A recombinant fusion protein and use thereof

Abstract

The application provides a fusion protein with multiple biological activities, and a preparation method and application thereof. The fusion protein is a recombinant fusion polypeptide of collagen, fibronectin and elastin, and the amino acid sequence is shown as SEQ ID NO. 1. The recombinant fusion polypeptide has multiple biological activities, and the activity of promoting cell adhesion and cell proliferation of the fusion polypeptide is more significant compared with single molecules of collagen, fibronectin and elastin, and the fusion polypeptide can be applied to biological skin care and medical devices, and has obvious repair effect.

Description

Technical Field

[0001] This specification relates to the field of genetic engineering, and in particular to a recombinant fusion protein and its applications. Background Technology

[0002] Collagen is the most abundant protein in all animals, especially found in the extracellular matrix (ECM) of fibrous connective tissues such as skin and tendons, where it is responsible for the structural integrity of the tissue. Type I collagen is the most abundant protein in human skin, accounting for about 70% of the skin's dry weight. It is an insoluble fibrous protein with a molecular weight of 138.9 kDa. Its basic structural unit is a triple helix composed of three α-polypeptide chains, exhibiting a highly ordered three-dimensional structure. The α-polypeptide chains are highly repetitive sequences, with repeating units of Gly-XY, where X and Y are usually proline (Pro) and hydroxyproline (Hyp). After collagen is secreted into the ECM by fibroblasts, the two propeptides at both ends are cleaved and cross-linked under the action of lysine oxidase, ultimately existing in the ECM as a fibrous network structure. Collagen fibers act as a stable scaffold, and are important structural substances for maintaining the plumpness and elasticity of the skin.

[0003] Elastin is an important extracellular matrix protein in animals, second only to collagen in abundance. It plays an irreplaceable role in tissue contraction and expansion, and resistance to deformation under external forces, its most prominent characteristic being its ability to impart elasticity to tissues. Tropoesterin, a type of elastin, cross-links to form a large molecular network structure, which then combines with microfibrils to assemble into elastic fibers; this is the main form of natural elastin. Elasticity bound to fibers is highly cross-linked, exhibits good thermal stability, a long half-life, and excellent resistance to enzymatic degradation. Simultaneously, elastin plays a crucial role in regulating various cellular functions, including promoting cell adhesion, proliferation, differentiation, chemotaxis, and migration. Natural elastin is 68.3 kDa in size and its sequence consists of two types of domains: hydrophobic domains and hydrophilic cross-linked regions. The hydrophobic domains use (VPGXG) as the basic repeating unit, where X can be any amino acid other than P, but usually the X position is valine (V), such as VPVAPG, KGGVG, VGGVG, GVGVP, LGAGGAG, and LGAGGAGVL. The hydrophilic regions are mainly composed of alanine (A) and lysine (K), with these two amino acids repeating irregularly and interspersed with a small number of hydrophilic cross-linked regions of other amino acids.

[0004] Fibronectin (FN) is a ubiquitous and important component of the extracellular matrix (ECM). There are over 20 types in the human body, widely distributed in blood, body fluids, and various tissues. It serves as a regulator of cellular activities and a crucial scaffold protein for maintaining and guiding tissue structure and ECM composition. In the body's life processes, FN participates in cell migration, adhesion, proliferation, hemostasis, tissue repair, and embryonic development. It acts as a growth factor, promoting cell proliferation, inducing epidermal cells to pass through granulation tissue, and promoting the remodeling of the basement membrane and normal keratinization. Fibronectin also participates in numerous pathological processes. FN is a glycoprotein with a molecular weight of approximately 450 kDa, consisting of a dimer formed by two subunits linked by disulfide bonds. The entire molecular structure is composed of two similar A and B chains, forming a V-shape. Analysis of amino acids, genes, and genomic DNA sequences indicates that FN is a multi-domain glycoprotein composed of a series of repetitive modular structures: 12 FNI type repeat sequences (FNI), 2 FNII type repeat sequences (FN II), 15 constitutive and 2 alternating splice (referred to in this paper as EIIIA and EIIIB) FN type III repeat sequences (FN III), and a non-homologous variable (V) or type III linker (IIICS) region. This multi-modal structure and inter-modal regions endow the FN molecule with flexibility, enabling it to participate in the regulation of its function.

[0005] The three proteins mentioned above are all essential components of the ECM (Extracellular Matrix), widely used in functional skincare, medical aesthetics, and other fields. They respectively endow the skin with certain tensile strength, elasticity, and cell adhesion, playing a crucial role in maintaining the stability of skin tissue; none can be dispensed with. Most of the three proteins used in the market are derived from natural sources, which are costly and pose a risk of carrying pathogens, thus presenting certain safety concerns. Furthermore, each protein is extracted independently and exerts its own biological activity individually. Summary of the Invention

[0006] To address the aforementioned technical issues, this application provides a recombinant fusion protein with multiple biological activities, its preparation method, and its applications. This reduces the cost of using existing collagen, fibronectin, and elastin simultaneously, improves ease of use and safety, and endows a single protein with multiple biological effects and more application areas.

[0007] This application provides a fusion protein, which comprises, from the N-terminus to the C-terminus, a collagen peptide fragment, a fibronectin fragment, and an elastin fragment.

[0008] This application also provides an isolated polynucleotide that encodes the aforementioned fusion protein.

[0009] This application also provides a construct containing the isolated polynucleotides described above.

[0010] This application also provides a cell comprising the above-described construct, or the cell genome integrating the above-described polynucleotides.

[0011] This application also provides a method for preparing the above-mentioned fusion protein, the method comprising culturing the above-mentioned cells to express the fusion protein.

[0012] This application also provides the use of the above-mentioned fusion protein in cosmetics, skin care products, tissue materials, or in the preparation of medical devices or pharmaceuticals that promote wound healing or repair.

[0013] This application also provides an active agent that promotes cell proliferation and / or adhesion, said active agent comprising the above-described fusion protein.

[0014] The beneficial effects of this application include, but are not limited to: This application unexpectedly discovered that the fusion protein obtained by combining collagen peptide fragments, fibronectin fragments, and elastin fragments in a specific order possesses multiple biological activities and can be expressed solublely in *E. coli* without forming inclusion bodies. This not only solves the disadvantages of natural proteins, such as excessively large molecular weight, difficulty in extraction from animal tissues, poor processability, and high cost, but also addresses the problem that natural full-length collagen, elastin, and fibronectin cannot be expressed in *E. coli*. Furthermore, it reduces rejection reactions and avoids the risk of infectious diseases, while combining the excellent biological activities of three natural proteins, making it more cost-effective than preparing three proteins individually. The recombinant fusion protein obtained in this application can promote cell growth, increase cell adhesion rate, and enhance cell metabolism. In addition, the recombinant fusion protein obtained in this application, when combined with appropriate excipients, can be further prepared into lyophilized powders, nanospheres, nanoliposomes, aqueous solutions, gels, and other semi-solid formulations, which can be used as active additives in tissue engineering, pharmaceuticals, and cosmetic skincare. The recombinant fusion protein prepared in this application has the outstanding advantages of humanization, low immunogenicity, and low likelihood of causing allergic reactions. Attached Figure Description

[0015] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein:

[0016] Figure 1 The image shows the plasmid map of the recombinant expression plasmid pET32a-CFE for Escherichia coli.

[0017] Figure 2The results show the expression test results of recombinant engineered Escherichia coli. Lane 1 contains pre-induction bacterial protein of the recombinant engineered bacteria, lane 2 contains post-induction bacterial protein of the recombinant engineered bacteria, lane 3 contains post-induction lysed and centrifuged precipitated protein of the recombinant engineered bacteria, and lane 4 contains post-induction lysed and centrifuged supernatant protein of the recombinant engineered bacteria.

[0018] Figure 3 The results show the fermentation expression of recombinant engineered Escherichia coli. Lane 1 shows the bacterial protein before fermentation induction, lane 2 shows the bacterial protein 2 hours after fermentation induction, and lane 3 shows the bacterial protein 3.6 hours after fermentation induction.

[0019] Figure 4 The results show the purification of the fusion protein. Lane 1 is the TrxA-CFE sample, lane 2 is the TrxA-CFE enzyme digestion test sample, lane 3 is sample 1 separated after TrxA-CFE enzyme digestion, lane 4 is sample 2 separated after TrxA-CFE enzyme digestion, lane 5 is sample 3 separated after TrxA-CFE enzyme digestion, and lanes 6 and 7 are the final purified CFE samples.

[0020] Figure 5 The results show the activity of the fusion protein in promoting the adhesion of NHDF-positive human skin fibroblasts.

[0021] Figure 6 The results show the activity of the fusion protein in promoting the proliferation of BALB / 3T3 cells. Detailed Implementation

[0022] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0023] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0024] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0025] This application provides a fusion protein, which comprises, from the N-terminus to the C-terminus, a collagen peptide fragment, a fibronectin fragment, and an elastin fragment.

[0026] In some embodiments, the amino acid sequence of the collagen fragment may be as shown in SEQ ID NO.3.

[0027] The term "sequence" in this article should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the amino acid sequence, unless a more specific interpretation is required in this article.

[0028] SEQ ID NO.3

[0029] GERGAPGFRGPAGPNGIPGEKGPAGERGAP

[0030] In some embodiments, the amino acid sequence of the fibronectin fragment may be as shown in SEQ ID NO.4.

[0031] SEQ ID NO.4

[0032] NAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPGRGDSP

[0033] In some embodiments, the amino acid sequence of the elastin fragment may be as shown in SEQ ID NO.5.

[0034] SEQ ID NO.5

[0035] GLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGVA

[0036] In some embodiments, the collagen fragments and fibronectin fragments, as well as the fibronectin fragments and elastin fragments, may be linked by linker peptides. Preferably, in some embodiments, the amino acid sequence of the linker peptide is as shown in SEQ ID NO. 6 and SEQ ID NO. 7.

[0037] SEQ ID NO.6: GGGGSGGGGSGGGGS

[0038] SEQ ID NO.7: GGGGSGGGGSGGGGS

[0039] In some embodiments, the N-terminus of the collagen fragment may also be attached with a TrxA tag.

[0040] In some embodiments, the fusion protein may include the amino acid sequence shown in SEQ ID NO.1.

[0041] SEQ ID NO.1

[0042] GERGAPGFRGPAGPNGIPGEKGPAGERGAPGGGGSGGGGSGGGGSNAPQPSHISKYILRWRPKNSVGRWKEATIPGHLNSYTIKGLKPGVVYEGQLISIQQYGHQEVTRFDFTTTSTSTPGRGDSPGGGGSGGGGSGGGGSGLVPGVGVAPGVGVAPGVGVAPGVGLAPGVGVAPGVGVAPGVGVAPGIGPGGVA**

[0043] This application also provides an isolated polynucleotide that encodes the aforementioned fusion protein.

[0044] The term "polynucleotide" refers to a polymer of nucleotides typically linked from one deoxyribose or ribose to another, and, depending on the context, refers to both DNA and RNA. The term "polynucleotide" in this application does not contain any size limitation and also includes polynucleotides containing modifications, particularly modified nucleotides.

[0045] In some embodiments, the isolated polynucleotide may include the sequence shown in SEQ ID NO.2.

[0046] SEQ ID NO.2

[0047] GGTGAAAGAGGTGCTCCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGTGAAAAGGGTCCAGCAGGAGAGAGAGGTGCACCTGGTGGAGGCGGTTCTGGAGGTGGCGGTTCCGGCGGTGGAGGATCCAACGCTCCACA ACCTTCTCATATTTCCAAGTACATTTGAGATGGCGTCCAAAGAACTCTGTTGGTAGATGGAAGGAAGCTACTATTCCAGGTCATTTGAACTCCTACACCATTAAGGGTTTGAAGCCAGGAGTTGTCTACGAAGGTCAATTGATTT CTATCCAACAGTACGGTCATCAAGAAGTTACTAGATTTGATTTCACTACCACATCTACTTCCACACCAGGTAGAGGTGATTCCCCAGGAGGAGGAGGTTCAGGTGGAGGTGGCTCCGGGGGAGGTGGTTCCGGATTGGTTCCAGGA GTAGGAGTCGCCCCCGGCGTTGGTGTCGCACCCGGCGTGGGTGTCGCCCCTGGCGTGGGTTTGGCCCCAGGTGTTGGTGTGCCCCAGGAGTCGGAGTCGCTCCAGGAGTGGGAGTCGCACCAGGTATTGGACCAGGAGGTGTCGCT

[0048] This application also provides a construct containing the isolated polynucleotides described above.

[0049] As used herein, "construction" refers to a polynucleotide capable of carrying at least one polynucleotide fragment. Constructs can deliver fragments of nucleic acids, or individual polynucleotides, into host cells. It may contain at least one expression cassette containing a regulatory sequence for the proper expression of the polynucleotide incorporated therein. The polynucleotide to be introduced into the cell (e.g., a polynucleotide encoding a target product or a selectable marker) can be inserted into the expression cassette of a vector for expression therefrom. Constructs according to this application may exist in circular or linear (linearized) form and also include construct expression fragments. The term "construction" also includes artificial chromosomes or similar individual polynucleotides that allow the transfer of exogenous nucleic acid fragments.

[0050] This application also provides a cell comprising the above-described construct, or the cell genome integrating the above-described polynucleotides.

[0051] This application also provides a method for preparing the above-mentioned fusion protein, the method comprising culturing the above-mentioned cells to express the fusion protein.

[0052] In some embodiments, the method may further include the following steps: 1) culturing and expressing a fusion protein with a TrxA tag; 2) removing the TrxA tag from the fusion protein.

[0053] Thioredoxin (TrxA or Trx1) is an oxidoreductase found in eukaryotic and prokaryotic organisms. It is composed of α-helices and β-sheets and has extremely high thermal stability. As a fusion tag protein, TrxA has the following main advantages: (1) promotes solubility; (2) has good thermal stability; (3) its N-terminus and C-terminus are exposed, allowing it to fuse with the target protein without affecting the target protein's structure and activity due to steric hindrance; at the same time, it acts as a valence mate, giving it enough time to complete the correct folding; (4) its small molecular weight of 11.6 kDa reduces the impact on the target protein and downstream applications.

[0054] This application also provides the use of the above-mentioned fusion protein in cosmetics, skin care products, tissue materials, or in the preparation of medical devices or pharmaceuticals that promote wound healing or repair.

[0055] This application also provides an active agent that promotes cell proliferation and / or adhesion, said active agent comprising the above-described fusion protein.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0057] Example 1 - Preparation of Recombinant Fusion Protein

[0058] 1. Construction of recombinant fusion protein engineered strains

[0059] The amino acid sequence encoding the recombinant fusion protein was referenced from humanized collagen, fibronectin, and elastin, as shown in SEQ ID NQ.1. The corresponding nucleotide coding sequences of the amino acid sequence shown in SEQ ID NQ.1 were modified, using codons that were frequently used in the *E. coli* expression system, while avoiding transcription factor binding regions, repetitive sequences, and higher-order RNA structures that might affect expression. The DNA coding sequence of the fusion protein obtained after codon optimization is shown in SEQ ID NO.2. The nucleotide sequence corresponding to the TrxA tag was added to the 5' end of SEQ ID NO.2 to synthesize this sequence.

[0060] Primers were designed to amplify the coding sequence of the recombinant fusion protein tagged with TrxA, and NcoI and NotI restriction sites were introduced. The amplified products were digested with NcoI and NotI and then ligated with the pET32a plasmid (purchased from Thermo Fishier). The digested fragments were ligated using T4 DNA ligase. The resulting recombinant plasmid was transformed into *E. coli* DH5α strain and cultured overnight at 37°C in an inverted position. The following day, a subset of clones were selected for PCR identification and sequencing. The successfully constructed recombinant plasmid was named pET32a-CFE.

[0061] The recombinant pET32a-CFE plasmid was transformed into *Escherichia coli* strain BL21(DE3). A subset of clones were selected for expression validation, and SDS-PAGE was used to verify the expression of the recombinant protein. The resulting engineered strain was named pET32a-CFE-BL21(DE3).

[0062] Expression results of pET32a-CFE-BL21(DE3) Figure 2 The results showed a clear heterologous protein expression band in the E. coli culture. The theoretical molecular weight of TrxA-CFE is 36.2 kDa, and the heterologous protein in the figure matches this theoretical molecular weight, indicating that the CFE protein is effectively expressed.

[0063] 2. Fermentation expression test of recombinant fusion protein engineered strain

[0064] The pET32a-CFE-BL21(DE3) expression strain preserved in glycerol tubes was inoculated into LB medium at a ratio of 1:1000 (v / v) and cultured in shake flasks for 16-24 hours. During the cell proliferation phase, the shake-flask culture was inoculated into fermentation medium at a ratio of 1:20 (v / v). Dissolved oxygen levels were maintained above 20% by adjusting the rotation speed, tank pressure, and aeration rate, allowing *E. coli* cells to grow for 3-4 hours. During the induction phase, IPTG was added, and dissolved oxygen levels were maintained above 20% by adjusting the rotation speed, tank pressure, and aeration rate. Fermentation was terminated after 4 hours. The fermented cells were collected after centrifugation for subsequent purification. SDS-PAGE analysis was performed on fermented cells collected at different time points. Figure 3 The results showed that the expression of TrxA-CFE protein increased continuously with the extension of induction time, and the expression level accounted for more than 20% of the total bacterial cells, which meets the requirements of large-scale production.

[0065] 3. Purification test of recombinant fusion protein

[0066] The fermented bacterial cells were added to Tris buffer at pH 7.5, homogenized at 400 bar, and centrifuged at 18,000 rpm for 20 min. The collected supernatant was purified by nickel column chromatography. After equilibration with Tris buffer at pH 7.5, imidazole gradient elution was performed, and the elution peak was collected at 150 mM. The eluted sample was desalted using G25 chromatography, and the high-purity sample was further digested with TEVProtease to remove the TrxA tag. The digested sample was purified by nickel column chromatography, and the eluted sample was further purified by Poros 50HQ chromatography to obtain the high-purity, low-endotoxin target protein CFE. The final purified sample is shown below. Figure 4 The sequence was identified as expected.

[0067] Example 2 - Activity Analysis of Fusion Protein

[0068] 1. Analysis of the cell adhesion-promoting activity of the fusion protein

[0069] Cell adhesion promotion experiments were conducted on the fusion protein of the present invention.

[0070] 1.1 Experimental Reagents and Materials

[0071] DMEM medium: GIBCO, catalog number 11965-084 (liquid), 12800017 (powder); FBS: CORNING, catalog number 35081CV or GIBCO catalog number 10091-148; Trypsin-EDTA (0.25%): GIBCO, catalog number 25200072; Penicillin-Streptomycin 100×: GIBCO, catalog number 15140-122; CCK-8 solution: Tongren Chemical; catalog number CK04; Sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4·12H2O), potassium dihydrogen phosphate (KH2PO4) were all domestically produced analytical grade reagents.

[0072] 96-well cell culture plate: Thermo, catalog number 167008; disposable pipettes (5ml, 10ml, 25ml): Thermo; 0.6ml centrifuge tubes (sterile); 1.5ml centrifuge tubes (sterile); 10μL pipette tips (sterile), 200μL pipette tips (sterile);

[0073] Cells: BALB / 3T3 cells, NIH / 3T3 cells, and NHDF normal human skin fibroblasts.

[0074] 1.2 Specific Implementation Plan

[0075] The fusion protein was pre-diluted to 0.1 mg / mL with PBS buffer. After pre-dilution, it was serially diluted 2-fold in 96-well cell culture plates, with a total of 9 dilutions. 100 μL of the fusion protein sample at different dilutions was used in each well. A negative control (PBS buffer) was also included. The coated 96-well cell culture plates were incubated at room temperature for 60 ± 10 min. Then, 1 × 10⁻⁶ cells were added. 4 / hole ~ 10×10 4 Cells were seeded at the specified density into the aforementioned 96-well cell culture plates and incubated at 37°C for 60 ± 10 min. After incubation, the liquid in the cell culture plates was discarded, and 200 μL of PBS buffer was added to each well for adhesion. The liquid in the plates was discarded again, and the washing was repeated twice. Finally, 100 μL of DEME complete medium was added to each well for adhesion, followed by 10 μL of CCK-8 solution. The plates were sealed with sterile water and incubated for another 1–4 h.

[0076] The absorbance of the cell plate was measured at 450 nm using an ELISA reader. The reference wavelength was 650 nm.

[0077] The test result (OD difference) is the average OD of the fusion protein wells minus the average OD of the PBS wells.

[0078] 1.3 Results Analysis

[0079] Based on the specific experimental procedures described above, after analyzing the data ( Figure 5 The study found that the fusion protein had significant cell adhesion-promoting activity. In this experimental method, the fusion protein showed the best cell adhesion-promoting activity compared to single-component collagen, fibronectin, elastin and commercially available collagen.

[0080] 2. Analysis of the cell proliferation-promoting activity of the fusion protein

[0081] Cell proliferation experiments were conducted on the fusion protein of the present invention.

[0082] 2.1 Experimental Reagents and Materials

[0083] DMEM medium: GIBCO, catalog number 11965-084 (liquid), 12800017 (powder); FBS: CORNING, catalog number 35081CV or GIBCO catalog number 10091-148; Trypsin-EDTA (0.25%), GIBCO, catalog number 25200072; Penicillin-Streptomycin 100×: GIBCO, catalog number 15140-122; CCK-8 solution: Tongren Chemical, catalog number CK04; Sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4·12H2O), potassium dihydrogen phosphate (KH2PO4) were all domestically produced analytical grade reagents.

[0084] 96-well cell culture plate: Thermo, catalog number 167008; disposable pipettes (5ml, 10ml, 25ml): Thermo; 0.6ml centrifuge tubes (sterile); 1.5ml centrifuge tubes (sterile); 10μL pipette tips (sterile), 200μL pipette tips (sterile).

[0085] Cells: BALB / 3T3 cells, NIH / 3T3 cells, and NHDF normal human skin fibroblasts.

[0086] 2.2 Specific Implementation Plan

[0087] Take fibroblasts in good growth condition, at 1×10 3 / hole ~ 4×10 3 Cells were seeded at a density of 100 μL per well in 96-well cell culture plates and incubated at 37±2℃ in a 5% CO2 incubator for 16-24 hours. The liquid in the cell culture plates was discarded, and 100 μL of 1% FBS-DMEM medium was added to each well. Subsequently, the fusion protein was pre-diluted to 0.1 mg / mL with PBS buffer. After pre-dilution, a 2-fold serial dilution was performed in the 96-well cell culture plates, for a total of 9 dilutions. 10 μL of different dilutions of the fusion protein sample was added to each well, and a negative control (PBS buffer) was included. The plates were then incubated for 60-72 hours. Finally, the 96-well cell culture plates were removed from the incubator, and 10 μL of CCK-8 solution was added to each well. The plates were then incubated at 37±2℃ in a 5% CO2 incubator for 1-4 hours.

[0088] The absorbance of the cell plate was measured at 450 nm using an ELISA reader. The reference wavelength was 650 nm.

[0089] The test result (OD difference) is the average OD of the recombinant fusion protein wells minus the average OD of the PBS wells.

[0090] 2.3 Results Analysis

[0091] Based on the specific experimental procedures described above, after analyzing the data ( Figure 6 The study found that the fusion protein had a very significant cell proliferation-promoting activity. In this experimental method, compared with single-component collagen, fibronectin and elastin, the fusion protein had the best cell proliferation-promoting activity and was significantly higher than commercially available collagen.

[0092] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0093] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0094] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0095] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. A fusion protein, wherein the fusion protein comprises, from the N-terminus to the C-terminus, a collagen peptide fragment, a fibronectin fragment, and an elastin fragment.

2. The fusion protein of claim 1, wherein, The amino acid sequence of the collagen fragment is shown in SEQ ID NO. 3; And / or, the amino acid sequence of the fibronectin fragment is as shown in SEQ ID NO.4; And / or, the amino acid sequence of the elastin fragment is as shown in SEQ ID NO.5; And / or, the collagen fragments and fibronectin fragments, as well as the fibronectin fragments and elastin fragments, are linked by a linker peptide, preferably, the amino acid sequence of the linker peptide is shown in SEQ ID NO.6 and SEQ ID NO.7; And / or, the N-terminus of the collagen fragment is also attached with a TrxA tag.

3. The fusion protein of claim 1, wherein, The fusion protein comprises the amino acid sequence shown in SEQ ID NO.

1.

4. An isolated polynucleotide, comprising, The isolated polynucleotide encodes the fusion protein as described in any one of claims 1 to 3.

5. The isolated polynucleotide of claim 4, wherein, The isolated polynucleotides include the sequence shown in SEQ ID NO.

2.

6. A construct, characterized in that, The construct contains the isolated polynucleotides as described in claim 4 or 5.

7. A cell comprising the construct of claim 6, or the cell genome integrating the polynucleotides of claim 4 or 5.

8. A method for preparing the fusion protein as described in any one of claims 1 to 3, the method comprising culturing cells as described in claim 7 to express the fusion protein.

9. The method of claim 8, wherein, Includes the following steps: 1) Culture and express fusion proteins with the TrxA tag; 2) Remove the TrxA tag from the fusion protein.

10. The use of the fusion protein as described in any one of claims 1 to 3 in cosmetics, skin care products, tissue materials, or in the preparation of medical devices or pharmaceuticals that promote wound healing or repair.

11. An active agent that promotes cell proliferation and / or adhesion, said active agent comprising the fusion protein as described in any one of claims 1 to 3.

Images