Recombinant type xvii collagen having triple helix structure and use thereof

Abstract

Provided in the present application is a recombinant type XVII collagen having a triple helix structure. The collagen has an amino acid sequence containing one or more amino acid residue repeating units, wherein each of the repeating units contains a sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the repetition number is n, where 1＜n≤60. The recombinant type XVII collagen provided in the present application can be used in the fields of food, health care products, cosmetics, medical instruments, etc.

Description

Recombinant type XVII collagen with triple helical structure and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 5, 2023, with application number "202311658196.9" and invention name "A recombinant type XVII collagen with a triple helix structure and its application", the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the field of biological protein technology, and in particular to a recombinant type XVII collagen with a triple helical structure and its application. Background Art

[0003] There are 28 types of collagen, accounting for approximately 30% of the total human protein. Collagen is primarily found in the skin, tendons, bones, blood vessels, and cornea, with molecular weights ranging from 50 to 340 kDa. Type XVII collagen, a transmembrane protein composed of three α chains, each 180 kDa, is primarily found in the skin and epithelium. Type XVII collagen mediates interactions between stem cells and surrounding cells and the matrix, regulating skin homeostasis, aging, and wound repair. It holds broad application prospects in cosmetics and medical devices.

[0004] In human skin, collagen plays a major supporting role, and the realization of this supporting role mainly depends on the triple helix structure of collagen. At present, the production of recombinant collagen is mainly based on microbial fermentation. For example, patents CN113185604A and CN110845603A use yeast cells and Escherichia coli cells to design and express recombinant type XVII collagen, respectively, but they do not show a triple helix structure. The use of synthetic biology technology to achieve collagen with the correct triple helix structure in microbial fermentation has high technical barriers. Therefore, the design and realization of recombinant type XVII collagen with a triple helix structure remains a technical problem that needs to be overcome in the production of recombinant collagen by microbial fermentation. Summary of the Invention

[0005] In order to solve the above problems, the present application aims to provide a recombinant type XVII collagen with a triple helical structure that is simple to prepare, has high expression level, good efficacy, and can achieve self-assembly.

[0006] On the one hand, the present application provides a recombinant type XVII collagen, the amino acid sequence of which comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.1, the number of repetitions being n, 1<n≤60.

[0007] Optionally, the value range of the repetition number n can be: 1<n≤60, 2≤n≤60, 3≤n≤60, 4≤n≤60, 5≤n≤60, 6≤n≤60, 7≤n≤60, 8≤n≤60, 9≤n≤60, 10≤n≤60, 11≤n≤60, 12≤n≤60, 13≤n≤60, 14≤n≤60, 15≤n≤ 60, 16≤n≤60, 17≤n≤60, 18≤n≤60, 19≤n≤60, 20≤n≤60, 21≤n≤60, 22≤n≤60, 23≤n≤60, 24≤n≤60, 25≤n≤60, 26≤n≤60, 27≤n≤60, 28≤n≤60, 29≤n≤60, 30≤n≤60, 31 ≤n≤60, 32≤n≤60, 33≤n≤60, 34≤n≤60, 35≤n≤60, 36≤n≤60, 37≤n≤60, 38≤n≤60, 39≤n≤60, 40≤n≤60, 41≤n≤60, 42≤n≤60, 43≤n≤60, 44≤n≤60, 45≤n≤60, 46≤n≤60 , 47≤n≤60, 48≤n≤60, 49≤n≤60, 50≤n≤60, 51≤n≤60, 52≤n≤60, 53≤n≤60, 54≤n≤60, 55≤n≤60, 56≤n≤60, 57≤n≤60, 59≤n≤60, 59≤n≤60, 1≤n≤10, 10≤n≤20, 20≤n≤ 30, 30≤n≤40, 40≤n≤50, 50≤n≤60, 5≤n≤15, 15≤n≤25, 25≤n≤35, 35≤n≤45, 45≤n≤55, 55≤n≤60, 1≤n≤10, 10≤n≤20, 20≤n≤30, 30≤n≤40, 40≤n≤50, 50≤n≤60, 5≤n≤ 15, 15≤n≤25, 25≤n≤35, 35≤n≤45, 45≤n≤55, 55≤n≤60, 10≤n≤30, 30≤n≤60, 5≤n≤30, 30≤n≤40, 40≤n≤50, 50≤n≤60, 5≤n≤15, 15≤n≤25, 25≤n≤35, 35≤n≤45, 45≤n ≤55, 55≤n≤60, 1≤n≤55, 5≤n≤55, 10≤n≤60, 15≤n≤60, 25≤n≤60, 35≤n≤60, 1≤n≤4, 2≤n≤4, 3≤n≤4, 1≤n≤8, 2≤n≤8, 3≤n≤8, 4≤n≤8, 5≤n≤8, 6≤n≤8, 7≤n≤8, 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≤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≤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≤35, 2≤n≤35, 3≤n≤35, 4≤n≤35, 5≤n≤35, 6≤n≤35, 7≤n≤35, 8≤n≤35, 9≤n≤35, 10≤n≤35, 11≤n≤35, 12≤n≤35, 13≤n≤35, 14≤n≤35, 15≤n≤35, 16≤n≤35, 17≤n≤35, 18≤n≤35, 19≤n≤35, 20≤n≤35, 21≤n≤35, 22≤n≤3 5, 23≤n≤35, 24≤n≤35, 25≤n≤35, 26≤n≤35, 27≤n≤35, 28≤n≤35, 29≤n≤35, 4≤n≤40, 4≤n≤24, 4≤n≤8, 4≤n≤16, 4≤n≤24, 8≤n≤16, 8≤n≤24, 8≤n≤40, 16≤n≤24, 16≤n≤40, 24≤n≤40, 30≤n≤35, 30≤n≤40, 35≤n≤40 and any range therebetween.

[0008] On the other hand, the present application also provides a recombinant type XVII collagen, the amino acid sequence of which comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises the sequence shown in SEQ ID NO.2, the number of repetitions being n, 1<n≤60.

[0009] Optionally, the value range of the repetition number n can be: 1<n≤60, 2≤n≤60, 3≤n≤60, 4≤n≤60, 5≤n≤60, 6≤n≤60, 7≤n≤60, 8≤n≤60, 9≤n≤60, 10≤n≤60, 11≤n≤60, 12≤n≤60, 13≤n≤60, 14≤n≤60, 15≤n≤ 60, 16≤n≤60, 17≤n≤60, 18≤n≤60, 19≤n≤60, 20≤n≤60, 21≤n≤60, 22≤n≤60, 23≤n≤60, 24≤n≤60, 25≤n≤60, 26≤n≤60, 27≤n≤60, 28≤n≤60, 29≤n≤60, 30≤n≤60, 31 ≤n≤60, 32≤n≤60, 33≤n≤60, 34≤n≤60, 35≤n≤60, 36≤n≤60, 37≤n≤60, 38≤n≤60, 39≤n≤60, 40≤n≤60, 41≤n≤60, 42≤n≤60, 43≤n≤60, 44≤n≤60, 45≤n≤60, 46≤n≤60 , 47≤n≤60, 48≤n≤60, 49≤n≤60, 50≤n≤60, 51≤n≤60, 52≤n≤60, 53≤n≤60, 54≤n≤60, 55≤n≤60, 56≤n≤60, 57≤n≤60, 59≤n≤60, 59≤n≤60, 1≤n≤10, 10≤n≤20, 20≤n≤ 30, 30≤n≤40, 40≤n≤50, 50≤n≤60, 5≤n≤15, 15≤n≤25, 25≤n≤35, 35≤n≤45, 45≤n≤55, 55≤n≤60, 1≤n≤10, 10≤n≤20, 20≤n≤30, 30≤n≤40, 40≤n≤50, 50≤n≤60, 5≤n≤ 15, 15≤n≤25, 25≤n≤35, 35≤n≤45, 45≤n≤55, 55≤n≤60, 10≤n≤30, 30≤n≤60, 5≤n≤30, 30≤n≤40, 40≤n≤50, 50≤n≤60, 5≤n≤15, 15≤n≤25, 25≤n≤35, 35≤n≤45, 45≤n ≤55, 55≤n≤60, 1≤n≤55, 5≤n≤55, 10≤n≤60, 15≤n≤60, 25≤n≤60, 35≤n≤60, 1≤n≤4, 2≤n≤4, 3≤n≤4, 1≤n≤8, 2≤n≤8, 3≤n≤8, 4≤n≤8, 5≤n≤8, 6≤n≤8, 7≤n≤8, 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≤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≤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≤35, 2≤n≤35, 3≤n≤35, 4≤n≤35, 5≤n≤35, 6≤n≤35, 7≤n≤35, 8≤n≤35, 9≤n≤35, 10≤n≤35, 11≤n≤35, 12≤n≤35, 13≤n≤35, 14≤n≤35, 15≤n≤35, 16≤n≤35, 17≤n≤35, 18≤n≤35, 19≤n≤35, 20≤n≤35, 21≤n≤35, 22≤n≤3 5, 23≤n≤35, 24≤n≤35, 25≤n≤35, 26≤n≤35, 27≤n≤35, 28≤n≤35, 29≤n≤35, 4≤n≤40, 4≤n≤24, 4≤n≤8, 4≤n≤16, 4≤n≤24, 8≤n≤16, 8≤n≤24, 8≤n≤40, 16≤n≤24, 16≤n≤40, 24≤n≤40, 30≤n≤35, 30≤n≤40, 35≤n≤40 and any range therebetween.

[0010] 48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,90,91,92,93,94,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115

[0011] In one embodiment, the repeating unit comprises the amino acid sequence shown in SEQ ID NO.2, and the repeating number n is in the range of 2≤n≤50.

[0012] In one embodiment, the repeating unit comprises the amino acid sequence shown in SEQ ID NO.2, and the repeating number n ranges from 3≤n≤45.

[0013] In one embodiment, the repeating unit comprises the amino acid sequence shown in SEQ ID NO.2, and the repeating number n ranges from 4≤n≤40.

[0014] In one embodiment, the amino acid residue sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 are derived from human type XVII collagen, and are taken from positions 609-638 and 695-724 of the amino acid sequence of human type XVII collagen, respectively.

[0015] Optionally, the collagen may comprise a sequence as shown in SEQ ID NO.1 and SEQ ID NO.2 or a sequence thereof having 80-100% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity repeating units. The individual repeating units may be directly connected. The collagen is a recombinant type XVII collagen triple helical structure collagen. The collagen described herein may have a triple helical structure or three identical chains (i.e., in trimer form). The sequence of each chain may be a sequence as described herein.

[0016] Optionally, the amino acids of the collagen comprise the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO.3.

[0017] Optionally, the amino acids of the collagen comprise the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO.4.

[0018] Optionally, the amino acids of the collagen comprise the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO.7.

[0019] Optionally, the amino acids of the collagen comprise the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO.8.

[0020] On the other hand, the present application provides a biomaterial comprising any one of the following:

[0021] A1) a nucleic acid molecule encoding the recombinant type XVII collagen;

[0022] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0023] A3) a recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2);

[0024] A4) A recombinant microorganism containing the nucleic acid molecule described in A1), the expression cassette described in A2) or the recombinant vector described in A3).

[0025] Optionally, the expression cassette refers to DNA capable of expressing the above-mentioned proteins in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all regulatory sequences necessary for expressing any of the above-mentioned proteins. The regulatory sequences are capable of directing the coding sequence to express any of the above-mentioned proteins in a suitable host cell under compatible conditions. The regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator. At a minimum, the regulatory sequences include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences to the coding region of the protein-encoding nucleic acid sequence, the regulatory sequences may be provided with linkers. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence recognized by the host cell in which the nucleic acid sequence is to be expressed. The promoter sequence contains transcriptional regulatory sequences that mediate protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to the host cell. A regulatory sequence may also be a suitable transcriptional terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that is functional in the selected host cell may be used in this application. A regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of an mRNA that is important for translation in the host cell. The leader sequence may be operably linked to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that is functional in the selected host cell may be used in this application. A regulatory sequence may also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of the protein that directs the encoded protein into the cell's secretory pathway. Any signal peptide coding region that directs the expressed protein into the secretory pathway of the selected host cell may be used in this application. It may also be desirable to add regulatory sequences that can regulate protein expression based on the growth conditions of the host cells. Examples of regulatory systems are those that can turn gene expression on or off in response to chemical or physical stimuli (including in the presence of regulatory compounds). Other examples of regulatory sequences are those that enable gene amplification. In these instances, the protein-encoding nucleic acid sequence should be operably linked to the regulatory sequences.

[0026] Alternatively, the vector may include a nucleic acid molecule encoding the above-mentioned protein, a promoter, and transcription and translation termination signals. When preparing a recombinant vector, the nucleic acid molecule encoding the above-mentioned protein may be located in the vector so as to be operably linked to an appropriate expression control sequence. The recombinant vector may be any vector (e.g., a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of the nucleic acid sequence. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is to be introduced. The vector may be a linear or closed-loop plasmid. The vector may be an autonomously replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may include any mechanism that ensures self-replication. Alternatively, the vector may be a vector that, when introduced into a host cell, will be integrated into the genome and replicated together with the chromosome into which it is integrated. In addition, a single vector or plasmid may be used, or two or more vectors or plasmids, or transposons, that collectively comprise the entire DNA that will be introduced into the host cell genome may be included. The vector may contain one or more selectable markers that are convenient for selecting transformed cells. A selectable marker is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or prototrophy to an auxotroph. Examples of bacterial selectable markers include the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. Vectors contain elements that enable the vector to be stably integrated into the host cell genome or to ensure autonomous replication of the vector in the cell, independent of the cellular genome. In the case of autonomous replication, the vector may also contain an origin of replication that enables the vector to replicate autonomously in the target host cell. The origin of replication may contain a mutation that renders it temperature-sensitive in the host cell (see, for example, Ehrlich, 1978, Proceedings of the National Academy of Sciences of the United States of America 75:1433). One or more copies of a nucleic acid molecule encoding any of the above-described proteins may be inserted into the host cell to increase the production of the gene product. The number of copies of the nucleic acid molecule can be increased by inserting at least one additional copy of the nucleic acid molecule into the host cell genome, or by inserting an amplifiable selectable marker along with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and, thereby, the additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selective agent. The procedures used to connect the above-mentioned elements to construct the recombinant expression vectors described herein are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0027] The term "operably linked" is defined herein as a configuration in which a regulatory sequence is appropriately positioned relative to the coding sequence of a DNA sequence such that the regulatory sequence directs the expression of a protein.

[0028] Alternatively, the microorganism comprises a polynucleotide of the present application operably linked to one or more control sequences that instruct the production of the collagen protein of the present application. The construct comprising the polynucleotide is introduced into the microorganism so that the construct is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector. The term "microorganism" encompasses any parental cell progeny that is not identical to the parental cell due to a sudden change that occurs during replication. The selection of microorganism will depend to a great extent on the gene encoding the collagen protein or polypeptide and its source.

[0029] Optionally, the microorganism is selected from any one of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and yeast.

[0030] On the other hand, the present application provides a method for obtaining the recombinant type XVII collagen, comprising the step of expressing the recombinant type XVII collagen using the biomaterial.

[0031] Alternatively, the method of obtaining the recombinant collagen may be to ferment the biomaterial with microorganisms. For example, the nucleic acid molecule encoding the recombinant type XVII collagen is introduced into a blank vector and then transferred into host cells, and the host cells are cultured for expression.

[0032] Optionally, the efficacy of the recombinant type XVII collagen is selected from one or more of the following:

[0033] 1) Improve cell adhesion;

[0034] II) regulating skin homeostasis;

[0035] III) Delay skin aging;

[0036] IV) promote wound healing;

[0037] 1) Promote hair follicle repair.

[0038] Optionally, the recombinant type XVII collagen has an electron microscopic structure, and further, a fibrous structure can be observed under an electron microscope.

[0039] On the other hand, the present application provides a composition comprising the recombinant type XVII collagen or the biomaterial.

[0040] Optionally, the composition may further include substances of the same type or with similar efficacy as the recombinant type XVII collagen, and other conventional adjuvants may also be added according to the desired effect.

[0041] Optionally, the efficacy of the composition is selected from one or more of the following:

[0042] 1) Improve cell adhesion;

[0043] II) regulating skin homeostasis;

[0044] III) Delay skin aging;

[0045] IV) promote wound healing;

[0046] V) Promote hair follicle repair.

[0047] On the other hand, the present application provides the recombinant type XVII collagen, or the recombinant type XVII collagen obtained by the method, or the composition for use in preparing products for improving cell adhesion and / or regulating skin homeostasis and / or delaying skin aging and / or promoting wound healing and / or firming and / or anti-wrinkle and / or soothing and repairing.

[0048] On the other hand, the present application provides the use of the recombinant type XVII collagen, or the recombinant type XVII collagen obtained by the method, or the composition in the preparation of products promoting hair follicle repair and growth and / or hair care and / or hair growth.

[0049] On the other hand, the present application provides the use of the recombinant type XVII collagen, or the recombinant type XVII collagen obtained by the method, or the composition in the preparation of hydrating and / or skin filling products.

[0050] In another aspect, the present application provides the use of the recombinant type XVII collagen, or the biomaterial, or the composition in the preparation of daily chemical products and / or medical devices, wherein the efficacy of the daily chemical products and / or medical devices is selected from one or more of the following:

[0051] 1) Improve cell adhesion;

[0052] II) regulating skin homeostasis;

[0053] III) Delay skin aging;

[0054] IV) promote wound healing;

[0055] V) Promote hair follicle repair.

[0056] In one embodiment, the recombinant type XVII collagen is used to support the intracellular or extracellular space of animal cells.

[0057] In one embodiment, the recombinant type XVII collagen is used at a temperature not higher than 60°C, optionally not higher than 50°C, and more optionally not higher than 40°C.

[0058] On the other hand, the present application provides a daily chemical product comprising the recombinant type XVII collagen or the biomaterial.

[0059] Optionally, the daily chemical products include but are not limited to cosmetics, cleaning products, and hair care products.

[0060] On the other hand, the present application provides a medical device comprising the recombinant type XVII collagen or the biomaterial.

[0061] Optionally, medical device supplies include but are not limited to medical beauty supplies, such as injections and freeze-dried powders used for beauty.

[0062] Compared with the prior art, this application has at least the following beneficial effects:

[0063] 1. This application designed and constructed a series of recombinant type XVII collagen proteins and successfully screened and obtained a recombinant type XVII collagen protein with a triple helical structure that can achieve self-assembly. The production process of this recombinant type XVII collagen protein is simple, does not require hydroxylation, can be obtained by microbial fermentation, and has a high expression level, making it easy to expand production.

[0064] 2. The recombinant type XVII collagen provided in this application has a sequence 100% derived from a human protein sequence, has low immunogenicity, is highly safe, and has good stability in its triple helix structure, and has a strong ability to support skin cells. In efficacy experiments, it has shown that it has higher cell adhesion, wound healing, and hair follicle repair capabilities than currently available type XVII collagen products on the market, and can be widely used in cosmetics, medical devices, and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0066] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. For those skilled in the art, other drawings can be obtained based on these drawings without inventive work. In the drawings:

[0067] FIG1 is a graph showing the CD test results of recombinant type XVII collagen and commercially available animal collagen in Example 3, wherein a to e are test curves at temperatures of 4° C., 25° C., 37° C., 60° C., and 90° C., respectively.

[0068] FIG2 is a graph showing the CD test results of the recombinant type XVII collagen of Example 4 at 4° C., 25° C., and 37° C., respectively, wherein a is the test result of XVII-2-1, and b is the test result of XVII-2-2.

[0069] FIG3 is a transmission electron micrograph of the recombinant collagen XVII-2 of Example 2. DETAILED DESCRIPTION

[0070] In order to more clearly illustrate the overall concept of the application, the following is described in detail in the form of embodiments. In the following description, a large amount of specific details are provided so that a more thorough understanding of the application is provided. However, it will be apparent to those skilled in the art that the application can be implemented without the need for one or more of these details. In other examples, in order to avoid confusion with the application, some technical features well known in the art are not described.

[0071] Unless otherwise specified, in the following embodiments, all reagents or instruments used without manufacturer indication are conventional products that can be purchased commercially. If specific conditions are not indicated in the examples, conventional conditions or conditions recommended by the manufacturer were followed.

[0072] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were commercial products, and specific procedures were performed according to the kit instructions. Unless otherwise noted, the experimental methods, detection methods, and preparation methods disclosed in this application all utilize conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, specifically those in Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0073] Example 1: Design of gene sequence

[0074] From human type XVII collagen (source: UniPort: Q9UMD9), amino acid residues 609-638 and 695-724 were selected as basic repeating units, with a repeat number of n. Two recombinant type XVII collagens were designed, named XVII-1 and XVII-2, respectively. See Table 1.

[0075] Table 1 Sequence selection position and repeat number

[0076] Example 2: Obtaining recombinant type XVII collagen

[0077] 1. Construction of genetically engineered bacteria:

[0078] The nucleic acid molecules for the two recombinant proteins XVII-1 and XVII-2 designed in Table 1 of Example 1 were ligated into the BamH1 and Xho1 sites of the pET32a vector, respectively, to generate recombinant plasmids pET32a-XVII-1 and pET32a-XVII-2. The recombinant plasmids were then transformed into E. coli BL21(DE3) hosts to generate genetically engineered strains T-XVII-1 and T-XVII-2, respectively. See Table 2.

[0079] Table 2 Strain design and target protein size

[0080] 2. Expression of recombinant type XVII collagen:

[0081] A single colony of the engineered strain was picked from the plate and inoculated into 5 mL of LB medium (containing 100 μg / ml Amp) and cultured overnight at 37°C and 220 rpm. The above culture solution was transferred to 20 mL of LB medium (containing 100 μg / ml Amp) at a ratio of 1:100 and cultured at 37°C and 220 rpm. The OD 600 When the pH reached 0.6-0.8, 0.2 mM isopropylthiogalactoside (IPTG) was added and cultured at 16°C, 220 rpm for 20 hours. 10 OD of cells were harvested and suspended in 1 ml of buffer (100 mM Tris-HCl, pH 7.0). The cells were disrupted by ultrasonication. 20 μL of the supernatant was collected and 20 μL of 2× loading buffer was added. The mixture was boiled for 10 minutes and centrifuged. The supernatant was collected and subjected to SDS-PAGE, quantified using bovine serum albumin (BSA). The results are shown in Table 3.

[0082] Table 3 Protein expression of strains

[0083] From the results in Table 3, it can be seen that the two designed recombinant proteins XVII-1 and XVII-2 can both be expressed in the host E. coli, among which XVII-2 has the highest expression level, reaching 0.19 g / L.

[0084] 3. Purification of recombinant type XVII collagen:

[0085] Single colonies of the engineered strains T-XVII-1 and T-XVII-2 were picked from the plates and inoculated into 5 mL of LB medium (containing 100 μg / ml Amp) and cultured overnight at 37°C and 220 rpm. The above culture solution was transferred to 100 mL of LB medium (containing 100 μg / ml Amp) at a ratio of 1:100 and cultured at 37°C and 220 rpm. The OD 600When the pH value reached 0.6-0.8, 0.2 mM isopropylthiogalactoside (IPTG) was added and cultured at 16°C and 220 rpm for 20 h. 10 OD of bacterial cells were collected and suspended in 1 ml of buffer (100 mM Tris-HCl, pH 7.0). The bacterial cells were broken by ultrasonic wave and centrifuged at 12000 rpm for 60 min to remove the precipitate. The supernatant was taken for enzyme protein purification and the supernatant was placed on Ni 2+ The agarose affinity chromatography column was washed with binding buffer (50 mmol / L Tris-HCl, 100 mmol / L NaCl, and 25 mmol / L imidazole, pH = 7.0) for 3-4 column volumes and eluted with elution buffer (50 mmol / L Tris-HCl, 400 mmol / L NaCl, and 250 mmol / L imidazole, pH = 7.0) to obtain the pure enzyme protein. The pure enzyme protein was digested with TEV protease overnight at 4°C and then passed through a nickel column to obtain the untagged purified protein.

[0086] Example 3: Structural Characterization of Recombinant Type XVII Collagen

[0087] Circular dichroism (CD) spectra were performed using samples of collagen derived from T-XVII-1, T-XVII-2, and commercially available animal collagen. The detection conditions were as follows: the detection wavelength was set at 180 nm to 400 nm; the samples were treated with 5 mM (pH = 7.0) NaCl-free PB buffer, and liquid CD detection was performed at different detection temperature gradients of 4°C, 25°C, 37°C, 60°C, and 90°C. The results are shown in Figure 1.

[0088] A negative peak at 195nm and a positive peak at 221nm indicate a triple helical structure. Figure 1 shows that at 4°C, recombinant proteins XVII-1 and XVII-2 maintain the same triple helical structure as animal collagen. At 25°C and 37°C, only XVII-2 retains the same triple helical structure.

[0089] Example 4: Electron Microscopic Characterization of Type XVII Collagen

[0090] The collagen prepared in Example 2 was examined using a transmission electron microscope, and the TEM image was measured using ImageJ. The results are shown in FIG3 .

[0091] As shown in FIG3 , the recombinant collagen XVII-2 prepared in Example 2 can form a high-grade fiber structure.

[0092] Example 5: Structural Characterization of Recombinant Type XVII Collagen with Different Repeat Numbers

[0093] Taking the recombinant protein XVII-2 as an optional example, multiple recombinant proteins were constructed by designing the repeating units with different repeat numbers, as shown in Table 4.

[0094] Table 4 Repeating units and repeat numbers

[0095] According to the design of the recombinant protein in Table 4, the recombinant protein was prepared and purified using the method of Example 2, and the structure of the recombinant protein was characterized using the detection method of Example 3. The results are shown in Figure 2.

[0096] As shown in FIG2 , the recombinant proteins XVII-2-1 and XVII-2-2 have a triple helical structure.

[0097] Example 6: Efficacy Experiment

[0098] 1. Cell Adhesion Detection

[0099] The cell adhesion ability of the collagen prepared in Example 2 and Example 5 was tested as follows:

[0100] The cells were cultured to the logarithmic growth phase, digested with 0.25% (m / v) trypsin, and suspended in serum-free DMEM medium to adjust the concentration to 5×10 5 Cells were seeded into 5 collagen-coated 24-well plates, and 500 μL of cell suspension was added to each well, so that the number of cells in each well was 2.5×10 5 Each group of cells was plated with 3 replicate wells, and then placed in a cell culture incubator (37°C, 5% CO2) for 24 hours. After 24 hours, the culture was stopped, the culture medium was discarded, and the cells were washed 2-3 times with PBS, and the PBS was discarded. 1 ml of PFA was added to each well, fixed for 15 minutes, and the fixative was discarded. 1 mL of 0.1% crystal violet staining solution was added to each well for 20 minutes, the stain was slowly washed away with running water, and cell adhesion was photographed under a microscope. ImageJ was used to analyze the cell staining area, and the values ​​of each replicate well were averaged. The calculation formula for the cell adhesion rate is as follows:

[0101] Cell adhesion rate = cell crystal violet staining area / total area × 100%.

[0102] The results are shown in Table 5:

[0103] Table 5 Cell adhesion rate

[0104] From the results in Table 5, it can be seen that the recombinant proteins prepared in Examples 2 and 5 have significantly better cell adhesion capabilities than commercially available recombinant collagen.

[0105] 2. Cell scratch repair detection

[0106] The collagen prepared in Example 2 and Example 5 was tested for its cell scratch repair ability, and the steps were as follows:

[0107] The cells were cultured to the logarithmic growth phase, digested with 0.25% (m / v) trypsin, and suspended in serum-free DMEM medium to adjust the concentration to 5×10 5 1 mL of cell suspension was added to each well of a 24-well plate, so that the number of cells in each well was 5 × 10 5 Each group had 3 replicate wells. After the cells were cultured in complete medium until they reached 80% confluence, the cells in the wells were scratched and human fibroblasts (HSF) were treated with five types of collagen. The confluence of the cells at the scratch site was photographed using an inverted microscope at 0h, 12h, and 24h. The cell area of ​​the scratch area was analyzed using ImageJ, and the percentage of the cell migration area was calculated. The values ​​of each replicate well were averaged. The calculation formula for the percentage of the cell migration area is as follows:

[0108] The proportion of cell migration area = cell confluence area / scratch area × 100%.

[0109] The results are shown in Table 6:

[0110] Table 6 Cell migration rate

[0111] From the results in Table 6, it can be seen that compared with commercially available recombinant collagen and the control group, the recombinant collagen provided in the present application has a higher cell migration rate and is suitable for preparing relevant functional products.

[0112] 3. Detection of gene regulation related to hair follicle repair

[0113] The recombinant collagen prepared in Example 2 was used to detect hair follicle-related genes using qPCR technology, and the steps were as follows:

[0114] The cells were cultured to the logarithmic growth phase, digested with 0.25% (m / v) trypsin, and suspended in serum-free DMEM medium to adjust the concentration to 5×10 5 1×10 cells / mL, and inoculated into 6-well plates, adding 2 mL of cell suspension to each well so that the number of cells in each well was 1×10 6Each group had 3 replicate wells. After the cells were cultured in complete medium until they reached 80% confluence, human dermal papilla cells were treated with 1 mg / ml collagen sample (37°C, 5% CO2) for 24 hours, and cell RNA was collected for qPCR. Total RNA was isolated from the cell pellet using Trizol reagent, and RNA was converted into cDNA using a cDNA reverse transcription kit. WNT10a, β-catenin, and VEGFA mRNA expression levels were measured and analyzed using SybrGreen mix and primers to prepare a reaction solution on a real-time fluorescence quantitative PCR system. GAPDH was used as an internal reference to analyze relative mRNA expression.

[0115] The results are shown in Table 7:

[0116] Table 7 Hair follicle repair-related functional genes

[0117] The results in Table 7 show that, compared to commercially available recombinant collagen, both recombinant proteins XVII-1 and XVII-2 can activate the hair follicle repair-related gene Wnt10a, activating hair follicle stem cells and shifting dormant hair follicles into the growth phase. Furthermore, XVII-1 and XVII-2 can also activate the β-catenin gene, promoting hair follicle repair. Furthermore, XVII-1 and XVII-2 can also upregulate the expression of vascular endothelial growth factor (VEGFA), inducing angiogenesis in the dermal papilla to regulate the cyclical growth of hair follicles and maintain and promote hair follicle growth. Therefore, recombinant proteins XVII-1 and XVII-2 possess excellent abilities to promote hair follicle repair and growth, making them suitable for the preparation of related efficacy products.

[0118] 4. Detection of the effect of recombinant collagen type XVII on the expression of other collagens

[0119] Taking XVII-2 collagen as an example, its effect on the content of Collagen I, Collagen III and MMP-9 was detected. The steps are as follows:

[0120] Human fibroblasts were used and irradiated when the cell plating rate in 24-well plates reached 40-60%. The negative control group, positive control group and sample group received 9J / cm 2 The blank control group was placed in the same environment (UVA radiation dose of 0 J / cm 2After irradiation, 1 ml of cell culture medium was added to each well of the blank control (BC) and negative control (NC) cells. 1 mL of culture medium containing 100 μg / mL VC and 7 μg / mL VE was added to each well of the positive control (PC). 1 mL of XVII-2 recombinant protein sample at varying concentrations was added to each well of the sample group. After 24 hours of incubation, the cell culture supernatant was collected into EP tubes and stored in a -80°C freezer. Collagen I, Collagen III, and MMP-9 levels were assayed according to the ELISA kit's instructions. The results are shown in Table 8:

[0121] Table 8 Effects of XVII-2 on other collagens and MMP-9

[0122] The results in Table 8 show that recombinant collagen XVII-2 significantly increased Collagen I content in human fibroblasts at different concentrations, promoted the expression of type III collagen, and significantly reduced the content of matrix metalloproteinase MMP-9 in human fibroblasts. Specifically, XVII-2 collagen promoted the expression of type I and type III collagens and significantly reduced the expression of MMP-9, demonstrating its anti-wrinkle and firming properties.

[0123] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. Recombinant type XVII collagen, wherein: The amino acid sequence of the collagen comprises one or more repeating units of amino acid residues, wherein the repeating unit comprises a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the number of repeats is n, 1<n≤60.

2. The recombinant type XVII collagen according to claim 1, wherein The repeating unit comprises an amino acid sequence as shown in SEQ ID NO.2, and the repeating number n ranges from 2≤n≤18.

3. The recombinant type XVII collagen according to claim 1 or 2, wherein The amino acids of the collagen include the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence that is at least 90% identical to SEQ ID NO.3, and / or the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence that is at least 90% identical to SEQ ID NO.4, and / or the amino acid sequence shown in SEQ ID NO.7 or an amino acid sequence that is at least 90% identical to SEQ ID NO.7, and / or the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence that is at least 90% identical to SEQ ID NO.

8.

4. Biomaterials, wherein Choose from any of the following: A1) a nucleic acid molecule encoding the recombinant type XVII collagen according to any one of claims 1 to 3; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2); A4) A recombinant microorganism containing the nucleic acid molecule described in A1), the expression cassette described in A2) or the recombinant vector described in A3).

5. A method for obtaining recombinant type XVII collagen, wherein: The method comprises the step of using the biomaterial as claimed in claim 4 to express the recombinant type XVII collagen.

6. A composition, wherein The composition comprises the recombinant type XVII collagen according to any one of claims 1 to 3 or the recombinant type XVII collagen obtained by the method according to claim 5.

7. Use of the recombinant type XVII collagen as described in any one of claims 1 to 3, or the recombinant type XVII collagen obtained by the method as described in claim 5, or the composition as described in claim 6 in the preparation of daily chemical products, medical device products, medicines and / or tissue engineering products.

8. Use of the recombinant type XVII collagen as described in any one of claims 1 to 3, or the recombinant type XVII collagen obtained by the method according to claim 5, or the composition as described in claim 6 in the preparation of products that improve cell adhesion and / or regulate skin homeostasis and / or delay skin aging and / or promote wound healing and / or tighten and / or anti-wrinkle and / or soothing and repairing.

9. Use of the recombinant type XVII collagen as claimed in any one of claims 1 to 3, or the recombinant type XVII collagen obtained by the method as claimed in claim 5, or the composition as claimed in claim 6 in the preparation of products for promoting hair follicle repair and growth and / or hair care and / or hair growth.

10. Use of the recombinant type XVII collagen according to any one of claims 1 to 3, or the recombinant type XVII collagen obtained by the method according to claim 5, or the composition according to claim 6 in the preparation of hydrating and / or skin filling products.

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