Mfp-sod recombinant protein, and preparation method and application thereof

Abstract

The application relates to the technical field of protein design, and particularly discloses a kind of Mfp-SOD recombinant proteins and a preparation method and application thereof.The recombinant protein takes the mussel mucin Mfp-3 as a skeleton, assists anchoring human-derived protein SOD, and obtains a new type of recombinant protein.The recombinant protein is fused by two kinds of proteins, has excellent skin surface affinity and functions of antioxidation, anti-aging, whitening, spot-fading, anti-inflammation and sun protection, and can achieve long-acting antioxidation and anti-aging effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein design, and more particularly to a Mfp-SOD recombinant protein and a preparation method and application thereof. BACKGROUND

[0002] Mussel adhesive protein is a special protein secreted by the foot gland of marine mussels, mainly including Mfp-1, Mfp-2, Mfp-3, Mfp-4, Mfp-5, Mfp-6, pCOL-D, etc. The mussel adhesive protein contains L-3,4-dihydroxyphenylalanine, and the dopa group is converted from tyrosine through post-translational modification, which has a positive charge and can adhere to cells with a negative charge through electrostatic adsorption. Related studies have shown that the dopa group rich in mussel adhesive protein can displace water molecules and directly bind to the interface in a humid or liquid environment, with high adhesion strength. At the same time, the mussel adhesive protein has high biocompatibility, low cytotoxicity and low allergenicity, and has broad development prospects in the field of skin care products.

[0003] At present, there are related skin care products containing mussel adhesive protein, which claim that the dopa group of mussel adhesive protein has antioxidant effect on the product. However, the actual antioxidant capacity of mussel adhesive protein is much lower than that of traditional antioxidants such as glutathione and superoxide dismutase (SOD). Moreover, when mussel adhesive protein is used as an antioxidant, the dopa group is destroyed after the antioxidant is reduced, and the mussel adhesive protein loses its adhesion ability, so the skin care product is easy to dry and fall off from the skin surface. Traditional antioxidants are extremely unstable in the atmospheric environment, and often lose their antioxidant ability before they can act on skin cells because of the reaction with oxygen in the air.

[0004] Based on the above situation, the present applicant urgently needs to develop a recombinant protein with excellent skin surface affinity and cell membrane antioxidant and anti-aging function. SUMMARY

[0005] In order to solve the problem of lack of recombinant protein with excellent skin surface affinity and cell membrane antioxidant and anti-aging function in the industry, the present application provides a Mfp-SOD recombinant protein and a preparation method and application thereof.

[0006] In a first aspect, the present application provides a Mfp-SOD recombinant protein, which adopts the following technical scheme:

[0007] A Mfp-SOD recombinant protein is formed by fusion of the C-terminal of mussel adhesive protein Mfp-3 and the N-terminal of human protein SOD.

[0008] Further, the amino acid sequence of Mfp3-SOD1 in the Mfp-SOD recombinant protein is shown in SEQ ID NO. 1.

[0009] Further, the amino acid sequence of Mfp3-SOD2 in the Mfp-SOD recombinant protein is shown in SEQ ID NO. 2.

[0010] Further, the amino acid sequence of Mfp3-SOD3 in the Mfp-SOD recombinant protein is shown in SEQ ID NO. 3.

[0011] The recombinant protein designed in the present application is a fusion protein of Mfp-3 and SOD, in which Mfp-3 is used as a scaffold to anchor SOD, and the C-terminal of Mfp-3 is fused with the N-terminal of SOD.

[0012] The basic repeat unit fragment of Mfp-3 is GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYY. After fusion with SOD, the Mfp-SOD recombinant protein still retains a large number of dopa groups after translation modification. The dopa groups can use their unique physicochemical properties to repel water molecules and impart hydrophobicity to the recombinant protein. The Mfp-SOD recombinant protein helps to maintain skin moisture and is not easily lost due to sweat secretion or water washing. The presence of the Mfp-SOD recombinant protein on the skin surface has a long-lasting effect. As an endogenous antioxidant, SOD neutralizes harmful free radicals such as ROS by catalyzing the disproportionation of superoxide anions into oxygen and hydrogen peroxide. At the same time, by activating the AMPK and Nrf2 / HO-1 signaling pathways, the generation of collagen is promoted, thereby preventing skin aging.

[0013] SOD is anchored on Mfp-3, and Mfp-3 provides a stable microenvironment for SOD, solving the defect that SOD cannot long-actingly exert the ROS scavenging function. The removal of ROS by SOD can provide a suitable microenvironment for Mfp-3 to realize its normal function. The Mfp-SOD recombinant protein, by fusing two proteins, has excellent skin surface affinity and cell membrane antioxidant and anti-aging functions, continuously and stably releases antioxidant protection, and resists inflammation and oxidative damage.

[0014] In the design process of the recombinant protein, Mfp-3 can be fused with human SOD through a peptide chain or without a peptide chain; the peptide chain linker can be a rigid peptide chain or a flexible peptide chain; in this application, a flexible peptide chain is preferably used for connection, and more specifically, the flexible peptide chain in this application is SRPVAT. The flexible peptide chain serves as a bridge to connect the domains of Mfp-3 and human SOD, reduces the mutual interference between the two domains, ensures the correct three-dimensional folding of the two proteins, and thus maintains the biological activity of each protein. At the same time, the flexible peptide chain provides the necessary freedom of movement for the two domains, allowing them to move and rotate relative to each other to optimally bind to their substrates or ligands.

[0015] In a second aspect, the application provides a preparation method of Mfp-SOD recombinant protein, which adopts the following technical scheme:

[0016] The preparation method of Mfp-SOD recombinant protein comprises the following steps:

[0017] Plasmid construction;

[0018] Transformation, amplification and sequencing verification;

[0019] Induced culture and recombinant protein expression detection;

[0020] The bacterial cells are subjected to lysis, ultrasonic crushing and separation treatment to obtain crude protein;

[0021] The crude protein is purified to obtain Mfp-SOD recombinant protein stock solution.

[0022] The applicant entrusts a cooperative party to construct a plasmid and provides a plasmid map; the constructed plasmid is transformed into competent BL21(DE3) for amplification and sequencing verification, a single colony is inoculated for further culture, an inducer is added to start the expression of the target recombinant protein, and SDS-PAGE electrophoresis is used to detect whether the expression meets the design expectation. Then the bacterial cells are put into a lysis solution for lysis, and the crude protein enters the supernatant through ultrasonic crushing and centrifugal separation. The supernatant is purified by affinity chromatography to obtain the recombinant protein stock solution.

[0023] In a third aspect, the application provides an application of a recombinant protein, which adopts the following technical scheme:

[0024] The application of the recombinant protein, wherein the aforementioned Mfp-SOD recombinant protein has an antioxidant function and is applied to cosmetics, medical devices, functional foods and health products.

[0025] The skin care product added with the recombinant protein mainly has an anti-aging function, and also has whitening, spot-fading, anti-inflammatory and sunscreen effects; at the same time, the recombinant protein can also be used in drugs or pharmaceutical excipients.

[0026] Further, the Mfp-SOD recombinant protein is applied to the human body in the form of smearing, microneedle intervention or microsphere coating for non-therapeutic or diagnostic purposes.

[0027] The present application has at least the following advantages:

[0028] The recombinant protein is subjected to functional verification in the later stage, and the content of DOPA group in the recombinant protein and the SOD activity are determined, which directly proves that the design of the recombinant protein can retain the DOPA group, and endow the recombinant protein with sufficient adhesion. The higher the content of DOPA group is, the stronger the adhesion of the recombinant protein is. The SOD activity determination result shows that the antioxidant capacity of the recombinant protein is relatively strong, and is close to or even exceeds the SOD activity value of the recombinant human protein SOD; it is shown that the recombinant protein still retains a relatively high antioxidant capacity after the human protein SOD and the mussel adhesive protein Mfp-3 are fused. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The plasmid map of the recombinant protein Mfp3-SOD1 of Example 1.

[0030] Figure 2 The SDS-PAGE electrophoretogram of the Mfp3-SOD1 recombinant protein expressed by using IPTG to induce protein expression in Example 1.

[0031] Figure 3 The SDS-PAGE electrophoretogram of the Mfp3-SOD1 recombinant protein after purification in Example 1.

[0032] Figure 4 The plasmid map of the recombinant protein Mfp3-SOD2 of Example 2.

[0033] Figure 5 The plasmid map of the recombinant protein Mfp3-SOD3 of Example 3. DETAILED DESCRIPTION

[0034] Example 1

[0035] A recombinant protein Mfp3-SOD1, the amino acid sequence of which is shown in SEQ ID NO. 1:

[0036] GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYYSRPVATMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ;

[0037] According to the following steps:

[0038] Plasmid construction: the plasmid construction is completed by a cooperative partner; the plasmid map is shown in Figure 1 ;

[0039] Transformation, amplification and sequencing verification: 1 μL of plasmid is added to 100 μL of competent BL21 (DE3) (from Beijing Yiqiao God Science and Technology Co., Ltd.), and placed on ice for 20 min;

[0040] The mixture containing the plasmid is warmed to 42°C, heat shocked for 90 s, quickly placed on ice for 5 min for continuous cultivation, and 600 μL of LB culture solution is added; then the LB culture medium is placed in a 37°C environment for recovery and shaking, the shaking frequency is 220 r / min, and shaking is performed for 1 h; after centrifugation, all are coated on an LB plate containing 50 μg / mL Amp, and the LB plate is placed in a 37°C environment for inverted cultivation overnight;

[0041] Induced culture and recombinant protein expression detection: a single colony on the transformation plate is inoculated in a test tube containing 3 mL of LB culture solution containing 50 μg / mL Amp (Kan), and placed in a 37°C environment for shaking overnight at a frequency of 220 r / min;

[0042] The next day, inoculate in 30 mL of TB culture solution containing 50 μg / mL Amp (Kan) at a volume ratio of 1:100, and place in a 37°C environment for shaking at a frequency of 220 r / min until the bacterial OD 600 is 0.6-0.8;

[0043] IPTG is added to the culture to a final concentration of 0.2 mM, and the culture is placed in a 15°C environment for shaking overnight at a frequency of 220 r / min to induce the expression of the fusion protein;

[0044] See Figure 2SDS-PAGE electrophoretogram, M is protein molecular weight standard; 1 is recombinant protein without IPTG induction; 2 is recombinant protein after IPTG induction; 3 is protein organization in supernatant after induction and disruption; 4 is protein organization in precipitate after induction and disruption;

[0045] SDS-PAGE electrophoretogram analysis showed that the recombinant protein met the design expectation;

[0046] Disruption: resuspend the bacterial pellet in 20 mL lysis buffer (20 mM Tris-HCl containing 1 mM PMSF and bacteria protease inhibitor cocktail, pH=8.0), and perform ultrasonic disruption on the lysed product, with a power of 400 W, 2-10 sec working and 2-10 sec interval, for a total of 20 min; centrifuge the ultrasonic disrupted lysate at 4°C at a speed of 10000 r / min for 20 min, collect the precipitate and reserve the supernatant;

[0047] Purification: use low-pressure chromatography system, and load the supernatant solution into a Ni-NTA Binding-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH=8.0) pre-equilibrated Ni-NTA-Sepharose Cl-6B affinity chromatography column at a flow rate of 1-3 min retention time;

[0048] Wash with Ni-NTA Binding-Buffer at a flow rate of 1-3 min retention time until the OD 280 value of the effluent reaches the baseline;

[0049] Elute the target protein 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, and collect the effluent as Mfp-SOD1 recombinant protein stock solution. The SDS-PAGE electrophoretogram of the purified recombinant protein is shown in Figure 3 ;

[0050] See Figure 3 , SDS-PAGE electrophoretogram, M is protein molecular weight standard; 1 is recombinant protein without IPTG induction; 2 is recombinant protein after IPTG induction; 3 is protein organization in supernatant after induction and disruption; 4 is protein organization in precipitate after induction and disruption; Figure 3 The purified recombinant protein met the design expectation, and this method obtained high-purity recombinant protein.

[0051] Example 2

[0052] A recombinant protein Mfp3-SOD2, the amino acid sequence of which is shown as SEQ ID NO. 2:

[0053] GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYYSRPVATMLSRAVCGTSRQLAPVLGYLGSRQKHSLPDLPYDYGALEPHINAQIMQLHHSKHHAAYVNNLNVTEEKYQEALAKGDVTAQIALQPALKFNGGGHINHSIFWTNLSPNGGGEPKGELLEAIKRDFGSFDKFKEKLTAASVGVQGSGWGWLGFNKERGHLQIAACPNQDPLQGTTGLIPLLGIDVWEHAYYLQYKNVRPDYLKAIWNVINWENVTERYMACKK;

[0054] According to the following steps:

[0055] Plasmid construction: the plasmid construction is completed by a cooperative partner; the plasmid map is shown in Figure 4 ;

[0056] Transformation, amplification and sequencing verification: 1 μL of the plasmid is added to 100 μL of competent BL21 (DE3) (from Beijing Yewo God State Technology Co., Ltd.) and placed on ice for 20 min;

[0057] The mixture containing the plasmid is warmed to 42°C, heat shocked for 90 s, quickly placed on ice for 5 min and continuously cultured, 600 μL of LB culture solution is added; then the LB culture medium is placed in a 37°C environment for recovery and shaking at a frequency of 220 r / min for 1 h, after centrifugation, all are coated on an LB plate containing 50 μg / mL Amp, the LB plate is placed in a 37°C environment and incubated overnight upside down;

[0058] Induced culture and recombinant protein expression detection: a single colony on the transformation plate is inoculated in a test tube containing 3 mL of LB culture solution containing 50 μg / mL Amp (Kan) and placed in a 37°C environment, shaken at a frequency of 220 r / min overnight;

[0059] The next day, it is inoculated in 30 mL of TB culture solution containing 50 μg / mL Amp (Kan) at a volume ratio of 1:100, placed in a 37°C environment and shaken at a frequency of 220 r / min until the bacterial OD 600 is 0.6-0.8;

[0060] The IPTG was added to the culture to a final concentration of 0.2 mM, and the culture was placed in an environment at 15°C and shaken at a frequency of 220 r / min overnight to induce expression of the fusion protein;

[0061] Lysis and disruption: The bacterial pellet was resuspended in 20 mL lysis buffer (20 mM Tris-HCl containing 1 mM PMSF and bacteria protease inhibitor cocktail, pH=8.0), and the lysed product was subjected to ultrasonic disruption at a power of 400 W for 2-10 seconds of work and 2-10 seconds of interval, for a total of 20 minutes. The lysed product after ultrasonic disruption was centrifuged at 4°C at a speed of 10000 r / min for 20 minutes, and the precipitate was collected and the supernatant was reserved;

[0062] Purification: The supernatant was loaded onto a Ni-NTA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-NTA Binding-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH=8.0) at a flow rate of 1-3 min retention time;

[0063] The column was washed with Ni-NTA Binding-Buffer at a flow rate of 1-3 min retention time until the OD 280 value reached the baseline;

[0064] 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, and the eluate was collected as the Mfp-SOD2 recombinant protein stock solution.

[0065] Example 3

[0066] A recombinant protein Mfp3-SOD3, the amino acid sequence of which is shown in SEQ ID NO. 3:

[0067] GYGYDGYNAWYNNGYYGYNGYNGYHGRYGWNKGWNSGWGGSYYGNKGYYSRPVATMLALLCSCLLLAAGASDAWTGEDSAEPNSDSAEWIRDMYAKVTEIWQEVMQRRDDDGALHAACQVQPSATLDAAQPRVTGVVLFRQLAPRAKLDAFFALEGFPTEPNSSSRAIHVHQFGDLSQGCESTGPHYNPLAVPHPQHPGDFGNFAVRDGSLWRYRAGLAASLAGPHSIVGRAVVVHAGEDDLGRGGNQASVENGNAGRRLACCVVGVCGPGLWERQAREHSERKKRRRESECKAA

[0068] According to the following steps:

[0069] Plasmid construction: the plasmid construction is completed by a cooperative partner; the plasmid map is shown in Figure 5 ;

[0070] Transformation, amplification and sequencing verification: 1 μL of the plasmid is added to 100 μL of competent BL21 (DE3) (from Beijing Yewo Genetech Co., Ltd.) and placed on ice for 20 min;

[0071] The mixture containing the plasmid is warmed to 42°C, heat shocked for 90 s, quickly placed on ice for 5 min and continuously cultured, 600 μL of LB culture solution is added; then the LB culture medium is placed in a 37°C environment for recovery and shaking at a frequency of 220 r / min for 1 h, after centrifugation, all are coated on an LB plate containing 50 μg / mL Amp, the LB plate is placed in a 37°C environment and incubated overnight upside down;

[0072] Induced culture and recombinant protein expression detection: a single colony on the transformation plate is inoculated in a 3 mL test tube containing 50 μg / mL Amp (Kan) LB culture solution and placed in a 37°C environment for shaking at a frequency of 220 r / min overnight;

[0073] The next day, it is inoculated in a 30 mL TB culture solution containing 50 μg / mL Amp (Kan) at a volume ratio of 1:100, placed in a 37°C environment and shaken at a frequency of 220 r / min until the bacterial OD 600 is 0.6-0.8;

[0074] IPTG is added to the culture to a final concentration of 0.2 mM, the culture is placed in a 15°C environment and shaken at a frequency of 220 r / min overnight to induce the expression of the fusion protein;

[0075] Cracking and breaking: resuspend the bacteria pellet in 20 mL lysis solution (20 mM Tris-HCl containing 1 mM PMSF and bacteria protease inhibitor cocktail, pH=8.0), and perform ultrasonic cracking on the cracked product, with a power of 400 W, 2-10 sec working and 2-10 sec interval, for a total of 20 min; centrifuge the ultrasonic-cracked product at 4°C for 20 min at a speed of 10000 r / min, collect the precipitate and reserve the supernatant;

[0076] Purification: use a low-pressure chromatography system, and load the supernatant into a Ni-NTA Binding-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH=8.0) pre-equilibrated Ni-NTA-Sepharose Cl-6B affinity chromatography column at a flow rate of 1-3 min retention time;

[0077] Wash with Ni-NTA Binding-Buffer at a flow rate of 1-3 min retention time, until the OD 280 value of the effluent reaches the baseline;

[0078] Elute the target protein 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, and collect the effluent as the Mfp-SOD3 recombinant protein stock solution.

[0079] Recombinant protein functional verification assay:

[0080] 1. DOPA group content detection:

[0081] 1.1 Reagents used in the test:

[0082] A. Hydrochloric acid solution (0.012 mol / L): dilute 0.2 mL hydrochloric acid with pure water to 200 mL;

[0083] B. DOPA standard solution (200 ug / mL): weigh 20 mg DOPA standard and dilute with hydrochloric acid solution to 100 mL, and prepare immediately before use;

[0084] C. Acidic reagent (0.516 mol / L): take 4.3 mL hydrochloric acid and dilute with pure water to 100 mL;

[0085] D. Alkaline reagent (1 mol / L): weigh 4.0 g sodium hydroxide (NaOH) and dilute with pure water to 100 mL;

[0086] E. Nitrous acid reagent: Weigh 10 g of sodium molybdate dihydrate and 10 g of sodium nitrite, dissolve them with pure water and make up to 100 ml;

[0087] 1.2 Test steps:

[0088] 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 reserve.

[0089] b. Take 1 mL of the above series of standard solutions respectively, add 0.5 mL of acid reagent to each test tube, then add 1.5 mL of nitrous acid reagent and 2 mL of alkaline reagent (the alkaline reagent should be added within 5 minutes after the addition of the nitrous acid reagent) to each test tube in turn, and shake well.

[0090] c. Take 1 mL of the sample to be tested in a 10 mL test tube and follow the above method to operate. Take the No. 0 tube as the blank, and measure the absorbance of the prepared standard solution and sample solution with a 1 cm cuvette at a wavelength of 500 nm. If the absorbance of the sample to be tested exceeds the highest value of the standard curve, the sample needs to be diluted.

[0091] d. Take the DOPA concentration as the abscissa and the absorbance as the ordinate to draw a standard curve, and then calculate the percentage content of DOPA in the sample according to the measured absorbance of the sample solution.

[0092] 1.3 The detection results are as follows:

[0093] Taking recombinant Mfp-3 as the control group, the quantitative analysis results are as follows:

[0094] 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 control 2.1

[0095] 1.4 Detection conclusion:

[0096] The DOPA content of Mfp3-SOD1, Mfp3-SOD2, and Mfp3-SOD3 is close to that of recombinant mussel mucus protein Mfp-3, indicating that the recombinant protein obtained by fusing Mfp3 with the three human-derived proteins SOD still retains a relatively rich dopa group, and the dopa group endows the recombinant protein with excellent adhesion.

[0097] 2. SOD activity detection:

[0098] 2.1 Test steps:

[0099] According to the method of pyrogallol autoxidation in GB / T5009.171-2003, the enzyme activity is determined;

[0100] S1, at about 25°C, in a 10 mL cuvette, sequentially add A liquid (pH = 8.20, 0.1 mol / L Tris-hydrochloric acid buffer, containing 1 mmol / L EDTA·2Na) 2.35 mL, distilled water 2 mL, B liquid (4.5 mmol / L pyrogallol hydrochloride solution) 0.15 mL, immediately after adding B liquid, mix and pour into a cuvette, respectively measure the absorbance at 325 nm wavelength at the initial time and after 1 min, the difference between the two is the autoxidation rate of pyrogallol ΔA 325 (min -1 ). The test determines that ΔA 325 (min -1 ) is 0.060;

[0101] S2, take 1 g of sample, the sample is derived from Mfp3-SOD1 obtained in Example 1, Mfp3-SOD2 obtained in Example 2 and Mfp3-SOD3 obtained in Example 3, dilute with 9 mL of normal saline to 10 mL, centrifuge at 4000 r / min for 15 min, and take the supernatant as the sample solution for determination;

[0102] S3, according to step S1, measure the sample solution, and calculate according to the following formula:

[0103] SOD activity (U / g) = [(ΔA 325 - ΔA' 325 ) / ΔA 325 × 100%] / 50%×4.5×D / V×V1 / m;

[0104] Wherein, V is the volume of the sample solution;

[0105] ΔA' 325 is the inhibition rate of the sample solution on the autoxidation of pyrogallol;

[0106] ΔA 325 is the autoxidation rate of pyrogallol;

[0107] D is the dilution multiple of the sample solution;

[0108] V1 is the total volume of the sample solution;

[0109] m is the mass of the sample;

[0110] 4.5 is the total volume of the reaction solution.

[0111] 2.2 The detection results are as follows:

[0112] Taking recombinant SOD as the control group, the quantitative analysis results are as follows:

[0113] Detection object 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 control 22010

[0114] 2.3 Detection Conclusion:

[0115] Mfp3-SOD1, Mfp3-SOD2 and Mfp3-SOD3 were detected by the method of autoxidation of pyrogallol, and according to the detection results, the SOD activity of the recombinant protein after fusion was close to or even higher than that of the recombinant SOD, indicating that the recombinant protein had excellent free radical scavenging efficacy and could realize anti-aging function.

[0116] At the same time, due to the antioxidant activity of the recombinant protein, it can be added to skin care products, which can help to improve skin color and reduce pigmentation, thereby playing a whitening and spot-fading role; and the recombinant protein can reduce the damage of free radicals to tissue cells and inhibit the further development of inflammation, thereby playing an anti-inflammatory role; the recombinant protein can also inhibit the formation of free radicals induced by ultraviolet and visible light, thereby playing a sunscreen role.

[0117] 3. Long-term antioxidant capacity detection:

[0118] 3.1 Test procedure:

[0119] The sample was exposed to air for 6h, and after exposure, the SOD activity was detected according to the test procedure of 2.1;

[0120] 3.2 The detection results are as follows:

[0121] The recombinant SOD was used as a control group, and the quantitative analysis results are as follows:

[0122] Detection object 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 control 19853 9.8

[0123] 3.3 Detection Conclusion:

[0124] Mfp3-SOD1, Mfp3-SOD2, Mfp3-SOD3 and recombinant SOD were all exposed to air, and after 6h of air oxidation, the antioxidant capacity of the above proteins was reduced to different degrees, but the recombinant SOD had a significant decrease in antioxidant capacity and was easily lost after contacting with oxygen in the air. Although the antioxidant capacity of Mfp3-SOD1, Mfp3-SOD2 and Mfp3-SOD3 recombinant proteins was reduced, the decrease was relatively low, and they still had certain antioxidant capacity, indicating that Mfp3-SOD1, Mfp3-SOD2 and Mfp3-SOD3 recombinant proteins had long-term antioxidant capacity.

[0125] The technical features of the above-described embodiments can be combined in any way. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described, but as long as the combinations of the technical features do not exist contradictions, they should be considered as the scope of the present disclosure.

[0126] And, the above-mentioned embodiments only express several implementation manners of the present application, the description is more specific and detailed, but it cannot be understood as the limitation of the patent scope of the present application. It should be pointed out that for ordinary skilled in the art, without departing from the concept of the present application, several modifications and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. A Mfp-SOD recombinant protein, characterized in that, The amino acid sequence of the obtained recombinant protein is shown as SEQ ID NO. 1, or the amino acid sequence of the obtained recombinant protein is shown as SEQ ID NO. 2, or the amino acid sequence of the obtained recombinant protein is shown as SEQ ID NO.

3.

2. The method for preparing the Mfp-SOD recombinant protein according to claim 1, characterized in that: The method comprises the following steps: plasmid construction; transformation, amplification and sequencing verification; induction culture and recombinant protein expression detection; cell lysis, ultrasonic crushing and separation treatment to obtain crude protein; crude protein purification to obtain Mfp-SOD recombinant protein stock solution.

3. The use of the Mfp-SOD recombinant protein according to claim 1, characterized in that: The Mfp-SOD recombinant protein has an antioxidant function and is applied to cosmetics.

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