Recombinant VII type collagen as well as preparation method and application thereof

Abstract

The invention provides recombinant VII type collagen as well as a preparation method and application thereof, and relates to the technical field of bioengineering. According to the invention, the human-derived VII type collagen is designed and screened, so that a new functional repetitive unit with biological activity of the VII type humanized collagen is successfully obtained, and a new direction is provided for research of the VII type collagen. In the application, three new high-yield recombinant VII type humanized collagens are successfully obtained by repeating the functional repeating units. The recombinant VII type humanized collagen has the effect of improving cell viability, and the protein is proved to be high in safety. The protein also can promote cell adhesion, so that the protein has an excellent effect of treating skin injury. The protein can also improve the cell migration rate and has excellent transdermal absorption performance. In addition, the protein also has the effect of promoting collagen expression, and provides a new functional component for anti-aging related products.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to a recombinant type VII collagen, its preparation method, and its application. Background Technology

[0002] Type VII collagen is a major component of anchoring fibrils and acts at the junction of the epidermis and dermis. Besides the skin, small amounts of type VII collagen are also found in the epithelial tissues of other organs such as the retina and digestive tract. Clinical studies have confirmed that type VII collagen can be used for subcutaneous injection to treat malnutrition-related bullous epidermolysis. Furthermore, type VII collagen participates in skin homeostasis, including wound healing, ulcer treatment, and prevention of skin aging problems such as wrinkles and sagging.

[0003] Type VII collagen in the human body is mainly formed by keratinocytes and fibroblasts, accounting for 0.001% of total collagen. Human type VII collagen has a helical structure formed by three identical α chains, each chain containing 2,944 amino acids. Currently, there are few reports on the in vitro recombinant expression of type VII collagen, both domestically and internationally. Most studies focus on obtaining small amounts of type VII collagen through tissue or organ extraction or lentiviral transfection for mechanistic research. However, these two methods have limitations such as high preparation difficulty, low purity, and complex control, making them difficult to apply to commercial production. Due to the large molecular weight of human type VII collagen, in vitro recombinant expression is difficult. Therefore, in research, the triple helix sequence of type VII collagen is often extracted for recombinant expression to achieve large-scale preparation of type VII collagen. Summary of the Invention

[0004] This invention designs and screens human type VII collagen to successfully obtain high-yield recombinant type VII humanized collagen. The production process of this protein is simple and easy to scale up, and it can be widely used in cosmetics and medical devices.

[0005] On the one hand, this application provides recombinant type VII collagen, said recombinant type VII collagen comprising at least one or more of the following A1)-A2):

[0006] A1) The amino acid sequence of the recombinant type VII collagen contains one or more repeating units of amino acid residues, the repeating unit containing the sequence shown in SEQ ID NO.2, the number of repeats being n, 1≤n≤20;

[0007] A2) The amino acid sequence of the recombinant type VII collagen contains one or more repeating units of amino acid residues, the repeating units containing the sequence shown in SEQ ID NO.3, with a repeat number of n, 10≤n≤20.

[0008] Preferably, the recombinant type VII collagen is human recombinant type VII collagen.

[0009] Preferred,

[0010] A1) The amino acid sequence of the recombinant type VII collagen contains one or more repeating units of amino acid residues, said repeating unit comprising, as shown in SEQ ID NO. The sequence shown in NO.2 has a repetition count of n, where n is a natural number. Optionally, the repetition count n can take the following values: 1≤n≤20, 2≤n≤20, 3≤n≤20, 4≤n≤20, 5≤n≤20, 6≤n≤20, 7≤n≤20, 8≤n≤20, 9≤n≤20, 10≤n≤20, 11≤n≤20, 12≤n≤20, 13≤n≤20, 14≤n≤20, 15≤n≤20, 16≤n≤20, 17≤n≤20, 18≤n≤20, 19≤n≤20, 1≤n≤19, 2≤n≤19, 3≤n≤19, 4≤n≤19, 5≤n≤19, 6≤n≤19, 7≤n≤19, 8≤n≤ 19, 9≤n≤19, 10≤n≤19, 11≤n≤19, 12≤n≤19, 13≤n≤19, 14≤n≤19, 15≤n≤19, 16≤n≤19, 17≤n≤19, 18≤n≤19, 1≤n≤18, 2≤n≤18, 3≤n≤18, 4≤n≤18, 5≤n≤18, 6≤n≤18, 7≤n≤18, 8≤n≤18, 9≤n≤18, 10≤n≤18, 11≤n≤18, 12≤n≤18, 13≤n≤18, 14≤n≤18, 15≤n≤18, 16≤n≤18, 17≤n≤18, 1≤n≤17, 2≤n≤17, 3≤n≤17, 4≤n≤17 5≤n≤17, 6≤n≤17, 7≤n≤17, 8≤n≤17, 9≤n≤17, 10≤n≤17, 11≤n≤17, 12≤n≤17, 13≤n≤17, 14≤n≤17, 15≤n≤17, 16≤n≤17, 1≤n≤16, 2≤n≤16, 3≤n≤16, 4≤n≤16, 5≤n≤16, 6≤n≤16, 7≤n≤16, 8≤n≤16, 9≤n≤16, 10≤n≤16, 11≤n≤16, 12≤n≤16, 13≤n≤16, 14≤n≤16, 15≤n≤16, 1≤n≤15, 2≤n≤15, 3≤n≤15, 4≤n≤15, 5≤n≤1 5, 6≤n≤15, 7≤n≤15, 8≤n≤15, 9≤n≤15, 10≤n≤15, 11≤n≤15, 12≤n≤15, 13≤n≤15, 14≤n≤15, 1≤n≤14, 2≤n≤14, 3≤n≤14, 4≤n≤14, 5≤n≤14, 6≤n≤14, 7≤n≤14, 8≤n≤14, 9≤n≤14, 10≤n≤14, 11≤n≤14, 12≤n≤14, 13≤n≤14, 1≤n≤13, 2≤n≤13, 3≤n≤13, 4≤n≤13, 5≤n≤13, 6≤n≤13, 7≤n≤13, 8≤n≤13, 9≤n≤13, 10≤n≤1311≤n≤13, 12≤n≤13, 1≤n≤12, 2≤n≤12, 3≤n≤12, 4≤n≤12, 5≤n≤12, 6≤n≤12, 7≤n≤12, 8≤n≤12, 9≤n≤12, 10≤n≤12, 11≤n≤12, 1≤n≤11, 2≤n≤11, 3≤n≤11, 4≤n≤11, 5≤n≤11, 6≤n≤11, 7≤n≤11, 8≤n≤11, 9≤n≤11, 10≤n≤11, 1≤n≤10, 2≤n≤10, 3≤n≤10, 4≤n≤10, 5≤n≤10, 6≤n≤10, 7≤n≤10, 8≤n≤10, 9 ≤n≤10, 1≤n≤9, 2≤n≤9, 3≤n≤9, 4≤n≤9, 5≤n≤9, 6≤n≤9, 7≤n≤9, 8≤n≤9, 1≤n≤8, 2≤n≤8, 3≤n≤8, 4≤n≤8, 5≤n≤8, 6≤n≤8, 7≤n≤8, 1≤n≤7, 2≤n≤7, 3≤n≤7, 4≤n≤7, 5≤n≤7, 6≤n≤7, 1≤n≤6, 2≤n≤6, 3≤n≤6, 4≤n≤6, 5≤n≤6, 1≤n≤5, 2≤n≤5, 3≤n≤5, 4≤n≤5, 1≤n≤4, 2≤n≤4, 3≤n≤4, 1≤n≤3, 2≤n≤3, 1≤n≤2;

[0011] For example, the value of n, or the upper limit of n, or the lower limit of n, can be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and any value between them.

[0012] Preferably, the number of repetitions is 4 and / or 16.

[0013] A2) The amino acid sequence of the recombinant type VII collagen contains one or more repeating units of amino acid residues, said repeating unit comprising, as shown in SEQ ID NO. The sequence shown in NO.3 has a repetition count of n, where n is a natural number. Optionally, the repetition count n can take the following values: 10≤n≤20, 11≤n≤20, 12≤n≤20, 13≤n≤20, 14≤n≤20, 15≤n≤20, 16≤n≤20, 17≤n≤20, 18≤n≤20, 19≤n≤20, 10≤n≤19, 11≤n≤19, 12≤n≤19, 13≤n≤19, 14≤n≤19, 15≤n≤19, 16≤n≤19, 17≤n≤19, 18≤n≤19, 10≤n≤18, 11≤n≤18, 12≤n≤18, 13≤n≤18, 14≤n≤18, 15≤n≤18. 16≤n≤18, 17≤n≤18, 10≤n≤17, 11≤n≤17, 12≤n≤17, 13≤n≤17, 14≤n≤17, 15≤n≤17, 16≤n≤17, 10≤n≤16, 11≤n≤16, 12≤n≤16, 13≤n≤16, 14≤n≤16, 15≤n≤16, 10≤n≤15, 11≤n≤15, 12≤n≤15, 13≤n≤15, 14≤n≤15, 10≤n≤14, 11≤n≤14, 12≤n≤14, 13≤n≤14, 10≤n≤13, 11≤n≤13, 12≤n≤13, 10≤n≤12, 11≤n≤12, 10≤n≤11.

[0014] For example, the value of n, or the upper limit of n, or the lower limit of n, can be selected from 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and any value between them.

[0015] Preferably, the number of repetitions is 16.

[0016] In a preferred embodiment, the recombinant type VII collagen is obtained by directly linking amino acid residue repeating units. Those skilled in the art will understand that known linkers can be added between the amino acid residue repeating units for connection, and that the recombinant type VII collagen can also undergo known modifications, including phosphorylation, acetylation, ubiquitination, and glycosylation, without affecting the protein's function.

[0017] Further, the recombinant type VII collagen has an amino acid sequence as shown in SEQ ID NO.4 or an amino acid sequence having at least 98% identity with SEQ ID NO.4, and / or an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence having at least 98% identity with SEQ ID NO.5, and / or an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence having at least 98% identity with SEQ ID NO.6.

[0018] Preferred,

[0019] The recombinant type VII collagen comprises the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having 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 with SEQ ID NO.4. More preferably, the nucleic acid molecule encoding the recombinant type VII collagen comprises: a nucleotide sequence as shown 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%, or 99.9% identity with SEQ ID NO.7.

[0020] The recombinant type VII collagen comprises the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having 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 with SEQ ID NO. 5. More preferably, the nucleic acid molecule encoding the recombinant type VII collagen comprises: a nucleotide sequence as shown 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 with SEQ ID NO. 8.

[0021] The recombinant type VII collagen comprises an amino acid sequence as shown in SEQ ID NO. 6 or an amino acid sequence having 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 with SEQ ID NO. 6. More preferably, the nucleic acid molecule encoding the recombinant type VII collagen comprises: a nucleotide sequence as shown in SEQ ID NO.9 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 with SEQ ID NO.9.

[0022] It is understood that those skilled in the art can select a suitable gene editing system and gene editing method to complete the construction of the above-mentioned recombinant type VII collagen according to the actual situation, or they can choose to use a general method to prepare the recombinant type VII collagen.

[0023] Those skilled in the art will understand that known modifications and optimizations can be made without affecting the function of the recombinant type VII collagen, and this application does not impose any mandatory limitations in this regard.

[0024] Preferably, the recombinant type VII collagen is recombinant type VII humanized collagen.

[0025] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following B1)-B5):

[0026] B1) A nucleic acid molecule containing a nucleotide sequence encoding the recombinant type VII collagen;

[0027] B2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in B1);

[0028] B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2);

[0029] B4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3);

[0030] B5) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3).

[0031] Further, the nucleic acid molecule includes: 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, and / or a nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having at least 80% identity with SEQ ID NO.9.

[0032] More preferably, the nucleic acid molecule comprises: a nucleotide sequence as shown 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%, or 99.9% identity with SEQ ID NO.7.

[0033] More preferably, the nucleic acid molecule comprises: a nucleotide sequence as shown 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 with SEQ ID NO. 8.

[0034] More preferably, the nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.9 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 with SEQ ID NO.9.

[0035] Those skilled in the art will recognize that other commonly used expression elements can be adaptively added to the expression cassette described in this application to assist in the expression of the target gene, such as adding tags, fluorescent protein markers, and resistance selection markers.

[0036] Optionally, the recombinant vector may include a nucleic acid molecule encoding the aforementioned protein, a promoter, and transcription and translation termination signals. The promoters or terminators used to regulate expression in this application may be general promoters or terminators, or they may be host-specific promoters or terminators. Those skilled in the art can make adaptive selections according to the actual situation. This application does not impose mandatory limitations on the sequences of promoters or terminators.

[0037] The recombinant vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, microchromosomes, or artificial chromosomes. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, can be used, or the total DNA to be introduced into the host cell's genome can be included. Preferably, the recombinant vector can be the pPIC9k plasmid and / or the pPICZαA plasmid.

[0038] Furthermore, the recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

[0039] Preferably, the recombinant microorganism is Pichia pastoris.

[0040] In a preferred embodiment, the Pichia pastoris is Pichia pastoris GS115.

[0041] Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain, as long as they can complete the expression of the recombinant type VII collagen described in this application. No specific strain is limited here.

[0042] In one optional 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, as will be known to those skilled in the art, the nucleic acid molecule can be selectively inserted into the genome of the starting strain or can exist on a free plasmid, as long as the expression of the nucleic acid molecule or the synthesis of recombinant type VII collagen can be achieved.

[0043] In one optional embodiment, the recombinant vector and recombinant microorganism contain an resistance selection marker gene. Those skilled in the art can choose according to the actual situation. This application does not impose mandatory limitations on the resistance selection marker gene.

[0044] A resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or a protrophic auxotrophic phenotype. Examples of bacterial selection markers include the dal gene in Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline.

[0045] On the other hand, this application also provides a method for preparing recombinant type VII collagen, the method comprising: constructing a recombinant microorganism expressing the recombinant type VII collagen, and culturing the recombinant microorganism.

[0046] Furthermore, the recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

[0047] Preferably, the recombinant microorganism is Pichia pastoris.

[0048] In a preferred embodiment, the Pichia pastoris is Pichia pastoris GS115.

[0049] In a preferred embodiment, the preparation method includes the following steps:

[0050] Step 1: Construct recombinant microorganisms expressing the recombinant type VII collagen;

[0051] Step 2: Inoculate the recombinant microbial cells into the fermentation medium. The culture conditions are 25℃-37℃, 100-300rpm, and fermentation time of 24-72h. Add methanol at a final concentration of 1%-5% every 24h.

[0052] In a preferred embodiment, the preparation method includes the following steps:

[0053] Step 1: Construct recombinant microorganisms expressing the recombinant type VII collagen;

[0054] Step 2: Inoculate the recombinant microbial cells into the fermentation medium. The culture conditions are 25℃-37℃, pH 5-7, air flow rate of 1-5L / min, stirring power of 300-900rpm, and dissolved oxygen is activated to maintain the dissolved oxygen concentration at about 30% for fermentation.

[0055] Preferably, the fermentation process is divided into three stages: the first stage, fermentation for 10-20 hours; the second stage, adding 10%-50% glycerol for about 10-20 hours to allow the OD to reach its maximum. 600 Accumulate to 150-180; in the third stage, methanol containing 1.2% PTM1 is added in a gradient, with the methanol feed rate gradually increasing from 2 mL / h to 12 mL / h.

[0056] The fermentation medium can be BMMY medium.

[0057] Preferably, step two further includes a purification step. The purification described above can be performed using common methods.

[0058] On the other hand, this application also provides a composition containing the recombinant type VII collagen.

[0059] Those skilled in the art will understand that the composition may contain excipients. As physiologically acceptable media, their composition, dosage, etc., can be selected by those skilled in the art based on their knowledge, depending on the type of application required.

[0060] On the other hand, this application also provides the use of the recombinant type VII collagen or the biomaterial or the composition described herein in the preparation of products that improve cell adhesion and / or cell viability and / or cell migration and / or promote collagen expression.

[0061] Preferably, the cells are keratinocytes and / or fibroblasts.

[0062] Preferably, the collagen includes type I collagen and / or type IV collagen.

[0063] In a preferred embodiment, the concentration of the recombinant type VII collagen is at least about 0.001%, and can be 0.001%-10%; preferably, 0.05%-0.5%.

[0064] The concentration of the recombinant type VII collagen can be any value of 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or higher.

[0065] Those skilled in the art can adjust the protein concentration according to the actual situation, and no specific restrictions are imposed in this application.

[0066] On the other hand, this application also provides the use of the recombinant type VII collagen, the biomaterial, or the composition thereof in products for repairing skin damage and / or anti-wrinkle and / or firming and / or improving transdermal absorption.

[0067] in,

[0068] The improved transdermal absorption performance is achieved by increasing the cumulative permeability of collagen.

[0069] The repair of skin damage is achieved by increasing cell migration, cell adhesion, or cell growth.

[0070] The anti-wrinkle and firming effects are achieved by promoting collagen expression and increasing cell migration or cell adhesion.

[0071] On the other hand, this application also provides the use of the recombinant type VII collagen, the biomaterial, or the composition in daily chemical and / or pharmaceutical and / or engineered tissue materials.

[0072] The present invention has the following beneficial effects:

[0073] This invention, through the design and screening of human type VII collagen, successfully obtained novel bioactive functional repeating units for type VII humanized collagen, providing a new direction for type VII collagen research. This application also successfully obtained three new high-yield recombinant type VII humanized collagens by repeating these functional repeating units.

[0074] This recombinant type VII humanized collagen protein enhances cell viability, demonstrating its high safety profile and broad applicability across various scenarios. It also promotes cell adhesion, resulting in excellent therapeutic effects for skin lesions. Furthermore, it increases cell migration, effectively improving the transdermal absorption of collagen products. Additionally, it promotes collagen expression, providing a novel active ingredient for anti-aging products.

[0075] This invention also provides a method for preparing recombinant type VII humanized collagen. The production process is simple and easy to scale up, making it widely applicable in cosmetics and medical devices. Attached Figure Description

[0076] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0077] Figure 1 The pPIC9k-Ⅶ-X plasmid map;

[0078] Figure 2 The pPICZαA-Ⅶ-8-16 plasmid map;

[0079] Figure 3 This is a schematic diagram of a type VII collagen freeze-dried sample. Detailed Implementation

[0080] 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 studies.

[0081] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0082] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0083] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0084] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0085] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0086] Free expression: Free expression is the expression of target genes using free plasmids. Free plasmids are independent DNA molecules that exist in cells and have the ability to replicate and be transmitted independently. They are widely used in genetic engineering and molecular biology research.

[0087] Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0088] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. For embodiments where specific conditions are not specified, follow standard conditions or conditions recommended by the manufacturer. The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercially available products, and specific operations were performed according to the kit instructions.

[0089] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0090] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0091] All plasmids used in this invention were designed using conventional molecular biology techniques, and constructed through PCR and vector fragment recombination. All recombinant plasmids were verified by sequencing to be completely consistent with the target sequence.

[0092] MD medium: glucose 20 g / L, amino-free yeast basic nitrogen source 13.4 g / L, 4 × 10⁻⁵% biotin, balance water;

[0093] YPD medium: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast extract, with the remainder being water. For solid medium, an additional 15 g / L of agar powder needs to be added.

[0094] BMMY medium: 20 g / L peptone, 13.4 g / L amino-free yeast basal nitrogen source, 10 g / L yeast extract, 4 × 10⁻⁵% biotin, 0.5% methanol, 100 mM potassium phosphate buffer, balance water, adjust pH to 6.0.

[0095] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0096] In the following examples, unless otherwise specified, % means wt%, i.e., weight percentage.

[0097] Example 1: Design of recombinant type VII humanized collagen and preparation of its recombinant strain

[0098] (1) Design and construction of expression plasmids

[0099] Type VII collagen (Uniprot Q02388, amino acid sequence as shown in SEQ ID NO.1) was selected. In this embodiment, its physicochemical properties and protein structure were predicted. Some sequences were selected as repeating units, as shown in SEQ ID NO.2 and SEQ ID NO.3, and these sequences were repeated several times to obtain the amino acid sequences of recombinant type VII humanized collagen (as shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6). Pichia pastoris codon optimization was performed on the DNA sequences encoding sequences VII-8-4, VII-8-16, and VII-11-16. The optimization results are shown in SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9. Specific sequence information is recorded in Table 1.

[0100] The optimized DNA sequences were synthesized by GenScript and then incorporated into the α-factor secretion signal peptide of the pPIC9k plasmid, resulting in plasmids pPIC9k-VII-8-4, pPIC9k-VII-8-16, and pPIC9k-VII-11-16, respectively. Figure 1 As shown, Figure 1 The yellow color block "VII-X" can be VII-8-4, VII-8-16, or VII-11-16.

[0101] Table 1. Amino acid sequence of recombinant type VII humanized collagen

[0102]

[0103] (2) Construction and screening of recombinant strains

[0104] Plasmids pPIC9k-VII-8-4, pPIC9k-VII-8-16, and pPIC9k-VII-11-16 were linearized using the restriction endonuclease Sac I, and then transformed into wild-type Pichia pastoris GS115 competent cells (purchased from Beijing Huayueyang Biotechnology Co., Ltd.). The cells were plated on MD (medium-density broth) and incubated at 30°C for 48-72 h. Single colonies on the plates were identified by colony PCR. Correctly identified single colonies were streaked onto YPD (medium-density broth) containing 4 mg / mL genimycin (G418) and incubated at 30°C for 48-72 h. The fastest-growing single colonies were selected as the high-copy-rate recombinant type VII humanized collagen strains obtained through screening and named Col-VII-8-4, Col-VII-8-16, and Col-VII-11-16.

[0105] GenScript synthesized the VII-8-16 DNA sequence and constructed it into the αfactor secretion signal peptide of the pPICZαA plasmid, obtaining the plasmid pPICZαA-VII-8-16. Figure 2 As shown. The plasmid pPICZαA-VII-8-16 was linearized with restriction endonuclease Sac I, and then transformed into Col-VII-8-16 competent cells by electroporation. The cells were plated on YPD (plate) medium containing Zeocin resistance (100 mg / L) and incubated at 30°C for 48-72 h. Single colonies on the plates were identified by colony PCR. Single colonies with correct identification results were picked and streaked onto YPD (plate) medium containing Zeocin resistance (4 mg / mL) and incubated at 30°C for 48-72 h. The single colonies with the fastest growth rate were selected as the high-copy recombinant type VII humanized collagen strain obtained through screening and named C-VII-8-16.

[0106] Example 2: Shake-flask fermentation and 5L fermenter fermentation of recombinant type VII humanized collagen

[0107] (1) Shake-flask fermentation of recombinant strains Col-VII-8-4, Col-VII-8-16, Col-VII-11-16 and C-VII-8-16

[0108] Single clones of Col-VII-8-4, Col-VII-8-16, Col-VII-11-16, and C-VII-8-16 were picked from the plate and inoculated into test tubes containing 10 mL of fresh YPD medium. After 36 h, the cells were collected by centrifugation and transferred to 250 mL baffle shake flasks containing 25 mL of BMMY medium. The culture conditions were 30 °C, 220 rpm, and fermentation time was 72 h. Methanol was added every 24 h to a final concentration of 1%. After fermentation, the supernatant was collected by centrifugation and SDS protein electrophoresis was performed for quantitative experiments. The fermentation yield is shown in Table 2 below.

[0109] Table 2 Yield of Recombinant Type VII Humanized Collagen by Shake-Flavor Fermentation

[0110] strains Shake flask yield (g / L) Col-VII-8-4 0.5 Col-VII-8-16 0.163 Col-VII-11-16 0.102 C-Ⅶ-8-16 0.6

[0111] (2) Fermentation of recombinant strains C-VII-8-16, Col-VII-8-4, and Col-VII-11-16 in a 5L fermenter

[0112] C-VII-8-16, Col-VII-8-4, and Col-VII-11-16 from the glycerol tubes were streaked onto MD (plate) medium and incubated at 30°C for 48-72 h. Single colonies from the plates were picked and inoculated into 200 mL of YPD (shake flask) medium. After incubation at 30°C for 36 h, all colonies were transferred to a 5 L fermenter containing 1.8 L of BMMY medium. Initial OD... 600The pH should be around 1. Set the temperature to 30℃ and add ammonia via a peristaltic pump to maintain the pH at around 6.0. Set the airflow rate to 2L / min, the stirring power to 300-900rpm, and activate dissolved oxygen to maintain a dissolved oxygen concentration of around 30%.

[0113] The fermentation process consists of three stages. In the first stage, the yeast grows in the fermenter for approximately 16 hours until the glycerol in the tank is depleted. In the second stage, 50% glycerol is continuously added for about 10 hours to increase the OD (Oxygen Demand). 600 Accumulated to about 180; in the third stage, methanol containing 1.2% PTM1 was added in a gradient, with the methanol feeding rate gradually increasing from 2 mL / h to 12 mL / h. Samples were taken at 24 h, 48 h and 72 h during the methanol addition stage. After centrifugation, the fermentation supernatant was used for SDS protein electrophoresis quantification. The results are shown in Table 3.

[0114] Table 3. Protein yield of strains C-VII-8-16 and Col-VII-8-4 in a 5L fermenter.

[0115]

[0116] Example 3: Purification of recombinant type VII humanized collagen

[0117] Fermentation broth from 5L fermenters of recombinant strains C-VII-8-16, Col-VII-8-4, and Col-VII-11-16 obtained in Example 2 was collected and centrifuged at 5000 rpm for 20 min to collect the supernatant. The supernatant was subjected to hollow fiber microfiltration (purchased from Cytiva, catalog number 56-4102-68), with the permeation pressure controlled below 0.6 MPa, and the permeate was collected. The collected hollow fiber filtrate was concentrated by ultrafiltration using a membrane packing (purchased from Combat, catalog number UFELA0005010P) and then subjected to medium exchange. SPFF cation exchangers (purchased from Cytiva, catalog number 17051010) were used for injection, and the target protein bound to the cation exchanger. After sample loading, elution was performed with a buffer containing NaCl, and the target peak was collected. The collected eluent was concentrated by ultrafiltration using a membrane packing and then subjected to medium exchange, followed by lyophilization. The lyophilized sample appeared as a white fibrous sponge, as shown in the image. Figure 3 As shown.

[0118] Example 4: Efficacy determination of recombinant type VII humanized collagen

[0119] In this embodiment, the freeze-dried samples VII-8-16, VII-8-4 and VII-11-16 obtained in Example 3 were used to perform the following efficacy determinations.

[0120] 1. Cell adhesion detection

[0121] Human immortalized keratinocytes and human fibroblasts were cultured to the logarithmic growth phase, respectively. The cells were digested with trypsin and then suspended in serum-free medium (Cell Applications, catalog number 116F-500) to adjust the concentration to 5 × 10⁻⁶. 4 Cells / mL were seeded into 96-well plates coated with lyophilized collagen samples. The blank control (BC) was an uncoated 96-well plate washed with serum-free medium, while the negative control (NC) was a coated 96-well plate washed with serum-free medium. The plates were then incubated in a cell culture incubator (37℃, 5% CO2). After 6 hours of incubation, culture was stopped, the culture medium was discarded, and the plates were washed 2-3 times with PBS, which was then discarded. 100 μL of 0.5 mg / mL MTT solution was added, and the plates were incubated at 37℃ in the dark for 2 hours. After incubation, the supernatant was discarded, and 50 μL of LDMSO was added to each well. The OD value was read at 490 nm. Cell adhesion rate was calculated as: Cell adhesion rate = (sample well OD / total cell well OD) * 100%. Specific treatment concentrations and results are shown in Table 4.

[0122] Table 4. Results of cell adhesion

[0123]

[0124] Note: Statistical analysis was performed using the two-tailed text test method. For comparisons between the NC and BC groups, significance is indicated by # (p-value < 0.05, p-value < 0.01, p-value < 0.0 ...

[0125] Table 4 shows that, for the keratinocyte experimental group, the keratinocyte adhesion rate in the NC group was significantly increased compared to the BC group, indicating that the well plate coating was effective. Furthermore, compared to the NC group, the recombinant type VII humanized collagen VII-8-16 and VII-8-4 in the samples significantly promoted keratinocyte adhesion and demonstrated repair efficacy.

[0126] In the human fibroblast experimental group, the keratinocyte adhesion rate was significantly increased in the NC group compared to the BC group, indicating that the well plate coating was effective. Compared with the NC group, type VII collagen VII-8-16 and VII-8-4 in the sample significantly promoted fibroblast adhesion.

[0127] 2. Cell viability detection

[0128] Collect logarithmic growth phase cells from immortalized human keratinocytes, at a cell density of 1×10⁻⁶. 5 / wells were seeded into 24-well culture plates. The plates were incubated in an incubator (37℃, 5% CO2) for 24 h. 200 μL of culture medium containing 10% PBS was added to the control group, and 200 μL of culture medium containing the corresponding concentration of lyophilized sample was added to the sample group (specific concentrations are shown in Table 5). After 72 h, the supernatant was discarded, and 500 μL of culture medium containing 0.5 mg / mL MTT was added. The plates were incubated at 37℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 300 μL of DMSO was added to each well. The OD value was read at 490 nm.

[0129] Collect human fibroblasts in the logarithmic growth phase, at a cell density of 8 × 10⁻⁶ cells / year. 4 / wells were seeded into 24-well culture plates. After incubation in an incubator (37℃, 5% CO2) for 24 h, samples were loaded. 200 μL of culture medium containing 10% PBS was added to the control group, and 200 μL of culture medium containing the corresponding concentration of lyophilized sample was added to the sample group. 48 h after loading, the supernatant was discarded, and 500 μL of culture medium containing 0.5 mg / mL MTT was added. The plates were incubated at 37℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 300 μL of DMSO was added to each well. OD values ​​were read at 490 nm.

[0130] The formula for calculating cell viability is as follows:

[0131]

[0132] The results are shown in Table 5.

[0133] Table 5 Cell viability results

[0134]

[0135]

[0136] Compared with the control group, the significance is indicated by *, p<0.05 is indicated by *, p<0.01 is indicated by **, and p<0.001 is indicated by ***.

[0137] The results showed that in the keratinocyte experimental group, type VII collagen VII-8-16, VII-8-4, and VII-11-16 could enhance keratinocyte activity, thus proving that type VII collagen VII-8-16, VII-8-4, and VII-11-16 have repair effects.

[0138] In the human fibroblast experimental group, compared with the control group, type VII collagen VII-8-16 and VII-8-4 significantly enhanced fibroblast viability.

[0139] 3. Cell migration detection

[0140] Logarithmic growth phase cells of immortalized human keratinocytes were collected, at a cell density of 1.5 × 10⁻⁶. 5 Cells were seeded per well into 24-well culture plates. After culturing for 24 h in an incubator (37℃, 5% CO2), horizontal scratches were made perpendicular to the 24-well plate using a 200 μL pipette tip. Cells were washed once with PBS to remove the scratched cells. The plates were then incubated for another 24 h in an incubator (37℃, 5% CO2). Each group was divided into three replicates. The control group received 200 μL of culture medium containing 10% PBS, while the sample groups received 200 μL of culture medium containing the corresponding concentration of lyophilized sample. The migrating cells in each group were photographed using an inverted microscope, and the average scratch area was calculated using Image Pro Plus software.

[0141] Collect human fibroblasts in logarithmic growth phase, at a cell density of 2 × 10⁻⁶ cells. 5 Cells were seeded per well into 24-well culture plates. After culturing for 24 hours in an incubator (37℃, 5% CO2), horizontal scratches were made perpendicular to the 24-well plate using a 200 μL pipette tip. Cells were washed once with PBS to remove the scratched cells. The plates were then incubated for another 24 hours in an incubator (37℃, 5% CO2). Each group was divided into three replicates. The control group received 200 μL of culture medium containing 10% PBS, while the sample groups received 200 μL of culture medium containing the corresponding concentration of lyophilized sample. The migrating cells in each group were photographed using an inverted microscope, and the average scratch area was calculated using Image Pro Plus software. The healing rate was calculated as follows:

[0142]

[0143] The specific treatment concentrations and results are shown in Table 6.

[0144] Table 6. Results of cell migration detection experiments

[0145]

[0146]

[0147] Compared with the control group C, the significance of the sample group is indicated by *, p<0.05 is indicated by *, p<0.01 is indicated by **, and p<0.001 is indicated by ***.

[0148] The results showed that, compared with the control group, type VII collagen VII-8-16 significantly improved the cell healing rate of keratinocytes in the keratinocyte experimental group; VII-8-4 and VII-11-16 also significantly improved the cell healing rate of keratinocytes. Therefore, type VII collagen VII-8-16, VII-8-4, and VII-11-16 can promote cell healing of keratinocytes and have a repair effect.

[0149] In the human fibroblast experimental group, compared with the control group, type VII collagen VII-8-16 and VII-8-4 significantly promoted fibroblast migration; type VII collagen VII-11-16 at a concentration of 0.5% or higher significantly promoted fibroblast migration.

[0150] 4. Type VII collagen promotes the expression of type I and type IV collagen.

[0151] According to the appropriate inoculation density (8×10 4 Human fibroblasts were seeded into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2). The experiment included a blank control group, a negative control group, a positive control group, and a sample group. Three concentration gradients were set up for the sample group. Irradiation was performed when the cell deposition rate in the 24-well plates reached 40%-60%. The negative control group and the sample group received a total dose of 9 J / cm². 2 UVA radiation was applied. Meanwhile, the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm²). 2 After irradiation, 1 mL of cell culture medium was added to each well of the blank control group and negative control group, and 1 mL of culture medium containing the corresponding concentration of lyophilized sample was added to each well of the sample group. Cell collection: After incubation for 24 h, 0.5 mL of lysis buffer was added to each well, and the cells were incubated at room temperature for 5 min to allow for complete lysis. The cells were then transferred to 1.5 mL RNase-free Eppendorf tubes, RNA was extracted, and then reverse transcribed to synthesize cDNA. qRT-PCR was performed to detect the Collagen I and Collagen IV genes, and the results are shown in Table 7.

[0152] Table 7. Relative expression levels of Collagen I and Collagen IV genes

[0153]

[0154]

[0155] Note: Statistical analysis was performed using the two-tailed text test method. For comparisons between the NC and BC groups, significance is indicated by # (p-value < 0.05, p-value < 0.01, p-value < 0.0 ...

[0156] The results showed that, compared with the NC group, type VII collagen VII-8-16 significantly promoted the gene expression of type I collagen in fibroblasts. Type VII collagen VII-8-4 significantly promoted the gene expression of type I collagen in fibroblasts.

[0157] Compared to the NC group, type VII VII-8-16 and VII-8-4 collagen significantly increased the expression level of the Collagen IV gene in human fibroblasts. Type VII VII-11-16 collagen also significantly increased the expression level of the Collagen IV gene in human fibroblasts.

[0158] 5. Permeability assay of recombinant type VII humanized collagen

[0159] (1) Collagen fluorescent labeling method:

[0160] ① Prepare a 10 mg / mL dye stock solution by dissolving TAMRA (5(6)-carboxytetramethylrhodamine succinimide ester) dye in DMSO;

[0161] ② Prepare a 0.5% collagen stock solution by dissolving collagen (VII-8-16, VII-8-4, and VII-11-16) in pH 6.8 PBS buffer solution;

[0162] ③ Mix the collagen stock solution and the dye stock solution at a certain mass ratio so that the dye / collagen ratio is 5 / 1 (molar ratio), and shake overnight;

[0163] ④ Add water equal to collagen mother liquor to the solution after reaction, and centrifuge for 10 minutes to remove excess dye;

[0164] ⑤ Dialyze the supernatant in water overnight (using a 1kDa dialysis bag), changing the water once during the process. This will give you the labeled collagen stock solution. Dilute it to a concentration of 0.1% before use.

[0165] (2) Transdermal test method for piglet skin:

[0166] The in vitro transdermal permeation of the active ingredient was determined using the Franz diffusion method.

[0167] ① Experimental conditions: Temperature controlled at (37±2)℃; rotation speed 300 rpm; diffusion cell area 2.2 cm². 2 The receiving cell volume is 7 mL.

[0168] ② Experimental steps: Thaw the frozen pig skin naturally, cut it to the size suitable for the receiving pool, soak it in physiological saline for 30 minutes beforehand, place the skin flat between the diffusion pool and the receiving pool, keeping the outer side of the stratum corneum facing the diffusion pool and the inner side facing the receiving pool, and use metal clamps to stabilize the device. Use phosphate buffer solution with pH=6.8 as the solvent for the receiving and supply solutions. The supply solution includes the collagen stock solution labeled in (1) (collagen concentration of 0.5%). Ensure that air bubbles are expelled during the addition process, and add a magnetic stir bar. Seal the supply pool with plastic wrap. Take 0.5 mL samples at 2, 4, 6, 8 and 24 hours after the start of the experiment, and add an equal volume of isothermal phosphate buffer solution with pH=6.8.

[0169] ③ Test method: Quantitative analysis using fluorescence intensity method, with excitation light at 543 nm and absorption light at 572 nm.

[0170] ④ Data processing method: The formula for calculating the unit cumulative permeability (Qn) is as follows:

[0171]

[0172]

[0173] Where A: area of ​​the diffusion cell; V: volume of the receiving cell; Vi: volume of the i-th sample; Ci: sample concentration in the i-th sample. Qn is the cumulative permeation amount of active ingredient per unit area, Q is the total amount of active ingredient per unit area in the supply cell, and η is the cumulative permeation rate of collagen.

[0174] The transdermal results for VII-8-16, VII-8-4, and VII-11-16 are shown in Table 8.

[0175] Table 8

[0176]

[0177] The results showed that type VII collagen (VII-8-16 and VII-8-4VII) possesses excellent transdermal absorption capabilities, enabling it to penetrate deeper into the skin more effectively, thereby enhancing its bioavailability and the actual efficacy of the product. This superior transdermal performance successfully overcomes many difficulties in transdermal absorption of traditional collagens, making type VII collagen an ideal choice for personal care and pharmaceutical applications.

[0178] 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 VII collagen, characterized in that: The recombinant type VII collagen comprises at least one or more of the following A1)-A2): A1) the amino acid sequence of the recombinant type VII collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.2, and the number of repeats is n, 1<n≤20; A2) The amino acid sequence of the recombinant type VII collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.3, and the number of repeats is n, 10<n≤20.

2. The recombinant type VII collagen according to claim 1, characterized in that The recombinant type VII collagen comprises an amino acid sequence as shown in SEQ ID NO.4 or an amino acid sequence that is at least 98% identical to SEQ ID NO.4, and / or an amino acid sequence as shown in SEQ ID NO.5 or an amino acid sequence that is at least 98% identical to SEQ ID NO.5, and / or an amino acid sequence as shown in SEQ ID NO.6 or an amino acid sequence that is at least 98% identical to SEQ ID NO.

6.

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

4. The biomaterial according to claim 3, characterized in that The nucleic acid molecule includes: 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, and / or a nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having at least 80% identity with SEQ ID NO.

9.

5. The biomaterial according to claim 3 or 4, characterized in that: The recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

6. A method for preparing recombinant type VII collagen, characterized in that: The method comprises: constructing a recombinant microorganism expressing the recombinant type VII collagen according to claim 1 or 2, and culturing the recombinant microorganism.

7. A composition, characterized in that The composition contains the recombinant type VII collagen according to claim 1 or 2.

8. Use of the recombinant type VII collagen as claimed in claim 1 or 2, or the biomaterial as claimed in any one of claims 3 to 5, or the composition as claimed in claim 7 in the preparation of products for improving cell adhesion and / or cell viability and / or cell mobility and / or promoting collagen expression.

9. Use of the recombinant type VII collagen as claimed in claim 1 or 2, or the biomaterial as claimed in any one of claims 3 to 5, or the composition as claimed in claim 7 in products for repairing skin damage and / or anti-wrinkle and / or firming and / or improving transdermal absorption performance.

10. Use of the recombinant type VII collagen as claimed in claim 1 or 2, or the biomaterial as claimed in any one of claims 3 to 5, or the composition as claimed in claim 7 in daily chemicals and / or medical devices and / or engineered tissue materials.

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