.2, ascorbic acid (Shanghai Sangon Biotechnology), 1 mM FeSO₄ (Shanghai Sangon Biotechnology), and 1 mM IPTG (Biosharp) are added to a final concentration of 10 mg / ml to induce expression at 16°C, 180 rpm, for 18-24 hours. The cells are harvested by centrifugation for further purification or frozen at -20°C or below.

[0071] 3. Protein Purification

[0072] 3.1 After the induction, keep a sample of the bacterial solution for gel running and centrifuge at 8000 rpm for 20 minutes to collect the bacteria.

[0073] 3.2 Preparation of Ni-NTA (Sangon Biotech Co., Ltd.) purification buffer:

[0074] Lysis buffer: 50 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, 5% glycerol, pH 7.0;

[0075] Wash buffer: 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, 5% glycerol, pH 7.0;

[0076] Elution buffer: 50 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, 5% glycerol, pH 7.0.

[0077] 3.3 Preparation of lysis buffer

[0078] Lysis buffer components: PMSF (MIKX) at a final concentration of 1 mM, protease inhibitor (MCE), super nuclease (Biyuntian), and MgCl2 (Sangon Biotechnology) at a final concentration of 1 mM.

[0079] According to the weight of the bacteria, add the corresponding volume of lysis buffer at a ratio of 1:10, use a mixer to resuspend the bacteria, and mix by pipetting.

[0080] 3.4 Affinity purification

[0081] Pre-cool the high-pressure homogenizer (Dongning Biotech). Pour the bacterial suspension into the column and recycle three times at 700-800 bar. Collect the broken bacterial suspension. Centrifuge in a high-speed refrigerated centrifuge (Beckman) at 4°C, 20,000 rpm, for 1 hour. After centrifugation, transfer the supernatant to a new 50ml centrifuge tube and filter through a 0.45μm filter. Remove the Ni-NTA gravity column (1ml Ni-NTA), rinse with 5 column volumes of filtered pure water, rinse with 3 column volumes of lysis buffer, and load the column using a peristaltic pump. Rinse the peristaltic pump tubing extensively with pure water, then rinse with lysis buffer. Then, load the filtered supernatant onto the Ni-NTA gravity column using a peristaltic pump and collect the flow-through. Wash the Ni-NTA column with 100ml of wash buffer and collect the wash buffer. Elute the target protein with elution buffer, adding 1ml at a time, incubate for 5 minutes, and collect the eluate.

[0082] 3.5 Ion exchange purification

[0083] The target protein was dialyzed into buffer A (20mM PB, pH 7.0). The protein sample was filtered using a 0.22μm, 13mm filter membrane. Cation exchange was performed using AKTApure (5ml SP column). Linear elution was performed using buffer B (20mM PB and 1M NaCl, pH 7.0) to obtain the target protein of high purity. The protein molecular weight and purity were verified by SDS-PAGE electrophoresis. The protein sample was mixed with protein loading buffer (DTT) and placed in a metal bath. The sample was then boiled at 95°C for 10 minutes before running on the gel. The protein concentration was determined by the BCA method to estimate the protein yield. Depending on the purity of the protein, the protein was concentrated or lyophilized for storage.

[0084] 4. Results

[0085] Figures 1-6 The SDS-PAGE electrophoresis diagram of the recombinant type XVII collagen of the present invention after expression and purification in Escherichia coli.

[0086] Example 2 Expression (Pichia pastoris expression system) and purification of the recombinant type XVII collagen LY1701 of the present invention

[0087] 1. LY1701 sequence synthesis

[0088] GenScript Biotech was commissioned to synthesize a gene fragment encoding the amino acid sequence (SEQ ID NO. 7) of the recombinant type XVII collagen LY1701 of the present invention and to construct it into the pPICZalphaA Pichia pastoris expression vector. GenScript Biotech was commissioned to synthesize a gene fragment encoding the hydroxylase BaP4H and to construct it into the pPIC9k vector.

[0089] 2. Construction of Pichia pastoris expression system

[0090] 2.1 Electroporation

[0091] The type XVII collagen plasmid pPICZalphaA-LY1701 was linearized at the SacI site by PCR amplification, followed by PCR fragment purification using the Thermo GeneJET PCR Purification Kit. GS115 competent cells were thawed on ice, and 5-10 μg of the linearized plasmid was added. The cells were incubated on ice for 15 minutes. The plasmid and competent cells were then added to a pre-chilled electroporation cuvette. Electroporation was performed using a voltage of 2000 V, a resistance of 200 Ω, a capacitance of 25 μF, 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 cells were transferred to a sterile 1.5 ml centrifuge tube and activated at 230 rpm at 30°C for 2 hours. Centrifuge at 5000 g for 1 min, discard 300 μl of supernatant, resuspend the cells and spread on YPDSZ solid plates (20 g / L tryptone, 10 g / L yeast extract, 182.1 g / L D-sorbitol, 20% glucose, 100 μg / ml bleomycin), and culture at 30°C and 230 rpm for 48 h.

[0092] 2.2 Remove the plate with colonies, pick out single clones from it, and replicate them on YPD solid medium with high concentration (400 μg / ml or 800 μg / ml) of bleomycin for screening.

[0093] 2.3 Remove the plate containing colonies and select a single colony. Inoculate each colony into 50 ml of YPD liquid medium (containing 100 μg / ml zeocin) and culture overnight at 30°C, 230 rpm. Amplify the overnight culture by inoculating the seed solution into 1 L of MGY medium (20 g / L tryptone, 10 g / L yeast extract, 11.73 g / L potassium dihydrogen phosphate (KH2PO4), 2.4 g / L potassium dihydrogen phosphate (K2HPO4), 10 g / L glycerol, 134 g / L YNB, and 1× biotin) at a ratio of 1:50. Incubate at 30°C, 230 rpm for 48 h. The BMGY seed culture was centrifuged at 3000 rpm for 5 minutes, the supernatant discarded, and the suspension resuspended in sterile water. The suspension was centrifuged again at 3000 rpm for 5 minutes, the supernatant discarded, and the suspension resuspended in 1 L of BMMY medium (without methanol). 1% methanol was added for induction, and 1% methanol was added every 24 hours. After 72 hours of induction, the supernatant was centrifuged and purified.

[0094] 2.4 Ion exchange purification

[0095] The target protein was dialyzed into buffer A (20mM PB, pH = 7.0). The protein sample was filtered using a filter membrane with a pore size of 0.22μm and a diameter of 13mm. Cation exchange was performed using AKTApure (5ml SP column). Buffer B (20mM PB and 1M NaCl, pH = 7.0) was used for linear elution to obtain the target protein with high purity. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the protein. The protein sample was added with protein loading buffer (with DTT added), mixed, placed in a metal bath, heated at 95°C for 10min, centrifuged at 1500rpm for 1min, and the supernatant was taken for SDS-PAGE detection. The protein concentration was determined by BCA method to estimate the protein yield. Depending on the purity of the protein, it was concentrated or lyophilized for storage.

[0096] 2.5 Preparation of yeast competent cells containing LY1701 expression plasmid

[0097] Select LY1701 monoclonal colonies with high expression yield and culture them in 5 ml YPD medium (bleomycin resistance) at 30°C and 230 rpm overnight. Inoculate them into 500 ml fresh YPD medium (bleomycin resistance) at a ratio of 1:200 and culture them overnight until OD 600 = between 1.3 and 1.5. Harvest the cells by centrifugation at 1500 g for 5 min at 4°C. Resuspend the cells in 500 ml of pre-chilled sterile water. Centrifuge again at 1500 g for 5 min at 4°C to harvest the cells. Wash three times with pre-chilled sterile water. Resuspend the cells in 20 ml of 1 M sorbitol. Discard the supernatant. Repeat the wash process two to three times. Harvest the cells by centrifugation at 1500 g for 5 min at 4°C. Resuspend the cells in 500 μl of 1 M sorbitol and store at -80°C at 100 μl per tube.

[0098] 2.6BaP4H-pPIC9k linearization and electroporation

[0099] Design primers to linearize the SalI site of pPIC9k using PCR, and recover the PCR fragments using the Thermo GeneJET PCR Purification Kit. Thaw the yeast competent cells containing the LY1701 expression plasmid obtained in Example 2.4 on ice, add 5-10 μg of the linearized plasmid, and incubate on ice for 15 minutes. Add the plasmid and competent cells to a pre-chilled electroporation cuvette. Electroporation: voltage 2000 V, resistance 200 Ω, capacitance 25 μF, electroporation cuvette diameter 0.2 mm, breakdown time 5 ms. Immediately after electroporation, add 400 μl of pre-chilled sorbitol, transfer to a sterile 1.5 ml centrifuge tube, and activate at 30°C, 230 rpm for 2 hours. The cells were centrifuged at 5000 g for 1 min, 300 μl of supernatant was discarded, and the cells were resuspended and spread on YPDSZ solid plates (20 g / L tryptone, 10 g / L yeast extract, 20% glucose, 4 mg / ml G418), and cultured at 30°C, 230 rpm for 48 h.

[0100] 2.7 Inducible Expression

[0101] Select a single colony that has grown well on the resistant plate and inoculate it into 1 L of BMGY medium. Incubate at 30°C and 230 rpm for 48 hours. Centrifuge at 3000 rpm for 5 minutes and discard the supernatant. Resuspend the culture in 500 ml of sterile water and centrifuge again at 3000 rpm for 5 minutes. Discard the supernatant. Resuspend the culture in 1 L of BMMY medium (without methanol) and transfer it to an Erlenmeyer flask. Add 1% methanol, and add 1% methanol every 24 hours. After 72 hours of induction, centrifuge and collect the supernatant for purification.

[0102] 2.8 Ion exchange purification

[0103] The target protein was dialyzed into buffer A (20mM PB, pH = 7.0). The protein sample was filtered using a filter membrane with a pore size of 0.22μm and a diameter of 13mm. Cation exchange was performed using AKTApure (5ml SP column). Buffer B (20mM PB and 1M NaCl, pH = 7.0) was used for linear elution to obtain the target protein with high purity. SDS-PAGE electrophoresis was used to verify the molecular weight and purity of the protein. The protein sample was added with protein loading buffer (with DTT added), mixed, placed in a metal bath, heated at 95°C for 10min, centrifuged at 1500rpm for 1min, and the supernatant was taken for SDS-PAGE detection. The protein concentration was determined by BCA method to estimate the protein yield. Depending on the purity of the protein, it was concentrated or lyophilized for storage.

[0104] 3. Results

[0105] Figure 7This is the SDS-PAGE electrophoresis diagram of the recombinant type XVII collagen LY1701 of the present invention after expression and purification in Pichia pastoris. The molecular weight is 49 KDa (theoretical value 33.75 KDa).

[0106] Example 3 Detection of the triple helical conformation of type XVII collagen by circular dichroism (CD)

[0107] The type XVII collagen lyophilized powder LY1701 prepared in Example 1 was dissolved in 20 mM PB buffer (pH 7.4) to a concentration of 0.5 mg / ml. The sample concentration was further diluted to 0.01 mg / ml and the volume was 3 ml. The sample to be tested was transferred to a 10 mm × 10 mm sample cell of a circular dichroism spectrometer. The scanning wavelength range was set to 190 nm-260 nm, the scanning speed was set to 100 nm / min, and the scanning temperature was set to room temperature. The CD spectra were all averaged over three scans. The results are shown in FIG. Figure 8 As shown, the recombinant type XVII collagen of the present invention has a maximum characteristic positive peak at 221 nm and a negative peak at less than 200 nm. According to the circular dichroism characteristics of the known collagen triple helix structure, it is judged that the collagen sample has a triple helix structure.

[0108] Example 4 Circular Dichroism (CD) Detection of Thermal Stability of Type XVII Collagen

[0109] The collagen sample was diluted according to the method described in Example 3, and the thermal stability of the protein was detected by real-time temperature increase at a rate of 1°C / min. The CD spectra were the average of three scans. The data were analyzed and calculated on the software, and the relationship between the molar ellipticity of the recombinant collagen and temperature was measured at 221 nm. The results are shown in Figure 2. Figure 9 As shown, based on the disappearance of the characteristic positive peak at 221 nm, the thermal stability of the preferred LY1701 can be maintained at 40°C.

[0110] Example 5 Cell scratch assay to detect the cell migration activity of type XVII collagen

[0111] The cell scratch test is a commonly used method to detect the cell migration activity of collagen. The higher the migration rate, the better the biological activity of the collagen. The specific steps are as follows:

[0112] 1. Resuscitate and passage mouse 3T3 cells (Starfish Bio) 3 days in advance to maintain a good state and viability. When the cell viability reaches above 98%, inoculate them into 6-well plates at a density of 5×10 5 The cells were plated at a volume of 2 ml and cultured in a 37°C, 5% CO2 incubator for 24 h. When the cells filled the 6-well plate (more than 90%), the next step of the scratch test was performed.

[0113] 2. Dissolve the test type XVII collagen lyophilized powder LY1701 (SEQ ID NO. 7) in the experimental group, BSA in the control group, and commercially available recombinant collagen products (competitors 1 and 2) in 20 mM PB buffer (pH 7.4). Detect the protein concentration after dissolution and dilute it to 0.5 mg / ml.

[0114] 3. Use a ruler that has been exposed to ultraviolet light for 30 minutes and a black marker to draw parallel horizontal lines with a distance of 0.5-1 cm on the bottom of the 6-well plate. Draw at least 3 lines in each well. After drawing the horizontal lines, mark the plate separately for subsequent observation.

[0115] 4. Use a cell scraper or a 300ul pipette tip perpendicular to the surface of the 6-well plate to make a scratch perpendicular to the bottom of the 6-well plate in the confluent monolayer of cells. Try to ensure that the scratch width is consistent during the process. Wash with PBS three times to remove free cells and cell debris caused by the scratch. Add 2ml of DMEM serum-free medium and the prepared protein solution to the wells, and ensure that the final concentration of the protein is 50μg / ml. Continue to culture in a 37℃, 5% CO2 incubator.

[0116] 5. Observe the cell scratches under a microscope and take photos at 0 h and 12 h. Process the images of cell migration using Image J 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 - blank area after cell migration / initial scratch area) * 100%.

[0117] The results are as follows Figure 10 As shown, after only 12 hours of treatment, the scratch recovery area of ​​LY1701 cells treated with the recombinant type XVII collagen of the present invention was significantly reduced compared with the control group, and the cell aggregation phenomenon in the blank area was more significant than that in other groups.

[0118] Data analysis such as Figure 11 As shown, the recovery area of ​​the scratch of the BSA-treated group was 23.61%, the recovery area of ​​the recombinant type XVII collagen LY1701 cells of the present invention was 39.68%, while that of the collagen-treated group of the comparative product 1 was 24.88%, and that of the collagen-treated group of the comparative product 2 was 28.92%.

[0119] It can be seen that the recombinant type XVII collagen LY1701 of the present invention has an excellent effect of promoting cell migration.

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that Its amino acid sequence is shown in SEQ ID NO:

1.

2. A recombinant type XVII collagen, characterized in that: Its amino acid sequence is formed by repeated series connection of n core units, the amino acid sequence of the core unit is shown in SEQ ID NO: 1, and n is selected from any one of 3, 4, 5, 6, 7, and 8.

3. A gene encoding the recombinant type XVII collagen according to claim 2.

4. The recombinant vector expressing the recombinant type XVII collagen according to claim 2, characterized in that: Comprising the gene according to claim 3.

5. The recombinant cell expressing the recombinant type XVII collagen according to claim 2, characterized in that: The recombinant cell is transfected with the recombinant vector according to claim 4.

6. The method for preparing recombinant type XVII collagen according to claim 2, characterized in that: The following steps are involved: 1) Recombining the gene described in claim 3 into the vector pET28a to obtain a recombinant vector; 2) Transfecting the recombinant vector into Escherichia coli BL21 (DE3) to obtain recombinant cells; 3) culturing the recombinant cells, centrifuging, and collecting the supernatant; 4) Separating and obtaining the recombinant type XVII collagen from the supernatant.

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

8. The preparation method according to claim 6, characterized in that The culture in step 3) includes adding ascorbic acid with a final concentration of 10 mg / ml, 1 mM FeSO4 and 1 mM IPTG to induce the expression of the target protein.

9. The method for preparing recombinant type XVII collagen according to claim 2, characterized in that: The following steps are involved: 1) Recombining the gene described in claim 3 into the vector pPICZαA to obtain a recombinant vector; 2) transfecting the recombinant vector into Pichia pastoris GS115 to obtain recombinant cells; 3) culturing the recombinant cells, centrifuging, and collecting the supernatant; 4) Separating and obtaining the recombinant type XVII collagen from the supernatant.

10. The preparation method according to claim 9, characterized in that The method also includes constructing a gene fragment encoding hydroxylase BaP4H into an expression vector pPIC9k to obtain a recombinant vector pPIC9k-BaP4H, and transfecting the recombinant vector into Pichia pastoris GS115.

11. Use of the recombinant type XVII collagen according to claim 2 or the recombinant type XVII collagen prepared by the preparation method according to claim 6 or 9 in any of the following: i) preparing cosmetics; ii) Preparation of medical materials.

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