Multi-functional domain fusion extracellular matrix protein as well as coding gene, preparation method and application thereof

Abstract

The invention discloses a multi-functional domain fusion extracellular matrix protein as well as a coding gene, a preparation method and application thereof, and belongs to the fields of biotechnology, medical cosmetology and wound repair. The multi-functional-domain fusion extracellular matrix protein provided by the invention contains core functional domains of human-derived III-type collagen, elastin and fibronectin, integrates key biological functions of the human III-type collagen, elastin and fibronectin, and is obtained by expression of mammalian cells. The method can promote formation of a collagen triple helix structure, is simple in process, high in yield and easy for industrial production, and can be widely applied to the fields of effective skin care, skin aging resistance, medical cosmetology, wound repair and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, medical cosmetology and wound repair, and relates to a multifunctional domain fusion extracellular matrix protein, a coding gene thereof, a preparation method and application, in particular to a triple helix fusion matrix protein with multiple biological activities of collagen type III, elastin and fibronectin and a coding gene thereof, a preparation method and application. BACKGROUND

[0002] Skin is the first line of defense of the body. Extracellular matrix, as an important component of skin structure, often refers to the non-cellular components in tissues other than cells, which is a complex network structure and an invisible gel structure filling the network structure, containing a large number of signal molecules, participating in the regulation and control of cell growth, regeneration, repair, migration and metabolism. Among them, extracellular matrix proteins are mainly composed of collagen, elastin and fibronectin, which are key proteins for maintaining the mechanical properties and physiological functions of the skin and play a key role in skin anti-aging and wound repair.

[0003] The dermis of the skin is mainly composed of collagen and elastin, and collagen is the most abundant extracellular matrix protein, mainly type I and type III collagen, which constitutes most of the skin material and plays an important role in maintaining the normal physiological functions of cells, tissues and organs and damage repair. Elastin and collagen are associated in tissues, especially type III collagen, which gives the skin a smooth and delicate appearance, and elastin gives the skin elasticity. Both have excellent biocompatibility, biodegradability and absorbability, promote cell formation, and are widely used in skin care products, medical cosmetology, plastic repair and regenerative medicine fields. Among them, natural type III collagen is a superhelix structure composed of 3 alpha helix peptide chains, which is currently expressed by E. coli, Pichia pastoris, insect cells and mammalian cells. E. coli and Pichia pastoris have no endogenous proline hydroxylase, and the expressed collagen cannot form the triple helix structure required for function. Although the three-dimensional structure and physicochemical properties of recombinant collagen molecules prepared by P4H co-expression of Pichia pastoris are closer to those of natural collagen, the abnormal mannose glycosylation of yeast-expressed proteins has high immunogenicity, which brings potential safety hazards to recombinant collagen products,

[0004] Fibronectin is a natural material for inducing tissue repair and regeneration, commonly known as biological adhesive, which provides stable signal transmission and structural support for tissues and organs, has good biocompatibility and endogenous tissue induction capacity. Studies have shown that fibronectin forms a core functional domain through the RGD motif, which is an important determinant of the adhesion activity of various cells, is conducive to cell adhesion and growth, and regulates the production and construction of extracellular matrix by cells, accelerates the tissue repair process, can be used for tissue filling, induces tissue regeneration, and repairs damaged tissues, and is called a new generation of biological regenerative material, which has great advantages in wound repair, beauty and skin care, and anti-aging. Traditional fibronectin is mainly obtained by step-by-step separation and purification from plasma, which is time-consuming, complex, and low in yield, and is difficult to produce on a large scale, and has the biological safety risk of virus transmission.

[0005] For example, patent document CN116535520A (hereinafter referred to as document 1) discloses a fusion protein of extracellular matrix protein fused with humanized type I collagen, humanized type III collagen, fibronectin and elastin, which has good effects of promoting cell adhesion, promoting cell migration, promoting cell proliferation, promoting collagen secretion and antioxidant, and each effect is better than that of single protein. However, the structure and amino acid sequence of the fusion protein disclosed in document 1 are different from those of the present application. Specifically, the fragments of the fusion protein in document 1 are connected by a connecting peptide, and have a histidine tag, which introduces an exogenous amino acid sequence, so the natural sequence of the corresponding matrix protein is different. In addition, the fusion protein disclosed in document 1 is expressed by Saccharomyces cerevisiae cells. Since the yeast cells do not have endogenous proline hydroxylase, the proline in the collagen cannot be hydroxylated to form the triple helix required for function. At the same time, the abnormal glycosylation inherent in yeast cell-expressed proteins leads to high immunogenicity, which brings potential safety hazards to the recombinant collagen product. For example, patent document CN117143256A (hereinafter referred to as document 2) discloses a recombinant extracellular matrix protein for hair repair, which is a fusion protein of fibronectin, type III collagen, elastin and insulin-like growth factor expressed by Saccharomyces cerevisiae. It can significantly promote the recovery and healing of hair and surrounding skin damage caused by chemical reagents, and promote hair growth, and can also reflect the efficacy on animal models. However, the structure and amino acid sequence of the fusion protein disclosed in document 2 are different from those of the present application. Specifically, the fragments of the fusion protein in document 2 are connected by a connecting peptide, which introduces an exogenous amino acid sequence, so the natural sequence of the corresponding matrix protein is different. In addition, the fusion protein disclosed in document 2 is also expressed by Saccharomyces cerevisiae cells. Since the yeast cells do not have endogenous proline hydroxylase, the proline in the collagen cannot be hydroxylated to form the triple helix required for function. At the same time, the abnormal glycosylation inherent in yeast cell-expressed proteins leads to high immunogenicity, which brings potential safety hazards to the recombinant collagen product. SUMMARY

[0006] The present application aims at the bottleneck problem existing in the current extracellular matrix proteins and their preparation process, and provides a brand-new structural design and preparation method, i.e. a high-activity, multi-functional, triple-helix fusion matrix protein (also referred to as multi-functional domain fusion extracellular matrix protein, also simply referred to as fusion matrix protein) integrating the core functions of collagen type III, elastin and fibronectin, as well as a technical method for preparing the fusion matrix protein by using a double-promoter expression vector and human cell culture, so as to provide a new biological regenerative material with "one product with multiple functions" for efficacy skin care, medical cosmetology and plastic repair.

[0007] Specifically, the present application is mainly realized by the following technical solutions.

[0008] In a first aspect, the present application provides a multi-functional domain fusion extracellular matrix protein, which comprises specific domains of human collagen type III, elastin and fibronectin, and has key biological functions of collagen, elastin and fibronectin.

[0009] In some embodiments, the multi-functional domain fusion extracellular matrix protein has a connection order of collagen type III-elastin-fibronectin from N-terminus to C-terminus, and each element is directly connected.

[0010] In some embodiments, the multi-functional domain fusion extracellular matrix protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

[0011] In some embodiments, the encoding gene of the multi-functional domain fusion extracellular matrix protein comprises or consists of the nucleotide sequence shown in SEQ ID NO: 2.

[0012] In some embodiments, the multi-functional domain fusion extracellular matrix protein is obtained by expression of mammalian cells, which can be CHO cells or human embryonic kidney cells HEK293.

[0013] In a second aspect, the present application provides a polynucleotide encoding the multi-functional domain fusion extracellular matrix protein provided in the first aspect of the present application.

[0014] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence shown in SEQ ID NO: 2.

[0015] In a third aspect, the present application provides a double-promoter mammalian cell expression vector comprising the polynucleotide provided in the second aspect of the present application.

[0016] In some embodiments, the double-promoter mammalian cell expression vector comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0017] In a fourth aspect, the present application provides a transgenic cell line or host bacteria comprising the polynucleotide of the second aspect of the present application or the dual-promoter mammalian cell expression vector of the third aspect of the present application.

[0018] In a fifth aspect, the present application provides a method for successfully preparing the multifunctional domain fusion extracellular matrix protein of the first aspect of the present application, comprising the following steps:

[0019] 1) introducing the expression vector of the multifunctional domain fusion extracellular matrix protein into mammalian cells for expression to obtain supernatant containing the multifunctional domain fusion extracellular matrix protein; and

[0020] 2) purifying the supernatant to obtain the multifunctional domain fusion extracellular matrix protein.

[0021] In some embodiments, the expression vector of the multifunctional domain fusion extracellular matrix protein in step 1) is the dual-promoter mammalian cell expression vector of the third aspect of the present application.

[0022] The use of the multifunctional domain fusion extracellular matrix protein, the polynucleotide, the dual-promoter mammalian cell expression vector, or the transgenic cell line or host bacteria provided by the present application in the preparation of a medicament for skin anti-aging and / or wound repair also belongs to the content of the present application.

[0023] In a sixth aspect, the present application also provides a medicament for skin anti-aging and / or wound repair, which comprises the multifunctional domain fusion extracellular matrix protein of the first aspect of the present application as the main active ingredient.

[0024] In some embodiments, the medicament for skin anti-aging and / or wound repair further comprises one or more of the following: a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant.

[0025] Based on the above technical solutions, the present application provides a completely new structural design and preparation method, i.e. the multifunctional domain fusion extracellular matrix protein integrating the core functions of collagen type III, elastin and fibronectin, and the technical method for preparing the multifunctional domain fusion extracellular matrix protein by using a dual-promoter expression vector and human cell culture, which provides a new type of biological regenerative material with "one product with multiple functions" for efficacy skincare, medical cosmetology and plastic repair.

[0026] In addition, in the multifunctional domain fusion extracellular matrix protein provided by the present application, the elements (type III collagen, elastin and fibronectin) are directly connected without being connected by a connecting peptide, and do not contain a histidine tag, so that no foreign amino acid sequence is introduced, so that the sequence is the same as that of the corresponding matrix protein, and thus the excellent biocompatibility, safety and low immunogenicity are more advantageous. Furthermore, the multifunctional domain fusion extracellular matrix protein provided by the present application is obtained by expression from a double promoter expression vector and mammalian cells (such as CHO and human embryonic kidney cells HEK293), which can promote the formation of collagen triple helix structure on the one hand, and can avoid the defect of high immunogenicity caused by abnormal glycosylation inherent in the use of yeast cells to express proteins in the prior art (such as the above-mentioned document 1 and document 2), and further can avoid the potential safety hazard of recombinant collagen protein product, and the method of the present application is simple in process, high in yield, easy to industrialize, and can be widely used in the fields of efficacy skin care, skin anti-aging, medical cosmetology and wound repair. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 A 12% SDS-PAGE electrophoresis identification gel map of the supernatant containing the fusion matrix protein expressed in HEK293 in Example 1 is shown.

[0028] Figure 2 A 12% SDS-PAGE electrophoresis identification gel map of the target fusion matrix protein obtained after expression and purification in HEK293 in Example 1 is shown.

[0029] Figure 3 A Tm value determination result curve of the fusion matrix protein obtained in Example 1 is shown.

[0030] Figure 4 A circular dichroism detection curve of type III collagen (A) and fusion matrix protein (B) obtained in Example 1 is shown.

[0031] Figure 5 A cell migration experiment result of the fusion matrix protein and type III collagen obtained in Example 1 is shown. DETAILED DESCRIPTION

[0032] The present application aims to provide a multifunctional domain fusion matrix protein with biological activities of type III collagen, elastin and fibronectin. A method for successfully preparing the multifunctional domain fusion matrix protein is also provided. The present application is specifically implemented through the following specific embodiments.

[0033] (I) The present application provides a preferred combination of multifunctional domain fusion extracellular matrix protein, which mainly includes the core functional domain of type III collagen, elastin and fibronectin.

[0034] Specifically, the fusion matrix protein is one of the following amino acid residue sequences:

[0035] 1) the amino acid residue sequence shown in SEQ ID NO: 1 in the sequence listing;

[0036] 2) a protein having the amino acid residue sequence shown in SEQ ID NO: 1 in the sequence listing with one to ten amino acid residues substituted, deleted or added and having extracellular matrix protein activity.

[0037] SEQ ID NO: 1 in the sequence listing consists of 906 amino acid residues, the first 292 (292 aa) amino acid residues from the amino terminal are collagen type III functional domain, the 293-632 (340 aa) amino acid residues from the amino terminal are elastin functional domain, and the 633-906 (274 aa) amino acid residues from the amino terminal are fibronectin functional domain. In the fusion matrix protein, the fragments are not connected by exogenous connecting peptides, nor do they contain histidine tags, thus no exogenous amino acid sequences are introduced, so that the fusion matrix protein can be the same as the natural sequence of the corresponding matrix protein, and is more conducive to its excellent biocompatibility, safety and low immunogenicity. In addition, test results prove that the above structural design of the multi-functional domain fusion extracellular matrix protein can ensure that the fusion matrix protein can be correctly assembled and expressed, and can play a synergistic role without impairing the core biological functions of the three.

[0038] Further, in one aspect, the polypeptide fragments, derivatives and analogs of the above-mentioned fusion matrix protein also belong to the present application, which have the same biological functions or activities as the above-mentioned fusion matrix protein, wherein the polypeptide fragments are defined as: 1) a polypeptide substituted by one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and such substituted amino acid residues can or can not be encoded by genetic code; 2) a polypeptide having a substituent group in one or more amino acid residues; 3) a polypeptide formed by chimerization of a mature polypeptide with another compound; 4) a polypeptide formed by chimerization of an additional amino acid sequence to the polypeptide sequence (such as a sequence for purifying the polypeptide, or a chimeric toxin of an antibody fragment or other antigen ligand sequence), or a coding sequence of a chimeric polypeptide can be obtained by chimerization of a nucleic acid sequence (or a part thereof) encoding another polypeptide with a nucleic acid sequence (or a part thereof) of the present application, and the coding sequence of the chimeric polypeptide is expressed to produce the chimeric polypeptide. The techniques for producing chimeric polypeptides are well known in the art, including linking the coding sequences of the polypeptides so that they are in the same reading frame, and the expression of the chimeric polypeptide is controlled by the same promoter and terminator.

[0039] The differences between the above-mentioned analogs of the fusion matrix protein and the fusion matrix protein can be differences in the amino acid sequence, or differences in the form of modification that does not affect the sequence, or both. The analogs include natural or induced genetic variants. The induced variants can be obtained by various techniques, such as random mutagenesis by irradiation or exposure to mutagens, and by site-directed mutagenesis or other known molecular biology techniques. It should be understood that the amino acid residue sequence of the chimeric toxin of the present application is not limited to the above-mentioned representative sequences.

[0040] Further, in another aspect, the fusion matrix protein can also be a modified fusion matrix protein polypeptide, or a fusion matrix protein polypeptide that has been modified to improve its proteolytic resistance or to optimize its solubility. The forms of modification (which generally do not change the primary structure) include: 1) chemical derivatization of the polypeptide in vivo or in vitro, such as acetylation or carboxylation; 2) glycosylation, such as those polypeptides that have been modified by glycosylation during synthesis and processing or further processing steps, which modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation (such as a mammalian glycosylation enzyme or deglycosylation enzyme); 3) sequences having phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, phosphothreonine).

[0041] The gene encoding the above-mentioned fusion matrix protein also belongs to the present application, and more specifically:

[0042] The gene encoding the fusion matrix protein is one of the following nucleotide sequences:

[0043] 1) the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing;

[0044] 2) a nucleotide sequence encoding the amino acid residue sequence shown in SEQ ID NO: 1 in the Sequence Listing;

[0045] 3) a nucleotide sequence having more than 90% homology with the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing and encoding a protein having HP immunogenicity;

[0046] 4) a nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO: 2 in the Sequence Listing under high stringency conditions.

[0047] The high stringency conditions are washing the membrane with a solution containing 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS at 65°C after hybridization.

[0048] SEQ ID NO: 2 in the sequence listing consists of 2718 bases, the coding sequence of which is from the 1st to the 2718th base at the 5' end, encoding a protein having the amino acid residue sequence shown in SEQ ID NO: 2 in the sequence listing, from the 1st to the 876th base at the 5' end encoding a collagen type III functional domain, from the 877th to the 1896th base at the 5' end encoding an elastin functional domain, and from the 1897th to the 2718th base at the 5' end encoding a fibronectin functional domain.

[0049] The polynucleotide encoding the fusion matrix protein of the present application can be in the form of DNA or RNA. The DNA form includes cDNA or artificially synthesized DNA, which can be single-stranded or double-stranded, and can be the coding strand or the non-coding strand.

[0050] Further, the variant of the polynucleotide encoding the fusion matrix protein of the present application also belongs to the present application, which encodes a polypeptide or a polypeptide fragment, an analog and a derivative having the same amino acid sequence as the fusion matrix protein. The variant of the polynucleotide can be a naturally occurring allelic variant or a non-naturally occurring variant, and can include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which can be a substitution, deletion or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.

[0051] The expression vector containing the gene of the present application, the transgenic cell line and the host bacteria all belong to the present application.

[0052] (II) The present application also provides a double-promoter mammalian cell expression vector for promoting the formation of collagen triple helix, and a method for preparing recombinant fusion matrix protein using the vector, specifically, transforming or transducing a host cell (such as a mammalian cell) with a recombinant expression vector containing the coding gene of the fusion matrix protein, culturing the host cell, separating and purifying the protein from the culture medium or the cell to obtain the fusion matrix protein.

[0053] In the method, the recombinant expression vector containing the coding gene of the fusion matrix protein is a recombinant expression vector into which the coding gene of the fusion matrix protein or the variant gene thereof is inserted. The starting vector for constructing the recombinant expression vector includes but is not limited to vectors that can be expressed in mammalian cells. In general, any plasmid and vector can be used as long as it can replicate and be stable in the host (mammalian cell host in the present application). An important feature of the starting vector is that it usually contains a replication point, a promoter, a marker gene and a translation control element.

[0054] The recombinant expression vector containing the coding gene of the fusion matrix protein, which is constructed using pSQl.0(+) as the starting vector, is named SQl.0 / COEFN. The fusion matrix protein expressed in a host cell (e.g., a mammalian cell, which can be CHO or human embryonic kidney cell HEK293) using the above-mentioned recombinant expression vector is named COEFN, the amino acid sequence of which is shown as SEQ ID NO: 1 in the sequence listing, and the nucleotide sequence encoding the amino acid sequence is shown as SEQ ID NO: 2 in the sequence listing.

[0055] The recombinant expression vector can be constructed using methods well known to those skilled in the art, such as in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology, etc. (Sambrook, et al Molecular cloning, a Laboratory Manual. Cold spring harbor laboratory. New York, 1989). The DNA sequence of the coding gene of the fusion matrix protein can be operably linked to a suitable promoter in the expression vector to direct the synthesis of mRNA. The promoter can be a CMV promoter or other promoters that can control the expression of genes in eukaryotic cells. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0056] In addition, the recombinant expression vector can also contain one or more selective marker genes to provide a phenotypic form for selecting transformed host cells, such as a dihydrofolate reductase gene for eukaryotic cell culture, a neomycin resistance gene, and a green fluorescent protein (GFP) gene or a tetracycline or ampicillin resistance gene for E. coli, etc. When the coding gene of the fusion matrix protein of the present application is expressed in higher eukaryotic cells, an enhancer sequence can also be inserted into the recombinant expression vector to enhance transcription. Enhancers are cis-acting factors of DNA, usually 10-300 base pairs in length, which act on promoters to enhance gene transcription. Examples include the SV40 enhancer of about 100-270 base pairs in length on the late side of the replication origin, the polyoma enhancer on the late side of the replication origin, or the adenovirus enhancer, etc.

[0057] In this method, the transformed or transduced host cell can be a mammalian cell such as CHO, COS, 293 (HEK293) cell, etc.

[0058] The recombinant expression vector can be transformed into a host cell using conventional techniques well known to those skilled in the art, the transformant can be cultured, the expression of the target protein (i.e., the fusion matrix protein COEFN) can be induced, and the target protein can be isolated and purified.

[0059] The culture medium and culture conditions for culturing the host cell containing the fusion matrix protein COEFN having the extracellular matrix protein activity are the same as those for culturing the host cell.

[0060] When the fusion matrix protein COEFN is prepared, the fusion matrix protein COEFN can be present in a prokaryotic expression vector.

[0061] The present application also provides a skin anti-aging and / or wound repair medicament containing the fusion matrix protein COEFN as the main active ingredient. If necessary, one or more pharmaceutically acceptable carriers can be added to the medicament. The carriers include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, and adsorption carriers, etc. that are conventional in the pharmaceutical field.

[0062] The medicament of the present application can be prepared in various forms such as injection solution or lyophilized powder. The medicaments in the above-mentioned various forms can be prepared according to the conventional methods in the pharmaceutical field.

[0063] The methods used in the following examples are conventional methods unless otherwise specified. The specific steps can be found in "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0064] The percentage concentrations are mass / volume (W / V) percentage concentrations or volume / volume (V / V) percentage concentrations unless otherwise specified.

[0065] The primers, DNA sequence synthesis, and DNA sequence determination were completed by Nanjing Kingsriver Biotech Co., Ltd.

[0066] The obtaining routes of the various biomaterials described in the examples are only provided as a route for obtaining experiments to achieve the specific purposes disclosed, and should not be considered as a limitation on the sources of the biomaterials of the present application. In fact, the sources of the biomaterials used are extensive, and any biomaterial that can be obtained without violating laws and moral ethics can be used according to the hints in the examples; in industrial implementation, the various cells derived from rats, mice, pigs, or humans, etc. mammals are all ex vivo, and include those obtained from cell banks, or commercially purchased, and those prepared according to the descriptions in the existing literature, and those induced from various stem cells that can be commercially obtained using known methods.

[0067] The embodiments are implemented on the premise of the technical solutions of the present application, and detailed implementation modes and specific operation processes are given. The embodiments will help to understand the present application, but the scope of the present application is not limited to the following embodiments.

[0068] Example 1, expression and purification of fusion matrix protein COEFN in human embryonic kidney cells HEK293

[0069] The expression and purification of the fusion matrix protein COEFN in HEK293 designed in this example include the following operations.

[0070] 1.1, construction of fusion matrix protein expression vector

[0071] The fusion matrix protein COEFN coding gene (SEQ ID NO: 2) was synthesized by Nanjing Kingsriver Biotechnology Co., Ltd. according to the preferred codon of CHO, and the expression vector pSQ1.0 / COEFN was constructed as a spare plasmid.

[0072] The core nucleotide sequence of the expression vector pSQ1.0 / COEFN is shown in SEQ ID NO: 3 in the sequence listing, which consists of 7470 bases, wherein the first-660 bases from the 5'-end are CMV promoter 1, the 661-677 bases are Kozak sequence, the 678-734 bases from the 5'-end encode signal peptide sequence, the 735-3452 bases from the 5'-end are fusion matrix protein COEFN coding gene, the 3453-3458 bases from the 5'-end are stop codon, the 3459-4047 bases from the 5'-end are CMV promoter 2, the 4048-4064 bases from the 5'-end are Kozak sequence 2, the 4065-5639 bases from the 5'-end are P4Hβ sequence, the 5640-5717 bases from the 5'-end are T2A sequence, the 5718-7241 bases from the 5'-end are P4Hα sequence, and the 7242-7470 bases from the 5'-end are SV40 PolyA sequence.

[0073] 1.2, expression and purification of fusion matrix protein in HEK293

[0074] 1.2.1 Expression: adjust the density of 293 cells to 1×10 6The cells were cultured in 37°C, 5% CO2. 30 μg of the plasmid constructed in step 1.1 was diluted in 0.5 ml of serum-free medium and mixed well; 150 μl of transfection reagent (plasmid: transfection reagent = 1:5) was diluted in 0.5 ml of serum-free medium and mixed well, and incubated at room temperature for 5 min; the transfection reagent dilution was added to the DNA dilution, mixed well, and incubated at room temperature for 30 min, and then added to the cell suspension, which was cultured at 37°C, 5% CO2, 120 rpm. The supernatant was collected: on the 6th day after transfection, centrifuged at 4400 rpm for 10 min to collect the supernatant. Figure 1 A 12% SDS-PAGE gel is shown for identification of the fusion matrix protein in the obtained supernatant.

[0075] 1.2.2 Purification: SP column was used, and the column was washed with ultrapure water for 5-10 column volumes, and then washed with the equilibration buffer (Buffer A) for 5-10 column volumes until the baseline was stable; sample loading: the sample was adjusted for pH and conductivity, and loaded at 1 mL / min; re-equilibration: the column was washed with the equilibration buffer (Buffer A) for 5-10 column volumes until the baseline was stable; elution: different concentrations of Buffer B were used for elution, and the elution products were collected; identification: SDS-PAGE detection. Figure 2 A 12% SDS-PAGE gel is shown for identification of the purified fusion matrix protein, which proves that the COEFN protein of interest was successfully obtained in this example.

[0076] In this example, type III collagen (with an amino acid sequence of SEQ ID NO: 1 from the 1st to 292nd (292 aa) amino acid residues from the amino terminus), elastin (with an amino acid sequence of SEQ ID NO: 1 from the 293rd to 632nd (340 aa) amino acid residues from the amino terminus), and fibronectin (with an amino acid sequence of SEQ ID NO: 1 from the 633rd to 906th (274 aa) amino acid residues from the amino terminus) were also constructed, expressed, and purified according to the above procedures, respectively.

[0077] Example 2, Tm value determination: differential scanning fluorometry (DSF)

[0078] To further evaluate the stability of the fusion matrix protein, the inventors used differential fluorescence scanning to detect the Tm value of the protein. The specific operation was as follows: the sample (COEFN protein purified sample obtained in Example 1) was diluted to 1 mg / ml with ultrapure water; 2 μl of the fluorescence developer Sypro Orange (5000x) (GIBCO, S6650) was mixed thoroughly, 48 μl of ultrapure water (diluted 25 times) was added to obtain the fluorescence developer Sypro Orange (200x) sample. 90 μl (1 mg / ml) of the sample was taken, 10 μl of the fluorescence developer Sypro Orange (200x) was added and mixed. 30 μl of this solution was added to a 96-well PCR plate for determination, and 3 replicates of the control and each sample were determined. The reaction program was set to 25°C for 5 minutes, and then the temperature was increased to 99°C at a rate of 0.5%. After the program ended, the lowest point temperature value of the curve in the "Melt Curve" graph was the Tm value of the sample. Figure 3 The Tm value determination results of the COEFN protein prepared in Example 1 are shown as an example (Tm = 55°C).

[0079] Example 3, Circular Dichroism Detection

[0080] The COEFN protein purified sample obtained in Example 1 and the type III collagen protein purified sample (as a positive collagen control) were used for circular dichroism detection, and the specific method included:

[0081] 1) Each 400 μL sample (COEFN protein purified sample obtained in Example 1 or type III collagen protein purified sample) was taken in a new EP tube, and 20 mM PB-50 mM NaCl (pH 7.3) was used as a blank control for machine detection;

[0082] 2) Far ultraviolet detection: the parameters were set as follows: starting wavelength (Begin) 180 nm, ending wavelength (End) 260 nm, scanning speed (Scanning speed) 50 nm / min, response (Response) 4 sec, bandwidth (Band Width) 1 nm, detection times (Accumulation) 3 times, and cell width 1 mm.

[0083] 3) The blank control solution (20 mM PB-50 mM NaCl) was loaded, the loading amount was 300 μL, and the data was saved after measurement. Then the sample was tested, the loading amount was 300 μL, and the data was saved after testing.

[0084] The results are shown in Figure 4 , where A shows the circular dichroism detection curve of the positive control collagen (type III collagen), and B shows the circular dichroism detection curve of the COEFN protein. Figure 4The results can be seen from the circular dichroism detection curve of collagen type III shown in Figure A, and the circular dichroism detection curve of COEFN protein shown in Figure B is more regular, with a minimum negative peak at 195-200 nm and a maximum positive peak at 220-230 nm, showing the typical characteristics of triple helix structure. The above results show that the presence of elastin and fibronectin functional domains in COEFN protein promotes the assembly and ordered arrangement of collagen type III, which in turn helps to promote the formation of triple helix of collagen type III.

[0085] Example 4, cell adhesion promoting experiment of fusion matrix protein

[0086] The purified sample of COEFN protein obtained in Example 1 above and the purified sample of collagen type III (as a positive collagen control) were used to detect their effects on promoting cell adhesion of human keratinocyte cell line HACAT, respectively, and the specific method included the following steps:

[0087] 1) Protein adsorption: adjust the concentration of COEFN or positive control collagen protein to 0.125 mg / ml, 0.25 mg / ml, 0.5 mg / ml, 1.0 mg / ml respectively with PBS (pH 7.4), 200 μl per well, add to 96-well plates, incubate at 37°C for 2 hours, then discard the buffer for standby.

[0088] 2) Dilute the logarithmic growth phase of HACAT cells to 1.5 x 10 4 / mL, add to 96-well plates, 200 μl per well (3000 cells per well) (the first row does not add cells), incubate at 37°C, 5% CO2 for 2, 4, 8, 12, 24 hours respectively.

[0089] 3) Discard the culture medium in the cell culture well, add 100 μl of 0.5% crystal violet solution (prepared with 20% methanol), incubate at room temperature for 30 minutes, wash off the dye and air dry.

[0090] 4) Add 150 μl of Sorenson's buffer (0.1 M sodium citrate, pH 4.2, 50% ethanol), incubate at room temperature for 60-90 minutes, measure the absorbance at 630 nm.

[0091] 5) Measure the absorbance value (OD value) of each well, calculate the proliferation rate according to the formula: (average OD value of experimental wells / average OD value of negative control wells) x 100%, and draw the adherent cell number curve.

[0092] The results show that both COEFN protein and collagen type III can promote the adhesion of HACAT cells, but COEFN protein promotes the adhesion of HACAT cells more significantly than collagen type III.

[0093] Example 5, Cell proliferation experiment of fusion matrix protein

[0094] The purified sample of COEFN protein obtained in Example 1 above and the purified sample of collagen type III (as a positive collagen control) were used to test the proliferation effect of both on HACAT, and the specific method included the following steps:

[0095] 1) Dilute the HACAT cells in logarithmic growth phase to 1.5 x 10 4 / mL, add to a 96-well plate, 200 μl per well (3000 cells per well) (the first row does not add cells), incubate at 37°C, 5% CO2 overnight. Dilute COEFN and positive control collagen in the 96-well plate at a gradient of 1:5 (the starting concentration is 1 μM), and the final volume per well is 200 μl. Discard the culture medium in the culture wells, add different concentrations of COEFN and positive control collagen (the first row adds culture medium, and the second row does not add COEFN and positive control collagen), and incubate at 37°C, 5% CO2 for 24 and 48 hours, respectively.

[0096] 2) Discard the culture medium in the cell culture wells, add 100 μl of 0.5% crystal violet solution (prepared with 20% methanol), incubate at room temperature for 30 minutes, wash off the dye, and air dry.

[0097] 3) Add 150 μl of Sorenson's buffer (0.1M sodium citrate, pH 4.2, 50% ethanol), incubate at room temperature for 60-90 minutes, and measure the absorbance at 630 nm.

[0098] 4) Measure the absorbance value (OD value) of each well, calculate the survival rate according to the formula: (average OD value of experimental wells / average OD value of negative control wells) x 100%, and draw a cell survival curve.

[0099] The results show that both COEFN protein and positive control collagen can promote the proliferation of HACAT cells, but the proliferation activity of COEFN protein is significantly higher than that of positive control collagen, proving that the COEFN protein provided by the present application can better enhance the viability of skin cells and has the effect of anti-wrinkle and firming.

[0100] Example 6, Cell migration experiment of fusion matrix protein

[0101] 6.1 Test principle

[0102] When the cells grow to a state of fusion into a monolayer, a blank area is artificially created on the fused monolayer cells, called "scratch". The cells at the edge of the scratch will gradually enter the blank area to heal the "scratch". During the cell migration process, images are captured at the beginning and periodically, and the cell migration rate is determined by comparing the images.

[0103] 6.2 Test steps

[0104] 6.2.1 First, use a marker pen to align the ruler on the back of the 6-well plate and draw horizontal lines evenly, approximately every 0.5 to 1 cm, across the holes.

[0105] 6.2.2 Add 1.5×10 4 2 ml of HACAT cell suspension with a concentration of 100 μg / mL.

[0106] 6.2.3 On the second day, observe that all cells in the 6-well plate have grown into a complete monolayer. Use the tip of the pipette to measure the ruler and make two scratches perpendicular to the horizontal line on the back of the plate.

[0107] 6.2.4 Wash the cells three times with PBS to remove the suspended cells.

[0108] 6.2.5 Add 1.8 ml of serum-free culture medium to the wells according to grouping, followed by 200 μl of sample (the purified COEFN protein sample obtained in Example 1). Add an equal amount of purified type III collagen sample, purified elastin sample, or purified fibronectin sample to the cell control wells.

[0109] 6.2.6 Place in a 37°C 5% CO2 incubator and incubate. Take a photo at 0:00 and record the photo location within each well. Observe and photograph the fixed location during subsequent observations.

[0110] The results are as follows Figure 5 As shown, it exemplifies photos of 0 and 24 hours after treatment with COEFN protein and CTR (type III collagen). It can be seen that 24 hours after COEFN protein treatment, the cells at the edge of the scratch have entered about 40% of the blank area to heal the "scratch", while 24 hours after CTR treatment, only a small amount of cells at the edge of the scratch entered the blank area (about 15%). Similar to CTR, when elastin and fibronectin were used to treat respectively for 24 hours, only a small amount of cells at the edge of the scratch entered the blank area, of which only about 6% of the blank area was entered after elastin treatment, and only about 8% of the blank area was entered after fibronectin treatment. The above results can prove that COEFN protein can play a synergistic role with type III collagen, elastin and fibronectin, significantly promoting the migration of HACAT cells, so that the COEFN protein provided by the present invention can play a good wound repair effect. In addition, the various elements (type III collagen, elastin and fibronectin) in the COEFN protein are directly connected without being connected by connecting peptides, and do not contain a histidine tag, so no exogenous amino acid sequence is introduced, making it identical to the natural sequence of the corresponding matrix protein, which is more conducive to its excellent biocompatibility, safety and low immunogenicity.

[0111] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent ones. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A multifunctional domain-fused extracellular matrix protein, characterized in that: The multifunctional domain-fused extracellular matrix protein comprises specific structural domains of human type III collagen, elastin and fibronectin, and has the key biological functions of collagen, elastin and fibronectin.

2. The multifunctional domain-fused extracellular matrix protein according to claim 1, characterized in that The connection order of the multifunctional domain fused extracellular matrix protein from N-terminus to C-terminus is type III collagen-elastin-fibronectin, and each element is directly connected; Optionally, the multifunctional domain-fused extracellular matrix protein comprises or consists of the amino acid sequence shown in SEQ ID NO:

1.

3. The multifunctional domain-fused extracellular matrix protein according to claim 1 or 2, characterized in that: The gene encoding the multifunctional domain-fused extracellular matrix protein comprises or consists of the nucleotide sequence shown in SEQ ID NO:

2.

4. The multifunctional domain-fused extracellular matrix protein according to any one of claims 1 to 3, characterized in that The multifunctional domain-fused extracellular matrix protein is expressed by mammalian cells, and can be optionally expressed by CHO cells or human embryonic kidney cells HEK293.

5. A polynucleotide encoding the multifunctional domain-fused extracellular matrix protein according to any one of claims 1 to 4; optionally, the polynucleotide comprises or consists of the nucleotide sequence shown in SEQ ID NO:

2.

6. A dual-promoter mammalian cell expression vector comprising the polynucleotide according to claim 5; Optionally, the dual-promoter mammalian cell expression vector comprises the nucleotide sequence shown in SEQ ID NO: 3; Further optionally, the mammalian cells include but are not limited to CHO and HEK293 cells.

7. A transgenic cell line or host bacteria, characterized in that: The transgenic cell line or host bacteria comprises the polynucleotide according to claim 5 or the dual-promoter mammalian cell expression vector according to claim 6.

8. A method for preparing the multifunctional domain-fused extracellular matrix protein according to any one of claims 1 to 4, comprising the following steps: 1) transferring the expression vector of the multifunctional domain-fused extracellular matrix protein into mammalian cells for expression, and obtaining a supernatant containing the multifunctional domain-fused extracellular matrix protein; and 2) purifying the supernatant to obtain the multifunctional domain-fused extracellular matrix protein; Optionally, the expression vector for the multifunctional domain fused extracellular matrix protein in step 1) is the dual-promoter mammalian cell expression vector according to claim 6; Further optionally, the mammalian cells in step 1) include but are not limited to CHO and HEK293 cells.

9. Use of the multifunctional domain-fused extracellular matrix protein according to any one of claims 1 to 4, the polynucleotide according to claim 5, the dual-promoter mammalian cell expression vector according to claim 6, or the transgenic cell line or host bacteria according to claim 7 in the preparation of a medicament for skin anti-aging and / or wound repair.

10. A skin anti-aging and / or wound repair drug comprising the multifunctional domain-fused extracellular matrix protein according to any one of claims 1 to 4 as a main active ingredient; Optionally, the skin anti-aging and / or wound repair drug further comprises one or more of the following: a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption accelerator, and a surfactant.

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