High-activity recombinant human superoxide dismutase as well as preparation method and application thereof

Abstract

The invention provides high-activity recombinant human superoxide dismutase as well as a preparation method and application thereof. The recombinant human superoxide dismutase with high activity comprises an amino acid sequence as shown in SEQ ID No.1, is prepared through coding expression of an optimized nucleotide sequence as shown in SEQ ID No.2, has a relatively good expression quantity, has excellent thermal stability and acid-base stability, and has good stability at 60 DEG C and good stability at pH of 3.7-12; an excellent cell light aging resisting effect is also achieved; the product can be used for resisting cell photoaging.

Description

Technical Field

[0001] This application belongs to the field of bioengineering technology, and in particular relates to a highly active recombinant human superoxide dismutase, its preparation method and application. Background Technology

[0002] Superoxide dismutase (SOD) is a crucial metallo-oxidizing enzyme in living organisms that catalyzes the oxidation of superoxide anion radicals (O2·4O3 ... - It undergoes a disproportionation reaction to produce oxygen and hydrogen peroxide, forming the body's first line of defense against oxidative stress. It shows great potential in treating various diseases caused by free radicals, such as inflammatory diseases, ischemia-reperfusion injury, radiation sickness, and anti-aging, thus possessing extremely high clinical application value and market prospects.

[0003] Human superoxide dismutase (hSOD) mainly includes intracellular Cu / Zn-SOD (SOD1) and mitochondrial Mn-SOD (SOD2). Among them, recombinant human Cu / Zn-SOD (rhSOD1) has become a focus of research and development due to its stable structure and well-defined physiological functions. Currently, the main technical route for producing rhSOD1 is to use genetic engineering technology to introduce the gene encoding human SOD1 into prokaryotic or eukaryotic expression systems for heterologous expression. However, existing recombinant preparation methods still have a series of significant technical bottlenecks, which severely limit the industrial application of rhSOD and its clinical efficacy:

[0004] First, expression yield is low and activity is insufficient. Especially in prokaryotic expression systems, exogenous proteins readily form insoluble inclusion bodies. Although the yield of inclusion bodies may be high, subsequent complex denaturation and renaturation steps are required, a process that is inefficient and prone to protein misfolding, resulting in extremely low final yields of active rhSOD. Second, stability is poor. rhSOD1 has a short half-life in vivo, is rapidly cleared by the kidneys, and is sensitive to temperature and pH, leading to a significant reduction in its bioavailability and making it difficult to maintain effective therapeutic concentrations at the target site.

[0005] Therefore, developing a recombinant human superoxide dismutase that can be expressed efficiently, has high specific activity, and exhibits excellent stability, and expanding its application in new drug development and biomedicine, has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] This application provides a method for preparing and applying highly active recombinant human superoxide dismutase to solve the problems existing in related technologies. The technical solution is as follows:

[0007] In a first aspect, embodiments of this application provide a highly active recombinant human superoxide dismutase, the highly active recombinant human superoxide dismutase comprising:

[0008] a) The amino acid sequence as shown in SEQ ID No. 1; or

[0009] b) An amino acid sequence in which one or more amino acid residues are added, substituted, dropped or inserted in the amino acid sequence shown in SEQ ID NO.1, and which retains the collagen activity of the amino acid sequence of SEQ ID NO.1.

[0010] SEQ ID No. 1:

[0011] MAIHAVCVLKGDSPVTGTIHLKEEGDMVTVTGEITGLTPGKHGFHVHEFGDNTNGCTSAGGHFNPHGKEHGAPEDENRHAGDLGNVVAGEDGKAVINMKDKLVKLTGPDSVIGRTLVVHVDEDDLGRGGHEQSKITGNAGGRLACGVIGITKE.

[0012] Secondly, embodiments of this application provide a nucleotide sequence encoding the aforementioned highly active recombinant human superoxide dismutase; the nucleotide is shown in SEQ ID No. 2;

[0013] SEQ ID No. 2:

[0014] ATGGCAATTCATGCAGTTTGCGTCCTGAAAGGTGACTCCCCGGTAACCGGTACCATCCACCTGAAAGAGGAAGGCGACATGGTCACTGTGACTGGTGAGATCACCGGCCTGACCCCGGGCAAACACGGTTTCCACGTTCACGAATTTGGCGACAATACCAACGGCTGCACTTCTGCTGGCGGTCACTTCAACCCGCATGGTAAAGAACATGGCGCTCCGGAGGACGAAAA CCGTCATGCAGGTGACCTGGGCAACGTAGTTGCAGGCGAGGATGGTAAAGCCGTCATTAACATGAAGGACAAACTGGTGAAGCTGACCGGCCCGGATTCTGTTATCGGTCGTACCCTGGTGGTACACGTAGATGAGGACGACCTGGGTCGTGGTGGTCACGAGCAGTCCAAAATCACCGGTAACGCAGGTGGCCGTCTGGCATGTGGTGTAATCGGTATCACTAAAGAA.

[0015] The gene sequence of the highly active recombinant human superoxide dismutase of this invention has been codon-optimized. After constructing a recombinant plasmid, it was expressed using an E. coli expression system. The expression of the highly active recombinant human superoxide dismutase nucleotide acid reached 25.7%. It was free of protein purification tags and had no impact on protein structure or function, possessing the complete structure and function of superoxide dismutase.

[0016] Thirdly, embodiments of this application provide a recombinant plasmid containing the aforementioned nucleotides.

[0017] In one embodiment, the plasmid vector is rHuSOD-1-pET-30a(+).

[0018] Fourthly, embodiments of this application provide a host cell for efficient expression, wherein the host cell for efficient expression comprises the nucleotides described above; or the recombinant plasmids described above.

[0019] In one embodiment, the host cell for high-efficiency expression is Escherichia coli BL21(DE3) competent cells.

[0020] In one embodiment, the highly efficient expression host cell is obtained by introducing the recombinant plasmid described above into the host.

[0021] Fifthly, embodiments of this application provide a method for preparing highly active recombinant human superoxide dismutase, characterized in that...

[0022] The highly active recombinant human superoxide dismutase was expressed in the host cells described above, and then separated and purified to obtain the highly active recombinant human superoxide dismutase.

[0023] Sixthly, embodiments of this application provide a cell photoaging resistance agent, the cell photoaging resistance agent containing the above-mentioned highly active recombinant human superoxide dismutase; or the above-mentioned highly active recombinant human superoxide dismutase encoded by nucleotides; or the above-mentioned highly active recombinant human superoxide dismutase expressed by host cells with high efficiency.

[0024] Seventhly, embodiments of this application provide the application of the above-mentioned highly active recombinant human superoxide dismutase; or the above-mentioned highly active recombinant human superoxide dismutase encoded by nucleotides; or the above-mentioned highly active recombinant human superoxide dismutase expressed by host cells with high efficiency in anti-photoaging food, cosmetic, health product or medical device products.

[0025] The advantages or beneficial effects of the above technical solutions include at least the following:

[0026] The highly active recombinant human superoxide dismutase of this application is prepared by expression of an optimized gene sequence, exhibiting good expression levels. Furthermore, the recombinant human superoxide dismutase not only possesses excellent thermal and acid-base stability, with good stability at 60℃ and between pH 3.7 and 12, but also demonstrates excellent resistance to cell photoaging, making it suitable for use in products designed to combat cell photoaging.

[0027] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0028] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0029] Figure 1 Map of the recombinant expression vector rHuSOD-1-pET-30a(+);

[0030] Figure 2 The image shows the enzyme digestion verification pattern of the recombinant plasmid of the positive clone.

[0031] Figure 3 Electrophoresis images of recombinant human superoxide dismutase expression before and after induction;

[0032] Figure 4 This is an electrophoresis image of the purified recombinant human superoxide dismutase.

[0033] Figure 5 The enzyme activity of recombinant human superoxide dismutase after heat treatment at different temperatures is shown in the graph.

[0034] Figure 6 The enzyme activity of recombinant human superoxide dismutase after treatment at different pH values ​​is shown in the graph.

[0035] Figure 7 Figure showing the cell positivity rate results for β-galactosidase staining to detect cell photoaging.

[0036] Figure 8 Images of fibroblasts treated with different concentrations of recombinant human superoxide dismutase. Detailed Implementation

[0037] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0038] This application provides a highly active recombinant human superoxide dismutase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0039] This application also provides a nucleotide encoding the above-mentioned highly active recombinant human superoxide dismutase; the nucleotide is shown in SEQ ID No. 2.

[0040] In the above specific implementation scheme, nucleic acid molecules are a collective term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are biological macromolecules composed of many nucleotide monomers and are one of the most basic substances of life. A nucleotide sequence refers to the arrangement of bases in DNA or RNA. Nucleic acid molecules contain cDNA. In some cases, nucleic acid molecules can be modified for use in the vectors of this application, such as for codon optimization. In some cases, for the purpose of cloning into a vector, the sequence can be designed to contain terminal restriction sites. Nucleic acid molecules can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of nucleic acids encoded by one or more given cells or isolated from said one or more given cells.

[0041] This application also provides expression vectors carrying the aforementioned nucleic acid molecules.

[0042] In a specific implementation, an expression vector is a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) into a recipient cell. As the most commonly used and simplest vector in genetic engineering, it must include three parts: a genetic marker gene, a replication region, and the target gene. Besides the commonly used *E. coli* plasmid vector, many other artificially constructed plasmid vectors suitable for microorganisms, yeast, plants, etc., have been developed. Vectors include, but are not limited to: single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, or without free ends (e.g., circular); nucleic acid molecules containing DNA, RNA, or both; and other polynucleotide species known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, through standard molecular cloning techniques. Some vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) integrate into the host cell's genome after introduction and thus replicate along with the host genome. Furthermore, some vectors can direct the expression of the target gene. Such vectors are referred to herein as “expression vectors”. Recombinant expression vectors may contain a form suitable for expressing nucleic acids in host cells, which means that recombinant expression vectors include one or more regulatory elements that can be selected based on the host cell used for expression and which can be operatively linked to the nucleic acid sequence to be expressed.

[0043] This application also provides host cells expressing the aforementioned highly active recombinant human superoxide dismutase.

[0044] In one specific implementation, "host cell" refers to any cell type susceptible to transformation, transfection, transduction, etc., of a nucleic acid construct or expression vector containing the polynucleotides of this application. "Host cell" encompasses any progeny of the parent cell that is not entirely identical to the parent cell due to mutations during replication. The host cell can be any cell useful in the production of the highly active recombinant human superoxide dismutase of this application. To produce highly active recombinant human superoxide dismutase, the nucleic acid encoding the highly active recombinant human superoxide dismutase can be isolated and inserted into one or more vectors for further cloning and / or expression in the host cell. This nucleic acid can be easily isolated and sequenced using conventional techniques, e.g., by using oligonucleotide probes capable of specifically binding to genes encoding recombinant collagen. The host cell refers to a cell in which exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny may not be entirely identical to the parent cell in terms of nucleic acid content but may contain mutations. Methods for introducing vectors into host cells are well-known, such as electroporation, transfection, microinjection, gene gun technology, and liposome-mediated methods. The host cells are prokaryotic or eukaryotic cells. The host cells are selected from *Escherichia coli* BL21(DE3) competent cells.

[0045] Example 1

[0046] 1. Construction of recombinant plasmids:

[0047] via GenSmart TM Online codon optimization tool; restriction enzyme sites XhoI: CTCGAG and NdeI: CATATGA;

[0048] The unoptimized gene sequence is shown in SEQ ID No. 3.

[0049] SEQ ID No. 3:

[0050] ATGGCTATTCATGCTGTCTGTGTCTTGAAAGGTGACTCTCCAGTGACCGGCACGATTCATCTAAAGGAAGAGGGTGACATGGTTACCGTGACTGGTGAGATAACTGGTCTTACTCCTGGTAAGCACGGTTTTCATGTGCATGAATTCGGTGACAACACGAATGGATGTACTAGTGCCGGCGGTCATTTTAACCCACACGGAAAAGAGCACGGTGCACCTGAGGACGAGAACAGGCATGCAGGAGACTTGGGAAATGTTGTTGCTGGTGAAGACGGTAAAGCAGTCATTAACATGAAGGATAAGCTAGTTAAGCTAACTGGCCCAGACTCTGTTATAGGTAGAACTCTAGTGGTCCATGTAGATGAAGACGACTTAGGCAGAGGAGGACACGAGCAGAGTAAAATCACGGGTAATGCCGGAGGAAGACTAGCTTGTGGAGTAATCGGTATAACCAAGGAA。

[0051] The optimized gene sequence is shown in SEQ ID No.2, and the optimized CG content is 56.21%:

[0052] SEQ ID No.2:

[0053] ATGGCAATTCATGCAGTTTGCGTCCTGAAAGGTGACTCCCCGGTAACCGGTACCATCCACCTGAAAGAGGAAGGCGACATGGTCACTGTGACTGGTGAGATCACCGGCCTGACCCCGGGCAAACACGGTTTCCACGTTCACGAATTTGGCGACAATACCAACGGCTGCACTTCTGCTGGCGGTCACTTCAACCCGCATGGTAAAGAACATGGCGCTCCGGAGGACGAAAA CCGTCATGCAGGTGACCTGGGCAACGTAGTTGCAGGCGAGGATGGTAAAGCCGTCATTAACATGAAGGACAAACTGGTGAAGCTGACCGGCCCGGATTCTGTTATCGGTCGTACCCTGGTGGTACACGTAGATGAGGACGACCTGGGTCGTGGTGGTCACGAGCAGTCCAAAATCACCGGTAACGCAGGTGGCCGTCTGGCATGTGGTGTAATCGGTATCACTAAAGAA.

[0054] The rHuSOD-1-pET-30a vector, constructed using SnapGene software with primers 5'-CTCGAGCTCG and 3'-TATGCATATG, is shown below. Figure 1 As shown.

[0055] The constructed plasmid was transformed into E. coli BL21(de3) competent cells (purchased from Thermo Scientific). TM (Catalog No.: EC0114, Lot No. 2723635), competent cells were selected as single clones for plasmid restriction enzyme digestion pattern verification; the restriction enzyme digestion verification pattern is shown below. Figure 2 shown. Lane M: DNA Marker; Lane 1: Plasmid digested by ApaLI; Lane 2: Uncutplasmid DNA.

[0056] 2. Expression screening and expression product validation

[0057] The correctly identified positive bacterial strains were activated by inoculating a 1:1000 plasmid powder solution into 30 ml of LB liquid medium containing 50 μg / L kanamycin and incubating at 37°C with shaking at 200 rpm for 12-16 h. The next day, the bacterial culture was inoculated at a 1:100 ratio into 30 ml of TB medium containing 50 μg / L kanamycin and incubated at 37°C with shaking at 200 rpm until the OD600 reached 0.6-0.8. IPTG (1 mM / ml) was then added for induction for 4 h. Samples before and after induction were analyzed by SDS-PAGE electrophoresis. Results are as follows: Figure 3 As shown, there is significant expression at 17-20 kDa, consistent with the predicted molecular weight. Where M: protein marker; 1: uninduced recombinant bacteria; 2: induced whole recombinant bacteria; 3: induced recombinant bacteria supernatant; 4: induced recombinant bacteria precipitate.

[0058] 3. Fermentation by engineered bacteria

[0059] The working strain underwent primary seed culture, secondary seed culture, and fermentation with a 5% inoculum. Fermentation pH was 6.8-7.2, aeration rate was 1-2 VVM, dissolved oxygen was controlled at 20%-30%, and fermentation temperature was 37℃. When OD was measured... 600 At 10-12 pm, add 1 mM IPTG for induction at 16°C for 16 hours. After fermentation, centrifuge to collect the cells.

[0060] The fermentation medium consisted of 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 170 mM KH2PO4, 720 mM K2HPO4, and trace elements. The trace elements were as follows: CoCl2·6H2O (2.5 mg / L), CuCl2·2H2O (1.5 mg / L), H3BO3 (3 mg / L), Na2MoO4·2H2O (2.5 mg / L), Zn(CH3COO)2·2H2O (8 mg / L), Titriplex III (8.4 mg / L), Fe(III)citrate (60 mg / L), and MnCl2·2H2O (12.3 mg / L).

[0061] 4. Purification of recombinant expression products

[0062] Dissolve the precipitate with 20 mmol / L Tris at a ratio of 1:10, set the pressure of the high-pressure homogenizer to 700-900 Pa, homogenize the bacterial cells under high pressure, repeat 5-8 times, centrifuge at 8000 rpm, 4℃ for 30 min, and collect the supernatant and precipitate.

[0063] The precipitate was added to inclusion body washing solution and homogenized and washed using a homogenizer. It was then centrifuged at 8000 rpm and 4°C for 30 min, and the supernatant was collected. This process was repeated three times.

[0064] The washed inclusion bodies were denatured and renatured; high-purity SOD was obtained by Ni column affinity chromatography and DEAE ion exchange chromatography. Its SDS-PAGE electrophoresis results are as follows: Figure 4 As shown, M: protein marker; 1: SOD-g purified and diluted 10 times; 2: SOD-g purified and diluted 20 times; 3: SOD-g purified and diluted 30 times; 4: SOD-g purified and diluted 10 times for grayscale analysis; 5: SOD-g purified and diluted 20 times for grayscale analysis; 6: SOD-g purified and diluted 30 times for grayscale analysis.

[0065] The protein band grayscale analysis performed using ImageJ is shown in Table 1.

[0066] Table 1

[0067]

[0068] As can be seen from Table 2, the purity of the obtained human superoxide dismutase is above 95.7%.

[0069] Comparative Example 1

[0070] The fermentation induction temperature was 37°C, and the other conditions were the same as in Example 1.

[0071] Comparative Example 2

[0072] The fermentation medium consisted of 12 g / L tryptone, 24 g / L yeast extract, and 4 mL / L glycerol. The concentrations were 170 mM KH₂PO₄ and 720 mM K₂HPO₄. No trace elements were added. All other conditions were the same as in Example 1.

[0073] (1) Detection of expression level and enzyme activity

[0074] The enzyme expression levels and enzyme activity were detected by the methods of Example 1, Comparative Example 1 and Comparative Example 2, and the results are shown in Table 2.

[0075] The enzyme activity was determined according to GB / T41906-2022 "Method for Detection of Superoxide Dismutase Activity"; the blank rate (ΔA / min)A was adjusted to 0.070±0.002 according to the following formula; the sample concentration was adjusted to the rate (ΔA / min)A' was 0.035±0.002; N is the dilution factor; V is the volume (ml);

[0076]

[0077] Table 2

[0078] Testing items Example Comparative Example 1 Comparative Example 2 SOD expression level 25.7％ 18.9％ 24.9％ SOD enzyme activity detection 23000U / mL 17250U / mL 2560U / mL

[0079] As shown in Table 1, the method and design of this application yielded recombinant human superoxide dismutase through nucleotide expression, achieving an expression level of 25.7%. Fermentation temperature and the composition of the fermentation medium had a certain impact on the expression level and enzyme activity.

[0080] (2) Temperature stability test results

[0081] The recombinant human superoxide dismutase obtained in Example 1 was treated at 60℃ and 80℃ for 30 min, 60 min, 90 min, and 120 min, respectively. Enzyme activity was measured at different temperatures and times, following the method described in GB / T41906-2022, "Methods for Determination of Superoxide Dismutase Activity". The results are as follows: Figure 5 As shown.

[0082] The enzyme activity decreased by 95% after heat treatment at 80℃, but remained stable at around 90% after heat treatment at 60℃. Therefore, SOD enzyme has good stability at 60℃.

[0083] (3) Results of acid-base stability test

[0084] The recombinant human superoxide dismutase obtained in Example 1 was treated at different pH values ​​of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, and 15. Enzyme activity was measured at different pH values. The enzyme activity assay method followed GB / T41906-2022, "Methods for Determination of Superoxide Dismutase Activity". The results are as follows: Figure 6 As shown.

[0085] The activity of this SOD enzyme is basically inactivated at pH 3.5 and pH 12, relatively stable at pH 6-11, and highest at pH 4-5. SOD does not become inactivated between pH 3.7 and pH 12, which verifies that the SOD enzyme has a wide range of operating conditions and its activity is enhanced under acidic conditions.

[0086] (4) Highly active SOD is used to resist cell photoaging.

[0087] β-Galactosidase staining method for detecting photoaging of cells: Normal human skin fibroblasts were seeded into 24-cell plates and cultured for 18-24 hours. The cell layer was washed twice with PBS. Maintenance medium containing different concentrations of the test substance and maintenance medium without the sample were added according to the group, with 3 replicates per group, and exposed for 24 hours. The culture medium containing the test substance and the maintenance medium without the sample were removed. After washing with PBS 2-3 times, the cells were covered with a small amount of PBS and irradiated with UVA. After irradiation, the cells were washed twice with PBS. The negative control group and the model control group were replaced with maintenance medium, while the other groups were added with maintenance medium containing the sample or positive control and incubated for 48 hours. The cell culture medium was removed, and the cells were washed once with 1 mL of PBS. 0.5 mL of staining fixative was added and fixed at room temperature for 15 minutes. The cell fixative was removed, and the cells were washed three times with 1 mL of PBS for 3 minutes each time. The PBS was removed, and the staining working solution was prepared according to the instructions. 0.5 mL of staining working solution was added to each well. The cells were sealed and incubated overnight at 37°C.

[0088] Under a regular optical microscope, positive cells appeared dark blue. The number of positive cells in three fields of view was counted for each group, and the positive rate (%) was calculated; the results are shown in Table 3. Figure 7 As shown in the image. (Image of fibroblasts) Figure 8 As shown.

[0089] Table 3. Results of β-galactosidase staining

[0090]

[0091]

[0092] # indicates a statistically significant difference compared to the negative control group (NT) (p<0.05);

[0093] * indicates a statistically significant difference compared to the model control group (M) (p<0.05).

[0094] The experimental results showed that compared with the negative control group (NT), the number of β-galactosidase-positive cells in the model group (M) was significantly increased (p<0.05); compared with the model group, the number of β-galactosidase-positive cells in the positive control group was significantly decreased (p<0.05), indicating successful model establishment. Compared with the model group, the number of β-galactosidase-positive cells at SOD sample concentrations of 0.20% (w / w), 0.10% (w / w), and 0.05% (w / w) was significantly reduced (p<0.05). This demonstrates that SOD sample concentrations of 0.20% (w / w), 0.10% (w / w), and 0.05% (w / w) have significant anti-photoaging effects.

[0095] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0096] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0097] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A highly active recombinant human superoxide dismutase, characterized in that, The highly active recombinant human superoxide dismutase includes: a) The amino acid sequence as shown in SEQ ID No. 1; or b) An amino acid sequence in which one or more amino acid residues are added, substituted, dropped or inserted in the amino acid sequence shown in SEQ ID NO.1, and which retains the collagen activity of the amino acid sequence of SEQ ID NO.

1.

2. A nucleotide, characterized in that, The nucleotide acid encodes the highly active recombinant human superoxide dismutase of claim 1.

3. A nucleotide according to claim 2, characterized in that, The sequence of the nucleotide molecule is shown in SEQ ID NO.

2.

4. A recombinant plasmid, characterized in that, The recombinant plasmid comprises the nucleotides described in claim 2 or 3.

5. The recombinant plasmid according to claim 4, characterized in that, The plasmid vector is rHuSOD-1-pET-30a(+).

6. A host cell for efficient expression, characterized in that, The host cell for efficient expression contains the nucleotides of claim 2 or 3; or the recombinant plasmids of claim 4 or 5.

7. The host cell for high-efficiency expression according to claim 6, characterized in that, The host cell for high-efficiency expression is Escherichia coli BL21(DE3) competent cells.

8. The method for preparing the highly active recombinant human superoxide dismutase according to claim 1, characterized in that, The highly active recombinant human superoxide dismutase was expressed using the host cell with high efficiency of expression as described in claim 6, and then separated and purified to obtain the highly active recombinant human superoxide dismutase.

9. A cell photoaging resistance agent, characterized in that, The cell photoaging resistance agent contains the highly active recombinant human superoxide dismutase of claim 1; or the highly active recombinant human superoxide dismutase encoded by nucleotides of claim 2 or 3; or the highly active recombinant human superoxide dismutase expressed by host cells with high efficiency of expression as described in claim 6.

10. The use of the highly active recombinant human superoxide dismutase of claim 1; or the highly active recombinant human superoxide dismutase encoded by nucleotides of claim 2 or 3; or the highly active recombinant human superoxide dismutase expressed by host cells with high efficiency of expression of claim 6 in food, cosmetic, health product or medical device products that resist cell photoaging.

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