Recombinant type XVII collagen having triple helical structure and its use

Abstract

The present disclosure provides recombinant type XVII collagen having a triple helix structure and uses thereof, which belong to the field of protein engineering technology. The recombinant type XVII collagen has an amino acid sequence formed by tandem repeats of n or more core units, where n is an integer of 1 or more, and the core unit has the amino acid sequence shown in SEQ ID NO: 1. The present disclosure also provides methods for constructing multiple expression systems and methods for preparing recombinant type XVII collagen. provide.

Description

[Technical Field]

[0001] The present disclosure belongs to the field of protein engineering technology, and in particular relates to recombinant type XVII collagen having a triple helix structure and uses thereof. [Background technology]

[0002] Although type XVII collagen exists in the body in very small amounts, its direct extraction is extremely difficult and expensive. Currently, large-scale production of type XVII collagen in vitro is primarily achieved through recombinant methods. Commonly used in vitro recombinant protein expression systems include E. coli, yeast, and mammalian cells.

[0003] Native 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 1,497 amino acids long and contains intracellular, membrane, and extracellular domains, which are further divided into 16 non-triple-helical and 15 triple-helical regions. Recombinant expression of type XVII collagen in vitro is problematic due to low yields and facile degradation. It is even more difficult to incorporate expressed type XVII collagen into the non-triple-helical and triple-helical native structures.

[0004] In Chinese Patent Application Publication CN110845603A, a truncated sequence of the C15 helical region and approximately 130 amino acids from the C-terminus of type XVII collagen were selected for expression and validation using an Escherichia coli expression system, resulting in good yields without post-translational modification activity. In Chinese Patent Application Publication CN116640205A, a Pichia pastoris expression system was used, and a truncated sequence of the non-helical region NC16 and the C15 helical region and the C-terminus of type XVII collagen were selected for splicing and assembly. In Chinese Patent Application Publication CN116751282A, a Saccharomyces cerevisiae expression system was used, and a truncated sequence of the C15 helical region and the C1 helical region were selected for splicing and assembly. However, none of the above techniques resulted in the formation of the native triple helix structure of collagen.

[0005] Collagen has a natural micellar structure and a large molecular weight, making it difficult for it to penetrate the skin barrier and exert its effects on the dermis when used as an ingredient in skin care products.

[0006] In summary, the prior art has the following problems: 1) Recombinant collagen is not subject to post-translational modifications due to limitations imposed by traditional prokaryotic expression systems; 2) High-order triple helix structures are not formed; 3) Low biological activity; 4) Low stability; 5) Natural collagen has a large molecular weight, making it difficult to penetrate the skin. Summary of the Invention

[0007] The primary objective of the present disclosure is to provide recombinant type XVII collagen having a triple helical structure. The recombinant collagen provided by the present disclosure has high purity, excellent thermal stability, high biological activity, and a molecular weight much smaller than that of natural full-length collagen. Experiments have shown that recombinant type XVII collagen has multiple biological activities, such as promoting cell migration.

[0008] In the present disclosure, recombinant type XVII collagen has an amino acid sequence formed by tandem repeats of n or more core units, where n is an integer greater than or equal to 1, and the core unit has the amino acid sequence set forth in SEQ ID NO:1.

[0009] Preferably, n is any number selected from 3, 4, 5, 6, 7, or 8. Most preferably, n is 8.

[0010] Tandem repeats may be formed by connecting core units head-to-tail, or by connecting core units in tandem via a linker, which preferably contains glycine (Gly) and / or serine (Ser), and preferably contains 1 to 5 amino acids.

[0011] Furthermore, the core unit of the present invention may be joined to a plurality of amino acids, for example, 1 to 2 amino acids, at the head (N-terminus) or tail (C-terminus) of the consecutively formed amino acid sequence.

[0012] In the present disclosure, recombinant collagen XVII may be used as a domain or may be linked to additional domains from other sources to form fusion proteins to construct and express proteins with multiple functions. Recombinant collagen XVII may also be linked to a label to aid in the expression, purification, or detection of the protein.

[0013] In this disclosure, the introduction of an amino acid at the N-terminus of recombinant collagen XVII may refer to the method described in CN117511978A, which improves the stability of RNA secondary structures and slows down the protein translation rate, thereby allowing the peptide chain to better form the target protein and increasing the protein yield.

[0014] It should be understood that the mass production of the recombinant type XVII collagen of the present disclosure in the form of a single chain structure is also within the scope of protection of the present disclosure.

[0015] The present disclosure provides a gene encoding the recombinant type XVII collagen.

[0016] Encoding consists of transcribing a DNA molecule to form an RNA product, which is then translated into a protein, or transcribing a DNA molecule to form an RNA product, processing the RNA product to provide a processed RNA product, which is then translated into a protein.

[0017] The present disclosure provides a recombinant vector for expressing the recombinant type XVII collagen, comprising the gene.

[0018] A vector includes any nucleic acid molecule (e.g., a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single- or double-stranded DNA or RNA nucleic acid molecule) derived from any source and capable of genomic integration or autonomous replication. A vector includes one or more operably linked nucleic acid molecules. A vector may include, for example, one or more selectable markers, one or more origins of replication (e.g., prokaryotic and eukaryotic origins), one or more multiple cloning sites, and / or elements for stable integration of the construct into the genome of a host cell. Desirable vectors include, but are not limited to, pET28a, pCDFDuet-1, pPICZαA, and pPIC9k.

[0019] In the present disclosure, recombinant human XVII The type 1 collagen gene may be cloned into a vector alone and used to express recombinant human collagen. XVII The type 2 collagen gene may be cloned into the same vector together with the proline hydroxylase-encoding gene and / or the lysine hydroxylase-encoding gene.

[0020] The present disclosure provides recombinant cells that express recombinant collagen XVII, wherein the recombinant cells are transformed with the recombinant vector.

[0021] Recombinant cells include, but are not limited to, E. coli and Pichia pastoris.

[0022] A second object of the present disclosure is to provide a method for expressing recombinant collagen XVII.

[0023] In a preferred embodiment, recombinant collagen XVII is expressed in E. coli, preferably E. coli BL21(DE3), comprising the following steps:

[0024] 1) Ligating the gene into the vector pET28a to obtain a recombinant vector;

[0025] 2) Transform E. coli BL21(DE3) with the recombinant vector to obtain recombinant cells;

[0026] 3) culturing the recombinant cells, followed by centrifugation and collection of the supernatant;

[0027] 4) Recombinant collagen XVII is isolated, purified, and obtained from the supernatant.

[0028] More preferably, the method further includes incorporating gene segments encoding hydroxylases L593 and L230 into expression vector pCDFDuet-1 to obtain recombinant vector pCDFDuet-1-L593-L230, and transforming E. coli BL21(DE3) into the recombinant vector.

[0029] More preferably, the culturing in step 3) includes inducing expression of the protein of interest by adding ascorbic acid to a final concentration of 10 mg / ml, FeSO to a final concentration of 1 mM, and IPTG to a final concentration of 1 mM, which can further improve the thermostability of the protein of interest.

[0030] In another preferred embodiment, the recombinant collagen XVII is Pichia pastoris The vector is expressed in, preferably Pichia pastoris GS115, and has the following steps:

[0031] 1) Into the vector pPICZαA Encoding recombinant collagen XVII to obtain a recombinant vector,

[0032] 2) Transform Pichia pastoris GS115 with the recombinant vector to obtain recombinant cells;

[0033] 3) culturing the recombinant cells, followed by centrifugation and collection of the supernatant;

[0034] 4) Isolate and obtain recombinant collagen XVII from the supernatant.

[0035] More preferably, the method further comprises incorporating a gene segment encoding the hydroxylase BaP4H into an expression vector pPIC9k to obtain a recombinant vector pPIC9k-BaP4H, and transforming Pichia pastoris GS115 into the recombinant vector.

[0036] Collagen XVII, an important factor in skin aging and wound repair, also plays an important role in maintaining hair follicle stem cells to prevent hair loss and graying. Col17A1 is highly expressed in hair follicle stem cells (HFSCs), and Col17A1 is essential for the maintenance of hair follicle stem cells and melanocyte stem cells (MSCs) (Shintaro Tanimura, 2011). Due to its excellent biocompatibility and biodegradability, collagen has been widely studied and applied in the field of biomaterials, such as tissue engineering scaffolds and absorbable sutures.

[0037] Therefore, a third object of the present disclosure is to provide the recombinant type XVII collagen, and to provide recombinant type XVII collagen obtained by the preparation method and used for the following purposes:

[0038] i) promoting cell proliferation;

[0039] ii) promoting cell migration;

[0040] iii) manufacturing cosmetic products;

[0041] iv) Manufacturing medical supplies.

[0042] Promoting cell proliferation can refer to the use of this compound as a base component of a culture medium to promote cell proliferation, including in vitro culture and storage of hair follicle stem cells and hair follicle tissue.

[0043] Promoting cell migration can refer to the use of this compound as a base component of media, including in vitro culture and storage of hair follicle stem cells and hair follicle tissue.

[0044] Cosmetics include, but are not limited to, skin care lotions, skin care creams, essences, facial masks, scalp essences, shampoos, and the like.

[0045] Medical materials include fillers, repair materials, implants, tissue engineering scaffolds, hemostatic agents, drug release carriers, etc. Repair materials include, but are not limited to, bone repair materials, wound dressings, suture materials, etc.

[0046] The present disclosure further provides compositions comprising a cosmetically or pharmaceutically effective amount of at least one of the above proteins and at least one additive or cosmetically or pharmaceutically acceptable adjuvant. In some embodiments, the compositions are in dosage forms including, but not limited to, creams, lotions, aqueous solutions, gels, oils, powders, muds, patches, films, or lyophilized products. Furthermore, patch products can be manufactured using solid carriers such as nonwoven fabrics to facilitate transdermal absorption of collagen molecules. Patch products are applied to the face to extend the time the collagen solution is in contact with the skin surface. The recombinant type XVII collagen of the present disclosure can be prepared in combination with bioactive ingredients, such as recombinant or natural type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, fibronectin, or human epidermal growth factor, before use. Alternatively, the recombinant type XVII collagen can be prepared separately from the above-mentioned bioactive ingredients and then mixed.

[0047] Compared with the prior art, the present disclosure has the following beneficial effects:

[0048] 1) The recombinant type XVII collagen of the present disclosure has a theoretical molecular weight of approximately 33 kDa, which is much smaller than that of natural full-length collagen (150 kDa), thereby reducing the obstacles to collagen passing through the skin barrier to perform its biological functions and promoting cell migration.

[0049] 2) The recombinant type XVII collagen of the present disclosure has a stable molecular structure, a higher-order triple helix structure, and can maintain its thermal stability at 40°C.

[0050] 3) The recombinant type XVII collagen disclosed herein is a new recombinant type XVII collagen sequence that was created by optimizing and selecting segments with high biological activity and stable higher-order structure based on the original amino acid sequence of human type XVII collagen, and has properties such as good biocompatibility.

[0051] 4) The recombinant type XVII collagen of the present disclosure has excellent cell migration-promoting activity. [Brief explanation of the drawings]

[0052] [Figure 1] FIG. 1 is an SDS-PAGE image of the recombinant type XVII collagen LY1701 (n=3) of the present disclosure expressed from an E. coli expression system.

[0053] [Figure 2] 1 shows SDS-PAGE analysis of the recombinant type XVII collagen LY1701 (n=4) of the present disclosure expressed from an E. coli expression system.

[0054] [Figure 3] 1 shows SDS-PAGE analysis of the recombinant type XVII collagen LY1701 (n=5) of the present disclosure expressed from an E. coli expression system.

[0055] [Figure 4] 1 shows SDS-PAGE analysis of recombinant type XVII collagen LY1701 (n=6) of the present disclosure expressed from an E. coli expression system.

[0056] [Figure 5] 1 shows SDS-PAGE analysis of recombinant type XVII collagen LY1701 (n=7) of the present disclosure expressed from an E. coli expression system.

[0057] [Figure 6] 1 shows SDS-PAGE analysis of recombinant collagen XVII LY1701 (n=8) of the present disclosure expressed from an E. coli expression system.

[0058] [Figure 7] 1 shows SDS-PAGE analysis of the recombinant type XVII collagen LY1701 (n=8) of the present disclosure expressed from a Pichia pastoris expression system.

[0059] [Figure 8] 1 shows the circular dichroism spectra of recombinant type XVII collagen LY1701 (n=3, 4, 5, 6, 7, or 8) of the present disclosure at room temperature.

[0060] [Figure 9] 1 shows circular dichroism spectra of recombinant type XVII collagen LY1701 (n=3, 4, 5, 6, 7, or 8) of the present disclosure at various temperatures.

[0061] [Figure 10] 1 is a photograph showing a cell scratch assay of the recombinant type XVII collagen LY1701 (n=8) of the present disclosure.

[0062] [Figure 11] 1 is a graph showing cell scratch recovery area analysis of recombinant type XVII collagen LY1701 (n=8) of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0063] In the present disclosure, genes can be synthesized by biotechnology companies. In the present disclosure, the method for producing recombinant vectors and recombinant cells is not particularly limited, and conventional methods for producing recombinant vectors and recombinant cells can be used.

[0064] In the present disclosure, the separation and purification method is not particularly limited, and any conventional protein separation and purification method may be used. A preferred technical configuration is described in the following examples.

[0065] In this disclosure, the structure of collagen is characterized by circular dichroism (CD), a commonly used spectroscopic technique in the art. CD is a spectroscopic technique used to derive the structure of compounds with chiral structures that generate differential left-right optical rotational absorption. It is primarily used to determine the asymmetry of molecular structures. Because biopolymers generally contain chiral groups and structures, CD is often used to measure and observe the structure and changes of biopolymers. The triple helix structure of collagen generally exhibits CD characteristics, with a positive absorption peak near 221 nm and a negative absorption peak near 195 nm (industry standard YY / T1849-2022). The position of the absorption peak changes depending on the amino acid sequence and its length. The thermal stability of collagen is related to the thermal contraction temperature (Ts) and thermal denaturation temperature (Td) of collagen fibers. The thermal contraction temperature of collagen fibers is the temperature at which collagen fibers contract axially when heated, shortening by approximately 5% of their original length. The thermal denaturation temperature of collagen is the temperature at which 50% of the triple helix is ​​unwound into a single chain when collagen is heated in a medium. Therefore, CD spectroscopy can be used to study the helical structure of collagen and its thermal denaturation process.

[0066] In the examples below, LY1701 represents a series of proteins formed from tandem repeats of the core unit (SEQ ID NO:1), each of which has the core unit repeated 3, 4, 5, 6, 7, or 8 times.

[0067] The technical solutions provided in the present disclosure are described in detail below with examples, but this description does not limit the protection scope of the present disclosure.

[0068] Example 1: Expression (Escherichia coli expression system), isolation, and purification of recombinant type XVII collagen LY1701 of the present disclosure

[0069] 1. Synthesis of LY1701 sequence

[0070] Gene segments encoding the amino acid sequence of the recombinant type XVII collagen LY1701 of the present disclosure (SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7) were synthesized by GenScript Biotech Co., Ltd. and constructed into the pET28a E. coli expression vector. Gene segments encoding the hydroxylases L593 and L230 were synthesized by GenScript Biotech Co., Ltd. and inserted into the expression vector pCDFDuet-1 downstream of two promoters.

[0071] 2. Construction of E. coli Expression System

[0072] 2.1 Conversion

[0073] Type XVII collagen has a gene encoding pET28a-LY1701 (kanamycin resistance) and hydroxylase has a gene encodingThe two plasmids, pCDFDuet-1-L593-L230 (streptomycin resistance), were cotransformed into competent BL21(DE3) cells (AlpalifeBio, Shenzhen). The cells were placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 45 seconds, and immediately placed on ice for 2–3 minutes. Then, 900 μl of SOC or LB medium (without antibiotics) was added. The cells were grown for 1 hour at 37°C on a shaker at 200–250 rpm. Simultaneously, LB solid medium plates (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar, 100 μg / ml kanamycin antibiotic) that support kanamycin and streptomycin resistance were preheated in a 37°C incubator. After incubation on a shaker, the cells were centrifuged at 5000 rpm (2400 x g) for 5 minutes, and 900 μl of the supernatant was discarded. The bacteria were resuspended in the remaining medium and evenly spread onto a preheated, double-strength plate using a sterile spreader. The plate was then incubated upside down in a 37°C incubator for 12–16 hours until colonies were clearly visible.

[0074] 2.2 Plates with colonies were removed, and single clone colonies were selected and inoculated into 10 ml of LB liquid medium (containing kanamycin and streptomycin antibiotics). The medium was grown overnight at 37°C and 220 rpm, and then added to 1 L of LB medium containing kanamycin and streptomycin antibiotics at a ratio of 1:100. After 3-5 hours of growth at 37°C and 220 rpm, the OD value was monitored. 600 When the value reached 1.0 to 1.2, ascorbic acid (Sangon Biotech, Shanghai) was added to a final concentration of 10 mg / ml, FeSO4 (Sangon Biotech, Shanghai) to a final concentration of 1 mM, and IPTG (Biosharp) to a final concentration of 1 mM, and expression was induced for 18–24 h at 16°C and 180 rpm. Bacteria were collected by centrifugation for the next purification step or for frozen storage at −20°C or below.

[0075] 3. Protein purification

[0076] 3.1 After sampling and performing gel electrophoresis for later use, the induced bacterial solution was centrifuged at 8000 rpm for 20 minutes to collect the bacteria.

[0077] 3.2 Preparation of Ni-NTA (Sangon Biotech) purification buffer

[0078] Lysis buffer: 50 mM Tris-HCl, 500 mM NaCl, 10 mM imidazole, and 5% glycerol, pH 7.0

[0079] Wash buffer: 50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, and 5% glycerol, pH 7.0

[0080] Elution buffer: 50 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, and 5% glycerol, pH 7.0

[0081] 3.3 Preparation of lysis buffer

[0082] Lysis buffer components: PMSF (MIKX) at a final concentration of 1 mM, protease inhibitor (MCE), Supernuclease (Beyotime), and MgCl2 (Sangon Biotech) at a final concentration of 1 mM

[0083] Lysis buffer was added at a ratio of 1:10 according to the weight of the bacteria, and the bacteria were resuspended using a homogenizer and mixed well with a pipette.

[0084] 3.4 Affinity purification

[0085] A high-pressure homogenizer (Duoning Biotech) was pre-operated and pre-cooled. The bacterial solution was added and homogenized three times at 700-800 bar. The disrupted bacterial solution was then collected. The bacterial solution was centrifuged at 20,000 rpm for 1 hour at 4°C in a high-speed refrigerated centrifuge (Beckman). After centrifugation, the supernatant was transferred to a new 50 ml centrifuge tube and filtered through a 0.45 μm filter. A Ni-NTA gravity column (1 ml Ni-NTA) was washed with 5 column volumes of filtered pure water and 3 column volumes of lysis buffer. The sample was then loaded onto the column using a peristaltic pump, whose channel was washed with a large amount of pure water and rinsed with lysis buffer. The filtered supernatant was then loaded onto the Ni-NTA gravity column using a peristaltic pump, and the flow-through solution was collected. The Ni-NTA was washed with 100 ml of wash buffer, and the wash solution was collected. The target protein was eluted by adding 1 ml of elution buffer each time and incubating for 5 min, and the eluate was collected.

[0086] 3.5 Purification by ion exchange chromatography

[0087] The target protein was dialyzed against buffer A (20 mM PB, pH 7.0). The protein sample was filtered through a 0.22 μm, 13 mM filter membrane and subjected to cation exchange using the AKTA pure system (5 ml SP column). Subsequently, linear elution with buffer B (20 mM PB and 1 M NaCl, pH 7.0) yielded the target protein at a higher purity. The molecular weight and purity of the protein were verified by SDS-PAGE electrophoresis. The protein sample was mixed with protein loading buffer (containing DTT), placed in a metal bath, heated at 95°C for 10 minutes, and then loaded for gel electrophoresis. Protein concentration was determined by the BCA method to estimate the protein yield. Depending on the purity, the protein was concentrated or lyophilized for storage.

[0088] result

[0089] 1-6 show SDS-PAGE of the recombinant collagen XVII of the present disclosure after E. coli expression and purification. result is.

[0090] Example 2 Expression, isolation, and purification of recombinant type XVII collagen LY1701 (in a Pichia pastoris expression system) of the present disclosure

[0091] 1. Synthesis of LY1701 sequence

[0092] A gene segment encoding the amino acid sequence of the recombinant type XVII collagen LY1701 (SEQ ID NO:7) of the present disclosure was synthesized by GenScript Biotech Co., Ltd. and constructed into the pPICZalphaA Pichia pastoris expression vector. A gene segment encoding the hydroxylase BaP4H was synthesized and constructed into the vector pPIC9k.

[0093] 2. Construction of the Pichia pastoris expression system

[0094] 2.1 Electroporation

[0095] Type XVII collagen has a gene encodingpPICZalphaA-LY1701 was linearized at the SacI site by PCR amplification, followed by PCR fragment recovery (Thermo GeneJET PCR Purification Kit). Competent cells GS115 were thawed on ice, and 5-10 μg of linearized plasmid was added and incubated on ice for 15 minutes. The plasmid and competent cells were then added to a pre-chilled electroporation cuvette. Electroporation was performed at a voltage of 2000 V, a resistance of 200 Ω, a capacitance of 25 μF, a cuvette diameter of 0.2 mm, and a time constant of 5 ms. Immediately after electroporation, 1 ml of chilled sorbitol was added, and the mixture was transferred to a 1.5 ml sterile centrifuge tube and incubated at 30°C and 230 rpm for 2 hours. The mixture was centrifuged at 5000 g for 1 minute, and 300 μl of the supernatant was discarded. The cells were resuspended and spread onto YPDSZ solid plates (tryptone 20 g / L, yeast extract fermentate 10 g / L, D-sorbitol 182.1 g / L, 20% glucose, and 100 μg / ml bleomycin) and grown at 30°C and 230 rpm for 48 h.

[0096] 2.2 Plates with colonies were removed, and single clone colonies were selected and replicated onto YPD solid medium containing high concentrations (400 μg / ml or 800 μg / ml) of bleomycin for screening.

[0097] 2.3 Plates with colonies were removed, and single clones were selected and inoculated into 50 ml of YPD liquid medium (containing 100 μg / ml bleomycin). The seed solution was grown overnight at 30°C and 230 rpm. The overnight culture was inoculated into 1 L of BMGY medium (20 g / L tryptone, 10 g / L yeast extract fermentate, 11.73 g / L potassium phosphate monobasic (KH2PO4), 2.4 g / L potassium phosphate monobasic (K2HPO4), 10 g / L glycerol, 134 g / L YNB, and 1x biotin) at a 1:50 ratio and grown at 30°C and 230 rpm for 48 hours. The BMGY-grown seed solution was centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in sterile water and centrifuged again at 3000 rpm for 5 minutes, and the supernatant was discarded. Cells were resuspended in 1 L of BMMY medium (methanol-free) and supplemented with 1% methanol, followed by additional 1% methanol every 24 hours. After 72 hours of induction, the induced fluid was centrifuged and the supernatant was collected and purified.

[0098] 2.4 Purification by ion exchange chromatography

[0099] The target protein was dialyzed against Buffer A (20 mM PB, pH = 7.0). The protein sample was filtered through a 0.22 μm, 13 mm diameter filter membrane and subjected to cation exchange using the AKTA pure system (5 ml SP column). Subsequently, linear elution with Buffer B (20 mM PB and 1 M NaCl, pH = 7.0) yielded the target protein at a higher purity. The molecular weight and purity of the protein were verified by SDS-PAGE electrophoresis. The protein sample was mixed with protein loading buffer (containing DTT), placed in a metal bath, heated at 95°C for 10 minutes, and centrifuged at 1500 rpm for 1 minute. The supernatant was then sampled and analyzed by SDS-PAGE. Protein concentration was determined by the BCA method to estimate protein yield. Depending on the purity, the protein was concentrated or lyophilized for storage.

[0100] 2.5 Preparation of yeast competent cells containing the LY1701 expression plasmid

[0101] LY1701 monoclonal colonies with high expression yields were selected and grown overnight at 30°C and 230 rpm in 5 ml of YPD medium (bleomycin resistant). The culture was inoculated into 500 ml of fresh YPD medium (bleomycin resistant) at a ratio of 1:200, and the OD 600 The cells were cultured overnight until the RI reached 1.3 to 1.5. Cells were harvested by centrifugation at 4°C and 1500g for 5 minutes, washed in 500 ml of chilled sterile water, and resuspended. The resuspended cells were harvested by centrifugation at 4°C and 1500g for 5 minutes, washed three times with chilled sterile water, and resuspended in 20 ml of 1 M sorbitol. Centrifuge the resuspended cells The supernatant was discarded. After washing twice or three times, the cells were collected by centrifugation at 1500 g for 5 minutes at 4°C, resuspended in 500 μl of 1 M sorbitol, and stored in 100 μl aliquots in tubes at −80°C in a freezer.

[0102] 2.6 Linearization and electroporation of BaP4H-pPIC9k

[0103] Primers were designed for linearization of pPIC9k at the SalI site by PCR, and the PCR fragment was recovered (Thermo GeneJET PCR Purification Kit). Competent yeast cells containing the LY1701 expression plasmid obtained in Example 2.4 were thawed on ice, and 5–10 μg of linearized plasmid was added and incubated on ice for 15 minutes. The plasmid and competent cells were then added to a pre-chilled electroporation cuvette. Electroporation was performed at a voltage of 2000 V, a resistance of 200 Ω, a capacitance of 25 μF, a cuvette diameter of 0.2 mm, and a time constant of 5 ms. 400 μl of chilled sorbitol was added immediately after electroporation, and the mixture was transferred to a 1.5 ml sterile centrifuge tube and incubated at 30°C and 230 rpm for 2 hours. The mixture was centrifuged at 5000 g for 1 minute, and 300 μl of the supernatant was discarded. The cells were resuspended and spread onto YPDSZ solid plates (20 g / L tryptone, 10 g / L yeast extract fermentate, 20% glucose, and 4 mg / ml G418) and grown at 30°C and 230 rpm for 48 h.

[0104] 2.7 Induction of expression

[0105] A single colony that grew well on the resistant plate was selected and inoculated into 1 L of BMGY medium and cultured at 30°C and 230 rpm for 48 hours. The culture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 500 ml of sterile water and centrifuged again at 3000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1 L of BMMY medium (without methanol), and the mixture was transferred to a conical flask. Subsequently, 1% methanol was added, and 1% methanol was added every 24 hours. After 72 hours of induction, the culture was centrifuged, and the supernatant was collected and purified.

[0106] 2.8 Purification by ion exchange chromatography

[0107] The target protein was dialyzed against Buffer A (20 mM PB, pH = 7.0), filtered through a 0.22 μm, 13 mm diameter filter membrane, and subjected to cation exchange using the AKTA pure system (5 ml SP column). Subsequently, linear elution with Buffer B (20 mM PB and 1 M NaCl, pH = 7.0) was performed to obtain the target protein with higher purity. The molecular weight and purity of the protein were verified by SDS-PAGE electrophoresis. The protein sample was mixed with protein loading buffer (containing DTT), placed in a metal bath, heated at 95°C for 10 minutes, and centrifuged at 1500 rpm. The supernatant was then collected for SDS-PAGE analysis. Protein concentration was determined by the BCA method to estimate the protein yield. Depending on the purity, the protein was concentrated or lyophilized for storage.

[0108] result

[0109] Figure 7 shows SDS-PAGE of recombinant collagen XVII of the present disclosure after Pichia pastoris expression and purification. result Recombinant collagen XVII has a molecular weight of 49 kDa (theoretical value is 33.75 kDa).

[0110] Example 3 Determination of the triple helix structure of type XVII collagen by circular dichroism (CD)

[0111] The freeze-dried type XVII collagen 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 was further diluted to a volume of 3 ml and a concentration of 0.01 mg / ml. The sample to be tested was transferred to a 10 mm x 10 mm sample cuvette in a circular dichroism spectrometer with a scanning wavelength range of 190 nm to 260 nm, a scanning speed of 100 nm / min, and a scanning temperature of room temperature. CD spectra were averaged from three scans. As shown in Figure 8, the recombinant type XVII collagen of the present disclosure has a characteristic positive peak with a maximum at 221 nm and a negative peak below 200 nm. The circular dichroism characteristics of the known collagen triple helix structure allow us to determine that the collagen sample has a triple helix structure.

[0112] Example 4 Determination of thermal stability of type XVII collagen by circular dichroism (CD)

[0113] Collagen samples were diluted according to the method described in Example 3 and heated in real time at a rate of 1°C / min to test the thermal stability of the protein. CD spectra were averaged from three scans. Data were analyzed and calculated using software. The relationship between the molar ellipticity of the recombinant collagen and temperature was determined at 221 nm. The results are shown in Figure 9, which indicates that the favorable thermal stability of LY1701 is maintained at 40°C based on the disappearance of the characteristic positive peak at 221 nm.

[0114] Example 5 Determination of the cell migration activity of type XVII collagen by cell scratch assay

[0115] A cell scratch assay is commonly used to detect collagen-induced cell migration activity. The higher the biological activity of collagen, the higher the migration rate. The specific steps are as follows:

[0116] 1. Mouse 3T3 cells (Haixing Biotech) were resuscitated and subcultured 3 days prior to the start of the experiment to maintain good condition and viability. When the cell viability exceeded 98%, 5 × 10 5 Cells were seeded into 6-well plates at a density of 100 cells / ml in a plating volume of 2 ml. The cells were cultured in an incubator at 37°C and 5% CO2 for 24 hours. When the cells reached a confluency of over 90% in the 6-well plates, the following scratch test was performed.

[0117] 2. The lyophilized type XVII collagen powder LY1701 (SEQ ID NO: 7) to be tested in the experimental group, and BSA and commercially available recombinant collagen products (Competitor 1 and 2) in the control group were dissolved in 20 mM PB buffer (pH 7.4). The protein concentrations were measured after dissolution and diluted to a concentration of 0.5 mg / ml.

[0118] 3. Parallel horizontal lines were drawn at 0.5-1 cm intervals on the bottom of a 6-well plate using a ruler and black marker exposed to UV light for 30 minutes. At least three lines were drawn in each well. After drawing the horizontal lines, the plate was marked for subsequent observation.

[0119] 4. A wound perpendicular to the line on the bottom of a 6-well plate was created in the confluent monolayer of cells using a cell scraper or the tip of a 300 μl pipette perpendicular to the surface of the 6-well plate. The width of the wound was kept as constant as possible during wounding. The 6-well plate was washed three times with PBS to remove loose cells and cell debris from the wound. 2 ml of DMEM serum-free medium and the prepared protein solution were added to the wells to achieve a final protein concentration of 50 μg / ml. The plate was placed in an incubator at 37°C and 5% CO2 for incubation.

[0120] 5. The cell wounds were observed under a microscope at 0 and 12 hours and photographed. The photos of cell migration were processed using "Image J" software to obtain the initial wound area and the blank area after cell migration. The migration rate was calculated as follows: Migration rate = (initial wound area - blank area after cell migration) / initial wound area × 100%

[0121] The results are shown in Figure 10. area The cell aggregation phenomenon in the blank area was more pronounced than in the other groups after only 12 hours of treatment.

[0122] As shown in Figure 11, data analysis revealed that the recovery area of ​​the cell scratch was 23.61% for the BSA-treated group and 39.68% for the recombinant type XVII collagen LY1701 group, while the recovery area of ​​the cell scratch was 24.88% for the Competitor 1 collagen-treated group and 28.92% for the Competitor 2 collagen-treated group.

[0123] It is clear that the type XVII collagen LY1701 of the present disclosure has an excellent effect of promoting cell migration.

[0124] The above description is merely an example of the embodiment of the present disclosure. Improvements and modifications can be made by those skilled in the art without departing from the principle of the present disclosure. These improvements and modifications should be considered within the protection scope of the present disclosure.

Claims

1. A recombinant type XVII collagen having an amino acid sequence formed by tandem repeats of n or more core units, where n is an integer of 1 or more, and the core unit has the amino acid sequence shown in SEQ ID NO:

1.

2. The recombinant type XVII collagen of claim 1, wherein n is any number selected from 3, 4, 5, 6, 7, or 8.

3. A gene encoding the recombinant type XVII collagen of claim 1 or 2.

4. A recombinant vector for expressing the recombinant type XVII collagen of claim 1 or 2, which has the gene of claim 3.

5. A recombinant cell expressing the recombinant type XVII collagen of claim 1, which is transformed with the recombinant vector of claim 4.

6. 2. The method for producing recombinant type XVII collagen according to claim 1, 1) recombining the gene of claim 3 into vector pET28a to obtain a recombinant vector; 2) transforming E. coli BL21(DE3) with the recombinant vector to obtain recombinant cells; 3) culturing the recombinant cells followed by centrifugation and collection of the supernatant; 4) isolating and obtaining recombinant collagen XVII from the supernatant.

7. 7. The method of claim 6, further comprising the step of: inserting the gene segments encoding hydroxylases L593 and L230 into an expression vector pCDFDuet-1 to obtain a recombinant vector pCDFDuet-1-L593-L230; and transforming E. coli BL21 (DE3) into the recombinant vector.

8. In step 3), the culture was performed with ascorbic acid at a final concentration of 10 mg / ml and FeSO 4 and IPTG to a final concentration of 1 mM to induce expression of the protein of interest.

9. 2. The method for producing recombinant type XVII collagen according to claim 1, 1) recombining the gene of claim 3 into vector pPICZαA to obtain a recombinant vector; 2) transforming Pichia pastoris GS115 with the recombinant vector to obtain recombinant cells; 3) culturing the recombinant cells followed by centrifugation and collection of the supernatant; 4) isolating and obtaining recombinant collagen XVII from the supernatant.

10. 10. The method of claim 9, further comprising the step of: inserting a gene segment encoding hydroxylase BaP4H into an expression vector pPIC9k to obtain a recombinant vector pPIC9k-BaP4H, and transforming Pichia pastoris GS115 into the recombinant vector.

11. 10. Recombinant type XVII collagen according to claim 1 or obtained by the method according to claim 6 or 9, i) promoting cell proliferation; ii) promoting cell migration; iii) manufacturing cosmetic products; or iv) Recombinant collagen XVII, used to manufacture medical materials.

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