A recombinant human type VII collagen, its preparation method and application

Abstract

This invention discloses a recombinant human type VII collagen, its preparation method, and its applications, belonging to the field of biochemical technology. The recombinant human type VII collagen of this invention contains multiple repeating amino acid residue units, as shown in SEQ ID NO.1. Repeating these units multiple times yields a recombinant human type VII collagen with a smaller molecular weight compared to natural type VII collagen. After sequence optimization, it is easier to efficiently secrete and express in the Pichia pastoris system, exhibiting characteristics of high yield, low cost, simple operation, and ease of large-scale production. It also possesses excellent antioxidant activity, promotes skin moisturizing and repair, and accelerates wound healing. Furthermore, being derived from human collagen, it significantly reduces the risk of allergic reactions and rejection, demonstrating extremely high safety. Therefore, this recombinant collagen can be widely used as a core active ingredient in cosmetics, pharmaceuticals, and other fields, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and more specifically, to a recombinant human type VII collagen, its preparation method, and its applications. Background Technology

[0002] Collagen is the most abundant protein in the human body and a major component of the extracellular matrix, essential for maintaining the structural integrity and elasticity of the skin. Type VII collagen is a major component of anchoring fibers, and its function is to anchor the basement membrane to the underlying dermis, playing a key role in the stability and resilience of the skin.

[0003] Traditional animal-derived collagen faces risks such as viral contamination, potential immune rejection, and batch-to-batch quality instability. Producing collagen using recombinant DNA technology can effectively avoid these problems. However, full-length type VII collagen has a large molecular weight and complex structure (especially its numerous triple-helix regions and complex post-translational modifications), making it difficult to express efficiently and fold correctly in prokaryotic or simple eukaryotic expression systems.

[0004] Because of the aforementioned bottlenecks in full-length natural type VII collagen, researchers have turned their attention to recombinant collagen, which is humanized collagen produced in large quantities using genetic engineering (such as expression systems based on E. coli, yeast, and CHO cells). Among these, the Pichia pastoris expression system offers advantages such as high-density fermentation, low cost, and ease of scale-up, making it an ideal platform for recombinant protein production. However, the complexity of the natural sequence of type VII collagen makes achieving efficient secretory expression in Pichia pastoris extremely challenging. Therefore, developing a short-fragment recombinant type VII collagen that can be efficiently expressed in the Pichia pastoris system while retaining the core biological functions of type VII collagen has significant industrial value.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a recombinant human type VII collagen, its preparation method, and its application. This recombinant human type VII collagen can be efficiently secreted and expressed in a Pichia pastoris expression system. The recombinant human type VII collagen can produce good antioxidant activity, skin moisturizing and repair, and wound healing effects.

[0007] In a first aspect, the present invention provides a recombinant human type VII collagen, the amino acid sequence of which is any one of the following (A1) to (A3): (A1) The amino acid sequence of the above recombinant human type VII collagen contains multiple amino acid residue repeating units, as shown in SEQ ID NO.1, with a repeating number of n, 1 < n ≤ 20; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant human type VII collagen described in (A1); (A3) has an amino acid sequence that is at least 90% identical to the recombinant human type VII collagen described in (A1) or the fusion protein described in (A2) and has antioxidant effects.

[0008] Secondly, the present invention provides biomaterials related to the above-mentioned recombinant human type VII collagen, which are any one of items (B1) to (B4): (B1) The nucleic acid molecule encoding the above-mentioned recombinant human type VII collagen; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A host cell containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

[0009] Thirdly, the present invention provides a method for preparing the above-mentioned recombinant human type VII collagen, which includes: introducing a nucleic acid molecule encoding the above-mentioned recombinant human type VII collagen into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then separating and purifying it after fermentation culture to obtain recombinant human type VII collagen.

[0010] Fourthly, the present invention provides a composition comprising the above-described recombinant human type VII collagen.

[0011] Fifthly, the present invention provides the application of the above-mentioned recombinant human type VII collagen in the preparation of antioxidant products.

[0012] In a sixth aspect, the present invention provides the application of the above-mentioned recombinant human type VII collagen in the preparation of products that promote wound healing and repair.

[0013] In a seventh aspect, the present invention provides the application of the above-mentioned recombinant human type VII collagen in the preparation of products that promote skin moisturizing and repair.

[0014] The present invention has the following beneficial effects: (1) This invention obtains a recombinant human type VII collagen by screening a core sequence from the functional region fragment of natural human type VII collagen and repeating it multiple times. Compared with natural type VII collagen, the recombinant human type VII collagen of this invention has a smaller molecular weight, and its sequence is more easily and efficiently secreted and expressed in the Pichia pastoris system after optimization. It has the characteristics of high yield, low cost, simple operation and easy large-scale production.

[0015] (2) It has been verified that the recombinant human type VII collagen of the present invention has excellent antioxidant activity, the ability to promote skin moisturizing and repair and promote wound healing. Therefore, the recombinant collagen can be widely used as a core active ingredient in high-end skin care products, functional cosmetics and medical skin repair dressings, and has good application prospects.

[0016] (3) The repeating units of the recombinant human type VII collagen of the present invention are derived from human collagen, which can significantly reduce the risk of allergies and rejection reactions and has extremely high safety. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is an electrophoresis image of the induced fermentation results (2 μl sample loading) of recombinant collagen VII-1520 in Example 1. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] Unless otherwise defined below, all technical and scientific terms used in the specific embodiments of this invention are intended to have the same meaning as commonly understood by those skilled in the art. While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

[0021] As used in this invention, the terms “comprising,” “including,” “having,” “containing,” or “involving” are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term “consisting of” is considered a preferred embodiment of the term “comprising.” If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists only of those embodiments.

[0022] When referring to a singular noun, the indefinite or definite article used, such as "a" or "a kind of," "the," includes the plural form of the noun.

[0023] The following is provided merely to aid in understanding the invention. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made by any person within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0025] Because natural full-length type VII collagen is a giant trimer molecule with a very large molecular weight, it places a huge burden on the protein synthesis capacity of the expression system. Furthermore, the formation of the collagen triple helix structure in this protein is an extremely precise and energy-intensive process. In heterologous expression systems, high-speed, high-volume expression can easily lead to peptide chain misfolding or aggregation, forming insoluble inclusion bodies. Moreover, natural type VII collagen requires various complex and synergistic modifications, while simple expression systems lack all the tools to perform these modifications. These problems prevent the efficient production of natural type VII collagen in simple expression systems (such as bacteria or yeast).

[0026] To address the aforementioned issues, this invention provides a type VII recombinant collagen (abbreviated as VII-1520), which is formed by multiple repeating units connected in series. The amino acid sequence of the repeating unit is: GQKGAPGPKGFKGDP (SEQ ID NO.1).

[0027] The main function of type VII collagen is to anchor fibers. The inventors of this invention selected a polypeptide fragment as shown in SEQ ID NO.1 from the triple helix functional region of the α1 chain of natural type VII collagen as the core sequence, and used this fragment as a repeating unit to obtain a new type VII recombinant collagen through repeated repetition. It has been verified that this type VII recombinant collagen can not only be produced in high quantities through microbial fermentation, but also has good antioxidant activity, promotes skin moisturizing and repair, and promotes wound healing.

[0028] The repetition frequency n of the aforementioned repeating unit can be 5, 10, 15, or 20, with 20 being the preferred frequency. The recombinant collagen composed of these repeating units chained together 20 times consists of 300 amino acids, with the following specific amino acid sequence: GQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAP GPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDPGQKGAPGPKGFKGDP (SEQ ID NO. 2).

[0029] To facilitate subsequent separation and purification, the aforementioned type VII recombinant collagen can also be tagged at the N-terminus and / or C-terminus to obtain a fusion protein. Protein tags can be broadly categorized into three types based on their function: detection tags, expression and purification tags, and tracer tags. Commonly used expression and purification tags include: His, GST, MBP, CBD, Strep-tag, Halo-Tag, SNAP-tag, SUMO, NusA, TrxA, DsbA, Flag, and c-Myc. This invention does not limit the type of tag; those skilled in the art can adjust it as needed.

[0030] In some embodiments, the amino acid sequence of the above-mentioned recombinant human type VII collagen may also be: an amino acid sequence that has at least 90% identity with the above-mentioned recombinant human type VII collagen or fusion protein and has antioxidant, wound healing and skin moisturizing and repair effects.

[0031] The percentage of sequence identity is the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with a reference polypeptide sequence, introducing vacancies where necessary to obtain the maximum percentage of sequence identity, and without considering any conserved substitutions as part of the sequence identity. Alignments used to determine the percentage of amino acid sequence identity can be performed in various ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0032] In some embodiments, having at least 90% sequence identity can specifically be a range of any one or any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0033] This invention provides a nucleic acid molecule that encodes the aforementioned type VII recombinant collagen.

[0034] Nucleic acid molecules are a collective term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), biological macromolecules composed of many nucleotide monomers, and are one of the most fundamental substances of life. A nucleotide sequence refers to the arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA, and in some cases, nucleic acid molecules can be modified for use in the vectors of this invention, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of encoding nucleic acids from one or more given cells or isolated from said one or more given cells.

[0035] In specific implementation schemes, the aforementioned nucleic acid molecules can be optimized according to the codon preferences of the host cell before being used to artificially synthesize gene fragments. It should be understood that all nucleic acid molecules capable of being translated into the aforementioned amino acid sequences are within the scope of protection of this invention.

[0036] In some embodiments, the nucleotide sequence of the above-mentioned nucleic acid molecule is shown in SEQ ID NO.3.

[0037] This invention provides an expression cassette comprising the aforementioned nucleic acid molecule. The expression cassette also contains regulatory sequences, such as promoters and terminators.

[0038] An expression cassette is a nucleic acid construct containing coding and regulatory sequences that are operable upon introduction into a host cell, leading to transcription and / or translation of RNA or polypeptide, respectively. An expression cassette should be understood to include a promoter that allows transcription to begin, an open reading frame of the target gene, and a transcription terminator. Typically, the promoter sequence is placed upstream of the target gene, at a distance compatible with expression control.

[0039] In a specific implementation plan, the expression cassette comprises one or more “promoter-target gene-transcription terminator” structural units from upstream to downstream.

[0040] The present invention provides an expression vector comprising the above-mentioned nucleic acid molecule or expression cassette.

[0041] An expression vector is a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) into a recipient cell. Besides the commonly used *E. coli* plasmid vector, many other artificially constructed plasmid vectors suitable for microorganisms, yeast, plants, etc., have been developed. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide types known in the art. The most commonly used vector type is the "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques. Some vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) integrate into the host cell's genome after introduction and replicate along with the host genome. Furthermore, some vectors can direct the expression of a target gene. Such vectors are referred to herein as "expression vectors." Recombinant expression vectors may contain a form of nucleic acid suitable for expression in host cells, which means that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0042] In some embodiments, the expression vector contains the aforementioned nucleic acid molecule and a promoter (e.g., AOX1 promoter) and a signal peptide sequence (e.g., Pichia pastoris α-mating factor signal peptide) operably linked to the nucleotide molecule and adapted to guide expression in a specific host cell.

[0043] The present invention provides a host cell comprising the above-mentioned nucleic acid molecule or the above-mentioned expression cassette or the above-mentioned expression vector, and the host cell is capable of expressing the above-mentioned type VII recombinant collagen.

[0044] The host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc., of a nucleic acid construct or expression vector containing the polynucleotides of the present invention. The host cell can be any cell useful in the production of type VII recombinant collagen according to the present invention. To produce type VII recombinant collagen, the nucleic acid encoding type VII recombinant collagen can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. Methods for introducing the vector into the host cell are well known, such as using electroporation, and can also include transfection, microinjection, gene gun technology, liposome-mediated methods, etc.

[0045] In a specific implementation plan, the host cell can be a prokaryotic cell or a eukaryotic cell, including: bacterial hosts such as *Escherichia coli*, *Bacillus subtilis*, and *Bacillus licheniformis*; and eukaryotic hosts such as *Pichia pastoris*, *Saccharomyces cerevisiae*, animal cells, and plant cells. Preferably, the host cell is *Pichia pastoris*, and more preferably, the host cell is... Pichia pastoris GS115 or Pichia pastoris X-33.

[0046] This invention provides a method for preparing type VII recombinant collagen, comprising: introducing the nucleic acid molecule of the above-mentioned recombinant human type VII collagen into an expression vector, then introducing the obtained recombinant plasmid into a host cell, and then separating and purifying the recombinant human type VII collagen after fermentation culture.

[0047] In the specific implementation plan, the preparation of type VII recombinant collagen using host cells refers to fermenting and culturing host cells, and the culture medium and culture conditions are well known to those skilled in the art.

[0048] In some embodiments, fermentation culture involves inoculating recombinant engineered strains into a culture medium for batch fermentation or high-density fermentation culture.

[0049] The present invention does not impose any restrictions on the expression mode of the host cell, which can be selected and adjusted as needed. For example, the expression can be one or a combination of two of the following: compositional expression and inducible expression. The inducing agent for inducible expression can be IPTG, β-galactoside, methanol, ethanol, etc.

[0050] In some embodiments, type VII recombinant collagen is obtained by inducing expression in the host cells described above. Specifically, when the cell density reaches a certain value, methanol is added to induce expression, and parameters such as temperature, pH and dissolved oxygen are controlled during the induction period.

[0051] As for the methods of separation and purification, they include one or a combination of several of the following: filtration, salting out, chromatographic chromatography, affinity chromatography, acid-base precipitation, membrane separation, etc.

[0052] In some embodiments, the separation and purification process involves: after fermentation, centrifuging to collect the supernatant, and then performing steps such as filtration and chromatography on the supernatant. Preferably, the chromatography method is ion exchange chromatography and molecular sieve chromatography.

[0053] This invention provides the application of the aforementioned type VII recombinant collagen in the preparation of products for anti-oxidation, wound healing and repair, and skin moisturizing and repair. These products include pharmaceuticals, cosmetics, or medical devices; more preferably, they include cosmetics and medical devices. The medical devices include medical skin repair dressings.

[0054] This specification is intended to enable those skilled in the art to practice the invention. Various modifications to the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0055] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0056] Example 1 This example describes the preparation of recombinant human type VII collagen in a yeast expression system. The specific method is as follows: 1. Preparation of recombinant yeast (1) Preparation of shuttle plasmids According to the amino acid sequence shown in SEQ ID No. 2, the codons of the yeast expression system were optimized to obtain the target gene sequence VII-1520. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was ligated into the pPicZαA plasmid to obtain the pPicZαA-I-α1 plasmid.

[0057] (2) Preparation of yeast expression strains pPicZαA-I-α1 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Transformants were screened using bleomycin resistance as a selection marker to obtain the yeast expression strain.

[0058] 2. Induced expression of the target protein (1) Select a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and incubate at 30℃ and 200 rpm for 48 h to activate it; (2) Transfer 1% of the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium), and incubate at 30°C and 200 rpm for 24 h to serve as the seed for the next flask. (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter. Sterilize at 121℃ for 20 min. After cooling to 30℃, adjust the pH to 5.0. Add the seed prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture. (4) Cultivate to OD 600nm Start adding 50% glycerin when the OD reaches 70, until... 600nm Once the dissolved oxygen level reaches approximately 120, stop adding more methanol and wait for it to rebound to 100% before starting to add methanol for induction. (5) During the induction process, the dissolved oxygen should be controlled to be no less than 30% and the pH should be around 5.0. After induction for 40 h, the fermentation was stopped. The culture medium was centrifuged at 12000 rpm for 2 min. The supernatant was collected and the protein yield and purity were detected by BCA method and SDS-PAGE method.

[0059] Full-length type VII collagen is present in extremely small amounts in organisms, far less abundant than type I and type III collagen. Extracting specific types of type VII collagen from animal tissues is not only technically challenging but also extremely costly. Furthermore, since full-length type VII collagen is difficult to express in microorganisms, obtaining it from natural sources is even more difficult. However, the recombinant collagen VII-1520 prepared using the above method achieved a yield as high as 15 g / L according to BCA analysis in the fermenter, and the SDS-PAGE electrophoresis results were as follows... Figure 1 As shown, the target protein accounts for more than 30%, and the protein purity after ultrafiltration through the hollow fiber column can reach more than 80%.

[0060] Example 2 This embodiment describes the identification of the antioxidant activity of recombinant human type VII collagen, as detailed below: This experiment used the DPPH method to test antioxidant activity: solutions of the recombinant type VII collagen of this invention at different concentrations (0.1, 0.5, 1.0, 2.0, 5.0 mg / mL) were prepared. 2 mL of the sample solution was mixed with 2 mL of 0.1 mM DPPH ethanol solution, and reacted in the dark for 30 minutes. The absorbance was then measured at 517 nm. Vitamin C (Vc) was used as a positive control. The clearance rate was calculated using the formula: Clearance rate (%) = [1 - (A... 样品 – A 空白 ) / A 对照 ] × 100%.

[0061] Table 1 Antioxidant Activity Results

[0062] The results in Table 1 show that the recombinant type VII collagen provided by this invention can achieve a maximum DPPH free radical scavenging rate of 88.9% in the concentration range of 0.1-5.0 mg / mL, with an IC50 value of 1.4 mg / mL, which fully demonstrates that the recombinant protein has significant antioxidant activity.

[0063] This inherent antioxidant activity means that when this collagen is applied to skincare products, it can not only play a basic role in moisturizing and repairing, but also directly eliminate free radicals and reduce the damage of oxidative stress to the skin. Thus, it can play a synergistic role in anti-aging and soothing sensitivity, surpassing the function of natural type VII collagen, which only passively enhances antioxidant activity as a structural protein.

[0064] Example 3 This example illustrates the effect of recombinant human type VII collagen on cell migration, as detailed below: Scratch assays were performed using human-like immortalized keratinocytes (HaCaT). Cells were cultured in 6-well plates until confluence exceeded 90%, and straight scratches were made on a monolayer of cells using a 200 μL pipette tip. After washing with PBS, serum-free medium (negative control), medium containing 10 ng / mL EGF (positive control), and medium containing 0.1 mg / mL of the recombinant type VII collagen of this invention were added, respectively. The cells were observed under a microscope at 0 h and 24 h.

[0065] The results showed that the positive control was approximately 65.5%, the migration rate of the recombinant type VII collagen of the present invention at 0.1 mg / mL was approximately 63.8%, and compared with the negative control group of approximately 47.9%, the experimental group with added recombinant type VII collagen of the present invention showed a significant increase in scratch healing area after 24 hours, indicating that it can effectively promote the migration of HaCaT cells and has the potential to promote skin wound healing and repair.

[0066] Example 4 This example demonstrates the identification of the moisturizing properties of recombinant human type VII collagen, as detailed below: This experiment used an in vitro hygroscopicity test for identification: a certain amount of sample was placed in a desiccator with constant humidity, weighed periodically, and its hygroscopicity was calculated. The high molecular weight HA in the control group was 1800 KD, and the low molecular weight HA was 50 KD. Specific hygroscopicity results are shown in Table 2. Table 2. Results of moisture absorption rate tests for each group

[0067] As shown in Table 2, the recombinant type VII collagen of the present invention exhibits superior water-holding capacity compared to sodium hyaluronate (HA) within 48 hours.

[0068] Based on the above embodiments, it can be found that the recombinant type VII collagen of the present invention can be expressed at a high level in Pichia pastoris, is easy to purify, and has good antioxidant, cell migration promotion and moisturizing repair functions. It can be used as an excellent active ingredient in cosmetics and skin repair products.

[0069] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant human type VII collagen, characterized in that, The amino acid sequence of the recombinant human type VII collagen is shown in SEQ ID NO.

2.

2. A biomaterial related to the recombinant human type VII collagen as described in claim 1, characterized in that, It can be any one of items (B1) to (B4): (B1) A nucleic acid molecule encoding the recombinant human type VII collagen as described in claim 1; (B2) An expression cassette containing the nucleic acid molecule described in (B1); (B3) A recombinant vector containing the nucleic acid molecule described in (B1) or the expression cassette described in (B2); (B4) A host cell containing the nucleic acid molecule described in (B1), the expression cassette described in (B2), or the recombinant vector described in (B3).

3. The method for preparing recombinant human type VII collagen as described in claim 1, characterized in that, include: The nucleic acid molecule encoding the recombinant human type VII collagen as described in claim 1 is introduced into an expression vector, and then the obtained recombinant plasmid is introduced into a host cell. After fermentation culture, the recombinant human type VII collagen is separated and purified to obtain the recombinant human type VII collagen.

4. The preparation method according to claim 3, characterized in that, The host cell includes Pichia pastoris.

5. A composition, characterized in that, Includes the recombinant human type VII collagen as described in claim 1.

6. The use of the recombinant human type VII collagen as described in claim 1 in the preparation of antioxidant products.

7. The use of the recombinant human type VII collagen according to claim 1 in the preparation of products that promote wound healing and repair.

8. The use of the recombinant human type VII collagen according to claim 1 in the preparation of products that promote skin moisturizing and repair.

9. The application according to any one of claims 6 to 8, characterized in that, The products include cosmetics and pharmaceuticals.

10. The application according to any one of claims 6 to 8, characterized in that, The products include skincare products.

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