Mfp-SOD recombinant protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of protein design, and particularly discloses an Mfp-SOD recombinant protein as well as a preparation method and application thereof. According to the recombinant protein, mussel mucin Mfp-3 is used as a skeleton to assist anchoring of human-derived protein SOD, and a novel recombinant protein is obtained. According to the recombinant protein, two proteins are fused, so that the recombinant protein has excellent skin surface affinity and the functions of oxidation resistance, aging resistance, whitening, spot fading, inflammation resistance, sunscreen and the like, and the effects of oxidation resistance and aging resistance can be exerted for a long time.

Description

Technical Field

[0001] This invention relates to the field of protein design technology, and more specifically, to an Mfp-SOD recombinant protein, its preparation method, and its applications. Background Technology

[0002] Mussel adhesive proteins are a special type of protein secreted by the foot glands of marine mussels, mainly including Mfp-1, Mfp-2, Mfp-3, Mfp-4, Mfp-5, Mfp-6, and pCOL-D. Mussel adhesive proteins contain L-3,4-dihydroxyphenylalanine, whose dopa group is derived from tyrosine through post-translational modification and carries a positive charge. This dopa group can adhere to negatively charged cells through electrostatic adsorption. Related studies have shown that the dopa group abundant in mussel adhesive proteins can utilize its unique physicochemical properties in moist or liquid environments to displace water molecules and directly bind to the interface, resulting in high adhesion strength. Furthermore, mussel adhesive proteins possess high biocompatibility, low cytotoxicity, and low allergenicity, making them promising for development in the skincare industry.

[0003] Currently, some skincare products containing mussel adhesive protein claim that the dopa groups in the protein contribute to the product's antioxidant effects. However, the actual antioxidant capacity of mussel adhesive protein is far lower than that of traditional antioxidants such as glutathione and superoxide dismutase (SOD). Furthermore, when mussel adhesive protein is used as an antioxidant, the dopa groups are destroyed upon reduction, causing the protein to lose its adhesive properties and making the skincare product easily dry and peel off the skin. Traditional antioxidants are extremely unstable in the atmospheric environment; when applied to the skin, they often lose their antioxidant capacity due to reaction with oxygen in the air before they can even act on skin cells.

[0004] Based on the above, the applicant urgently needs to develop a recombinant protein that has both excellent skin surface affinity and cell membrane antioxidant and anti-aging functions. Summary of the Invention

[0005] To address the industry's lack of recombinant proteins that possess both excellent skin surface affinity and cell membrane antioxidant and anti-aging functions, this application provides an Mfp-SOD recombinant protein, its preparation method, and its applications.

[0006] In a first aspect, this application provides an Mfp-SOD recombinant protein, employing the following technical solution: A recombinant Mfp-SOD protein is formed by fusing the C-terminus of mussel adhesive protein Mfp-3 with the N-terminus of human protein SOD.

[0007] Furthermore, the amino acid sequence of Mfp3-SOD1 in the Mfp-SOD recombinant protein is shown in SEQ ID NO.1.

[0008] Furthermore, the amino acid sequence of Mfp3-SOD2 in the Mfp-SOD recombinant protein is shown in SEQ ID NO.2.

[0009] Furthermore, the amino acid sequence of Mfp3-SOD3 in the Mfp-SOD recombinant protein is shown in SEQ ID NO.3.

[0010] The recombinant protein designed in this application uses mussel adhesive protein Mfp-3 as a backbone to help anchor human protein SOD, and is formed by fusing the C-terminus of Mfp-3 with the N-terminus of human protein SOD. The basic repeating unit fragment of mussel adhesive protein Mfp-3 is: GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYY. After this repeating unit fragment is fused with human SOD protein and undergoes translational modification, the Mfp-SOD recombinant protein still retains a large number of dopa groups. Dopa groups, with their unique physicochemical properties, can repel water molecules, giving the recombinant protein hydrophobicity. Mfp-SOD recombinant protein helps retain skin moisture and is not easily lost due to sweat or washing, thus having a long-lasting effect on the skin surface. Human SOD protein, as an endogenous antioxidant, neutralizes harmful free radicals such as ROS by catalyzing the dismutation of superoxide anions into oxygen and hydrogen peroxide. Simultaneously, it promotes collagen production by activating the AMPK and Nrf2 / HO-1 signaling pathways, thereby preventing skin aging.

[0011] Human-derived superoxide dismutase (SOD) is anchored to Mfp-3, which provides a stable microenvironment for SOD, overcoming the limitation of SOD's inability to sustainably scavenge reactive oxygen species (ROS). Conversely, SOD's ROS scavenging provides a suitable microenvironment for Mfp-3 to function properly. This Mfp-SOD recombinant protein, through the fusion of these two proteins, possesses both excellent skin surface affinity and cell membrane antioxidant and anti-aging functions, continuously and stably releasing antioxidant protection to resist inflammation and oxidative damage.

[0012] During the design of recombinant proteins, Mfp-3 and the human protein SOD can be connected via peptide chain fusion or not. The peptide chain linker can be a rigid or flexible peptide chain; in this application, a flexible peptide chain is preferred, and more specifically, SRPVAT is selected as the flexible peptide chain. The flexible peptide chain acts as a bridge, connecting the domains of Mfp-3 and the human protein SOD, reducing interference between the two domains, ensuring that the two protein structures can undergo correct three-dimensional folding, thereby maintaining their respective biological activities. Simultaneously, the flexible peptide chain provides the necessary degrees of freedom for movement between the two domains, allowing them to move and rotate relative to each other, binding optimally to their substrates or ligands.

[0013] Secondly, this application provides a method for preparing Mfp-SOD recombinant protein, using the following technical solution: A method for preparing Mfp-SOD recombinant protein includes the following steps: Plasmid construction; Transformation, amplification, and sequencing validation; Induction culture and detection of recombinant protein expression; The bacterial cells were lysed, ultrasonically disrupted, and separated to obtain crude protein; The crude protein was purified to obtain the Mfp-SOD recombinant protein stock solution.

[0014] The applicant commissioned a collaborator to construct a plasmid and provided a plasmid map. The constructed plasmid was transformed into competent BL21(DE3) cells for amplification and sequencing verification. Single-clone colonies were inoculated and cultured further. An inducer was added to initiate the expression of the target recombinant protein, and SDS-PAGE electrophoresis was used to detect whether the expression met the design expectations. The bacterial cells were then lysed in lysis buffer, and the protein was sonicated and centrifuged. The crude protein was collected in the supernatant, which was then purified by affinity chromatography to obtain the recombinant protein stock solution.

[0015] Thirdly, this application provides an application of a recombinant protein, employing the following technical solution: An application of a recombinant protein, the aforementioned Mfp-SOD recombinant protein, which has antioxidant function, is applied in cosmetics, medical devices, functional foods and health products.

[0016] Skincare products containing this recombinant protein primarily function to combat aging, and also offer whitening, spot-fading, anti-inflammatory, and sun protection benefits. Furthermore, this recombinant protein can also be used in pharmaceuticals or as a pharmaceutical excipient.

[0017] Furthermore, the Mfp-SOD recombinant protein is introduced into the human body's internal and external microenvironments via topical application, microneedle intervention, or microsphere encapsulation for non-therapeutic or diagnostic purposes.

[0018] This application has at least the following advantages: The recombinant protein underwent functional validation, with measurements of the dopa (DOPA) content and SOD activity. This directly demonstrated that the recombinant protein design retained the dopa group, endowing it with sufficient adhesiveness; the higher the dopa group content, the stronger the adhesiveness. SOD activity measurements showed that the recombinant protein possessed strong antioxidant capacity, approaching or even exceeding the SOD activity value of recombinant human SOD protein. This indicates that after fusing human SOD protein with mussel adhesive protein Mfp-3, the recombinant protein still retains high antioxidant capacity. Attached Figure Description

[0019] Figure 1 This is a plasmid map of the recombinant protein Mfp3-SOD1 from Example 1.

[0020] Figure 2 The expression of the Mfp3-SOD1 recombinant protein, induced by IPTG in Example 1, is shown in the SDS-PAGE electrophoresis pattern of the recombinant protein expression.

[0021] Figure 3 This is the SDS-PAGE electrophoresis pattern of the purified Mfp3-SOD1 recombinant protein from Example 1.

[0022] Figure 4 This is a plasmid map of the recombinant protein Mfp3-SOD2 from Example 2.

[0023] Figure 5 This is a plasmid map of the recombinant protein Mfp3-SOD3 in Example 3. Detailed Implementation

[0024] Example 1

[0025] A recombinant protein, Mfp3-SOD1, has the amino acid sequence shown in SEQ ID NO.1: GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYYSRPVATMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ; Prepare according to the following steps: Plasmid construction: Plasmid construction was outsourced to a partner; see plasmid map for details. Figure 1 ; Transformation, amplification, and sequencing validation: Add 1 μL of plasmid to 100 μL of competent cells BL21(DE3) (from Beijing Yiqiao Shenzhou Technology Co., Ltd.) and place on ice for 20 min; The mixture containing plasmids was heated to 42°C and heat-shocked for 90 seconds. It was then immediately placed on ice for 5 minutes and cultured for another 5 minutes. 600 μL of LB medium was added. Subsequently, the LB medium was placed in a 37°C environment and shaken at a frequency of 220 r / min for 1 hour. After centrifugation, the entire mixture was spread onto LB plates containing 50 μg / mL Amp. The LB plates were then placed in a 37°C environment and incubated upside down overnight. Induction culture and recombinant protein expression detection: Single clones from the transformation plate were picked and inoculated into test tubes containing 3 mL LB medium containing 50 μg / mL Amp (Kan), and placed in an environment of 37℃ and shaken at a frequency of 220 r / min overnight; The following day, the culture medium was inoculated at a volume ratio of 1:100 into 30 mL of TB culture medium containing 50 μg / mL Amp (Kan), and incubated at 37°C with shaking at 220 rpm until the bacterial cell OD reached its maximum. 600 It is 0.6-0.8; IPTG was added to the culture to a final concentration of 0.2 mM, and the culture was placed in an environment of 15°C and shaken at a frequency of 220 r / min overnight to induce the expression of the fusion protein. See Figure 2 In the SDS-PAGE electrophoresis pattern, M represents the molecular weight standard of the protein; 1 represents the recombinant protein without IPTG induction; 2 represents the recombinant protein after IPTG induction; 3 represents the protein structure in the supernatant after induction and disruption; and 4 represents the protein structure in the precipitate after induction and disruption. SDS-PAGE electrophoresis analysis showed that the recombinant protein met the design expectations. Lysis and disruption: The bacterial precipitate was resuspended in 20 mL of lysis buffer (20 mM Tris-HCl containing 1 mM PMSF and bacteria protease inhibitor cocktail, pH=8.0). The lysed products were ultrasonically disrupted at a power of 400 W for 2–10 seconds, followed by a 2–10 second interval, for a total of 20 min. The ultrasonically disrupted lysate was then placed in a 4°C environment and centrifuged at 10,000 r / min for 20 min. The precipitate was collected and the supernatant was retained. Purification: Using a low-pressure chromatography system, the supernatant was loaded onto a Ni-NTA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-NTA-Sepharose Cl-6B at a retention time of 1–3 min. Rinse with Ni-NTA Binding-Buffer at a flow rate of 1–3 min for a retention time until the effluent OD 280 The value reaches the baseline; The target protein was eluted with Ni-NTA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH=8.0) at a flow rate of 1–3 min retention time. The effluent was collected as the Mfp-SOD1 recombinant protein stock solution. The SDS-PAGE electrophoresis results of the purified recombinant protein are shown below. Figure 3 As shown; See Figure 3 In the SDS-PAGE electrophoresis pattern, M represents the molecular weight standard of the protein; 1 represents the purified recombinant protein; and 2 represents bovine serum albumin. Figure 3 This indicates that the purified recombinant protein meets the design expectations, and the method yields a high-purity recombinant protein.

[0026] Example 2

[0027] A recombinant protein, Mfp3-SOD2, has the amino acid sequence shown in SEQ ID NO.2: GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYYSRPVATMLSRAVCGTSRQLAPVLGYLGSRQKHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNVTEEKYQEALAKGDVTAQIAL QPALKFNGGGHINHSIFWTNLSPNGGGEPKGELLEAIKRDFGSFDKFKEKLTAASVGVQGSGWGWLGFNKERGHLQIAACPNQDPLQGTTGLIPLLGIDVWEHAYYLQYKNVRPDYLKAIWNVINWENVTERYMACKK; Prepare according to the following steps: Plasmid construction: Plasmid construction was outsourced to a partner; see plasmid map for details. Figure 4 ; Transformation, amplification, and sequencing validation: Add 1 μL of plasmid to 100 μL of competent BL21(DE3) cells (from Beijing Yiqiao Shenzhou Technology Co., Ltd.) and place on ice for 20 min; The mixture containing plasmids was heated to 42°C and heat-shocked for 90 seconds. It was then immediately placed on ice for 5 minutes and cultured for another 5 minutes. 600 μL of LB medium was added. Subsequently, the LB medium was placed in a 37°C environment and shaken at a frequency of 220 r / min for 1 hour. After centrifugation, the entire mixture was spread onto LB plates containing 50 μg / mL Amp. The LB plates were then placed in a 37°C environment and incubated upside down overnight. Induction culture and recombinant protein expression detection: Single clones from the transformation plate were picked and inoculated into test tubes containing 3 mL LB medium containing 50 μg / mL Amp (Kan), and placed in an environment of 37℃ and shaken at a frequency of 220 r / min overnight; The following day, the culture medium was inoculated at a volume ratio of 1:100 into 30 mL of TB culture medium containing 50 μg / mL Amp (Kan), and incubated at 37°C with shaking at 220 rpm until the bacterial cell OD reached its maximum. 600 It is 0.6-0.8; IPTG was added to the culture to a final concentration of 0.2 mM, and the culture was placed in an environment of 15°C and shaken at a frequency of 220 r / min overnight to induce the expression of the fusion protein. Lysis and disruption: The bacterial precipitate was resuspended in 20 mL of lysis buffer (20 mM Tris-HCl containing 1 mM PMSF and bacteria protease inhibitor cocktail, pH=8.0). The lysed products were ultrasonically disrupted at a power of 400 W for 2–10 seconds, followed by a 2–10 second interval, for a total of 20 min. The ultrasonically disrupted lysate was then placed in a 4°C environment and centrifuged at 10,000 r / min for 20 min. The precipitate was collected and the supernatant was retained. Purification: Using a low-pressure chromatography system, the supernatant was loaded onto a Ni-NTA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-NTA-Sepharose Cl-6B at a retention time of 1–3 min. Rinse with Ni-NTA Binding-Buffer at a flow rate of 1–3 min for a retention time until the effluent OD 280 The value reaches the baseline; The target protein was eluted with Ni-NTA Elution-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH=8.0) at a flow rate of 1-3 min retention time, and the effluent was collected as the Mfp-SOD2 recombinant protein stock solution.

[0028] Example 3

[0029] A recombinant protein, Mfp3-SOD3, has the amino acid sequence shown in SEQ ID NO.3: GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYYSRPVATMLALLCSCLLLAAGASDAWTGEDSAEPNSDSAEWIRDMYAKVTEIWQEVMQRRDDDGALHAACQVQPSATLDAAQPRVTGVVLFRQLAPRAKL DAFFALEGFPTEPNSSSRAIHVHQFGDLSQGCESTGPHYNPLAVPHPQHPGDFGNFAVRDGSLWRYRAGLAASLAGPHSIVGRAVVVHAGEDDLGRGGNQASVENGNAGRRLACCVVGVCGPGLWERQAREHSERKKRRRESECKAA; Prepare according to the following steps: Plasmid construction: Plasmid construction was outsourced to a partner; see plasmid map for details. Figure 5 ; Transformation, amplification, and sequencing validation: Add 1 μL of plasmid to 100 μL of competent BL21(DE3) cells (from Beijing Yiqiao Shenzhou Technology Co., Ltd.) and place on ice for 20 min; The mixture containing plasmids was heated to 42°C and heat-shocked for 90 seconds. It was then immediately placed on ice for 5 minutes and cultured for another 5 minutes. 600 μL of LB medium was added. Subsequently, the LB medium was placed in a 37°C environment and shaken at a frequency of 220 r / min for 1 hour. After centrifugation, the entire mixture was spread onto LB plates containing 50 μg / mL Amp. The LB plates were then placed in a 37°C environment and incubated upside down overnight. Induction culture and recombinant protein expression detection: Single clones from the transformation plate were picked and inoculated into test tubes containing 3 mL LB medium containing 50 μg / mL Amp (Kan), and placed in an environment of 37℃ and shaken at a frequency of 220 r / min overnight; The following day, the culture medium was inoculated at a volume ratio of 1:100 into 30 mL of TB culture medium containing 50 μg / mL Amp (Kan), and incubated at 37°C with shaking at 220 rpm until the bacterial cell OD reached its maximum. 600 It is 0.6-0.8; IPTG was added to the culture to a final concentration of 0.2 mM, and the culture was placed in an environment of 15°C and shaken at a frequency of 220 r / min overnight to induce the expression of the fusion protein. Lysis and disruption: The bacterial precipitate was resuspended in 20 mL of lysis buffer (20 mM Tris-HCl containing 1 mM PMSF and bacteria protease inhibitor cocktail, pH=8.0). The lysed products were ultrasonically disrupted at a power of 400 W for 2–10 seconds, followed by a 2–10 second interval, for a total of 20 min. The ultrasonically disrupted lysate was then placed in a 4°C environment and centrifuged at 10,000 r / min for 20 min. The precipitate was collected and the supernatant was retained. Purification: Using a low-pressure chromatography system, the supernatant was loaded onto a Ni-NTA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-NTA-Sepharose Cl-6B at a retention time of 1–3 min. Rinse with Ni-NTA Binding-Buffer at a flow rate of 1–3 min for a retention time until the effluent OD 280 The value reaches the baseline; The target protein was eluted with Ni-NTA Elution-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH=8.0) at a flow rate of 1-3 min retention time, and the effluent was collected as the Mfp-SOD3 recombinant protein stock solution.

[0030] Functional validation assays for recombinant proteins: 1. Detection of dopa group content: 1.1 Reagents used in the experiment: A. Hydrochloric acid solution (0.012 mol / L): Take 0.2 mL of hydrochloric acid and dilute it with pure water to a final volume of 200 mL; B. DOPA standard solution (200ug / mL): Weigh 20mg of DOPA standard and dilute to 100mL with hydrochloric acid solution. Prepare fresh before use. C. Acidic reagent (0.516 mol / L): Take 4.3 mL of hydrochloric acid and dilute it with pure water to a final volume of 100 mL; D. Alkaline reagent (1 mol / L): Weigh 4.0 g of sodium hydroxide (NaOH) and dilute with pure water to a final volume of 100 mL; E. Nitrite reagent: Weigh 10g of sodium molybdate dihydrate and 10g of sodium nitrite, dissolve them in pure water and bring the volume to 100ml; 1.2 Test Procedure: a. Preparation of DOPA standard solution series: Take 0 mL, 0.1 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL and 3 mL of DOPA standard solution respectively, dilute with hydrochloric acid solution to 10 mL, shake well and set aside.

[0031] b. Take 1 mL of the above series of standard solutions, 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 minutes after the nitrite reagent is added), and shake well.

[0032] c. Take 1 mL of the sample to be tested into a 10 mL test tube and proceed as described above. Using tube 0 as a blank, measure the absorbance of the prepared standard solution and sample solution at a wavelength of 500 nm using a 1 cm cuvette. If the absorbance of the test solution exceeds the highest value of the standard curve, the sample needs to be diluted.

[0033] d. Plot a standard curve with DOPA concentration on the x-axis and absorbance on the y-axis, and then calculate the percentage content of DOPA in the sample based on the measured absorbance of the sample solution.

[0034] 1.3 The test results are as follows: Using recombinant Mfp-3 as a control group, the quantitative analysis results are as follows: Detection object DOPA content / % Mfp3-SOD1 obtained in Example 1 2.2 Mfp3-SOD2 obtained in Example 2 2.0 Mfp3-SOD3 obtained in Example 3 2.0 Recombinant Mfp-3 reference standard 2.1 1.4 Test Results: The DOPA content of Mfp3-SOD1, Mfp3-SOD2, and Mfp3-SOD3 is close to that of recombinant mussel adhesive protein Mfp-3, indicating that the recombinant protein obtained by fusing Mfp3 with three human SOD proteins still retains a relatively rich amount of dopa groups. The dopa groups endow the recombinant protein with excellent adhesion.

[0035] 2. SOD activity test: 2.1 Test Procedure: Enzyme activity was determined according to the method of pyrogallol autoxidation in GB / T5009.171-2003; S1. At approximately 25℃, add 2.35 mL of solution A (pH=8.20, 0.1 mol / L Tris-hydroxymethylaminomethane (Tris)-hydrochloric acid buffer, containing 1 mmol / L EDTA·2Na), 2 mL of distilled water, and 0.15 mL of solution B (4.5 mmol / L pyrogallol hydrochloric acid solution) sequentially to a 10 mL colorimetric tube. Mix immediately after adding solution B and pour into a cuvette. Measure the absorbance at the initial wavelength of 325 nm and after 1 minute. The difference between the two values ​​is the pyrogallol self-oxidation rate ΔA. 325 (min -1This experiment determined ΔA. 325 (min -1 The value is 0.060; S2. Take 1g of sample, which is derived from Mfp3-SOD1 obtained in Example 1, Mfp3-SOD2 obtained in Example 2 and Mfp3-SOD3 obtained in Example 3. Dilute with 9mL of physiological saline and bring the volume to 10mL. After centrifugation at 4000r / min for 15min, take the supernatant as the sample solution for determination. S3. Determine the sample solution according to step S1, and calculate according to the following formula: SOD activity (U / g) = [(ΔA 325 -ΔA' 325 ) / ΔA 325 ×100%] / 50%×4.5×D / V×V1 / m; Where V is the volume of the sample solution added; ΔA' 325 To inhibit the auto-oxidation rate of pyrogallol in the sample solution; ΔA 325 The auto-oxidation rate of pyrogallol; D is the dilution factor of the sample solution; V1 is the total volume of the sample solution; m is the sample mass; 4.5 represents the total volume of the reaction solution.

[0036] 2.2 The test results are as follows: Using recombinant SOD as a control group, the quantitative analysis results are as follows: Detection object <![CDATA[SOD activity / U·g -1 > Mfp3-SOD1 obtained in Example 1 23709 Mfp3-SOD2 obtained in Example 2 22496 Mfp3-SOD3 obtained in Example 3 21993 Recombinant SOD Reference Standard 22010 2.3 Test Results: The enzyme activities of Mfp3-SOD1, Mfp3-SOD2, and Mfp3-SOD3 were determined by the pyrogallol auto-oxidation method. According to the test results, after fusion, the SOD activity of the recombinant protein was close to or even higher than that of the recombinant SOD, indicating that the recombinant protein has excellent free radical scavenging effect and can achieve anti-aging function.

[0037] Meanwhile, due to the antioxidant activity of recombinant proteins, when added to skincare products, they can help improve skin tone and reduce pigmentation, thus achieving whitening and fading of dark spots. Furthermore, recombinant proteins can reduce the damage of free radicals to tissue cells and inhibit the further development of inflammation, thus playing an anti-inflammatory role. Recombinant proteins can also inhibit the formation of free radicals caused by ultraviolet and visible light, thus playing a sun protection role.

[0038] 3. Long-term antioxidant capacity testing: 3.1 Test Procedure: The samples were exposed to air for 6 hours. After exposure, the SOD activity was tested according to the test procedure in 2.1. 3.2 The test results are as follows: Using recombinant SOD as a control group, the quantitative analysis results are as follows: Detection object <![CDATA[SOD activity / U·g -1 > Decrease / % Mfp3-SOD1 obtained in Example 1 23211 2.1 Mfp3-SOD2 obtained in Example 2 21866 2.8 Mfp3-SOD3 obtained in Example 3 21333 3.0 Recombinant SOD Reference Standard 19853 9.8 3.3 Test Results: All Mfp3-SOD1, Mfp3-SOD2, Mfp3-SOD3, and recombinant SOD were exposed to air. After 6 hours of air oxidation, the antioxidant capacity of all these proteins decreased to varying degrees, but the decrease was more significant for recombinant SOD, indicating that it easily lost its antioxidant effect upon contact with oxygen in the air. While the antioxidant capacity of recombinant proteins such as Mfp3-SOD1, Mfp3-SOD2, and Mfp3-SOD3 was weakened, the decrease was less severe, and they still possessed a certain degree of antioxidant capacity, suggesting that recombinant proteins such as Mfp3-SOD1, Mfp3-SOD2, and Mfp3-SOD3 have long-lasting antioxidant capabilities.

[0039] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0040] Furthermore, the above-described embodiments merely illustrate several implementation methods of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A recombinant Mfp-SOD protein, characterized in that, It is formed by fusing the C-terminus of mussel adhesive protein Mfp-3 with the N-terminus of human protein SOD.

2. The Mfp-SOD recombinant protein as described in claim 1, characterized in that: The amino acid sequence of Mfp3-SOD1 in the Mfp-SOD recombinant protein is shown in SEQ ID NO.

1.

3. The Mfp-SOD recombinant protein as described in claim 1, characterized in that: The amino acid sequence of Mfp3-SOD2 in the Mfp-SOD recombinant protein is shown in SEQ ID NO.

2.

4. The Mfp-SOD recombinant protein as described in claim 1, characterized in that: The amino acid sequence of Mfp3-SOD3 in the Mfp-SOD recombinant protein is shown in SEQ ID NO.

3.

5. The Mfp-SOD recombinant protein as described in claim 1, characterized in that: The Mfp-3 and the human protein SOD can be fused through a flexible peptide chain.

6. The Mfp-SOD recombinant protein as described in claim 5, characterized in that: The flexible peptide chain is SRPVAT.

7. A method for preparing an Mfp-SOD recombinant protein according to any one of claims 1-6, characterized in that: Includes the following steps: Plasmid construction; Transformation, amplification, and sequencing validation; Induction culture and detection of recombinant protein expression; The bacterial cells were lysed, ultrasonically disrupted, and separated to obtain crude protein; The crude protein was purified to obtain the Mfp-SOD recombinant protein stock solution.

8. The application of the Mfp-SOD recombinant protein according to any one of claims 1-6, characterized in that: The Mfp-SOD recombinant protein has antioxidant functions and can be used in cosmetics, medical devices, functional foods and health products.

9. The application of the Mfp-SOD recombinant protein according to claim 8, characterized in that: The Mfp-SOD recombinant protein is introduced into the human body's internal and external microenvironments via topical application, microneedle intervention, or microsphere encapsulation for non-therapeutic or diagnostic purposes.

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