High-purity human SOD (superoxide dismutase) as well as preparation method and application thereof

Abstract

The invention provides high-purity human SOD (superoxide dismutase) as well as a preparation method and application thereof, and the amino acid sequence of the human SOD is SEQ ID NO: 1. The human-derived SOD provided by the invention has the advantages of good stability, good biological safety, uniform molecular weight, high purity, no virus transmission hidden danger, low rejection reaction and the like, is a safe and effective biomedical material, is high in biological activity, can be effectively, stably and massively expressed, and has a good application prospect. The method can be widely applied to the fields of medical instruments, cosmetics, food, biomedical materials and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical products, and specifically relates to a high-purity human SOD and a preparation method and use thereof. Background Art

[0002] Superoxide dismutase (SOD) is a class of enzymes commonly found in animals, plants, and microorganisms. It is the first line of defense of the biological antioxidant system and the only antioxidant enzyme in the body that can specifically remove oxygen free radicals. Under the action of various enzymes, exogenous compounds in the human body will produce oxygen free radicals, which will be converted into hydrogen peroxide under the catalytic action of SOD. The catalase (CAT) and peroxidase (POD) in the body will immediately decompose it into completely harmless water. When the organism does not have enough SOD, reactive oxygen will accumulate in large quantities. If it cannot be removed in time, it will cause oxidative damage, leading to damage to the body, such as base mutations, DNA chain breaks, membrane lipid peroxidation, and protein damage, thereby accelerating aging or causing disease.

[0003] Superoxide dismutase (SOD) has an important antioxidant effect and is an important raw material for the production of medical devices, cosmetics, food, and pharmaceutical industries. The medical devices, cosmetics, food, and pharmaceutical industries have a very high demand for highly efficient, highly active, and easily absorbed superoxide dismutase. Animal or plant-derived SOD has immunogenicity problems for the human body and is difficult to absorb.

[0004] Human SOD prepared by recombinant expression has the advantages of single molecular weight, high purity, and no risk of viral transmission. Therefore, it has begun to be used to replace animal and plant SOD in medical devices, cosmetics, food, biomedical materials and other fields. Therefore, recombinant human SOD has great room for development in future market applications.

[0005] Recombinant human SOD is constructed by using genetic engineering technology to express engineered bacteria containing human SOD genes, and then synthesize SOD through engineered bacteria, and finally use a specific purification process to obtain high-purity recombinant SOD. Human SOD has no hidden dangers of viruses and low immune rejection reactions. It not only retains the original efficacy of SOD, but also gives it new functions, such as processability, water solubility, quality controllability, etc.

[0006] In the field of human biological research, Escherichia coli is the first microorganism to achieve in vitro expression of foreign genes in prokaryotes. Due to the simplicity of Escherichia coli gene manipulation, this expression system has been continuously developed in the direction of protein expression strains. The process of Escherichia coli expressing heterologous proteins is orderly, and the steps are divided into sequence analysis, expression strategy and cloning method, and finally form Escherichia coli expression clones. In order to obtain recombinant strains with high expression efficiency, the construction of expression vectors is the key to success. Escherichia coli BL21 (DE3) and pET expression vectors containing T7 promoters, which are widely used for heterologous protein expression in Escherichia coli, are the most representative recombinant protein expression systems. The advantage of pET expression vectors is that when inducing protein expression, nutrients and inducers are fully utilized and the yield is high. Escherichia coli BL21 (DE3) removes Lon protease and OmpT exoproteinase, which can effectively prevent the expression product from being degraded by the host itself, maintain a high expression level of the target protein, and inhibit the expression of host proteins. The pET expression vector with T7 promoter can make the cloned gene express independently and efficiently. The selected pET-S1 is a commonly used T7 promoter plasmid vector. The advantages of this recombinant protein expression system are low production cost, high production rate, simple operation, clear genetic background, etc. Therefore, the use of genetic engineering technology to construct genetically engineered strains is of great significance for the preparation of high-quality human SOD. Summary of the invention

[0007] The purpose of the present invention is to provide a high-purity human SOD and a preparation method and use thereof, wherein SOD is recombined into human SOD through genetic engineering technology, has no virus hidden dangers and low immune rejection reaction, and can be effectively and stably expressed in large quantities.

[0008] The present invention first provides a human superoxide dismutase SOD, whose amino acid sequence is as follows: MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ (SEQ ID NO: 1).

[0009] The present invention also provides a nucleic acid fragment encoding the human SOD, and the specific sequence of the nucleic acid fragment is as follows (SEQ ID NO: 2):

[0010] ATGGCAACCAAAGCAGTGTGCGTGCTGAAAGGTGACGGCCCGGTTCAGGGCATTATTAATTTTGAACAGAAAGAAAGCAACGGTCCGGTGAAAGTGTGGGGCAGTATTAAGGCCTGACCGAAGGTCTGCATGGCTTTCATGTTCATGAATTTGGTGACAATACCGCAGGTTGCACCAGCGCCGGCCCGCATTTTAATCCGCTGAGCCGTAAACATGGTGGTCCGAAAGATGA AGAACGTCATGTGGGCGATCTGGGTAATGTTACCGCAGATAAAGATGGTGTTGCAGATGTTAGCATTGAAGATAGTGTTATTAGTCTGAGCGGCGATCATTGTATTATTGGCCGTACCCTGGTTGTTCATGAAAAAGCAGATGATCTGGGCAAAGGCGGTAATGAAGAAAGCACCAAAACCGGTAATGCCGGTAGCCGTCTGGCCTGTGGCGTGATTGGCATTGCACAGTAA.

[0011] The present invention also provides a recombinant expression vector, into which a nucleic acid fragment for recombinantly expressing the human SOD is inserted.

[0012] The vector, as specifically described in the examples, is a pet-S1 expression vector;

[0013] In another aspect, the present invention provides a recombinant engineering strain, wherein the recombinant engineering strain carries the recombinant expression vector.

[0014] The recombinant engineering strain, as specifically described in the examples, is the Escherichia coli BL21 (DE3) strain.

[0015] The recombinant human SOD provided by the present invention is used for preparing products for repairing oxidative damage.

[0016] The present invention also provides a product for repairing oxidative damage, which contains the human SOD in a pharmacologically effective concentration.

[0017] The human SOD provided by the present invention has the advantages of good stability, good biosafety, uniform molecular weight, high purity, no hidden danger of virus transmission and low rejection reaction. It is a safe and effective biomedical material with high biological activity, can be effectively and stably expressed in large quantities, and can be widely used in medical devices, cosmetics, food, biomedical materials and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : pet-S1 vector map;

[0019] Figure 2 :HuSOD protein expression diagram;

[0020] Figure 3 : SDS-PAGE electrophoresis detection results, lanes 1-10 are the supernatant and precipitate with ammonium sulfate concentrations of 20%, 25%, 30%, 35%, and 40% respectively;

[0021] Figure 4 : SDS-PAGE electrophoresis detection results, lanes 11-19 are samples, supernatants and precipitates with ammonium sulfate concentrations of 45%, 50%, 55%, and 60% respectively;

[0022] Figure 5 : SDS-PAGE electrophoresis detection results, lanes 20-28 are samples, supernatants and precipitates with ammonium sulfate concentrations of 65%, 70%, 75%, and 80% respectively;

[0023] Figure 6 :The effect of recombinant human SOD on cell activity;

[0024] Figure 7 :Chart of recombinant human SOD oxidative damage repair performance;

[0025] Figure 8 :Expression of recombinant human SOD pro-inflammatory factors. DETAILED DESCRIPTION

[0026] The present invention will be further described in the following embodiments, but the embodiments are only used to illustrate the present invention rather than to limit the present invention.

[0027] Example 1: Design and recombinant expression of recombinant human SOD

[0028] The amino acid sequence of the recombinant human SOD is shown in SEQ ID NO.1: MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ (SEQ ID NO.1).

[0029] The nucleotide sequence of recombinant human SOD is shown in SEQ ID Shown in NO.2: ATGGCAACCAAAGCAGTGTGCGTGCTGAAAGGTGACGGCCCGGTTCAGGGCATTATTAATTTTGAACAGAAAGAAAGCAACGGTCCGGTGAAAGTGTGGGGCAGTATTAAGGGCCTGACCGAAGGTCTGCATGGCTTTCATGTTCATGAATTTGGTGACAATACCGCAGGTTGCACCAGCGCCGGCCCGCATTTTAATCCGCTGAGCCGTAAACATGGTGGTCCGAAAGAT GAAGAACGTCATGTGGGCGATCTGGGTAATGTTACCGCAGATAAAGATGGTGTTGCAGATGTTAGCATTGAAGATAGTGTTATTAGTCTGAGCGGCGATCATTGTATTATTGGCCGTACCCTGGTTGTTCATGAAAAAGCAGATGATCTGGGCAAAGGCGGTAATGAAGAAAGCACCAAAACCGGTAATGCCGGTAGCCGTCTGGCCTGTGGCGTGATTGGCATTGCACAGTAA (SEQ ID NO.2).

[0030] Construction of genetically engineered Escherichia coli expressing recombinant human SOD: The coding gene of the above-mentioned recombinant human SOD (SEQ ID NO.2) was synthesized, and the target fragment and the vector were double-digested and purified before cloning into the pET-S1 vector. After sequencing verification, the plasmid was extracted and transformed into the competent Escherichia coli BL21 (DE3), and positive recombinants were obtained after screening with 50ug / ml kanamycin.

[0031] Example 2: Expression and identification of recombinant human SOD in genetically engineered Escherichia coli

[0032] 1. Expression and identification of recombinant bacteria

[0033] Pick a single clone from the transformation plate, inoculate it into 5 ml LB medium, culture it at 37°C, 250 rpm overnight, transfer it at 1:100, induce it in a shake flask at 30°C for 16-18 hours, add copper and zinc and continue to culture it for 2 hours, collect the wet cells by centrifugation, resuspend the wet cells with PBS buffer; repeat ultrasonic disruption 3 times (ultrasonication for 5 seconds, stop for 8 seconds, cycle for 10 minutes); heat denaturation treatment at 65°C for 10 minutes; centrifuge at 10000 rpm / min for 20 minutes, and collect the supernatant. At this time, the solution contains a large amount of target human SOD protein, which can be subjected to SDS-PAGE electrophoresis. Figure 2 As shown, the target band is visible, and the recombinant human SOD is expressed in a soluble manner.

[0034] 2. Purification of Cu and Zn-SOD

[0035] 1) Ammonium sulfate graded precipitation

[0036] Weigh 383.5g of ammonium sulfate powder into a 1L beaker, add 500mL of water, heat in a microwave oven to fully dissolve the ammonium sulfate, and filter with a single layer of filter paper. The next day, ammonium sulfate crystals will precipitate. Before use, adjust the pH of the saturated ammonium sulfate solution to 7.0-7.5 with ammonia water.

[0037] Take a certain amount of sample and add ammonium sulfate to make the ammonium sulfate saturation reach 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% and 80%.

[0038] After mixing evenly, let it stand at room temperature for 2-5 hours, centrifuge at 12,000rpm and 4℃ for 20 minutes, and take the supernatant and precipitate for SDS-PAGE electrophoresis. The sample preparation method is 80μL sample + 20μL Loading Buffer, boil the sample at 100℃ for 10 minutes, take the supernatant and precipitate after centrifugation, and load them separately, with a sample volume of 10μL. The electrophoresis results are as follows Figure 3-5 .

[0039] Example 3: Evaluation of cytotoxicity of recombinant human SOD

[0040] Skin fibroblasts in good culture state were placed in different concentrations of recombinant human SOD solution prepared in 10% serum culture medium as the experimental group, 10% serum culture medium as the blank control group, and 10% zinc diethyldithiocarbamate (ZDEC) solution prepared in 10% serum culture medium as the positive control group. Each group was set up with 6 replicates, and 100 μL of recombinant human SOD solution of different concentrations, blank control culture medium, and ZDEC solution were added to each well and incubated at 37°C for 24 hours. MTT was used to detect cell activity. Figure 6 It can be seen that recombinant human SOD has no cytotoxicity.

[0041] Example 5: Oxidative damage repair performance of recombinant human SOD

[0042] Skin fibroblasts cultured in good condition were placed in H2O2 solution to establish an oxidative damage model. The oxidative solution was discarded and recombinant human SOD solution of different concentrations was added. Sterile water for injection was used as a blank control and 10% vitamin C was used as a positive control. Six replicates were set up in each group. The cells were incubated at 37°C for 24 hours, and the relative cell proliferation rate was detected by the MTT method.

[0043] Figure 7 The results showed that recombinant human SOD had a good effect in repairing oxidative damage.

[0044] Example 6: Detection of expression of pro-inflammatory factors of recombinant human SOD

[0045] Skin fibroblasts in good culture condition were placed in different concentrations of recombinant human SOD solution prepared in 10% serum culture medium as the experimental group, 10% serum culture medium as the blank control group, and 10% wild SOD solution prepared in 10% serum culture medium as the positive control group. Each group was set up with 6 replicates, and 100 μL of recombinant human SOD solution of different concentrations, blank control culture medium and wild SOD solution were added to each well. The cells were incubated at 37°C for 24 hours, and the expression of pro-inflammatory factors IL-6, TNF-α and IFN-γ in each group of cells was detected by ELISA. Figure 8 It can be seen that there is no significant difference in pro-inflammatory factors between the human SOD at each concentration and the blank group, which confirms that human SOD does not cause inflammatory response.

[0046] In summary, the human SOD provided by the present invention has good stability, no disease risk caused by mutation, will not cause immune rejection when applied to the human body, and has good biosafety; compared with SOD from animal sources, it has the advantages of uniform molecular weight, high purity, no hidden danger of virus transmission and low rejection reaction, etc. It is a safe and effective biomedical material with high biological activity, and is widely used in medical devices, cosmetics, food, biomedical materials and other fields.

Claims

1. A human superoxide dismutase SOD, characterized in that: The amino acid sequence of the human superoxide dismutase SOD is SEQ ID NO:

1.

2. A gene, characterized in that The gene encodes the human superoxide dismutase SOD described in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence of the gene is SEQ ID NO:

2.

4. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with a nucleic acid fragment for recombinantly expressing the human superoxide dismutase SOD described in claim 1.

5. The recombinant expression vector according to claim 4, characterized in that The recombinant expression vector is a pet-S1 expression vector.

6. A recombinant engineering strain, characterized in that: The recombinant engineering strain carries the recombinant expression vector according to claim 3.

7. The recombinant engineered strain according to claim 6, characterized in that The recombinant engineering strain is the Escherichia coli BL21 (DE3) strain.

8. Use of the human superoxide dismutase SOD according to claim 1 in the preparation of products for repairing oxidative damage.

9. A product for repairing oxidative damage, characterized in that: The product for repairing oxidative damage contains the human superoxide dismutase SOD according to claim 1 at a pharmacologically effective concentration.

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