Synthetic process of a recombinant humanized SOD

Abstract

This invention discloses a recombinant humanized SOD synthesis process, belonging to the field of biosynthesis technology. The method includes optimizing the gene sequence encoding human copper-zinc superoxide dismutase using E. coli codon preference, activating engineered bacteria and inoculating them in a fermentation medium, inducing humanized SOD expression through temperature, collecting the fermentation cells expressing humanized SOD, cleaving and purifying the fermentation cells to obtain purified humanized SOD, and modifying the humanized SOD to obtain modified recombinant humanized SOD. This invention successfully achieves high-activity and high-conformation-fidelity recombinant humanized SOD expression using an E. coli expression system. Furthermore, endotoxins can be deeply removed through synergistic treatment of nickel chromatography, ion chromatography, and molecular sieve chromatography. Through the synergistic effect of betaine, sodium ascorbate phosphate, and linoleic acid stabilizers in the fermentation medium, SOD maintains excellent enzyme activity under a wide range of conditions, including below 80℃ and pH 2-12.

Description

Technical Field

[0001] This invention belongs to the field of biosynthesis technology, specifically relating to a recombinant humanized SOD synthesis process. Background Technology

[0002] Superoxide dismutase (SOD) is a class of enzymes ubiquitous in animals, plants, and microorganisms. It serves as the first line of defense in biological antioxidant systems and is the only antioxidant enzyme in the body capable of specifically scavenging oxygen free radicals. Currently, SOD is mainly classified into three types: Cu / Zn-SOD, Mn-SOD, and Fe-SOD. In the human body, endogenous and exogenous compounds generate oxygen free radicals under the action of various enzymes. These oxygen free radicals are converted into hydrogen peroxide under the catalysis of SOD. Catalase (CAT) and peroxidase (POD) in the body immediately decompose this into completely harmless water. Therefore, superoxide dismutase (SOD) is the core antioxidant enzyme for scavenging superoxide free radicals in organisms, playing a crucial role in delaying aging, reducing inflammation, and repairing oxidative damage.

[0003] Currently, the main sources of SOD include animal tissue extraction, plant extraction, and microbial recombinant expression. Among these, animal-derived SOD has significant limitations: on the one hand, raw materials are limited and purification is difficult, resulting in low product purity and high cost; on the other hand, animal-derived SOD carries a high risk of pathogen contamination, severely restricting its safety in clinical applications. Plant-derived SOD, on the other hand, is difficult to apply on a large scale due to complex extraction processes and low activity.

[0004] With advancements in genetic engineering technology, recombinant expression of SOD using microbial systems such as E. coli has become an alternative. However, existing technologies still have the following problems: Due to insufficient enzyme activity and stability, recombinant SOD often exists as inactive inclusion bodies or has significantly lower enzyme activity than natural SOD. For example, Chinese patent CN115896048B discloses a recombinant human Cu / Zn-SOD with high enzyme activity and good stability, as well as its preparation method and uses. The enzyme activity of the recombinant human Cu / Zn-SOD provided is about twice that of natural human Cu / Zn-SOD, and its enzyme activity stability is higher than that of natural human Cu / Zn-SOD. However, the long-term stability of this recombinant human Cu / Zn-SOD is still not ideal. Some studies have improved its heat resistance through fusion expression or point mutation, but this is often accompanied by loss of activity or conformational changes, affecting its functional consistency with human SOD.

[0005] Endotoxin residue problem: E. coli expression systems are prone to introducing endotoxins, which are difficult to completely remove using conventional purification processes, resulting in products that fail to meet the safety standards for injectable drugs or implantable medical devices.

[0006] Furthermore, current technologies mostly focus on optimizing a single performance aspect, failing to comprehensively address the combined needs of high enzyme activity, low endotoxin levels, and long-term stability. To address these issues, we propose a recombinant humanized SOD synthesis process. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a recombinant humanized SOD synthesis process.

[0008] This invention is implemented as follows: a recombinant humanized SOD synthesis process includes the following steps: S10, the gene sequence encoding human copper-zinc superoxide dismutase was optimized by Escherichia coli codon preference to obtain the optimized gene sequence. The nucleotide sequence of the optimized gene is shown in SEQ ID NO:1. Based on the optimized gene sequence, the whole gene was synthesized to obtain the nucleic acid fragment encoding human copper-zinc superoxide dismutase. The Escherichia coli vector for gene expression was constructed to obtain the recombinant expression plasmid. The methods for constructing E. coli vectors for gene expression include: Based on the human SOD whole gene sequence downloaded from NCBI, at least one set of candidate SOD whole genes was obtained. According to the codon usage frequency table of Escherichia coli BL21(DE3) strain, the rare codons in the candidate SOD whole genes were replaced with high-frequency codons of Escherichia coli to obtain the optimized candidate gene sequence. Obtain at least one set of optimized candidate gene sequences. With the goal of minimizing continuous repeating bases and removing internal restriction sites, optimize the secondary structure of the candidate gene sequences and output the optimized gene sequences. The nucleotide sequence of the optimized gene is shown in SEQ ID NO:1, and the optimized gene sequence has ≤80% homology with the known sequence. The optimized gene sequence was chemically synthesized to obtain a nucleic acid fragment encoding human copper-zinc superoxide dismutase. The target fragment was recovered by double digestion with Nco I and Xho I, and ligated overnight at 16°C with the pET-28a(+) vector digested with the same enzymes in the presence of T4 DNA ligase to obtain the enzyme digestion and ligation product. The enzyme digestion and ligation products were transformed into E. coli DH5α competent cells, plated on LB plates containing 50 μg / mL kanamycin, and cultured at 37°C for 12 hours. Single clones were selected for colony PCR verification. Positive clones were extracted and subjected to double enzyme digestion verification and full-length sequencing confirmation to obtain the recombinant expression plasmid pET-28a-hSOD-His.

[0009] S20, the recombinant expression plasmid is transformed into Escherichia coli, the expression level of E. coli containing the recombinant expression plasmid is screened and activated to obtain activated engineered bacteria, the activated engineered bacteria are inoculated in fermentation medium and cultured, and humanized SOD is expressed by temperature induction, and the fermentation cells expressing humanized SOD are collected; The method for screening expression levels and activating E. coli containing recombinant expression plasmids includes: E. coli containing the recombinant expression plasmid were plated on agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 14 hours. Twenty morphologically robust single colonies were selected and inoculated into 5 mL of LB medium, and cultured at 37°C and 220 rpm until OD500. 600 ≥0.6; 0.1 mM IPTG was added to LB medium and induced at 25°C for 6 hours. The bacterial cells in LB medium were collected, centrifuged, and the bacterial cells in the centrifuged liquid were ultrasonically disrupted. The expression level of the target protein was detected by SDS-PAGE, and the bacterial cells with the highest protein expression level were selected as candidate bacterial cells. The specific activity of candidate bacterial cells was measured, and the candidate bacterial cell with the highest specific activity was selected as the target engineered bacteria. The target engineered bacteria were continuously passaged in LB medium for 15 generations. The plasmid retention rate and protein expression level were verified every 5 generations. If the plasmid retention rate and protein expression level error were within ±5%, the target engineered bacteria were determined to be the final selected target engineered bacteria. The target engineered bacteria strain was preserved in glycerol. The target engineered bacterial strain preserved in glycerol was inoculated into 10 mL of LB medium and activated at 37°C and 250 rpm for 12 hours. It was then transferred at a 1:150 ratio to 250 mL of LB medium and cultured until OD (October Expectation) reached. 600 ≥0.9, activated engineered bacteria are obtained.

[0010] S30, the fermentation cells are broken and purified to obtain purified humanized SOD. The humanized SOD is then modified to obtain modified recombinant humanized SOD. The amino acid sequence of the recombinant humanized SOD is shown in SEQ ID NO:2. The modified recombinant humanized SOD is then subjected to ultrafiltration, concentration and freeze-drying to obtain recombinant humanized SOD freeze-dried powder.

[0011] Preferably, the method for inoculating the activated engineered bacteria into a fermentation medium includes: Place the fermentation medium in a fermenter and sterilize the fermenter for 30-35 minutes. Inoculate the activated engineered bacteria into the fermentation medium at an inoculum rate of 5-8%. Adjust the pressure inside the fermenter to 0.04-0.05 MPa and stir the fermentation medium at 220-250 rpm. Control the temperature inside the fermenter at 37℃, dissolved oxygen at 25-30%, and pH at 6.9-7.3 to cultivate the activated engineered bacteria. When the OD of the activated engineered bacteria... 600 The bacterial cell amplification is completed when the temperature reaches 15-20°C. The fermenter is cooled to 24-26℃ at a rate of 0.2-0.8℃ / min, the pressure inside the fermenter is 0.15MPa, the low-temperature induction program for activated engineered bacteria is started, the dissolved oxygen in the fermentation medium is controlled at 40-42%, and IPTG and copper sulfate are added to the fermentation medium. The amount of IPTG added is 0.08-0.12mM / L, the final concentration of copper sulfate is 0.15mM, and the low-temperature induction time is 2-2.2 hours. The fermenter was heated to 28℃ at a rate of 0.2℃ / min, and the pressure inside the fermenter was 0.12MPa. The temperature induction program for activated engineered bacteria was started, the dissolved oxygen in the fermentation medium was controlled at 25%, and zinc sulfate was added to the fermentation medium at a final concentration of 0.15mM. The temperature induction time was 2-4 hours. Carbon source supplement was added during temperature induction to make the specific growth rate 0.1-0.12 / h. The fermenter was heated to 30℃ at a rate of 0.2℃ / min, and the pressure inside the fermenter was 0.15MPa. The temperature induction program for activating engineered bacteria was started. The dissolved oxygen in the fermentation medium was controlled at 50%, and carbon source supplement was added to the fermentation medium to make the specific growth rate 0.1-0.12 / h. The temperature induction time was 5-10 hours. After the induction was completed, the fermentation cells expressing humanized SOD were collected by centrifugation.

[0012] Preferably, the fermentation medium comprises: 10-15 g / L glycerol, 5-10 g / L maltodextrin, 8-12 g / L soybean peptone, 15-20 g / L yeast extract, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L dipotassium hydrogen phosphate, 5-10 g / L sodium glycerophosphate, 1-5 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L stabilizer, 0.5-1 g / L mixed amino acids, and 0.01-0.04 g / L defoamer.

[0013] Preferably, the fermentation medium comprises: 12 g / L glycerol, 8 g / L maltodextrin, 10 g / L soybean peptone, 18 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L dipotassium hydrogen phosphate, 8 g / L sodium glycerophosphate, 3 g / L potassium dihydrogen phosphate, 0.3 g / L stabilizer, 0.6 g / L mixed amino acids, and 0.02 g / L defoamer.

[0014] Preferably, the stabilizing agent includes betaine, sodium ascorbate phosphate, and linoleic acid, with a weight ratio of betaine, sodium ascorbate phosphate, and linoleic acid of 10:7:1. The mixed amino acids include L-cysteine ​​and L-histidine, with a weight ratio of L-cysteine ​​and L-histidine of 1:1. The defoaming agent includes rice bran oil and vitamin E, with a weight ratio of rice bran oil and vitamin E of 2:1.

[0015] Preferably, the carbon source supplement includes 200-250 g / L glycerol, 100-120 g / L glucose, and 0.5 g / L metal auxiliary material, wherein the metal auxiliary material includes copper glycinate and zinc glycinate, and the weight ratio of copper glycinate to zinc glycinate is 1:1.

[0016] Preferably, the method for cleaving and purifying fermentation cells includes: The fermentation cells were washed with a washing buffer at pH 8 to obtain a wet fermentation broth. The washing buffer consisted of 20 mM Tris-HCl, 400 mM sodium chloride, 20 mM imidazole, and 20% (v / v) glycerol. The wet fermentation broth was then subjected to high-pressure homogenization at a pressure of 900-1000 bar, repeated 3-5 times, and in a water bath at 4-10℃. The homogenized liquid was collected and centrifuged to obtain the supernatant. The nickel column was washed with 3-5 times the amount of ultrapure water and equilibrated with 10-20 mM imidazole solution. At 4°C, the supernatant was loaded onto the equilibrated nickel column at a flow rate of 1-1.5 mL / min, and after elution with 30-40 mM imidazole solution to remove impurities, it was eluted with 100-200 mM imidazole solution, and the protein eluent was collected. The protein eluent was loaded into an ion chromatography column, washed with 150 mM sodium chloride, and then purified with 250-300 mM sodium chloride and 0.1% sodium deoxycholate. The purified humanized SOD was obtained by elution with 450-500 mM sodium chloride. The medium of the ion chromatography column was Q Sepharose FF.

[0017] Preferably, the method for modifying humanized SOD includes: A 10% (v / v) rice bran powder solution was prepared using deionized water, and the pH was adjusted to 8.0-8.2. Then, alkaline protease was added to the rice bran powder solution at 55℃ for 2-2.5 hours for hydrolysis. After enzyme inactivation treatment at 95℃ for 10 minutes, the supernatant was collected by centrifugation. The supernatant was then filtered sequentially through 10kDa, 5kDa, and 3kDa ultrafiltration membranes to obtain rice bran polypeptide filtrate with a molecular weight of 1-3kDa. The purified humanized SOD was obtained and diluted with 20 mM sodium phosphate buffer to make the concentration of humanized SOD in the SOD dilution solution 10 mg / mL. 150 mM sodium chloride, 0.5 mM DTT and 7% (v / v) glycerol were added to the SOD dilution solution at the same time. The rice bran peptide filtrate was diluted with 20 mM sodium phosphate buffer to obtain a peptide-modified solution with a final concentration of 30 mg / mL. 10 mM EDC and 6 mM NHS were added as activators and activated at 25°C in the dark for 30-40 min. β-mercaptoethanol was added until the final concentration of β-mercaptoethanol was 10 mM to terminate the activation reaction, thus obtaining the activated peptide-modified solution. The peptide modification solution and 10 mg / mL hyaluronic acid solution were simultaneously added dropwise to the SOD dilution solution. The weight ratio of the peptide modification solution, hyaluronic acid solution and SOD dilution solution was 1:1:5. The reaction was carried out at 300-350 rpm and pH 7-7.2 for 6-7 hours. After the reaction was completed, glycine was added to the mixture to terminate the reaction. The mixture was then allowed to stand at 4°C to obtain the SOD modification solution. Obtain the SOD modification solution, wash the molecular sieve column with 3-5 times the volume of equilibration buffer, concentrate the SOD modification solution to a protein concentration of 10-40 mg / mL using ultrafiltration membrane, filter it through a 0.45 μm filter membrane, load the filtered SOD modification solution into the molecular sieve column, and elute the molecular sieve column with 5-6 times the volume of equilibration buffer to obtain the modified recombinant humanized SOD.

[0018] Preferably, the medium of the molecular sieve column is Sephacryl S-200 HR, the column height is 80-100cm, the diameter is 3-5cm, and the equilibration buffer includes 30mM sodium phosphate buffer, 150mM sodium chloride, 10% (v / v) glycerol, and 2mM DTT.

[0019] Compared with the prior art, the embodiments of this application have the following main advantages: This invention optimizes the gene sequence encoding human copper-zinc superoxide dismutase (SOD) using E. coli codon preference and successfully achieves high-activity, high-conformation-fidelity recombinant human SOD expression using an E. coli expression system. This effectively solves the problems of inclusion body formation, low enzyme activity, and conformational distortion in traditional techniques. The resulting recombinant human SOD lyophilized powder exhibits high specific activity, and endotoxins are deeply removed through synergistic processing using nickel chromatography, ion chromatography, and molecular sieve chromatography, ensuring endotoxin levels meet the standards for injectable and implantable medical devices. Furthermore, the synergistic effect of betaine, sodium ascorbate phosphate, and linoleic acid stabilizers in the fermentation medium allows SOD to maintain excellent enzyme activity over a wide range of conditions, including below 80°C and pH 2-12. The product maintains stability at room temperature for more than two years. The combination of rice bran peptides and hyaluronic acid further enhances its long-lasting effect and tissue targeting, enabling it to demonstrate significant therapeutic and protective effects in various reactive oxygen species (ROS)-related scenarios, including ophthalmic photoaging protection, relief of aseptic oxidative damage in joint inflammation, and skin and mucous membrane protection during radiotherapy and chemotherapy. This overcomes the limitations of existing single-performance optimization technologies in achieving a balance between high enzyme activity, low endotoxin levels, and long-term stability. It possesses the comprehensive advantages of high activity, low endotoxin, and high stability, making it widely applicable in injectable pharmaceuticals, implantable medical devices, and topical medical device preparations. It holds particular value in medical scenarios related to ROS damage, such as ophthalmic photoaging protection, joint inflammation treatment, and skin and mucous membrane protection during radiotherapy and chemotherapy.

[0020] In this embodiment of the invention, when the activated engineered bacteria are inoculated into the fermentation medium, cell amplification is carried out under suitable dissolved oxygen conditions to ensure rapid biomass accumulation and provide sufficient cell numbers for subsequent expression. Subsequently, low-temperature induction is initiated by slowly cooling to 24-26℃ and adding low doses of IPTG and copper sulfate. This reduces the damage to the SOD conformation caused by high temperature and promotes the stable binding of copper ions to the protein active site, thereby enhancing enzyme activity. Then, during the temperature variation stage at 28℃, zinc sulfate is added and carbon source is used to maintain a suitable specific growth rate, further optimizing zinc ion incorporation and protein folding, and reducing inclusion body formation. Then, the temperature is raised to 30℃ and dissolved oxygen and carbon source supply are increased for long-term induction to promote efficient SOD secretion and accumulation, while maintaining a stable oxidative environment to ensure high product activity and high purity. By employing a multi-stage controlled temperature, oxygen, and inducer method for cultivation, the problems of high-temperature inactivation, uneven incorporation of metal cofactors, and low yield in traditional E. coli expression were effectively solved. Furthermore, by precisely controlling temperature, pressure, dissolved oxygen, and inducer addition in stages, the efficient amplification of activated engineered bacteria and the controllable expression of recombinant humanized SOD were achieved, significantly improving the specific activity and stability of the product.

[0021] In this embodiment of the invention, the composite fermentation medium, through the scientific ratio and synergistic effect of its components, achieves a balance between high-density fermentation and high-activity SOD expression. The slow-release carbon source combination of glycerol and maltodextrin utilizes the rapid availability of glycerol to meet the initial growth requirements of the cells, while the slow degradation of maltodextrin maintains a stable carbon source supply in the later stages of induction, avoiding concentration fluctuations and metabolic imbalances caused by a single carbon source, thus stabilizing the specific growth rate within the ideal range. Using soybean peptone as a plant-derived nitrogen source not only provides abundant amino acids and growth factors, but more importantly, significantly reduces the endotoxin background compared to animal-derived peptone, reducing the endotoxin load of the final product from the source. The organic-inorganic complex composed of sodium glycerophosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate... The phosphorus source system provides ample phosphorus while reducing metal ion precipitation through the chelation of organophosphorus compounds, enhancing buffering capacity and maintaining pH stability during fermentation. A stabilizing agent composed of betaine, sodium ascorbate phosphate, and linoleic acid works synergistically to protect the SOD active site from oxidative stress damage during fermentation. This is achieved through the osmotic pressure regulation and protein stabilization of betaine, the antioxidant protection of sodium ascorbate phosphate, and the membrane stability maintenance of linoleic acid. A natural defoamer composed of rice bran oil and vitamin E effectively controls foam while avoiding the cytotoxicity and product contamination that chemical defoamers may cause. Furthermore, the antioxidant properties of vitamin E further enhance the stability of SOD, resulting in significantly better performance than traditional LB medium or simple composite mediums.

[0022] In this embodiment of the invention, a dual modification strategy using rice bran peptides and hyaluronic acid was employed to comprehensively enhance the stability, targeting, and functionality of SOD. The rice bran peptides were obtained through controlled hydrolysis by alkaline protease and three-stage ultrafiltration, preserving the antioxidant active peptides in rice bran. As a modifier, they not only provide steric hindrance protection but also form a synergistic antioxidant effect with SOD. The synergistic effect of the dual modification strategy using rice bran peptides and hyaluronic acid significantly improves the thermal stability of the final product, broadens the pH tolerance range to 2-12, and extends the room temperature storage stability to more than 2 years. At the same time, the CD44 receptor targeting provided by hyaluronic acid enriches it at inflammatory sites, and the synergistic antioxidant effect of rice bran peptides enhances the overall free radical scavenging ability. Attached Figure Description

[0023] Figure 1 The diagram shows the thermal stability test results of the recombinant humanized SOD prepared in Examples 1-5 and Comparative Examples 1-4 of this invention.

[0024] Figure 2 The diagram shows the pH stability test results of the recombinant humanized SOD prepared in Examples 1-5 and Comparative Examples 1-4 of this invention.

[0025] Figure 3 The diagram shows the activity test results of the recombinant humanized SOD enzyme prepared in Example 5 and Comparative Examples 1-4 of the present invention.

[0026] Figure 4 The diagram shows the animal efficacy results of the recombinant humanized SOD prepared in Example 5 and Comparative Examples 1-4 of this invention in alleviating rheumatoid arthritis. Detailed Implementation

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0028] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0029] Example 1 This invention provides a recombinant humanized SOD synthesis process, which includes the following steps: S10, the gene sequence encoding human copper-zinc superoxide dismutase was optimized by Escherichia coli codon preference to obtain the optimized gene sequence. The nucleotide sequence of the optimized gene is shown in SEQ ID NO:1. Based on the optimized gene sequence, the whole gene was synthesized to obtain the nucleic acid fragment encoding human copper-zinc superoxide dismutase. The Escherichia coli vector for gene expression was constructed to obtain the recombinant expression plasmid. The methods for constructing E. coli vectors for gene expression include: S101. Based on the human SOD whole gene sequence downloaded from NCBI, at least one set of candidate SOD whole genes was obtained. According to the codon usage frequency table of E. coli BL21(DE3) strain, rare codons in the candidate SOD whole genes were replaced with high-frequency codons of E. coli to obtain optimized candidate gene sequences. Among them, the tRNA abundance in the BL21(DE3) strain is low, which can easily lead to translation pause and ribosome shedding. After codon optimization, the codons were replaced with high-frequency codons, which can improve the translation speed and reduce the truncated proteins that are not fully translated. In this embodiment, the BL21(DE3) strain-specific codon usage frequency table should be used to avoid the deviation between the general table and the actual strain tRNA library, and only rare codons with a usage frequency of <10% are replaced. S102, obtain at least one set of optimized candidate gene sequences, optimize the secondary structure of the candidate gene sequences with the goal of minimizing continuous repeating bases and removing internal restriction sites, and output the optimized gene sequences. The nucleotide sequence of the optimized gene is shown in SEQ ID NO:1, and the optimized gene sequence has ≤80% homology with the known sequence. It retains the amino acid sequence and reduces the difficulty of synthesis through base rearrangement, avoiding deletions / mutations in the synthesis of long fragments. S103, the optimized gene sequence was chemically synthesized to obtain a nucleic acid fragment encoding human copper zinc superoxide dismutase. The target fragment was recovered by double digestion with Nco I and Xho I, and ligated overnight at 16°C with the pET-28a(+) vector digested with the same enzymes in the presence of T4 DNA ligase to obtain the enzyme digestion and ligation product. S104. The enzyme digestion and ligation product was transformed into E. coli DH5α competent cells. E. coli DH5α competent cells could efficiently take up the ligation product. The product was plated on LB agar plates containing 50 μg / mL kanamycin and cultured at 37°C for 12 hours. Single clones were selected for colony PCR verification. The plasmid was extracted from positive clones and verified by double enzyme digestion and full-length sequencing to obtain the recombinant expression plasmid pET-28a-hSOD-His.

[0030] In this embodiment of the invention, when constructing the E. coli vector for gene expression, a recombinant expression plasmid pET-28a-hSOD-His, which is homologous to human SOD sequence, codon-adapted to E. coli, free from internal enzyme digestion interference, and has high genetic stability, was successfully constructed. This provides a core gene vector for the subsequent fermentation expression of highly active and conformationally faithful SOD, thereby laying the molecular foundation for the industrialization of injectable / implantable SOD products.

[0031] S20 involves transforming recombinant expression plasmids into *E. coli*, screening and activating the *E. coli* containing the recombinant expression plasmids to obtain activated engineered bacteria. These activated engineered bacteria are then inoculated into fermentation medium and cultured. Humanized SOD is expressed through temperature induction, and the fermentation cells expressing humanized SOD are collected. High-expression, genetically stable engineered strains are obtained through expression screening and activation. A three-stage gradient induction strategy (low temperature, variable temperature, and temperature increase), combined with an optimized fermentation medium containing a combination of slow-release carbon sources, plant-derived nitrogen sources, stabilizers, and mixed amino acids, along with precise feedstock control of carbon source supplements and metal additives, enables high-density fermentation and high-level soluble expression of the activated engineered bacteria. The method for screening expression levels and activating E. coli containing recombinant expression plasmids includes: S201: E. coli containing the recombinant expression plasmid were plated on agar plates containing 50 μg / mL kanamycin and incubated at 37°C for 14 hours. Twenty morphologically robust single colonies were selected and inoculated into 5 mL of LB medium, and cultured at 37°C and 220 rpm until OD500. 600 ≥0.6; S202, 0.1 mM IPTG was added to LB medium and induced at 25℃ for 6 hours. The bacterial cells in LB medium were collected, centrifuged, and the bacterial cells in the centrifuged liquid were ultrasonically disrupted. The expression level of the target protein was detected by SDS-PAGE, and the bacterial cells with the highest protein expression level were selected as candidate bacterial cells. S203, measure the specific activity of candidate bacterial cells, and select the candidate bacterial cell with the highest specific activity as the target engineered bacteria. The target engineered bacteria are continuously passaged in LB medium for 15 generations. Every 5 generations, the plasmid retention rate and protein expression level are verified. If the plasmid retention rate and protein expression level error are within ±5%, it is determined to be the final selected target engineered bacteria. The target engineered bacteria strain is preserved in glycerol. S204: The target engineered bacterial strain preserved in glycerol was inoculated into 10 mL of LB medium and activated at 37°C and 250 rpm for 12 hours. It was then transferred at a 1:150 ratio to 250 mL of LB medium and cultured until OD (October Expectation) reached. 600 ≥0.9, activated engineered bacteria are obtained.

[0032] S30, the fermentation cells are broken and purified to obtain purified humanized SOD. The humanized SOD is then modified to obtain modified recombinant humanized SOD. The amino acid sequence of the recombinant humanized SOD is shown in SEQ ID NO:2. The modified recombinant humanized SOD is then subjected to ultrafiltration, concentration and freeze-drying to obtain recombinant humanized SOD freeze-dried powder.

[0033] In this embodiment of the invention, the method for inoculating the activated engineered bacteria into a fermentation medium includes: S301: Place the fermentation medium in the fermenter, sterilize the fermenter for 30 minutes, and inoculate the activated engineered bacteria at a rate of 5% into the fermentation medium. Adjust the pressure inside the fermenter to 0.04 MPa, stir the fermentation medium at 220 rpm, and control the temperature inside the fermenter at 37℃, dissolved oxygen at 25%, and pH at 6.9-7.3 to cultivate the activated engineered bacteria. When the OD of the activated engineered bacteria... 600 The bacterial cell amplification was completed at 15:00. S302, the fermenter was cooled to 24℃ at a rate of 0.2℃ / min, the pressure inside the fermenter was 0.15MPa, the low-temperature induction program for activated engineered bacteria was started, the dissolved oxygen in the fermentation medium was controlled at 40%, and IPTG and copper sulfate were added to the fermentation medium. The amount of IPTG added was 0.08mM / L, and the final concentration of copper sulfate was 0.15mM. The low-temperature induction time was 2 hours. Among them, the addition of copper sulfate during the low-temperature stage is conducive to the stable coordination of copper ions with cysteine / histidine residues in the active site of SOD, improving the binding rate of metal cofactors, thereby significantly improving the specific enzyme activity. S303, the fermenter is heated to 28℃ at a rate of 0.2℃ / min, the pressure inside the fermenter is 0.12MPa, the temperature induction program for activated engineered bacteria is started, the dissolved oxygen in the fermentation medium is controlled at 25%, and zinc sulfate is added to the fermentation medium with a final concentration of 0.15mM. The temperature induction time is 2 hours. Carbon source supplement is added during temperature induction to make the specific growth rate 0.1 / h. Controlling the specific growth rate avoids excessive cell proliferation and consumption of nutrients, thereby ensuring that carbon source and energy are preferentially used for SOD expression and modification. S304, the fermenter was heated to 30℃ at a rate of 0.2℃ / min, the pressure inside the fermenter was 0.15MPa, the temperature induction program for activating engineered bacteria was started, the dissolved oxygen in the fermentation medium was controlled at 50%, and carbon source supplement was added to the fermentation medium to make the specific growth rate 0.1 / h, the temperature induction time was 5 hours, and after the induction was completed, the fermentation cells expressing humanized SOD were collected by centrifugation.

[0034] In this embodiment of the invention, when the activated engineered bacteria are inoculated into the fermentation medium, cell amplification is carried out under suitable dissolved oxygen conditions to ensure rapid biomass accumulation and provide sufficient cell numbers for subsequent expression. Subsequently, low-temperature induction is initiated by slowly cooling to 24-26℃ and adding low doses of IPTG and copper sulfate. This reduces the damage to the SOD conformation caused by high temperature and promotes the stable binding of copper ions to the protein active site, thereby enhancing enzyme activity. Then, during the temperature variation stage at 28℃, zinc sulfate is added and carbon source is used to maintain a suitable specific growth rate, further optimizing zinc ion incorporation and protein folding, and reducing inclusion body formation. Then, the temperature is raised to 30℃ and dissolved oxygen and carbon source supply are increased for long-term induction to promote efficient SOD secretion and accumulation, while maintaining a stable oxidative environment to ensure high product activity and high purity. By employing a multi-stage controlled temperature, oxygen, and inducer method for cultivation, the problems of high-temperature inactivation, uneven incorporation of metal cofactors, and low yield in traditional E. coli expression were effectively solved. Furthermore, by precisely controlling temperature, pressure, dissolved oxygen, and inducer addition in stages, the efficient amplification of activated engineered bacteria and the controllable expression of recombinant humanized SOD were achieved, significantly improving the specific activity and stability of the product.

[0035] In this embodiment of the invention, the fermentation medium comprises: 10 g / L glycerol, 5 g / L maltodextrin, 8 g / L soybean peptone, 15 g / L yeast extract, 0.5 g / L magnesium sulfate heptahydrate, 1 g / L dipotassium hydrogen phosphate, 5 g / L sodium glycerophosphate, 1 g / L potassium dihydrogen phosphate, 0.1 g / L stabilizer, 0.5 g / L mixed amino acids, and 0.01 g / L defoamer.

[0036] The stabilizer includes betaine, sodium ascorbate phosphate, and linoleic acid, with a weight ratio of 10:7:1. The mixed amino acids include L-cysteine ​​and L-histidine, with a weight ratio of 1:1. The defoamer includes rice bran oil and vitamin E, with a weight ratio of 2:1.

[0037] In this embodiment of the invention, the composite fermentation medium, through the scientific ratio and synergistic effect of its components, achieves a balance between high-density fermentation and high-activity SOD expression. The slow-release carbon source combination of glycerol and maltodextrin utilizes the rapid availability of glycerol to meet the initial growth requirements of the cells, while the slow degradation of maltodextrin maintains a stable carbon source supply in the later stages of induction, avoiding concentration fluctuations and metabolic imbalances caused by a single carbon source, thus stabilizing the specific growth rate within the ideal range. Using soybean peptone as a plant-derived nitrogen source not only provides abundant amino acids and growth factors, but more importantly, significantly reduces the endotoxin background compared to animal-derived peptone, reducing the endotoxin load of the final product from the source. The organic-inorganic complex composed of sodium glycerophosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate... The phosphorus source system provides ample phosphorus while reducing metal ion precipitation through the chelation of organophosphorus compounds, enhancing buffering capacity and maintaining pH stability during fermentation. A stabilizing agent composed of betaine, sodium ascorbate phosphate, and linoleic acid works synergistically to protect the SOD active site from oxidative stress damage during fermentation. This is achieved through the osmotic pressure regulation and protein stabilization of betaine, the antioxidant protection of sodium ascorbate phosphate, and the membrane stability maintenance of linoleic acid. A natural defoamer composed of rice bran oil and vitamin E effectively controls foam while avoiding the cytotoxicity and product contamination that chemical defoamers may cause. Furthermore, the antioxidant properties of vitamin E further enhance the stability of SOD, resulting in significantly better performance than traditional LB medium or simple composite mediums.

[0038] In preparing the fermentation medium, 4L of deionized water was added, and then the mixture was stirred at 100rpm. Based on the formulation of the fermentation medium, glycerol, maltodextrin, soybean peptone, and yeast extract were added sequentially while stirring to ensure complete dissolution. Then, based on the formulation, magnesium sulfate heptahydrate, dipotassium hydrogen phosphate, sodium glycerophosphate, stabilizer, mixed amino acids, and defoamer were added. After the formulation was completed, the volume was adjusted to 5L with deionized water, and the mixture was stirred at 100rpm for 10 minutes. The pH was measured and adjusted to 7.1 using hydrochloric acid or sodium hydroxide solution to obtain the fermentation medium.

[0039] In this embodiment of the invention, the carbon source supplement includes 200 g / L glycerol, 100 g / L glucose, and 0.5 g / L metal auxiliary material. The metal auxiliary material includes copper glycinate and zinc glycinate, and the weight ratio of copper glycinate and zinc glycinate is 1:1.

[0040] In this embodiment of the invention, the method for cleaving and purifying fermentation cells includes: S401, the fermentation cells were washed with a washing buffer at pH 8 to obtain a wet cell fermentation broth. The washing buffer included 20 mM Tris-HCl, 400 mM sodium chloride, 20 mM imidazole, and 20% (v / v) glycerol. The wet cell fermentation broth was then subjected to high-pressure homogenization at a pressure of 900 bar, repeated 3 times, and in a 4°C water bath. The high-pressure homogenized liquid was collected and centrifuged to obtain the supernatant. S402, the nickel column was washed with three times the amount of ultrapure water as the nickel column, and the nickel column was equilibrated with a 10 mM imidazole solution; S403, at 4℃, the supernatant was loaded onto the equilibrated nickel column at a flow rate of 1 mL / min, and after elution with 30 mM imidazole solution to remove impurities, it was eluted with 100 mM imidazole solution and the protein eluent was collected. S404, the protein eluent is loaded into the ion chromatography column, the ion chromatography column is washed with 150 mM sodium chloride, then impurities are removed with 250 mM sodium chloride and 0.1% sodium deoxycholate, and purified humanized SOD is obtained by elution with 450 mM sodium chloride. The medium of the ion chromatography column is Q Sepharose FF.

[0041] In this embodiment of the invention, during the lysis and purification of fermentation cells, the cell wall / membrane is destroyed by physical shearing force, allowing for the full release of intracellular SOD. At the same time, the low-temperature environment inhibits protease activity and oxidative stress, preventing the degradation or inactivation of the active site. After centrifugation, a high-yield supernatant containing SOD is obtained. The His-tagged SOD is efficiently captured and preliminarily purified by nickel column affinity chromatography. The ion chromatography column uses Q Sepharose FF medium. The endotoxin removal strategy involves preliminary washing with 150mM sodium chloride and a combination of 250-300mM sodium chloride and 0.1% sodium deoxycholate. The high efficiency of endotoxin removal is achieved by utilizing the property of sodium deoxycholate to destroy lipopolysaccharide aggregates.

[0042] In this embodiment of the invention, the method for modifying humanized SOD includes: S501 uses deionized water to prepare a 10% (v / v) rice bran powder solution, adjusts the pH to 8.0, then adds alkaline protease to the rice bran powder solution at 55℃ for 2 hours for hydrolysis, and after enzyme inactivation treatment at 95℃ for 10 minutes, centrifuges to collect the supernatant. The supernatant after centrifugation is then filtered through 10kDa, 5kDa, and 3kDa ultrafiltration membranes to obtain rice bran peptide filtrate with a molecular weight of 2kDa. In this process, the rice bran protein is hydrolyzed in a controlled manner by alkaline protease, which can release small molecule peptides rich in hydrophobic amino acids and active groups. These small molecule peptides have potential antioxidant, membrane protection, and inflammation regulation effects, and can form a functional synergy with SOD. Moreover, the small molecule peptides are more likely to form multi-point binding with the surface of SOD and hyaluronic acid, increasing the modification density and stability, while reducing the influence of steric hindrance on the active site of SOD. S502, obtain purified humanized SOD, dilute humanized SOD with 20mM sodium phosphate buffer to make the concentration of humanized SOD in the SOD dilution solution 10mg / mL, and add 150mM sodium chloride, 0.5mM DTT and 7% (v / v) glycerol to the SOD dilution solution at the same time. S503, the rice bran peptide filtrate was diluted with 20mM sodium phosphate buffer to obtain a peptide modification solution with a final concentration of 30mg / mL. 10mM EDC and 6mM NHS were added as activators, and the mixture was activated at 25℃ in the dark for 30min. β-mercaptoethanol was added until the final concentration of β-mercaptoethanol was 10mM to terminate the activation reaction, resulting in the activated peptide modification solution. EDC / NHS can activate the carboxyl group of rice bran peptide to form an amine-reactive ester, which enables it to efficiently crosslink with the amino groups of SOD and hyaluronic acid in subsequent steps to form a stable amide bond. S504: Peptide modification solution and 10 mg / mL hyaluronic acid solution were simultaneously added dropwise to SOD dilution solution. The weight ratio of peptide modification solution, hyaluronic acid solution and SOD dilution solution was 1:1:5. The reaction was carried out at 300 rpm and pH 7 for 6 hours. After the reaction was completed, glycine was added to the mixture to terminate the reaction. The mixture was allowed to stand at 4°C to obtain SOD modification solution. In this solution, bran peptide provides antioxidant / anti-inflammatory auxiliary functions, while hyaluronic acid provides moisturizing, lubrication, targeting and barrier penetration capabilities. The co-modification of both with SOD achieves a synergistic effect of antioxidant and barrier protection. The resulting modified recombinant humanized SOD has high activity, high stability, targeting and biocompatibility, and is suitable for diversified application scenarios such as pharmaceutical injections, medical device implants and topical preparations. S505. Obtain the SOD modification solution. Wash the molecular sieve column with 3-5 times the volume of equilibration buffer. Concentrate the SOD modification solution using ultrafiltration to a protein concentration of 20 mg / mL, and filter it through a 0.45 μm filter membrane. Load the filtered SOD modification solution into the molecular sieve column and elute the column with 5 times the volume of equilibration buffer to obtain the modified recombinant humanized SOD. The medium of the molecular sieve column is Sephacryl S-200 HR, with a column height of 80 cm and a diameter of 4 cm. The equilibration buffer includes 30 mM sodium phosphate buffer, 150 mM sodium chloride, 10% (v / v) glycerol, and 2 mM DTT. The use of a high-strength Sephacryl S-200 HR column combined with the buffer system can effectively separate the free modifier and the modification product, thereby obtaining modified SOD with uniform molecular weight and low dispersion. The synergistic effect of the dual modification strategy significantly improves the thermal stability of the final product, broadens the pH tolerance range to 2-12, and extends the room temperature storage stability to more than 2 years. At the same time, the CD44 receptor targeting of hyaluronic acid makes it accumulate at the site of inflammation, and the antioxidant synergistic effect of rice bran peptides enhances the overall free radical scavenging ability.

[0043] In this embodiment of the invention, a dual modification strategy using rice bran peptides and hyaluronic acid was employed to comprehensively enhance the stability, targeting, and functionality of SOD. The rice bran peptides were obtained through controlled hydrolysis by alkaline protease and three-stage ultrafiltration, preserving the antioxidant active peptides in rice bran. As a modifier, they not only provide steric hindrance protection but also form a synergistic antioxidant effect with SOD. The synergistic effect of the dual modification strategy using rice bran peptides and hyaluronic acid significantly improves the thermal stability of the final product, broadens the pH tolerance range to 2-12, and extends the room temperature storage stability to more than 2 years. At the same time, the CD44 receptor targeting provided by hyaluronic acid enriches it at inflammatory sites, and the synergistic antioxidant effect of rice bran peptides enhances the overall free radical scavenging ability.

[0044] This invention optimizes the gene sequence encoding human copper-zinc superoxide dismutase (SOD) using E. coli codon preference and successfully achieves high-activity, high-conformation-fidelity recombinant human SOD expression using an E. coli expression system. This effectively solves the problems of inclusion body formation, low enzyme activity, and conformational distortion in traditional techniques. The resulting recombinant human SOD lyophilized powder exhibits high specific activity, and endotoxins are deeply removed through synergistic processing using nickel chromatography, ion chromatography, and molecular sieve chromatography, ensuring endotoxin levels meet the standards for injectable and implantable medical devices. Furthermore, the synergistic effect of betaine, sodium ascorbate phosphate, and linoleic acid stabilizers in the fermentation medium allows SOD to maintain excellent enzyme activity over a wide range of conditions, including below 80°C and pH 2-12. The product maintains stability at room temperature for more than two years. The combination of rice bran peptides and hyaluronic acid further enhances its long-lasting effect and tissue targeting, enabling it to demonstrate significant therapeutic and protective effects in various reactive oxygen species (ROS)-related scenarios, including ophthalmic photoaging protection, relief of aseptic oxidative damage in joint inflammation, and skin and mucous membrane protection during radiotherapy and chemotherapy. This overcomes the limitations of existing single-performance optimization technologies in achieving a balance between high enzyme activity, low endotoxin levels, and long-term stability. It possesses the comprehensive advantages of high activity, low endotoxin, and high stability, making it widely applicable in injectable pharmaceuticals, implantable medical devices, and topical medical device preparations. It holds particular value in medical scenarios related to ROS damage, such as ophthalmic photoaging protection, joint inflammation treatment, and skin and mucous membrane protection during radiotherapy and chemotherapy.

[0045] Example 2 In this embodiment of the invention, the recombinant humanized SOD synthesis process steps are the same as in Example 1. The fermentation medium includes: 15 g / L glycerol, 10 g / L maltodextrin, 12 g / L soybean peptone, 20 g / L yeast extract, 1 g / L magnesium sulfate heptahydrate, 5 g / L dipotassium hydrogen phosphate, 10 g / L sodium glycerophosphate, 5 g / L potassium dihydrogen phosphate, 0.5 g / L stabilizer, 1 g / L mixed amino acids, and 0.04 g / L defoamer.

[0046] Example 3 In this embodiment of the invention, the recombinant humanized SOD synthesis process steps are the same as in Example 1. The fermentation medium includes: 11 g / L glycerol, 6 g / L maltodextrin, 9 g / L soybean peptone, 16 g / L yeast extract, 0.6 g / L magnesium sulfate heptahydrate, 2 g / L dipotassium hydrogen phosphate, 6 g / L sodium glycerophosphate, 1.5 g / L potassium dihydrogen phosphate, 0.2 g / L stabilizer, 0.6 g / L mixed amino acids, and 0.02 g / L defoamer.

[0047] Example 4 In this embodiment of the invention, the recombinant humanized SOD synthesis process steps are the same as in Example 1. The fermentation medium includes: 14 g / L glycerol, 9 g / L maltodextrin, 11 g / L soybean peptone, 19 g / L yeast extract, 0.9 g / L magnesium sulfate heptahydrate, 4 g / L dipotassium hydrogen phosphate, 9 g / L sodium glycerophosphate, 4 g / L potassium dihydrogen phosphate, 0.5 g / L stabilizer, 1 g / L mixed amino acids, and 0.04 g / L defoamer.

[0048] Example 5 In this embodiment of the invention, the recombinant humanized SOD synthesis process steps are the same as in Example 1. The fermentation medium includes: 12 g / L glycerol, 8 g / L maltodextrin, 10 g / L soybean peptone, 18 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L dipotassium hydrogen phosphate, 8 g / L sodium glycerophosphate, 3 g / L potassium dihydrogen phosphate, 0.3 g / L stabilizer, 0.6 g / L mixed amino acids, and 0.02 g / L defoamer.

[0049] Comparative Example 1 In this comparative example, when the activated engineered bacteria were inoculated into the fermentation medium, the fermentation medium was LB medium, and everything else was the same as in Example 1.

[0050] Comparative Example 2 In this comparative example, when the activated engineered bacteria were inoculated into the fermentation medium, the fermentation medium was LB medium, the carbon source supplement was glycerol 240g / L, and the rest was the same as in Example 1.

[0051] Comparative Example 3 In this comparative example, in step S10, the gene sequence encoding human copper-zinc superoxide dismutase was not optimized for E. coli codon preference, and the gene sequence encoding human copper-zinc superoxide dismutase was a natural human SOD sequence, with the amino acid sequence being 100% identical to the SOD produced by the human body. Other aspects were the same as in Example 1.

[0052] Comparative Example 4 In this comparative example, the recombinant humanized SOD is a commercially available product.

[0053] Performance testing: Thermal stability test: Recombinant humanized SOD prepared in Examples 1-5 and Comparative Examples 1-4 of this invention were placed at -20°C for 2 hours, then incubated in an 80°C water bath for 2 hours. After five cycles, the SOD enzyme activity in the recombinant humanized SOD was tested. The SOD enzyme activity was detected according to the method provided in GB / T41906-2022 Superoxide Dismutase Activity Detection Method. The enzyme activity test results (relative SOD enzyme activity retention rate) after five cycles are shown in Table 1 and... Figure 1 As shown.

[0054] Table 1 As shown in Table 1, the recombinant humanized SOD prepared in Examples 1-5 of this invention retained a relative activity of ≥80% after 5 cycles at -20℃ / 80℃. The thermal stability among the comparative examples showed a trend of Comparative Example 1 > Comparative Example 2 > Comparative Example 3 > Comparative Example 4, indicating that this invention can significantly improve the freeze-thaw-thermal shock stability of recombinant humanized SOD by optimizing the gene sequence, the special fermentation medium, and the stabilizing agent system. In addition, the thermal stability of the products in the comparative examples that relied only on conventional culture medium or natural gene sequence gradually decreased with the increase of process defects. Therefore, this invention has significant advantages in terms of high activity retention, long shelf life, and applicability to multiple scenarios. It can solve the technical bottlenecks of existing recombinant SOD that are prone to thermal inactivation and require strict cold chain during storage and use, and provide a solid quality foundation for its industrial application in the fields of injections, implantable devices, and topical preparations.

[0055] pH stability test: Take the recombinant humanized SOD prepared in Examples 1-5 and Comparative Examples 1-4 of this invention, reconstitute it with 20mM sodium phosphate buffer and dilute it to 1mg / mL, dispense it into several equal portions and avoid repeated freeze-thaw cycles, and then prepare a series of buffer solutions according to the pH range (2.0-12.0). The buffer solutions were divided into acidic (pH=2), neutral (pH=7), and alkaline (pH=12) groups. PBS (pH=7.0) served as a negative control, and untreated SOD was used as an initial activity reference. The aliquoted SOD samples were added to buffer solutions of different pH values ​​at a volume ratio of 1:9 to maintain a final protein concentration of 0.1 mg / mL. The sample buffer solutions at different pH values ​​were incubated in a 25°C water bath for 24 hours. Using the PBS group's activity at 0 hours as a baseline of 100%, the relative activity retention rate of each pH group after 24 hours of incubation was calculated. The pH stability test results after 24 hours (relative SOD enzyme activity retention rate of each pH group after 24 hours) are shown in Table 2. Figure 2 As shown.

[0056] Table 2 As shown in Table 2, the recombinant humanized SOD prepared in Examples 1-5 of this invention exhibited better relative activity retention rates than comparative examples 1-4 after incubation at pH 2.0, 7.0, and 12.0 for 24 hours. The recombinant humanized SOD prepared in these examples also maintained over 70% activity under extreme acid / alkaline conditions. This indicates that the rice bran polypeptide-hyaluronic acid double-modified layer forms a physicochemical barrier and buffers the direct attack of extreme pH on the protein's active site; the codon optimization ensures the correct folding structure, conferring inherent conformational stability to the protein; and the robust Cu / Zn coordination with in-situ metal integration prevents cofactor detachment within the pH range of 2-12. In contrast, Comparative Example 1 lacked modification protection, Comparative Example 2 suffered from increased pH sensitivity due to metabolic damage, Comparative Example 3's inclusion body heterogeneity led to rapid dissociation at extreme pH levels, and Comparative Example 4, a conventional industrial product, exhibited the worst pH tolerance due to the accumulation of multiple defects. These results demonstrate that the present invention, by optimizing the gene sequence, the dedicated fermentation medium, and the stabilizing and protective agent system, can significantly improve the conformational and activity retention capabilities of recombinant humanized SOD over a wide pH range, thus solving the technical problems of easy inactivation and limited applicability of existing processes and commercially available products under acidic / alkaline environments.

[0057] Endotoxin and purity tests: The recombinant humanized SOD prepared in Examples 1-5 and Comparative Examples 1-4 of this invention were tested for purity and endotoxin. The purity of the recombinant humanized SOD was determined by SDS-PAGE qualitative method, and the endotoxin of the recombinant humanized SOD was determined by dynamic turbidimetric assay with Limulus amebocyte lysate (LAL) reagent. The results of the endotoxin (EU / mL) and purity (%) tests are shown in Table 3.

[0058] Table 3 As shown in Table 3, the purity and endotoxin detection effects of Examples 1-5 of the present invention are significantly better than those of Comparative Examples 1-4. The embodiments of the present invention use plant-derived culture medium to reduce endotoxin load from the source, sodium deoxycholate ion exchange chromatography to achieve specific removal, and molecular sieve chromatography to ensure final purity. Through synergistic cooperation, the endotoxin level is reduced.

[0059] Enzyme activity test: The recombinant humanized SOD prepared in Example 5 and Comparative Examples 1-4 of this invention was incubated in an 80℃ water bath for 4 hours. The activity was tested according to the method provided in GB / T41906-2022, specifically the initial specific activity (U / mg), the specific activity after 2 hours of incubation, and the specific activity after 4 hours of incubation. The enzyme activity test results are shown in Table 4. Figure 3 As shown.

[0060] Table 4 Table 4 shows that the initial specific activity of Example 5 was significantly higher than that of Comparative Examples 1-4. After 4 hours of high temperature, the enzyme activity could still retain more than 80% of the initial activity. In contrast, Comparative Examples 1-4 suffered more activity loss due to one or more process defects. Therefore, the present invention achieves the initial specific activity and high temperature stability of recombinant humanized SOD through the synergistic combination of gene optimization, special culture medium, stabilizing agent, and multi-step purification.

[0061] Animal remission experiment of rheumatoid arthritis: A model of rheumatoid arthritis was established using Lewis rats (sensitive to adjuvants, easy to model, with an immune mechanism similar to human RA). The rats were male, weighing 180-200g. 0.1 mL of the drug was injected subcutaneously into the right hind paw of each rat. CFA emulsion was injected to avoid puncturing the plantar fascia and reduce mechanical damage to swelling. Swelling rate was measured on days 7, 14, and 21 after modeling. A swelling rate ≥50% indicated successful modeling. Then, recombinant humanized SOD prepared in Example 5 and Comparative Examples 1-4 of this invention was administered. The administration method was once daily, 5000U each time, with an interval of five days between injections. The remission rate was tested after 28 days. A comprehensive remission score (0-10 points) was calculated based on foot and claw score (0-4 points), functional score (0-3 points), and histological score (0-3 points). The remission rate per animal was calculated as (1 − comprehensive score / 10) × 100%, and the group remission rate was the mean remission rate of all groups. The remission rate test results are shown in Table 5. Figure 4 As shown.

[0062] The swelling score for the paw (0-4 points / paw, taking the average of the left and right hind feet) is as follows: 0 points - swelling rate ≤10% (basically back to normal); 1 point - swelling rate 11-25%; 2 points - swelling rate 26-50%; 3 points - swelling rate 51-75%; 4 points - swelling rate >75%.

[0063] Joint function score (0-3 points / animal): 0 points - free movement, no lameness, can grasp and climb normally; 1 point - mild lameness, slight discomfort when running; 2 points - obvious lameness, limited jumping, often lifts the affected foot; 3 points - severe lameness, unable to bear weight, difficulty moving.

[0064] Histological improvement score (0-3 points / animal, based on joint sections on day 28): 0 points - synovium is basically normal, with no inflammatory cell infiltration and no bone erosion; 1 point - mild synovial hyperplasia, a small amount of lymphocyte infiltration, no / very slight bone erosion; 2 points - moderate synovial hyperplasia, obvious inflammatory infiltration, and small-scale bone erosion; 3 points - severe synovial hyperplasia, a large number of inflammatory cells, and extensive bone destruction.

[0065] Table 5 As shown in Table 5, the remission rate of Example 5 of the present invention is higher than that of Comparative Examples 1-4 within a 28-day dosing cycle. This indicates that the high specific activity of the present invention ensures sufficient effective dose; while the high thermal stability ensures long-term sustained release in vivo, maintaining an effective concentration even after dosing at 5-day intervals; and the low endotoxin avoids interference from exogenous inflammation, thus solving the technical problem of insufficient remission rate caused by low activity and poor stability in existing processes and commercially available products.

[0066] In summary, this invention provides a recombinant humanized SOD synthesis process. By optimizing the gene sequence encoding human copper-zinc superoxide dismutase using E. coli codon preference, this invention successfully achieves high-activity and high-conformation-fidelity recombinant humanized SOD expression using an E. coli expression system. This effectively solves the problems of inclusion body formation, low enzyme activity, and conformational distortion in traditional techniques. The resulting recombinant humanized SOD lyophilized powder has high specific activity, and endotoxins can be deeply removed through synergistic treatment by nickel chromatography, ion chromatography, and molecular sieve chromatography, ensuring endotoxin levels meet the standards for injectable and implantable medical devices. Simultaneously, the synergistic effect of betaine, sodium ascorbate phosphate, and linoleic acid stabilizers in the fermentation medium allows for a wide range of SOD activity below 80℃ and at pH 2-12. It maintains excellent enzyme activity under various conditions and its stability at room temperature can be maintained for more than two years. Combined with the dual modification of rice bran peptides and hyaluronic acid, it further enhances the long-term efficacy and tissue targeting. This process enables the product to show significant therapeutic and protective effects in various reactive oxygen species (ROS) damage-related scenarios, such as ophthalmic photoaging protection, relief of aseptic oxidative damage in joint inflammation, and skin and mucous membrane protection during radiotherapy and chemotherapy. It overcomes the problem that existing single-performance optimization technologies cannot coordinate high enzyme activity, low endotoxin, and long-term stability. It has the comprehensive advantages of high activity, low endotoxin, and high stability, and can be widely used in pharmaceutical injections, medical device implants, and medical device topical preparations. It has important application value, especially in medical scenarios related to ROS damage, such as ophthalmic photoaging protection, treatment of joint inflammation, and skin and mucous membrane protection during radiotherapy and chemotherapy.

[0067] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0068] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions are also within the scope of protection of the present invention.

Claims

1. A process for synthesizing recombinant humanized SOD, characterized in that, Includes the following steps: S10, the gene sequence encoding human copper-zinc superoxide dismutase was optimized by Escherichia coli codon preference to obtain the optimized gene sequence. The nucleotide sequence of the optimized gene is shown in SEQ ID NO:

1. Based on the optimized gene sequence, the whole gene was synthesized to obtain the nucleic acid fragment encoding human copper-zinc superoxide dismutase. The Escherichia coli vector for gene expression was constructed to obtain the recombinant expression plasmid. S20, the recombinant expression plasmid is transformed into Escherichia coli, the expression level of E. coli containing the recombinant expression plasmid is screened and activated to obtain activated engineered bacteria, the activated engineered bacteria are inoculated in fermentation medium and cultured, and humanized SOD is expressed by temperature induction, and the fermentation cells expressing humanized SOD are collected; S30, the fermentation cells are broken and purified to obtain purified humanized SOD. The humanized SOD is then modified to obtain modified recombinant humanized SOD. The amino acid sequence of the recombinant humanized SOD is shown in SEQ ID NO:

2. The modified recombinant humanized SOD is then subjected to ultrafiltration, concentration and freeze-drying to obtain recombinant humanized SOD freeze-dried powder.

2. The recombinant humanized SOD synthesis process as described in claim 1, characterized in that: The method for inoculating the activated engineered bacteria into a fermentation medium includes: Place the fermentation medium in a fermenter and sterilize the fermenter for 30-35 minutes. Inoculate the activated engineered bacteria into the fermentation medium at an inoculum rate of 5-8%. Adjust the pressure inside the fermenter to 0.04-0.05 MPa and stir the fermentation medium at 220-250 rpm. Control the temperature inside the fermenter at 37℃, dissolved oxygen at 25-30%, and pH at 6.9-7.3 to cultivate the activated engineered bacteria. When the OD of the activated engineered bacteria... 600 The bacterial cell amplification is completed when the temperature reaches 15-20°C. The fermenter is cooled to 24-26℃ at a rate of 0.2-0.8℃ / min, the pressure inside the fermenter is 0.15MPa, the low-temperature induction program for activated engineered bacteria is started, the dissolved oxygen in the fermentation medium is controlled at 40-42%, and IPTG and copper sulfate are added to the fermentation medium. The amount of IPTG added is 0.08-0.12mM / L, the final concentration of copper sulfate is 0.15mM, and the low-temperature induction time is 2-2.2 hours. The fermenter was heated to 28℃ at a rate of 0.2℃ / min, and the pressure inside the fermenter was 0.12MPa. The temperature induction program for activated engineered bacteria was started, the dissolved oxygen in the fermentation medium was controlled at 25%, and zinc sulfate was added to the fermentation medium at a final concentration of 0.15mM. The temperature induction time was 2-4 hours. Carbon source supplement was added during temperature induction to make the specific growth rate 0.1-0.12 / h. The fermenter was heated to 30℃ at a rate of 0.2℃ / min, and the pressure inside the fermenter was 0.15MPa. The temperature induction program for activating engineered bacteria was started. The dissolved oxygen in the fermentation medium was controlled at 50%, and carbon source supplement was added to the fermentation medium to make the specific growth rate 0.1-0.12 / h. The temperature induction time was 5-10 hours. After the induction was completed, the fermentation cells expressing humanized SOD were collected by centrifugation.

3. The recombinant humanized SOD synthesis process as described in claim 2, characterized in that: The fermentation medium comprises: 10-15 g / L glycerol, 5-10 g / L maltodextrin, 8-12 g / L soybean peptone, 15-20 g / L yeast extract, 0.5-1 g / L magnesium sulfate heptahydrate, 1-5 g / L dipotassium hydrogen phosphate, 5-10 g / L sodium glycerophosphate, 1-5 g / L potassium dihydrogen phosphate, 0.1-0.5 g / L stabilizer, 0.5-1 g / L mixed amino acids, and 0.01-0.04 g / L defoamer.

4. The recombinant humanized SOD synthesis process as described in claim 3, characterized in that: The fermentation medium comprises: 12 g / L glycerol, 8 g / L maltodextrin, 10 g / L soybean peptone, 18 g / L yeast extract, 0.8 g / L magnesium sulfate heptahydrate, 2 g / L dipotassium hydrogen phosphate, 8 g / L sodium glycerophosphate, 3 g / L potassium dihydrogen phosphate, 0.3 g / L stabilizer, 0.6 g / L mixed amino acids, and 0.02 g / L defoamer.

5. The recombinant humanized SOD synthesis process as described in claim 4, characterized in that: The stabilizer includes betaine, sodium ascorbate phosphate, and linoleic acid, with a weight ratio of 10:7:

1. The mixed amino acids include L-cysteine ​​and L-histidine, with a weight ratio of 1:

1. The defoamer includes rice bran oil and vitamin E, with a weight ratio of 2:

1.

6. The recombinant humanized SOD synthesis process as described in claim 5, characterized in that: The carbon source supplement includes 200-250 g / L of glycerol, 100-120 g / L of glucose, and 0.5 g / L of metal auxiliary material. The metal auxiliary material includes copper glycinate and zinc glycinate, and the weight ratio of copper glycinate and zinc glycinate is 1:

1.

7. The recombinant humanized SOD synthesis process as described in claim 1, characterized in that: The method for cleaving and purifying fermentation cells includes: The fermentation cells were washed with a washing buffer at pH 8 to obtain a wet cell fermentation broth. The wet cell fermentation broth was then subjected to high-pressure homogenization to break it up, and the high-pressure homogenate was collected. The high-pressure homogenate was then centrifuged to collect the supernatant. The nickel column was washed with 3-5 times the amount of ultrapure water and equilibrated with 10-20 mM imidazole solution. At 4°C, the supernatant was loaded onto the equilibrated nickel column at a flow rate of 1-1.5 mL / min, and after elution with 30-40 mM imidazole solution to remove impurities, it was eluted with 100-200 mM imidazole solution, and the protein eluent was collected. The protein eluent was loaded into an ion chromatography column, washed with 150 mM sodium chloride, and then purified with 250-300 mM sodium chloride and 0.1% sodium deoxycholate. The purified humanized SOD was obtained by elution with 450-500 mM sodium chloride.

8. The recombinant humanized SOD synthesis process as described in claim 7, characterized in that: The method for modifying humanized SOD includes: A 10% (v / v) rice bran powder solution was prepared using deionized water, and the pH was adjusted to 8.0-8.

2. Then, alkaline protease was added to the rice bran powder solution at 55℃ for 2-2.5 hours for hydrolysis. After enzyme inactivation treatment at 95℃ for 10 minutes, the supernatant was collected by centrifugation. The supernatant was then filtered sequentially through 10kDa, 5kDa, and 3kDa ultrafiltration membranes to obtain rice bran polypeptide filtrate with a molecular weight of 1-3kDa. The purified humanized SOD was obtained and diluted with 20 mM sodium phosphate buffer to make the concentration of humanized SOD in the SOD dilution solution 10 mg / mL. The rice bran peptide filtrate was diluted with 20 mM sodium phosphate buffer to obtain a peptide-modified solution with a final concentration of 30 mg / mL. 10 mM EDC and 6 mM NHS were added as activators and activated at 25°C in the dark for 30-40 min. β-mercaptoethanol was added until the final concentration of β-mercaptoethanol was 10 mM to terminate the activation reaction, thus obtaining the activated peptide-modified solution. The peptide modification solution and 10 mg / mL hyaluronic acid solution were simultaneously added dropwise to the SOD dilution solution. The reaction was carried out at 300-350 rpm and pH 7-7.2 for 6-7 hours. After the reaction was completed, glycine was added to the mixture to terminate the reaction. The mixture was then allowed to stand at 4°C to obtain the SOD modification solution. Obtain the SOD modification solution, wash the molecular sieve column with 3-5 times the volume of equilibration buffer, concentrate the SOD modification solution to a protein concentration of 10-40 mg / mL using ultrafiltration membrane, filter it through a 0.45 μm filter membrane, load the filtered SOD modification solution into the molecular sieve column, and elute the molecular sieve column with 5-6 times the volume of equilibration buffer to obtain the modified recombinant humanized SOD.

9. The recombinant humanized SOD synthesis process as described in claim 8, characterized in that: The molecular sieve column uses Sephacryl S-200 HR as its medium, with a column height of 80-100 cm and a diameter of 3-5 cm.

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