Recombinant collagen type iv and uses thereof

Abstract

The application provides a recombinant collagen IV and application thereof, and relates to the technical field of protein engineering.Amino acid residue repeating unit with good biological activity and stability in human collagen IV is found in the application, and the repeating unit is repeated and used as a core to construct a recombinant collagen IV, and then a new human recombinant collagen IV with biological activity is obtained.The human recombinant collagen IV provided in the application is a small molecular weight protein, is easy to be absorbed by a human body, has good safety, can significantly promote secretion of self collagen, has the effects of promoting cell adhesion and promoting cell migration, and has a good application prospect in basement membrane repair and remodeling.The application provides a new functional ingredient for products for repairing skin, tightening skin, resisting aging, resisting wrinkles, moisturizing and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein engineering, and particularly relates to recombinant type IV collagen and application thereof. BACKGROUND

[0002] Collagen is a kind of biological macromolecular fibrous protein, which is widely distributed in the extracellular matrix of human connective tissue and is also the most abundant and widely distributed functional protein in the human body, accounting for 25-30% of the total amount of protein. At present, 29 different types of collagen have been identified, which can be divided into seven categories according to their structures, i.e. fibrillar collagen, FACIT family collagen, reticular collagen, beaded collagen, transmembrane collagen, anchored collagen and multiple triple helix collagen. Among them, type IV collagen has a non-fibrillar reticular structure and is composed of six genetically different subtypes, i.e. α1(IV)-α6(IV), in which the α1 chain and the α2 chain construct the main subunit of type IV collagen, and the six subunits form three heterotrimer molecules α1α1α2(IV), α3α4α5(IV) and α5α5α6(IV). Type IV collagen forms a reticular structure between the true epidermis and is combined with nidogen, perlecan and laminin to form a basement membrane network. As a key element of the basement membrane, type IV collagen helps the epidermis layer to combine with the dermis layer, has a supporting capacity for cells, and at the same time provides a stable framework for soluble glycoproteins, proteoglycans and growth factors, can recognize and interact with specific receptors on the cell surface, and then activate the intracellular signal transduction pathway, participate in important physiological processes such as cell adhesion, migration, growth, proliferation and differentiation, and plays a decisive role in maintaining the stability of cells and the connection of tissues. The lack of type IV collagen will lead to abnormal structure and function of the basement membrane, and then affect the normal physiological activities of cells.

[0003] At present, collagen raw materials are mainly animal collagen extracted from terrestrial animals such as pigs and cows and marine organisms. Animal-derived collagen is a highly cross-linked biological material, which has limited solubility, large batch-to-batch difference, immunogenicity and pathogen transmission risk, and the molecules are easy to break during the processing, causing changes in physical and chemical properties, losing the original biological activity, and being limited in application. Type IV collagen has a low content in animals, and the existing technology cannot extract type IV collagen single component product from animals. Traditionally, type IV collagen is mainly separated from human placenta, and its extraction process is complex, which has limitations such as low extraction rate, high cost and low purity. With the continuous development of genetic technology, biosynthetic collagen has become the best substitute for animal-derived collagen in biomedical and tissue engineering, and recombinant humanized collagen has the advantages of scalability, consistency and safety, and has been widely used in the fields of medicine and cosmetics.

[0004] In recent years, technicians have begun to try to express recombinant humanized type IV collagen in E. coli and yeast. However, the structure of natural human type IV collagen is complex, and the full-length human type IV collagen has not yet been expressed. Although some truncated type IV collagen can be recombinantly expressed, the yield is low and the biological activity is low. Large-scale production has become an important issue. At present, there are no humanized recombinant type IV collagen products on the market in China that can be produced on a large scale. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for preparing and applying a recombinant type IV humanized collagen protein with high expression yield and good biological activity in Pichia pastoris.

[0006] On the one hand, this application provides recombinant type IV collagen, wherein the amino acid sequence of said recombinant type IV collagen includes any one or more of the following A1)-A2):

[0007] A1) The amino acid sequence of the recombinant type IV collagen contains one or more repeating units of amino acid residues, the repeating units containing sequences as shown in SEQ ID NO.1 and / or SEQ ID NO.5, with a repeat number of n, 1≤n≤30;

[0008] A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type IV collagen described in A1).

[0009] Furthermore, the amino acid sequence of the recombinant type IV collagen includes one or more repeating units of amino acid residues, the repeating unit including the sequence shown in SEQ ID NO.5, with a repeat number of n, 1≤n≤10.

[0010] Preferred,

[0011] The amino acid sequence of the recombinant type IV collagen includes any one or more of the following: A1)-A3):

[0012] A1) The amino acid sequence of the recombinant type IV collagen contains one or more repeating units of amino acid residues, the repeating unit containing the sequence shown in SEQ ID NO.1, the number of repeats being n, 1≤n≤30;

[0013] A2) The amino acid sequence of the recombinant type IV collagen contains one or more repeating units of amino acid residues, the repeating unit containing the sequence shown in SEQ ID NO.5, the number of repeats being n, 1≤n≤10;

[0014] A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type IV collagen described in A1) or A2).

[0015] Preferably, the recombinant type IV collagen is human recombinant type IV collagen.

[0016] Wherein, the amino acid sequence of the recombinant type IV collagen described in A1) comprises one or more repeating units of amino acid residues, the repeating units comprising, as shown in SEQ ID The sequence shown in NO.1 has a repetition count of n, where n is a natural number. The repetition count n can optionally take the following values: 1≤n≤30, 2≤n≤30, 3≤n≤30, 4≤n≤30, 5≤n≤30, 6≤n≤30, 7≤n≤30, 8≤n≤30, 9≤n≤30, 10≤n≤30, 11≤n≤30, 12≤n≤30, 13≤n≤30, 14≤n≤30, 15≤n≤30, 16≤n≤30, 17≤n≤30, 18≤n≤30, 19≤n≤30, 20≤n≤30, 21≤n≤30, 22≤n≤30, 23≤n≤30, 24≤n≤30, 25≤n≤30, 26≤n ≤30, 27≤n≤30, 28≤n≤30, 29≤n≤30, 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, 1 0≤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≤15, 6≤n≤157≤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≤13, 11≤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≤1 1. 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; preferably, 1≤n≤20; more preferably, 1≤n≤16;

[0017] For example, the value of n, or the upper limit of n, or the lower limit of n, can be selected from any value between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30;

[0018] A2) The amino acid sequence of the recombinant type IV collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.5, and the number of repeats is n, where n is a natural number. Optionally, the value range of the number of repeats n can be: 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; preferably, 1≤n≤5; more preferably, 1≤n≤4;

[0019] 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 and any value between them;

[0020] A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type IV collagen described in A1) or A2).

[0021] In a preferred embodiment, the recombinant type IV 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 IV collagen can also undergo known modifications, including phosphorylation, acetylation, ubiquitination, and glycosylation, without affecting the protein's function.

[0022] Furthermore, the amino acid sequence of the recombinant type IV collagen includes any one or more of B1-B2:

[0023] B1) contains the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7;

[0024] The sequence described in B2) is an amino acid sequence that has at least 95% identity with the amino acid sequence described in B1).

[0025] Preferably, the amino acid sequence of the recombinant type IV collagen includes any one or more of B1)-B7):

[0026] B1) Contains the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 95% identity with SEQ ID NO.1;

[0027] B2) Contains the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 95% identity with SEQ ID NO.2;

[0028] B3) Contains the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 95% identity with SEQ ID NO.3;

[0029] B4) Contains the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 95% identity with SEQ ID NO.4;

[0030] B5) contains the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 95% identity with SEQ ID NO.5;

[0031] B6) contains the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having at least 95% identity with SEQ ID NO. 6;

[0032] B7) contains the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence that is at least 95% identical to SEQ ID NO.7.

[0033] in,

[0034] B1) Contains the amino acid sequence shown in SEQ ID NO. 1 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 1; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 8 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 99.8%, or 99.9% identity with SEQ ID NO. 1; NO.8 has nucleotide sequences with 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%, and 99.9% identity.

[0035] B2) Contains the amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 2; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 9 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 99.8%, or 99.9% identity with SEQ ID NO. 2; NO.9 has nucleotide sequences with 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%, and 99.9% identity.

[0036] B3) Contains the amino acid sequence shown in SEQ ID NO. 3 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 3; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 10 or an amino acid sequence with 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 99.8%, or 99.9% identity with SEQ ID NO. 3; NO.10 has nucleotide sequences with 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%, and 99.9% identity.

[0037] B4) Contains the amino acid sequence shown in SEQ ID NO. 4 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 4; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 11 or an amino acid sequence with SEQ ID NO. 4. NO.11 has nucleotide sequences with 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%, and 99.9% identity.

[0038] B5) Contains the amino acid sequence shown in SEQ ID NO. 5 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 5; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 12 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 99.8%, or 99.9% identity with SEQ ID NO. 5; NO.12 has nucleotide sequences with 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%, and 99.9% identity.

[0039] B6) Contains the amino acid sequence shown in SEQ ID NO. 6 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 6; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 13 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 99.8%, or 99.9% identity with SEQ ID NO. 6; NO.13 has nucleotide sequences with 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%, and 99.9% identity.

[0040] B7) Contains the amino acid sequence shown in SEQ ID NO. 7 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 7; preferably, the nucleic acid molecule sequence encoding the recombinant type IV collagen contains the nucleotide sequence shown in SEQ ID NO. 14 or an amino acid sequence with 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 99.8%, or 99.9% identity with SEQ ID NO. 7; NO.14 has nucleotide sequences with 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%, and 99.9% identity.

[0041] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a repeating unit of one amino acid residue, the repeating unit comprising the sequence shown in SEQ ID NO.1, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.1, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.8.

[0042] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a two-amino acid residue repeating unit, the repeating unit comprising the sequence shown in SEQ ID NO.1, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.2, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.9.

[0043] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a repeating unit of 4 amino acid residues, the repeating unit comprising the sequence shown in SEQ ID NO.1, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.3, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.10.

[0044] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a 16-amino acid residue repeating unit, the repeating unit comprising the sequence shown in SEQ ID NO.1, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.4, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.11.

[0045] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a repeating unit of one amino acid residue, the repeating unit comprising the sequence shown in SEQ ID NO. 5, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO. 5, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO. 12.

[0046] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a two-amino acid residue repeating unit, the repeating unit comprising the sequence shown in SEQ ID NO.5, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.6, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO.13.

[0047] In a preferred embodiment, the amino acid sequence of the recombinant type IV collagen comprises a repeating unit of 4 amino acid residues, the repeating unit comprising the sequence shown in SEQ ID NO. 5, the amino acid sequence of the recombinant type IV collagen comprising the sequence shown in SEQ ID NO. 7, and the nucleotide sequence encoding the recombinant type IV collagen comprising the sequence shown in SEQ ID NO. 14.

[0048] 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 IV collagen according to the actual situation, or they can choose to use a general method to prepare the recombinant type IV collagen.

[0049] Those skilled in the art will understand that known modifications and optimizations can be made without affecting the function of the recombinant type IV collagen, and this application does not impose any mandatory limitations in this regard.

[0050] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following C1)-C5):

[0051] C1) A nucleic acid molecule, wherein the nucleic acid molecule contains a nucleic acid molecule encoding the recombinant type IV collagen;

[0052] C2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in C1);

[0053] C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or the expression cassette described in C2);

[0054] C4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3);

[0055] C5) Recombinant cells containing the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3).

[0056] Furthermore, the nucleic acid molecule sequence includes at least one or more of the following D1)-D4):

[0057] D1) The nucleic acid molecule sequence includes sequences such as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14;

[0058] The nucleic acid molecule sequence described in D2) has at least 85% identity with the nucleotide sequence described in D1);

[0059] D3) hybridizes with any of the specified nucleic acid sequences from D1) to D2) under stringent conditions;

[0060] Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in D4 and D1-D3.

[0061] Preferably, the nucleic acid molecule sequence includes at least one or more of the following D1)-D9):

[0062] D1) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO.8 or a nucleotide sequence having at least 90% identity with SEQ ID NO.8;

[0063] D2) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO.9 or a nucleotide sequence having at least 90% identity with SEQ ID NO.9;

[0064] D3) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 10 or a nucleotide sequence having at least 90% identity with SEQ ID NO. 10;

[0065] D4) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 11 or a nucleotide sequence having at least 90% identity with SEQ ID NO. 11;

[0066] D5) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO.12 or a nucleotide sequence having at least 90% identity with SEQ ID NO.12;

[0067] D6) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO.13 or a nucleotide sequence having at least 90% identity with SEQ ID NO.13;

[0068] D7) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO.14 or a nucleotide sequence having at least 90% identity with SEQ ID NO.14;

[0069] D8) Hybridizes with any of the specified nucleic acid sequences from A1) to A7) under stringent conditions;

[0070] Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in D9 and A1-A8.

[0071] in,

[0072] D1) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 8 or a nucleotide sequence having 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.

[0073] D2) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 9 or a nucleotide sequence having 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.

[0074] D3) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 10 or a nucleotide sequence having 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. 10.

[0075] D4) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 11 or a nucleotide sequence having 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. 11.

[0076] D5) The nucleic acid molecule sequence comprises the nucleotide sequence shown in SEQ ID NO. 12 or a nucleotide sequence having 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. 12.

[0077] D6) The nucleic acid molecule sequence comprises a nucleotide sequence as shown in SEQ ID NO. 13 or a nucleotide sequence having 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. 13.

[0078] D7) The nucleic acid molecule sequence comprises the nucleotide sequence shown in SEQ ID NO. 14 or a nucleotide sequence having 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. 14.

[0079] D8) Hybridizes with any of the specified nucleic acid sequences from A1) to A7) under stringent conditions;

[0080] Nucleic acid molecules that are complementary to any of the defined nucleic acid sequences in D9 and A1-A8.

[0081] 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.

[0082] Optionally, the recombinant vector may include a nucleic acid molecule encoding the aforementioned protein, a promoter, and transcription and translation termination signals. The promoter or terminator used to regulate expression in this application may be a universal promoter or terminator or a host-specific promoter or terminator. Those skilled in the art can make adaptive selections according to the actual situation. This application does not impose mandatory limitations on the sequence of the promoter or terminator. In a preferred embodiment, the promoter may be pAOX1.

[0083] 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 vector is a secretory vector containing a secretory signal α-factor. In a preferred embodiment, the recombinant vector may be the pPIC9K plasmid and / or the pPICZB plasmid.

[0084] Furthermore, the recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

[0085] Preferably, the recombinant microorganism is Pichia pastoris.

[0086] In a preferred embodiment, the Pichia pastoris is Pichia pastoris GS115.

[0087] 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 IV collagen described in this application. No specific strain is limited here.

[0088] 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 IV collagen can be achieved.

[0089] 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.

[0090] 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.

[0091] In a preferred embodiment, the resistance selection marker gene may be a herbicides resistance gene and / or a bleomycin resistance gene.

[0092] Preferably, in the recombinant microorganism, the copy number of the recombinant type IV collagen gene is greater than or equal to 1.

[0093] More preferably, the copy number is 2.

[0094] Preferably, in the recombinant microorganism, the recombinant type IV collagen gene is expressed in a free manner and / or integratedly.

[0095] Those skilled in the art can use known gene editing methods to complete the above construction, and no specific restrictions are imposed here.

[0096] On the other hand, this application also provides a method for preparing recombinant type IV collagen, the method comprising: constructing a recombinant microorganism expressing the recombinant type IV collagen, and culturing the recombinant microorganism.

[0097] Preferably, in the recombinant microorganism, the copy number of the recombinant type IV collagen gene is greater than or equal to 1.

[0098] More preferably, the copy number is 2.

[0099] Preferably, in the recombinant microorganism, the recombinant type IV collagen gene is expressed in a free manner and / or integratedly.

[0100] Furthermore, the recombinant microorganism is selected from one or more of Pichia pastoris, Streptococcus, Bacillus, Escherichia coli, Corynebacterium, and Saccharomyces cerevisiae.

[0101] Preferably, the recombinant microorganism is Pichia pastoris.

[0102] In a preferred embodiment, the Pichia pastoris is Pichia pastoris GS115.

[0103] Those skilled in the art can use conventional methods to ferment the above-mentioned recombinant microorganisms to obtain recombinant type IV collagen.

[0104] In a preferred embodiment, the preparation method includes the following steps:

[0105] Step 1: Construct recombinant microorganisms expressing the recombinant type IV collagen;

[0106] Step 2: Inoculate the recombinant microbial cells into the fermentation medium. The culture conditions are 25℃-37℃, 100-300 rpm, and methanol is added every 24 h to a final concentration of 1%-5%. Fermentation is considered complete when the product content no longer increases.

[0107] Preferably, the preparation method includes the following steps:

[0108] Step 1: Construct recombinant microorganisms expressing the recombinant type IV collagen;

[0109] Step 2: Inoculate the recombinant microbial monoclonal culture medium and culture at 25℃-37℃ and 100-300rpm for 12-48 h to obtain the primary seed liquid;

[0110] Step 3: Inoculate the primary seed culture at an inoculation rate of 1%-5% into the secondary seed culture medium, and culture at 25℃-37℃ and 100-300 rpm for 12-48 h to obtain the secondary seed culture.

[0111] Step 3: Centrifuge the secondary seed liquid, collect the bacterial cells, inoculate them into the fermentation medium, and ferment at 25℃-37℃ and 100-300 rpm. Add 1%-5% methanol every 24 hours until the product content no longer increases, which is considered the end of fermentation.

[0112] The primary seed culture is YPD medium, the secondary seed culture is BMGY medium, and the fermentation medium is BMMY medium.

[0113] The yield of recombinant type IV collagen prepared by fermentation using this method is greater than or equal to 0.15 g / L; preferably, it is greater than or equal to 0.4 g / L.

[0114] In a preferred embodiment, the preparation method includes the following steps:

[0115] Step 1: Construct recombinant microorganisms expressing the recombinant type IV collagen;

[0116] Step 2: Inoculate the recombinant microbial cells into the fermentation medium at an inoculation rate of 1%-5%. Fermentation conditions include a pH of 6.0-7.0, a temperature of 25℃-37℃, and dissolved oxygen control at 30%-40%. Start adding glycerol when dissolved oxygen rebounds. When the OD of the fermentation broth... 600 When the value reaches 100-200, stop adding glycerol, starve the product for 0.5-1 h, then add methanol to induce fermentation. Fermentation is considered complete when the product content no longer increases.

[0117] Preferably, the preparation method includes the following steps:

[0118] Step 1: Construct recombinant microorganisms expressing the recombinant type IV collagen;

[0119] Step 2: Inoculate the recombinant microbial monoclonal culture medium and culture at 25℃-37℃ and 100-300rpm for 12-48 h to obtain the primary seed liquid;

[0120] Step 3: Inoculate the primary seed culture at an inoculation rate of 1%-5% into the secondary seed culture medium, and culture at 25℃-37℃ and 100-300 rpm for 12-48 h to obtain the secondary seed culture.

[0121] Step 3: Inoculate the secondary seed culture at an inoculation rate of 1%-5% into the fermentation medium. Fermentation conditions include a pH of 6.0-7.0, a temperature of 25℃-37℃, and dissolved oxygen control at 30%-40%. When dissolved oxygen rebounds, begin adding glycerol at a final concentration of 40%-60%. When the OD of the fermentation broth... 600When the value reaches 100-200, stop adding glycerol, starve the product for 0.5-1 h, then add methanol to induce fermentation at a flow rate of 5-10 mL / min. Fermentation is considered complete when the product content no longer increases.

[0122] The primary seed culture is YPD medium, the secondary seed culture is BMGY medium, and the fermentation medium is BMMY medium.

[0123] The yield of recombinant type IV collagen prepared by fermentation using this method is greater than or equal to 2 g / L; preferably, it is greater than or equal to 4 g / L.

[0124] Preferably, the method further includes a purification step.

[0125] Those skilled in the art can purify recombinant type IV collagen using conventional methods.

[0126] The method may include one or more of the following: preliminary impurity removal using hollow fiber columns, membrane exchange solution, and cation exchange column chromatography.

[0127] The recombinant type IV collagen can also be dried according to the needs of those skilled in the art; preferably, it also includes a freeze-drying step.

[0128] The recombinant type IV humanized collagen obtained by this invention has a purity of over 95%, and exhibits good protein activity after water refolding.

[0129] On the other hand, this application also provides a composition containing the recombinant type IV collagen.

[0130] 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.

[0131] On the other hand, this application also provides a product containing the recombinant type IV collagen or the composition.

[0132] The products include one or more of the following: products that improve cell adhesion, skin repair products, products that promote cell migration, skin firming products, products that promote cell proliferation, anti-wrinkle products, anti-aging products, products that increase collagen content, transdermal products, and moisturizing products.

[0133] Furthermore, the products mentioned include daily chemical products, hyaluronic acid injections, and other medical devices.

[0134] On the other hand, this application also provides the application of the recombinant type IV collagen or the biomaterial or the recombinant type IV collagen obtained by the preparation method described above in the preparation of products that improve cell adhesion function.

[0135] Cell adhesion function is closely related to tissue repair, maintaining the integrity and normal function of tissues and organs, regulating normal cell growth and behavior, and the structure and function of the basement membrane.

[0136] In a preferred embodiment, the cells are keratinocytes and / or fibroblasts.

[0137] On the other hand, this application also provides the application of the recombinant type IV collagen or the recombinant type IV collagen obtained by the biomaterial or the preparation method described above in the preparation of skin repair and / or cell migration promotion products.

[0138] Preferably, the skin repair is achieved by enhancing cell migration and / or cell adhesion functions.

[0139] In a preferred embodiment, the cells are keratinocytes and / or fibroblasts.

[0140] On the other hand, this application also provides the use of the recombinant type IV collagen or the recombinant type IV collagen obtained by the biomaterial or the preparation method described above in the preparation of skin-tightening and / or cell proliferation-promoting products.

[0141] Preferably, the skin firming effect is achieved by promoting cell proliferation and / or promoting collagen expression.

[0142] The recombinant type IV collagen provided in this application can effectively promote cell proliferation, which also demonstrates the safety and non-toxicity of this collagen.

[0143] In a preferred embodiment, the cells are keratinocytes and / or fibroblasts.

[0144] On the other hand, this application also provides the application of the recombinant type IV collagen or the recombinant type IV collagen obtained by the biomaterial or the preparation method described above in the preparation of anti-wrinkle and / or anti-aging and / or collagen-enhancing products.

[0145] Preferably, the anti-wrinkle and / or anti-aging effects are achieved by increasing collagen content.

[0146] Preferably, increasing collagen content includes promoting collagen expression.

[0147] More preferably, the collagen is type IV collagen.

[0148] The recombinant type IV collagen proposed in this application can not only supplement type IV collagen externally, but also promote the body's own synthesis of type IV collagen, thereby achieving the goal of increasing collagen content through two pathways and effectively combating aging.

[0149] In a preferred embodiment, the cells are keratinocytes and / or fibroblasts.

[0150] On the other hand, this application also provides the application of the recombinant type IV collagen or the recombinant type IV collagen obtained by the biomaterial or the preparation method described above in the preparation of transdermal and / or moisturizing products.

[0151] The transdermal and / or moisturizing effects are achieved by increasing the cumulative permeability of collagen.

[0152] The recombinant type IV collagen obtained in this application has excellent transdermal absorption capacity, promoting collagen to penetrate through the epidermis to the dermis, making it easier for the body to absorb and use.

[0153] Preferably, in the composition or product or in the above-described applications, the concentration of the recombinant type IV collagen is greater than or equal to 0.001% by mass; more preferably, 0.001%-10%; more preferably, 0.005%-0.5%.

[0154] The concentration of the recombinant type IV collagen, expressed as a percentage by mass, may be any value or range from 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%, to 10%.

[0155] Those skilled in the art can adjust the concentration used according to the actual situation, and no specific limitations are made here.

[0156] The present invention has the following beneficial effects:

[0157] In this invention, a repeating amino acid residue unit with good biological activity and stability was found in human type IV collagen. Based on this unit, repetition was performed to construct recombinant type IV collagen, thereby obtaining a new biologically active human recombinant type IV collagen.

[0158] The recombinant human type IV collagen provided in this invention is a low molecular weight protein, easily absorbed by the human body, and has good safety. It can significantly promote the secretion of the body's own collagen and also promotes cell adhesion and migration, showing promising application prospects in basement membrane repair and remodeling. It provides a new functional ingredient for products that repair, tighten, fight aging, reduce wrinkles, and moisturize the skin.

[0159] This invention also provides a method for preparing recombinant human type IV collagen and a method for constructing engineered bacteria expressing recombinant human type IV collagen, thereby realizing the large-scale production of bioactively stable type IV collagen. Attached Figure Description

[0160] 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:

[0161] Figure 1 The pPIC9K-Y4-6-3-6P plasmid map;

[0162] Figure 2 Image of Tricine-SDS-PAGE electrophoresis results for shake-flask fermentation products of Col-IV-63-6P strain;

[0163] Figure 3 Image of Tricine-SDS-PAGE electrophoresis results of fermentation products of Col-IV-63-6P strain in fermenter;

[0164] Figure 4 Morphological image of a freeze-dried sample of recombinant type IV humanized collagen;

[0165] Figure 5 Image showing the results of Tricine-SDS-PAGE electrophoresis of IV-63-6P collagen;

[0166] Figure 6 The image shows the fluorescence results of the transdermal absorption assay for IV-63-6P collagen. Blue represents the nuclear DAPI fluorescence, and red represents the sample's own fluorescence. The magnification is 15x. Detailed Implementation

[0167] Technical terms:

[0168] 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.

[0169] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0170] 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.

[0171] 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.

[0172] Recombinant microorganisms: bacterial cell lines that use genetic engineering methods to achieve efficient expression of foreign genes.

[0173] 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.

[0174] 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.

[0175] Integrated expression: Expression that occurs when a gene is integrated into the genome.

[0176] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0177] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0178] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0179] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0180] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0181] 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).

[0182] 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).

[0183] 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.

[0184] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0185] The culture media involved in the following examples are as follows:

[0186] MD plate: Yeast basic nitrogen source (YNB) 13.4 g / L, biotin 0.4 mg / L, glucose 20 g / L, agar powder 15 g / L, balance water.

[0187] YPD plate: yeast extract 10 g / L, peptone 20 g / L, glucose 10 g / L, agar powder 20 g / L, balance water.

[0188] YPD liquid culture medium: yeast extract 10 g / L, peptone 20 g / L, glucose 10 g / L, with the remainder being water.

[0189] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, potassium phosphate buffer (pH 6.0) 100 mmol / L, YNB 1.34%, biotin (4 × 10⁻⁶) -5 1% glycerol, the remainder is water.

[0190] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, potassium phosphate buffer (pH 6.0) 100 mmol / L, YNB 1.34%, biotin (4 × 10⁻⁶) -5 )%, methanol 3%, and the remainder is water.

[0191] Serum-free culture medium: high glucose DMEM medium (Gibco).

[0192] Example 1: Construction and expression of recombinant type IV humanized collagen

[0193] 1. Design of amino acid sequences

[0194] Using computer-aided protein structure prediction, functional regions of human type IV collagen (Gene ID 1286) that are easy to recombinantly express in vitro, readily form structures, and exhibit good stability were screened. The resulting sequence fragments were optimized through n-fold repetition ligation to ensure that the molecular weight of the recombinant type IV humanized collagen remained within a certain range and that it was easy to purify and stabilize. The final results are shown in Table 1.

[0195] Table 1. Amino acid sequence of recombinant type IV humanized collagen

[0196]

[0197] 2. Synthesis and construction of expression vectors

[0198] The nucleic acid sequences of the functional region of type IV collagen selected in step 1 were tandemly repeated at different multiples. After codon optimization by Pichia pastoris, gene fragments SEQ ID NO. 8-14 were obtained. The gene sequences were synthesized by Nanjing Genscript Biotech Co., Ltd. The synthesized gene fragments SEQ ID NO. 8-14 were recombined into the pPIC9K empty vector. After cloning, the 30 bp sequence at positions 1216-1345 in the vector was replaced, so that the target fragment was accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor, and a vector plasmid expressing recombinant type IV humanized collagen was obtained. The plasmid map is shown below. Figure 1 As shown in Table 2 (taking recombinant collagen IV-63-6P as an example), the plasmid information is as follows.

[0199] Table 2

[0200]

[0201] 3. Construction of Pichia pastoris expression strain

[0202] The recombinant type IV collagen expression plasmid obtained in step 2 was transformed into Pichia pastoris and multiple copy strains were screened. The specific steps are as follows:

[0203] 1) Take 5 μg of the above-mentioned recombinant expression vector plasmids with different sequences, and use 5 U Sac I Enzyme digestion was performed in a 30℃ water bath for 1 h. The linearized plasmid fragment was recovered using a DNA recovery kit (omega, D6492-02). The linearized plasmid fragment was then added to Pichia pastoris GS115 competent cells thawed on ice, along with 5 μL of carrier DNA and 500 μL of Pichia pastoris transformation medium. The mixture was incubated at 30℃ for 30 min, followed by the addition of 20 μL of dimethyl sulfoxide and incubation at 42℃ for 15 min. The mixture was then centrifuged briefly at 12000 rpm to remove the supernatant. 1 mL of YPD liquid culture medium was added, and the mixture was incubated on a shaker at 30℃ for 1 h. The mixture was then plated on MD plates and incubated upside down in a 30℃ incubator for 2-3 days. Transformants were then screened.

[0204] 2) After the clones grow on the MD plate, select single clones for colony PCR verification and sequencing to verify that the selected recombinant gene fragments have been successfully integrated into the pAOX1 promoter of GS115 cells.

[0205] 3) The selected correctly integrated monoclonal strains were streaked onto YPD plates with G418 (Geneticin) at concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL as the screening drug. The plates were then inverted and incubated in a 30°C incubator for 1-2 days. High-copy integrated strains that could grow on high-concentration G418 plates were preferentially screened.

[0206] 4. Shake-flask fermentation and screening of yeast expression strains

[0207] 1) The type IV humanized collagen high-copy integrative yeast strain selected in step 3 was streaked on MD plates for resurrection. Single clones were picked and inoculated into test tubes containing 5 mL of YPD liquid medium and cultured at 30°C and 220 rpm for 24 h.

[0208] 2) Transfer the bacterial culture from the test tube after the culture in step 1) at a ratio of 1% to a shake flask containing 50 mL of BMGY medium, and incubate at 30°C and 220 rpm for 24 h on a shaker.

[0209] 3) Centrifuge the bacterial culture from step 2) at 4000 rpm to collect the bacterial cells. Transfer the bacterial cells to a shake flask containing 50 mL of BMMY medium and continue culturing at 30°C and 220 rpm for 96 h. During this period, add 1% methanol to the shake flask every 24 h for induction.

[0210] 4) During the induced culture in BMMY medium, samples were taken every 24 h, and the supernatant was collected by centrifugation for Tricine-SDS-PAGE electrophoresis to detect the expression of different recombinant type IV collagen sequences, as shown in Table 3. Shake-flask expression screening showed that the recombinant type IV humanized collagen sequences SEQ ID NO. 1-7 were successfully secreted and expressed in Pichia pastoris chassis strains.

[0211] Table 3. Expression of type IV collagen strains in shake flasks

[0212]

[0213] 5. Optimization and screening of yeast strains capable of expression

[0214] After screening strains that can express type IV collagen sequences well in Pichia pastoris through shake-flask fermentation in step 4 above, the expression level of type IV collagen in the engineered strains is gradually increased through screening, and the specific steps are as follows:

[0215] 1) Using pPICZB plasmid as a template, bleomycin resistance gene expression cassette was amplified by PCR and integrated into pPIC9K vector plasmid containing the target gene sequences of each type IV collagen. The HIS4 position on pPIC9K vector plasmid was replaced to confer bleomycin resistance gene to the expression vector.

[0216] 2) Sac I The vector plasmid containing the bleomycin resistance gene and the target protein gene was digested with enzymes, the linearized plasmid fragment was recovered, and electroporated into the yeast chassis strain with good expression of the corresponding target protein selected in the first round of screening in step 4. The transformation solution was then plated on YPD plates with 150 μg / mL zeocin as the screening drug and incubated upside down in a 30℃ constant temperature incubator for 2-3 days.

[0217] 3) After single colonies grow on the transformation plate, the successfully integrated transformants are selected by PCR and sequencing verification, and streaked onto YPD plates containing 5 mg / mL and 6 mg / mL G418 screening drugs, respectively, to screen for high-copy integrated strains.

[0218] 4) The high-copy integration strains were verified by shake-flask fermentation, and double-copy yeast engineered strains with significantly increased expression of type IV collagen were obtained.

[0219] The fermentation and detection methods are the same as in step 4. The expression yields of each strain in shake flasks are shown in Table 4. Taking IV-63-6P in the shake flask expression supernatant of the Col-IV-63-6P strain as an example, the Tricine-SDS-PAGE electrophoresis results are as follows: Figure 2 As shown in the figure, the selected clone can successfully express recombinant type IV humanized collagen. The target protein is effectively secreted in the supernatant of Pichia pastoris fermentation broth, and the electrophoretic bands are clear and single.

[0220] Table 4. Expression of Type IV Collagen in Shake Flasks

[0221]

[0222] Example 2: Fermentation and purification of recombinant type IV humanized collagen

[0223] 1. Fermentation of high-expression engineered yeast strains in a 5 L tank

[0224] The Col-IV-63-6P, Col-IV-64-3P, and Col-IV-64-12P strains from Example 1 were fermented in a 5 L tank. The specific steps are as follows:

[0225] 1) Select the selected yeast high-expression monoclonal strains into test tubes containing 5 mL of YPD liquid medium and incubate at 30℃ and 220 rpm for 24 h to activate them;

[0226] 2) Transfer the bacterial culture obtained in step 1) to a shake flask containing 50 mL of YPD liquid medium at an inoculation rate of 1%, and incubate at 30°C and 220 rpm for 24 h to obtain the seed culture for the upper tank.

[0227] 3) Prepare 1.8 L of BMMY medium in a 5 L fermenter, sterilize and cool it, adjust the pH to 6.0, and inoculate the seed liquid obtained in step 2) into the fermenter at an inoculation rate of 1% for fermentation culture, control the dissolved oxygen at 30% and the fermentation temperature at 30℃.

[0228] 4) When dissolved oxygen rebounds, begin adding 50% glycerol. When the OD of the fermentation broth... 600 When the value reaches about 150, stop adding glycerol, starve the plant for half an hour, and start adding methanol for induction at a flow rate of 8 mL / min.

[0229] 5) Methanol was continuously added for induction, and samples were taken for monitoring every 24 hours. After a cumulative induction of 96 hours, fermentation was stopped. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the fermentation supernatant was collected. Samples taken at each stage were analyzed by Tricine-SDS-PAGE electrophoresis and quantified by BSA.

[0230] The fermentation yields of each strain in a 5 L tank are shown in Table 5. Among them, the Col-IV-63-6P strain achieved a target protein yield of 4.86 g / L after 72 h of induction. The fermentation results of the Col-IV-63-6P strain are shown below. Figure 3 As shown, recombinant IV humanized collagen can be well secreted in the fermentation supernatant.

[0231] Table 5. Fermentation yield of strain IV in 5 L tanks

[0232]

[0233] 2. Purification and sample collection of recombinant type IV humanized collagen

[0234] The fermentation supernatant was purified by hollow fiber column chromatography, membrane exchange, and cation exchange chromatography. The cation exchange sample was desalted by water exchange and then freeze-dried to obtain a high-purity recombinant type IV humanized collagen freeze-dried sample. The freeze-dried sample is shown below. Figure 4 As shown. 10 mg of lyophilized collagen was dissolved in 10 mL of ultrapure water to prepare a recombinant collagen aqueous solution. The protein purity was determined using SDS-PAGE electrophoresis and liquid chromatography. The purity of the recombinant type IV humanized collagen obtained by this invention can reach over 95%. Taking IV-63-6P collagen as an example, with bovine serum albumin (BSA) as a control, the electrophoresis results after purification are shown in the example below. Figure 5 As shown.

[0235] Example 3: Bioactivity assay of recombinant type IV humanized collagen

[0236] In this embodiment, recombinant type IV humanized collagen IV-63-6P, IV-64-3P, and IV-64-12P purified in Example 2 were used as samples for the following tests.

[0237] 1. Cell adhesion test

[0238] (1) Using human immortalized keratinocytes (HaCaT) and humanized fibroblasts as detection models, the cell adhesion rate of the recombinant type IV humanized collagen sample purified in Example 2 was evaluated by the MTT assay.

[0239] The specific steps are as follows: Immortalized human keratinocytes and humanized fibroblasts are cultured to the logarithmic growth phase, the cells are digested with trypsin, and the cells are suspended in serum-free medium to adjust the cell concentration to 5 × 10⁶. 4Cells / mL were seeded into 96-well plates coated or uncoated with 10 μg / mL Collagen I. In the experimental group, collagen samples IV-63-6P, IV-64-3P, and IV-64-12P were added after coating, with a loading concentration of 0.1%. Three replicates were set up. After loading, the wells were placed in a cell culture incubator (37℃, 5% CO2) and cultured for 6 h. Culture was stopped, the culture medium was discarded, and unattached cells were washed away with PBS buffer (plate washing). The total cell group was coated in 96-well plates without washing; the blank control (BC) was uncoated in 96-well plates and washed; the negative control (NC) was coated in 96-well plates and washed. No sample treatment was added to the total cell group, blank control, and negative control; all other procedures were consistent with the experimental group. Add 100 μL of serum-free medium containing 0.5 mg / mL MTT and incubate at 37°C in the dark for 2 h. After incubation, discard the supernatant, add 50 μL of DMSO to each well, and read the OD value at 490 nm. Calculate the cell adhesion rate using the formula below. The results are shown in Table 6, and the data were analyzed for significance.

[0240] Cell adhesion rate = .

[0241] Table 6 Results of cell adhesion assay for recombinant type IV humanized collagen

[0242]

[0243] Note: Compared with the BC group, the significance of the NC 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 ###; compared with the NC group, the significance of the sample 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 ***.

[0244] The experimental results are shown in Table 6. Based on the human immortalized keratinocyte (HaCaT) model and the human fibroblast model, the recombinant type IV humanized collagen samples IV-63-6P and IV-64-12P significantly improved the cell adhesion rate, and the difference was statistically significant compared with the BC group (p<0.05). This indicates that the IV-63-6P and IV-64-12P samples can promote the adhesion of keratinocytes and fibroblasts and have repair effects.

[0245] 2. Cell migration test

[0246] Promoting cell proliferation and migration is a key measure for skin damage repair. Using human immortalized keratinocytes (HaCaT) and humanized fibroblasts as research subjects, the efficacy of the samples in repairing skin barrier damage was evaluated by detecting the migration effect of the purified recombinant type IV humanized collagen sample from Example 2.

[0247] The specific experimental steps are as follows:

[0248] 1) Collect cells in the logarithmic growth phase, at a cell density of 1×10⁻⁶. 5 Inoculate one well per well into a 24-well culture plate.

[0249] 2) After culturing in an incubator (37℃, 5% CO2) for 24 h, use a 200 μL pipette tip to make horizontal scratches perpendicular to the 24-well plate, wash the cells once with PBS buffer, and remove the scratched cells.

[0250] 3) Place the sample in an incubator (37℃, 5% CO2) and incubate for 24 h. Set up 3 replicates for each group, with a sample concentration of 0.1%. Use the control group without sample as a blank control (BC).

[0251] 4) 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 then calculated using the formula below, and the results are shown in Table 7. A significance analysis was performed on the data.

[0252] Healing rate (%) = .

[0253] Table 7 Results of the cell migration-promoting assay using recombinant humanized type IV collagen

[0254]

[0255] Note: The significance of the sample group compared with group BC 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 ***.

[0256] The experimental results are shown in Table 7. Based on the human immortalized keratinocyte (HaCaT) model, after 24 h of treatment with collagen samples IV-63-6P and IV-64-12P, the cell healing rate of keratinocytes was significantly improved, and there was a statistically significant difference compared with the BC group (p<0.05). This indicates that collagen samples IV-63-6P and IV-64-12P can promote cell healing of keratinocytes and have a repairing effect.

[0257] Based on the human fibroblast model, after 24 h of treatment with collagen samples IV-63-6P and IV-64-12P, the cell healing rate of human fibroblasts was improved, and there was a statistically significant difference compared with the BC group (p<0.05), indicating that the collagen samples V-63-6P and IV-64-12P can promote cell healing of human fibroblasts and have a repair effect.

[0258] 3. Cell proliferation test

[0259] Based on the human immortalized keratinocyte (HaCaT) model and the humanized fibroblast model, the repair efficacy of recombinant type IV humanized collagen samples was evaluated by detecting their effect on cell proliferation. The specific steps are as follows:

[0260] 1) Collect cells in the logarithmic growth phase, at a cell density of 1×10⁻⁶. 5 / well inoculated into a 24-well culture plate.

[0261] 2) Sample loading: After incubation in an incubator (37℃, 5% CO2) for 24 h, samples were loaded. Each group was set up in 3 replicates with a sample concentration of 0.1%. The control group without sample was used as a blank control (BC).

[0262] 3) Detection: 48 h after sample loading, the supernatant was discarded, and 500 μL of serum-free culture medium containing 0.5 mg / mL MTT was added. The mixture was incubated at 37°C 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.

[0263] 4) Calculate cell viability using the formula below. The results are shown in Table 8. Perform significance analysis on the data. .

[0264] Table 8 Results of cell proliferation assay using recombinant humanized type IV collagen

[0265]

[0266] Note: The significance of the sample group compared with group BC 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 ***.

[0267] Table 8 shows that after treatment with recombinant humanized collagen IV-63-6P and IV-64-12P samples at a loading concentration of 0.1% for 48 h, the cell viability of keratinocytes and fibroblasts was significantly improved, and there was a statistically significant difference compared with the BC group (p<0.05). This indicates that the collagen samples IV-63-6P and IV-64-12P can improve the viability of keratinocytes and fibroblasts and have repair and firming effects.

[0268] 4. Type IV collagen expression enhancement test

[0269] Using human immortalized keratinocytes (HaCaT) as the testing system, this study evaluated the anti-wrinkle and firming effects of collagen samples IV-63-6P and IV-64-12P by investigating changes in the gene expression of type IV collagen.

[0270] The specific experimental steps are as follows:

[0271] 1) Cell seeding: at 1×10 5 Cells were seeded at a density of cells per well into 24-well plates and incubated overnight in an incubator (37°C, 5% CO2).

[0272] 2) Solution preparation: Prepare working solutions of the test substances (IV-63-6P and IV-64-12P collagen samples) at test concentrations of 0.005%, 0.05%, and 0.1%, with 3 replicate wells for each concentration gradient. The solvent is serum-free culture medium.

[0273] 3) Drug administration: Drug administration is performed when the cell deposition rate in the 24-well plate reaches 40%-60%. For the blank control group, add 1 mL of serum-free culture medium to each well; for the sample group, add 1 mL of serum-free culture medium containing the corresponding concentration of the test substance to each well.

[0274] 4) Cell collection: After incubation for 24 h, add 0.5 mL of lysis buffer to each well, incubate at room temperature for 5 min to allow for complete lysis, transfer to 1.5 mL RNase-free Eppendorf tubes, and extract RNA according to the RNA extraction kit procedure.

[0275] 5) Reverse transcription: Synthesize cDNA according to the instructions of the RNA reverse transcription kit.

[0276] 6) The Collagen IV gene was detected by qRT-PCR, and the results are shown in Table 9. The significance of the data was analyzed.

[0277] Table 9 Results of recombinant type IV humanized collagen promoting Collagen IV gene expression

[0278]

[0279] Note: The significance of the sample group compared with group BC 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 ***.

[0280] The experimental results are shown in Table 9. Based on the human immortalized keratinocyte (HaCaT) model, the IV-63-6P and IV-64-12P collagen samples at concentrations of 0.005%, 0.050%, and 0.100% (m / V) all showed a significant increase in the expression level of the Collagen IV gene in keratinocytes, and the difference was statistically significant compared with the BC group (p<0.01), indicating that the IV-63-6P and IV-64-12P collagen samples have good anti-wrinkle and firming effects.

[0281] 5. Transdermal absorption detection

[0282] Preparation of fluorescently labeled collagen samples: A 10 mg / mL dye stock solution was prepared by dissolving TAMRA (5(6)-carboxytetramethylrhodamine succinimide) dye in DMSO; a 0.5% collagen stock solution was prepared by dissolving collagen in pH 6.8 PBS buffer; the collagen stock solution and dye stock solution were mixed at a certain mass ratio, such that the dye / collagen ratio was 5 / 1 (molar ratio), and shaken overnight; an equal amount of water as the collagen stock solution was added to the reaction solution, and the solution was centrifuged for 10 min to remove excess dye; the supernatant was dialyzed overnight in water (using a 1 kDa dialysis bag), with one water change during the process, to obtain the labeled collagen stock solution, which was then diluted to a concentration of 0.1% for use.

[0283] Transdermal absorption detection: Based on the piglet skin-Franz cell diffusion cell system, Franz diffusion cell permeability experiments were conducted on samples using piglet back skin to investigate the transdermal ability of the samples to isolated piglet back skin. The in vitro transdermal behavior was described by measuring the fluorescence signal intensity of the samples in the piglet skin. The specific steps included:

[0284] 1) The Franz cell diffusion chamber is divided by the skin and includes a supply chamber and a receiving chamber. Add receiving solution to the receiving chamber: Use a pipette to draw 7.0 mL of receiving solution (physiological saline) and inject it into the receiving chamber, and place the matching magnetic stir bar inside the receiving chamber.

[0285] 2) Assembly and fixation of the test model: The piglet skin was fixed between the diffusion chamber and the receiving chamber of the Franz cell diffusion cell, with the stratum corneum of the piglet skin facing the diffusion chamber and the dermis facing the receiving chamber. After fixing the piglet skin, 0.5 mL of receiving solution (physiological saline) was added to the sampling tube using a pipette according to the liquid level in the sampling tube, so that the dermis of the piglet skin was in close contact with the receiving solution, and the total volume of the receiving solution was 7.5 mL.

[0286] 3) Fix the Franz cell diffusion cell in the transdermal absorption diffusion apparatus, turn on the electromagnetic stirrer and stir at a speed of 300 rpm, maintain a constant temperature water bath of (32±1)℃, and ensure that there are no air bubbles in the water bath jacket.

[0287] 4) Sample loading: After the water bath temperature of the diffuser has stabilized, the sample loading process is carried out. Use a pipette to draw 400 μL of IV-63-6P sample (with built-in fluorescent label) and add it to the surface of piglet skin.

[0288] 5) Qualitative analysis: Pig skin samples were collected at 0 h, 8 h, 12 h, and 24 h for frozen sectioning, counterstained with DAPI, and photographed using a fluorescence microscope.

[0289] Experimental results are as follows Figure 6 As shown in the figure, transdermal absorption tests revealed that IV-63-6P collagen penetrated the epidermis and reached the dermis at 12 h and 24 h. In the figure, blue represents nuclear DAPI fluorescence, and red represents the sample's own fluorescence. This result demonstrates that IV-63-6P collagen has excellent transdermal absorption capabilities.

[0290] The above description is merely an embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of this application should be included within the scope of the claims of this application.

Claims

1. Recombinant type IV collagen, characterized in that, The amino acid sequence of the recombinant type IV collagen is any one of the following A1)-A3): A1) The amino acid sequence of the recombinant type IV collagen is a repeating unit of multiple amino acid residues, as shown in SEQ ID NO.1, with a repeat number of 2; A2) The amino acid sequence of the recombinant type IV collagen is a repeating unit of multiple amino acid residues, as shown in SEQ ID NO.5, with a repeating number of 4; A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the recombinant type IV collagen described in A1) or A2).

2. The recombinant type IV collagen according to claim 1, characterized in that, The amino acid sequence of the recombinant type IV collagen is shown in SEQ ID NO.2 or SEQ ID NO.

7.

3. A biomaterial, characterized in that, The biomaterial includes any one of the following C1)-C5): C1) A nucleic acid molecule, wherein the nucleic acid molecule is a nucleic acid molecule encoding the recombinant type IV collagen as described in claim 1 or 2; C2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in C1); C3) A recombinant vector containing the nucleic acid molecule described in C1) and / or the expression cassette described in C2); C4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3); C5) Recombinant cells containing the nucleic acid molecule described in C1), the expression cassette described in C2), and / or the recombinant vector described in C3).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid molecule sequence is shown in SEQ ID NO.9 or SEQ ID NO.

14.

5. The use of the recombinant type IV collagen as described in claim 1 or 2, or the biomaterial as described in claim 3 or 4, in the preparation of products that enhance cell adhesion function.

6. The use of the recombinant type IV collagen as described in claim 1 or 2, or the biomaterial as described in claim 3 or 4, in the preparation of skin repair and / or cell migration promotion products.

7. The use of the recombinant type IV collagen as described in claim 1 or 2, or the biomaterial as described in claim 3 or 4, in the preparation of skin-tightening and / or cell proliferation-promoting products.

8. The use of the recombinant type IV collagen as described in claim 1 or 2 or the biomaterial as described in claim 3 or 4 in the preparation of anti-wrinkle and / or anti-aging and / or collagen-enhancing products.

9. The use of the recombinant type IV collagen as described in claim 1 or 2, or the biomaterial as described in claim 3 or 4, in the preparation of transdermal and / or moisturizing products.

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