Small-molecule recombinant XVII type collagen as well as preparation method and application thereof

Abstract

The invention provides a micromolecular recombinant XVII type collagen as well as a preparation method and application thereof, and relates to the technical field of protein engineering. The novel recombinant human XVII type collagen is obtained by optimizing and screening a plurality of spiral region sequences such as a collagen structural domain, a C-terminal region and a middle region of the human XVII type collagen. The amino acid composition of the recombinant XVII type collagen is consistent with that of a natural collagen alpha1 chain, immunological rejection is not generated when the recombinant XVII type collagen is applied to a human body, the recombinant XVII type collagen can be widely applied to surgical medical treatment and medical beauty industries, the molecular weight of the collagen is small, efficient secretion soluble expression in eukaryotic host cells such as pichia pastoris can be achieved, and the recombinant XVII type collagen has a good application prospect. And industrial large-scale production is carried out. According to the present invention, the verification results show that the micromolecular recombinant XVII type collagen further has basement membrane anti-aging activity, wherein the basement membrane anti-aging activity specifically comprises the improvement of the expression levels of the IV type collagen, the VII type collagen and the XVII type collagen;

Description

Technical Field

[0001] The present application relates to the field of protein engineering technology, and in particular to a small molecule recombinant type XVII collagen and a preparation method and application thereof. Background Art

[0002] Collagen is a structural protein in the extracellular matrix (ECM). It is the primary component of animal connective tissue and the most abundant and widely distributed functional protein in mammals. A white polysaccharide containing small amounts of galactose and glucose, it is the primary component of the extracellular matrix (ECM), accounting for approximately 85% of the solid collagen fibers. Collagen is a ubiquitous macromolecular protein found primarily in connective tissues (bone, cartilage, skin, tendons, and ligaments), accounting for 25%-30% of the protein content in mammals, equivalent to 6% of body weight. In many marine organisms, such as fish skin, it accounts for over 80% of the protein content. The current market is dominated by high-molecular-weight collagens. For example, animal-derived collagen generally has a molecular weight of over 100 kDa, while recombinant collagens are mostly over 20 kDa. Development of low-molecular-weight collagens is relatively limited. For skin moisturizing and firming effects, higher-molecular-weight collagens may be more suitable, as they form a protective barrier on the skin's surface, preventing water loss. For deeper repair and revitalization effects, collagen with smaller molecular weight may be more advantageous because they can penetrate deeper into the skin more easily, stimulating collagen synthesis and cell activation.

[0003] For a long time, people have been extracting and hydrolyzing collagen from raw materials such as animal skins and bones using acid or alkaline methods. However, these acid-base methods for hydrolyzing collagen are associated with high pollution, high energy consumption, and poor hydrolysis specificity, and most of the collagen is degraded into free amino acids. With the continuous upgrading of the collagen peptide industry, the preparation process of collagen peptides has also undergone significant improvements and gradually matured. The original acid-base method has gradually been replaced by protease hydrolysis technology. Enzymatic hydrolysis for the preparation of collagen peptides has the advantages of mild conditions, low pollution, and high specificity. The molecular weight of the prepared collagen peptides generally ranges from several hundred to several thousand Daltons. The technical difficulty of this method is the ability to produce single, active small-molecule collagen.

[0004] Therefore, in this technical field regarding the expression of recombinant small molecule collagen, an effective solution is urgently needed to screen small molecular weight, biologically active collagen. Summary of the Invention

[0005] To address these issues with existing technologies, the present invention has designed a variety of recombinant type XVII collagen active products with smaller molecular weights and recombinantly expressed them using genetic engineering techniques. This method effectively avoids viral risks and immune rejection, maintains a defined molecular weight, and undergoes mild production conditions, effectively preserving biological activity.

[0006] In one aspect, the present application provides a small molecule recombinant type XVII collagen, wherein the recombinant type XVII collagen comprises at least one or more of the following A1)-A5):

[0007] A1) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 95% identity with SEQ ID NO. 1;

[0008] A2) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 95% identity with SEQ ID NO. 2;

[0009] A3) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence having at least 95% identity with SEQ ID NO. 3;

[0010] A4) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having at least 95% identity with SEQ ID NO. 4;

[0011] A5) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the recombinant type XVII collagen described in A1) or A2) or A3) or A4).

[0012] in,

[0013] A1) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence of SEQ ID NO. 1, or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 1;

[0014] A2) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence of SEQ ID NO. 2, or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 2;

[0015] A3) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence of SEQ ID NO. 3, or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 3;

[0016] A4) The amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 4, or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to SEQ ID NO. 4.

[0017] Preferably, the molecular weight of the recombinant type XVII collagen is less than 10 kD, more preferably, less than or equal to 5 kD.

[0018] Preferably, the recombinant type XVII collagen is recombinant humanized type XVII collagen.

[0019] On the other hand, the present application also provides a biomaterial, which comprises any one of the following B1) to B5):

[0020] B1) a nucleic acid molecule comprising a nucleotide sequence encoding the recombinant type XVII collagen;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector comprising the nucleic acid molecule described in B1) and / or the expression cassette described in B2);

[0023] B4) a recombinant microorganism comprising the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3);

[0024] B5) A recombinant cell comprising the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3).

[0025] Those skilled in the art will appreciate that the expression cassette described herein may also include functional elements such as promoters, terminators, and marker genes. Those skilled in the art can make routine selections based on actual conditions, as long as they can achieve expression of the nucleic acid molecule encoding the protein. Other commonly used expression elements may be adaptively added to the expression cassette described in this application to assist in the expression of the target gene, such as adding a label, a fluorescent protein marker, a resistance screening marker, etc. The structure and composition of the expression cassette are not further restricted herein.

[0026] The recombinant vector described herein refers to a vector capable of transporting exogenous DNA or a gene of interest into host cells for amplification and expression. Such vectors can be either cloning or expression vectors, including but not limited to plasmids, bacteriophages (such as lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), or viral vectors. In a preferred embodiment, the vector is the Pichia pastoris expression vector pPIC9k.

[0027] Those skilled in the art will appreciate that conventional fermentation strains or any known industrial strains can be used as the starting strain, as long as they can achieve the expression of recombinant type XVII collagen described in this application. No particular limitation is imposed on the specific strains herein.

[0028] Preferably, the recombinant vector and the recombinant microorganism contain a resistance selection marker gene, which can be selected by those skilled in the art according to actual conditions.

[0029] In an alternative embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or introduced into a recombinant microorganism using a recombinant vector. It should be noted that those skilled in the art will appreciate that the nucleic acid molecule can be inserted into the genome of the starting strain or can be present on a free plasmid, as long as the expression of the nucleic acid molecule or the synthesis of collagen can be achieved.

[0030] It is understandable that those skilled in the art can select known gene editing systems and gene editing methods to construct the above-mentioned biomaterials according to actual conditions.

[0031] Further, the nucleic acid molecule includes: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having at least 80% identity with SEQ ID NO.5, and / or a nucleotide sequence as shown in SEQ ID NO.6 or a nucleotide sequence having at least 80% identity with SEQ ID NO.6, and / or a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having at least 80% identity with SEQ ID NO.7, and / or a nucleotide sequence as shown in SEQ ID NO.8 or a nucleotide sequence having at least 80% identity with SEQ ID NO.8.

[0032] in,

[0033] The nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO.5, and / or,

[0034] The nucleotide sequence as set forth in SEQ ID NO.6, or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO.6, and / or

[0035] The nucleotide sequence as set forth in SEQ ID NO.7, or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity to SEQ ID NO.7, and / or,

[0036] The nucleotide sequence as set forth in SEQ ID NO.8, or a nucleotide sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity thereto.

[0037] In a preferred embodiment,

[0038] The recombinant type XVII collagen comprises at least one or more of the following A1)-A5):

[0039] A1) the amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.5;

[0040] A2) the amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 6;

[0041] A3) the amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 7;

[0042] A4) the amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO. 4, and the nucleotide sequence is shown in SEQ ID NO. 8;

[0043] A5) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the recombinant type XVII collagen described in A1) or A2) or A3) or A4).

[0044] Furthermore, the recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

[0045] Preferably, Pichia.

[0046] In a preferred embodiment, the Pichia pastoris may be Pichia pastoris, preferably, Pichia pastoris GS115.

[0047] On the other hand, the present application also provides a method for preparing recombinant type XVII collagen, which comprises: constructing a recombinant microorganism that expresses the recombinant type XVII collagen, and culturing the recombinant microorganism.

[0048] Conventional culture methods can be selected for culture according to the characteristics of the recombinant microorganism.

[0049] Preferably, the recombinant microorganism is Pichia pastoris.

[0050] In one embodiment, a method for preparing recombinant type XVII collagen comprises:

[0051] Step 1, constructing a recombinant microorganism expressing the recombinant type XVII collagen;

[0052] Step 2: culturing the recombinant microorganism in a seed culture medium at 25°C-37°C and 100-300 rpm for 20-24 hours to obtain a first-level seed solution;

[0053] Step 3: Inoculate the first-level seed solution into the seed culture medium at an inoculum size of 1%-5%, and culture at 25°C-37°C at 100-300 rpm for 20-24 hours to obtain the second-level seed solution;

[0054] Step 4: The secondary seed liquid is transferred into the fermentation medium at an inoculum rate of 1%-10% for fermentation. The fermentation conditions include: 25°C-37°C, pH 5-7, and dissolved oxygen controlled at 10%-30% to obtain fermentation liquid. The supernatant after centrifugation of the fermentation liquid is purified and freeze-dried to obtain recombinant type XVII collagen.

[0055] Preferably, when the dissolved oxygen rises suddenly, glycerol is added to maintain the dissolved oxygen at 15%-20%. When the wet bacterial weight of the fermentation broth reaches 180-220 mg / mL, feeding is stopped, starvation is performed, and methanol is added to induce fermentation. The dissolved oxygen is kept above 15% by adjusting the rotation speed, tank pressure and ventilation volume.

[0056] The seed culture medium may be a YPD medium, and the fermentation medium may be a (main fermentation) basal salt medium.

[0057] The above purification process can be carried out using general methods.

[0058] In a preferred embodiment, purification is performed using a cationic chromatography column.

[0059] On the other hand, the present application also provides a composition containing the recombinant type XVII collagen.

[0060] The composition of the present application may also contain excipients, which may be appropriate solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc.

[0061] The composition of the present application can be prepared by general methods, wherein one or more diluents or carriers can be added, such as pills, tablets, capsules, granules, powders, lozenges, syrups, emulsions, suspensions, etc.

[0062] On the other hand, the present application also provides the use of the recombinant type XVII collagen or the biomaterial or the preparation method or the composition in preparing type XVII collagen, increasing the yield of type XVII collagen and / or increasing the activity of type XVII collagen.

[0063] On the other hand, the present application also provides the use of the recombinant type XVII collagen, the biomaterial, the preparation method, or the composition in the preparation of a product that promotes cell proliferation.

[0064] In this application, experiments have verified that the recombinant type XVII collagen has no potential cytotoxicity, and its relative cell proliferation rate is higher than that of commercially available products, with the highest relative proliferation rate reaching 120%. However, commercially available recombinant type XVII collagen has no obvious effect in promoting cell proliferation, which proves that the recombinant type XVII collagen is safe.

[0065] In a preferred embodiment, the cells are immortalized epidermal keratinocytes.

[0066] Those skilled in the art can adjust the concentration according to actual needs, and this application does not impose too many restrictions on this.

[0067] In a preferred embodiment, the collagen concentration is 0.001-10 mg / mL, more preferably 0.1 mg / mL.

[0068] On the other hand, the present application also provides the use of the recombinant type XVII collagen or the biomaterial or the preparation method or the composition in promoting the synthesis of endogenous collagen and / or preparing products that promote the synthesis of endogenous collagen; preferably, the endogenous collagen includes at least type IV collagen, type VII collagen and / or type XVII collagen.

[0069] Endogenous collagen is naturally occurring collagen, synthesized by the body's own cells (primarily fibroblasts and chondrocytes) through gene expression, translation, and secretion. It exists naturally in the human and animal body and is a key structural and functional component of connective tissues such as skin, bone, cartilage, tendons, and blood vessels.

[0070] The present application demonstrates that the recombinant type XVII collagen has the biological activity of promoting the synthesis of endogenous collagen, especially promoting the synthesis of type IV collagen, type VII collagen and type XVII collagen.

[0071] Regarding type IV collagen, recombinant type XVII collagen comprising the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and / or SEQ ID NO.4 can effectively promote the increase in the expression level of type IV collagen.

[0072] Regarding type VII collagen, recombinant type XVII collagen comprising the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2 and / or SEQ ID NO.3 can effectively promote the increase in the expression level of type VII collagen.

[0073] Regarding type XVII collagen, recombinant type XVII collagen comprising the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and / or SEQ ID NO.4 can effectively promote the increase in the expression level of type XVII collagen.

[0074] Those skilled in the art can adjust the concentration according to actual needs, and this application does not impose too many restrictions on this.

[0075] In a preferred embodiment, the promoting endogenous collagen synthesis is promoting endogenous collagen synthesis in cells, and the cells are immortalized epidermal keratinocytes.

[0076] In a preferred embodiment, the collagen concentration is 0.001-10 mg / mL, more preferably 0.1 mg / mL.

[0077] On the other hand, the present application also provides the use of the recombinant type XVII collagen or the biomaterial or the preparation method or the composition in anti-aging; preferably, the anti-aging at least includes anti-basement membrane aging.

[0078] The basement membrane zone is located at the junction of the epidermis and dermis. It is composed of keratinocytes in the basal layer of the epidermis and the dermis, and is a highly specialized structural and functional connection. Studies have shown that the morphology of the basement membrane changes during aging, and the protein composition also changes accordingly.

[0079] Type IV collagen is a key component of the basement membrane, primarily distributed in the dense layer of the basement membrane. Together with laminin, it forms a network structure that provides stability to the basement membrane. Type VII collagen is primarily distributed in the dense sublayer of the basement membrane. It binds to nidogen and laminin to form a collagen network, which further interacts with type IV collagen, resulting in a tight bond to the basement membrane. Type XVII collagen is a transmembrane glycoprotein expressed on epidermal keratinocytes and is a key transmembrane component of the epidermal anchoring complex. It binds to laminin and type IV collagen in the basement membrane through ligands, maintaining a tight connection between the epidermis and dermis.

[0080] On the one hand, the anti-basement membrane aging is achieved by increasing the synthesis level of endogenous collagen, and the endogenous collagen includes type IV collagen, type VII collagen and / or type XVII collagen.

[0081] Type XVII collagen, a transmembrane protein found in the basement membrane, has two primary functions: anchoring epidermal stem cells to the basement membrane and promoting their orderly differentiation. Therefore, supplementing type XVII collagen can delay aging or repair damage to the basement membrane zone.

[0082] On the other hand, the present application also provides the use of the recombinant type XVII collagen or the biomaterial or the preparation method or the composition in food, cosmetics, health products and / or pharmaceutical and medical products.

[0083] The present invention has the following beneficial effects:

[0084] The present invention optimizes and screens the sequences of multiple helical regions such as the collagen domain, C-terminal region, and middle region of human type XVII collagen, and then combines the sequences to obtain a new recombinant human type XVII collagen. The amino acid composition of the recombinant type XVII collagen is consistent with the α1 chain of natural collagen. It will not produce immune rejection when applied to the human body and can be widely used in surgical medicine and medical beauty industries. Moreover, the collagen has a small molecular weight and can be efficiently secreted and soluble in eukaryotic host cells such as Pichia pastoris for industrial large-scale production. The present invention has been verified to have the anti-aging activity of the small molecule recombinant type XVII collagen in the basement membrane, specifically including promoting the increase in the expression levels of type IV collagen, type VII collagen, and type XVII collagen.

[0085] The present invention also provides a preparation method of the recombinant type XVII collagen, which uses Pichia pastoris as a fermentation engineering bacterium, does not contain endotoxin, has low production cost and high production efficiency, and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0087] Figure 1 is the plasmid map of the recombinant expression vector pPIC9K-COL in Example 2, wherein COL is recombinant type XVII collagen;

[0088] Figure 2 This is the SDS-PAGE electrophoresis diagram of the fermentation supernatant of recombinant type XVII collagen in Example 5, where 72 h and 96 h are the fermentation times;

[0089] Figure 3 This is a graph showing the results of recombinant type XVII collagen promoting cell proliferation in Example 7;

[0090] Figure 4 This is a graph showing the results of Example 8 in which recombinant type XVII collagen promoted the synthesis of basement membrane collagen. DETAILED DESCRIPTION

[0091] Technical terms:

[0092] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution research.

[0093] Recombination: In a broad sense, any gene exchange process that causes genotype changes is called recombination.

[0094] Expression cassette: An expression cassette is a set of DNA sequences consisting of a promoter, target gene, and reporter gene that can be expressed in specific tissues and easily detected.

[0095] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0096] Recombinant microorganisms: Fungal cell lines that use genetic engineering methods to efficiently express exogenous genes.

[0097] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not completely identical to the parent cell due to mutations that occur during replication.

[0098] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0099] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0100] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0101] Unless otherwise specified, in the following embodiments, all reagents or instruments used without manufacturer indication are conventional products that can be purchased commercially. If specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0102] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were commercial products, and the specific operations were performed according to the kit instructions.

[0103] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0104] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0105] In the following examples, unless otherwise specified, % represents wt%, i.e., percentage by weight.

[0106] Example 1: Sequence design of recombinant type XVII collagen

[0107] Natural human type XVII collagen α1 chain protein (reference sequence number NG_007069) was obtained from the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). Functional protein sequences were optimized and screened from multiple helical region sequences of human type XVII collagen, including the collagen domain, C-terminal region, and middle region. New proteins were designed by combining sequences and combining functional fragments. Combined with computer modeling analysis, multiple functional protein sequences were finally designed with a controlled molecular weight range of ≤5 kD to obtain recombinant type XVII collagen COL17-4-1, COL17-5, COL17-6, and COL17-7.

[0108] The specific sequence of recombinant type XVII collagen COL17-4-1 is shown in SEQ ID NO.1, the specific sequence of COL17-5 is shown in SEQ ID NO.2, the specific sequence of COL17-6 is shown in SEQ ID NO.3, and the specific sequence of COL17-7 is shown in SEQ ID NO.4.

[0109] Example 2: Gene synthesis of recombinant type XVII collagen

[0110] According to the codon preference of Pichia pastoris for expressing heterologous proteins, the nucleotide sequences of the four recombinant type XVII collagen proteins described in Example 1 were designed and artificially synthesized and subcloned into the pPIC9K plasmid. The specific sequences are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. The whole gene synthesis was completed by Nanjing GenScript Biotech Co., Ltd. The recombinant plasmid vectors were named pPIC9K-COL17-4-1, pPIC9K-COL17-5, pPIC9K-COL17-6, and pPIC9K-COL17-7. The recombinant plasmid structures are shown in Figure 2. Figure 1 and as shown in Table 1.

[0111] Table 1

[0112]

[0113] Example 3: Electrotransformation of Pichia pastoris

[0114] First, the pPIC9K-COL17-4-1, pPIC9K-COL17-5, pPIC9K-COL17-6, and pPIC9K-COL17-7 recombinant plasmids constructed in Example 2 were linearized with Sac I (purchased from Thermo Fisher). The reaction system is shown in Table 2.

[0115] Table 2

[0116]

[0117] Mix the reagents in Table 2 and incubate at 37°C in a water bath for 2 h. Before terminating the reaction, remove 5 μL and verify complete digestion by electrophoresis on a 1.0% agarose gel. After confirming completion of the reaction, recover the linearized pPIC9K-COL17-4-1, pPIC9K-COL17-5, pPIC9K-COL17-6, and pPIC9K-COL17-7 plasmids using a DNA purification kit.

[0118] 5 μg of pPIC9K-COL17-4-1, pPIC9K-COL17-5, pPIC9K-COL17-6, and pPIC9K-COL17-7 plasmids linearized with Sac I endonuclease were mixed with 100 μL of Pichia pastoris GS115 competent cells, transferred to an ice-cold electroporation cuvette with an electrode spacing of 0.2 cm, and electroporated for 4-10 milliseconds. 1 mL of ice-cold 1 M sorbitol solution was added to mix the cells, and the cells were spread on MD (Minimal Dextrase; 1 L MD contains 13.4 g YNB, 0.2 g biotin, 20 g glucose, 20 g agar powder, and the balance is water) medium plates and inverted cultured at 30°C for 2-3 days until colonies grow on the MD medium plates.

[0119] Example 4: Screening of multi-copy insertion recombinants

[0120] Colonies grown on the MD medium plates in Example 3 were inoculated with sterile toothpicks onto YPD (yeast extract peptone dextrose; 1 L contains 10 g yeast extract, 20 g tryptone, 20 g glucose, 20 g agar powder, and the balance is water) medium plates containing 0.5 g / L, 1 g / L, and 2 g / L G418, respectively. The plates were incubated at 30°C and screened with high-concentration antibiotics to obtain transformants. If a Pichia pastoris transformant can grow on a plate containing high-concentration G418, it indicates that the transformant contains multiple copies of the target gene, meaning that multiple recombinant fragments have entered the Pichia pastoris and integrated into its chromosomes through homologous recombination. This screening step yields high-copy, highly efficient expression of COL17-4-1, COL17-5, COL17-6, and COL17-7 recombinant Pichia pastoris engineered strains.

[0121] Example 5: Fermentation of recombinant type XVII collagen

[0122] The engineered strain screened in Example 4 was inoculated into 50 mL of YPD medium and cultured with shaking at 30°C, 220 rpm for 24 h. The strain was then transferred to 350 mL of YPD medium at a 2% inoculum as a primary seed and cultured at 30°C, 220 rpm for 24 h. The strain was then transferred to a 10 L tank containing 3.5 L of a main fermentation basal salt medium (1 L containing 40 g of glycerol, 18.2 g of K2SO4, 26.7 mL of H3PO4, 0.93 g of CaSO4·2H2O, 14.9 g of MgSO4, 4.13 g of KOH, and the remainder being water) at a 6% inoculum as a secondary seed for fermentation. During the fermentation process, the growth temperature is 26°C, the pH value is 6, and the dissolved oxygen is controlled at about 20%. When the dissolved oxygen rises sharply, it indicates that the glycerol in the basal salt medium has been exhausted. Glycerol with a mass percentage concentration of 50% is added at an initial rate of 15 mL / L / h. The 50% mass percentage glycerol contains 15 mL / L of trace elements PTM1 (1 L contains 6 g of CuSO4·5H2O, 0.08 g of NaI, 3 g of MnSO4·H2O, 0.2 g of Na2MoO4·H2O, 0.02 g of H3BO3, 5 mL of H2SO4, 0.5 g of CoCl2·6H2O, 20 g of ZnCl2, 75 g of FeSO4·7H2O, 0.2 g of biotin, and the balance is water), and the dissolved oxygen is maintained at 15%-20%. When the wet bacterial weight of the fermentation broth reaches 180-220 mg / mL, stop feeding, starve for 1 hour, and exhaust the glycerol. Then, add methanol to induce fermentation. The methanol contains 15 mL / LPTM1 trace elements and is added at a rate of 2 mL / L / h for 2-3 hours. Then, increase the rate to 10.9 mL / L / h for continued fermentation. By adjusting the speed, tank pressure, and ventilation volume to make the dissolved oxygen greater than 15%, induce fermentation for 96 hours to obtain the fermentation broth. The total protein concentration of the fermentation broth supernatant can reach up to 2 g / L. The electrophoresis results of the fermentation broth supernatant are shown in Figure 2 Lane M is a protein marker, and the remaining lanes are COL17-4-1, COL17-5, COL17-6, and COL17-7, respectively. This proves that recombinant type XVII collagen COL17-4-1, COL17-5, COL17-6, and COL17-7 were obtained by fermentation in this example.

[0123] Example 6: Purification of recombinant type XVII collagen

[0124] The fermentation broth obtained in Example 5 was further purified in this example, and the specific method is as follows.

[0125] The fermentation broth was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected. After adjusting the supernatant pH to 6.0, it was filtered using a 0.22 μm filter membrane. Sample purification was performed using a cationic chromatography column (5 ml SP column). First, the column was balanced with 5 column volumes of buffer A until the conductivity baseline was stable. The treated supernatant was loaded onto the column, the flow-through liquid was collected, and the column was repeated 2-3 times. The column was washed with 5 column volumes of buffer A until the UV baseline was stable, the impurities were washed away, and the effluent was collected. The column was eluted with 5 column volumes of eluent B, and the effluent was collected, which was the purified target protein solution. The target protein solution was ultrafiltered and freeze-dried, and the freeze-dried product was finally collected to obtain the purified recombinant type XVII collagen COL17-4-1, COL17-5, COL17-6, and COL17-7.

[0126] Buffer A includes 50 mM phosphate buffer, pH=6.0; eluent B includes 50 mM phosphate buffer, 3 M sodium chloride, pH=6.0; and each buffer is prepared with deionized water.

[0127] Example 7: Recombinant Type XVII Collagen Promoting Cell Proliferation / Cytotoxicity Experiment

[0128] The following experiment was conducted using the purified recombinant type XVII collagen COL17-4-1, COL17-5, COL17-6, and COL17-7 obtained in Example 6 as samples.

[0129] HaCaT cells in logarithmic growth phase (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) were cultured at 1×10 4Cells were seeded in 96-well plates at a density of 100 μL per well and divided into control and experimental groups. Cells were cultured in a CO2 incubator at 37°C and 5% CO2 for 24 hours. Five recombinant type XVII collagen samples (COL17-4-1, COL17-5, COL17-6, COL17-7, and commercially available type XVII collagen) were prepared in serum-free culture medium (purchased from Jiangsu KeyGen Biotech Co., Ltd.) at a concentration of 0.1 mg / mL. The solutions were sterilized by filtration through a 0.22 μm filter. After 24 hours of conventional cell culture, the old culture medium was discarded. The negative control group received 100 μL of serum-free culture medium, the positive control group received 100 μL of serum-free culture medium containing 10% FBS, and the experimental group received 100 μL of recombinant type III collagen sample solution. Six replicates were added to each group. After 24 hours of continuous culture, the culture medium was discarded and 100 μL of CCK-8 diluted 10-fold with serum-free culture medium (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was added to each well. The cells were placed in a cell culture incubator and incubated for another 2 hours. The relative cell proliferation rate was determined using the CCK-8 assay, and the absorbance was measured at a wavelength of 450 nm using a microplate reader. The relative cell proliferation rate (RGR) % = absorbance value of the experimental group / absorbance value of the normal control group × 100%. The results are shown in Table 3 and Figure 3 shown.

[0130] Table 3

[0131]

[0132]

[0133] Note: * indicates p < 0.05 compared with negative control, ** indicates p < 0.01, *** indicates p < 0.001;

[0134] # indicates p < 0.05, ## indicates p < 0.01, and ### indicates p < 0.001 compared with commercial collagen.

[0135] As shown in Table 3 and Figure 3 The recombinant type XVII collagens COL17-4-1, COL17-5, COL17-6, COL17-7, and a positive control were treated with HaCaT cells for 24 hours. The relative cell proliferation rates of COL17-4-1, COL17-5, COL17-6, COL17-7, and the positive control were significantly higher than those of the negative control, increasing by 14%, 17%, 20%, 10%, and 48%, respectively. Commercially available type XVII collagen had no significant promoting effect, and COL17-4-1, COL17-5, COL17-6, and COL17-7 were significantly superior to commercially available type XVII collagen in their ability to promote cell proliferation.

[0136] Example 8: Effect of recombinant type XVII collagen on basement membrane collagen synthesis

[0137] The following experiment was conducted using the purified recombinant type XVII collagen COL17-4-1, COL17-5, COL17-6, and COL17-7 obtained in Example 6 as samples.

[0138] HaCaT cells in logarithmic growth phase were collected and plated at 3×10 5 Cells / mL were seeded in 6-well plates and cultured in a constant temperature incubator at 37°C, 5% CO2 and 95% humidity for 24 hours. COL17-4-1, COL17-5, COL17-6, COL17-7 and commercially available type XVII collagen solution (N=3, concentration 0.1 mg / mL) were prepared using serum-free DMEM medium to treat cells. The control group was serum-free DMEM medium. After 24 hours of culture, the cells were washed 2-3 times with PBS, and then the cells were collected with RNA-easyTM Isolation Reagent reagent, transferred to a centrifuge tube and frozen at -80°C. Total RNA was extracted and reverse transcribed from the collected cells, and finally SYBR Green I was used for real-time fluorescence quantitative PCR to explore the expression levels of type IV, type VII, and type XVII collagen. The results were analyzed and processed by Excel. Table 4 and Figure 4 shown.

[0139] Table 4

[0140]

[0141] Note: * indicates comparison with control, p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001;

[0142] # indicates p < 0.05, ## indicates p < 0.01, and ### indicates p < 0.001 compared with commercial collagen.

[0143] Table 4 and Figure 4 The results showed that the addition of recombinant type XVII collagen COL17-4-1, COL17-5, COL17-6, and COL17-7 could significantly promote the synthesis of type IV collagen gene, with the increase ratios being 35%, 25%, 53%, and 33%, respectively. COL17-6 was significantly superior to commercially available type XVII collagen.

[0144] Regarding type VII collagen gene expression, the promotion ratios of COL17-4-1 and COL17-6 were 32% and 69%, respectively. COL17-6 was significantly higher than commercially available type XVII collagen.

[0145] At the same time, COL17-4-1, COL17-5, COL17-6, COL17-7 and commercially available type XVII collagen can significantly promote type XVII collagen gene expression, with the increase ratios being 41%, 34%, 89%, 22% and 20% respectively. COL17-6 is significantly higher than commercially available type XVII collagen.

[0146] Therefore, COL17-4-1, COL17-5, COL17-6, and COL17-7 can significantly increase the synthesis of type IV, type VII, and type XVII collagen, thereby exerting an anti-aging effect on the basement membrane.

[0147] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. Recombinant type XVII collagen, characterized in that The recombinant type XVII collagen comprises at least one or more of the following A1)-A5): A1) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having at least 95% identity with SEQ ID NO. 1; A2) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having at least 95% identity with SEQ ID NO. 2; A3) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence having at least 95% identity with SEQ ID NO. 3; A4) the amino acid sequence of the recombinant type XVII collagen comprises the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having at least 95% identity with SEQ ID NO. 4; A5) A fusion protein obtained by connecting a tag to the N-terminus or / and C-terminus of the recombinant type XVII collagen described in A1) or A2) or A3) or A4).

2. Biomaterial, characterized in that The biological material includes any one of the following B1) to B5): B1) a nucleic acid molecule comprising a nucleotide sequence encoding the recombinant type XVII collagen according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector comprising the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) a recombinant microorganism comprising the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3); B5) A recombinant cell comprising the nucleic acid molecule described in B1), the expression cassette described in B2) and / or the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that The nucleic acid molecule includes: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having at least 80% identity with SEQ ID NO.5, and / or a nucleotide sequence as shown in SEQ ID NO.6 or a nucleotide sequence having at least 80% identity with SEQ ID NO.6, and / or a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having at least 80% identity with SEQ ID NO.7, and / or a nucleotide sequence as shown in SEQ ID NO.8 or a nucleotide sequence having at least 80% identity with SEQ ID NO.

8.

4. The biomaterial according to claim 2, characterized in that The recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

5. A method for preparing recombinant type XVII collagen, characterized in that: The method comprises: constructing a recombinant microorganism expressing the recombinant type XVII collagen according to claim 1, and culturing the recombinant microorganism.

6. A composition characterized in that The composition contains the recombinant type XVII collagen according to claim 1.

7. Use of the recombinant type XVII collagen according to claim 1, or the biomaterial according to any one of claims 2 to 4, or the preparation method according to claim 5, or the composition according to claim 6 in preparing type XVII collagen, increasing the yield of type XVII collagen, and / or increasing the activity of type XVII collagen.

8. Use of the recombinant type XVII collagen according to claim 1, or the biomaterial according to any one of claims 2 to 4, or the preparation method according to claim 5, or the composition according to claim 6 in the preparation of a product that promotes cell proliferation.

9. Use of the recombinant type XVII collagen according to claim 1, or the biomaterial according to any one of claims 2 to 4, or the preparation method according to claim 5, or the composition according to claim 6 in the preparation of a product for promoting endogenous collagen synthesis; preferably, the endogenous collagen includes at least type IV collagen, type VII collagen and / or type XVII collagen.

10. Use of the recombinant type XVII collagen according to claim 1, or the biomaterial according to any one of claims 2 to 4, or the preparation method according to claim 5, or the composition according to claim 6 in anti-aging products; preferably, the anti-aging effect at least includes anti-basement membrane aging.

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