An antioxidant fusion protein and a preparation method and application thereof

Abstract

The present application relates to an antioxidant fusion protein, a preparation method and application thereof, an amino acid sequence of the antioxidant fusion protein comprising an amino acid sequence shown in SEQ ID No: 1, and / or an amino acid sequence having at least 85% homology with SEQ ID No: 1. The antioxidant fusion protein of the present application can be used for preparing an antioxidant stress preparation for preventing and / or treating ischemia-hypoxia encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

Description

Technical Field

[0001] This invention relates to the technical field of antioxidant proteins, and in particular to an antioxidant fusion protein, its preparation method, and its application. Background Technology

[0002] Superoxide dismutase (SOD) is an antioxidant metalloenzyme present in living organisms. It catalyzes the dismutation of superoxide anion free radicals into oxygen and hydrogen peroxide, playing a crucial role in the body's oxidation-antioxidant balance and being closely related to the occurrence and development of many diseases. Cu / Zn-SOD is present in most primitive invertebrate cells, while vertebrates generally contain both Cu / Zn-SOD and Mn-SOD. Cu / Zn-SOD is found in erythrocytes and hepatocytes of humans, mice, pigs, and cattle, primarily in the cytoplasm, but also between the inner and outer mitochondrial membranes. Mn-SOD has also been purified from human and animal hepatocytes and is generally found in the mitochondrial matrix.

[0003] Skin aging and damage are important characteristics of human aging, which is a consequence of the accumulation or impaired clearance of reactive oxygen species (ROS). Excess free radicals in the body can cause cell damage and pigmentation. Since human skin is in direct contact with oxygen, it is susceptible to aging and damage. Supplementation with exogenous Superoxide Dismutase (SOD) can help delay skin aging, provide antioxidant benefits, and reduce pigmentation. Therefore, many cosmetic manufacturers both domestically and internationally add a certain proportion of SOD to their products. Furthermore, because SOD is a specific dismutation catalyst acting on superoxide anion free radicals, it has significant therapeutic effects as a pharmaceutical product in treating inflammation, autoimmune diseases, and cardiovascular diseases caused by free radical activity. SOD can also inhibit various types of inflammation, such as arthritis, pleurisy, and acute bronchitis, through its antioxidant properties.

[0004] Currently available antioxidant proteins, particularly SOD, are primarily monofunctional, lacking the ability to function both externally and internally, and thus limiting their antioxidant activity. Furthermore, antioxidant proteins exhibit poor cell membrane penetration, restricting their ability to exert antioxidant effects within cells. Therefore, there is an urgent need to develop an antioxidant fusion protein with dual antioxidant functions, enabling it to exert different antioxidant activities and broaden its applicability. This would allow for both external application (skin nourishment, anti-aging, wrinkle prevention, etc.) and internal application (injection) for treating ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and combating oxidative stress-induced bodily damage. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the first objective of the present invention is to provide an antioxidant fusion protein for the treatment of ischemic encephalopathy, arteriosclerosis, neurodegenerative diseases, skin aging, etc., and to resist the damage to the body caused by oxidative stress.

[0006] The second objective of this invention is to provide a method for preparing an antioxidant fusion protein, which has the advantages of simple steps and high product recovery rate.

[0007] The third objective of this invention is to provide an application of an antioxidant fusion protein for the treatment of ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, skin aging, etc., and to resist oxidative stress-induced bodily damage.

[0008] To achieve the first objective mentioned above, the present invention provides the following technical solution:

[0009] An antioxidant fusion protein, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID No:1, and / or an amino acid sequence having at least 85% homology with SEQ ID No:1.

[0010] By adopting the above technical solution, the amino acid sequence of the antioxidant fusion protein (GLS) is composed of 6×his-rhGrx-linker-rhSOD-15RQ. When constructing the sequence, firstly, the human superoxide dismutase mRNA sequence is used as a template for gene construction. By analyzing the types, ratios and specific sequences of its constituent amino acids, the N-terminus is a 6×his-linked rhGrx, the C-terminus is a SOD containing a membrane-penetrating peptide, and 9 Gly molecules are used as a linker in the middle to form an antioxidant fusion protein. Among them, rhGrx is a member of the glutoreductin family distributed in brain tissue. This protein plays an important role in cellular redox homeostasis, thiol homeostasis and anti-oxidative stress defense. This protein fuses rhGrx with SOD to improve the antioxidant and free radical scavenging performance of the rhSOD sequence segment. Compared with natural SOD, it can catalyze the dismutation of superoxide anion free radicals to generate oxygen and hydrogen peroxide more quickly.

[0011] Secondly, the self-designed linker (G9, GGGGGGGGG) is flexible and not easily degraded by proteases. It can fuse and express rhSOD and rhGrx genes, so that the antioxidant fusion protein can catalyze the redox reaction between glutathione (GSH) and protein disulfide bonds, repair protein activity, participate in maintaining the redox state of cells and provide a reducing environment in the cytoplasm, and have the effect of resisting and treating ROS-induced cellular oxidative stress damage.

[0012] Third, 6×His was added to the N-terminus to facilitate affinity purification, and a self-designed transmembrane peptide sequence (RRQRRQRRQRRQRRR, abbreviated as 15RQ) was added to the C-terminus to guide the antioxidant fusion protein to cross the membrane and enter the cell, thereby playing a better role in anti-oxidative stress.

[0013] In summary, this allows the antioxidant fusion protein to treat ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging, and to resist oxidative stress-induced bodily damage.

[0014] Furthermore, the amino acid sequence of the antioxidant fusion protein comprises the amino acid sequence shown in SEQ ID No:1, and / or n repeating sequences having at least 85% homology with the amino acid sequence of SEQ ID No:1, where n≥1.

[0015] Furthermore, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer greater than or equal to 2, the repeating sequences are directly connected.

[0016] Furthermore, the amino acid sequence of the antioxidant fusion protein has at least 88%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% homology with the amino acid sequence of SEQ ID No:1.

[0017] To achieve the second objective mentioned above, the present invention provides the following technical solution:

[0018] A method for preparing an antioxidant fusion protein includes the following steps:

[0019] S1 synthesized the nucleotide sequence SEQ ID No:2 of the antioxidant fusion protein using artificial whole-genome synthesis and inserted it between the XhoI and EcoRI restriction enzyme sites of the pPIC 9K vector to obtain the expression vector;

[0020] S2 linearizes the expression vector obtained in S1 by SalI single enzyme digestion, then introduces it into Pichia pastoris by electroporation. After fermentation expression and post-treatment, the antioxidant fusion protein is obtained.

[0021] By adopting the above technical solution, the preparation steps are simple. Using the above method, an antioxidant fusion protein with a yield of ≥800mg and a purity of ≥98% can be obtained in every 1L of fermentation broth.

[0022] Further, in S2, the Pichia pastoris cells are Pichia pastoris GS115. The Pichia pastoris GS115 are first preliminarily screened on MD plates, and then the clones grown on the MD plates are picked onto YPD plates containing different concentrations of antibiotic G418 (1-20 mg / mL) for resistance screening. Finally, highly resistant colonies are selected and identified using PCR technology.

[0023] Furthermore, during the fermentation expression of S2, the amount of primary seed culture BMGY was controlled at 1.0–2.0 L, the amount of secondary seed culture BMGY was controlled at 10–20 L, the fermentation medium was BMMY medium, the fermentation vessel was a 300 L fermenter with an actual filling volume of 100–200 L, the inoculation ratio was 1:(8–12), the culture temperature was 25–35 °C, the inducer was 1% methanol, the induction time was 70–75 h, the pH was adjusted to 5.5–6.5 with ammonia water, and 8–12 L of 10% peptone was added every 24 h as feed.

[0024] Furthermore, in the post-processing of S2, the fermentation broth obtained from fermentation expression is first centrifuged, with the centrifugation temperature controlled at 3-5℃, the rotation speed at 3500-4500 rpm, and the time at 25-35 min. The supernatant after centrifugation is collected and then sequentially subjected to His-tag affinity chromatography, cation exchange chromatography, and ultrafiltration desalting concentration to obtain the recombinant human collagen peptide.

[0025] To achieve the third objective mentioned above, the present invention provides the following technical solution:

[0026] The application of an antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0027] Specifically, in this invention, hypoxic-ischemic encephalopathy (HIE) refers to brain lesions caused by ischemia and hypoxia of brain tissue due to various reasons. The most common is neonatal hypoxic-ischemic encephalopathy, but it can also occur at other ages. Neonatal hypoxic-ischemic encephalopathy is a brain lesion caused by hypoxia in newborns during the perinatal period. Common causes include fetal distress in utero due to various reasons, such as umbilical cord entanglement, abnormal amniotic fluid, etc. It is also common in asphyxia during delivery and after birth, and rarely in brain damage caused by other reasons. This disease mostly occurs in full-term infants, but can also occur in premature infants. During delivery, the fetal heart rate may increase or decrease, or the second stage of labor may be prolonged, the amniotic fluid may be contaminated with meconium, there may be a history of asphyxia at birth, and after resuscitation, there may still be changes in consciousness, muscle tone, respiratory rhythm, reflexes, etc., and even seizures may occur. In contrast, hypoxic-ischemic encephalopathy in the non-neonatal period is seen in severe cerebral ischemia and hypoxia caused by various reasons, commonly with respiratory and cardiac arrest, and can also be seen in shock, CO poisoning, status epilepticus, myasthenia gravis, etc.

[0028] The term atherosclerosis (AS) refers to the fact that atherosclerosis is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease. Lipid metabolism disorders are the pathological basis of atherosclerosis. Its characteristics include that the lesions of the affected arteries begin in the intima, generally with the accumulation of lipids and complex carbohydrates, hemorrhage, and thrombosis, followed by fibrosis and calcification, and gradual degeneration and calcification of the arterial media, leading to thickening and hardening of the arterial wall and narrowing of the vascular lumen. The lesions often involve large and medium-sized muscular arteries. Once they develop to the point of obstructing the arterial lumen, the tissues or organs supplied by that artery will become ischemic or necrotic. Because the lipids accumulated in the arterial intima appear as a yellow porridge, it is called atherosclerosis.

[0029] The term "neurodegenerative disease" refers to a condition caused by the loss of neurons and / or their myelin sheaths, which worsens over time and results in functional impairment; non-limiting examples include Alzheimer's disease (AD), Parkinson's disease (PD), or amyotrophic lateral sclerosis (ALS).

[0030] Specifically, the dosage form of the antioxidant stress agent is a liquid dosage form or an injectable dosage form. The liquid dosage form includes solvent-based, aromatic aqueous, tincture, elixir, colloidal solution, gel, suspension, or emulsion. The injectable dosage form includes injectable water, injectable oil, traditional Chinese medicine injection, or sterile powder for injection.

[0031] Furthermore, the antioxidant stress agent comprises 40-60% oil phase and 40-60% aqueous phase; wherein,

[0032] The oil phase is made from raw materials comprising the following weight percentages: 0.5-1.0% emulsifier, 0.5-5.0% olive oil, 0.05-0.50% vitamin E, with the balance being injection oil;

[0033] The aqueous phase is made from raw materials comprising the following weight percentages: 0.1-1.0% antioxidant fusion protein and 0.5% sodium hyaluronate.

[0034] 1–2.0%, glycerol 1–10%, sodium carboxymethyl cellulose 0.05–0.50%, balance is water for injection.

[0035] Furthermore, in the oil phase, the emulsifier is Tween-40.

[0036] Furthermore, the oil phase further includes 0.1-1.0% petrolatum, 0.1-1.0% glyceryl monostearate, and 0.05-0.50% methylparaben.

[0037] Furthermore, the aqueous phase also includes 0.15–0.25% of *Gnaphalium affine* extract, 0.15–0.20% of black goji berry extract, 0.10–0.20% of black grape seed extract, 0.05–0.10% of black berry fruit extract, 0.05–0.10% of galactooligosaccharides, and 0.05–0.10% of stachyose. *Gnaphalium affine* is rich in emodin, chlorophyll, isoflavones, SOD, and trace elements such as selenium, iron, and magnesium, which can provide nutrients to cells. Black goji berries, black grape seeds, and black berry fruits are all extremely rich in proanthocyanidins and polysaccharides. Extraction can largely preserve these two active substances. The synergistic effect of stachyose and galactooligosaccharides with the above-mentioned active components can promote the absorption of these active substances, achieving the effects of scavenging free radicals and inhibiting the release of inflammatory factors.

[0038] Specifically, the black goji berry extract, black grape seed extract, and black berry extract were obtained by supercritical CO2 low-temperature extraction of black goji berries, black grape seeds, and black berry fruits, respectively. The extraction temperature was 40–42℃, the extraction pressure was 35–36 MPa, and the extraction time was 110–130 min. Under these extraction conditions, the active substances can be effectively extracted. The supercritical CO2 low-temperature extraction technology uses carbon dioxide gas in a supercritical state to contact black goji berries, black grape seeds, and black berry fruits, respectively. Proanthocyanidins and polysaccharides dissolve in the supercritical fluid, achieving separation of the active substances from the raw materials. Then, by reducing the pressure or increasing the temperature, the active substances extracted in the supercritical fluid are separated in a separator, thus obtaining high-quality active substances.

[0039] Furthermore, the preparation process of the antioxidant stress agent includes,

[0040] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0041] Antioxidant fusion protein, leafwort extract, black goji berry extract, black grape seed extract, black fruit extract, galactooligosaccharide, and stachyose were dissolved in water for injection, and then sodium hyaluronate, glycerin, and sodium carboxymethyl cellulose were added in sequence and stirred evenly to obtain an aqueous phase.

[0042] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified to obtain an oil-in-water emulsion of the antioxidant stress agent.

[0043] Alternatively, in the oil phase, the emulsifier is lecithin.

[0044] Furthermore, the aqueous phase also includes 6-10% mannitol and 3-5% trehalose.

[0045] Furthermore, the preparation process of the antioxidant stress agent includes,

[0046] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0047] The antioxidant fusion protein, mannitol, and trehalose were dissolved in water for injection. The pH was adjusted to 7.0-7.5 with 20 mM Tris-Cl. Then, sodium hyaluronate, glycerol, and sodium carboxymethyl cellulose were added in sequence and stirred until homogeneous to obtain the aqueous phase.

[0048] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified. The emulsion obtained is sterilized and then freeze-dried to obtain a lyophilized powder of the antioxidant stress agent.

[0049] In summary, the beneficial technical effects of the present invention are as follows:

[0050] 1. The antioxidant fusion protein of the present invention fuses rhGrx and rhSOD through a linker to form an antioxidant fusion protein, thereby exerting the functions of both proteins in resisting oxidative stress, scavenging free radicals, and maintaining cellular homeostasis.

[0051] 2. The antioxidant fusion protein of the present invention has 6 His linked at its N-terminus and a self-designed cell-penetrating peptide 15RQ (RRQRRQRRQRRQRRR) fused at its C-terminus. This facilitates purification and the cell-penetrating effect of the recombinant antioxidant fusion protein, enabling it to quickly enter the cell and exert its antioxidant effect. It can also cross the blood-brain barrier, which is convenient for the treatment of ischemic stroke.

[0052] 3. The antioxidant fusion protein of the present invention has a wide range of effects. It can be used externally for skin anti-aging and repair of UV-damaged skin, and internally for the treatment of ischemic hypoxic encephalopathy, arteriosclerosis, neurodegenerative diseases, etc. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of the structure of the antioxidant fusion protein in Embodiment 1 of the present invention.

[0054] Figure 2 This is a flowchart of the method provided in Embodiment 2 of the present invention.

[0055] Figure 3 This is a diagram showing the results of the antioxidant fusion protein of Example 1 of the present invention repairing UV-induced skin damage at different concentrations.

[0056] Figure 4This is a graph showing the therapeutic results of the lyophilized powder of the antioxidant stress preparation of Example 7 of the present invention on cerebral ischemia-reperfusion injury (CIRI) in rats.

[0057] Figure 5 This is a neurological function score chart of CIRI rats after treatment with the lyophilized powder of the antioxidant stress preparation of Example 7 of the present invention; wherein, * P<0.05, ## P<0.01; Scoring criteria: 0 points, no obvious defect; 1 point, forelimb flexion; 2 points, reduced reverse lateral thrust, no circle; 3 points, same behavior as 2 points, but with one circle; 4 points, difficulty walking or inability to walk. Detailed Implementation

[0058] To make the technical means, creative features, objectives and effects of this invention clearer and easier to understand, the invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0059] Example

[0060] Example 1: Refer to Figure 1 This invention discloses an antioxidant fusion protein, the amino acid sequence of which includes the amino acid sequence shown in SEQ ID No:1.

[0061] SEQ ID No:1(HHHHHHMAQEFVNCKIQPGKVVVFIKPTCPYCRRAQEILSQLPIKQGLL EFVDITATNHTNEIQDYLQQLTGARTVPRVFIGKDCIGGCSDLVSLQQSGELLTRLKQIGALQGGGGGGGGMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVH EFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHSIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQRRQRRQRRQRRQRRR)

[0062] Example 2: Refer to Figure 2 This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses a method for preparing the antioxidant fusion protein, including the following steps:

[0063] S1: The nucleotide sequence SEQ ID No:2 encoding the antioxidant fusion protein is artificially fully gene-synthesized, and inserted between the XhoI and EcoRI restriction endonuclease sites of the pPIC9K vector to obtain an expression vector;

[0064] SEQ ID No:2(CATCATCACCATCACCACATGGCCCAAGAGTTCGTTAACTGTAAGATC CAGCCAGGTAAGGTCGTCGTGTTCATCAAGCCAACTTGTCCATACTGTAGACGTGCCCAAGAGATCTTGTCCCAGTTGCCTATTAAGCAGGGTCTGTTGGAGTTCGTTGACATCACTGCTACCAACCACACTAACGAGATCCAGGACTACTTGCAGCAATTGACTGGTGCTAGAACCGTGCCAAGAGTGTTCATTGGTAAGGACTGTATCGGTGGTTGCTCCGACTTGGTTTCCTTGCAACAATCTGGTGAGTTGCTGACCAGACTGAAGCAAATTGGTGCATTGCAAGGTGGTGGTGGCGGAGGTGGCGGTGGTATGGCTACAAAAGCTGTTTGTGTCCTGAAAGGTGACGGTCCAGTTCAGGGTATTATCAACTTCGAGCAGAAAGAGTCCAACGGTCCTGTTAAGGTTTGGGGTTCCATCAAGGGTTTGACTGAAGGATTGCACGGTTTCCACGTTCACGAGTTCGGTGATAATACTGCTGGTTGTACTTCTGCCGGTCCACACTTTAACCCATTGTCCAGAAAACACGGTGGTCCAAAGGACGAAGAAAGACACGTTGGTGACTTGGGTAACGTCACTGCTGATAAGGATGGTGTTGCTGACGTTTCCATTGAGGACTCCGTTATTTCTTTGTCCGGTGACCACTCCATCATCGGTAGAACTTTGGTTGTTCACGAGAAGGCTGACGACCTTGGTAAAGGTGGTAACGAAGAGTCCACTAAGACTGGTAACGCTGGTTCCAGATTGGCCTGTGGTGTTATTGGTATTGCTCAGAGAAGGCAAAGAAGGCAGCGTAGACAAAGACGTCAGAGAAGAAGATAA)

[0065] S2 The expression vector obtained in S1 was linearized by SalI single enzyme digestion, and then electroporated into Pichia pastoris. After fermentation expression and post-treatment, the antioxidant fusion protein was obtained.

[0066] S21 Pichia pastoris cells were Pichia pastoris GS115. Pichia pastoris GS115 cells were first preliminarily screened on MD plates, and then clones grown on MD plates were picked onto YPD plates containing different concentrations of antibiotic G418 (1-20 mg / mL) for resistance screening. Finally, highly resistant colonies were selected and identified using PCR technology.

[0067] The S22 fermentation expression process adopted a two-stage fermentation. The amount of BMGY seed culture in the primary stage was controlled at 1.5L, and the amount of BMGY seed culture in the secondary stage was 15L. The fermentation medium was BMMY medium. The fermentation vessel was a 300L fermenter with an actual filling volume of 150L. The inoculation ratio was 1:10, the culture temperature was 30℃, the inducer was 1% methanol, the induction time was 72h, the pH was adjusted to 6.0 with ammonia water, and 10L of 10% peptone was added every 24h as feed.

[0068] After the S23 fermentation expression was completed, the fermentation broth obtained from the fermentation expression was first centrifuged. The centrifugation temperature was controlled at 4℃, the rotation speed at 4000rpm and the time at 30min. The supernatant after centrifugation was collected and then subjected to His-tag affinity chromatography, cation exchange chromatography and ultrafiltration desalting concentration in sequence to obtain the recombinant human collagen peptide.

[0069] Example 3: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0070] Specifically, the antioxidant stress preparation comprises a 50% oil phase and a 50% aqueous phase, and its dosage form is an oil-in-water emulsion. The oil phase is made from raw materials containing the following weight percentages: emulsifier (Tween-40) 0.8%, olive oil 0.3%, vitamin E 0.3%, petrolatum 0.8%, glyceryl monostearate 0.8%, methylparaben 0.3%, and the balance being injection oil.

[0071] The aqueous phase is made from the following ingredients in weight percentage: 0.8% antioxidant fusion protein, 1.5% sodium hyaluronate, 6% glycerin, 0.3% sodium carboxymethyl cellulose, 0.18% *Gynostemma pentaphyllum* extract, 0.18% black goji berry extract, 0.15% black grape seed extract, 0.08% black berry fruit extract, 0.08% galactooligosaccharides, 0.08% stachyose, with the balance being water for injection.

[0072] Correspondingly, the preparation process of antioxidant stress agents includes,

[0073] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0074] Black goji berries, black grape seeds, and black fruit glandular rib flowers were extracted using supercritical CO2 at low temperature. The extraction temperature was 41℃, the extraction pressure was 35MPa, and the extraction time was 120min, respectively, to obtain black goji berry extract, black grape seed extract, and black fruit glandular rib flower extract.

[0075] Antioxidant fusion protein, edible leaf grass extract, black goji berry extract, black grape seed extract, black fruit aralia extract, galactooligosaccharide, and stachyose were dissolved in water for injection, and then sodium hyaluronate, glycerin, and sodium carboxymethyl cellulose were added in sequence and stirred evenly to obtain an aqueous phase.

[0076] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified to obtain an oil-in-water emulsion of the antioxidant stress agent.

[0077] Example 4: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0078] Specifically, the antioxidant stress preparation comprises a 45% oil phase and a 55% aqueous phase, and its dosage form is an oil-in-water emulsion. The oil phase is made from raw materials containing the following weight percentages: emulsifier (Tween-40) 0.7%, olive oil 0.2%, vitamin E 0.2%, petrolatum 0.4%, glyceryl monostearate 0.4%, methylparaben 0.2%, and the balance being injection oil.

[0079] The aqueous phase is made from the following ingredients in weight percentage: 0.4% antioxidant fusion protein, 0.5% sodium hyaluronate, 8% glycerin, 0.2% sodium carboxymethyl cellulose, 0.15% *Gnaphalium affine* extract, 0.15% black goji berry extract, 0.1% black grape seed extract, 0.05% black berry fruit extract, 0.05% galactooligosaccharides, 0.05% stachyose, with the balance being water for injection.

[0080] Correspondingly, the preparation process of antioxidant stress agents includes,

[0081] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0082] Black goji berries, black grape seeds, and black fruit glandular rib flowers were extracted using supercritical CO2 at low temperature. The extraction temperature was 40℃, the extraction pressure was 36MPa, and the extraction time was 110min, respectively, to obtain black goji berry extract, black grape seed extract, and black fruit glandular rib flower extract.

[0083] Antioxidant fusion protein, edible leaf grass extract, black goji berry extract, black grape seed extract, black fruit aralia extract, galactooligosaccharide, and stachyose were dissolved in water for injection, and then sodium hyaluronate, glycerin, and sodium carboxymethyl cellulose were added in sequence and stirred evenly to obtain an aqueous phase.

[0084] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified to obtain an oil-in-water emulsion of the antioxidant stress agent.

[0085] Example 5: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0086] Specifically, the antioxidant stress preparation comprises a 40% oil phase and a 60% aqueous phase, and its dosage form is an oil-in-water emulsion. The oil phase is made from raw materials containing the following weight percentages: emulsifier (Tween-40) 0.5%, olive oil 0.05%, vitamin E 0.05%, petrolatum 0.1%, glyceryl monostearate 0.1%, methylparaben 0.05%, with the balance being injection oil.

[0087] The aqueous phase is made from the following ingredients in weight percentage: 0.1% antioxidant fusion protein, 0.1% sodium hyaluronate, 1% glycerin, 0.05% sodium carboxymethyl cellulose, 0.17% *Gnaphalium affine* extract, 0.17% black goji berry extract, 0.12% black grape seed extract, 0.1% black berry fruit extract, 0.1% galactooligosaccharide, 0.1% stachyose, with the balance being water for injection.

[0088] Correspondingly, the preparation process of antioxidant stress agents includes,

[0089] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0090] Black goji berries, black grape seeds, and black fruit glandular ribbed flowers were extracted using supercritical CO2 at low temperature. The extraction temperature was 42℃, the extraction pressure was 35MPa, and the extraction time was 130min, respectively, to obtain black goji berry extract, black grape seed extract, and black fruit glandular ribbed flower extract.

[0091] Antioxidant fusion protein, edible leaf grass extract, black goji berry extract, black grape seed extract, black fruit aralia extract, galactooligosaccharide, and stachyose were dissolved in water for injection, and then sodium hyaluronate, glycerin, and sodium carboxymethyl cellulose were added in sequence and stirred evenly to obtain an aqueous phase.

[0092] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified to obtain an oil-in-water emulsion of the antioxidant stress agent.

[0093] Example 6: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0094] Specifically, the antioxidant stress preparation comprises a 60% oil phase and a 40% aqueous phase, and its dosage form is an oil-in-water emulsion. The oil phase is made from raw materials containing the following weight percentages: emulsifier (Tween-40) 1%, olive oil 0.5%, vitamin E 0.5%, petrolatum 1%, glyceryl monostearate 1%, methylparaben 0.5%, and the balance being injectable oil.

[0095] The aqueous phase is made from the following ingredients in weight percentage: 1% antioxidant fusion protein, 2% sodium hyaluronate, 10% glycerin, 0.5% sodium carboxymethyl cellulose, 0.2% *Gnaphalium affine* extract, 0.2% black goji berry extract, 0.2% black grape seed extract, 0.06% black berry fruit extract, 0.06% galactooligosaccharides, 0.06% stachyose, with the balance being water for injection.

[0096] Correspondingly, the preparation process of antioxidant stress agents includes,

[0097] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0098] Black goji berries, black grape seeds, and black fruit glandular rib flowers were extracted using supercritical CO2 at low temperature. The extraction temperature was 41℃, the extraction pressure was 36MPa, and the extraction time was 125min, resulting in black goji berry extract, black grape seed extract, and black fruit glandular rib flower extract.

[0099] Antioxidant fusion protein, leafwort extract, black goji berry extract, black grape seed extract, black fruit extract, galactooligosaccharide, and stachyose were dissolved in water for injection, and then sodium hyaluronate, glycerin, and sodium carboxymethyl cellulose were added in sequence and stirred evenly to obtain an aqueous phase.

[0100] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified to obtain an oil-in-water emulsion of the antioxidant stress agent.

[0101] Example 7: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0102] Specifically, the antioxidant stress preparation comprises a 50% oil phase and a 50% aqueous phase, and its dosage form is a lyophilized powder. Among them,

[0103] The oil phase is made from raw materials comprising the following weight percentages: emulsifier (lecithin) 0.8%, olive oil %, vitamin E %, and the balance being injection oil;

[0104] The aqueous phase is made from the following ingredients in weight percentage: 0.8% antioxidant fusion protein, 1.5% sodium hyaluronate, 6% glycerol, 0.3% sodium carboxymethyl cellulose, 8% mannitol, 4% trehalose, and the balance being water for injection.

[0105] Correspondingly, the preparation process of antioxidant stress agents includes,

[0106] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0107] The antioxidant fusion protein, mannitol, and trehalose were dissolved in water for injection. The pH was adjusted to 7.2 with 20 mM Tris-Cl. Then, sodium hyaluronate, glycerol, and sodium carboxymethyl cellulose were added in sequence and stirred until homogeneous to obtain the aqueous phase.

[0108] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified. The emulsion obtained is sterilized and then freeze-dried to obtain a lyophilized powder of the antioxidant stress agent.

[0109] Example 8: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0110] Specifically, the antioxidant stress preparation comprises a 45% oil phase and a 55% aqueous phase, and its dosage form is a lyophilized powder. Among them,

[0111] The oil phase is made from raw materials comprising the following weight percentages: emulsifier (lecithin) 0.7%, olive oil %, vitamin E %, and the balance being injection oil;

[0112] The aqueous phase is made from the following ingredients in weight percentage: 0.4% antioxidant fusion protein, 0.5% sodium hyaluronate, 8% glycerol, 0.2% sodium carboxymethyl cellulose, 6% mannitol, 3% trehalose, and the balance being water for injection.

[0113] Correspondingly, the preparation process of antioxidant stress agents includes,

[0114] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0115] The antioxidant fusion protein, mannitol, and trehalose were dissolved in water for injection. The pH was adjusted to 7 with 20 mM Tris-Cl. Then, sodium hyaluronate, glycerol, and sodium carboxymethyl cellulose were added in sequence and stirred until homogeneous to obtain the aqueous phase.

[0116] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified. The emulsion obtained is sterilized and then freeze-dried to obtain a lyophilized powder of the antioxidant stress agent.

[0117] Example 9: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0118] Specifically, the antioxidant stress preparation comprises a 40% oil phase and a 60% aqueous phase, and its dosage form is a lyophilized powder. Among them,

[0119] The oil phase is made from raw materials containing the following weight percentages: emulsifier (lecithin) 0.5%, olive oil %, vitamin E %, and the balance being injection oil;

[0120] The aqueous phase is made from the following ingredients in weight percentage: 0.1% antioxidant fusion protein, 0.1% sodium hyaluronate, 1% glycerol, 0.05% sodium carboxymethyl cellulose, 7% mannitol, 5% trehalose, and the balance being water for injection.

[0121] Correspondingly, the preparation process of antioxidant stress agents includes,

[0122] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0123] The antioxidant fusion protein, mannitol, and trehalose were dissolved in water for injection. The pH was adjusted to 7.5 with 20 mM Tris-Cl. Then, sodium hyaluronate, glycerol, and sodium carboxymethyl cellulose were added in sequence and stirred until homogeneous to obtain the aqueous phase.

[0124] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified. The emulsion obtained is sterilized and then freeze-dried to obtain a lyophilized powder of the antioxidant stress agent.

[0125] Example 10: This invention discloses an antioxidant fusion protein, which differs from Example 1 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0126] Specifically, the antioxidant stress preparation comprises 60% oil phase and 40% aqueous phase, and its dosage form is lyophilized powder. Among them,

[0127] The oil phase is made from raw materials comprising the following weight percentages: emulsifier (lecithin) 1%, olive oil %, vitamin E %, and the balance being injection oil;

[0128] The aqueous phase is made from the following ingredients in weight percentage: 1% antioxidant fusion protein, 2% sodium hyaluronate, 10% glycerol, 0.5% sodium carboxymethyl cellulose, 10% mannitol, 4% trehalose, and the balance being water for injection.

[0129] Correspondingly, the preparation process of antioxidant stress agents includes,

[0130] After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase.

[0131] The antioxidant fusion protein, mannitol, and trehalose were dissolved in water for injection. The pH was adjusted to 7.4 with 20 mM Tris-Cl. Then, sodium hyaluronate, glycerol, and sodium carboxymethyl cellulose were added in sequence and stirred until homogeneous to obtain the aqueous phase.

[0132] The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified. The emulsion obtained is sterilized and then freeze-dried to obtain a lyophilized powder of the antioxidant stress agent.

[0133] Comparative Example

[0134] Comparative Example 1: This is an antioxidant fusion protein disclosed in this invention. The difference from Example 3 or 7 is that the rhGrx sequence is removed, and the amino acid sequence of the antioxidant fusion protein contains the amino acid sequence shown in SEQ ID No:1.

[0135] SEQ ID No:3(HHHHHHGGGGGGGGGGMATKAVCVLKGDGPVQGIINFEQKESNGPVKV WGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADK DGVADVSIEDSVISLSGDHSIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQR RQRRQRRQRRQRRR)

[0136] Comparative Example 2: This is an antioxidant fusion protein disclosed in this invention. The difference from Example 3 or 7 is that the linker sequence has been removed, and the amino acid sequence of the antioxidant fusion protein contains the amino acid sequence shown in SEQ ID No:4.

[0137] SEQ ID No:4(HHHHHHMAQEFVNCKIQPGKVVVFIKPTCPYCRRAQEILSQLPIKQGLL EFVDITATNHTNEIQDYLQQLTGARTVPRVFIGKDCIGGCSDLVSLQQSGELLTRLKQIGALQMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGD NTAGCTSAGHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHSIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQRRQRRQRRQRRQRRR)

[0138] Comparative Example 3: This is an antioxidant fusion protein disclosed in this invention. The difference from Example 3 or 7 is that the rhSOD sequence is removed, and the amino acid sequence of the antioxidant fusion protein contains the amino acid sequence shown in SEQ ID No:5.

[0139] SEQ ID No:6(HHHHHHMAQEFVNCKIQPGKVVVFIKPTCPYCRRAQEILSQLPIKQGLL EFVDITATNHTNEIQDYLQQLTGARTVPRVFIGKDCIGGCSDLVSLQQSGELLTRLKQIGALQGGGGGGGGRRQRRQRRQRRQRRR)

[0140] Comparative Example 4: This invention discloses an antioxidant fusion protein, which differs from Example 3 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0141] Specifically, the formulation of the antioxidant stress agent is an oil-in-water emulsion. The aqueous phase does not contain the herb extract.

[0142] Comparative Example 5: This invention discloses an antioxidant fusion protein, which differs from Example 3 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0143] Specifically, the formulation of the antioxidant stress agent is an oil-in-water emulsion. The aqueous phase does not contain black goji berry extract, black grape seed extract, or black fruit extract.

[0144] Comparative Example 6: This invention discloses an antioxidant fusion protein, which differs from Example 3 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0145] Specifically, the formulation of the antioxidant stress agent is an oil-in-water emulsion. The aqueous phase does not contain galactooligosaccharides or stachyose.

[0146] Comparative Example 7: This invention discloses an antioxidant fusion protein, which differs from Example 7 in that it also discloses the application of the antioxidant fusion protein in the preparation of antioxidant stress agents for the prevention and / or treatment of hypoxic-ischemic encephalopathy, atherosclerosis, neurodegenerative diseases, and skin aging.

[0147] Specifically, the dosage form of the antioxidant stress agent is a lyophilized powder. The aqueous phase does not contain mannitol or trehalose.

[0148] Performance testing

[0149] (1) Detection of the activity of recombinant antioxidant fusion protein on rat skin fibroblasts

[0150] Rat skin fibroblasts were injected at a concentration of 1.0 × 10⁻⁶. 4 / wells were seeded into 96-well culture plates. After 24 hours of culture, the medium was replaced with fresh medium, and the cells were divided into three groups: control group (no phorate oxidative damage, no drug administration), model group (phorate oxidative damage, no drug administration), and drug administration group (phorate oxidative damage, given 1 mL of the oil-in-water emulsion of the antioxidant stress preparations from Examples 3, 1, 2, 3, 4, 5, and 6). Cells were incubated at 37°C in a 5% CO2 incubator for another 24 hours (6 replicates per concentration). Before termination, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 hours. The supernatant was then discarded, and the cells were dissolved in 100 μL of DMSO. The optical density (OD) was measured using a microplate reader after 10 minutes. 570nm The cell viability was calculated by taking the OD value of the treatment group and the OD value of the control group [Viability = (OD value of the treatment group / OD value of the control group) × 100%], and the results are shown in Table 1.

[0151] Table 1. Effects of antioxidant fusion protein on the activity of skin fibroblasts in phoxim-exposed rats (n=6, x±s)

[0152]

[0153]

[0154] Among them, # compared with the control group, P<0.05; * compared with the phorate oxidative damage group, P<0.05.

[0155] (2) Protective effect of recombinant antioxidant fusion protein against skin damage caused by ultraviolet radiation.

[0156] Twelve male rats (weighing 200–300 g) were routinely cultured for seven days before the experiment. Before the experiment, the fur on the rats' backs was shaved clean, and the shaved area was divided into four regions (1.5 cm × 1.5 cm). The drug administration components were: a low-dose region (2.5 μM antioxidant fusion protein), a medium-dose region (5 μM antioxidant fusion protein), and a high-dose region (10 μM antioxidant fusion protein). The remaining area served as a blank control. The rats' bodies were covered with specially made cardboard, leaving only the four regions exposed. They were irradiated with ultraviolet light (UV wavelength 280–320 nm) for 10 minutes (at a distance of 5–7 cm), and the drug was administered immediately after irradiation. Different doses of the antioxidant fusion protein (1 mL volume) were applied to the irradiated areas on the rats' backs using a fine glass rod. The color changes of the rat skin after UV radiation were studied using a colorimeter and the Lab colorimetric system. Erythema intensity (α value) was measured to represent the red content in the skin. The erythema reduction rate was expressed as the α value of each group minus the value of the blank control group. A higher α value indicated more severe erythema and greater UV radiation damage. The results are shown below. Figure 3 As shown.

[0157] (3) Protection of rat cardiomyocytes from hypoxia-reoxygenation injury by recombinant antioxidant fusion protein

[0158] H9c2 cardiomyocytes were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. An H9C2 cell injury model was established using the hypoxia / reoxygenation method: cells were washed with PBS buffer, then added to serum-free low-glucose DMEM medium, and placed in a 37°C saturated humidity anaerobic incubator containing 85% N2, 10% H2, and 5% CO2. After 4 hours of culture, the cells were removed to complete the hypoxia process. Then, fresh DMEM medium containing 15% FBS was added, and the cells were placed in a CO2 incubator for another 2 hours to complete the reoxygenation process.

[0159] The cultured cells were divided into four groups: a control group (normal cultured cells), a model group (after hypoxia / reoxygenation, without drug administration), and a drug administration group (after hypoxia / reoxygenation, administered 1 mL, 10 μM of the lyophilized powder of the antioxidant stress agents from Examples 7, 1, 2, 3, 6, and 7), and incubated together for 30 min. The apoptosis index was detected using the TUNEL assay, and the total ATPase activity and MDA content in mitochondria were measured using a kit. The results are shown in Table 2.

[0160] Table 2

[0161]

[0162]

[0163] Among them, # compared with the normal group, P<0.05; * compared with the hypoxia / reoxygenation model group, P<0.05.

[0164] (4) Therapeutic effect of recombinant antioxidant fusion protein on MCAO model rats

[0165] Eighteen experimental rats were randomly divided into three groups: a sham-operated group, a model group, and a drug-treated group. The rats were fasted for 12 hours prior to surgery but allowed free access to water. The following day, left middle cerebral artery occlusion surgery was performed according to the previously established modeling method. Model rats were anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.3 mL / 100 g). The left common carotid artery (CCA) and external carotid artery (ECA) were separated and ligated. The internal carotid artery (ICA) was separated, and a suture was inserted through the CCA into the ICA to the proximal end (18–20 mm) of the anterior cerebral artery. The insertion was stopped to block blood flow to the middle cerebral artery on that side, inducing cerebral ischemia. The suture was removed 1 hour after occlusion to achieve reperfusion. The sham-operated group underwent the same procedure as the modeling group, except that no suture was inserted. After modeling, the rats were further divided into a model group (injected with 7 mg / (kg·d) saline) and a drug-treated group (injected with 7 mg / (kg·d) 10 μM of the lyophilized powder of the antioxidant stress agent from Example 7), with six rats in each group. One hour after perfusion, rats in the sham-operated group and the model group were intraperitoneally injected with the drug, and an equal volume of 0.9% sodium chloride solution was injected intraperitoneally. The drug administration volume was 0.6 mL / 100 g, once daily for 7 consecutive days. Results are as follows: Figure 3 and Figure 4 As shown.

[0166] from Figure 3 As can be seen, compared with the model group rats, rats treated with the antioxidant fusion protein showed a significant reduction in the area of ​​white infarcts following middle cerebral artery occlusion, and neurological function scores indicated this reduction. Figure 4 The neurological function score sheet shows that the rats' neurological function scores improved significantly after treatment.

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An antioxidant fusion protein, characterized in that: The amino acid sequence of the antioxidant fusion protein is the amino acid sequence shown in SEQ ID No:

1.

2. The method for preparing an antioxidant fusion protein according to claim 1, characterized in that: Includes the following steps, S1 synthesized the nucleotide sequence SEQ ID No:2 of the antioxidant fusion protein using artificial whole-genome synthesis and inserted it between the XhoI and EcoRI restriction enzyme sites of the pPIC9K vector to obtain the expression vector; S2 linearizes the expression vector obtained in S1 by SalI single enzyme digestion, then introduces it into Pichia pastoris by electroporation. After fermentation expression and post-treatment, the antioxidant fusion protein is obtained.

3. The application of the antioxidant fusion protein according to claim 1 in the preparation of an antioxidant stress agent for treating skin damage caused by ultraviolet radiation.

4. The application of the antioxidant fusion protein according to claim 3, characterized in that: The antioxidant stress agent comprises 40-60% oil phase and 40-60% aqueous phase; wherein, The oil phase is made from raw materials comprising the following weight percentages: 0.5-1.0% emulsifier, 0.5-5.0% olive oil, 0.05-0.50% vitamin E, with the balance being injection oil; The aqueous phase is made from raw materials comprising the following weight percentages: 0.1-1.0% antioxidant fusion protein, 0.1-2.0% sodium hyaluronate, 1-10% glycerol, 0.05-0.50% sodium carboxymethyl cellulose, with the balance being water for injection.

5. The application of the antioxidant fusion protein according to claim 4, characterized in that: In the oil phase, the emulsifier is Tween-40.

6. The application of the antioxidant fusion protein according to claim 5, characterized in that: The oil phase also includes 0.1-1.0% petrolatum, 0.1-1.0% glyceryl monostearate, and 0.05-0.50% methylparaben.

7. The application of the antioxidant fusion protein according to claim 5, characterized in that: The aqueous phase also includes 0.15-0.25% of *Gnaphalium affine* extract, 0.15-0.20% of black goji berry extract, 0.10-0.20% of black grape seed extract, 0.05-0.10% of black berry fruit extract, 0.05-0.10% of galactooligosaccharides, and 0.05-0.10% of stachyose.

8. The application of the antioxidant fusion protein according to claim 5, characterized in that: The preparation process of the antioxidant stress agent includes, After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase. Antioxidant fusion protein, leafwort extract, black goji berry extract, black grape seed extract, black fruit extract, galactooligosaccharide, and stachyose were dissolved in water for injection, and then sodium hyaluronate, glycerin, and sodium carboxymethyl cellulose were added in sequence and stirred evenly to obtain an aqueous phase. The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified to obtain an oil-in-water emulsion of the antioxidant stress agent.

9. The application of the antioxidant fusion protein according to claim 4, characterized in that: In the oil phase, the emulsifier is lecithin.

10. The application of the antioxidant fusion protein according to claim 9, characterized in that: The preparation process of the antioxidant stress agent includes, After dissolving the emulsifier in the injection oil, olive oil, vitamin E, petrolatum, glyceryl monostearate, and methylparaben are added in sequence and stirred until homogeneous to obtain the oil phase. The antioxidant fusion protein, mannitol, and trehalose were dissolved in water for injection. The pH was adjusted to 7.0-7.5 with 20 mM Tris-Cl. Then, sodium hyaluronate, glycerol, and sodium carboxymethyl cellulose were added in sequence and stirred until homogeneous to obtain the aqueous phase. The aqueous phase is added to the oil phase and treated with a colloid mill. Then, water for injection is added to make up the volume, and the mixture is emulsified. The emulsion obtained is sterilized and then freeze-dried to obtain a lyophilized powder of the antioxidant stress agent.

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