SOD (superoxide dismutase) and CAT (catalase) double-enzyme-activity fusion protein and construction method and application thereof

Abstract

The invention provides an SOD (superoxide dismutase) and CAT (catalase) double-enzyme-activity fusion protein as well as a construction method and application thereof, and belongs to the technical field of mixed enzymes. The invention provides a fusion protein of SOD (superoxide dismutase) enzyme and CAT (catalase) enzyme connected by a connecting peptide. The invention provides a recombinant expression vector and a recombinant expression method for expressing the fusion protein, and also verifies that the fusion protein has relatively strong stability and acid-base tolerance after being expressed, and the fusion protein has relatively high SOD enzyme activity and CAT enzyme activity. The fusion protein provides a new choice for combined application of SOD and CAT, and can be widely applied to food, beauty and biological medicine industries.

Description

Technical Field

[0001] The invention belongs to the technical field of mixed enzymes, and in particular relates to a SOD and CAT dual-enzyme activity fusion protein and a construction method and application thereof. Background Art

[0002] Superoxide dismutase (SOD) is an antioxidant enzyme that carries metal ions. It catalyzes the conversion of harmful superoxide anion free radicals into molecular oxygen and hydrogen peroxide, thereby protecting cells from oxidative damage, preventing oxidative stress, maintaining intracellular redox balance, and regulating signal transduction. According to the different metal ions of SOD, it is mainly divided into copper-zinc SOD (Cu / Zn-SOD), manganese SOD (Mn-SOD) and iron SOD (Fe-SOD). Cu / Zn-SOD is mainly distributed in the form of homodimers in plant cytoplasm and chloroplasts, animal cell nuclei, lysosomes, peroxisomes, cytoplasm and extracellular matrix, and the periplasmic space of prokaryotes. Fe-SOD mostly exists in the form of homodimers or tetramers, mainly distributed in some plant chloroplasts and prokaryotic cytoplasm. Mn-SOD usually exists in the form of homodimers or tetramers in the mitochondria of eukaryotes and the periplasmic space of prokaryotes.

[0003] Catalase (CAT) is an important antioxidant enzyme widely present in organisms, which can convert hydrogen peroxide into water and oxygen. CAT can maintain the redox balance in cells and defend against oxidative stress damage caused by ROS. According to the different structures of the protein catalytic center, CAT can be divided into two categories: (1) CAT with an iron porphyrin structure, usually called iron catalase (FeCAT), more than 85% of the CATs discovered so far belong to this category; (2) CAT with a porphyrin structure that uses manganese ions instead of iron ions, called manganese catalase (MnCAT).

[0004] Since both SOD and CAT are important antioxidant enzymes in the human body, and CAT can convert low-toxic hydrogen peroxide produced by SOD in the process of removing harmful free radicals into harmless water and oxygen, SOD and CAT are often used in combination to achieve the best antioxidant effect. Some studies have co-encapsulated SOD and CAT proteins using nanocarriers to enhance the cascade reaction between SOD and CAT, but this will increase the cost of raw materials accordingly, and the effect of the combination of the two is limited. Summary of the invention

[0005] The present invention provides a SOD and CAT dual enzyme activity fusion protein and a construction method and application thereof. The fusion protein has high stability and higher antioxidant capacity, and provides a potential stable protein drug for antioxidant treatment of related diseases.

[0006] The invention provides a fusion protein, comprising a SOD enzyme and a CAT enzyme connected by a connecting peptide.

[0007] In a specific embodiment of the present invention, the connecting peptide comprises GGGGS or EAAAK.

[0008] In a specific embodiment of the present invention, the SOD enzyme may be Fe-SOD, Cu / ZnSOD and Mn-SOD;

[0009] The CAT enzyme is FeCAT, and may also be MnCAT.

[0010] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is shown as SEQ ID No.1 to SEQ ID No.3.

[0011] The present invention also provides a nucleic acid sequence encoding the fusion protein.

[0012] The present invention also provides a recombinant expression vector comprising the nucleic acid sequence and expressing the fusion protein.

[0013] In a specific embodiment of the present invention, the basic backbone vector of the recombinant expression vector includes a prokaryotic expression vector.

[0014] The present invention also provides a method for expressing the above fusion protein, comprising the following steps: connecting the coding gene of the above fusion protein to a basic skeleton vector to construct a recombinant expression vector;

[0015] Transforming a host cell with the recombinant expression vector to obtain a recombinant expression cell;

[0016] The recombinant expression cells are cultured and the expression of the fusion protein is induced, and the supernatant contains the fusion protein.

[0017] The present invention also provides a SOD and CAT dual enzyme activity fusion protein expressed by the method.

[0018] The present invention also provides the use of the above fusion protein or the above SOD and CAT dual enzyme activity fusion protein in the preparation of drugs for preventing and / or treating oxidation-related diseases.

[0019] The present invention also provides a medicine for preventing and / or treating oxidation-related diseases, wherein the effective ingredients include the above fusion protein or the above SOD and CAT dual-enzyme fusion protein, and also include pharmaceutically acceptable excipients.

[0020] Beneficial effects: The present invention provides a fusion protein of a SOD enzyme and a CAT enzyme connected by a connecting peptide. After expression, the fusion protein has strong stability and tolerance, and the fusion protein has high SOD enzyme activity and CAT enzyme activity. In the embodiment of the present invention, the SOD enzyme of the CAT-G3-AcSOD11 fusion protein has the strongest tolerance to high temperature and acid-base. The SOD still maintains about 80% of its enzyme activity within 120 minutes of high temperature heating at 80°C. At the same time, its SOD enzyme activity remains relatively stable at pH 3 to 10, reaching the highest at pH 9; the CAT enzyme of the CAT-G3-AcSOD11 fusion protein also shows a certain tolerance to high temperature and acid-base. The fusion protein of the present invention provides a new option for the combined application of SOD and CAT, and can be widely used in the food, beauty and biopharmaceutical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a schematic diagram of the construction of the recombinant plasmid expressing the AcSOD11 and FeCAT fusion protein, in which A represents the plasmid map, B represents the linearized plasmid of pET-28a-AcSOD11, C represents the amplified band of CAT, and D represents the sequencing result of AcSOD11-G3-CAT;

[0022] Figure 2 Figure 1 is an SDS-PAGE electrophoresis analysis of AcSOD11 and CAT fusion proteins, in which A represents AcSOD11-G3-CAT; B represents CAT-G3-AcSOD11; and C represents CAT-E3-AcSOD11.

[0023] Figure 3 This is a diagram showing the results of the enzyme activity assay of the fusion protein of the present invention, in which A represents SOD and B represents CAT;

[0024] Figure 4 This is the result of the thermal stability test of the fusion protein, in which A represents SOD and B represents CAT;

[0025] Figure 5 This is a graph showing the results of the pH stability test of the fusion protein, where A represents SOD and B represents CAT. DETAILED DESCRIPTION

[0026] The invention provides a fusion protein, comprising a SOD enzyme and a CAT enzyme connected by a connecting peptide.

[0027] The SOD enzyme described in the present invention includes multiple types, such as Fe-SOD, Cu / ZnSOD and Mn-SOD. In the embodiments, Fe-SOD is used as an example for illustration. In one embodiment, AcSOD11 contained in the Escherichia coli BL21 (DE3) -pET28a-AcSOD11 strain containing iron ion superoxide dismutase (AcSOD11, a fusion protein of the C-terminus of the AcSOD protein fused with a SPIT peptide) is used as an example for illustration, wherein the Escherichia coli BL21 (DE3) -pET28a-AcSOD11 is obtained by transforming the pET28a-AcSOD11 plasmid into Escherichia coli BL21 (DE3) competent cells, and the construction method of the pET28a-AcSOD11 plasmid has been disclosed in Chinese patent CN202411126051.9, and the nucleotide sequence of AcSOD11 in the present invention is shown in SEQ ID No. 4, which is exactly the same as AcSOD-SPIT in CN202411126051.9.

[0028] The CAT enzyme of the present invention includes multiple types, such as FeCAT and MnCAT. FeCAT is used as an example for illustration in the embodiments. For example, the GenBank number of FeCAT in one embodiment is WP_012845258.1.

[0029] The present invention uses a flexible connecting peptide to connect the SOD enzyme and the CAT enzyme, and when the connection is made, the CAT enzyme can be fused to the C-terminus or N-terminus of the SOD enzyme, and the connecting peptide can be a flexible connecting peptide or a rigid connecting peptide, wherein the flexible connecting peptide includes GGGGS, and the rigid connecting peptide includes EAAAK. In one embodiment of the present invention, three fusion proteins are constructed: using (GGGGS)3 to fuse the FeCAT protein to the C-terminus of AcSOD11, named AcSOD11-G3-CAT; using (GGGGS)3 to fuse the FeCAT protein to the N-terminus of AcSOD11, named CAT-G3-AcSOD11; using (EAAAK)3 to fuse the FeCAT protein to the C-terminus of AcSOD11, named CAT-E3-AcSOD11.

[0030] In the examples of the present invention, the enzyme activities of the above three fusion proteins were also determined. The results showed that there was no significant difference in the SOD enzyme activity of the three fusion proteins, but there was a significant difference in the CAT enzyme activity. Among them, CAT-G3-AcSOD11 had the highest CAT enzyme activity (531U / mg CAT enzyme activity), and CAT-E3-AcSOD11 had the worst enzyme activity. The enzyme stability study found that both CAT and SOD of the CAT-G3-AcSOD11 fusion protein showed significant high temperature and acid-base tolerance.

[0031] In a specific embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID No.1 to SEQ ID No.3, wherein the amino acid sequence of the CAT-G3-AcSOD11 fusion protein is shown in SEQ ID No.1; the amino acid sequence of AcSOD11-G3-CAT is shown in SEQ ID No.2; the amino acid sequence of CAT-E3-AcSOD11 is shown in SEQ ID No.3; and the nucleotide sequence of the AcSOD11 is shown in SEQ ID No.4.

[0032] The present invention also provides a nucleic acid sequence molecule encoding the fusion protein.

[0033] The present invention also provides a recombinant expression vector comprising the nucleic acid sequence molecule and expressing the fusion protein.

[0034] In a specific embodiment of the present invention, the basic backbone vector of the recombinant expression vector includes a prokaryotic expression vector. In one embodiment, pET-28a is selected, and the coding genes of the above three fusion proteins are connected between the NcoI and EcoR I sites of pET-28a.

[0035] The present invention also provides a method for expressing the above fusion protein, comprising the following steps: connecting the coding gene of the above fusion protein to a basic skeleton vector to construct a recombinant expression vector;

[0036] Transforming a host cell with the recombinant expression vector to obtain a recombinant expression cell;

[0037] The recombinant expression cells are cultured and the expression of the fusion protein is induced, and the supernatant contains the fusion protein.

[0038] In one embodiment of the present invention, the pET-28a-AcSOD11 plasmid is first extracted from Escherichia coli BL21 (DE3)-pET28a-AcSOD11, and the pET-28a-AcSOD11 plasmid is used as a template, and primers Fe-F and Fe-R are used to perform PCR to obtain a linearized vector band. The pMUT1-MLH plasmid is used as a template, and primers CAT-F and CAT-R are used to perform PCR amplification to obtain the target gene CAT. The present invention uses a flexible connecting peptide (GGGGS) 3 as a connector between the linearized vector band obtained by the above amplification and the CAT gene, and the two PCR products recovered by the gel are seamlessly cloned, transformed into Escherichia coli competent cells, and the positive monoclonal clones are selected and sequenced to obtain the correct plasmid pET-28a-AcSOD11-G3-CAT.

[0039] Then, pET-28a-AcSOD11 was used as a template, and primers XCL-F and XXX-R, XXX-F and XXX-R were used to perform PCR amplification to obtain a linearized vector, and then pET-28a-CAT plasmid was used as a template, and primers CA-F and CL-R, CA-F and CR-R were used to perform PCR amplification to obtain the target gene. Then, nucleic acid electrophoresis was performed, and the rubber recovery kit was used for recovery, seamless cloning, heat shock transformation of Escherichia coli competent cells, and positive monoclonal strains were picked. After sequencing, pET-28a-CAT-G3-AcSOD11 and pET-28a-CAT-E3-AcSOD11 plasmids were obtained. The pET-28a-CAT plasmid of the present invention is a recombinant plasmid obtained by connecting FeCAT (WP_012845258.1) between the Nco I and EcoR I sites of pET-28a.

[0040] Table 1 Primer sequences

[0041]

[0042]

[0043] In one embodiment of the present invention, the fusion protein is produced based on a prokaryotic expression method, specifically comprising transforming the three correctly sequenced plasmids into BL21 (DE3) competent cells to construct three recombinant bacteria: BL21 (DE3) -pET-28a-AcSOD11-G3-CAT, BL21 (DE3) -pET-28a-CAT-G3-AcSOD11 and BL21 (DE3) -pET-28a-CAT-E3-AcSOD11. The present invention uses IPTG to induce the expression of the fusion protein in the recombinant bacteria, and after the bacterial solution is broken, the supernatant is collected by centrifugation, and the supernatant contains the target fusion protein.

[0044] The present invention also provides a SOD and CAT dual enzyme activity fusion protein expressed by the method.

[0045] The SOD and CAT dual enzyme activity fusion protein of the present invention has the enzyme activities of SOD and CAT at the same time, and it has been verified that there is no significant difference in the SOD specific enzyme activity among the three fusion proteins AcSOD11-G3-CAT, CAT-G3-AcSOD11 and CAT-E3-AcSOD11, which are 179.5U / mg, 171.6U / mg and 190.4U / mg respectively; but the specific enzyme activity of the CAT enzyme varies greatly, among which the CAT specific enzyme activity of the CAT-G3-AcSOD11 fusion protein reaches the highest 531U / mg, which is significantly improved compared with the other two fusion proteins.

[0046] The present invention also provides the use of the fusion protein or the SOD and CAT dual-enzyme activity fusion protein in the preparation of an antioxidant agent.

[0047] The SOD and CAT dual enzyme activity fusion protein of the present invention has significant high temperature stability and acid-base tolerance, can be used as a combined application reagent of SOD and CAT, and is widely used in food, beauty and biomedicine industries. The SOD and CAT dual enzyme activity fusion protein of the present invention is added to food as a natural antioxidant to extend the shelf life of food, prevent oil oxidation and food deterioration; the SOD and CAT dual enzyme activity fusion protein is used in the production of health food to improve the antioxidant capacity of the human body and prevent diseases. At the same time, since the SOD and CAT dual enzyme activity fusion protein has the effect of scavenging free radicals, it also has potential applications in anti-aging and beauty products. In the field of medicine, the combined enzyme of SOD and CAT can be used in antioxidant therapy and anti-aging. Therefore, the SOD and CAT dual enzyme activity fusion protein of the present invention can be used to treat diseases caused by oxidative stress, such as cardiovascular disease, diabetes, inflammatory diseases, etc.

[0048] The present invention also provides an antioxidant agent, the active ingredients of which include the above fusion protein or the above SOD and CAT dual enzyme activity fusion protein, and also include auxiliary materials.

[0049] CAT is often used in combination with SOD in the treatment strategy of oxidative stress-related diseases. The main function of SOD is to catalyze the conversion of harmful ROS into molecular oxygen and hydrogen peroxide. At low concentrations, hydrogen peroxide, as an important signaling molecule, participates in a variety of biological processes such as cell proliferation, apoptosis and inflammatory response. However, when the concentration of hydrogen peroxide is high, the Fenton reaction will occur, combining with soluble Fe(II) to generate highly active hydroxyl radicals, leading to damage of proteins, lipids and DNA, and then inducing cell dysfunction and death. At this time, CAT converts hydrogen peroxide into harmless water and oxygen, maintains the redox balance in the cell, and defends against oxidative stress damage caused by ROS. Studies have shown that high doses of SOD may increase the risk of reperfusion injury, and the coupled use of SOD and CAT can effectively reduce the level of free hydrogen peroxide, reduce the occurrence of the Fenton reaction, and thus reduce cell damage. However, there are still some problems in the combination of two separate enzyme proteins, such as the need to extract and purify SOD and CAT proteins separately, which increases the application cost of the two enzymes; at the same time, the synergistic effect of the two separate enzyme proteins is uncertain, and it is difficult to ensure that the two separate enzymes can play a synergistic role at the same time. Studies have shown that the cascade reaction between SOD and CAT can be enhanced by embedding separate SOD and CAT with metal nanomaterials, but the effect of the combination of the two is also limited, and the cost of raw materials will increase accordingly. The construction of fusion protein can obtain SOD and CAT dual enzyme activity protein by one extraction and purification, which greatly reduces the application cost of the two enzymes; at the same time, SOD and CAT dual enzyme activity fusion protein can ensure the synergistic effect of the two antioxidant enzymes and increase the antioxidant effect of the combination of the two.

[0050] To further illustrate the present invention, a SOD and CAT dual enzyme activity fusion protein provided by the present invention, as well as its construction method and application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Unless otherwise specified, the materials and reagents used in the embodiments of the present invention are conventional commercial preparations in the art: Escherichia coli BL21 (DE3)-pET28a-AcSOD11 strains containing iron superoxide dismutase (AcSOD11 or AcSOD-SPIT) were obtained by transforming Escherichia coli BL21 (DE3) competent cells with pET28a-AcSOD11 plasmids and deposited in the Biochemical Molecular Laboratory of Zhejiang University of Technology. The construction method of the pET28a-AcSOD11 plasmid has been disclosed in Chinese patent CN202411126051.9; the pMUT1-MLH plasmid was constructed and preserved in this laboratory, and the construction method was as follows: first, the pMUT1 cryptic plasmid was extracted from wild-type EcN cells, and linearized and amplified by primers pMUT1-F / pMUT1-R. Then, the RtSOD-KanR fragment was amplified using the plasmid pGEX-4T-1-M-RtSOD as a template by using primers pMUT1-RtSOD-F / pMUT1-RtSOD-R. The pGEX-4T-1-M-RtSOD plasmid was synthesized by a biological company and the RtSOD gene (WP_012843426.1) was inserted into the GTATTC site downstream of the pGEX-4T-1 plasmid operon, and obtained by mutating the operon. The linearized pMUT1 and the RtSOD-KanR fragment were connected by non-cloning technology to obtain the recombinant plasmid pMUT1-RtSOD. The plasmid pMUT1-RtSOD was further linearized and amplified by the PCR method using primers pMRtSOD-XF and pMRtSOD-XR. The pMRtSOD-XR primer introduced the flexible linker peptide (GGGGS) 3 coding sequence. At the same time, the CAT gene fragment was amplified by PCR using primers L-FeCAT-F and L-FeCAT-R with plasmid pET28a-CAT as template. The linearized pMUT1-RtSOD was connected to the CAT fragment by seamless cloning technology to obtain the recombinant plasmid pMUT1-MLH; the open reading frame (ORF) of FeCAT (GenBank No.: WP_012845258.1) was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. and then cloned into the plasmid to obtain the recombinant plasmid pET28a-CAT; primer synthesis came from Hangzhou Youkang Biological Company; BCA protein concentration determination kit came from Shanghai Biyuntian Biotechnology Co., Ltd.; hydrogen peroxide (CAT) activity detection kit came from Beijing Solebow Technology Co., Ltd.; FastPure Plasmid Mini Kit came from Nanjing Novogene Biotechnology Co., Ltd.; kanamycin sulfate came from Sangon Biotechnology (Shanghai) Co., Ltd.; ultrasonic disruptor (Fangru Technology (Shanghai) Co., Ltd.); NannoDrop 1000 spectrophotometer (Thermo); vertical electrophoresis instrument (Bio-Rad), etc.

[0052] The present invention uses GraphPadPrism software (version 8.0) for statistical analysis and graphical representation. The differences between groups were evaluated by t test or one-way ANOVA (n≥3). In the statistical significance analysis, p<0.05 indicates a significant difference, p<0.01 indicates a very significant difference, and p<0.001 indicates an extremely significant difference, respectively marked with *p<0.05, **p<0.01, ***p<0.001, and each row mean is with SEM.

[0053] Example 1

[0054] 1. Design of AcSOD11 and FeCAT fusion protein and construction of its recombinant expression vector

[0055] According to the three-dimensional structure of AcSOD11 protein and FeCAT protein, the present invention uses (GGGGS)3 or (EAAAK)3 to fuse FeCAT to the C-terminus or N-terminus of AcSOD11, and designs three recombinant fusion proteins CAT-G3-AcSOD11 (SEQ ID No.1), AcSOD11-G3-CAT (SEQ ID No.2) and CAT-E3-AcSOD11 (SEQ ID No.3) ( Figure 1 (A).

[0056] First, the pET-28a-AcSOD11 plasmid was extracted from Escherichia coli BL21 (DE3)-pET28a-AcSOD11 using a plasmid extraction kit. The pET-28a-AcSOD11 was used as a template. The template was linearized by PCR using primers Fe-F and Fe-R in Table 1. Figure 1 Using pMUT1-MLH as a template, primers CAT-F and CAT-R were used to amplify the target gene CAT with the upstream (GGGGS)3 coding sequence ( Figure 1 C). A flexible peptide (GGGGS)3 was used as a connector between the AcSOD11 and CAT genes. Then the gel recovery kit was used for recovery, and the PCR product was used for seamless cloning and heat shock method for transformation of E. coli DH5α competent cells. After spot picking, it was expanded to 5mL culture medium at 37℃, 220rpm, cultured for 12h, and then sent to Youkang Biological Company for sequencing, and the recombinant bacteria containing the correct plasmid pET-28a-AcSOD11-G3-CAT were obtained, named DH5α-pET-28a-AcSOD11-G3-CAT ( Figure 1 (middle D).

[0057] Then, pET-28a-AcSOD11 was used as a template, and primers XCL-F and XXX-R, XXX-F and XXX-R were used to perform PCR to obtain a linearized vector. Then, pET-28a-CAT was used as a template, and primers CA-F and CL-R, CA-F and CR-R were used to perform PCR amplification to obtain the target gene. Then, nucleic acid electrophoresis was performed, the gel recovery kit was used for recovery, seamless cloning was performed, and heat shock transformation was performed into DH5α. Positive monoclonal strains were selected and sent to Youkang Biological Company for sequencing. Recombinant bacteria containing the correct plasmids pET-28a-CAT-G3-AcSOD11 and pET-28a-CAT-E3-AcSOD11 were obtained, respectively, and named DH5α-pET-28a-CAT-G3-AcSOD11 and DH5α-pET-28a-CAT-E3-AcSOD11, respectively.

[0058] 2. Prokaryotic expression of the fusion protein of AcSOD11 and FeCAT

[0059] Take 5mL of DH5α-pET-28a-AcSOD11-G3-CAT, DH5α-pET-28a-CAT-G3-AcSOD11 and DH5α-pET-28a-CAT-E3-AcSOD11 cultured for 12 hours, and extract the corresponding plasmids using a plasmid extraction kit. Take out 100μL of BL21 (DE3) competent cells from the refrigerator and put them in an ice box to thaw. After thawing, take 10μL of the above extracted plasmids and add them to the competent cells, incubate on ice for 30min, turn on the 42℃ water bath, and heat shock at 42℃ for 90S. Quickly add 900μL LB culture medium in the clean bench and culture it in a shaker at 37℃, 220rpm for 1h. Take out the prepared Kan-resistant plate from the refrigerator, centrifuge the cultured centrifuge tube at 12000rpm for 2min, remove 900μL of supernatant, suspend the remaining solution and apply it to the plate, culture at 37℃ for 12h, pick a single colony and put it into 5mL LB medium with Kan resistance and culture at 37℃ for 12h to obtain the corresponding BL21(DE3) strain.

[0060] The BL21(DE3)-pET-28a-AcSOD11-G3-CAT, BL21(DE3)-pET-28a-CAT-G3-AcSOD11 and BL21(DE3)-pET-28a-CAT-E3-AcSOD11 strains obtained above were inoculated into 5 mL LB medium, cultured in a shaker at 37°C, 220 rpm for 6 h, and then 5 μL 1MIPTG was added, and induced culture was carried out in a shaker at 37°C, 220 rpm for 12 h. The bacterial liquid was collected into centrifuge tubes and resuspended with 500 μL 1×PBS. The centrifuge tubes were placed on ice and placed in an ultrasonic crusher. The ultrasonic conditions were crushing for 3s, pausing for 5s, 90% power, and crushing for 2min. After the ultrasonication was complete, the bacterial liquid changed from turbid to clear. Subsequently, the supernatant was taken by centrifugation at 12000 rpm for 3 min, and the corresponding protein solution was obtained by filtering with a 0.45 nm filter membrane. The induced products were made into uninduced whole bacteria, induced whole bacteria, induced broken supernatant and induced broken precipitate for SDS-PAGE protein electrophoresis.

[0061] The results are as follows Figure 2 As shown, the IPTG-induced sample showed a specific target protein band, indicating that the molecular weight of the three fusion proteins AcSOD11-G3-CAT, CAT-G3-AcSOD11 and CAT-E3-AcSOD11 was 93.9KDa, and the expression was successful, and there was no significant difference in the expression amount. Further ultrasonic fragmentation experiments were performed to identify the protein expression pattern, and the results showed that the three fusion proteins were all soluble.

[0062] 3. Comparison of enzyme activities of AcSOD11 and FeCAT fusion proteins

[0063] Take 50 μL of recombinant bacteria BL21(DE3)-pET-28a-AcSOD11-G3-CAT, BL21(DE3)-pET-28a-CAT-G3-AcSOD11 and BL21(DE3)-pET-28a-CAT-E3-AcSOD11 and add them to 5 mL LB medium. Put them in a shaker at 37°C, 220 rpm and culture for 6 hours. Add 5 μL 1M IPTG to induce for 12 hours. Centrifuge at 12000 rpm for 3 minutes, resuspend and wash once with 500 μL 1×PBS, and resuspend with 500 μL 1×PBS. Put the centrifuge tube on ice, put it in an ultrasonic crusher, and ultrasonically crush it. The SOD and CAT enzyme activities of the corresponding proteins were determined by the pyrogallol self-oxidation method (Chinese national standard: GBT5009.171) and the hydrogen peroxide activity detection kit, and compared. The BCA protein concentration assay kit was then used to measure the supernatant protein concentrations of AcSOD11-G3-CAT, CAT-G3-AcSOD11 and CAT-E3-AcSOD11, respectively. The SOD or CAT specific enzyme activity (U / mg) = SOD or CAT enzyme activity (U / mL) / protein concentration (mg / mL) was then used to calculate the SOD or CAT specific enzyme activity of the fusion protein.

[0064] The results are as follows Figure 3 As shown, there is no significant difference in the SOD activity of the three fusion proteins, which are 179.5U / mg, 171.6U / mg and 190.4U / mg respectively. However, the CAT activity of the three fusion proteins varies greatly, among which the CAT activity of the CAT-G3-AcSOD11 fusion protein is the highest, reaching 531U / mg, the CAT activity of the AcSOD11-G3-CAT fusion protein is in the middle, at 235U / mg, and the CAT activity of the CAT-E3-AcSOD11 fusion protein is the lowest, at 86U / mg.

[0065] 4. Heat resistance test of AcSOD11 and FeCAT fusion protein

[0066] Take three kinds of SOD and CAT fusion protein crude enzyme solutions, divide them into 21 centrifuge tubes containing 100 μL crude enzyme solutions, heat them in a 80℃ metal bath for 20, 40, 60, 80, 100, and 120 min, take samples every 20 min, and use pyrogallol auto-oxidation method and hydrogen peroxide activity detection kit to determine SOD enzyme activity or CAT enzyme activity at each time point. There are 3 parallel samples in each group. The unheated SOD or CAT enzyme activity is taken as 100%, and the relative SOD enzyme and CAT enzyme activity at different heating temperatures of 80℃ are calculated.

[0067] The results of SOD activity of CAT-G3-AcSOD11 fusion protein heated at 80℃ for 120min are as follows Figure 4 As shown in A, it remains relatively stable and still retains about 80% of the enzyme activity, showing high heat resistance. However, when AcSOD11-G3-CAT and CAT-E3-AcSOD11 fusion proteins were heated at 80°C for 20 minutes, the SOD activity rapidly dropped to about 50%, and then slowly decreased, showing low heat resistance. The different order of connecting AcSOD11 and CAT by flexible peptide (GGGGS) 3 has a great influence on the heat resistance of SOD enzyme activity. Compared with AcSOD11-G3-CAT, the thermal stability of CAT-G3-AcSOD11 enzyme activity is greatly improved.

[0068] The results of CAT enzyme activity and thermal stability test are as follows Figure 4 As shown in B, after the AcSOD11-G3-CAT, CAT-E3-AcSOD11 and CAT-G3-AcSOD11 fusion proteins were heated at 80°C for 20 min, the CAT activity rapidly decreased to about 50%, and then slowly decreased, indicating that the heat resistance of the CAT enzyme activity of the three fusion proteins was not much different.

[0069] Among the three fusion proteins, the SOD activity of CAT-G3-AcSOD11 protein is relatively stable in heat resistance, and the CAT activity of CAT-E3-AcSOD11 protein decreases the slowest in heat resistance. In summary, considering the overall heat resistance of SOD and CAT activity, the CAT-G3-AcSOD11 fusion protein has the strongest tolerance to high temperature and is an ideal SOD and CAT dual-enzyme activity high heat stability protein.

[0070] 5. Acid and alkali resistance test of AcSOD11 and FeCAT fusion protein

[0071] Take three kinds of crude enzyme solutions of SOD and CAT fusion proteins and divide them into 33 centrifuge tubes containing 100 μL crude enzyme solution. Dilute the crude enzyme solution 10 times with a series of pH buffers (pH 2-12), heat it in a 37℃ metal bath for 30 minutes, centrifuge it, and take the supernatant for SOD and CAT enzyme activity determination. There are 3 parallel samples in each group. Taking the SOD and CAT enzyme activities at pH 7 as 100%, calculate the relative SOD and CAT enzyme activities after treatment with different pH buffers.

[0072] AcSOD11-G3-CAT has SOD activity in the range of pH 2 to 12. Figure 5As shown in A, the SOD activity of CAT-G3-AcSOD11 remains relatively stable within pH 3 to 10, remaining above 90%, showing a high acid-base tolerance. The SOD activity of CAT-E3-AcSOD11 remains relatively stable within pH 4 to 9, but the SOD activity decreases significantly under extreme acid-base conditions, showing a certain acid-base tolerance. The results of the acid-base stability test of CAT enzyme activity are shown in Figure 5 As shown in B, AcSOD11-G3-CAT has the highest CAT activity at pH 7, and the CAT activity rapidly decreases to about 10% under higher acid and alkaline conditions, showing low acid and alkaline tolerance. CAT-E3-AcSOD11 and CAT-G3-AcSOD11 have the highest CAT activity at pH 6, and can still maintain about 50% of the CAT activity under strong acid conditions, while the CAT activity slowly decreases to 20% with the stronger alkalinity within pH 6-12.

[0073] The above results show that among the three fusion proteins, the acid-base resistance of the SOD enzyme of the CAT-G3-AcSOD11 protein is the most stable, and the acid-base resistance of the CAT enzyme of the CAT-G3-AcSOD11 and CAT-E3-AcSOD11 fusion proteins decreases the slowest. In summary, considering the acid-base resistance of the SOD enzyme and the CAT enzyme as a whole, the CAT-G3-AcSOD11 fusion protein has the strongest acid-base tolerance and is an ideal SOD and CAT dual enzyme activity acid-base stable protein.

[0074] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that It includes SOD enzyme and CAT enzyme connected by connecting peptide.

2. The fusion protein according to claim 1, characterized in that The connecting peptide includes GGGGS or EAAAK.

3. The fusion protein according to claim 1, characterized in that The SOD enzymes include Fe-SOD, Cu / ZnSOD and Mn-SOD; the CAT enzymes include FeCAT and MnCAT.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID No.1 to SEQ ID No.

3.

5. A nucleic acid sequence encoding the fusion protein according to any one of claims 1 to 4.

6. A recombinant expression vector comprising the nucleic acid sequence of claim 5 and expressing the fusion protein of any one of claims 1 to 4.

7. A method for expressing the fusion protein according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: connecting the editing gene of the fusion protein according to any one of claims 1 to 4 to a basic backbone vector to construct a recombinant expression vector; Transforming a host cell with the recombinant expression vector to obtain a recombinant expression cell; The recombinant expression cells are cultured and the expression of the fusion protein is induced, and the supernatant contains the fusion protein.

8. The SOD and CAT dual enzyme activity fusion protein expressed by the method of claim 7.

9. Use of the fusion protein according to any one of claims 1 to 4 or the SOD and CAT dual enzyme activity fusion protein according to claim 8 in the preparation of an antioxidant agent.

10. An antioxidant agent, characterized in that The active ingredients include the fusion protein according to any one of claims 1 to 4 or the SOD and CAT dual enzyme activity fusion protein according to claim 8, and also include auxiliary materials.

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