Human-derived superoxide dismutase-catalase fusion protein as well as preparation method and application thereof

Abstract

The invention discloses a human-derived superoxide dismutase-catalase fusion protein as well as a preparation method and application thereof. The amino acid sequence of the fusion protein sequentially comprises superoxide dismutase, connecting peptide and catalase from an N terminal to a C terminal, the connecting peptide comprises 1 to 5 repetitive units, and the amino acid sequence of each repetitive unit is Glu-Ala-Ala-Ala-Lys. The in-vivo half-life period of the superoxide dismutase-catalase fusion protein is greatly prolonged compared with that of monomers of two enzymes, and the superoxide dismutase-catalase fusion protein has good performance in enzyme activity level and protein expression level and is more suitable for clinical medicine.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a human superoxide dismutase-catalase fusion protein and a preparation method and application thereof. Background Art

[0002] Superoxide dismutase (SOD), a copper-containing protein first isolated from bovine blood, has the biological activity of catalyzing the dismutation reaction of superoxide anions to generate oxygen and hydrogen peroxide. Clinically, it is primarily used to alleviate local inflammation, particularly in the treatment of rheumatoid arthritis, chronic polyarthritis, and inflammation following radiotherapy. SOD has a significant protective effect on the white blood cells of patients undergoing radiotherapy and chemotherapy, effectively maintaining white blood cell count and thus accelerating the treatment process. In addition to the aforementioned medical applications, another major application of SOD is in cosmetics and skin care. SOD has the biological activity of scavenging free radicals in the human body, inhibiting the damage of oxygen free radicals to skin connective tissue, protecting collagen in skin tissue from attack by oxygen free radicals, enhancing skin elasticity and reducing wrinkles, and reducing the production of lipofuscin on the skin surface and pigmentation.

[0003] Catalase (CAT) is an enzyme containing an iron-porphyrin structure, widely present in the human body and in plant and animal cells. As its name suggests, catalase's primary biological activity is the decomposition of hydrogen peroxide into water and oxygen. Together with superoxide dismutase (SOD), catalase forms the cellular antioxidant system, clearing the accumulation of reactive oxygen species (superoxide anions and hydrogen peroxide) produced during metabolism, thereby significantly enhancing the body's antioxidant and anti-aging capabilities.

[0004] When organisms are exposed to environmental stimuli or disease, they produce large amounts of reactive oxygen species (ROS), causing damage. CAT, along with superoxide dismutase (SOD), forms an oxidative defense system to scavenge excess ROS, counteracting oxidative stress and maintaining body homeostasis. SOD plays the primary role in ROS scavenging, catalyzing the rapid dismutation of superoxide anions (a type of ROS) into hydrogen peroxide. CAT then decomposes excess hydrogen peroxide (a type of ROS) in the body into water and oxygen. Although SOD and CAT are widely used in clinical medicine, daily chemicals, and food, in practice, their inherent protein properties mean they are rapidly metabolized in the body, resulting in extremely short half-lives in their natural state. Studies have explored the use of phospholipid liposomes to encapsulate SOD to extend its in vivo half-life, but liposome preparation is expensive and may compromise SOD's bioavailability. Summary of the Invention

[0005] The present invention aims to address at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a fusion protein that has a significantly improved in vivo half-life compared to the monomers of SOD and CAT, and exhibits good performance in both enzyme activity and protein expression levels.

[0006] The present invention also provides a biomaterial.

[0007] The present invention also provides a method for preparing the fusion protein.

[0008] The present invention also provides applications of the above fusion protein, biological material or preparation method.

[0009] The present invention also provides a product.

[0010] According to the first aspect of the present invention, a fusion protein is proposed, the amino acid sequence of which includes, from N-terminus to C-terminus, SOD, a connecting peptide and CAT; the connecting peptide includes 1 to 5 repeating units, and the amino acid sequence of the repeating unit is Glu-Ala-Ala-Ala-Lys.

[0011] In some embodiments of the present invention, the amino acid sequence of the SOD is shown in SEQ ID NO: 2.

[0012] In some embodiments of the present invention, the amino acid sequence of CAT is shown in SEQ ID NO: 3.

[0013] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:4.

[0014] In some embodiments of the present invention, the molecular weight of the fusion protein is 70-80 kDa.

[0015] In some preferred embodiments of the present invention, the molecular weight of the fusion protein is about 75 kDa.

[0016] According to a second aspect of the present invention, a biomaterial is provided, wherein the biomaterial is any one of the following (1) to (8):

[0017] (1) a nucleic acid molecule encoding the fusion protein according to the first aspect of the present invention;

[0018] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0019] (3) a recombinant vector containing the nucleic acid molecule described in (1);

[0020] (4) a recombinant vector containing the expression cassette described in (2);

[0021] (5) A transgenic cell line containing the nucleic acid molecule described in (1);

[0022] (6) a transgenic cell line containing the expression cassette described in (2);

[0023] (7) A transgenic cell line containing the recombinant vector described in (3);

[0024] (8) A transgenic cell line containing the recombinant vector described in (4).

[0025] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:5.

[0026] In some preferred embodiments of the present invention, the primer set for amplifying the nucleic acid molecule encoding the nucleic acid molecule includes an upstream primer sequence as shown in SEQ ID NO: 7 and a downstream primer sequence as shown in SEQ ID NO: 8.

[0027] In some embodiments of the present invention, the promoter in the expression cassette comprises AOX1.

[0028] In some embodiments of the present invention, the recombinant vector comprises pPICZαA.

[0029] In some embodiments of the present invention, the recombinant microorganism includes Escherichia coli and Pichia pastoris.

[0030] In some preferred embodiments of the present invention, the Escherichia coli includes Escherichia coli TOP10.

[0031] In some preferred embodiments of the present invention, the Pichia pastoris includes Pichia pastoris X33 and Pichia pastoris SMD1168H.

[0032] According to the third aspect of the present invention, a method for preparing the fusion protein as described in the first aspect of the present invention is proposed, the preparation method comprising the following steps: transferring a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 5 into a host cell for inducing expression.

[0033] In some embodiments of the present invention, the host cell comprises Pichia pastoris.

[0034] In some preferred embodiments of the present invention, the Pichia pastoris includes Pichia pastoris X33 and Pichia pastoris SMD1168H.

[0035] In some more preferred embodiments of the present invention, the Pichia pastoris is Pichia pastoris X33.

[0036] In some embodiments of the present invention, the primer set for amplifying the nucleic acid molecule encoding the nucleic acid molecule includes a forward primer sequence as shown in SEQ ID NO: 7 and a reverse primer sequence as shown in SEQ ID NO: 8.

[0037] In some embodiments of the present invention, the vector used to carry the nucleic acid molecule includes pPICZαA.

[0038] According to the fourth aspect of the present invention, the use of the fusion protein described in the first aspect of the present invention, the biomaterial described in the second aspect of the present invention, or the preparation method described in the third aspect of the present invention in the preparation of antioxidant and / or anti-inflammatory products is proposed.

[0039] In some embodiments of the present invention, the products include medicines and cosmetics.

[0040] In some preferred embodiments of the present invention, the dosage form of the drug includes at least one of injection, tablet, capsule, solution, syrup, emulsion, suspension, granule, ointment, suppository and aerosol.

[0041] In some preferred embodiments of the present invention, the drug further comprises pharmaceutically acceptable excipients.

[0042] In some more preferred embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant or a preservative.

[0043] In some preferred embodiments of the present invention, the cosmetics include at least one of a facial cleanser, a toner, a skin care lotion, a facial mask, a facial cream, a foundation, a primer, or a sunscreen.

[0044] According to a fifth aspect of the present invention, a product is provided, wherein the product comprises the fusion protein according to the first aspect of the present invention or the biomaterial according to the second aspect of the present invention.

[0045] In some embodiments of the present invention, the products include medicines and cosmetics.

[0046] In some preferred embodiments of the present invention, the dosage form of the drug includes at least one of injection, tablet, capsule, solution, syrup, emulsion, suspension, granule, ointment, suppository and aerosol.

[0047] In some preferred embodiments of the present invention, the drug further comprises pharmaceutically acceptable excipients.

[0048] In some more preferred embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a disintegrant, a diluent, a lubricant, a binder, a wetting agent, a flavoring agent, a suspending agent, a surfactant or a preservative.

[0049] In some more preferred embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, cross-linked polyvinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium or alginic acid.

[0050] In some more preferred embodiments of the present invention, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate or crystalline cellulose.

[0051] In some more preferred embodiments of the present invention, the lubricant is selected from at least one of micropowder silica gel, magnesium stearate, calcium stearate, stearic acid, talc or anhydrous silica gel.

[0052] In some more preferred embodiments of the present invention, the binder is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methyl cellulose or polyvinyl pyrrolidone.

[0053] In some more preferred embodiments of the present invention, the wetting agent is sodium lauryl sulfate.

[0054] In some more preferred embodiments of the present invention, the flavoring agent is at least one of aspartame, stevioside, sucrose, maltitol or citric acid.

[0055] In some more preferred embodiments of the present invention, the suspending agent is selected from at least one of gum arabic, gelatin, methyl cellulose, sodium carboxymethyl cellulose, hydroxymethyl cellulose or aluminum stearate gel.

[0056] In some more preferred embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate or glyceryl monostearate.

[0057] In some more preferred embodiments of the present invention, the preservative is selected from at least one of methylparaben and propylparaben.

[0058] In some preferred embodiments of the present invention, the cosmetics include at least one of a facial cleanser, a toner, a skin care lotion, a facial mask, a facial cream, a foundation, a primer, or a sunscreen.

[0059] The present invention has at least the following beneficial effects:

[0060] (1) The present invention provides a novel bifunctional fusion protein with both human SOD and CAT activities. The fusion protein is obtained by organically connecting human SOD and CAT through a rigid connecting peptide (EAAAK)3 obtained by extensive screening. The N-terminus of the rigid connecting peptide is SOD and the C segment is CAT. The use of the rigid connecting peptide can effectively avoid the influence between the two enzymes, enabling efficient and stable expression in Pichia pastoris. The enzyme activity far exceeds that of fusion proteins using other connecting peptides, and the protein bands are complete and clear.

[0061] (2) The fusion protein provided by the present invention uses a rigid connecting peptide to connect SOD and CAT. The rigid connecting peptide can prevent the fusion protein from being hydrolyzed by hydrolases during the fermentation process and can also better protect the active center of the enzyme;

[0062] (3) The molecular weight of the fusion protein provided by the present invention is 75 kDa, which effectively avoids the fusion protein from being rapidly excreted through renal filtration in the body, thereby significantly extending its half-life in the body;

[0063] (4) The present invention provides a nucleic acid molecule encoding the above-mentioned fusion protein, which is obtained by codon optimization of SOD and CAT genes, so that it can be successfully expressed in Pichia pastoris, avoiding translation barriers caused by rare codons in human genes in yeast;

[0064] (5) The present invention provides a method for preparing the above-mentioned SOD-CAT fusion protein. The method uses the pPICZαA vector with AOX1 as the promoter, and its expression effect is far better than the pGAPZαA vector with GAP as the promoter. The method also uses Pichia pastoris X33 as the host bacteria, and its expression effect is better than Pichia pastoris SMD1168H.

[0065] (6) The preparation method of the SOD-CAT fusion protein provided by the present invention connects the target gene to the back of the first XhoI restriction site of the vector, and then uses the α signal peptide on the vector to secrete and express the target protein. The signal peptide is then removed by the Kex2 enzyme in yeast, so that the product is secreted outside the cell without any excess amino acid residues. Since the product is secreted outside the cell, the accumulation of the target protein can be increased, and the crude enzyme solution can be obtained by simple centrifugation, which is more conducive to downstream purification, can shorten the preparation time and reduce production costs.

[0066] (7) The preparation method of the SOD-CAT fusion protein provided by the present invention utilizes the Pichia pastoris eukaryotic expression system. Compared with the prokaryotic expression system, this system has the function of post-translational modification of recombinant protein expression. The structure of the expressed protein is similar to that of human protein, has better compatibility, and is suitable for drug production. The target gene of the scheme of the present invention is integrated into the yeast chromosome, the strain has good genetic stability, and is easy to ferment at high density. In addition, the new bifunctional fusion protein expressed by the yeast eukaryotic system does not contain endotoxins compared with the prokaryotic system, and is more suitable for clinical medical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The present invention will be further described below with reference to the accompanying drawings and embodiments, in which:

[0068] Figure 1 Figure 1 is a diagram showing the verification results of the STEA gene fragment and the pPICZαA plasmid during the preparation process in Example 1 of the present invention; wherein, Figure A is an electrophoresis result diagram after amplification of the STEA fragment, and lanes 1 and 2 are both STEA fragment samples; Figure B is an electrophoresis result diagram after extraction of the pPICZαA plasmid, and lanes 1 to 3 are pPICZαA plasmids; Figure C is an electrophoresis result diagram after double enzyme digestion of the pPICZαA plasmid, lane 1 is pPICZαA without enzyme digestion, and lanes 2 and 3 are both pPICZαA after double enzyme digestion; Figure D is a positive identification result diagram of Escherichia coli containing the recombinant plasmid pPICZαA-STEA, and lanes 1 to 5 are all Escherichia coli samples containing the recombinant plasmid pPICZαA-STEA; Figure E is an electrophoresis result diagram after extraction of the recombinant plasmid pPICZαA-STEA, and lanes 1 and 2 are both recombinant plasmid pPICZαA-STEA samples;

[0069] Figure 2 Schematic diagram of the recombinant plasmid pPICZαA-STEA in Example 1 of the present invention;

[0070] Figure 3 Figure 1 is a diagram showing the verification results of the recombinant plasmids pPICZαA-STEA and XP-STEA recombinant yeast in Example 1 of the present invention; wherein, Figure A is an electrophoresis result diagram of the recombinant plasmid pPICZαA-STEA after linearization, lanes 1 and 3 are recombinant plasmid pPICZαA-STEA linearized samples, and lanes 2 and 4 are undigested recombinant plasmid pPICZαA-STEA samples; Figure B is a diagram showing the screening results of XP-STEA recombinant yeast, wherein a total of 9 different monoclonal strain samples were detected, namely XP-STEA-1 to 9, which are located in lanes 5, 6, 8, 10, 13, 14, 18, 19 and 20, respectively;

[0071] Figure 4This is a graph showing the results of the SP-STEA recombinant yeast screening in Example 2 of the present invention, wherein a total of 9 different monoclonal strain samples were tested, namely SP-STEA-1 to SP-STEA-9, located in lanes 5, 9, 10, 12, 13, 14, 15, 16, and 19, respectively;

[0072] Figure 5 Figure 1 is a diagram showing the verification results of the pGAPZαA plasmid and the pGAPZαA-STEA recombinant plasmid in the preparation process in Comparative Example 1 of the present invention; wherein, Figure A is an electrophoresis result diagram after extraction of the pGAPZαA plasmid, and lanes 4 to 6 are pGAPZαA plasmids; Figure B is an electrophoresis result diagram after double enzyme digestion of the pGAPZαA plasmid, lane 1 is pGAPZαA without enzyme digestion, and lane 2 is pGAPZαA after double enzyme digestion; Figure C is a positive identification result diagram of Escherichia coli containing the pGAPZαA-STEA recombinant plasmid, and lanes 1 to 5 are all Escherichia coli samples containing the recombinant plasmid pGAPZαA-STEA; Figure D is an electrophoresis result diagram after extraction of the pGAPZαA-STEA recombinant plasmid, and lanes 1 to 2 are all pGAPZαA-STEA recombinant plasmid samples;

[0073] Figure 6 Schematic diagram of the pGAPZαA-STEA recombinant plasmid in Comparative Example 1 of the present invention;

[0074] Figure 7 Figure 1 is a diagram showing the verification results of the construction of the XG-STEA recombinant yeast in Comparative Example 1 of the present invention; Figure A is an electrophoresis result diagram after linearization of the pGAPZαA-STEA recombinant plasmid, lane 1 is a linearized pGAPZαA-STEA recombinant plasmid sample, and lane 2 is an undigested pGAPZαA-STEA recombinant plasmid sample; Figure B is a diagram showing the screening results of the XG-STEA recombinant yeast, in which a total of 9 different monoclonal strain samples, namely XG-STEA-1 to XG-STEA-9, were tested, located in lanes 1, 2, 6, 11, 12, 13, 14, 15 and 16, respectively;

[0075] Figure 8 This is a graph showing the results of SG-STEA recombinant yeast screening in Comparative Example 2 of the present invention, wherein a total of 9 different monoclonal strain samples were tested, namely SG-STEA-1 to SG-STEA-9, located in lanes 4, 5, 6, 9, 10, 12, 16, 17, and 18, respectively;

[0076] Figure 9Figure 3 is a diagram showing the verification results of the STGS gene fragment and the pPICZαA-STGS recombinant plasmid during the preparation process in Comparative Example 3 of the present invention; wherein, Figure A is an electrophoresis result diagram after STGS fragment amplification, and lanes 1 to 3 are STGS fragment samples; Figure B is a positive identification result diagram of Escherichia coli containing the pPICZαA-STGS recombinant plasmid, and lanes 1 to 6 are Escherichia coli samples containing the recombinant plasmid pPICZαA-STGS; Figure C is an electrophoresis result diagram after pPICZαA-STGS recombinant plasmid extraction, and lanes 1 to 2 are pPICZαA-STGS recombinant plasmid samples;

[0077] Figure 10 Schematic diagram of the pPICZαA-STGS recombinant plasmid in Comparative Example 3 of the present invention;

[0078] Figure 11 Figure 3 is a diagram showing the verification results of the construction of XP-STGS recombinant yeast in Comparative Example 3 of the present invention; Figure A is an electrophoresis result diagram after linearization of the pPICZαA-STGS recombinant plasmid, lanes 1 and 3 are linearized samples of the pGAPZαA-STEA recombinant plasmid, and lanes 2 and 4 are undigested pPICZαA-STGS recombinant plasmid samples; Figure B is a diagram showing the screening results of the XP-STGS recombinant yeast, in which a total of 9 different monoclonal strain samples, namely XP-STGS-1 to 9, were detected, located in lanes 2, 3, 4, 5, 6, 8, 10, 11 and 17, respectively;

[0079] Figure 12 This is a graph showing the results of SP-STGS recombinant yeast screening in Comparative Example 4 of the present invention, in which a total of 7 different monoclonal strain samples were detected, namely SP-STGS-1 to SP-STGS-7, which are located in lanes 1, 4, 5, 6, 7, 8, and 10, respectively;

[0080] Figure 13 Figure 5 is a diagram showing the verification results of the pGAPZαA-STGS recombinant plasmid preparation in Comparative Example 5 of the present invention; Figure A is a diagram showing the electrophoresis results after extraction of the pGAPZαA-STGS recombinant plasmid, and lanes 1 to 2 are all pGAPZαA-STGS recombinant plasmid samples; Figure B is a diagram showing the positive identification results of Escherichia coli containing the pGAPZαA-STGS recombinant plasmid, and lanes 1 to 10 are all pGAPZαA-STGS recombinant plasmid samples;

[0081] Figure 14 Schematic diagram of the pGAPZαA-STGS recombinant plasmid in Comparative Example 5 of the present invention;

[0082] Figure 15Figure 5 is a diagram showing the verification results of the construction of the XG-STGS recombinant yeast in Comparative Example 5 of the present invention; wherein, Figure A is an electrophoresis result diagram after linearization of the pGAPZαA-STGS recombinant plasmid, lane 1 is a linearized sample of the pGAPZαA-STGS recombinant plasmid, and lane 2 is an undigested pGAPZαA-STGS recombinant plasmid sample; Figure B is a diagram showing the screening results of the XG-STGS recombinant yeast, wherein a total of 9 different monoclonal strain samples, namely XG-STGS-1 to 9, were tested, located in lanes 1, 2, 4, 14, 15, 16, 17, 18 and 20, respectively;

[0083] Figure 16 This is a diagram showing the results of the SG-STGS recombinant yeast screening in Comparative Example 6 of the present invention, in which a total of 9 different monoclonal strain samples, namely SG-STGS-1 to 9, were detected, located in lanes 1, 2, 3, 4, 5, 6, 7, 12 and 15, respectively;

[0084] Figure 17 Figure 7 is a diagram showing the verification results of the STPTTD gene fragment and the pPICZαA-STPTD recombinant plasmid during the preparation process in Comparative Example 7 of the present invention; wherein, Figure A is a diagram showing the electrophoresis result after amplification of the STPTTD fragment, and lanes 1 to 4 are all STPTTD fragment samples; Figure B is a diagram showing the positive identification result of Escherichia coli containing the pPICZαA-STPTD recombinant plasmid, and lanes 1 to 3 are all Escherichia coli samples containing the recombinant plasmid pPICZαA-STPTD; Figure C is a diagram showing the electrophoresis result after extraction of the pPICZαA-STPTD recombinant plasmid, and lanes 6 to 7 are all pPICZαA-STPTD recombinant plasmid samples;

[0085] Figure 18 Schematic diagram of the pPICZαA-STPTD recombinant plasmid in Comparative Example 7 of the present invention;

[0086] Figure 19 It is a diagram of the verification results of the construction of XP-STPTD recombinant yeast in Comparative Example 7 of the present invention; wherein, Figure A is the electrophoresis result diagram after linearization of the pPICZαA-STPTD recombinant plasmid, lanes 2 and 4 are pPICZαA-STPTD recombinant plasmid linearization samples, and lanes 1 and 3 are undigested pPICZαA-STPTD recombinant plasmid samples; Figure B is the XP-STPTD recombinant yeast screening result diagram, in which a total of 9 different monoclonal strain samples, namely XP-STPTD-1 to 9, were detected, located in lanes 1, 3, 4, 5, 6, 7, 11, 14 and 15, respectively;

[0087] Figure 20This is a graph showing the results of SP-STPTD recombinant yeast screening in Comparative Example 8 of the present invention, wherein a total of 9 different monoclonal strain samples were detected, namely SP-STPTD-1 to SP-STPTD-9, located in lanes 2, 5, 7, 11, 12, 13, 14, 15, and 19, respectively;

[0088] Figure 21 Figure 9 is a diagram showing the verification results of the pGAPZαA-STPTD recombinant plasmid during the preparation process in Comparative Example 9 of the present invention; Figure A is a diagram showing the positive identification results of Escherichia coli containing the pGAPZαA-STPTD recombinant plasmid, and lanes 7 to 11 are all Escherichia coli samples containing the recombinant plasmid pPICZαA-STPTD; Figure B is a diagram showing the electrophoresis results after the pGAPZαA-STPTD recombinant plasmid is extracted;

[0089] Figure 22 Schematic diagram of the pGAPZαA-STPTD recombinant plasmid in Comparative Example 9 of the present invention;

[0090] Figure 23 It is a diagram showing the verification results of the construction of the XG-STPTD recombinant yeast in Comparative Example 9 of the present invention; wherein, Figure A is an electrophoresis result diagram after linearization of the pGAPZαA-STPTD recombinant plasmid, lanes 1 and 3 are pGAPZαA-STPTD recombinant plasmid linearization samples, and lanes 2 and 4 are undigested pGAPZαA-STPTD recombinant plasmid samples; Figure B is a diagram showing the screening results of the XG-STPTD recombinant yeast, in which a total of 9 different monoclonal strain samples, namely XG-STPTD-1 to 9, were detected, located in lanes 1, 3, 7, 8, 12, 15, 17, 20 and 23, respectively;

[0091] Figure 24 This is a diagram showing the results of screening the SG-STPTD recombinant yeast in Comparative Example 10 of the present invention, wherein a total of 6 different monoclonal strain samples were tested, namely SG-STPTD-1 to 6, which are located in lanes 1, 4, 5, 6, 10 and 19, respectively;

[0092] Figure 25 Graphs showing the trend of SOD / CAT enzyme activity detection in the fermentation supernatants of the recombinant strains of Examples 1 to 2 and Comparative Examples 1 to 4 in the test examples of the present invention; wherein, Graph A shows the XP-STEA-6 strain described in Example 1; Graph B shows the SP-STEA-4 strain described in Example 2; Graph C shows the XG-STEA-6 strain described in Comparative Example 1; Graph D shows the SG-STEA-5 strain described in Comparative Example 2; Graph E shows the XP-STGS-8 strain described in Comparative Example 3; and Graph F shows the SP-STGS-2 strain described in Comparative Example 4.

[0093] Figure 26The present invention is a trend chart of the SOD / CAT enzyme activity detection in the fermentation supernatant of the recombinant strains of Comparative Examples 5 to 10 in the test examples; wherein, Figure A is the XG-STGS-2 strain described in Comparative Example 5; Figure B is the SG-STGS-6 strain described in Comparative Example 6; Figure C is the XP-STPTD-6 strain described in Comparative Example 7; Figure D is the SP-STPTD-6 strain described in Comparative Example 8; Figure E is the XG-STPTD-8 strain described in Comparative Example 9; and Figure F is the SG-STPTD-6 strain described in Comparative Example 10;

[0094] Figure 27 Figure 1 is a graph showing the SDS-PAGE electrophoresis results of the fermentation supernatant samples of the recombinant strains of Examples 1 to 2 and Comparative Examples 1 to 4 in the test examples of the present invention; wherein, Figure A shows the XP-STEA-1 to 9 strains described in Example 1; Figure B shows the SP-STEA-1 to 9 strains described in Example 2; Figure C shows the XG-STEA-1 to 9 strains described in Comparative Example 1; Figure D shows the SG-STEA-1 to 9 strains described in Comparative Example 2; Figure E shows the XP-STGS-1 to 9 strains described in Comparative Example 3; and Figure F shows the SP-STGS-1 to 7 strains described in Comparative Example 4.

[0095] Figure 28 Figure 1 is a graph showing the SDS-PAGE electrophoresis results of the fermentation supernatant samples of the recombinant strains of Comparative Examples 5 to 10 in the test examples of the present invention; wherein, Figure A is the XG-STGS-1 to 9 strains described in Comparative Example 5; Figure B is the SG-STGS-1 to 9 strains described in Comparative Example 6; Figure C is the XP-STPTD-1 to 9 strains described in Comparative Example 7; Figure D is the SP-STPTD-1 to 9 strains described in Comparative Example 8; Figure E is the XG-STPTD-1 to 9 strains described in Comparative Example 9; and Figure F is the SG-STPTD-1 to 6 strains described in Comparative Example 10;

[0096] Figure 29 These are the Western blot verification results of the XP-STEA-6 strain fermentation supernatant obtained in Example 1 and the half-life test results in rats in the test examples of the present invention; wherein, Figure A is the Western blot result of the XP-STEA-6 strain fermentation supernatant, and lanes 1 to 2 are both XP-STEA-6 strain fermentation supernatant samples; Figure B shows the CAT pharmacokinetics of the purified product of the XP-STEA-6 strain fermentation supernatant; Figure C shows the SOD pharmacokinetics of the purified product of the XP-STEA-6 strain fermentation supernatant. DETAILED DESCRIPTION

[0097] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0098] Example 1

[0099] In this example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein consisted of SOD-(EAAAK)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris X33 using the pPICZαA vector for induced expression. The specific preparation method of the fusion protein is as follows:

[0100] 1. Acquisition of target genes:

[0101] 1) The amino acid sequence information of human SOD (NP_000445.1) and human CAT (NP_001743.1) was searched through NCBI. The two enzymes were linked together using a (EAAAK)3 rigid linker peptide to obtain the amino acid sequence of the fusion protein; SOD was located at the N-terminus of the linker peptide, and CAT was located at the C-terminus of the linker peptide;

[0102] The amino acid sequence of the (EAAAK)3 rigid linker peptide is as follows: EAAAKEAAAKEAAAK (SEQ ID NO: 1);

[0103] The amino acid sequence of the SOD is as follows:

[0104] LEKRMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGC TSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ (SEQ ID NO: 2);

[0105] The amino acid sequence of the CAT is as follows:

[0106] MADSRDPASDQMQHWKEQRAAQKADVLTTGAGNPVGDKLNVITVGPRGPLLVQDVVFTDEMAHFDRERIPERVVHAKGAGAFGYFEVTHDITKYSKAKVFEHIGKKTPIAVRFSTVAGESGSADTVRDPRGFAVKFYTEDGNWDLVGNNTPIFFIRDPILFPSFIHSQKRNPQTHLKDPDMVWDFWSLRPESLHQVSFLFSDRGIPDGHRHMNGYGSHTFKLVNANGEAVYCKFHYKTDQGIKNLSVEDAARLSQEDPDYGIRDLFNAIATGKYPSWTFYIQVMTFNQAETFPFNPFDLTKVWPHKDYPLIPVGKLVLNRNPVNYFAEVEQIAFDPSNMPPGIEASPDKMLQGRLFAYPDTHRHRLGPNYLHIPVNCPYRARVANYQRDGPMCMQDNQGGAPNYYPNSFGAPEQQPSALEHSIQYSGEVRRFNTANDDNVTQVRAFYVNVLNEEQRKRLCENIAGHLKDAQIFIQKKAVKNFTEVHPDYGSHIQALLDKYNAEKPKNAIHTFVQSGSHLAAREKANL(SEQ ID NO:3);

[0107] The amino acid sequence of the fusion protein is specifically as follows:

[0108] *(SEQ ID NO:4);

[0109] The corresponding nucleotide sequence was obtained based on the amino acid sequence of the above fusion protein. At the same time, based on the restriction site information on the multiple cloning site of the pPICZαA vector, an XhoI restriction site and a Lys-Arg sequence (Kex2 restriction site) were added to the 5' end of the cDNA fragment of the target gene, and an XbaI restriction site was added to the 3' end. After 3D modeling and codon optimization (the XhoI and XbaI restriction sites were not optimized), the fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. to synthesize the corresponding gene, namely SOD-(EAAAK)3-CAT (the fusion gene was named STEA). The optimized nucleotide sequence of STEA is as follows:

[0110]

[0111] The nucleotide sequence of STEA before optimization is as follows:

[0112]

[0113] 2) Using the synthesized STEA gene fragment as a template, PCR amplification was performed using a high-fidelity enzyme (Novozyme, 2× Phanta Flash Master Mix) to obtain the target sequence. The PCR amplification reaction system is shown in Table 1; the PCR reaction procedure is shown in Table 2; the specific sequences of the primers used for amplification are as follows:

[0114] Upstream primer: 5′-GAAGAAGGGGTATCTCTCGAGAAGAGAATGGCTACTAAAGC-3′ (SEQ ID NO: 7);

[0115] Downstream primer: 5′-CTGAGATGAGTTTTTGTTCTAGATTAAAGGTTAGCCTTTTCTCTAGC-3′ (SE QID NO: 8).

[0116] Table 1 PCR reaction system (100 μL)

[0117]

[0118] Table 2 PCR reaction procedure

[0119]

[0120] 3) After the PCR amplification is completed, the amplified product is detected by 1.2% agarose gel electrophoresis. If the detected product is consistent with the expected result, the target fragment is cut and recovered. The results of the electrophoresis detection are as follows: Figure 1 As shown in A.

[0121] 2. Construction of yeast expression vector:

[0122] 1) Using the pPICZαA plasmid as the vector backbone, E. coli containing the pPICZαA plasmid was inoculated into LB medium containing bleomycin, and cultured in a shaking incubator at 37°C for 16 h. The pPICZαA plasmid was extracted using EndoFree Plasmid Maxi Kit, and the extracted plasmid was detected by 1.2% agarose gel electrophoresis. The electrophoresis results were as follows: Figure 1 As shown in lanes 1 to 3 in B.

[0123] 2) The vector pPICZαA obtained in step 1) was digested with a double enzyme digestion method. The double enzyme digestion system is shown in Table 3.

[0124] Table 3 Double enzyme digestion reaction system (500 μL)

[0125]

[0126] 3) Gel recovery of plasmid backbone: The plasmid backbone after double enzyme digestion in step 2) was detected by 1.2% agarose gel electrophoresis. The electrophoresis results were as follows: Figure 1 As shown in C; Figure 1 Lane 1 in C is pPICZαA without enzyme digestion, and lanes 2 and 3 are pPICZαA after double enzyme digestion. After the detection product meets the expectation, the gel is cut and recovered.

[0127] 4) Connect the target fragment to the vector: Use seamless ligation kit (Novozyme, The target gene fragment with homologous sequences obtained in step 1 and the vector backbone recovered after enzyme digestion in step 3) were ligated using an Ultra OneStep Cloning Kit. The molar ratio of plasmid vector to target gene fragment in the ligation reaction was approximately 1:2. The ligation system and reaction procedure are shown in Tables 4 and 5, respectively.

[0128] Table 4 Seamless connection system

[0129]

[0130] Table 5 Seamless connection reaction program

[0131]

[0132] 5) Transformation and identification of recombinant plasmid: The recombinant plasmid obtained in step 4) (product after seamless connection treatment) was transformed into competent Escherichia coli cells TOP10, 500 μL of LB liquid medium was added and placed on a shaker at 37°C and 200 r / min for 1 hour, and then plated (containing 100 μg / mL Zeocin). After a single colony grew, the bacteria were picked for culture, and then PCR positive identification of Escherichia coli containing the recombinant plasmid pPICZαA-STEA was performed. The reaction system and reaction procedure for PCR positive identification are shown in Tables 6 and 7, respectively; after PCR, nucleic acid electrophoresis was performed to verify the positive recombinant vector, and the results were as follows: Figure 1 D; the PCR primer sequences used in the positive identification are as follows:

[0133] Upstream primer: 5′-GCATCCTCCGCATTAGCTGCTCCAGTCAACAC-3′ (SEQ ID NO: 9);

[0134] Downstream primer: 5′-CTCTCAGGCAAATGGCATTCTGACATCCTCTTG-3′ (SEQ ID NO: 10);

[0135] Then the recombinant plasmid was extracted for double enzyme digestion verification. The plasmid with successful double enzyme digestion was sent for sequencing. Then the positive strain with correct sequencing was inoculated into 100 mL of LB liquid culture medium and cultured at 37°C and 200 r / min. After the appropriate concentration was reached, the recombinant plasmid pPICZαA-STEA was extracted and the recombinant plasmid was subjected to electrophoresis detection. The results are as follows: Figure 1 E; the map of the recombinant plasmid pPICZαA-STEA is shown in Figure 2 As shown;

[0136] Table 6 Recombinant vector PCR positive identification reaction system (10 μL)

[0137]

[0138] Table 7 Recombinant vector PCR positive identification reaction procedure

[0139]

[0140]

[0141] 3. Construction of recombinant Pichia pastoris strain (XP-STEA strain):

[0142] 1) The recombinant expression plasmid pPICZαA-STEA was linearized using NEB's SacI enzyme to convert the circular expression vector into a linear one. The linearization enzyme digestion system is shown in Table 8. CIAP enzyme was added to the linearized plasmid for dephosphorylation. The linearized plasmid was verified by nucleic acid electrophoresis. The results are shown in Table 8. Figure 3 As shown in A;

[0143] Table 8 Linearization enzyme digestion system (500 μL)

[0144]

[0145] 2) The linearized vector was extracted with chloroform-isoamyl alcohol and washed with 75% ethanol, dissolved in ultrapure water, and then electrotransformed into the competent Pichia pastoris X33 host bacteria. After the electrotransformed host Pichia pastoris was revived, it was inoculated into YPD solid medium containing 200 μg / mL bleomycin to screen for positive strains; then, single colonies were selected and the yeast genome was lysed by repeated freezing and thawing with liquid nitrogen and ultrasound for PCR identification. The reaction system for PCR identification is shown in Table 9, the reaction procedure for PCR identification is shown in Table 7, and the primer sequences used for identification are shown in SEQ ID NOs: 9-10; nucleic acid electrophoresis was used to verify the positive recombinant Pichia pastoris strains, and the results were shown in FIG. Figure 3 As shown in B, Figure 3 B A total of 9 different single clones of XP-STEA strain were detected; the verified positive recombinant strains were frozen for future use.

[0146] Table 9 Recombinant Pichia pastoris PCR identification reaction system (20 μL)

[0147]

[0148] 4. Induced expression of recombinant Pichia pastoris strains:

[0149] The recombinant positive strain was inoculated into a shake flask with BMMY medium at pH 6.0 and cultured at 30°C and 200 rpm. The expression was induced by an inducer (an equal volume mixture of methanol and 50% sorbitol). The expression was determined by OD 600 Calculate the amount of inducer to be added. Add the inducer once in the morning and evening. The amount to be added is calculated as follows: Amount of inducer added (μL) = OD 600 × dilution factor before detection × culture medium volume × 0.05 × 10; after the induction, the culture medium was collected and centrifuged to collect the supernatant to obtain the SOD-(EAAAK)3-CAT fusion protein.

[0150] Example 2

[0151] In this example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein consisted of SOD-(EAAAK)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris SMD1168H using the pPICZαA vector for induced expression. The specific preparation method of the fusion protein differed from that of Example 1 only in that the host strain Pichia pastoris X33 was replaced with an equal amount of Pichia pastoris SMD1168H. The results of nucleic acid electrophoresis verification of the positive recombinant Pichia pastoris strain (SP-STEA strain) were as follows: Figure 4 As shown, Figure 4 A total of 9 different SP-STEA strains were detected.

[0152] Comparative Example 1

[0153] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein was composed of SOD-(EAAAK)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris X33 using the pGAPZαA vector for induced expression. The specific preparation method of the fusion protein is as follows:

[0154] 1. Obtaining the target gene: The design and synthesis of the target gene cDNA were the same as in Example 1. The amino acid sequence of the (EAAAK)3 rigid linker peptide is shown in SEQ ID NO: 1, the amino acid sequence of SOD is shown in SEQ ID NO: 2, the amino acid sequence of CAT is shown in SEQ ID NO: 3, the amino acid sequence of the STEA fusion protein is shown in SEQ ID NO: 4, and the nucleotide sequence of the STEA fusion gene is shown in SEQ ID NO: 5.

[0155] 2. Construction of yeast expression vector:

[0156] 1) Using the pGAPZαA plasmid as the vector backbone, E. coli containing the pGAPZαA plasmid was inoculated into LB medium containing bleomycin, and cultured in a shaker at 37°C for 16 hours. The pGAPZαA plasmid was extracted using EndoFree Plasmid Maxi Kit, and the extracted plasmid was detected by 1.2% agarose gel electrophoresis. The electrophoresis results were as follows: Figure 5 Lanes 4 to 6 in A are shown.

[0157] 2) The vector pGAPZαA obtained in step 1) was digested with a double enzyme digestion method. The double enzyme digestion system is shown in Table 3.

[0158] 3) Gel recovery of plasmid backbone: The plasmid backbone after double enzyme digestion in step 2) was detected by 1.2% agarose gel electrophoresis. The electrophoresis results were as follows: Figure 5 As shown in B; Figure 5 Lane 1 in B is pPICZαA without enzyme digestion, and lane 2 is pGAPZαA after double enzyme digestion. After the detection product meets the expectation, the gel is cut and recovered.

[0159] 4) Ligation of target fragment and vector: The ligation system is the same as that in Example 1. The specific ligation system and reaction procedure are shown in Table 4 and Table 5, respectively.

[0160] 5) Transformation and identification of recombinant plasmid: The recombinant plasmid obtained in step 4) (product after seamless connection treatment) was transformed into competent Escherichia coli cells TOP10, 500 μL of LB liquid medium was added and placed on a shaker at 37°C and 200 r / min for 1 hour, and then plated (containing 100 μg / mL Zeocin). After a single colony grew, the bacteria were picked for culture, and then PCR positive identification of Escherichia coli containing the recombinant plasmid pGAPZαA-STEA was performed. The reaction system and reaction procedure for PCR positive identification are shown in Tables 6 and 7, respectively; after PCR, nucleic acid electrophoresis was performed to verify the positive recombinant vector, and the results were as follows: Figure 5C; the PCR primer sequences used for the positive identification are shown in SEQ ID NO: 9 and SEQ ID NO: 10;

[0161] Then the recombinant plasmid was extracted for double enzyme digestion verification. The plasmid with successful double enzyme digestion was sent for sequencing. Then the positive strain with correct sequencing was inoculated into 100 mL of LB liquid culture medium and cultured at 37°C and 200 r / min. After the appropriate concentration was reached, the recombinant plasmid pPICZαA-STEA was extracted and the recombinant plasmid was subjected to electrophoresis detection. The results are as follows: Figure 5 D; the map of the recombinant plasmid pGAPZαA-STEA is shown in Figure 6 As shown;

[0162] 3. Construction of recombinant Pichia pastoris strain (XG-STEA strain):

[0163] 1) The recombinant expression plasmid pGAPZαA-STEA was linearized using NEB's SacI enzyme to convert the circular expression vector into a linear one. The linearization enzyme digestion system is shown in Table 8. CIAP enzyme was added to the linearized plasmid for dephosphorylation. The linearized plasmid was verified by nucleic acid electrophoresis. The results are shown in Table 8. Figure 7 As shown in A;

[0164] 2) The method of linearized vector concentration and electroporation into yeast and PCR identification of positive recombinant yeast is the same as step 3 of Example 1. The positive verification PCR electrophoresis diagram is as follows: Figure 7 As shown in B, Figure 7 B A total of 9 different single clones of the XG-STEA strain were detected; the verified positive recombinant strains were frozen for future use.

[0165] 4. Induced expression of recombinant Pichia pastoris strains:

[0166] The recombinant positive strain was inoculated into a shake flask with BMMY medium at pH 6.0 and cultured at 30°C and 200 rpm. The expression was induced by an inducer (an equal volume mixture of 20% glucose and 50% sorbitol). The expression was determined by OD 600 Calculate the amount of inducer to be added. Add the inducer once in the morning and evening. The amount to be added is calculated as follows: Amount of inducer added (μL) = OD 600 × dilution factor before detection × culture medium volume × 0.05 × 10; after the induction, the culture medium was collected and centrifuged to collect the supernatant to obtain the SOD-(EAAAK)3-CAT fusion protein.

[0167] Comparative Example 2

[0168] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein was composed of SOD-(EAAAK)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris SMD1168H using the pGAPZαA vector for induced expression. The specific preparation method of the fusion protein was different from that of Comparative Example 1 only in that the host strain Pichia pastoris X33 was replaced with an equal amount of Pichia pastoris SMD1168H; wherein, the results of nucleic acid electrophoresis verification of the positive recombinant Pichia pastoris strain (SG-STEA strain) are as follows: Figure 8 As shown, Figure 8 A total of 9 different SG-STEA strains were detected.

[0169] Comparative Example 3

[0170] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein consisted of SOD-(GGGGS)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris X33 using the pPICZαA vector for induced expression. The specific preparation method of the fusion protein is as follows:

[0171] 1. Obtaining the target gene: The rigid connecting peptide (EAAAK) 3 in the fusion protein described in Example 1 was replaced with a flexible connecting peptide (GGGGS) 3 to obtain the fusion protein of this comparative example; the amino acid sequence of the flexible connecting peptide (GGGGS) 3 is as follows: GGGGSGGGGSGGGGS (SEQ ID NO: 11);

[0172] The amino acid sequence of the fusion protein is as follows:

[0173] *(SEQ ID NO:12);

[0174] The corresponding nucleotide sequence was obtained based on the amino acid sequence of the above fusion protein. At the same time, based on the restriction site information on the multiple cloning site of the pPICZαA vector, an XhoI restriction site and a Lys-Arg sequence (Kex2 restriction site) were added to the 5' end of the cDNA fragment of the target gene, and an XbaI restriction site was added to the 3' end. After 3D modeling and codon optimization (the XhoI and XbaI restriction sites were not optimized), the fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. to synthesize the corresponding gene, namely SOD-(GGGGS)3-CAT (the fusion gene was named STGS). The optimized nucleotide sequence of STGS is as follows:

[0175]

[0176] The nucleotide sequence of STGS before optimization is as follows:

[0177]

[0178] 2) Using the synthesized STGS gene fragment as a template, PCR amplification was performed using a high-fidelity enzyme (Novozyme, 2× Phanta Flash Master Mix) to obtain the target sequence. The PCR amplification reaction system is shown in Table 1; the PCR reaction procedure is shown in Table 2; the sequences of the primers used for amplification are as follows:

[0179] Upstream primer: 5′-GAAGAAGGGGTATCTCTCGAGAAGAGAATGGCTACTAAGGCTG-3′ (SEQ ID NO: 15);

[0180] Downstream primer: 5′-CTGAGATGAGTTTTTGTTCTAGATTACAAGTTAGCCTTTTCTCTAGC-3′ (SE QID NO: 16).

[0181] 3) After the PCR amplification is completed, the amplified product is detected by 1.2% agarose gel electrophoresis. If the detected product is consistent with the expected result, the target fragment is cut and recovered. The results of the electrophoresis detection are as follows: Figure 9 As shown in A.

[0182] 2. Construction of yeast expression vector:

[0183] 1) The plasmid extraction, double enzyme digestion and gel recovery of the plasmid backbone of the vector pPICZαA were the same as those described in Example 1, and the electrophoresis verification results were also consistent.

[0184] 2) Ligation of the target fragment and the vector: the same steps as described in Example 1.

[0185] 3) Recombinant plasmid transformation and identification: The recombinant plasmid obtained in step 2) (product after seamless connection treatment) was transformed into competent Escherichia coli cells TOP10, 500 μL of LB liquid medium was added and placed on a shaker at 37°C and 200 r / min for 1 hour, and then plated (containing 100 μg / mL Zeocin). After a single colony grew, the bacteria were picked and cultured, and then PCR positive identification of Escherichia coli containing the recombinant plasmid pPICZαA-STGS was performed. The reaction system and reaction procedure for PCR positive identification are shown in Tables 6 and 7, respectively; after PCR, nucleic acid electrophoresis was performed to verify the positive recombinant vector, and the results were as follows: Figure 9 B; the PCR primer sequences used for the positive identification are shown in SEQ ID NOs: 9 to 10;

[0186] Then the recombinant plasmid was extracted for double enzyme digestion verification. The plasmid with successful double enzyme digestion was sent for sequencing. Then the positive strain with correct sequencing was inoculated into 100 mL of LB liquid culture medium and cultured at 37°C and 200 r / min. After the appropriate concentration was reached, the recombinant plasmid pPICZαA-STGS was extracted and the recombinant plasmid was subjected to electrophoresis detection. The results are as follows: Figure 9 C; the map of the recombinant plasmid pPICZαA-STEA is shown in Figure 10 As shown;

[0187] 3. Construction of recombinant Pichia pastoris strain (XP-STGS strain):

[0188] 1) The recombinant expression plasmid pPICZαA-STGS was linearized using NEB's SacI enzyme to convert the circular expression vector into a linear one. The linearization enzyme digestion system is shown in Table 8. CIAP enzyme was added to the linearized plasmid for dephosphorylation. The linearized plasmid was verified by nucleic acid electrophoresis. The results are shown in Table 8. Figure 11 As shown in A;

[0189] 2) Extraction of linearized plasmid and electroporation of Pichia pastoris were performed according to the procedure described in step 3 of Example 1. The competent yeast for electroporation was X33. The nucleic acid electrophoresis pattern of the selected single colony for PCR positive identification was as follows: Figure 11 As shown in B.

[0190] 4. Induced expression of recombinant Pichia pastoris strain: the same as step 4 of Example 1.

[0191] Comparative Example 4

[0192] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein was composed of SOD-(GGGGS)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris SMD1168H using the pPICZαA vector for induced expression. The specific preparation method of the fusion protein was different from that of Comparative Example 3 only in that the host strain Pichia pastoris X33 was replaced with an equal amount of Pichia pastoris SMD1168H; wherein, the results of nucleic acid electrophoresis verification of the positive recombinant Pichia pastoris strain (SP-STGS strain) are as follows: Figure 12 As shown, Figure 12 A total of 7 different SP-STGS strains were detected.

[0193] Comparative Example 5

[0194] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein was composed of SOD-(GGGGS)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris X33 using the pGAPZαA vector for induced expression. The specific preparation method of the fusion protein was different from that of Comparative Example 1 only in that the target gene fragment was replaced by the STEA fusion gene (SEQ ID NO: 5) with the STGS fusion gene (SEQ ID NO: 13); wherein, the result of PCR nucleic acid electrophoresis verification of the positive recombinant vector (pGAPZαA-STGS) was as follows: Figure 13 As shown in A; the results of electrophoresis detection after extraction of recombinant plasmid are shown in Figure 13 B; the map of the recombinant plasmid pGAPZαA-STGS is shown in Figure 14 As shown; the electrophoresis verification results of the linearized recombinant plasmid are shown Figure 15 As shown in A; the results of nucleic acid electrophoresis verification of positive recombinant XG-STGS strains are as follows Figure 15 As shown in B, Figure 15 B A total of 9 different XG-STGS strain monoclones were detected.

[0195] Comparative Example 6

[0196] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein was composed of SOD-(GGGGS)3-CAT, and the corresponding fusion gene was transferred into Pichia pastoris SMD1168H using the pGAPZαA vector for induced expression. The specific preparation method of the fusion protein was different from that of Comparative Example 5 only in that the host strain Pichia pastoris X33 was replaced with an equal amount of Pichia pastoris SMD1168H; wherein, the results of nucleic acid electrophoresis verification of the positive recombinant Pichia pastoris strain (SG-STGS strain) are as follows: Figure 16 As shown, Figure 16 A total of 9 different SG-STGS strains were detected.

[0197] Comparative Example 7

[0198] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein consisted of a CAT-PTD connecting peptide-SOD, and the corresponding fusion gene was transferred into Pichia pastoris X33 using the pPICZαA vector for induced expression. The specific preparation method of the fusion protein is as follows:

[0199] 1. Obtaining the target gene: SOD (amino acid sequence shown in SEQ ID NO: 2) and CAT (amino acid sequence shown in SEQ ID NO: 3) were linked together using a PTD linker peptide, wherein CAT was located at the N-terminus of the linker peptide and SOD was located at the C-terminus of the linker peptide; the amino acid sequence of the PTD linker peptide was as follows: RKKRRQRRR (SEQ ID NO: 17);

[0200] The amino acid sequence of the fusion protein is as follows:

[0201] *(SEQ ID NO: 18);

[0202] The corresponding nucleotide sequence was obtained based on the amino acid sequence of the above fusion protein. At the same time, based on the restriction site information on the multiple cloning site of the pPICZαA vector, an XhoI restriction site and a Lys-Arg sequence (Kex2 restriction site) were added to the 5' end of the cDNA fragment of the target gene, and an XbaI restriction site was added to the 3' end. After 3D modeling and codon optimization (the XhoI and XbaI restriction sites were not optimized), the fragment was sent to Sangon Biotech (Shanghai) Co., Ltd. to synthesize the corresponding gene, namely CAT-RKKRRQRRR-SOD (the fusion gene was named STPTD). The optimized nucleotide sequence of STPTD is as follows:

[0203]

[0204] The nucleotide sequence of STPTD before optimization is as follows:

[0205]

[0206] 2) Using the synthesized STPTD gene fragment as a template, PCR amplification was performed using a high-fidelity enzyme (Novozyme, 2× Phanta Flash Master Mix) to obtain the target sequence. The PCR amplification reaction system is shown in Table 1; the PCR reaction procedure is shown in Table 2; the specific sequences of the primers used for amplification are as follows:

[0207] Upstream primer: 5′-CTAAAGAAGAAGGGGTATCTCTCGAGAAGCGAATGGCAGACTC-3′ (SEQ ID NO: 21);

[0208] Downstream primer: 5'-GAGATGAGTTTTTGTTCTAGATTACTGAGCTATACCTATAACC-3' (SEQ ID NO: 22).

[0209] 3) After the PCR amplification is completed, the amplified product is detected by 1.2% agarose gel electrophoresis. If the detected product is consistent with the expected result, the target fragment is cut and recovered. The results of the electrophoresis detection are as follows: Figure 17 As shown in A.

[0210] 2. Construction of yeast expression vector:

[0211] 1) The steps of plasmid extraction, plasmid double enzyme digestion, and plasmid backbone gel recovery were the same as step 2 of Example 1;

[0212] 2) Connect the target fragment to the vector: Use seamless ligation kit (Novozyme, The target gene STPTD with homologous sequence obtained in step 1 and the vector backbone pPICZαA recovered after enzyme digestion were ligated using the Ultra OneStep Cloning Kit. The molar ratio of plasmid vector to target gene fragment in the ligation reaction was approximately 1:2. The ligation system and reaction procedure are shown in Tables 4 and 5, respectively.

[0213] 3) Recombinant plasmid transformation and identification: The recombinant plasmid obtained in step 2) (product after seamless connection treatment) was transformed into competent Escherichia coli cells TOP10, 500 μL of LB liquid medium was added and placed on a shaker at 37°C and 200 r / min for 1 hour, and then plated (containing 100 μg / mL Zeocin). After a single colony grew, the bacteria were picked for culture, and then PCR positive identification of Escherichia coli containing the recombinant plasmid pPICZαA-STPTD was performed. The reaction system and reaction procedure for PCR positive identification are shown in Tables 6 and 7, respectively; after PCR, nucleic acid electrophoresis was performed to verify the positive recombinant vector, and the results were as follows: Figure 17 B; the PCR primer sequences used for the positive identification are shown in SEQ ID NOs: 9 to 10;

[0214] Then the recombinant plasmid was extracted for double enzyme digestion verification. The plasmid with successful double enzyme digestion was sent for sequencing. Then the positive strain with correct sequencing was inoculated into 100 mL of LB liquid culture medium and cultured at 37°C and 200 r / min. After the appropriate concentration was reached, the recombinant plasmid pPICZαA-STPTD was extracted and the recombinant plasmid was subjected to electrophoresis detection. The results are as follows: Figure 17 C; the map of the recombinant plasmid pPICZαA-STPTD is shown in Figure 18 As shown;

[0215] 3. Construction of recombinant Pichia pastoris strain (XP-STPTD strain):

[0216] 1) The recombinant expression plasmid pPICZαA-STPTD was linearized using NEB's SacI enzyme to convert the circular expression vector into a linear one. The linearization enzyme digestion system is shown in Table 8. CIAP enzyme was added to the linearized plasmid for dephosphorylation. The linearized plasmid was verified by nucleic acid electrophoresis. The results are shown in Table 8. Figure 19 As shown in A;

[0217] 2) The steps of extraction of linearized plasmid and electroporation of Pichia pastoris were the same as step 3 of Example 1. The competent yeast for electroporation was X33. Single colonies were selected for PCR identification of positive strains. The results were as follows: Figure 19 As shown in B, Figure 19 B A total of 9 different single clones of the XP-STPTD strain were detected; the primer sequences used for identification are shown in SEQ ID NOs: 9-10.

[0218] 4. Induced expression of recombinant Pichia pastoris strain: the same as step 4 of Example 1.

[0219] Comparative Example 8

[0220] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein consisted of a CAT-PTD connecting peptide-SOD, and the corresponding fusion gene was transferred into Pichia pastoris SMD1168H using the pPICZαA vector for induced expression. The specific preparation method of the fusion protein differed from that of Comparative Example 7 only in that the host strain Pichia pastoris X33 was replaced with an equal amount of Pichia pastoris SMD1168H. The results of nucleic acid electrophoresis verification of the positive recombinant Pichia pastoris strain (SP-STPTD strain) were as follows: Figure 20 As shown, Figure 20 A total of 9 different SP-STPTD strains were detected.

[0221] Comparative Example 9

[0222] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein consisted of a CAT-PTD connecting peptide-SOD, and the corresponding fusion gene was transferred into Pichia pastoris X33 using the pGAPZαA vector for induced expression. The specific preparation method of the fusion protein differed from that of Comparative Example 1 only in that the target gene fragment was replaced by the STEA fusion gene (SEQ ID NO: 5) with the STPTD fusion gene (SEQ ID NO: 19); wherein, the result of PCR nucleic acid electrophoresis verification of the positive recombinant vector (pGAPZαA-STPTD) was as follows: Figure 21 As shown in A; the results of electrophoresis detection after extraction of recombinant plasmid are shown in Figure 21 B; the map of the recombinant plasmid pGAPZαA-STGS is shown in Figure 22 As shown; the electrophoresis verification results of the linearized recombinant plasmid are shown Figure 23 As shown in A; the results of nucleic acid electrophoresis verification of positive recombinant XG-STPTD strains are as follows Figure 23 As shown in B, Figure 23 B A total of 9 different XG-STPTD strain monoclones were tested.

[0223] Comparative Example 10

[0224] In this comparative example, a superoxide dismutase-catalase fusion protein was prepared. The fusion protein was composed of a CAT-PTD connecting peptide-SOD, and the corresponding fusion gene was transferred into Pichia pastoris SMD1168H using the pGAPZαA vector for induced expression. The specific preparation method of the fusion protein was different from that of Comparative Example 9 only in that the host strain Pichia pastoris X33 was replaced with an equal amount of Pichia pastoris SMD1168H; wherein, the results of nucleic acid electrophoresis verification of the positive recombinant Pichia pastoris strain (SG-STPTD strain) are as follows: Figure 24 As shown, Figure 24 A total of 6 different SG-STPTD strain monoclones were detected.

[0225] Test example

[0226] This test example tested the performance of the fusion proteins prepared in Examples 1 to 2 and Comparative Examples 1 to 10. The specific test steps and results are as follows:

[0227] 1. SOD and CAT enzyme activity detection

[0228] The SOD and CAT enzyme activities of the supernatant samples at various time points (1 to 10 days, sampling every day) during the fermentation process of Examples 1 to 2 and Comparative Examples 1 to 10 were detected using Nanjing Jiancheng SOD kit (Article No.: A001-3) and CAT kit (Article No.: A007-1-1). The specific enzyme activity detection reaction systems are shown in Tables 10 and 11. The test results are shown in Tables 10 and 11. Figures 25-26 As shown;

[0229] The SOD enzyme activity calculation formula is:

[0230] The CAT enzyme activity calculation formula is: Among them, 235.65 is the reciprocal of the slope; V 样 is the sample volume - 0.01mL; T is the reaction time - 60s; N is the dilution factor of the sample before testing; ΔA = A 对照 -A 测定 .

[0231] Table 10 SOD enzyme activity detection reaction system

[0232]

[0233] Table 11CAT enzyme activity detection reaction system

[0234]

[0235] Depend on Figures 25 and 26 As can be seen, the SOD / CAT enzyme activities of Examples 1-2 were significantly improved compared to Comparative Examples 1-10, demonstrating that the use of a rigid linker peptide (EAAAK) 3 in the SOD-CAT fusion protein is far superior to the flexible linker peptide (GGGGS) 3 and the PTD short peptide. The optimal combination was the X33-pPICZαA-STEA recombinant strain. The maximum enzyme activities detected in Examples 1-2 and Comparative Examples 1-10 are shown in Table 12.

[0236] Table 12 Maximum statistics of SOD / CAT enzyme activity (U / mL)

[0237]

[0238] 2. SDS-PAGE Analysis of Fusion Protein

[0239] Prepare protein gel of relevant concentration according to the molecular weight of the protein, take 20 μL of the supernatant after centrifugation, add 5×portein buffer, and boil in water for 5 minutes; load the sample for electrophoresis, stain with Coomassie brilliant blue, decolorize, and observe and analyze the electrophoresis results; the Coomassie brilliant blue staining results obtained in Examples 1 to 2 and Comparative Examples 1 to 10 are as follows: Figures 27-28 shown.

[0240] At the same time, this experimental example further verified the fusion protein provided in Example 1 by Western blot. The specific steps are as follows: prepare a protein gel of relevant concentration according to the molecular weight of the protein, take 20 μL of the supernatant after centrifugation and add 5× portein buffer, and boil in a boiling water bath for 5 minutes; after SDS electrophoresis, transfer the membrane and place it in a blocking solution (TBST containing 5% skim milk powder) for 2 hours. After incubation with the primary antibody (D121245, BBI) and the secondary antibody (D110065, BBI), wash the membrane with TBST, and expose and develop: prepare the developer according to the instructions of the Millipore HRP kit, drop it on the membrane, protect it from light for 5 minutes, and then use the chemiluminescence development of the chemiluminescence imaging system to take pictures. The results are shown as follows: Figure 29 As shown in A.

[0241] Combined with the results of the above-mentioned SDS-PAGE protein electrophoresis and Western blot experiments, the expression level and molecular weight of the fusion protein prepared by the present invention were successfully verified.

[0242] 3. Half-life test in rat model

[0243] The pharmacokinetic half-life of the STEA fusion protein prepared in Example 1 was tested in an SD rat animal model. The specific steps are as follows:

[0244] The femoral artery and vein of rats were cannulated and the purified STEA fusion protein was injected intravenously. Blood was collected from the femoral artery of the rats at 0 min, 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h and 8 h after administration. 0.3 mL of blood sample was collected at each time point and immediately centrifuged at 3500 rpm for 5 min. The supernatant was collected and stored at 4°C for later use. The information of the experimental rats is shown in Table 13. The results are shown in Table 13. Figure 29 B and Figure 29 As shown in C.

[0245] Depend on Figure 29 From B to C, it can be seen that when the CAT dosage unit is 810.45U, according to the CAT enzyme activity value, the C max =66.02U / mL, half-life (T 1 / 2 ) value is 5.14±3.36h; when the SOD dosage unit is 3558.58U, according to the SOD enzyme activity value, it reaches C at 0min. max =694.16 U / mL, half-life (T 1 / 2 ) value is 1.33±0.68h, which is much longer than the half-life of SOD and CAT enzymes in vivo (CAT half-life T 1 / 2 The value is 30~60min; SOD half-life T 1 / 2value is 5 to 10 minutes).

[0246] Table 13 Experimental rat information

[0247] serial number Weight (g) Injection of recombinant enzyme (mL) Injection rate (mL / min) 1 320 3.0 0.3 2 337 1.8 0.3

[0248] In summary, the rigid connecting peptide (EAAAK) 3 used in the SOD-CAT fusion protein of the present invention is far superior to the flexible connecting peptide (GGGGS) 3 and the PTD (RKKRRQRRR) short peptide. During the culture and fermentation process, the new bifunctional fusion enzyme SOD-(EAAAK) 3-CAT has a stable structure and intact protein. Its enzymatic activity is also significantly better than the connection mode of the flexible connecting peptide (GGGGS) 3 and the PTD (RKKRRQRRR) short peptide. At the same time, the half-life of the bifunctional fusion enzyme SOD-(EAAAK) 3-CAT in rats is greatly extended, far exceeding the half-life of the single enzyme in vivo.

[0249] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A fusion protein, characterized in that The amino acid sequence of the fusion protein includes superoxide dismutase, a connecting peptide and catalase from the N-terminus to the C-terminus; the connecting peptide includes 1 to 5 repeating units, and the amino acid sequence of the repeating unit is Glu-Ala-Ala-Ala-Lys.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the superoxide dismutase is shown in SEQ ID NO: 2; And / or, the amino acid sequence of the catalase is shown in SEQ ID NO: 3; And / or, the amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

3. Biomaterial, characterized in that The biomaterial is any one of the following (1) to (8): (1) a nucleic acid molecule encoding the fusion protein according to claim 1 or 2; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1); (4) a recombinant vector containing the expression cassette described in (2); (5) A transgenic cell line containing the nucleic acid molecule described in (1); (6) a transgenic cell line containing the expression cassette described in (2); (7) A transgenic cell line containing the recombinant vector described in (3); (8) A transgenic cell line containing the recombinant vector described in (4).

4. The biomaterial according to claim 3, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

5.

5. A method for preparing the fusion protein according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: transferring a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 5 into a host cell and inducing expression.

6. Use of the fusion protein according to claim 1 or 2, the biomaterial according to claim 3 or 4, or the preparation method according to claim 5 in the preparation of antioxidant and / or anti-inflammatory products.

7. The use according to claim 6, characterized in that The products include pharmaceuticals and cosmetics.

8. A product, characterized in that The product comprises the fusion protein according to claim 1 or 2 or the biomaterial according to claim 3 or 4.

9. The product according to claim 8, characterized in that The products include pharmaceuticals and cosmetics.

10. The product according to claim 9, characterized in that The medicine also includes pharmaceutically acceptable excipients.

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