Humanized recombinant X VII collagen for promoting hair follicle stem cell repair and resisting aging as well as preparation method and application of humanized recombinant X VII collagen

Abstract

The invention provides humanized recombinant XVII collagen for promoting hair follicle stem cell repair and resisting aging as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The humanized recombinant XVII type collagen comprises polypeptide derived from the XVII type collagen or repeated protein formed by N times of repeated series connection of the polypeptide serving as a unit, n is an integer from 2 to 4; the polypeptide derived from the XVII type collagen comprises a polypeptide A and / or a polypeptide B; the amino acid sequence of the A polypeptide is as shown in SEQ ID NO: 1; and the amino acid sequence of the B polypeptide is as shown in SEQ ID NO: 2. The humanized recombinant XVII type collagen not only can be massively and stably recombined and expressed, but also has cell adhesion activity, cell proliferation promotion activity, cell migration promotion activity and excellent hair follicle repair and regeneration promotion activity, and has no sensitization side effect. The invention provides a new way for preparing collagen products with rich functions.

Description

Technical Field

[0001] The present invention belongs to the technical field of collagen, and specifically relates to a humanized recombinant type XVII collagen for promoting the repair of hair follicle stem cells and anti-aging, and a preparation method and application thereof. Background Art

[0002] Collagen is a biological macromolecular fibrin, which is one of the most abundant and widely distributed functional proteins in the extracellular matrix, accounting for about 1 / 3 of the total protein. In the composition of human skin proteins, collagen plays an important role. It can form a structural scaffold for tissues or wound healing, promote cell adhesion and chemotaxis, contribute to tissue repair, and provide strength and support for the skin. At present, at least 49 different collagen polypeptide chains have been discovered, and only 28 types of collagen can be artificially synthesized.

[0003] Human type XVII (encoded by the COL17A1 gene) collagen is a transmembrane non-fibroblast collagen, which is a homogeneous trimer composed of three identical α1 (type XVII) chains, and is divided into three major domains: intracellular, transmembrane, and extracellular. The COL17A1 protein is a component of hemidesmosomes in cells and plays an important role in the epithelial cell-basement membrane interaction. It can regulate the adhesion, separation, development and differentiation of epithelial cells, and has an important role in the differentiation and regeneration of hair follicle cells. However, unfortunately, due to the presence of a helical region in human type XVII collagen, which has strong thermal stability, it is difficult to be secreted extracellularly to play a role, which severely limits the industrial application of human type XVII collagen. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a humanized recombinant type XVII collagen, which is obtained by separating a specific region from the type XVII collagen sequence for fusion, and the obtained fusion protein can be successfully recombinantly expressed and has biological activity.

[0005] The present invention provides a humanized recombinant type XVII collagen, which includes a polypeptide derived from type XVII collagen or a repetitive protein formed by repeating the polypeptide as a unit N times in series; N is an integer from 2 to 4;

[0006] The polypeptide sequence derived from type XVII collagen includes an A polypeptide sequence and / or a B polypeptide sequence;

[0007] The amino acid sequence of the A polypeptide sequence is as shown in SEQ ID NO:1;

[0008] The amino acid sequence of the B polypeptide sequence is as shown in SEQ ID NO:2.

[0009] Preferably, the recombinant protein includes JY001 and JY002;

[0010] The amino acid sequence of JY001 is as shown in SEQ ID NO:3;

[0011] The amino acid sequence of JY002 is as shown in SEQ ID NO:4.

[0012] The present invention provides a nucleic acid molecule encoding the humanized recombinant type XVII collagen.

[0013] Preferably, it includes a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO:5 to SEQ ID NO:8.

[0014] The present invention provides a gene-derived product expressing the humanized recombinant type XVII collagen.

[0015] Preferably, the gene-derived product includes at least one of the following: gene expression cassette, recombinant vector, recombinant cell, and recombinant virus strain;

[0016] Preferably, the backbone vector of the recombinant vector includes pSUMO3;

[0017] The cloning sites of the nucleic acid molecule encoding the humanized recombinant type XVII collagen in pSUMO3 include BamHⅠ and XhoⅠ.

[0018] The present invention provides a method for preparing the humanized recombinant type XVII collagen, comprising the following steps:

[0019] Clone the nucleic acid molecule encoding the humanized recombinant type XVII collagen into an expression vector to construct a recombinant expression vector;

[0020] Transfer the recombinant expression vector into a host bacterium for culture to obtain a recombinant strain;

[0021] Perform recombinant expression of the protein on the recombinant strain, separate and purify the recombinant protein to obtain the humanized recombinant type XVII collagen.

[0022] Preferably, for the construction method of the recombinant expression vector, the nucleic acid molecule encoding the humanized recombinant type XVII collagen with BamHⅠ restriction enzyme site and XhoⅠ restriction enzyme site and pSUMO3 are respectively digested with BamHⅠ and XhoⅠ to obtain the digested nucleic acid molecule and the linear pSUMO3 vector;

[0023] Ligate the digested nucleic acid molecule and the linear pSUMO3 vector to obtain the recombinant expression vector.

[0024] The present invention provides the use of the humanized recombinant type XVII collagen or the humanized recombinant type XVII collagen prepared by the nucleic acid molecule, the gene derivative product or the preparation method in the preparation of a product for promoting cell migration and / or hair growth.

[0025] Preferably, the product includes medical supplies and / or daily necessities related to skin repair or anti-aging skin care of the face and head.

[0026] The present invention provides a humanized recombinant type XVII collagen, which includes a polypeptide derived from type XVII collagen or a repetitive protein formed by N tandem connections with the polypeptide as a unit; N is an integer from 2 to 4; the polypeptide derived from type XVII collagen includes polypeptide A and / or polypeptide B; the amino acid sequence of polypeptide A is as shown in SEQ ID NO:1; the amino acid sequence of polypeptide B is as shown in SEQ ID NO:2. In the present invention, the G767-844G region is intercepted from type XVII collagen (Q9UMD9) in the protein database to form polypeptide A, and at the same time, the G906-938S and G951-977P regions are intercepted and fused to form polypeptide B. The humanized recombinant type XVII collagen can not only be stably expressed in large quantities, but also has high cell adhesion activity, cell proliferation promoting activity, cell migration promoting activity and excellent biological activity of promoting hair follicle repair and regeneration; at the same time, through optimized screening from multiple helical region sequences of human type XVII collagen, the risk of sensitization can be reduced and virus infection can be avoided. The present invention enriches the types of collagen and provides a new way for the preparation of collagen products with rich functions. At the same time, the recombinant humanized type XVII collagen has low endotoxin, low cost and no immunogenicity, can be prepared in large quantities, effectively makes up for the market gap and enriches similar collagen products.

[0027] The present invention also provides a preparation method of the humanized recombinant type XVII collagen, including the following steps: cloning the nucleic acid molecule encoding the humanized recombinant type XVII collagen into an expression vector to construct a recombinant expression vector; transferring the recombinant expression vector into a host bacterium for culture to obtain a recombinant strain; performing recombinant expression of the protein on the recombinant strain, separating and purifying the recombinant protein to obtain the humanized recombinant type XVII collagen. The production process of the present invention is simple, rapid, easy to operate, has good repeatability, and has low cost and high yield, providing a production basis for the large-scale popularization of derivative products of humanized recombinant type XVII collagen. Description of the Drawings

[0028] Figure 1 It is the vector map of pSUMO3-JY001 in Example 1;

[0029] Figure 2Vector map of pSUMO3-JY002 in Example 1;

[0030] Figure 3 SDS-PAGE detection result graph of the expression of JY001 and JY002 in the supernatant of Escherichia coli in Example 1;

[0031] Figure 4 Microscopic observation test cell result graph of the promotion of cell migration by JY001, JY002 and human serum albumin in Example 1;

[0032] Figure 5 Result graph of the promotion of cell migration by JY001, JY002 and human serum albumin in Example 1;

[0033] Figure 6 Result graph of the hair growth experiment of JY001, JY002 and human serum albumin in mice in Example 1. Detailed implementation mode

[0034] The present invention provides a humanized recombinant type XVII collagen, which comprises a polypeptide derived from type XVII collagen or a repetitive protein formed by N - time repeated tandem of the polypeptide as a unit; N is an integer from 2 to 4; the polypeptide derived from type XVII collagen comprises polypeptide A and / or polypeptide B; the amino acid sequence of polypeptide A is as shown in SEQ ID NO:1 (GPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPA); the amino acid sequence of polypeptide B is as shown in SEQ ID NO:2 (GPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSGPPGPPGPQGPKGDKGDPGVPGALGIP).

[0035] In the present invention, the accession number of type XVII collagen in the UniProt protein database is Q9UMD9. The present invention screens the G767 - 844G region in the primary structure of type XVII collagen to form polypeptide A, and intercepts the G906 - 938S and G951 - 977P regions to fuse and form polypeptide B.

[0036] In the present invention, the recombinant protein formed by tandem repetition of the A polypeptide unit twice is JY001. The recombinant protein formed by tandem repetition of the B polypeptide unit twice is JY002. The amino acid sequence of JY001 is preferably as shown in SEQ ID NO:3 (GPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPAGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPA). The amino acid sequence of JY002 is preferably as shown in SEQ ID NO:4 (GPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSGPPGPPGPQGPKGDKGDPGVPGALGIP).

[0037] The present invention provides a nucleic acid molecule encoding the humanized recombinant type XVII collagen.

[0038] In the present invention, the nucleic acid molecule is preferably codon-optimized according to the species of the host to obtain a codon-optimized nucleic acid molecule. The nucleotide sequence of the nucleic acid molecule of polypeptide A is as shown in SEQ ID NO:5 (GGTCCTCCGGGCCCTTCTGGCGATCCGGGTAAACCAGGTCTGACCGGCC CGCAGGGTCCGCAAGGCCTGCCGGGTACCCCGGGCCGTCCGGGTATTAAAGGTGAACCAGGTGCCCCGGGCAAAATCGTAACCAGCGAAGGCTCTTCCATGCTGACTGTTCCGGGCCCGCCAGGCCCTCCAGGTGCTATGGGTCCGCCTGGTCCGCCAGGTGCACCGGGTCCTGCTGGTCCAGCA). The nucleotide sequence of the nucleic acid molecule of polypeptide B is as shown in SEQ ID NO:6 (GGTCCACCAGGCCCGCCGGGCCCACCAGGTCCAAAAGGTGACCAAGGT CCACCGGGCCCACGTGGTCATCAGGGTGAACAGGGCCTGCCGGGCTTCTC TGGCCCACCGGGTCCACCTGGTCCACAGGGCCCGAAAGGCGACAAAGGT GACCCGGGCGTGCCGGGTGCTCTGGGTATTCCG).

[0039] The nucleotide sequence of the nucleic acid molecule of JY001 is as shown in SEQ ID NO:7 (GGTCCTCCGGGTCCGTCTGGTGATCCGGGTAAACCTGGTCTGACTGGTC CACAAGGTCCACAGGGTCTGCCGGGTACCCCAGGTCGTCCAGGTATCAAAGGTGAACCGGGTGCGCCGGGTAAAATCGTGACTTCCGAAGGTTCTTCTATGCTGACTGTGCCGGGTCCTCCAGGTCCACCAGGTGCAATGGGTCCACCAGGTCCACCAGGTGCTCCTGGTCCGGCTGGTCCAGCAGGTCCACCTGGTCCTTCTGGTGATCCAGGTAAACCGGGTCTGACTGGTCCTCAGGGTCCTCAGGGCCTGCCTGGTACTCCAGGTCGTCCGGGTATCAAAGGCGAACCAGGTGCGCCGGGTAAAATTGTTACTTCTGAGGGTTCCTCCATGCTGACTGTTCCAGGTCCGCCAGGTCCTCCGGGTGCAATGGGTCCACCTGGTCCGCCAGGTGCTCCAGGTCCAGCTGGTCCTGCG).

[0040] The nucleotide sequence of the nucleic acid molecule of JY002 is as shown in SEQ ID NO:8 (GGTCCTCCGGGTCCACCTGGTCCACCTGGTCCAAAAGGTGATCAAGGCC CACCAGGTCCGCGCGGTCATCAAGGTGAACAGGGTCTGCCTGGCTTTTCTGGTCCTCCAGGTCCTCCAGGTCCTCAGGGTCCAAAGGGCGATAAAGGTGACCCGGGTGTTCCTGGTGCGCTGGGTATTCCTGGTCCACCGGGTCCACCAGGTCCACCAGGTCCAAAAGGTGATCAGGGTCCGCCGGGTCCACGTGGTCATCAAGGTGAGCAGGGTCTGCCTGGTTTTTCCGGTCCACCAGGTCCACCGGGTCCACAAGGTCCAAAGGGTGATAAGGGTGATCCGGGTGTACCAGGCGCCCTGGGTATTCCG).

[0041] The present invention provides a gene-derived product expressing the humanized recombinant type XVII collagen.

[0042] In the present invention, the gene-derived products preferably include at least one of the following: gene expression cassette, recombinant vector, recombinant cell, and recombinant virus strain. The gene expression cassette preferably consists of gene elements that promote the expression of a target gene and the target gene in series. The target gene includes a nucleic acid molecule encoding a humanized recombinant type XVII collagen. The gene elements include at least one of the following: promoter, enhancer, and terminator. The present invention does not impose any special restrictions on the type of the recombinant vector, and vectors applicable to prokaryotic expression systems well-known in the art, or vectors applicable to eukaryotic expression vectors or viral expression vectors can be used. In the embodiments of the present invention, the backbone vector of the recombinant vector preferably includes pSUMO3. The cloning sites of the nucleic acid molecule encoding the humanized recombinant type XVII collagen in pSUMO3 include BamHⅠ and XhoⅠ. The pSUMO3 contains a 6×His tag and a SUMO peptide segment. During recombinant expression, it is expressed in the form of a fusion protein formed by the humanized recombinant type XVII collagen, the 6×His tag, and the SUMO peptide segment. The 6×His tag facilitates the separation and purification of the recombinant protein, and the SUMO peptide segment can increase the expression level and solubility of the protein; after the fusion protein is treated with sentrin specific protease 2 (SENP2), the SUMO peptide segment is cleaved off by the enzyme, and only a serine residue (Ser) remains at the N-terminus of the target protein, reducing the impact on subsequent experiments and contributing to the purification of the protein.

[0043] In the present invention, the hosts of the recombinant cells preferably include prokaryotic expression systems and eukaryotic expression systems. The prokaryotic expression systems preferably include Escherichia coli, Bacillus expression systems, and Streptomyces expression systems, etc. The eukaryotic expression systems include yeast, insect cells, mammalian cells, etc.

[0044] The present invention provides a method for preparing the humanized recombinant type XVII collagen, comprising the following steps:

[0045] Clone the nucleic acid molecule encoding the humanized recombinant type XVII collagen into an expression vector to construct a recombinant expression vector;

[0046] Transfer the recombinant expression vector into a host bacterium for culture to obtain a recombinant strain;

[0047] Perform recombinant expression of the recombinant strain, separate and purify the recombinant protein to obtain the humanized recombinant type XVII collagen.

[0048] In the present invention, the nucleic acid molecule encoding the humanized recombinant type XVII collagen is cloned into an expression vector to construct a recombinant expression vector.

[0049] In the present invention, for the method of constructing the recombinant expression vector, preferably, the nucleic acid molecule encoding humanized recombinant type XVII collagen with BamHⅠ and XhoⅠ restriction sites and pSUMO3 are respectively digested with BamHⅠ and XhoⅠ to obtain the digested nucleic acid molecule and the linear pSUMO3 vector;

[0050] The digested nucleic acid molecule and the linear pSUMO3 vector are ligated to obtain the recombinant expression vector.

[0051] In the present invention, the nucleic acid molecule encoding humanized recombinant type XVII collagen with BamHⅠ and XhoⅠ restriction sites preferably includes those obtained by artificial synthesis or PCR amplification using specific primers with BamHⅠ and XhoⅠ restriction sites. The specific primer pairs for amplifying JY001 with BamHⅠ and XhoⅠ restriction sites are P1 (SEQ ID NO:9, GATCAAGGCCCACCAGGTC) and P2 (GGACCAGGAATAACCCAGCG, SEQ ID NO:10). The specific primer pairs for amplifying JY002 with BamHⅠ and XhoⅠ restriction sites are P3 (TCCGGGTCCGTCTGGTG, SEQ ID NO:11) and P4 (CATGGAGGAACCCTCAGAAGTA, SEQ ID NO:12).

[0052] After obtaining the recombinant expression vector, in the present invention, the recombinant expression vector is transferred into a host bacterium to obtain a recombinant strain.

[0053] The present invention has no special limitation on the method of transferring into the host bacterium, and the well-known transformation methods in the art can be used, such as heat shock method. The preferred culture conditions are culturing in LB medium and / or 2×YT medium at 36 - 38°C. The end point of the culture is when the OD of the bacterial solution 600 is 0.6 - 0.8.

[0054] After obtaining the recombinant strain, in the present invention, the recombinant strain is used for recombinant expression of the protein, and the recombinant protein is separated and purified to obtain humanized recombinant type XVII collagen.

[0055] In the present invention, the conditions for the recombinant expression of the protein are preferably culturing in 2×YT medium and inducing culture at 36 - 38°C. The preferred induction culture time is 24 - 72 h, which can be 36 - 48 h; the working concentration of IPTG during the induction culture is 0.4 - 0.6 M, which can be 0.5 M.

[0056] In the present invention, the method for separating recombinant protein includes collecting bacterial cells, resuspending them, disrupting the bacterial cells by a low-temperature high-pressure method, centrifuging, and collecting the supernatant to obtain the recombinant protein. The method for purifying the recombinant protein is preferably nickel ion affinity chromatography and ion exchange chromatography. In the nickel ion affinity chromatography process, the nickel ions chelated and immobilized on the packing material can form a coordination interaction with the imidazole groups on the histidine on the protein surface. Therefore, the recombinant protein carrying the His tag can bind to the nickel ion affinity chromatography column and can be competitively eluted by a high-concentration imidazole solution to achieve the purification purpose. After the nickel ion affinity chromatography, it is preferably further included to digest and remove the SUMO tag with SENP2 protease for reverse nickel ion affinity chromatography column. The purity of the purified recombinant protein is detected by SDS-PAGE. The results show that two single bands with the expected molecular weight sizes are formed in the SDS-PAGE electrophoresis pattern. This indicates that the present invention successfully recombinantly expresses and obtains recombinant humanized collagen XVII

[0057] In the present invention, the nucleic acid molecule encoding recombinant humanized collagen XVII is inserted into the pSUMO3 expression vector, transformed into Escherichia coli strains, and stable recombinant humanized collagen XVII is successfully expressed, and the yield of recombinant humanized collagen XVII is increased. The inventors of the present invention found that the structure of humanized collagen XVII is very complex. The structural feature of humanized collagen XVII is a triple helix structure formed by 3 peptide chains, that is, 3 α peptide chains form a protein in a right-handed superhelix manner. Such a triple helix region is called a collagen region. Each α peptide chain is composed of repeatedly occurring Gly-X-Y (X and Y represent any amino acid residues other than Gly, X is often Pro, and Y is often Hyp) peptide segments in the molecular structure to form a left-handed helix. Under the interaction of amino acid residues of the 3 chains, a stable triple helix structure is formed in a right-handed superhelix manner with the same axis as the center. Therefore, it is difficult for the sequence of humanized collagen XVII to spontaneously bind to form a stable triple helix structure to exert biological functions. The present invention selects to use the Escherichia coli system and adopts specific optimized sequence fragments for combined optimization, and successfully expresses stable humanized collagen XVII, and the process is simple and the operation is convenient.

[0058] In the present invention, in order to verify the biological activity of the prepared recombinant humanized collagen XVII, fibroblast migration tests and hair growth promotion tests are respectively carried out. The results of the fibroblast migration test show that JY001 and JY002 have the activity of promoting fibroblast migration relative to the human serum albumin control, and the migration rate is 72% - 78.01%. The hair growth promotion test shows that JY001 and JY002 can promote the resting follicles of mice to enter the growth phase in advance and have the activity of promoting hair growth.

[0059] Based on the characteristics that the humanized recombinant type XVII collagen has the properties of promoting cell migration and promoting hair growth, the present invention provides the application of the humanized recombinant type XVII collagen or the humanized recombinant type XVII collagen prepared by the nucleic acid molecule, the gene-derived product or the preparation method in the preparation of a product for promoting cell migration and / or hair growth.

[0060] In the present invention, the product preferably includes medical supplies and / or daily necessities related to skin repair or anti-aging skin care of the face and head. The medical supplies preferably include tissue engineering products, medical devices and drugs. The medical device is a wound dressing product. The daily necessities preferably include cosmetics and / or washing and care products. The cosmetics are cream, essence water and lotion. The washing and care products preferably include facial masks, scalp essence and shampoo.

[0061] The following is a detailed description of a humanized recombinant type XVII collagen and its preparation method and application provided by the present invention in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Example 1

[0063] A preparation method of recombinant XVII humanized collagen, comprising the following steps:

[0064] S1: The DNA sequences of XVII humanized collagen (JY001: SEQ ID NO:1, JY002: SEQ ID NO:2) were respectively amplified by PCR using primers with restriction sites (SEQ ID NO:9 - SEQ ID NO:12). The amplified products were respectively inserted into the pSUMO3 expression vector to construct the expression vectors of recombinant XVII humanized collagen. The specific steps were as follows: Using a plasmid extraction kit, extract the pSUMO3 empty plasmid according to the instructions; perform double digestion on the target gene and the pSUMO3 empty plasmid using QuickCutBamHⅠ and QuickCutXhoⅠ. The digestion system (100 μL) was: 10 μL each of QuickCutNotⅠ, QuickCutXbaⅠ, and 10×QuickCut Green Buffer, 70 μL of the pSUMO3 empty plasmid or the target gene; digest in a 37°C metal bath for 3 h; after digestion, perform electrophoresis on a 1.0% agarose gel, and respectively cut and recover the digested empty plasmid and the target gene fragment; ligate the recovered products through T4 DNALigase. The ligation system (10 μL) was: 5 μL of the target gene, 3 μL of the vector fragment, 1 μL each of T4 DNAligase and 10×Ligase buffer; transform the recombinant plasmid into Escherichia coli BL21(DE3) competent cells, pick positive transformants for culture after resistance screening, and send for sequencing after PCR identification; if the sequencing is correct, the new expression vectors pSUMO3 - JY001 (see Figure 1 ) and pSUMO3 - JY002 (see Figure 2 ) are obtained.

[0065] S2: Transfer the successfully constructed expression vector into Escherichia coli strain BL21(DE3) to construct the expression strain of recombinant XVII humanized collagen. The specific steps include the following steps:

[0066] M1: Add the successfully constructed expression plasmid into Escherichia coli BL21(DE3) competent cells and let it stand on ice for 30 min;

[0067] M2: Heat - shock the mixture in a 42°C water bath for 90 s, and then quickly place it on ice and let it stand for 3 - 4 min;

[0068] M3: Add 1 ml of LB without Amp to the mixture and culture it under shaking conditions at 37°C and 220 rpm for 1 h;

[0069] M4: Take 100 μl of the bacterial solution and evenly coat it on an LB plate containing 100 μg / ml concentration of Amp. Invert the coated plate and incubate it at 37°C until clear visible colonies grow.

[0070] S3: Cultivate the expression strain and isolate and purify recombinant humanized collagen XVII. The specific steps are as follows:

[0071] 1. Small-scale shaking culture for amplification: Pick a monoclonal colony after transformation into BL21(DE3) and transfer it to 100 mL of LB medium containing 100 μg / ml Amp. Culture it at 37 °C and 220 rpm for 12 h.

[0072] 2. Large-scale shaking culture for amplification: Add the small-scale cultured bacterial liquid to 1 L of 2YT medium containing 100 μg / ml Amp at a ratio of 1:100. Culture it at 37 °C and 220 rpm. After about 2.5 h, take 1 mL of the bacterial liquid in a laminar flow hood and detect the concentration of the bacterial liquid with NanoDrop 2000C. When OD 600 = 0.6 - 0.8, transfer the bacterial liquid to the induction temperature and culture it at 220 rpm.

[0073] 3. Induced expression: After 30 min, add 1 mL of 0.5 M IPTG to the medium to a final concentration of 0.5 mM, and continue to culture for 12 h to obtain the bacteria containing the recombinant expressed protein.

[0074] 4. Purification method: When constructing the pSUMO3-Protein plasmid, an 8×His tag and a SUMO peptide segment were fused to the N-terminus of the protein. The 8×His tag can be used for affinity chromatography, and the SUMO peptide segment can increase the expression level and solubility of the protein. After the fusion protein is treated with sentrin specific protease 2 (SENP2), the SUMO peptide segment is cleaved off, and only a serine residue (Ser) remains at the N-terminus of the target protein, reducing the impact on subsequent experiments and facilitating protein purification. The specific purification process is as follows:

[0075] 1) Bacterial liquid treatment:

[0076] a. Harvest bacteria: Centrifuge the bacterial liquid at 4500 rpm for 20 min, discard the supernatant, and collect the precipitate.

[0077] b. Resuspend bacteria: Resuspend the bacterial precipitate with Buffer A (0 - 30 mM imidazole).

[0078] c. Lyse bacteria: Use a low-temperature high-pressure cell disruptor to lyse the cells. After the disruption is completed, centrifuge the disrupted liquid at 4 °C and 15000 rpm for 1 h, and collect the supernatant for subsequent purification experiments.

[0079] 2) Nickel ion affinity chromatography

[0080] The nickel ions chelated and immobilized on the packing material of the nickel ion affinity chromatography column can form coordination interactions with the imidazole groups on the histidine on the protein surface. Therefore, the recombinant protein carrying the 8×His tag can bind to the nickel ion affinity chromatography column and can be competitively eluted by a high-concentration imidazole solution to achieve the purpose of purification.

[0081] (1) Nickel ion affinity chromatography: After equilibrating the nickel ion affinity chromatography column with 50 mL of Buffer A (the column volume is 5 mL), the supernatant after centrifugation of the lysate was loaded at a flow rate of 3 mL / min; then rinsed with 200 mL of Buffer A to wash away unbound or weakly bound miscellaneous proteins, and then eluted with 4 - 5 column volumes of Buffer B to elute the target protein. Take samples of each fraction and detect the protein purity by SDS-PAGE and Coomassie Brilliant Blue staining, collect the fractions containing the target protein, and add SENP2 protease to it, with the mass ratio of the target protein to SENP2 being 500:1, and digest overnight. At the same time, dialyze the protein solution into Buffer A for the next purification step.

[0082] (2) Reverse nickel ion affinity chromatography: Centrifuge the protein solution after digestion and dialysis at 4°C and 4000 rpm for 20 min, take the supernatant and flow it through the nickel ion affinity chromatography column equilibrated with Buffer A again, with a flow rate not exceeding 2 mL / min. The rinsing and elution volumes are the same as those in the first affinity chromatography. Take samples of each fraction and detect the protein purity by SDS-PAGE, and collect the flow-through fraction and dialyze it into 0.5×PBS.

[0083] 3) Ion exchange chromatography

[0084] For the first cation exchange chromatography, load the protein solution collected in the previous step onto a 1 mL cation exchange chromatography column that has been equilibrated with 0.5×PBS. On an AKTA purifier, perform a linear gradient elution with 0.5×PBS and 4×PBS, collect the elution fractions at 5 mL / tube, detect the purity of the elution fractions by SDS-PAGE, and combine the collection tubes containing the target protein. The results are shown in Figure 3 .

[0085] Example 2

[0086] Cell migration promotion assay

[0087] 1. Test principle

[0088] When the cells grow to a confluent monolayer state, an artificial blank area is created on the confluent monolayer cells, called a "scratch". The cells at the scratch edge will gradually enter the blank area to heal the "scratch". During the cell migration process, images are captured at the beginning and regularly, and the cell migration rate is determined by comparing the images.

[0089] 2. Test steps

[0090] 2.1 First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate with the help of a ruler. Draw a line every 0.5 cm, crossing the wells horizontally.

[0091] 2.2 Add 2 mL of 3T3 cell suspension with a concentration of 5×10 5 cells / mL to the wells of the 6-well plate.

[0092] 2.3 On the next day, when it is observed that the cells in the 6-well plate have all grown into a single layer, use a pipette tip against the ruler to make scratches perpendicular to the horizontal lines on the back, making 2 scratches in each well.

[0093] 2.4 Wash the cells 3 times with PBS to wash away the detached suspended cells.

[0094] 2.5 According to the grouping, add 1.8 mL of serum-free culture medium to the wells, and then add 200 μL of the JY001 and JY002 stock solutions prepared in Example 1 and control human albumin respectively. Add an equal amount of PBS solution to the cell control wells.

[0095] 2.6 Place it in an incubator at 37°C and 5% CO2 for cultivation. Take a photo at 0 hour and record the photo-taking position in each well. Observe and take photos of the fixed position during subsequent observations.

[0096] 3. Experimental results

[0097] According to the taken photos, calculate the cell migration rate. The results are shown in Table 3 and Figure 4 and Figure 5 .

[0098] Table 3 Results of cell migration experiment

[0099]

[0100]

[0101] Example 3

[0102] Mouse hair growth experiment

[0103] 1 Experimental purpose

[0104] In this experiment, by creating a male mouse alopecia model and using an aqueous solution containing protein, observe the hair growth of mice in each group.

[0105] 2 Experimental materials and methods

[0106] 2.1 Experimental animals

[0107] Select 18 healthy adult female C57BL / 6 mice with black hair. Raise them in a clean-grade environment, feed them with sufficient feed and water freely, feed and water them every day, and check the physiological activities of the animals.

[0108] 2.2 Test reagents

[0109] Protein stock solution, the protein stock solution prepared in the example, the concentration of which is adjusted to 200ug / ml; depilatory cream; 10% formalin.

[0110] 2.3 Test equipment

[0111] Fluorescence microscopy

[0112] 3 Experimental methods

[0113] 3.1 Experimental Grouping

[0114] Eighteen C57BL / 6 female and male mice (5 weeks old) were divided into three groups, with six mice in each group, half of which were male and half were female, and they were respectively the control blank group, the JY001 protein group and the JY002 protein group.

[0115] 3.2 Establishment of mouse model and intervention plan

[0116] Before the experiment, a depilatory cream was applied to the back of the mouse in a large area. After 15 minutes, the depilatory cream was wiped off to achieve the effect of plucking hair. The experimental group was smeared with the JY001 and JY002 protein stock solutions prepared in Example 1 and human serum albumin, and the control group was smeared with distilled water. The drug was administered once a day at a fixed time, 0.4 ml each time, for 18 consecutive days.

[0117] 3.3 Observation of hair growth

[0118] The hair growth of mice in the experimental group was observed on d 0, d 2, d 7, d 7, d 10, d 12, d 15, and d 18, and photos were taken under a fixed light source every day.

[0119] 4. Experimental Results

[0120] Observe the results through hair growth Figure 6 It can be concluded that the protein concentrate can promote the resting hair follicles of mice to enter the growth phase ahead of time and promote hair growth.

[0121] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A humanized recombinant type XVII collagen, characterized in that, A polypeptide sequence derived from type XVII collagen or a repetitive protein formed by tandemly repeating the polypeptide N times as a unit; N is an integer from 2 to 4; The polypeptide derived from type XVII collagen includes an A polypeptide sequence and / or a B polypeptide sequence; The amino acid sequence of the A polypeptide is as shown in SEQ ID NO: 1; The amino acid sequence of the B polypeptide is as shown in SEQ ID NO:

2.

2. The humanized recombinant type XVII collagen according to claim 1, wherein The recombinant protein includes JY001 and JY002; The amino acid sequence of JY001 is as shown in SEQ ID NO: 3; The amino acid sequence of JY002 is as shown in SEQ ID NO:

4.

3. A nucleic acid molecule encoding the humanized recombinant type XVII collagen according to claim 1 or 2.

4. The nucleic acid molecule according to claim 3, wherein A nucleic acid molecule including nucleotide sequences as shown in SEQ ID NO: 5 to SEQ ID NO:

8.

5. A gene-derived product expressing the humanized recombinant type XVII collagen according to claim 1 or 2.

6. The gene-derived product according to claim 5, wherein Including at least one of the following: a gene expression cassette, a recombinant vector, a recombinant cell, and a recombinant virus strain; The backbone vector of the recombinant vector includes pSUMO3; The cloning sites of the nucleic acid molecule encoding the humanized recombinant type XVII collagen according to claim 1 or 2 in pSUMO3 include BamHⅠ and XhoⅠ.

7. The preparation method of the humanized recombinant type XVII collagen according to claim 1 or 2, characterized in that, Including the following steps: Clone the nucleic acid molecule encoding the humanized recombinant type XVII collagen according to claim 1 or 2 into an expression vector to construct a recombinant expression vector; Transfer the recombinant expression vector into a host bacterium for culture to obtain a recombinant strain; Recombinantly express the protein in the recombinant strain, separate and purify the recombinant protein to obtain the humanized recombinant type XVII collagen.

8. The preparation method according to claim 7, characterized in that, The construction method of the recombinant expression vector: separately perform BamHⅠ and XhoⅠ digestion on the nucleic acid molecule encoding the humanized recombinant type XVII collagen with BamHⅠ restriction sites and XhoⅠ restriction sites and pSUMO3 to obtain the digested nucleic acid molecule and the linear pSUMO3 vector; Ligate the digested nucleic acid molecule and the linear pSUMO3 vector to obtain the recombinant expression vector.

9. The use of the humanized recombinant type XVII collagen according to claim 1 or 2 or the humanized recombinant type XVII collagen prepared by the preparation method according to claim 3, claim 5, or claim 7 or 8 in the preparation of a product for promoting cell migration and / or hair growth.

10. The application according to claim 9, wherein The product includes medical supplies and / or daily necessities related to skin repair or anti-aging skin care of the face and head.

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