Fusion protein for promoting proliferation of hair follicle stem cells as well as preparation method and application of fusion protein

Abstract

The invention provides a fusion protein for promoting proliferation of hair follicle stem cells as well as a preparation method and application of the fusion protein. The fusion protein is formed by fusion expression of a plurality of collagen structural domains capable of being combined with cell integrin and / or specific structural domains from certain functional proteins screened from collagen XVII and III type collagen. Nucleic acid molecules for coding the fusion protein are subjected to codon optimization and then recombined and stably expressed in escherichia coli to obtain the fusion protein. The fusion protein has excellent performance of promoting proliferation and migration of human hair follicle stem cells and human fibroblasts, can be used for repairing skin and hair follicle tissues and improving resistance of skin and hair follicles, and is suitable for popularization and application in the fields of beauty and skin care, medical equipment, tissue engineering and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of fusion protein preparation, and particularly relates to a fusion protein for promoting the proliferation of hair follicle stem cells, a preparation method thereof, and an application thereof. Background Art

[0002] Collagen XVII is a transmembrane protein located at the dermo-epidermal junction. As a key structural component in hemidesmosomes (a multi-protein complex located in the dermal-epidermal basement membrane zone that can mediate the adhesion of keratinocytes to the underlying membrane), it plays an important role in maintaining the tight connection between the basement membrane and the extracellular matrix. Some studies have shown that the expression of Collagen XVII can help inhibit the aging of hair follicle stem cells and thus delay hair loss. Collagen III is an important structural protein that is abundantly present in the human body and plays an important role in maintaining tissue structure and elasticity; Collagen III can promote cell adhesion and binding by binding to integrins on the cell surface; at the same time, Collagen III can bind to cell surface receptors and participate in regulating biological processes such as cell differentiation, proliferation, and development; some studies have shown that Collagen III can be degraded by special proteases and participate in tissue remodeling and repair processes. Some studies have shown that some functional proteins can also promote the activation of hair follicle stem cells, hair follicle development and regeneration, improve the hair follicle microenvironment, and play a crucial role in hair growth by directly or indirectly activating the Wnt / β-catenin signaling pathway and the TGF-β pathway. However, currently, collagen or functional proteins have a relatively large molecular weight and a single function, which is not conducive to simultaneously exerting the functions of promoting hair growth and tissue repair. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a fusion protein, which is formed by the fusion expression of collagen domains derived from Collagen XVII and Collagen III and specific segments from other functional proteins, and has the functions of promoting the proliferation, migration, and adhesion of hair follicle stem cells and fibroblasts, so as to achieve the purpose of scalp care.

[0004] The present invention provides a fusion protein, which comprises a fusion protein formed by the fusion expression of at least 3 of the following proteins in single-copy or double-copy: collagen domain 1, collagen domain 2, collagen domain 3, oligopeptide 1, oligopeptide 2, polypeptide 1, polypeptide 2, and polypeptide 3;

[0005] The amino acid sequences of the collagen domain 1, collagen domain 2, and collagen domain 3 are successively as shown in SEQ ID NO: 1 to SEQ ID NO: 3;

[0006] The amino acid sequence of the oligopeptide 1 is as shown in SEQ ID NO: 4;

[0007] The amino acid sequence of the oligopeptide 2 is as shown in SEQ ID NO: 5;

[0008] The amino acid sequence of the polypeptide 1 is as shown in SEQ ID NO: 6;

[0009] The amino acid sequence of the polypeptide 3 is as shown in SEQ ID NO: 7;

[0010] The amino acid sequence of the polypeptide 2 is as shown in SEQ ID NO: 8.

[0011] Preferably, the fusion protein comprises one or two copies of collagen domain 3, collagen domain 1 and at least one of the following proteins: polypeptide 1, polypeptide 3 and polypeptide 2.

[0012] Preferably, the fusion protein comprises a first fusion protein, a second fusion protein, a third fusion protein, a fourth fusion protein, a fifth fusion protein and a sixth fusion protein;

[0013] The first fusion protein is formed by sequentially concatenating one copy of collagen domain 1, collagen domain 2 and collagen domain 3;

[0014] The second fusion protein is formed by sequentially concatenating one copy of oligopeptide 1, collagen domain 1, collagen domain 2, collagen domain 3 and oligopeptide 2;

[0015] The third fusion protein is formed by sequentially concatenating polypeptide 1, collagen domain 1 and collagen domain 3;

[0016] The fourth fusion protein is formed by sequentially concatenating one copy of polypeptide 3, collagen domain 1 and two copies of collagen domain 3;

[0017] The fifth fusion protein is formed by sequentially concatenating one copy of polypeptide 2, two copies of collagen domain 1 and two copies of collagen domain 3.

[0018] The present invention provides a nucleic acid molecule encoding the fusion protein.

[0019] The present invention provides a gene-derived product expressing the fusion protein.

[0020] Preferably, it comprises at least one of the following: gene expression cassette, recombinant vector, recombinant strain and recombinant virus strain.

[0021] The present invention provides a method for preparing the fusion protein, comprising the following steps:

[0022] Clone the nucleic acid molecule encoding the fusion protein into a backbone vector to obtain a recombinant expression vector;

[0023] Transfer the recombinant expression vector into a host for recombinant expression, isolate and purify the recombinant protein to obtain a fusion protein.

[0024] Preferably, the backbone vector includes pET21M-SUMO3;

[0025] Preferably, the host includes Escherichia coli cells.

[0026] The present invention provides the application of the fusion protein or the fusion protein prepared from the nucleic acid molecule or the gene-derived product in the preparation of products for skin and / or hair follicle injury repair and promoting skin and / or hair follicle growth.

[0027] Preferably, the products include daily necessities and / or medical supplies.

[0028] The present invention provides a fusion protein, which includes a fusion protein formed by the fusion expression of at least 3 of the following proteins in single copy or double copy: collagen domain 1, collagen domain 2, collagen domain 3, oligopeptide 1, oligopeptide 2, polypeptide 1, polypeptide 2, and polypeptide 3; the amino acid sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 8 in sequence. The present invention intercepts several collagen domains and / or specific domains of functional proteins that bind to cell integrins from human type XVII collagen and human type III collagen and fuses them for expression to form a fusion protein. The fusion protein can significantly enhance the proliferation, migration, and adhesion abilities of human fibroblasts and human hair follicle stem cells. The fusion protein of the present invention can be used for the repair of skin and hair follicle tissues, improve the resistance of skin and hair follicles, and is suitable for the preparation of products for application in the fields of hair care, skin care, and related pharmaceuticals. Description of the Drawings

[0029] Figure 1 Spectrum of the recombinant vector for expressing the first fusion protein;

[0030] Figure 2 Spectrum of the recombinant vector for expressing the second fusion protein;

[0031] Figure 3 SDS-PAGE diagram of the first fusion protein and the second fusion protein, where lane 1: result of the first fusion protein, lane 2: result of the second fusion protein;

[0032] Figure 4 Results of the proliferation effects of the first fusion protein and the second fusion protein at different concentrations on human hair follicle stem cells;

[0033] Figure 5 Results of the proliferation effects of the first fusion protein and the second fusion protein at different concentrations on fibroblasts;

[0034] Figure 6Results of the migration rates of human hair follicle stem cells affected by the first fusion protein and the second fusion protein at different concentrations;

[0035] Figure 7 Results of the migration rates of fibroblasts affected by the first fusion protein and the second fusion protein at different concentrations;

[0036] Figure 8 SDS-PAGE diagram of the third fusion protein, the fourth fusion protein and the fifth fusion protein; where M is a protein marker of 245 kDa; RH3 represents the third fusion protein, RH4 represents the fourth fusion protein, and RH5 represents the fifth fusion protein;

[0037] Figure 9 Results of the proliferation experiments of the third fusion protein, the fourth fusion protein and the fifth fusion protein on hair follicle stem cells;

[0038] Figure 10 Results of the proliferation experiments of the third fusion protein, the fourth fusion protein and the fifth fusion protein on fibroblasts;

[0039] Figure 11 Results of the scratch experiments of the third fusion protein, the fourth fusion protein and the fifth fusion protein on hair follicle stem cells;

[0040] Figure 12 Results of the scratch experiments of the third fusion protein, the fourth fusion protein and the fifth fusion protein on fibroblasts. Detailed implementation mode

[0041] The present invention provides a fusion protein, which comprises a fusion protein formed by the fusion expression of at least 3 of the following proteins in single copy or double copy: collagen domain 1, collagen domain 2, collagen domain 3, oligopeptide 1, oligopeptide 2, polypeptide 1, polypeptide 2 and polypeptide 3.

[0042] In the present invention, collagen domain 1, collagen domain 2, and collagen domain 3 are all three basic collagen domains screened from human-derived type XVII collagen and human-derived type III collagen that can bind to cell integrins. The amino acid sequence of the collagen domain 1 is shown as SEQ ID NO: 1 (GERGAPGFRGPAGPNGIPGEKGPAGERGAP). The amino acid sequence of the collagen domain 2 is shown as SEQ ID NO: 2 (GPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPA). The amino acid sequence of the collagen domain 3 is shown as SEQ ID NO: 3 (GSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP), which is derived from the 906-977 region of type XVII collagen. Oligopeptide 1 and oligopeptide 2 have good biological effects in accelerating the synthesis of extracellular matrix proteins (laminin, type III and type XVII collagens), increasing the volume and length of hair follicles, and strengthening the repair of the epidermal-dermal connective tissue at the hair root. The amino acid sequence of the oligopeptide 1 is GHK; the amino acid sequence of the oligopeptide 2 is shown as SEQ ID NO: 5 (KLAKK).

[0043] In the present invention, the polypeptide 1 is the sequence of the 29-398 segment of functional protein 1. Functional protein 1 can promote angiogenesis by enhancing the migration and proliferation of vascular endothelial cells. This is crucial for hair follicle growth because the health and function of hair follicles depend on the rich blood supply around them. Angiogenesis can provide nutrients and oxygen to hair follicles while removing metabolic waste. The amino acid sequence of the polypeptide 1 is as shown in SEQ ID NO: 6 (DVDECVEGTDNCHIDAICQNTPRSYKCICKSGYTGDGKHCKDVDECEREDNAGCVHDCVNIPGNYRCTCYDGFHLAHDGHNCLDVDECAEGNGGCQQSCVNMMGSYECHCREGFFLSDNQHTCIQRPEEGMNCMNKNHGCAHICRETPKGGIACECRPGFELTKNQRDCKLTCNYGNGGCQHTCDDTEQGPRCGCHIKFVLHTDGKTCIETCAVNNGGCDSKCHDAATGVHCTCPVGFMLQPDRKTCKDIDECRLNNGGCDHICRNTVGSFECSCKKGYKLLINERNCQDIDECSFDRTCDHICVNTPGSFQCLCHRGYLLYGITHCGDVDECSINRGGCRFGCINTPGSYQCTCPAGQGRLHWNGKDCT).

[0044] In the present invention, the polypeptide 2 is derived from functional protein 2. Functional protein 2 promotes the generation and expansion of capillaries around hair follicles by stimulating the proliferation and migration of vascular endothelial cells, thereby providing support for the development and maintenance of hair follicles. Functional protein 2 is upregulated when hair follicles enter the growth phase, promoting the activity of hair follicle matrix cells and the blood flow around them. The amino acid sequence of the polypeptide 2 is as shown in SEQ ID NO: 8 (AKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR).

[0045] The polypeptide 3 is derived from the functional protein 3, which promotes the activation of hair follicle stem cells, hair follicle development and regeneration by activating the Wnt / β-catenin signaling pathway, and plays a crucial role in hair growth. The polypeptide 3 is optimized from the functional protein 3 (NEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFSFSMLAAGVMHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVTENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCK, SEQ ID NO: 14). The specific optimization method is to optimize the hydrophobic fragment (HAVATACSLGKLV, SEQ ID NO: 4) into a neutral amino acid sequence (HGVATACSLGKLV, SEQ ID NO: 15) with reduced aggregation tendency, and at the same time, part of the sequence is optimized into a hydrophilic sequence (GSSVQ, SEQ ID NO: 16) to reduce the aggregation tendency. The amino acid sequence of the polypeptide 3 is shown in SEQ ID NO: 7 (NEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRRPDITGSSVQGLHIAVHDCQHQLRDQRWNCGALSQGGRLPHHSAILKRGFRESAFSFSMLAAGVMHGVATACSLGKLVSGGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPGPGQQDSWEWGGCNHDMDFGEKFSRDFLDSREAPSGIQARMRVHNNSVGRQVVSEKLKRKCKCHGASGSCQYKTCWRDAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGQLVYFEKSPDFSERDPTSGSPGTSGRGACNKASRLLDGCGSLCCGRGHNVLRQTRVEKCHCRFHWCCYVLCDECKSTEWVNVCK).

[0046] In the present invention, when the fusion protein is formed by the fusion expression of three proteins, the fusion protein preferably includes a single copy or a double copy of collagen domain 3, collagen domain 1, and at least one of the following proteins: collagen domain 2, polypeptide 1, polypeptide 3, and polypeptide 2. When the fusion protein is formed by the fusion expression of five proteins, the fusion protein includes oligopeptide 1, collagen domain 1, collagen domain 2, collagen domain 3, and oligopeptide 2.

[0047] In an embodiment of the present invention, the fusion protein preferably includes a first fusion protein, a second fusion protein, a third fusion protein, a fourth fusion protein, a fifth fusion protein, and a sixth fusion protein;

[0048] The first fusion protein is preferably formed by the sequential tandem of a single copy of collagen domain 1, collagen domain 2, and collagen domain 3. The amino acid sequence of the first fusion protein is shown in SEQ ID NO: 9 (GERGAPGFRGPAGPNGIPGEKGPAGERGAPGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPAGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP).

[0049] The second fusion protein is preferably formed by the sequential tandem of a single copy of oligopeptide 1, collagen domain 1, collagen domain 2, collagen domain 3, and oligopeptide 2. The amino acid sequence of the second fusion protein is shown in SEQ ID NO: 10 (GHKGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKIVTSEGSSMLTVPGPPGPPGAMGPPGPPGAPGPAGPAGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIPKLAKK).

[0050] The third fusion protein is preferably formed by sequentially connecting polypeptide 1, collagen domain 1, and collagen domain 3 in series. The amino acid sequence of the third fusion protein is preferably as shown in SEQ ID NO: 11 (DVDECVEGTDNCHIDAICQNTPRSYKCICKSGYTGDGKHCKDVDECEREDNAGCVHDCVNIPGNYRCTCYDGFHLAHDGHNCLDVDECAEGNGGCQQSCVNMMGSYECHCREGFFLSDNQHTCIQRPEEGMNCMNKNHGCAHICRETPKGGIACECRPGFELTKNQRDCKLTCNYGNGGCQHTCDDTEQGPRCGCHIKFVLHTDGKTCIETCAVNNGGCDSKCHDAATGVHCTCPVGFMLQPDRKTCKDIDECRLNNGGCDHICRNTVGSFECSCKKGYKLLINERNCQDIDECSFDRTCDHICVNTPGSFQCLCHRGYLLYGITHCGDVDECSINRGGCRFGCINTPGSYQCTCPAGQGRLHWNGKDCTGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP).

[0051] The fourth fusion protein is preferably formed by sequentially concatenating a single copy of polypeptide 3, a collagen domain 1, and two copies of a collagen domain 3. The amino acid sequence of the fifth fusion protein is as shown in SEQ ID NO: 12 (NEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRRPDITGSSVQGLHIAVHDCQHQLRDQRWNCGALSQGGRLPHHSAILKRGFRESAFSFSMLAAGVMHGVATACSLGKLVSGGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPGPGQQDSWEWGGCNHDMDFGEKFSRDFLDSREAPSGIQARMRVHNNSVGRQVVSEKLKRKCKCHGASGSCQYKTCWRDAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGQLVYFEKSPDFSERDPTSGSPGTSGRGACNKASRLLDGCGSLCCGRGHNVLRQTRVEKCHCRFHWCCYVLCDECKSTEWVNVCKGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP).

[0052] The fifth fusion protein is preferably formed by sequentially concatenating a single copy of polypeptide 2, a single copy of collagen domain 1, and two copies of collagen domain 3. The amino acid sequence of the fifth fusion protein is as shown in SEQ ID NO: 13 (AKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVPCGPC SERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRRGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP).

[0053] In addition, the present invention has also constructed a variety of other fusion proteins, including the sixth fusion protein, the seventh fusion protein, and the eighth fusion protein. The sixth fusion protein: two copies of collagen domain 3 + two copies of collagen domain 1 + the 804-916 segment of functional protein 1, and the sequence is as follows: CGGELGEFTGYIESPNYPGNYPAGVECIWNINPPPKRKILIVVPEIFLPSEDECGDVLVMRKNSSPSSITTYETCQTYERPIAFTARSRKLWINFKTSEANSARGFQIPYVTGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP (SEQ ID NO: 17).

[0054] The seventh fusion protein is a single-copy collagen domain 3 + double-copy collagen domain 1 + the 804-916 segment of functional protein 1, and the sequence is as follows: GSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPCGGELGEFTGYIESPNYPGNYPAGVECIWNINPPPKRKILIVVPEIFLPSEDECGDVLVMRKNSSPSSITTYETCQTYERPIAFTARSRKLWINFKTSEANSARGFQIPYVT (SEQ ID NO: 18).

[0055] The eighth fusion protein is the original functional protein 3 + single-copy collagen domain 1 + double-copy collagen domain 3, and its specific amino acid sequence is: NEILGLKLPGEPPLTANTVCLTLSGLSKRQLGLCLRNPDVTASALQGLHIAVHECQHQLRDQRWNCSALEGGGRLPHHSAILKRGFRESAFSFSMLAAGVMHAVATACSLGKLVSCGCGWKGSGEQDRLRAKLLQLQALSRGKSFPHSLPSPGPGSSPSPGPQDTWEWGGCNHDMDFGEKFSRDFLDSREAPRDIQARMRIHNNRVGRQVVTENLKRKCKCHGTSGSCQFKTCWRAAPEFRAVGAALRERLGRAIFIDTHNRNSGAFQPRLRPRRLSGELVYFEKSPDFCERDPTMGSPGTRGRACNKTSRLLDGCGSLCCGRGHNVLRQTRVERCHCRFHWCCYVLCDECKVTEWVNVCKGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGSFLSNSETFLSGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPQGPKGDKGDPGVPGALGIP (SEQ ID NO: 19).

[0056] In the embodiments of the present invention, by recombinantly expressing different fusion proteins in a prokaryotic expression system, the combination of different collagen domains and the influence of the copy number cause some fusion proteins to fail to be successfully expressed to obtain recombinant proteins. The sixth to eighth fusion proteins cannot be normally expressed under the same recombinant expression method. The first to fifth fusion proteins can be normally expressed and obtained using a prokaryotic expression vector in a prokaryotic expression system.

[0057] The present invention provides a nucleic acid molecule encoding the fusion protein.

[0058] In the present invention, the nucleic acid molecule is codon-optimized according to the codon preference of the host. The nucleotide sequences of the codon-optimized collagen domain 1, collagen domain 2, and collagen domain 3 are shown as SEQ ID NO: 20 to SEQ ID NO: 22. The nucleotide sequence of the codon-optimized oligopeptide 2 is shown as SEQ ID NO: 23. The nucleic acid molecules encoding polypeptide 1, polypeptide 3, and polypeptide 2 after codon optimization preferably have the nucleotide sequences as shown in SEQ ID NO: 24 to SEQ ID NO: 26. The nucleic acid molecules encoding the first to fifth fusion proteins after codon optimization preferably have the nucleotide sequences as shown in SEQ ID NO: 27 to SEQ ID NO: 31.

[0059] The present invention provides a gene-derived product expressing the fusion protein.

[0060] In the present invention, the gene-derived product preferably includes at least one of the following: gene expression cassette, recombinant vector, recombinant strain, and recombinant virus strain. The gene expression cassette preferably includes a promoter, an enhancer, a target gene, and a terminator. The backbone vector of the recombinant vector preferably includes a prokaryotic expression vector and / or a eukaryotic expression vector and a viral expression vector. The backbone vector preferably includes pET21M-SUMO3. The host of the recombinant strain preferably includes Escherichia coli competent cells. The host virus of the recombinant virus strain preferably includes lentivirus or adeno-associated virus. The present invention has no special limitation on the construction method of the recombinant virus strain, and the well-known packaging method of the recombinant virus strain in the art can be used.

[0061] The present invention provides a method for preparing the fusion protein, including the following steps:

[0062] Clone the nucleic acid molecule encoding the fusion protein into a backbone vector to obtain a recombinant expression vector;

[0063] Transfer the recombinant expression vector into a host for recombinant expression, separate and purify the recombinant protein to obtain the fusion protein.

[0064] In the present invention, the backbone vector includes pET21M-SUMO3. The construction method of the recombinant vector preferably includes the following steps: seamlessly clone the nucleic acid molecule into pET21M-SUMO3, transfer the cloned product into Escherichia coli for expression, and obtain the recombinant vector after verification by colony PCR and sequencing.

[0065] After obtaining the recombinant expression vector, the present invention transfers the recombinant expression vector into a host for recombinant expression, separates and purifies the recombinant protein to obtain the fusion protein.

[0066] In the present invention, the type of the host is preferably Escherichia coli competent cells. The method for transferring the recombinant expression vector into the host preferably includes heat shock method. After transferring the recombinant expression vector into the host, it preferably further includes resistance screening and verification to obtain a recombinant strain. The resistance screening is preferably Amp resistance screening, and the screening concentration is preferably 100 μg / ml. The culture medium for the resistance screening preferably includes LB medium and 2YT medium. The culture conditions for the resistance screening are preferably culturing at 37°C and 220 rpm. The verification preferably includes colony PCR verification. The recombinant strain undergoes culturing and induced culturing for recombinant expression of the protein. The conditions for the induced culturing are preferably inducing expression with IPTG at 37°C for 24 to 72 h.

[0067] In the present invention, the method for isolating the recombinant protein preferably includes collecting the cells of the recombinant strain and disrupting them to collect the recombinant protein. His tag and SUMO peptide segment are fused to the N-terminus of the fusion protein in the recombinant protein. During purification, the His tag can be used for affinity chromatography; the SUMO peptide segment can increase the expression level and solubility of the protein; after separation by affinity chromatography, the obtained recombinant protein is treated with sentrin specific protease 2 (SENP2) to remove the SUMO peptide segment, and only a serine residue (Ser) remains at the N-terminus of the target protein, reducing the influence on subsequent experiments and facilitating protein purification.

[0068] The present invention provides the application of the fusion protein or the fusion protein prepared from the nucleic acid molecule or the gene-derived product in the preparation of products for skin and / or hair follicle injury repair and promoting skin and / or hair follicle growth.

[0069] In the present invention, the skin and / or hair follicle injury repair is based on the good ability of the fusion protein to promote the migration of fibroblasts and hair follicle stem cells. The promotion of skin and / or hair follicle growth is based on the good ability of the fusion protein to promote the proliferation of fibroblasts and hair follicle stem cells.

[0070] In the present invention, the products preferably include daily necessities and / or medical supplies. The daily necessities preferably include hair care daily necessities or skin care products. The medical supplies include drugs for treating folliculitis and skin injury repair and wound dressings for plastic surgery. The dosage forms of the drugs include external dosage forms and / or injection dosage forms.

[0071] The following examples are used to illustrate in detail a fusion protein for promoting the proliferation of hair follicle stem cells, its preparation method and application provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0072] Example 1

[0073] Construction of high-copy Escherichia coli genetic engineering bacteria:

[0074] (1) Using bioinformatics and protein structure prediction tools (NCBI, Uniprot, AlphaFold), four basic collagen domains that can bind to cell integrin were screened from human type XVII collagen and human type III collagen (collagen domain 1: amino acid sequence as SEQ ID NO: 1, collagen domain 2: SEQ ID NO: 2, collagen domain 3: SEQ ID NO: 3). At the same time, the oligopeptide 2 (GHK) sequence and oligopeptide 1 (SEQ ID NO: 5) were selected.

[0075] (2) The above four basic collagen domains were concatenated in the order of collagen domain 1 - collagen domain 2 - collagen domain 3 to obtain a first fusion protein with the amino acid sequence shown in SEQ ID NO: 9. At the same time, the four basic collagen domains, oligopeptide 1, and oligopeptide 2 were concatenated in the order of oligopeptide 1 - domain 1 - domain 2 - domain 3 - domain 4 - oligopeptide 2 to obtain a second fusion protein with the amino acid sequence shown in SEQ ID NO: 10.

[0076] (3) The nucleotide sequences of the above two fusion proteins were codon-optimized according to the Escherichia coli codon preference to obtain the optimized first fusion gene (nucleotide sequence as SEQ ID NO: 27) and the second fusion gene (nucleotide sequence as SEQ ID NO: 28).

[0077] (4) The first fusion gene and the second fusion gene were entrusted to Shanghai Nuojingqing Biotechnology Co., Ltd. for gene synthesis and seamlessly cloned into the pET21M-SUMO3 plasmid using Gibson assembly technology, with the restriction enzyme sites BamHI / XhoI, to construct the first recombinant expression vector (spectrum as Figure 1 shown) and the second recombinant expression vector (spectrum as Figure 2 shown).

[0078] After linearizing the first recombinant expression vector 1 and the second recombinant expression vector with SalI respectively by restriction digestion, they were electrotransformed into competent Escherichia coli cells. The electrotransformed cells were activated and cultured in YPD medium for 2 - 3 hours, and then spread on YNB selection medium and incubated in an inverted position in an incubator at 30 °C for 2 - 3 days. After the transformants grew out, colony PCR was used to verify the transformant monoclonal. When verifying by colony PCR, the detection primers P1 for expressing the recombinant first fusion protein: GTATTCCGGGCGAAAAAGGC (SEQ ID NO: 32); P2: GGTTTCGGAGTTGCTCAGGA (SEQ ID NO: 33); the detection primers P3 for expressing the recombinant first fusion protein: CATAAAGGCGAACGTGGTGC (SEQ ID NO: 34), P4: GGTTTCGGAGTTGCTCAGGA (SEQ ID NO: 35).

[0079] The verified transformants were respectively spread on screening plates containing 1 g / L, 2 g / L, 3 g / L, and 4 g / L Amp. After culturing for 2 - 4 days, the transformants with better growth on the Amp-containing plates were picked for purification and rescreening verification. After genomic extraction and correct verification of the transformants obtained by rescreening, they were stored in glycerol to obtain the high-copy Escherichia coli genetic engineering strain RH.1 expressing the first fusion protein and the high-copy Escherichia coli genetic engineering strain RH.2 expressing the second fusion protein.

[0080] Example 2

[0081] Methods for expressing and purifying the first fusion protein and the second fusion protein

[0082] 1. Small-scale shaking amplification: Single colonies of the strains RH.1 and RH.2 constructed in Example 1 were respectively picked and inoculated into 100 mL of LB medium containing Amp, and cultured at 37 °C and 220 rpm for 12 h;

[0083] 2. Large-scale shaking amplification: The small-scale shaking bacterial liquid was added to 1 L of 2YT medium containing Amp at a ratio of 1:100, and cultured at 37 °C and 220 rpm. After about 2.5 h, 1 mL of the bacterial liquid was taken in a laminar flow hood, and the concentration of the bacterial liquid was detected with NanoDrop 2000C. When OD 600 = 0.6 - 0.8, the bacterial liquid was transferred to the induction temperature and cultured at 220 rpm;

[0084] 3. Induced expression: After 30 min, 1 mL of 0.5 M IPTG was added to the medium to a final concentration of 0.5 mM, and the culture was continued for 12 h.

[0085] 4. Bacterial liquid treatment:

[0086] (1) Bacteria harvesting: Centrifuge the bacterial solution at 4000 rpm for 20 min, discard the supernatant and collect the precipitate.

[0087] (2) Bacteria resuspension: Resuspend the bacterial precipitate with Buffer A (0 - 30 mM imidazole).

[0088] (3) Cell lysis: Lyse the cells using a high-pressure cell disruptor at low temperature. After lysis, centrifuge the lysate at 4°C and 12000 rpm for 30 min, and collect the supernatant for subsequent purification experiments.

[0089] 5. Nickel ion affinity chromatography

[0090] The nickel ions chelated and immobilized on the packing of the nickel ion affinity chromatography column can form coordination interactions with the imidazole groups on the histidine of the protein surface. Therefore, the recombinant protein carrying an 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 purification purpose.

[0091] (1) Nickel ion affinity chromatography: After equilibrating the nickel ion affinity chromatography column with 50 mL of Buffer A (column volume is 5 mL), load the centrifuged supernatant of the lysate onto the column at a flow rate of 3 mL / min. Then wash with 200 mL of Buffer A to remove unbound or weakly bound impurities, and then elute the target protein with 4 - 5 column volumes of Buffer B. Take samples of each fraction to detect the protein purity by SDS-PAGE and Coomassie Brilliant Blue staining, collect the fractions containing the target protein, and add SENP2 protease to the collected fractions for enzymatic cleavage to remove the SUMO peptide segment. The mass ratio of the target protein to SENP2 protease is 500:1, and incubate overnight. At the same time, dialyze the protein solution into Buffer A for the next purification step.

[0092] (2) Reverse nickel ion affinity chromatography: Centrifuge the protein solution after enzymatic cleavage and dialysis at 4°C and 4000 rpm for 20 min, take the supernatant and pass it through the nickel ion affinity chromatography column equilibrated with Buffer A again at a flow rate not exceeding 2 mL / min. The washing and elution volumes are the same as the first affinity chromatography. Take samples of each fraction to detect the protein purity by SDS-PAGE, and collect the flow-through fraction and dialyze it into 0.5×PBS.

[0093] 6. Ion exchange chromatography

[0094] 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.

[0095] The results of SDS-PAGE detection are shown in Figure 3 . It can be seen from Figure 3 that the first fusion protein was recombinantly expressed in the genetically engineered Escherichia coli strain RH.1, and the second fusion protein was recombinantly expressed in the genetically engineered Escherichia coli strain RH.2.

[0096] Example 3

[0097] Applications of the first fusion protein and the second fusion protein in promoting cell proliferation

[0098] Using human hair follicle stem cells and fibroblasts as experimental cells respectively, the MTT method was used to detect the ability of the first fusion protein, the second fusion protein and human serum albumin at different concentrations (mass percentage 0.01%, 0.05% and 0.2%) to promote cell proliferation.

[0099] The results are shown in Figure 4 (results of human hair follicle stem cells) and Figure 5 (results of fibroblasts). It can be seen that the first fusion protein and the second fusion protein showed more excellent ability to promote cell proliferation compared with human serum albumin, and the second fusion protein had the highest ability to promote cell proliferation.

[0100] Example 4

[0101] Applications of the first fusion protein and the second fusion protein in promoting cell migration

[0102] The cell scratch method was used to determine cell migration movement. Using human hair follicle stem cells and fibroblasts as experimental cells respectively, the effects of the first fusion protein, the second fusion protein and human serum albumin at different concentrations (mass percentage 0.01%, 0.05% and 0.2%) on cell migration ability were detected.

[0103] The results are shown in Figure 6 (results of human hair follicle stem cells) and Figure 7 (results of fibroblasts). It can be seen that the first fusion protein and the second fusion protein showed more excellent ability to promote cell migration compared with human serum albumin, and the second fusion protein had the highest ability to promote cell migration.

[0104] Example 5

[0105] Recombinant expression methods of the third fusion protein, the fourth fusion protein and the fifth fusion protein

[0106] The third fusion protein: polypeptide 1 (29-398 segment of functional protein 1) (SEQ ID NO: 6) + collagen domain 1 + collagen domain 3, and the sequence is shown in SEQ ID NO: 11.

[0107] Fourth fusion protein: Polypeptide 3 (SEQ ID NO: 7) + Collagen domain 1 + 2 × Collagen domain 3, with the sequence shown in SEQ ID NO: 12.

[0108] Fifth fusion protein: Polypeptide 2 (SEQ ID NO: 8) + Collagen domain 1 + 2 × Collagen domain 3, with the sequence shown in SEQ ID NO: 13.

[0109] The nucleotide sequences encoding the third, fourth, and fifth fusion proteins were codon-optimized according to the codon preference of Escherichia coli to obtain the codon-optimized third fusion gene (SEQ ID NO: 29), fourth fusion gene (SEQ ID NO: 30), and fifth fusion gene (SEQ ID NO: 31). High-copy Escherichia coli genetic engineering bacteria were constructed according to the method of Example 1. Recombinant expression, purification, and detection of each fusion protein were performed according to the method of Example 2.

[0110] The results are shown in Figure 8 . The results showed that the molecular weight of the third fusion protein (denoted as RH3) was 49.3 kDa and the isoelectric point was 5.55. The molecular weight of the fourth fusion protein (denoted as RH4) was 58.34 kDa, which was consistent with the expectation. The molecular weight of the fifth fusion protein (denoted as RH4) was 41.4 kDa and the isoelectric point was 9.17. The concentrations of the third and fifth fusion proteins were greater than 1.5 g / L.

[0111] When the fifth fusion protein was recombinantly expressed, induction cultures were also carried out at 16 °C and 30 °C respectively. The results showed that the fifth fusion protein could not be normally induced and expressed at 16 °C and 30 °C.

[0112] Example 6

[0113] Proliferation experiments of hair follicle stem cells and fibroblasts were carried out with the third fusion protein (denoted as RH3), fourth fusion protein (denoted as RH4), and fifth fusion protein (denoted as RH5). The experimental steps were the same as those in Example 3.

[0114] The results are shown in Figure 9 and Figure 10 . All three proteins had good proliferative activities on hair follicle stem cells and fibroblasts. The third fusion protein had relatively balanced activities on the two types of cells. The fourth fusion protein showed higher activity on hair follicle stem cells, and the fifth fusion protein showed higher activity on fibroblasts.

[0115] Example 7

[0116] Migration experiments of the third fusion protein (denoted as RH3), the fourth fusion protein (denoted as RH4), and the fifth fusion protein (denoted as RH5) on hair follicle stem cells and fibroblasts were carried out. The experimental steps were the same as those in Example 4.

[0117] The results are shown in Figure 11 and Figure 12 . The results showed that all three proteins had good migratory promoting activities on hair follicle stem cells and fibroblasts. The third fusion protein had relatively balanced activities on the two types of cells. The fourth fusion protein showed higher activity on hair follicle stem cells, and the fifth fusion protein showed higher activity on fibroblasts.

[0118] Comparative Example 1

[0119] Recombinant expression method of the sixth fusion protein

[0120] Sixth fusion protein: Functional protein 1 (804 - 916) + Collagen domain 1 + Collagen domain 3, with the sequence shown in SEQ ID NO: 17, where the underlined part is Collagen domain 1.

[0121] The nucleotide sequence encoding the sixth fusion protein was codon-optimized according to the codon preference of Escherichia coli. The codon-optimized sixth fusion gene (SEQ ID NO: 36) was used to construct a high-copy Escherichia coli genetic engineering bacterium according to the method of Example 1. The sixth fusion protein was recombinantly expressed according to the method of Example 2.

[0122] SDS-PAGE was used to detect the recombinantly expressed protein. The results showed that no protein band of the expected size appeared on the electrophoresis gel, indicating that the sixth fusion protein was not successfully expressed.

[0123] Comparative Example 2

[0124] Recombinant expression method of the seventh fusion protein

[0125] Seventh fusion protein: Collagen domain 3×2 + Collagen domain 1×2 + Functional protein 1 (804 - 916), with the sequence shown in SEQ ID NO: 18, where the underlined part with gray background bases is the extracellular region, and the rest is the intracellular region).

[0126] The nucleotide sequence encoding the seventh fusion protein was codon-optimized according to the codon preference of Escherichia coli. The codon-optimized seventh fusion gene (SEQ ID NO: 37) was used to construct a high-copy Escherichia coli genetic engineering bacterium according to the method of Example 1. The seventh fusion protein was recombinantly expressed according to the method of Example 2, and SDS-PAGE was used to detect the recombinantly expressed protein.

[0127] The results showed that no protein band of the expected size appeared on the electrophoresis gel, indicating that the sixth fusion protein was not successfully expressed.

[0128] Comparative Example 3

[0129] Recombinant expression method of the eighth fusion protein

[0130] The eighth fusion protein: original functional protein 3 + collagen domain 1 + 2×collagen domain 3, with the sequence shown in SEQ ID NO: 19. The nucleotide sequence encoding the eighth fusion protein was codon-optimized according to the codon preference of Escherichia coli, and the codon-optimized eighth fusion gene (SEQ ID NO: 38) was used to construct a high-copy Escherichia coli genetic engineering bacterium according to the method of Example 1. The eighth fusion protein was recombinantly expressed according to the method of Example 2. The recombinantly expressed protein was detected by SDS-PAGE.

[0131] The results showed that no protein band of the expected size appeared on the electrophoresis diagram, indicating that the sixth fusion protein was not successfully expressed.

[0132] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein comprises a fusion protein formed by the expression of at least three of the following proteins in a single copy or double copy: collagen domain 1, collagen domain 2, collagen domain 3, oligopeptide 1, oligopeptide 2, polypeptide 1, polypeptide 2 and polypeptide 3; The amino acid sequences of the collagen domain 1, collagen domain 2, and collagen domain 3 are shown in SEQ ID NO: 1 to SEQ ID NO: 3, respectively; The amino acid sequence of the oligopeptide 1 is shown in SEQ ID NO: 4; The amino acid sequence of the oligopeptide 2 is shown in SEQ ID NO: 5; The amino acid sequence of polypeptide 1 is shown in SEQ ID NO: 6; The amino acid sequence of polypeptide 3 is shown in SEQ ID NO:7; The amino acid sequence of polypeptide 2 is shown in SEQ ID NO:

8.

2. The fusion protein according to claim 1, characterized in that The fusion protein includes a single copy or double copy of collagen domain 3, collagen domain 1 and at least one of the following proteins: polypeptide 1, polypeptide 2 and polypeptide 3.

3. The fusion protein according to claim 1, characterized in that The fusion proteins include a first fusion protein, a second fusion protein, a third fusion protein, a fourth fusion protein, a fifth fusion protein and a sixth fusion protein; The first fusion protein is formed by sequentially connecting a single copy of collagen domain 1, collagen domain 2, and collagen domain 3; The second fusion protein is formed by sequentially connecting a single copy of oligopeptide 1, collagen domain 1, collagen domain 2, collagen domain 3 and oligopeptide 2; The third fusion protein is formed by sequentially connecting polypeptide 1, collagen domain 1 and collagen domain 3; The fourth fusion protein is formed by sequentially connecting a single copy of polypeptide 3, collagen domain 1 and double copies of collagen domain 3; The fifth fusion protein is formed by sequentially connecting a single copy of polypeptide 2, a double copy of collagen domain 1 and a double copy of collagen domain 3.

4. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 3.

5. A gene-derived product expressing the fusion protein according to any one of claims 1 to 3.

6. The gene-derived product according to claim 5, characterized in that: It includes at least one of the following: a gene expression cassette, a recombinant vector, a recombinant bacterial strain and a recombinant virus strain.

7. A method for preparing the fusion protein according to any one of claims 1 to 3, characterized in that: The following steps are involved: Cloning a nucleic acid molecule encoding the fusion protein of any one of claims 1 to 3 into a backbone vector to obtain a recombinant expression vector; The recombinant expression vector is transferred into a host for recombinant expression, and the recombinant protein is separated and purified to obtain a fusion protein.

8. The preparation method according to claim 7, characterized in that: The backbone vector includes pET21M-SUMO3; The host includes competent Escherichia coli.

9. Use of the fusion protein according to any one of claims 1 to 3 or the fusion protein prepared from the nucleic acid molecule according to claim 4 or the gene-derived product according to claim 5 or 6 in the preparation of products for repairing skin and / or hair follicle damage and promoting skin and / or hair follicle growth.

10. The use according to claim 9, characterized in that: The products include daily necessities and / or medical supplies.

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