A recombinant collagen type Ⅶ and a preparation method and application thereof

Abstract

The application provides a kind of recombinant collagen type VII and its preparation method and application, it is related to the field of bioengineering technology.The application designs and selects human source collagen type VII, successfully obtains the new functional repeat unit of collagen type VII with biological activity, provides a new direction for collagen type VII research.In the application, three new high-yield recombinant collagen type VII humanization is successfully obtained by repeating the functional repeat unit.The recombinant collagen type VII humanization has the effect of improving cell viability, which proves that the protein has high safety.The protein can also promote cell adhesion, so it has excellent efficacy in treating skin damage.The protein can also improve cell migration rate and has excellent transdermal absorption performance.In addition, the protein also has the effect of promoting collagen expression, which provides a new functional ingredient for anti-aging related products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a recombinant type VII collagen protein as well as a preparation method and application thereof. BACKGROUND

[0002] Type VII collagen is an anchoring fibril major component, which acts on the junction of epidermis and dermis. In addition to the skin, a small amount of type VII collagen also exists in the epithelial tissues of the retina and digestive tract and other organs. Clinical studies have confirmed that type VII collagen can be used for subcutaneous injection to treat dystrophic epibullosis. In addition, type VII collagen is also involved in the establishment of skin homeostasis, including wound healing, ulcer treatment, prevention of wrinkles and skin aging problems such as relaxation.

[0003] Type VII collagen in the human body is mainly formed by keratinocytes and fibroblasts, accounting for 0.001% of the total collagen in the human body. Human type VII collagen is formed by 3 identical alpha chains in a helical structure, and each chain contains 2,944 amino acids. At present, there are few reports on the in vitro recombinant expression of type VII collagen at home and abroad, and most of the researches are focused on obtaining a small amount of type VII collagen through tissue organ extraction or lentivirus transfection for mechanism research. However, these two methods have limitations such as great difficulty in preparation, low purity and complex control, and are difficult to apply to commercial production. Since the molecular weight of human type VII collagen is large, it is difficult to express in vitro, and therefore, in the research process, the triple helix structure sequence in type VII collagen is often intercepted for recombinant expression to achieve large-scale preparation of type VII collagen. SUMMARY

[0004] The present application successfully obtains high-yield recombinant type VII humanized collagen protein by designing and screening human type VII collagen, and the protein production process is simple, easy to expand production, and can be widely applied in the fields of cosmetics and medical devices.

[0005] In one aspect, the present application provides a recombinant type VII collagen, which at least comprises any one or more of the following A1)-A2):

[0006] A1) the amino acid sequence of the recombinant type VII collagen comprises one or more amino acid residue repeat units, the repeat unit comprises a sequence as shown in SEQ ID NO. 2, and the repeat number n is 1≤n≤20;

[0007] A2) the amino acid sequence of the recombinant type VII collagen comprises one or more amino acid residue repeat units, the repeat unit comprises a sequence as shown in SEQ ID NO. 3, and the repeat number n is 10≤n≤20.

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

[0009] Preferably,

[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, 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 any of the above values.

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

[0013] A2) the amino acid sequence of the recombinant collagen type VII comprises one or more amino acid residue repeat units, the repeat unit comprises the sequence as shown in SEQ ID NO. 3, the number of repeats is n, n is a natural number, optionally, the number of repeats n can be in the range of 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, 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 repeats is 16.

[0016] In a preferred embodiment, the recombinant collagen type VII is obtained by directly connecting the amino acid residue repeat units. Those skilled in the art can understand that known linker peptides can be added between the amino acid residue repeat units for connection, and the recombinant collagen type VII can also be subjected to known modifications, including phosphorylation, acetylation, ubiquitination, glycosylation, etc., without affecting the function of the protein itself.

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

[0018] Preferably,

[0019] The recombinant collagen type VII has an amino acid sequence as set forth 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%, 99.9% identity to SEQ ID NO. 4. More preferably, the nucleic acid molecule encoding the recombinant collagen type VII comprises a 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.

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

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

[0022] It can be understood that those skilled in the art can select suitable gene editing systems and gene editing methods to complete the construction of the recombinant collagen type VII according to the actual situation, or a general method can be selected to prepare the recombinant collagen type VII.

[0023] It is understood by those skilled in the art that known modifications and optimizations can be made without affecting the function of the above-mentioned recombinant collagen type VII, which are not mandatory limited in the present application.

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

[0025] In another aspect, the present application also provides a biological material, which comprises any one of the following B1) to B5):

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

[0027] B2) an expression cassette containing the nucleic acid molecule of B1);

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

[0029] B4) a recombinant microorganism containing the nucleic acid molecule of B1), the expression cassette of B2), and / or the recombinant vector of B3);

[0030] B5) a recombinant cell containing the nucleic acid molecule of B1), the expression cassette of B2), and / or the recombinant vector of B3).

[0031] Further, the nucleic acid molecule comprises: 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%, 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 with 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. 8.

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

[0035] It can be conceived by those skilled in the art that other commonly used expression elements can be adaptively added to the expression cassette described in the present application to assist the expression of the target gene, such as adding tags, fluorescent protein markers, resistance screening markers, etc.

[0036] Optionally, the recombinant vector can comprise a nucleic acid molecule encoding the above-mentioned protein, a promoter and transcription and translation termination signals. The promoter or terminator for regulating expression in the present application can be a general promoter or terminator, or a specific promoter or terminator in the host bacteria. Those skilled in the art can adaptively select according to the actual situation, and the sequence of the promoter or terminator is not compulsorily limited in the present application.

[0037] The recombinant vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and expression of nucleic acid sequences. The choice of vector is typically dependent on the compatibility of the vector with the host cell into which it is to be introduced. The vector can be linear or closed circular. The vector can be an autonomously replicating vector (i.e., exists as an episome), e.g., a plasmid, a non-integrating viral vector, a mini-chromosome, or an artificial chromosome. The vector can comprise any mechanism for replication under the control of the host cell and can be autonomously replicating, integrated, or exist as an extra-chromosomal element. Alternatively, the vector can be one which, when introduced into a host cell, is integrated into the cell genome and replicated together with the chromosome sequences. Moreover, the vector can be a single vector or plasmid or two or more vectors or plasmids, which together comprise the total DNA to be introduced into the genome of the host cell, or a transposon. Preferably, the recombinant vector can be pPIC9k plasmid and / or pPICZ alpha A plasmid.

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

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

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

[0041] It is understood by those skilled in the art that a conventional fermentation strain or any known industrial strain can be used as a starting strain as long as the expression of recombinant collagen type VII can be achieved, and the specific strain is not limited herein.

[0042] In an alternative embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or introduced into the recombinant microorganism by using a recombinant vector. It is understood by those skilled in the art that the nucleic acid molecule can be inserted into the genome of the starting strain or present on a plasmid in a free state as long as the expression of the nucleic acid molecule or the synthesis of recombinant collagen type VII can be achieved.

[0043] In an alternative embodiment, 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 the actual situation, and the resistance selection marker gene is not compulsorily limited herein.

[0044] The resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers prototrophy to auxotrophs, etc. Examples of bacterial selection markers are the dal gene of Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline.

[0045] In another aspect, the present application also provides a method for preparing a recombinant collagen type VII, the method comprising: constructing a recombinant microorganism expressing the recombinant collagen type VII, and culturing the recombinant microorganism.

[0046] Further, 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 method for preparing a recombinant collagen type VII comprises the following steps:

[0050] Step one, constructing a recombinant microorganism expressing the recombinant collagen type VII;

[0051] Step two, inoculating the recombinant microorganism cells into a fermentation medium, and culturing under the conditions of 25-37°C, 100-300 rpm, and for 24-72 h, and adding 1-5% methanol every 24 h.

[0052] In a preferred embodiment, the method for preparing a recombinant collagen type VII comprises the following steps:

[0053] Step one, constructing a recombinant microorganism expressing the recombinant collagen type VII;

[0054] Step two, inoculating the recombinant microorganism cells into a fermentation medium, and culturing under the conditions of 25-37°C, pH 5-7, air flow rate of 1-5 L / min, stirring power set at 300-900 rpm, and linkage dissolved oxygen to maintain 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 h; the second stage, gradient feeding of 10-50% glycerol for about 10-20 h to make OD 600 accumulate to 150-180; and the third stage, gradient feeding of methanol containing 1.2% PTM1, with the methanol feeding rate increasing from 2 mL / h to 12 mL / h.

[0056] The fermentation medium can be BMMY medium.

[0057] Preferably, the step two further comprises a purification step. The above purification can be performed by a general method.

[0058] In another aspect, the present application also provides a composition containing the recombinant collagen type VII.

[0059] The skilled in the art can understand that the composition can comprise adjuvants. As physiologically acceptable media, their constitution, usage amount, etc. can be selected differently by the skilled in the art according to their mastered knowledge, as required by the type of application scenario.

[0060] In another aspect, the present application also provides the use of the recombinant collagen type VII or the biomaterial or the composition in the preparation of a product for improving cell adhesion and / or improving cell viability and / or improving cell migration rate and / or promoting collagen expression.

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

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

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

[0064] The concentration of the recombinant collagen type VII can be 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% and above any value.

[0065] The skilled in the art can adjust the concentration of the protein according to the actual situation, which is not specifically limited in the present application.

[0066] In another aspect, the present application also provides the use of the recombinant collagen type VII or the biomaterial or the composition in the preparation of a product for repairing skin damage and / or anti-wrinkle and / or tightening and / or improving transdermal absorption performance.

[0067] wherein,

[0068] The improved transdermal absorption performance is achieved by improving the cumulative permeation rate of collagen.

[0069] The repairing of skin damage is achieved by improving the cell migration rate or cell adhesion or cell growth.

[0070] The anti-wrinkle and tightening is achieved by promoting collagen expression, improving cell migration rate or cell adhesion.

[0071] In another aspect, the application also provides the use of the recombinant collagen type VII or the biomaterial or the composition in daily chemicals and / or medical devices and / or engineered tissue materials.

[0072] The application has the following beneficial effects:

[0073] The application designs and screens human-derived collagen type VII, successfully obtains a new functional repeat unit of bioactive human-derived collagen type VII, and provides a new direction for the research of collagen type VII.

[0074] The recombinant human-derived collagen type VII has the effect of improving cell viability, proves that the protein has high safety, and can be widely applied to various scenes. The protein can also promote cell adhesion, has excellent efficacy for treating skin damage. The protein can also improve cell migration rate, can effectively improve the transdermal absorption performance of collagen products. In addition, the protein also has the effect of promoting collagen expression, and provides a new efficacy component for anti-aging related products.

[0075] The application also provides a preparation method of the recombinant human-derived collagen type VII. BRIEF DESCRIPTION OF DRAWINGS

[0076] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and serve to explain the principles of the application, and do not limit the application in any way. In the drawings:

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

[0078] Figure 2 is a pPICZ alpha A-VII-8-16 plasmid map;

[0079] Figure 3 is a schematic diagram of a collagen type VII freeze-dried sample. DETAILED DESCRIPTION

[0080] Identity: refers to the similarity degree between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution research.

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

[0082] Expression cassette: Expression cassette refers to a set of DNA sequences composed of a promoter, a target gene and a reporter gene, which can be expressed in specific tissues and easily detected.

[0083] Recombinant vector: Recombinant vector is a vector in which the target gene is introduced into the basic skeleton of the cloning vector, so that the target gene can be expressed.

[0084] Recombinant microorganism: A strain of bacterial cells in which a foreign gene is highly expressed by genetic engineering methods.

[0085] 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 the parent cell that results from the replication of the parent cell that has occurred since the parent cell was originally derived, and which progeny has not undergone mutation or other alternation.

[0086] Free expression: Free expression is the expression of the target gene using a free plasmid. The free plasmid is an independent DNA molecule existing in the cell, which has the ability of independent replication and transmission, and is 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, in the following embodiments, the reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. The plasmids, endonucleases, PCR enzymes, column DNA extraction kits and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are carried out according to the kit instructions.

[0089] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present application all use conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields of conventional technology in the art. Specifically, they can be carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0090] In the present specification, the amino acids of the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, in which 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] In the present application, all the plasmids used are designed by primers, constructed by PCR and recombination of vector fragments using conventional molecular biology methods, and all the recombinant plasmids are verified by sequencing to be completely consistent with the target sequences.

[0092] MD medium: glucose 20 g / L, non-amino yeast basic nitrogen source 13.4 g / L, 4x10-5% biotin, the rest is water;

[0093] YPD medium: glucose 20 g / L, peptone 20 g / L, yeast extract 10 g / L, the rest is water, and 15 g / L agar powder needs to be added to the solid medium;

[0094] BMMY medium: peptone 20 g / L, non-amino yeast basic nitrogen source 13.4 g / L, yeast extract 10 g / L, 4x10-5% biotin, 0.5% methanol, potassium phosphate buffer with a final concentration of 100 mM, the rest is water, and the pH is adjusted to 6.0;

[0095] In addition, the "water" in the present application includes deionized water, distilled water, ion exchange water, double distilled water, high-purity water, pure water and any feasible water that can be used in the art.

[0096] In the following examples, % means wt%, i.e. weight percentage, if no other special instructions are given.

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

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

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

[0100] The optimized DNA sequences were synthesized by Kingsriver Company and constructed into the alpha factor secretion signal peptide of pPIC9k plasmid to obtain plasmids pPIC9k-VII-8-4, pPIC9k-VII-8-16, and pPIC9k-VII-11-16, as shown in Figure 1 Figure 1 The middle yellow block "VII-X" can be VII-8-4, VII-8-16, or VII-11-16.

[0101] Table 1 Recombinant human collagen type VII amino acid sequence

[0102]

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

[0104] The plasmids pPIC9k-VII-8-4, pPIC9k-VII-8-16, and pPIC9k-VII-11-16 were linearized with restriction enzyme Sac I, and then transformed into the competent cells of wild-type Pichia pastoris GS115 (purchased from Beijing Huaiyueyang Biological Technology Co., Ltd.). The cells were plated on MD (plate) medium and cultured at 30°C for 48-72 h. Colony PCR was performed on the single colonies on the plate, and the single colonies with correct identification results were streaked on YPD (plate) medium containing 4 mg / mL geneticin (G418) and cultured at 30°C for 48-72 h. The single colonies with fast growth were selected as high-copy strains of recombinant human collagen type VII, and were named Col-VII-8-4, Col-VII-8-16, and Col-VII-11-16.

[0105] The synthesized VII-8-16 DNA sequence was constructed into the alpha factor secretion signal peptide of pPICZαA plasmid by Kingsriver Company to obtain plasmid pPICZαA-VII-8-16, as shown in​Figure 2 The plasmid pPICZαA-VII-8-16 was linearized with restriction enzyme Sac I and transformed into Col-VII-8-16 competent cells by electroporation. The single colonies on the plates were identified by colony PCR, and the single colonies with correct identification results were inoculated on YPD (plate) medium containing Zeocin resistance (4 mg / mL) and cultured at 30°C for 48-72 h. The single colonies with fast growth were selected as the high-copy recombinant humanized collagen type VII strain C-VII-8-16.

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

[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] The single colonies of Col-VII-8-4, Col-VII-8-16, Col-VII-11-16 and C-VII-8-16 were inoculated into test tubes containing 10 mL of fresh YPD medium and cultured for 36 h. The bacterial cells were collected by centrifugation and transferred into 250 mL baffle shake flasks containing 25 mL of BMMY medium. The culture conditions were 30°C and 220 rpm, and the 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 subjected to SDS protein electrophoresis and quantitative experiments. The fermentation yields are shown in Table 2.

[0109] Table 2 Shake flask fermentation yield of recombinant humanized collagen type VII

[0110] Strains Shake flask yields (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) 5L fermenter fermentation of recombinant strains C-VII-8-16, Col-VII-8-4 and Col-VII-11-16

[0112] C-VII-8-16, Col-VII-8-4 and Col-VII-11-16 in the glycerol tube were inoculated into MD (plate) medium and cultured at 30°C for 48-72 h. The single colonies on the plates were inoculated into 200 mL of YPD (shaker) medium and cultured at 30°C for 36 h. Then, all the cultures were transferred into a 5L fermenter containing 1.8L of BMMY medium. The initial OD600 was 0.1, and the culture conditions were 30°C and 220 rpm. Methanol was added every 24 h to a final concentration of 1%. After fermentation, the supernatant was collected by centrifugation and subjected to SDS protein electrophoresis and quantitative experiments. The fermentation yields are shown in Table 3. 600The temperature was set at about 30°C, and the pH was maintained at about 6.0 by adding ammonia water through a peristaltic pump. The air flow rate was 2 L / min, and the stirring power was set at 300-900 rpm, and the dissolved oxygen was linked to maintain the dissolved oxygen concentration at about 30%.

[0113] The fermentation process was divided into three stages. In the first stage, the yeast grew in the fermenter for about 16 h, and the glycerol in the tank was depleted; in the second stage, 50% glycerol was continuously fed for about 10 h to make the OD 600 accumulate to about 180; in the third stage, methanol containing 1.2% PTM1 was fed at a gradient, and the methanol feeding rate was increased from 2 mL / h to 12 mL / h. Samples were taken at 24 h, 48 h, and 72 h, respectively, during the methanol feeding stage. After centrifugation, the fermentation supernatant was subjected to SDS protein electrophoresis for quantitative experiments, and the results are shown in Table 3.

[0114] Table 3 Protein yield of 5L fermenter of C-VII-8-16, Col-VII-8-4 strains

[0115]

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

[0117] The 5L fermenter broth of the recombinant strains C-VII-8-16, Col-VII-8-4, and Col-VII-11-16 obtained in Example 2 was collected, and the supernatant was collected by centrifugation at 5000 rpm for 20 min. The supernatant was subjected to hollow fiber microfiltration (purchased from Cytiva Company, item number 56-4102-68), and the permeate pressure was controlled to be below 0.6 Mpa. The filtrate obtained from the permeate end was concentrated by ultrafiltration using a membrane bag (purchased from Cytiva Company, item number UFELA0005010P) and then subjected to liquid exchange treatment. The target protein was combined with the cationic filler by loading SPFF cation (purchased from Cytiva Company, item number 17051010) as the exchange filler. After loading was completed, the target peak was collected by elution with a buffer containing NaCl. The eluate obtained was concentrated by ultrafiltration using a membrane bag and then subjected to liquid exchange treatment, followed by freeze-drying. The freeze-dried sample was white fibrous sponge-like, as shown in Figure 1. Figure 3

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

[0119] In this example, the freeze-dried samples of VII-8-16, VII-8-4, and VII-11-16 obtained in Example 3 were used for the following efficacy determination.

[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. 5Inoculate into 24-well culture plates. Incubate in an incubator (37°C, 5% CO2) for 24 h. Add 200 μL of culture solution containing 10% PBS to the control group, and add 200 μL of culture solution containing the corresponding concentration of freeze-dried sample to the sample group (the specific concentration is shown in Table 5). After 72 h, discard the supernatant, add 500 μL of culture medium containing 0.5 mg / mL MTT, and incubate at 37°C in the dark for 4 h. After incubation, discard the supernatant, add 300 μL of DMSO to each well, and read the OD value at 490 nm.

[0129] Collect human fibroblasts in the logarithmic growth phase, and inoculate at a cell density of 8 x 10 4 Inoculate into 24-well culture plates. After incubation in an incubator (37°C, 5% CO2) for 24 h, perform loading. Add 200 μL of culture solution containing 10% PBS to the control group, and add 200 μL of culture solution containing the corresponding concentration of freeze-dried sample to the sample group. After 48 h, discard the supernatant, add 500 μL of culture medium containing 0.5 mg / mL MTT, and incubate at 37°C in the dark for 4 h. After incubation, discard the supernatant, add 300 μL of DMSO to each well, and read the OD value at 490 nm.

[0130] The cell viability calculation formula is as follows:

[0131]

[0132] The results are shown in Table 5.

[0133] Table 5 Cell activity results

[0134]

[0135]

[0136] Significance is indicated by * for the sample group compared with the control group, p < 0.05, ** for p < 0.01, and *** for p < 0.001.

[0137] The results show that for the keratinocyte experimental group, collagen type VII Ⅶ-8-16, Ⅶ-8-4, and Ⅶ-11-16 can improve keratinocyte activity, which proves that collagen type VII Ⅶ-8-16, Ⅶ-8-4, and Ⅶ-11-16 have repair efficacy.

[0138] For the human fibroblast experimental group, collagen type VII Ⅶ-8-16 and Ⅶ-8-4 can significantly improve fibroblast activity compared with the control group.

[0139] 3. Cell migration detection

[0140] The logarithmic growth phase cells of human immortalized keratinocytes were collected and inoculated into 24-well culture plates at a cell density of 1.5 x 10 5 After 24 h of culture in an incubator (37 °C, 5% CO2), a horizontal line scratch was made on the 24-well plate with a 200 μL gun head, and the cells were washed once with PBS to remove the scratched cells. The cells were cultured in an incubator (37 °C, 5% CO2) for 24 h, and three parallels were set for each group. In the control group, 200 μL of culture solution containing 10% PBS was added, and in the sample group, 200 μL of culture solution containing the corresponding concentration of freeze-dried sample was added. The migrated cells in each group were photographed using an inverted microscope, and the average value of the scratch area was calculated using Image Pro Plus software.

[0141] The logarithmic growth phase cells of human fibroblasts were collected and inoculated into 24-well culture plates at a cell density of 2 x 10 5 After 24 h of culture in an incubator (37 °C, 5% CO2), a horizontal line scratch was made on the 24-well plate with a 200 μL gun head, and the cells were washed once with PBS to remove the scratched cells. The cells were cultured in an incubator (37 °C, 5% CO2) for 24 h, and three parallels were set for each group. In the control group, 200 μL of culture solution containing 10% PBS was added, and in the sample group, 200 μL of culture solution containing the corresponding concentration of freeze-dried sample was added. The migrated cells in each group were photographed using an inverted microscope, and the average value of the scratch area was calculated using Image Pro Plus software.

[0142]

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

[0144] Table 6 Results of cell migration detection experiment

[0145]

[0146]

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

[0148] The results show that for the keratinocyte experimental group, compared with the control group, collagen type VII-8-16 significantly improves the cell healing rate of keratinocytes; collagen type VII-8-4 significantly improves the cell healing rate of keratinocytes, and collagen type VII-11-16 significantly improves the cell healing rate of keratinocytes. Therefore, collagen type VII-8-16, VII-8-4 and VII-11-16 can promote the cell healing of keratinocytes and have repair efficacy.

[0149] For the human fibroblast experimental group, relative to the control group, collagen type VII-8-16 and VII-8-4 can significantly promote fibroblast migration; collagen type VII-11-16 can significantly promote fibroblast migration at a concentration of 0.5% and above.

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

[0151] Human fibroblasts were inoculated into a 24-well plate at an appropriate inoculation density (8 x 10 4 / well) and incubated in an incubator (37°C, 5% CO2) overnight. The experiment set up a blank control group, a negative control group, a positive control group, and a sample group. The sample group was set up at three concentration gradients. When the cell plating rate in the 24-well plate reached 40%-60%, irradiation was performed, and the negative control group and the sample group received a total dose of 9 J / cm 2 of UVA radiation. At the same time, the blank control group was placed in the same environment (UVA radiation dose of 0 J / cm 2 ). After irradiation, 1 mL of cell culture solution was added to each well of the blank control group and the negative control group, and 1 mL of culture medium containing the corresponding concentration of freeze-dried sample was added to each well of the sample group. Collect cells: after incubation for 24 h, 0.5 mL of lysis solution was added to each well, and it was placed at room temperature for 5 min to allow it to be fully lysed, transferred to a 1.5 mL RNase-free Eppendorf tube, and RNA was extracted, then reverse transcribed to synthesize cDNA. qRT-PCR was performed on the Collagen I (collagen type I) and Collagen IV (collagen type IV) genes, and the detection results are shown in Table 7.

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

[0153]

[0154]

[0155] Note: Statistical analysis was performed using the text two-tailed test method. The significance of the NC group compared to the BC group is indicated by #, p-value < 0.05 is indicated by #, p-value < 0.01 is indicated by ##, and p-value < 0.001 is indicated by ###. The significance of the sample group compared to the NC group is indicated by *, p < 0.05 is indicated by *, p < 0.01 is indicated by **, and p < 0.001 is indicated by ***.

[0156] The results show that: compared with the NC group, collagen type VII-8-16 can significantly promote the gene expression of fibroblasts to collagen type I. Collagen type VII-8-4 can significantly promote the gene expression of fibroblasts to collagen type I;

[0157] Compared with the NC group, collagen type VII-8-16 and VII-8-4 can significantly improve the gene expression of human fibroblasts to collagen type IV. Collagen type VII-11-16 can significantly improve the gene expression of human fibroblasts to collagen type IV.

[0158] 5. Transdermal permeability test of recombinant humanized collagen type VII

[0159] (1) Collagen fluorescent labeling method:

[0160] ① Prepare a 10 mg / mL dye stock solution by dissolving TAMRA (5(6)-carboxytetramethylrhodamine succinimidyl 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 with the dye stock solution at a certain mass ratio, so that the dye / collagen = 5 / 1 (molar ratio), and shake overnight;

[0163] ④ Add an equal amount of water to the reaction solution, centrifuge for 10 min to remove excess dye;

[0164] ⑤ Dialyze the supernatant in water overnight (1 k Da dialysis bag), and change the water halfway, to obtain the labeled collagen stock solution, and dilute the concentration to 0.1% for use.

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

[0166] Franz diffusion method was used to determine the in vitro transdermal penetration of active substances.

[0167] ① Experimental conditions: temperature control (37±2)℃; rotation speed 300 rpm; diffusion cell area 2.2 cm 2 ; receiving pool volume 7 mL.

[0168] (2) The experiment steps: the frozen pigskin was naturally thawed, cut into the size of the receiving pool, pre-soaked in physiological saline for 30 minutes, and placed between the diffusion pool and the receiving pool. The stratum corneum was kept outside the diffusion pool and inside the receiving pool, and the metal clamp was used to stabilize the device. The phosphate buffer solution with pH 6.8 was used as the receiving solution and the solvent of the supply solution, which included (1) the labeled collagen mother liquor (collagen concentration 0.5%) and a magnetic stirrer. The supply pool was sealed with plastic wrap. After 2, 4, 6, 8, and 24 hours, 0.5 mL of sample was taken, and an equal volume of isothermal phosphate buffer solution with pH 6.8 was added.

[0169] (3) Test method: fluorescence intensity method for quantitative determination, excitation light 543 nm, and absorption light 572 nm.

[0170] (4) Data processing method: the unit cumulative penetration quantity (Qn) calculation formula is as follows:

[0171]

[0172]

[0173] Wherein, A: diffusion pool area; V: receiving pool volume; Vi: volume of the ith sample; Ci: sample concentration in the ith sample. Qn is the cumulative penetration quantity of active material per unit area, Q is the total amount of active material per unit area in the supply pool, and η is the cumulative penetration rate of collagen.

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

[0175] Table 8

[0176]

[0177] The results show that the VII collagen of type VII-8-16 and VII-8-4 has excellent transdermal ability, can more effectively penetrate into the deep layer of the skin, and thus improve the bioavailability and the actual efficacy of the product. This excellent transdermal performance successfully overcomes many difficulties of traditional collagen in transdermal absorption, making the VII collagen an ideal choice in personal care and medical device applications.

[0178] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. A recombinant collagen type VII characterized in that, The recombinant collagen type VII has any one or more of the following A1) - A2): A1) the amino acid sequence of the recombinant collagen type VII is a plurality of amino acid residue repeat units, the repeat units are the sequence shown in SEQ ID NO. 2, and the number of repeats is n, n is selected from 4 or 16; A2) the amino acid sequence of the recombinant collagen type VII is a plurality of amino acid residue repeat units, the repeat units are the sequence shown in SEQ ID NO. 3, and the number of repeats is n, n is selected from 16.

2. The recombinant collagen of claim 1, wherein, The amino acid sequence of the recombinant collagen type VII is shown in SEQ ID NO. 4, or shown in SEQ ID NO. 5, or shown in SEQ ID NO.

6.

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

4. The biomaterial of claim 3, wherein, The nucleic acid molecule is shown in SEQ ID NO. 7, or shown in SEQ ID NO. 8, or shown in 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, Saccharomyces cerevisiae.

6. A method of producing a recombinant collagen type VII, characterized by, The method comprises constructing a recombinant microorganism expressing the recombinant collagen type VII of claim 1 or 2, and culturing the recombinant microorganism.

7. Composition, characterized in that, The composition contains the recombinant collagen type VII of claim 1 or 2.

8. Use of the recombinant collagen type VII according to claim 1 or 2 or the biomaterial according to any one of claims 3 to 5 or the composition according to claim 7 for the preparation of a product for increasing cell adhesion and / or increasing cell viability and / or increasing cell migration rate and / or promoting collagen expression, characterized in that, The cells are keratinocytes and / or fibroblasts.

9. Use of the recombinant collagen type VII of claim 1 or 2, or the biological material of any one of claims 3-5, or the composition of claim 7, in the preparation of a product for repairing skin damage and / or anti-wrinkle and / or tightening and / or improving transdermal absorption performance.

10. Use of the recombinant collagen type VII of claim 1 or 2, or the biological material of any one of claims 3-5, or the composition of claim 7, in the preparation of a daily chemical and / or medical device and / or engineered tissue material.

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