Recombinant XVII type collagen with triple helix structure and application of recombinant XVII type collagen

Abstract

The invention provides a recombinant XVII type collagen with a triple helix structure, and belongs to the technical field of biological proteins. The amino acid sequence of the collagen comprises one or more amino acid residue repetitive units, each repetitive unit comprises a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, the number of repetitions is n, and n is larger than 1 and smaller than or equal to 60. The recombinant XVII type collagen provided by the invention is simple in production process, relatively high in expression quantity, low in immunogenicity, high in safety, relatively good in stability of the triple-helix structure, and strong in supporting capacity for skin cells; efficacy experiments show that the collagen has higher cell adhesion, wound healing promotion and hair follicle repair promotion capability than XVII type collagen products in the market at present, and can be widely applied to the fields of food, health care products, cosmetics, medical instruments and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biological proteins, and in particular to a recombinant type XVII collagen with a triple helix structure and an application thereof. Background Art

[0002] There are 28 types of collagen, accounting for about 30% of the total protein in the human body. Collagen is mainly distributed in the skin, tendons, bones, blood vessels, cornea, etc., with molecular weights ranging from 50 to 340 kDa. Among them, type XVII collagen is mainly distributed in the skin and epithelium. It is a transmembrane protein composed of 3 α chains, each of which is 180 kDa. Type XVII collagen can mediate the interaction between stem cells and surrounding cells and the matrix, and has the functions of regulating skin homeostasis, aging and wound repair. It has broad application prospects in the fields of cosmetics and medical devices.

[0003] 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. There are high technical barriers to achieving collagen with a correct triple helix structure using synthetic biology technology in microbial fermentation. Therefore, the design and realization of recombinant type XVII collagen with a triple helix structure is still a technical problem that needs to be overcome in the production of recombinant collagen by microbial fermentation. Summary of the invention

[0004] In order to solve the above problems, the present invention aims to provide a recombinant type XVII collagen with a triple helical structure that is simple to prepare, has a high expression level, has good efficacy, and can achieve self-assembly.

[0005] 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, the repeating unit comprising the sequence shown in SEQ ID NO.1, the number of repetitions being n, 1<n≤60.

[0006] 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. ,

[0007] 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, the repeating unit comprising the sequence shown in SEQ ID NO.2, the number of repetitions being n, 1<n≤60.

[0008] 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. ,

[0009] 48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115

[0010] In one embodiment, the repeating unit comprises an amino acid sequence as shown in SEQ ID NO.2, and the repeating number n ranges from 2≤n≤50.

[0011] In one embodiment, the repeating unit comprises an amino acid sequence as shown in SEQ ID NO.2, and the repeating number n ranges from 3≤n≤45.

[0012] In one embodiment, the repeating unit comprises an amino acid sequence as shown in SEQ ID NO.2, and the repeating number n ranges from 4≤n≤40.

[0013] 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.

[0014] Optionally, the collagen may comprise a sequence 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. Each repeating unit 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 described herein.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] On the other hand, the present application provides a biomaterial comprising any one of the following:

[0020] A1) a nucleic acid molecule encoding the recombinant type XVII collagen;

[0021] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0022] A3) a recombinant vector containing the nucleic acid molecule described in A1) or the expression cassette described in A2);

[0023] 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).

[0024] Optionally, the expression cassette refers to a DNA capable of expressing the above-mentioned protein in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecule of any of the above-mentioned proteins. The regulatory sequence can guide the coding sequence to express any of the above-mentioned proteins in a suitable host cell under its compatible conditions. The regulatory sequence includes, but is 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 sequence should include a promoter and termination signals for transcription and translation. In order to introduce specific restriction enzyme sites of the vector so as to connect the regulatory sequence to the coding region of the nucleic acid sequence encoding the protein, a regulatory sequence with a joint can be provided. The regulatory sequence can be a suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter can be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular protein that is homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence may be operably linked to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a suitable leader sequence, i.e., an untranslated region of mRNA that is important for translation of 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 can function in the selected host cell may be used in the present invention. The regulatory sequence may also be a signal peptide coding region that encodes an amino acid sequence attached to the amino terminus of the protein that can guide the encoded protein into the cell secretory pathway. Signal peptide coding regions that can guide the expressed protein into the secretory pathway of the host cell used may be used in the present invention. It may also be desirable to add regulatory sequences that can regulate protein expression according to the growth of the host cell. Examples of regulatory systems are those that can respond to chemical or physical stimuli (including in the presence of regulatory compounds) to open or close gene expression. Other examples of regulatory sequences are those that can amplify genes. In these cases, the nucleic acid sequence encoding the protein should be operably linked to the regulatory sequences.

[0025] Optionally, 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 the 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 contain any mechanism that ensures self-replication. Alternatively, the vector is 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 generally contain all the DNA that will be introduced into the host cell genome may be included. The vector may contain one or more selection 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 confers prototrophy to an auxotroph, etc. Examples of bacterial selectable markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline. The vector contains elements that enable the vector to be stably integrated into the host cell genome, or that ensure that the vector replicates autonomously in the cell independently of the cell 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 carry a mutation that makes it temperature-sensitive in the host cell (see, e.g., fEhrlich, 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 proteins of the present invention may be inserted into the host cell to increase the yield 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 inserting an amplifiable selection marker together with the nucleic acid molecule, and selecting cells containing amplified copies of the selectable marker gene and thus the additional copies of the nucleic acid molecule by culturing the cells in the presence of a suitable selection agent. The operations for connecting the above-mentioned elements to construct the recombinant expression vector of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).

[0026] 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.

[0027] Optionally, the microorganism comprises a polynucleotide of the present invention operably linked to one or more control sequences that instruct the production of the collagen of the present invention. 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 parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The selection of microorganisms will depend to a large extent on the genes encoding collagen or polypeptides and their sources.

[0028] Optionally, the microorganism is selected from any one of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and yeast.

[0029] 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.

[0030] Alternatively, the method of obtaining the recombinant collagen may be to use the biomaterial for microbial fermentation. For example, the nucleic acid molecule encoding the recombinant type XVII collagen is introduced into a blank vector and then transferred into a host cell, and the host cell is cultured for expression.

[0031] Optionally, the efficacy of the recombinant type XVII collagen is selected from one or more of the following:

[0032] 1) Improve cell adhesion;

[0033] II) regulate skin homeostasis;

[0034] III) Delay skin aging;

[0035] IV) Promote wound healing;

[0036] I) Promote hair follicle repair.

[0037] Optionally, the recombinant type XVII collagen has an electron microscopic structure, specifically, a fibrous structure that can be observed under an electron microscope.

[0038] On the other hand, the present application provides a composition comprising the recombinant type XVII collagen or the biomaterial.

[0039] Optionally, the composition may also 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 required effect.

[0040] Optionally, the efficacy of the composition is selected from one or more of the following:

[0041] 1) Improve cell adhesion;

[0042] II) regulate skin homeostasis;

[0043] III) Delay skin aging;

[0044] IV) Promote wound healing;

[0045] V) Promote hair follicle repair.

[0046] 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 the preparation of 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.

[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 that promote hair follicle repair and growth and / or hair care and / or hair growth.

[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 hydrating and / or skin filling products.

[0049] On the other hand, 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 device products, wherein the efficacy of the daily chemical products and / or medical device products is selected from one or more of the following:

[0050] 1) Improve cell adhesion;

[0051] II) regulate skin homeostasis;

[0052] III) Delay skin aging;

[0053] IV) Promote wound healing;

[0054] V) Promote hair follicle repair.

[0055] In one embodiment, the recombinant type XVII collagen is used to support the intracellular or extracellular space of animal cells.

[0056] In one embodiment, the recombinant type XVII collagen is used at a temperature not higher than 60°C, preferably not higher than 50°C, more preferably not higher than 40°C.

[0057] On the other hand, the present application provides a daily chemical product, comprising the recombinant type XVII collagen or the biomaterial.

[0058] Optionally, the daily chemical products include but are not limited to cosmetics, cleaning products, and hair care products.

[0059] On the other hand, the present application provides a medical device, comprising the recombinant type XVII collagen or the biomaterial.

[0060] Optionally, medical device supplies include but are not limited to medical beauty supplies, such as injections and freeze-dried powders used for beauty.

[0061] Compared with the prior art, the present invention has at least the following beneficial effects:

[0062] 1. The present application designs and constructs a series of recombinant XVII collagen proteins, and successfully screens and obtains recombinant XVII collagen proteins with a triple helix structure that can achieve self-assembly. The production process of the recombinant XVII collagen protein is simple, and no hydroxylation is required. It can be obtained by microbial fermentation, and the expression level is high, which is easy to expand production.

[0063] 2. The recombinant type XVII collagen provided in the present 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 abilities to improve cell adhesion, promote wound healing, and promote hair follicle repair than the type XVII collagen products currently on the market, and can be widely used in the fields of cosmetics and medical devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0065] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the prior art descriptions. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0066] Figure 1 This 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.

[0067] Figure 2These are 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.

[0068] Figure 3 This is a transmission electron microscopy image of the recombinant collagen XVII-2 in Example 2. DETAILED DESCRIPTION

[0069] In order to more clearly explain the overall concept of the application, the following is described in detail by way of embodiment. In the following description, a large amount of specific details are provided so as to provide a more thorough understanding of the application. However, it is obvious to those skilled in the art that the application can be implemented without 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.

[0070] Unless otherwise specified, in the following embodiments, reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market. If no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer are followed.

[0071] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed according to the kit instructions.

[0073] It should be noted that the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0074] Example 1: Design of gene sequence

[0075] The amino acid residue sequences at positions 609-638 and 695-724 were selected from human type XVII collagen (source: UniPort: Q9UMD9) as basic repeating units, with a repeat number of n, and two recombinant type XVII collagens were designed, named XVII-1 and XVII-2, respectively. See Table 1 for details.

[0076] Table 1 Sequence selection position and repeat number

[0077]

[0078] Example 2: Obtaining recombinant type XVII collagen

[0079] 1. Construction of genetically engineered bacteria:

[0080] The nucleic acid molecules of the two recombinant proteins XVII-1 and XVII-2 designed in Table 1 of Example 1 were respectively connected to the BamH1 site and Xho1 site of the vector pET32a to obtain recombinant plasmids pET32a-XVII-1 and pET32a-XVII-2, and the recombinant plasmids were respectively transformed into the E. coli BL21 (DE3) host to obtain genetically engineered strains T-XVII-1 and T-XVII-2. See Table 2 for details.

[0081] Table 2 Strain design and target protein size

[0082] strain Plasmids Theoretical size of target protein (KDa) T-XVII-1 pET32a-XVII-1 48 T-XVII-2 pET32a-XVII-2 48

[0083] 2. Expression of recombinant type XVII collagen:

[0084] Pick a single colony of the engineering strain from the plate and inoculate it in 5mL of LB medium (containing 100μg / ml Amp) at 37℃, 220rpm overnight culture. Take the above culture solution 1:100 and transfer it to 20mL of LB medium (containing 100μg / ml Amp), culture at 37℃, 220rpm, and when OD reaches 0.6-0.8, add 0.2mM isopropylthiogalactoside (IPTG), and culture at 16℃, 220rpm for 20h. Collect 10OD bacteria, suspend with 1ml buffer (100mM Tris-HCl, pH7.0), ultrasonically break the bacterial cells, take 20μL of supernatant, add 20μL of 2× loading buffer, boil for 10min, centrifuge, take supernatant, perform SDS PAGE, and use bovine serum albumin (BSA) for quantification. The results are shown in Table 3.

[0085] Table 3 Protein expression of strains

[0086] strain Expression T-XVII-1 0.05g / L T-XVII-2 0.19g / L

[0087] From the results in Table 3, it can be seen that the two designed recombinant proteins XVII-1 and XVII-2 can be expressed in the host E. coli, among which XVII-2 has the highest expression level, reaching 0.19 g / L.

[0088] 3. Purification of recombinant type XVII collagen:

[0089] Single colonies of 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 at 37°C and 220 rpm overnight. 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. When the OD 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 bacteria were collected, suspended with 1 ml of buffer (100 mM Tris-HCl, pH 7.0), the bacterial cells were broken by ultrasonic wave, and the precipitate was removed by centrifugation at 12000 rpm for 60 min. The supernatant was taken for enzyme protein purification, and the supernatant sample was placed in Ni 2+ The agarose affinity chromatography column was washed with Binding buffer (50mmol / L Tris-HCl, 100mmol / L NaCl and 25mmol / L imidazole, pH = 7.0) for 3 to 4 column volumes, and finally eluted with Elution buffer (50mmol / L Tris-HCl, 400mmol / L NaCl and 250mmol / L imidazole, pH = 7.0) to obtain pure enzyme protein. The pure enzyme protein was digested with TEV protease at 4°C overnight, and then flowed through a nickel column to obtain purified protein without tags.

[0090] Example 3: Structural Characterization of Recombinant Type XVII Collagen

[0091] Circular dichroism spectra were tested using samples of collagen from T-XVII-1, T-XVII-2 and commercially available animals. The test conditions were as follows: the detection wavelength was set at 180 nm-400 nm; the samples were treated with 5 mM (pH = 7.0) PB buffer without NaCl, and liquid CD was tested at different detection temperature gradients of 4 ° C, 25 ° C, 37 ° C, 60 ° C, and 90 ° C. The results are shown in the figure. Figure 1 shown.

[0092] Among them, when the detection curve has a negative peak at 195nm and a positive peak at 221nm, it is a triple helix structure. Figure 1 The results showed that at 4°C, the recombinant proteins XVII-1 and XVII-2 had the same triple helical structure as animal collagen; at 25°C and 37°C, only XVII-2 still had the same triple helical structure as animal collagen.

[0093] Example 4: Electron microscopic characterization of type XVII collagen

[0094] The collagen prepared in Example 2 was detected using a transmission electron microscope; the TEM image was measured using ImageJ. The results obtained are as follows: Figure 3 shown.

[0095] Depend on Figure 3 It can be seen that the XVII-2 recombinant collagen prepared in Example 2 can form a high-order fiber structure.

[0096] Example 5: Structural characterization of recombinant type XVII collagen with different repeat numbers

[0097] Taking the recombinant protein XVII-2 as a preferred example, multiple recombinant proteins were designed with different repeat numbers of repeating units, as shown in Table 4.

[0098] Table 4 Repeating units and repeating numbers

[0099]

[0100] According to the design of the recombinant protein in Table 4, the method of Example 2 was used to prepare and purify the recombinant protein, and the detection method of Example 3 was used to characterize the structure of the recombinant protein. The results are shown in FIG. Figure 2 shown.

[0101] Depend on Figure 2 The results in show that the recombinant proteins XVII-2-1 and XVII-2-2 have a triple helical structure.

[0102] Example 6: Efficacy Experiment

[0103] 1. Cell Adhesion Detection

[0104] The cell adhesion ability of the collagen prepared in Example 2 and Example 5 was tested. The specific steps are as follows:

[0105] 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 5 500 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 divided into three 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, and the staining solution was slowly washed away with running water. 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:

[0106] Cell adhesion rate = cell crystal violet staining area / total area × 100%.

[0107] The results are shown in Table 5:

[0108] Table 5 Cell adhesion rate

[0109] Cell adhesion rate (%) Comparison 104.85％±4.78％ XVII-1 139.39％±9.99％ XVII-2 162.45％±12.07％ XVII-2-1 160.05％±9.08％ XVII-2-2 157.20％±11.02％ Commercially available recombinant collagen 41.79％±7.23％

[0110] 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 ability than commercially available recombinant collagen.

[0111] 2. Cell scratch repair detection

[0112] The cell scratch repair ability of the collagen prepared in Example 2 and Example 5 was tested. The specific steps are as follows:

[0113] 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 has 3 replicate wells. After the cells are cultured in complete medium until they reach 80% confluence, scratch the cells in the wells, treat human fibroblasts HSF with five types of collagen, and use an inverted microscope to photograph the confluence of the cells at the scratches at 0h, 12h, and 24h. Use ImageJ to analyze the cell area in the scratch area, calculate the percentage of the cell migration area, and take the average of the values ​​of each replicate well. The calculation formula for the percentage of the cell migration area is as follows:

[0114] The percentage of cell migration area = cell confluence area / scratch area × 100%.

[0115] The results are shown in Table 6:

[0116] Table 6 Cell migration rate

[0117]

[0118] It can be seen from the results in Table 6 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.

[0119] 3. Detection of gene regulation related to hair follicle repair

[0120] The recombinant collagen prepared in Example 2 was tested for hair follicle-related genes using qPCR technology. The specific steps are as follows:

[0121] 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. 2 mL of cell suspension was added to each well so that the number of cells in each well was 1×10 6 cells, with 3 replicates in each group. After the cells were cultured in complete medium until 80% confluence was reached, 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. The expression levels of WNT10a, β-catenin, and VEGFA mRNA were measured and analyzed on a real-time fluorescence quantitative PCR system using SybrGreenmix and primers to prepare a reaction solution. GAPDH was used as an internal reference to analyze relative mRNA expression.

[0122] The results are shown in Table 7:

[0123] Table 7 Hair follicle repair related functional genes

[0124]

[0125] As shown in Table 7, compared with commercially available recombinant collagen, recombinant proteins XVII-1 and XVII-2 can both activate the hair follicle repair-related gene Wnt10a, activate hair follicle stem cells, and cause dormant hair follicles to enter the growth phase. In addition, XVII-1 and XVII-2 can also activate the β-catenin gene to promote hair follicle repair. At the same time, XVII-1 and XVII-2 can also promote the up-regulation of vascular endothelial growth factor VEGFA, induce dermal hair papilla angiogenesis to regulate the periodic cycle of hair follicles, and maintain and promote hair follicle growth. Therefore, recombinant proteins XVII-1 and XVII-2 have a good ability to promote hair follicle repair and growth, and are suitable for the preparation of related efficacy products.

[0126] 4. Detection of the effect of type XVII recombinant collagen on the expression of other collagens

[0127] Taking XVII-2 collagen as an example, its effect on the content of Collagen I, Collagen III and MMP-9 was detected. The specific steps are as follows:

[0128] Human fibroblasts were used and irradiated when the cell plating rate reached 40-60% in a 24-well plate. The negative control group, positive control group, and sample group received 9 J / cm 2The blank control group was placed in the same environment (UVA radiation dose of 0 J / cm 2 ), after the irradiation, 1 ml of cell culture medium was added to each well of the blank control group (BC) and the negative control group (NC), 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), and 1 mL of XVII-2 recombinant protein samples of different concentrations were added to each well of the sample group; after incubation for 24 hours, the cell culture supernatant was collected in an EP tube and stored in a -80 refrigerator. The contents of Collagen I, Collagen III and MMP-9 were detected and analyzed according to the operating instructions of the ELISA detection kit, and the results are shown in Table 8:

[0129] Table 8 Effects of XVII-2 on other collagens and MMP-9

[0130]

[0131] From the results in Table 8, it can be seen that recombinant collagen XVII-2 can significantly increase the content of Collagen I in human fibroblasts at different concentrations, promote the expression of type III collagen, and significantly reduce the content of matrix metalloproteinase MMP-9 in human fibroblasts. That is, XVII-2 collagen can promote the expression of type I and type III collagen, and significantly reduce the expression of MMP-9, indicating that XVII-2 protein has anti-wrinkle and firming effects.

[0132] The above is only 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 modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. Recombinant type XVII collagen, characterized in that 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, characterized in that 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, characterized in that 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. Biomaterial, characterized in that 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, characterized in that: The method comprises the step of using the biomaterial as claimed in claim 4 to express the recombinant type XVII collagen.

6. A composition, characterized in that 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.

Images