Recombinant mussel foot protein and application thereof

Abstract

The invention discloses a recombinant mussel foot protein and application thereof. The amino acid sequence of the recombinant mussel foot protein is as shown in SEQ ID No.2. The recombinant mussel foot protein is subjected to dopa modification, experiments prove that the dopa modification rate of the recombinant mussel foot protein modified by the dopa reaches 4 wt% or above, the requirement of the industrial standard for the dopa content is completely met, the recombinant mussel foot protein modified by the dopa has high free radical scavenging capacity, and the free radical scavenging rate reaches 59.68%; the hydrogel can be dissolved with sodium hyaluronate, glycerol, EDTA (Ethylene Diamine Tetraacetic Acid) disodium and PEG (Polyethylene Glycol) 6000, so that the hydrogel has a good compounding potential; the dopa-modified recombinant mussel foot protein solution can promote migration of human-derived fibroblasts, so that the recombinant mussel foot protein has a cell repairing capability and a good repairing effect. Therefore, the recombinant mussel foot protein provided by the invention has an important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant mussel foot protein and its applications. Background Technology

[0002] Mussel adhesive protein (MAP), also known as mussel foot protein, is a protein secreted by mussels through their glands for attaching to various material surfaces. The adhesive properties of mussel adhesive protein have been shown to promote cell adhesion and crawling replacement. The dopa groups contained in mussel adhesive protein possess antioxidant and free radical scavenging capabilities. Furthermore, the oxidized dopaquinone and unoxidized dopa can form a self-crosslinking polymer network, which can prevent bacterial infection of wounds while allowing water vapor and air to pass through. These properties make mussel adhesive protein a promising candidate for applications in cosmetics and medical dressings.

[0003] However, the yield of natural mussel adhesive protein extracted from mussels is low, leading to high prices; in vitro passage culture of mussel foot gland cells has not yet been successful. Constructing engineered strains to express recombinant mussel adhesive protein using genetic engineering techniques is one effective method to increase its yield, but recombinant mussel adhesive protein has drawbacks compared to natural mussel adhesive protein, such as a lower dopa modification rate and poorer stability during in vitro modification. Obtaining proteins with high dopa content and possessing or partially possessing the functions of mussel adhesive protein presents certain technical challenges. Summary of the Invention

[0004] The purpose of this invention is to provide a protein that promotes cell adhesion or migration, thereby achieving a repair effect.

[0005] This invention first protects a mussel foot protein, which may be as follows: b1), b2), b3), or b4). b1) Proteins with amino acid sequences as shown in SEQ ID No. 4; b2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in b1); b3) A protein that has the same function as the mussel foot protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in b1) or b2). b4) A protein that has 80% or more identity with the amino acid sequence defined by b1) or b2) and has the same function as the mussel foot protein.

[0006] The proteins in b2) above are labeled as shown in Table 1.

[0007] Table 1. Sequence of Labels

[0008] The protein in b3) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0009] The proteins mentioned in b3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0010] The gene encoding the protein in b3) above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in SEQ ID No. 3, and / or by performing a missense mutation on one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0011] The mussel foot protein mentioned above can specifically be a recombinant mussel foot protein with an amino acid sequence as shown in SEQ ID No. 2.

[0012] Nucleic acid molecules encoding any of the mussel foot proteins described above are also within the scope of protection of this invention.

[0013] Any of the above-mentioned nucleic acid molecules may be DNA molecules as shown in c1), c2), c3), c4), c5), or c6): c1) The coding region is the DNA molecule shown in SEQ ID No. 3; c2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3; c3) The coding region is the DNA molecule shown in SEQ ID No. 1; c4) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 1; c5) and the DNA molecule defined by c1) or c2) or c3) or c4) have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity, and encode a DNA molecule that encodes any of the mussel foot proteins described above. c6) hybridizes under stringent conditions to a nucleotide sequence defined by c1) or c2) or c3) or c4) and encodes a DNA molecule that encodes any of the mussel foot proteins described above.

[0014] The stringent conditions were: hybridization in a 2×SSC, 0.1% SDS solution at 68°C with two washes of 5 min each, followed by hybridization in a 0.5×SSC, 0.1% SDS solution at 68°C with two washes of 15 min each.

[0015] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0016] Those skilled in the art can readily mutate the nucleotide sequence encoding any of the aforementioned mussel foot proteins using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides that possess 70% or higher identity with the nucleotide sequence of any of the aforementioned mussel foot proteins isolated by this invention, as long as they encode any of the aforementioned mussel foot proteins, are derived from and equivalent to the nucleotide sequence of this invention.

[0017] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 70% or higher, 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence encoding the amino acid sequence of the present invention, such as SEQ ID No. 4 or SEQ ID No. 2. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0018] Expression cassettes, recombinant vectors, or recombinant microorganisms containing any of the aforementioned nucleic acid molecules are also within the scope of protection of this invention.

[0019] An expression cassette containing any of the aforementioned nucleic acid molecules refers to DNA capable of expressing any of the aforementioned mussel foot proteins in host cells. This DNA may include not only promoters that initiate the coding gene for any of the aforementioned mussel foot proteins, but also terminators. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters.

[0020] Recombinant vectors containing expression cassettes of any of the aforementioned nucleic acid molecules can be constructed using existing expression vectors. These expression vectors include binary Agrobacterium vectors and vectors suitable for microbombardment, such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, or pCAMBIA1391-Xb (CAMBIA). The expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing the polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. When constructing expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The translation control signals and start codons are widely available and can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or a structural gene. The vector can be a plasmid, granule, bacteriophage, or viral vector.

[0021] The recombinant vector can be a recombinant plasmid obtained by inserting any of the above-mentioned nucleic acid molecules into the multiple cloning site of an expression vector or a cloning vector.

[0022] The expression vector may specifically be the vector pET28a(+).

[0023] The recombinant vector may specifically be the recombinant expression vector pET28(+)-M8. The recombinant expression vector pET28(+)-M8 may be obtained by replacing the small DNA fragment between the restriction endonucleases NdeI and HindIII of the vector pET28a(+) with the nucleotide sequence shown in SEQ ID No. 3, while keeping the remaining nucleotide sequence of the vector pET28a(+) unchanged.

[0024] The microorganism may be yeast, bacteria, algae, or fungi. The bacteria may be Escherichia coli. Specifically, the Escherichia coli may be Escherichia coli BL21(DE3).

[0025] The application of any of the mussel foot proteins, nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms described above in scavenging free radicals or in the preparation of products for scavenging free radicals is also within the scope of protection of this invention.

[0026] The application of any of the mussel foot proteins, nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms described above in repairing cells, promoting wound healing, and / or reducing scar formation is also within the scope of protection of this invention.

[0027] The use of any of the mussel foot proteins, nucleic acid molecules, or expression cassettes, recombinant vectors, or recombinant microorganisms described above in the preparation of products for repairing cells, promoting wound healing, and / or reducing scar formation is also within the scope of protection of this invention.

[0028] In any of the above-described applications, the mussel foot protein can be modified with dopa, i.e., dopa-modified mussel foot protein. In one embodiment of the present invention, the dopa modification rate of the dopa-modified recombinant mussel foot protein reaches more than 4 wt%, which far meets the industry standard requirements for dopa content.

[0029] In any of the above applications, the product may be a cosmetic or a pharmaceutical.

[0030] Experiments have shown that the dopamine-modified recombinant mussel foot protein possesses a high free radical scavenging ability, with a free radical scavenging rate reaching 59.68%. Furthermore, it exhibits good solubility within a pH range of 3.0-6.0, being miscible with sodium hyaluronate, glycerol, disodium EDTA, and PEG6000 (no protein flocculation or precipitation occurs when compounded with sodium hyaluronate), demonstrating good compounding potential. In addition, the dopamine-modified recombinant mussel foot protein solution can promote the migration of human fibroblasts, indicating that the recombinant mussel foot protein possesses cell repair capabilities and exhibits good repair efficacy. The recombinant mussel foot protein provided by this invention has significant application value. Attached Figure Description

[0031] Figure 1 The standard curve is plotted for measuring the dopa modification rate in step 1 of Example 2.

[0032] Figure 2 The color development of the 3-DOPA modified recombinant mussel foot protein solution in step one of Example 2 is shown.

[0033] Figure 3 The nitrocellulose membrane before NBT staining in step two of Example 2.

[0034] Figure 4 The nitrocellulose membrane after NBT staining in step two of Example 2.

[0035] Figure 5 This is the SDS-PAGE gel electrophoresis pattern of the dopa-modified recombinant mussel foot protein in step 1 of Example 3.

[0036] Figure 6 The results are obtained by liquid chromatography detection of recombinant mussel foot protein in step 2 of Example 3.

[0037] Figure 7The results are obtained by liquid chromatography from the dopa-modified recombinant mussel foot protein in step 2 of Example 3.

[0038] Figure 8 The UV and TIV spectra are those of the Trypsin enzymatic hydrolysis product in step 3 of Example 3.

[0039] Figure 9 The UV and TIC spectra of the Chymotrypsin enzymatic hydrolysis product in step 3 of Example 3 are shown.

[0040] Figure 10 The UV and TIC spectra of the Lys-C enzymatic hydrolysis product in step 3 of Example 3 are shown.

[0041] Figure 11 The images show the compounding of DOPA-modified recombinant mussel peduncle protein with commonly used cosmetic reagents in Example 5. From left to right, they are: DOPA-modified recombinant mussel peduncle protein solution, DOPA-modified recombinant mussel peduncle protein solution + 10% (w / v) PEG6000 solution, DOPA-modified recombinant mussel peduncle protein solution + 0.2% (w / v) disodium EDTA solution, DOPA-modified recombinant mussel peduncle protein solution + 20% (w / v) glycerol solution, and DOPA-modified recombinant mussel peduncle protein solution + 1.2% (w / v) sodium hyaluronate solution.

[0042] Figure 12 The following are examples of compounding commercially available recombinant mussel adhesive protein with commonly used cosmetic reagents in Example 5. From left to right, they are: commercially available recombinant mussel adhesive protein solution, commercially available recombinant mussel adhesive protein solution + 10% (w / v) PEG6000 solution, commercially available recombinant mussel adhesive protein solution + 0.2% (w / v) disodium EDTA solution, commercially available recombinant mussel adhesive protein solution + 20% (w / v) glycerol solution, and commercially available recombinant mussel adhesive protein solution + 1.2% (w / v) sodium hyaluronate solution.

[0043] Figure 13 The purpose of this study was to test the ability of dopa-modified recombinant mussel foot protein to migrate to human fibroblasts in Example 7.

[0044] Figure 14 The results show the statistical results of the migration rate of the dopa-modified recombinant mussel foot protein to human fibroblasts in Example 7. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0048] Example 1: Expression and purification of recombinant mussel foot protein I. Construction of the recombinant expression vector pET28(+)-M8 1. The inventors of this application obtained mussel foot protein with an amino acid sequence as shown in SEQ ID No.4 through extensive experiments.

[0049] 2. The amino acid sequence shown in SEQ ID No. 4 was translated into a nucleotide sequence and optimized according to the host to obtain the nucleotide sequence of mussel foot protein shown in SEQ ID No. 3.

[0050] 3. Replace the small DNA fragment between the restriction endonucleases NdeI and HindIII of the vector pET28a(+) (a product of Beijing Qingke Company) with the nucleotide sequence shown in SEQ ID No. 3, while keeping the rest of the nucleotide sequence of the vector pET28a(+) unchanged, to obtain the recombinant expression vector pET28(+)-M8.

[0051] The recombinant expression vector pET28(+)-M8 contains the fusion gene shown in SEQ ID No. 1, which is composed of the nucleotide sequence of mussel foot protein fused with six His tags on the vector pET28a(+). The fusion gene encodes recombinant mussel foot protein. The recombinant expression vector pET28(+)-M8 expresses the recombinant mussel foot protein. The amino acid sequence of the recombinant mussel foot protein is shown in SEQ ID No. 2.

[0052] II. Expression of recombinant mussel foot protein 1. The recombinant expression vector pET28(+)-M8 was transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli E. coli BL21(DE3) / pET28a(+)-M8.

[0053] 2. Recombinant E. coli E. coli BL21(DE3) / pET28a(+)-M8 was inoculated into 5 mL of TB medium containing kanamycin and cultured at 37℃ and 220 rpm for 24 h to obtain culture solution 1.

[0054] The solutes and their concentrations in TB medium were 12 g / L tryptone, 24 g / L yeast extract, 5 g / L glycerol, 16.43 g / L dipotassium hydrogen phosphate trihydrate and 2.31 g / L potassium dihydrogen phosphate, and the solvent was water.

[0055] 3. Inoculate culture solution 1 at a 2% inoculum rate into a 100 mL shake flask containing 50 mL of TB medium. Incubate at 37°C and 200 rpm until OD is obtained. 610nm Culture medium 2 reaches a pH of 4.0-6.0. Then, IPTG is added to culture medium 2 to make the concentration in the system 0.1mM, and cultured at 25℃ and 220 rpm for 24h. The culture medium is collected into centrifuge tubes, centrifuged at 5000 rpm for 5 min, and the bacterial cells are collected and placed at -20℃ for later use.

[0056] III. Purification and Dialysis of Recombinant Mussel Foot Protein by Nickel Ion Affinity Chromatography 1. Take 30 g of the bacterial cells collected in step 2 and resuspend them in 300 mL of PBS buffer to obtain the bacterial solution. Each time, homogenize 150 mL of the bacterial solution in a high-pressure homogenizer at 800 Pa for 30 min, centrifuge to remove impurities, and after cell lysis, centrifuge the sample at 4℃ and 8000 rpm for 15 min and collect the supernatant.

[0057] 2. Purify the supernatant collected in step 1 using a protein purification system, as follows: Medium-pressure column packing: Before packing the column, take the nickel ion affinity chromatography packing out of the refrigerator and cool it to room temperature. The medium-pressure column packing is 20 mL. The column is fixed with an iron stand or column clamp to ensure that the column tube is not tilted and is perpendicular to the ground.

[0058] Column loading procedure: Volumetric flow rate of 6 cm⁻¹ 2 / min. Wash the supernatant with PBS buffer for 5 column volumes until the λ280 absorbance is close to 0. Take a sample to measure the protein concentration in the supernatant. When the protein concentration is below 20 mg / L, elute with a 50 mM imidazole gradient until the λ280 absorbance stabilizes. Take the eluent to measure the protein concentration. When the protein concentration is below 20 mg / L, replace with a 250 mM imidazole gradient to elute the target protein until the λ280 absorbance stabilizes (at this point, the recombinant mussel foot protein solution is obtained). Wash with PBS buffer for 3-5 column volumes until the λ280 signal is close to the baseline, and then wash with 20% ethanol solution for two column volumes for storage.

[0059] The solutes and their concentrations in the PBS buffer are 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4, with water as the solvent and pH 7.4. When preparing a 1 L volume, the preparation method is as follows: Weigh 8 g of NaCl, 0.2 g of KCl, 1.42 g of Na2HPO4, and 0.27 g of KH2PO4, add them to 800 mL of deionized water to dissolve, adjust the pH to 7.4 with concentrated hydrochloric acid, and filter through a 0.45 µm water membrane.

[0060] 1 M imidazole: Weigh 68.07 g imidazole into a 1 L beaker, dissolve it in 800 mL PBS buffer, and after the imidazole is completely dissolved, adjust the pH to 7.4 with concentrated hydrochloric acid, and then bring the volume to 1 L.

[0061] Both 50 mM imidazole and 250 mM imidazole were obtained by diluting 1 M imidazole to the corresponding concentration with PBS buffer and then filtering with a 0.45 µm water membrane.

[0062] 3. Dialyze the 250mM imidazole aqueous solution containing recombinant mussel foot protein (i.e., recombinant mussel foot protein solution) using an MD4400 source leaf dialysis belt at 4°C for 4 hours in an aqueous solution with pH 6.0. Repeat the process three times and then measure the protein concentration to obtain the recombinant mussel foot protein solution (solvent is an aqueous solution with pH 6.0).

[0063] Example 2: Dopa Modification and Identification of Recombinant Mussel Foot Protein I. Dopa Modification and Detection of Recombinant Mussel Foot Protein 1. Preparation of the reaction system. The reaction system consisted of 10 ml of reaction mixture and 10 μL of 20 mg / L polyphenol oxidase (mushroom) cell lysis solution (a product of Shanghai Yuanye Biotechnology Co., Ltd., product catalog number S10171-100KU; the purpose is to modify tyrosine residues into dopa groups). The pH was adjusted to 6.0 with 2M NaOH solution. The solutes and their concentrations in the reaction mixture were 1 g / L recombinant mussel foot protein solution (solvent: pH 6.0, 0.01M PBS buffer), 2 μM CuSO4, and 30 mM ascorbic acid, with ddH2O as the solvent.

[0064] 2. The reaction system prepared in step 1 was reacted at 30°C and 100 rpm for 8 hours to obtain a reaction solution. Then, the reaction solution was purified and dialyzed by nickel ion affinity chromatography according to the method in step 3 of Example 1 to obtain a 1 g / L dopa-modified recombinant mussel foot protein solution (solvent was an aqueous solution with pH 6.0).

[0065] 3. Determination of dopa modification rate The modification rate of dopamine (DOPA) was determined according to Appendix B of the Y / T 1293.6-2020 Industry Standard for Mussel Adhesive Medical Dressings (Arrow Method). The basic principle is that substances containing the 3,4-dihydroxyphenylalanine (DOPA) structure react with sodium nitrite under acidic conditions to produce a yellow color, which turns deep orange-red when excess alkali is added. The specific steps are as follows: (1) Weigh 20 mg of DOPA standard (product of Aladdin Reagent (Shanghai) Co., Ltd., product catalog number D111048-100g) and dilute to 100 mL with hydrochloric acid solution to obtain a DOPA standard solution with a concentration of 200 μg / mL.

[0066] (2) Take 0 mL, 0.1 mL, 0.5 mL, 1.0 mL, 1.5 mL and 2.0 mL of DOPA standard solution respectively, dilute with hydrochloric acid solution to 10 mL, shake well to obtain DOPA series standard solutions.

[0067] Hydrochloric acid solution: Take 0.2 mL of hydrochloric acid and dilute it with pure water to a final volume of 200 mL to obtain a hydrochloric acid solution with a concentration of 0.012 mol / L.

[0068] (3) Take 1 mL of DOPA series standard solution into 10 mL test tubes respectively, add 0.5 mL of acidic reagent to each test tube, and then add 1.5 mL of nitrite reagent and 2 mL of alkaline reagent to each test tube in sequence (the alkaline reagent should be added within 5 min after the nitrite reagent is added), and shake well.

[0069] Acidic reagent: Take 4.3 mL of hydrochloric acid and dilute it with pure water to a final volume of 100 mL to obtain an acidic reagent with a concentration of 0.516 mol / L.

[0070] Nitrous acid reagent: Weigh 10 g of sodium molybdate dihydrate and 10 g of sodium nitrite, dissolve them in pure water and bring the volume to 100 mL to obtain nitrous acid reagent.

[0071] Alkaline reagent: Weigh 4.0 g of sodium hydroxide, dilute with pure water to a final volume of 100 mL, and obtain an alkaline reagent with a concentration of 1 mol / L.

[0072] (4) After completing step (3), the absorbance at a wavelength of 500 nm is measured. A standard curve is plotted with DOPA concentration on the x-axis and the corresponding absorbance on the y-axis; the expected regression value of the standard curve should be R. 2 >0.997.

[0073] The standard curve is shown below. Figure 1 The standard curve is y = 0.0121x - 0.0051, R0 2=0.9986; x is the concentration of dopa (mg / L) in the sample to be tested.

[0074] (5) Take 1 mL of the sample to be tested (the dopa-modified recombinant mussel foot protein solution obtained in step 2 (the solvent is an aqueous solution with pH 6.0), the commercially available recombinant mussel adhesive protein solution with a concentration of 1 g / L (the solvent is an aqueous solution with pH 6.0) or an aqueous solution with pH 6.0 (as a control)) into a 10 mL test tube, add 0.5 mL of acidic reagent to each test tube, and then add 1.5 mL of nitrite reagent and 2 mL of alkaline reagent to each test tube in sequence (the alkaline reagent should be added within 5 min after the nitrite reagent is added), and shake well; then detect the absorbance at a wavelength of 500 nm.

[0075] Substitute the absorbance of the sample to the standard curve to obtain the DOPA content of the sample. If the absorbance of the sample exceeds the maximum value of the standard curve, the sample needs to be diluted and retested. If there is interference with colorimetric analysis, centrifuge the sample at 3000 r / min for 10 min, and take the supernatant for colorimetric analysis.

[0076] Test results are shown Figure 2 (The left test tube shows the color development of the dopamine-modified recombinant mussel foot protein solution, and the right test tube shows the color development of the pH 6.0 aqueous solution) and Table 2. The test results show that the dopamine modification rate of the dopamine-modified recombinant mussel foot protein reaches more than 4 wt%, which is higher than the dopamine modification rate of commercially available recombinant mussel adhesive protein.

[0077] Table 2. Determination of dopa modification rate

[0078] Note: wt% refers to the mass of dopa / protein content.

[0079] II. Identification of the DOPA group in DOPA-modified recombinant mussel foot protein According to Appendix A of YY / T 1293.6-2020, the NBT staining method can be used to identify whether the protein side chain residues have dopa groups. In this embodiment, the dopa groups are specifically identified using the nitrocellulose membrane method, and the specific steps are as follows: 1. Place 2 µL of the sample to be tested (0.4 g / L dopa-modified recombinant mussel foot protein solution (solvent: pH 6.0 aqueous solution) or 0.2 g / L commercially available recombinant mussel adhesive protein solution (solvent: pH 6.0 aqueous solution)) onto a 5 cm × 5 cm 0.2 pm nitrocellulose membrane (NC membrane), and mark the sample position. After the sample is absorbed by the NC membrane, place the NC membrane containing the sample into a 500 mL beaker, add 300 mL of pure water, and sonicate for 10 min.

[0080] 2. After completing step 1, remove the NC membrane and place it in a culture dish. Add NBT staining solution and stain in the dark for 45 min.

[0081] NBT staining solution: Weigh 6 mg NBT, add 15 mL glycine-potassium buffer to dissolve, mix well, and use immediately.

[0082] Glycine-potassium buffer: Weigh 75 g of glycine, dissolve it in 400 mL of water, adjust the pH to 10 with solid potassium hydroxide, and add water to a final volume of 500 mL. After preparation, store at 2℃-8℃.

[0083] The basic principle of NBT staining is as follows: under alkaline conditions, with excess glycine as a reducing agent, the 1,2-benzenediol of the dopa (DOPA) residue in the dopa protein molecule can be oxidized into quinone compounds; after adding nitrotetrazole blue (NBT), insoluble blue-purple crystals formazan can be generated.

[0084] 3. After completing step 2, remove the NC membrane, rinse it twice with sodium borate solution, store it in sodium borate solution overnight, rinse it three times with pure water, and observe whether blue-purple spots are generated at the sample marking area.

[0085] Sodium borate solution: Weigh 2.5 g of sodium borate decahydrate, dissolve it in 40 mL of water, and sonicate it at 40°C until it is completely dissolved.

[0086] Nitrocellulose membrane before NBT staining is shown in the image. Figure 3 (The droplet on the left is a solution of recombinant mussel foot protein modified with dopa, and the droplet on the right is a solution of commercially available recombinant mussel adhesive protein.)

[0087] NBT-stained nitrocellulose membranes are shown Figure 4 (The droplet on the left is a solution of recombinant mussel foot protein modified with dopa, and the droplet on the right is a solution of commercially available recombinant mussel adhesive protein.)

[0088] The results showed that the dopa-modified recombinant mussel foot protein contained dopa groups, and the dopa group content was higher than that of commercially available recombinant mussel adhesive protein. This result was completely consistent with the dopa modification rate results in step one.

[0089] Example 3: Purity determination of dopa-modified recombinant mussel foot protein 1. Dilute the dopamine-modified recombinant mussel foot protein or commercially available recombinant mussel adhesive protein to 400 mg / L with sodium acetate solution at pH 4.0-5.0. Then add 20 μL of 5× protein loading buffer (Sangon Biotech Co., Ltd.), mix thoroughly, and heat in a 100°C water bath for 20 minutes. Prepare a 1.0 mm 10% SDS-PAGE gel using a protein gel electrophoresis kit (Bioss Co., Ltd.) for protein sample electrophoresis verification. Use TSINGKE TSP021 Trelief® Prestained Protein Ladder (Beijing Qingke Co., Ltd., catalog number TSP021) as the protein marker. After electrophoresis, stain the gel with Coomassie Rapid Brightness Stain G250 (170 rpm, 10 min), then discard the staining solution and add purified water for destaining for 30 min.

[0090] Test results are shown Figure 5 (Where M is the marker, and the band sizes from bottom to top are 180, 130, 95, 70, 55, 43, 33, 20, 17, and 10 kDa; lane 1 contains commercially available recombinant mussel adhesive protein, with a size of approximately 28 kDa; lane 2 contains dopamine-modified recombinant mussel foot protein, with a size of approximately 28 kDa, and the bands in lane 2 in the range of 40-53 kDa represent dimers of the dopamine-modified recombinant mussel foot protein). The results indicate that dopamine-modified recombinant mussel foot protein readily forms dimers under neutral conditions.

[0091] 2. Liquid chromatography (LC) was used to detect the purity of 1 g / L recombinant mussel paw protein solution (solvent: pH 3.0 acetic acid solution) and 1 g / L dopamine-modified recombinant mussel paw protein solution (solvent: pH 3.0 acetic acid solution) separately. LC separation and detection of protein purity is based on the fact that different types of proteins have different elution times. The elution time at 280 nm is determined by molecular weight. The purpose of the experiment was to confirm whether the dopamine-modified recombinant mussel paw protein contained other impurities.

[0092] Testing instruments and equipment: High performance liquid chromatograph: Shimadzu LC-2050C 3D chromatographic conditions Agilent Zobax SB300A C8 (4.6×250mm) column. Detection conditions: detection wavelength 280 nm, elution temperature 25 ℃, flow rate 0.9 mL / min, injection volume 20 μL.

[0093] The detection results of recombinant mussel foot protein solution are shown below. Figure 6 (Peak 1 represents recombinant mussel foot protein). The results showed that the purity of the recombinant mussel foot protein reached over 98%.

[0094] The detection results of the dopamine-modified recombinant mussel foot protein solution are shown in the figure. Figure 7 (Peak 4 represents dopa-modified recombinant mussel foot protein). The results showed that the purity of dopa-modified recombinant mussel foot protein reached over 98%.

[0095] 3. Coverage Analysis (1) Sample pretreatment Take an appropriate amount of dopa-modified recombinant mussel foot protein, denature and reduce it, then replace it with UreaTris buffer, add an appropriate amount of Trypsin, Chymotrypsin or Lys-C, and react at 37℃ for 18 hours to obtain the enzymatic hydrolysis product; accurately pipette the enzymatic hydrolysis product into a sample vial for instrumental testing.

[0096] (2) Liquid chromatography-mass spectrometry analysis After completing step (1), each enzymatic hydrolysis product was automatically loaded onto the chromatographic column by UPLC, and a suitable chromatographic gradient was set (0-65 min, 100% A solution; 65-65.5 min, 60% A solution and 40% B solution; 65.5-68.5 min, 10% A solution and 90% B solution; 68.5-75 min, 100% A solution; solution A is 0.1% FA aqueous solution, solution B is 0.1% FA acetonitrile solution) for desalting and separation. Each enzymatic hydrolysis product was separated by ultra-high performance liquid reverse phase chromatography (high performance liquid chromatograph model: Waters / ACQUITY Premier) and then subjected to mass spectrometry detection and analysis (high resolution mass spectrometer model: Waters / Xevo-G2-XS-Qtof). Finally, liquid chromatography-mass spectrometry data analysis was performed, and the raw data file was analyzed using the UNIFI (1.9, Waters) PeptideMap (ExactMassMS) program.

[0097] The parameters are set as follows: temperature 55℃; UV 214nm; flow rate 0.3ml / min; detection method positive ion (MSE) Scanrange (Full MS) 200-2000m / z.

[0098] UV and TIV spectra of trypsin hydrolysate are shown below. Figure 8 .

[0099] UV and TIC spectra of the Chymotrypsin hydrolysate are shown below. Figure 9 .

[0100] The UV and TIC spectra of the Lys-C enzymatic hydrolysate are shown below. Figure 10 .

[0101] The coverage analysis results are shown in Table 3. The results indicate that the peptide coverage is 100%, suggesting that the dopa-modified recombinant mussel foot protein solution is a high-purity protein.

[0102] Table 3

[0103] Example 4: pH range of dopa-modified recombinant mussel foot protein To determine the pH range for the use of dopa-modified recombinant mussel foot protein, the protein was diluted with 0.1M sodium acetate solution to a concentration of 1 g / L, and then the pH was adjusted to 3.0, 4.0, 5.0, or 6.0 using acetic acid or 2M NaOH. The solubility of the dopa-modified recombinant mussel foot protein was then observed.

[0104] Following the steps described above, replace the dopa-modified recombinant mussel foot protein with commercially available recombinant mussel adhesive protein, while keeping all other steps unchanged.

[0105] The solubility results are shown in Table 4. The results indicate that both proteins have good solubility within the pH range of 3.0-6.0. Based on the actual needs of cosmetic application, subsequent experiments will use protein solutions at pH 6.0.

[0106] Table 4

[0107] Example 5: Compounding of DOPA-modified recombinant mussel foot protein with commonly used cosmetic reagents The dopamine-modified recombinant mussel paw protein was diluted to a concentration of 1 g / L with 0.1 M sodium acetate solution to obtain a dopamine-modified recombinant mussel paw protein solution. Based on the maximum usage amounts of sodium hyaluronate, glycerol, disodium EDTA, and PEG6000, solutions of 1.2% (w / v) sodium hyaluronate, 20% (w / v) glycerol, 0.2% (w / v) disodium EDTA, and 10% (w / v) PEG6000 were prepared, respectively. Then, equal volumes of the dopamine-modified recombinant mussel paw protein solution and commonly used reagent solutions (1.2% (w / v) sodium hyaluronate solution, 20% (w / v) glycerol solution, 0.2% (w / v) disodium EDTA solution, or 10% (w / v) PEG6000 solution) were mixed and the pH was adjusted to 6.0 to form a compound solution. The solution was then observed to determine if it was clear and if any precipitation occurred.

[0108] Following the steps described above, replace the dopa-modified recombinant mussel foot protein with commercially available recombinant mussel adhesive protein, while keeping all other steps unchanged.

[0109] The test results are shown in Table 5. Figure 11 and Figure 12 The results showed that, compared with commercially available recombinant mussel adhesive protein, dopa-modified recombinant mussel foot protein was miscible with sodium hyaluronate and had good potential for compounding.

[0110] Table 5

[0111] Example 6: Free radical scavenging ability of dopa-modified recombinant mussel foot protein The free radical scavenging ability of dopa-modified recombinant mussel foot protein was determined according to the ABTS method of GB / T 38120-2019 (free radical scavenging rate (%) = (A... 空白 -A 测定 ) / A 空白 (×100%). The experiment was conducted by the Beijing Qingxi Technology Research Institute, and the results were averaged out of 8 replicates. The protein sample was a 2.4 g / L solution of recombinant mussel foot protein modified with dopa (solvent was pH 6.0 aqueous solution).

[0112] The test results are shown in Table 6. The results indicate that the free radical scavenging rate of the 2.4 g / L dopa-modified recombinant mussel foot protein solution reached 59.68%.

[0113] Table 6

[0114] Example 7: Detection of the migration ability of dopa-modified recombinant mussel foot protein on human fibroblasts. The effect of dopamine-modified recombinant mussel foot protein on the migration ability of human fibroblasts was investigated by Shanghai Microspection Testing Technology Group Co., Ltd., in accordance with T / ZHCA 020-2022 Cosmetic Repair Efficacy Test.

[0115] Each group was set up with 3 parallel experiments, and the results were averaged. The specific steps are as follows: 1. Human fibroblasts grown to the logarithmic growth phase were seeded into 24-well plates (approximately 2 × 10⁶ cells / well). 5 Cells / well), then add DMEM medium containing 10% FBS, and incubate at 37°C and 5% CO2 for 24 hours until monolayer confluence (approximately 80-90% density).

[0116] 2. Make horizontal scratches with the pipette tip perpendicular to the 24-well plate, gently wash three times with PBS buffer (to remove unadhered bacteria), take pictures using an inverted microscope, and calculate the scratch area using Image Pro Plus software to obtain the initial scratch area.

[0117] 3. Processing Blank control (BC group): 200 µL of DMEM solution containing human fibroblasts (cell density 2 × 10⁻⁶ cells / well) was added to each well. 5 (on the order of magnitude) and 2 µL of pH 6.0 aqueous solution, incubated at 37 °C and 5% CO2 for 24 h; Positive control (PC group): 200 µL of DMEM solution containing human fibroblasts and 10% fetal bovine serum was added to each well (cell density 2 × 10⁻⁶). 5 (on the order of magnitude) and 2 µL of pH 6.0 aqueous solution, incubated at 37 °C and 5% CO2 for 24 h; M-8-1.00% (sample group): Add 200µL of DMEM solution containing human fibroblasts (cell density 2×10⁻⁶) to each well. 5 The sample was incubated at 37°C and 5% CO2 for 24 h with 2 µL of a DOPA-modified recombinant mussel foot protein solution (solvent: pH 6.0 aqueous solution) and 2 µL of a DOPA-modified recombinant mussel foot protein solution (on the order of magnitude). The final concentration of the DOPA-modified recombinant mussel foot protein in the system was 0.07 g / L. The sample concentration in the sample group was 1.00% (v / v). Then, an inverted microscope was used to take pictures, and the scratch area was calculated using Image Pro Plus software to obtain the current scratch area.

[0118] 4. Migration rate After completing step 3, calculate the mobility using the following formula: .

[0119] Test results are shown Figure 13 , Figure 14 See Table 7. The results showed that a 0.07 g / L solution of recombinant mussel foot protein significantly promoted the migration of human fibroblasts. This indicates that recombinant mussel foot protein possesses cell repair capabilities and exhibits good repair efficacy.

[0120] Table 7

[0121] Note: The significance of the sample group and PC group compared with the BC group is as follows: * express, P-value < 0.05 Represented as * , P-value < 0.01 represents ** .

[0122] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A mussel foot protein, comprising the following: b1) or b2) or b3) or b4): b1) Proteins with amino acid sequences as shown in SEQ ID No. 4; b2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in b1); b3) A protein that has the same function as the mussel foot protein obtained by substituting and / or deleting and / or adding one or more amino acid residues of the protein shown in b1) or b2). b4) A protein that has 80% or more identity with the amino acid sequence defined by b1) or b2) and has the same function as the mussel foot protein.

2. The mussel foot protein according to claim 1, characterized in that: The mussel foot protein is a recombinant mussel foot protein with the amino acid sequence shown in SEQ ID No.

2.

3. A nucleic acid molecule encoding the mussel foot protein of claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid molecule is a DNA molecule as shown in c1), c2), c3), c4), c5), or c6): c1) The coding region is the DNA molecule shown in SEQ ID No. 3; c2) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 3; c3) The coding region is the DNA molecule shown in SEQ ID No. 1; c4) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 1; c5) has 75% or more identity with the nucleotide sequence defined by c1) or c2) or c3) or c4) and encodes a DNA molecule of the mussel foot protein as described in claim 1 or 2. c6) hybridizes under stringent conditions with the nucleotide sequence defined by c1) or c2) or c3) or c4) and encodes a DNA molecule of the mussel foot protein as described in claim 1 or 2.

5. An expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule of claim 3 or 4.

6. The use of the mussel foot protein of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or the expression cassette, recombinant vector or recombinant microorganism containing the nucleic acid molecule of claim 3 or 4 in scavenging free radicals or in the preparation of products for scavenging free radicals.

7. The use of the mussel foot protein of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or the expression cassette containing the nucleic acid molecule of claim 3 or 4, the recombinant vector, or the recombinant microorganism in repairing cells, promoting wound healing, and / or reducing scar formation.

8. The use of the mussel foot protein of claim 1 or 2, or the nucleic acid molecule of claim 3 or 4, or an expression cassette containing the nucleic acid molecule of claim 3 or 4, a recombinant vector, or a recombinant microorganism in the preparation of products for repairing cells, promoting wound healing, and / or reducing scar formation.

9. The application according to any one of claims 6 to 8, characterized in that: The mussel foot protein described in claim 1 or 2 can be modified with dopa.

10. The application according to any one of claims 6 to 8, characterized in that: The product in question is either a cosmetic or a pharmaceutical.

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