A gel complex loaded with a fusion protein of SOD and CAT, and a preparation method and application thereof

Abstract

The application discloses a fusion protein gel complex loaded with SOD and CAT, a preparation method and application thereof, and belongs to the technical field of biological medicines. The fusion protein gel complex loaded with SOD and CAT provided by the application comprises a superoxide dismutase, a catalase and a fusion protein support; the fusion protein support comprises a first fusion protein and a second fusion protein; the amino acid sequence of the first fusion protein is shown as SEQ ID NO:1; and the amino acid sequence of the second fusion protein is shown as SEQ ID NO:2. The fusion protein gel complex of the application significantly improves the stability of the superoxide dismutase and the catalase in the gastrointestinal environment, can target the enzyme to be delivered to an intestinal inflammation site, realizes effective enrichment and retention in an inflammation microenvironment, prolongs the action time of the enzyme at the lesion site, and has good biocompatibility, degradability and low cytotoxicity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a fusion protein gel complex loaded with SOD and CAT, its preparation method, and its application. Background Technology

[0002] Inflammatory bowel disease (IBD) is a disease characterized by chronic nonspecific inflammation of the intestines. Its pathogenesis is closely related to elevated levels of reactive oxygen species (ROS) and gut microbiota imbalance. Superoxide dismutase (SOD) and catalase (CAT) can convert ROS into water and oxygen through a cascade reaction, and have unique advantages in regulating oxidative stress.

[0003] However, the oral administration of natural antioxidant enzymes faces significant obstacles. The highly acidic conditions and abundant digestive enzymes in the gastrointestinal environment easily lead to enzyme protein denaturation and inactivation, making it difficult for simple enzyme preparations to maintain catalytic activity and reach the lesion site. While existing delivery strategies attempt to improve enzyme stability through polymer encapsulation or nanomaterial loading, chemical modification processes often result in enzyme activity loss, and traditional carriers have limited retention time in the intestine, making it difficult to achieve effective enrichment at the site of inflammation. Furthermore, most carrier systems are single-function and cannot respond to changes in reactive oxygen species levels in the inflammatory microenvironment to achieve controlled drug release. Summary of the Invention

[0004] In view of this, the present invention provides a fusion protein gel complex loaded with SOD and CAT, its preparation method and application, which solves the problems of poor stability, difficulty in effective enrichment and sustained action at intestinal inflammatory sites during oral delivery of antioxidant enzymes in the prior art.

[0005] In a first aspect, the present invention provides a fusion protein gel complex carrying SOD and CAT, comprising superoxide dismutase, catalase and a fusion protein scaffold; the fusion protein scaffold comprises a first fusion protein and a second fusion protein; The amino acid sequence of the first fusion protein is shown in SEQ ID NO: 1; the amino acid sequence of the second fusion protein is shown in SEQ ID NO: 2.

[0006] Preferably, the mass ratio of superoxide dismutase to catalase is 1:(0.5~2).

[0007] Preferably, the molar ratio of the first fusion protein to the second fusion protein is (1.5~2.5):1.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned fusion protein gel complex loaded with SOD and CAT, comprising the following steps: Superoxide dismutase and catalase were mixed and dissolved in a solvent to obtain a mother liquor; The mother liquor was mixed with the first fusion protein and the second fusion protein respectively to obtain the first reaction solution and the second reaction solution; The first and second reaction solutions are mixed and reacted to obtain the final product.

[0009] Preferably, the solvent is at least one of phosphate buffer (PBS), HEPES buffer, and Tris-HCl buffer.

[0010] Preferably, the concentration of superoxide dismutase in the mother liquor is 0.06~0.2 mg / mL, and the concentration of catalase is 0.06~0.2 mg / mL.

[0011] Preferably, the temperature of the mixing reaction is 35~40℃, and the mixing reaction time is 0.5~2h.

[0012] Preferably, the concentration of the first fusion protein in the first reaction solution is 4-12 wt%; and the concentration of the second fusion protein in the second reaction solution is 4-12 wt%.

[0013] Preferably, the method for preparing the first fusion protein or the second fusion protein includes: Construct recombinant expression vectors that express the first or second fusion protein; The recombinant expression vectors were introduced into host cells for induced expression. The first or second fusion protein is obtained by isolation and purification.

[0014] Thirdly, the present invention provides the application of the above-mentioned fusion protein gel complex loaded with SOD and CAT or the fusion protein gel complex loaded with SOD and CAT prepared by the above-mentioned preparation method in the preparation of a medicament for treating inflammatory bowel disease.

[0015] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention utilizes the self-assembly properties of fusion proteins (SEQ ID NO:1 and SEQ ID NO:2) to co-emulate superoxide dismutase (SOD) and catalase (CAT) with a fusion protein scaffold containing specific amino acid sequences. By forming a three-dimensional network structure, the two enzymes are embedded within a gel network, effectively avoiding degradation by gastric acid and digestive enzymes during oral administration. This significantly improves the stability of SOD and catalase in the gastrointestinal environment. Simultaneously, this fusion protein gel complex can target and deliver the loaded enzymes to sites of intestinal inflammation, achieving effective enrichment and retention in the inflammatory microenvironment. This prolongs the enzymes' action time at the lesion site, thereby more efficiently clearing excess reactive oxygen species in the intestinal tract and reducing pro-inflammatory factor levels, providing a foundation for improving the treatment efficacy of inflammatory bowel disease. Furthermore, this fusion protein gel complex exhibits good biocompatibility, degradability, and low cytotoxicity, showing promising application prospects in drug delivery and the treatment of inflammation and oxidative stress-related diseases. Attached Figure Description

[0016] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0017] Figure 1 These are macroscopic images (a) and (b) of TT and ULDC after mixing and reacting in Example 1 of the present invention. Figure 2 These are images of the injectability test results for the gel formed after the TCT and ULDC mixture reaction in Example 1 of this invention. Figure 3 This is a schematic flowchart of the preparation process of the fusion protein gel complex loaded with SOD and CAT in Embodiment 2 of the present invention; Figure 4 These are macroscopic images (b) of the fusion protein gel complex containing SOD and CAT in Example 2 of the present invention and (a) of the fusion protein gel without SOD and CAT. Figure 5 This is a cross-sectional scanning electron microscope image of the fusion protein gel complex containing SOD and CAT in Example 2 of the present invention; Figure 6 This is a test diagram of the release characteristics of the fusion protein gel complex loaded with Cy5-labeled enzyme in different release media in the experimental examples of this invention; Figure 7These are the enzyme activity protection efficacy test results in the experimental examples of this invention. Among them, A is the relationship between the scavenging effect of the gel complex on superoxide anions and the concentration of superoxide dismutase; B is the scavenging activity of different samples on superoxide anions; C is the relationship between the scavenging ability of the gel complex on hydrogen peroxide and the concentration of CAT; D is the scavenging activity of different samples on hydrogen peroxide; where Free CAT refers to free CAT, Free SOD refers to free SOD, Hydrogel refers to blank gel, and TUC@SC refers to the experimental group of gel complex with a final concentration of SOD enzyme or CAT enzyme of 20 μg / mL. Figure 8 The results show the intestinal retention performance of Cy5-labeled free enzyme (control group, FSC) and Cy5-labeled enzyme-loaded gel complex (experimental group, TUC@SC) in the experimental examples of this invention. Figure 9 This is the evaluation result of the preventive and therapeutic effects of the G1 group (normal control group), G2 group (model control group), G3 group (free enzyme group), G4 group (blank gel group), and G5 group (drug-loaded gel group) in the experimental examples of this invention. Among them, A is the change in mouse body weight; B is the DAI score; and C is the change in mouse colon length. Figure 10 These are pathological sections after H&E staining in the experimental examples of this invention: group G1 (normal control group), group G2 (model control group), group G3 (free enzyme group), group G4 (blank gel group), and group G5 (drug-loaded gel group). Figure 11 These are representative pathological sections of major organs after H&E staining in the experimental examples of this invention, including groups G1 (normal control group), G2 (model control group), G3 (free enzyme group), G4 (blank gel group), and G5 (drug-loaded gel group). Figure 12 The relative expression levels of inflammatory factors (TNF-α, IL-6, IL-1β) in the colon of mice in the experimental cases of this invention are G1 group (normal control group), G2 group (model control group), G3 group (free enzyme group), G4 group (blank gel group) and G5 group (drug-loaded gel group). Figure 13 These are the results of dihydroethidium (DHE) staining of colon tissue from groups G1 (normal control group), G2 (model control group), G3 (free enzyme group), G4 (blank gel group), and G5 (drug-loaded gel group) in the experimental examples of this invention. Detailed Implementation

[0018] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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 invention pertains.

[0019] Inflammatory bowel disease (IBD) is a disease characterized by chronic nonspecific inflammation of the intestines, and its pathogenesis is closely related to elevated levels of reactive oxygen species (ROS) in the intestines. Superoxide dismutase (SOD) and catalase can convert ROS into water and oxygen through a cascade reaction, exhibiting unique advantages in regulating oxidative stress. However, the oral administration of natural antioxidant enzymes faces significant obstacles. The highly acidic conditions and large amounts of digestive enzymes in the gastrointestinal environment easily lead to enzyme protein denaturation and inactivation, making it difficult to maintain catalytic activity and reach the lesion site.

[0020] This invention reveals that the key to improving the oral delivery efficiency of antioxidant enzymes lies in constructing a delivery system that can effectively protect enzyme activity and achieve targeted enrichment at inflammatory sites. The SpyTag / SpyCatcher system is a protein / peptide pair with specific covalent binding capabilities, where the SpyCatcher protein can spontaneously form irreversible heteropeptide bonds with the SpyTag peptide under mild conditions. Further research found that introducing a Coil helical domain between SpyTags can form a SpyTag-Coil-SpyTag fusion protein (amino acid sequence SEQ ID NO: 1). The physical interaction between these domains can complement the covalent cross-linking of SpyTag / SpyCatcher, enhancing the mechanical strength of the gel network. Simultaneously, the ULD domain has a natural tendency to tetramerize, serving as a physical cross-linking point to further stabilize the gel network.

[0021] Based on the above research findings, this invention constructs a gel complex that can efficiently encapsulate antioxidant enzymes and achieve targeted delivery to inflammatory sites by blending superoxide dismutase and catalase with a scaffold containing ULD-SpyCatcher fusion protein (amino acid sequence SEQ ID NO: 2) and SpyTag-Coil-SpyTag fusion protein, and utilizing a triple network structure formed by SpyTag / SpyCatcher covalent crosslinking, Coil-Coil physical crosslinking, and ULD tetramerization.

[0022] Specifically, the present invention provides a fusion protein gel complex carrying SOD and CAT, comprising superoxide dismutase, catalase and a fusion protein scaffold; the fusion protein scaffold comprises a first fusion protein and a second fusion protein; the amino acid sequence of the first fusion protein is shown in SEQ ID NO: 1; the amino acid sequence of the second fusion protein is shown in SEQ ID NO: 2.

[0023] The amino acid sequence shown in SEQ ID NO: 2 is a ULD-SpyCatcher fusion protein, wherein the ULD domain can form a tetramer, serving as a physical cross-linking site; the SpyCatcher domain can specifically bind to SpyTag to form a covalent bond. The amino acid sequence shown in SEQ ID NO: 1 is a SpyTag-Coil-SpyTag fusion protein, wherein the two SpyTag polypeptides can bind to the two SpyCatcher proteins respectively, forming cross-linking sites; the central Coil domain can interact with other Coil domains through coil-coil interactions, forming physical cross-links.

[0024] In the SOD and CAT fusion protein gel complex of the present invention, superoxide dismutase and catalase exist in a three-dimensional network structure formed by the fusion protein scaffold in a physically embedded form. This embedding method avoids the influence of chemical modification on enzyme activity, while the gel network can effectively block gastric acid and proteases from the external environment, protecting enzyme activity. The content of superoxide dismutase and catalase in the gel complex can be flexibly adjusted according to actual application requirements, but it must be ensured that both enzymes are effectively embedded in the gel network structure and maintain their inherent catalytic activity after embedding. In the present invention, the mass ratio of superoxide dismutase to catalase is 1:(0.5~2), more preferably 1:(0.8~1.5), and most preferably 1:1.

[0025] In some embodiments of the present invention, the molar ratio of the first fusion protein to the second fusion protein is (1.5~2.5):1. Preferably, the molar ratio of the first fusion protein to the second fusion protein is (1.8~2.2):1, more preferably (1.9~2.1):1. More preferably, the molar ratio of the first fusion protein to the second fusion protein is 2:1. This molar ratio is determined based on the reaction equivalence of SpyTag and SpyCatcher. One SpyTag-Coil-SpyTag fusion protein contains two SpyTags. Since ULD is a tetramer, the constructed second fusion protein contains four SpyCatchers, therefore the preferred molar ratio is 2:1.

[0026] In some embodiments of the present invention, the mass concentration of the fusion protein scaffold in the gel complex is 4-12 wt%. Preferably, the mass concentration of the fusion protein scaffold is 6-10 wt%. More preferably, the mass concentration of the fusion protein scaffold is 8 wt%.

[0027] The present invention also provides a method for preparing the above-mentioned fusion protein gel complex loaded with SOD and CAT, comprising the following steps: Superoxide dismutase and catalase were mixed and dissolved in a solvent to obtain a mother liquor; The mother liquor was mixed with the first fusion protein and the second fusion protein respectively to obtain the first reaction solution and the second reaction solution; The first and second reaction solutions are mixed and reacted to obtain the final product.

[0028] It should be noted that the above preparation method involves first mixing the enzyme with the two fusion proteins separately, and then mixing the two reaction solutions. This avoids cross-linking of the fusion proteins before they are fully mixed with the enzyme, ensuring that the enzyme is evenly distributed in the gel network. After mixing the first and second reaction solutions, the SpyCatcher in the first fusion protein and the SpyTag in the second fusion protein undergo specific covalent binding to form a cross-linked network. Simultaneously, the Coil domains in the second fusion protein exhibit coil-coil interactions, and the ULD domain in the first fusion protein undergoes tetramerization, collectively forming a stable triple network structure that encapsulates superoxide dismutase and catalase within the network.

[0029] In some embodiments of the present invention, the solvent is at least one selected from phosphate buffer, HEPES buffer, and Tris-HCl buffer. Preferably, the solvent is phosphate buffer (PBS) with a pH of 7.2-7.5. Phosphate buffer can maintain the pH stability of the reaction system, providing a suitable physiological environment for the enzyme and fusion protein, which is beneficial for maintaining enzyme activity and promoting the correct folding and interaction of proteins.

[0030] In some embodiments of the present invention, the concentration of superoxide dismutase in the mother liquor is 0.06~0.2 mg / mL, and the concentration of catalase is 0.06~0.2 mg / mL.

[0031] In some embodiments of the present invention, the concentration of the first fusion protein in the first reaction solution is 4-12 wt%, more preferably 6-10 wt%, and even more preferably 8 wt%; the concentration of the second fusion protein in the second reaction solution is 4-12 wt%, more preferably 6-10 wt%, and even more preferably 8 wt%. Suitable concentration ranges ensure that the fusion protein scaffold forms a three-dimensional network structure while possessing suitable rheological properties, injectability, and release characteristics.

[0032] In some embodiments of the present invention, the mixing reaction temperature is 35-40°C, and the mixing reaction time is 0.5-2 hours. Preferably, the mixing reaction temperature is 36-38°C, and the mixing reaction time is 0.8-1.5 hours. More preferably, the mixing reaction temperature is 37°C, and the mixing reaction time is 1 hour. 37°C simulates physiological temperature, which is conducive to the correct folding and interaction of proteins; too short a reaction time may lead to incomplete cross-linking, while too long a reaction time may increase production costs.

[0033] In some embodiments of the present invention, the method for preparing the first fusion protein or the second fusion protein includes: Construct recombinant expression vectors that express the first or second fusion protein; The recombinant expression vectors were introduced into host cells for induced expression. The first or second fusion protein is obtained by isolation and purification.

[0034] Specifically, when constructing a recombinant expression vector expressing the first or second fusion protein, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2 can be cloned into a suitable expression vector, such as the pET series vectors (e.g., pET32a). To facilitate purification, a tag sequence, such as a 6×His tag, can be introduced at the C-terminus of the fusion protein.

[0035] When introducing recombinant expression vectors into host cells, conventional transformation methods in the art can be used, such as heat shock or electroporation. The host cell can be an *E. coli* expression system, such as... E. coli BL21(DE3) strain. Transformed host cells are cultured in a suitable medium, such as LB medium. The culture temperature can be 35–38°C, preferably 37°C. When the OD600 of the bacterial culture reaches 0.6–0.8, an inducer is added to induce expression. The inducer can be isopropyl-β-D-thiogalactoside (IPTG), with a final concentration of 0.05–0.5 mM, preferably 0.1 mM. The induction temperature can be 15–20°C, preferably 16°C; the induction time can be 18–24 h, preferably 20 h. Lower temperatures are beneficial for soluble protein expression and reduce inclusion body formation.

[0036] After induction of expression, the bacterial cells were collected by centrifugation and resuspended in a suitable buffer, such as 50mM HEPES buffer (pH 7.5) containing 50mM NaCl. Cells were lysed using methods such as sonication or high-pressure homogenization, and the supernatant was collected by centrifugation. Since the fusion protein carries a His tag, it can be purified using immobilized metal affinity chromatography, such as using nickel ion chelating resin (Ni-NTA). The bound protein was eluted with an imidazole-containing buffer, and the eluent was collected.

[0037] The purified protein solution is dialyzed to remove salt ions and small molecule impurities. The dialysis buffer can be deionized water or a low-salt buffer. The dialyzed protein solution is then freeze-dried to obtain a fusion protein powder. Freeze-drying conditions include pre-freezing at -50°C to -80°C, followed by drying in a freeze dryer until a loose, powdery product is obtained.

[0038] The present invention also provides the application of the above-mentioned fusion protein gel complex loaded with SOD and CAT or the fusion protein gel complex loaded with SOD and CAT prepared by the above preparation method in the preparation of a drug for treating inflammatory bowel disease.

[0039] Inflammatory bowel disease includes ulcerative colitis and Crohn's disease. The fusion protein gel complex of this invention, when taken orally, can protect superoxide dismutase and catalase from degradation by gastric acid and digestive enzymes in the gastrointestinal tract, reaching the site of inflamed intestines. In the highly reactive oxygen species (ROS) microenvironment at the site of inflammation, the gel network degrades, releasing superoxide dismutase and catalase. These two enzymes work synergistically to convert superoxide anion radicals into hydrogen peroxide, which is then converted into water and oxygen, thereby scavenging excess ROS, reducing oxidative stress levels, alleviating inflammation, and achieving the therapeutic effect on inflammatory bowel disease.

[0040] The fusion protein gel complex of the present invention showed significant therapeutic effects in an animal model of colitis induced by dextran sulfate sodium, and was able to alleviate weight loss, reduce disease activity index, restore colon length, reduce histopathological damage, reduce pro-inflammatory factor levels, increase anti-inflammatory factor levels, and effectively remove reactive oxygen species in colon tissue.

[0041] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not impose any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used.

[0042] Example 1 This embodiment provides the expression and purification of recombinant fusion proteins.

[0043] (1) Gene construction: Gene sequences encoding SpyTag-SpyTag (TT), SpyTag-Coil-SpyTag (TCT) and ULD-SpyCatcher (ULDC) were synthesized and cloned into the pET32a expression vector to construct recombinant expression plasmids pET32a-TT, pET32a-TCT and pET32a-ULDC, with nucleotide sequences as shown in SEQ ID NO: 6, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; each fusion protein has a 6×His tag at its C-terminus.

[0044] (2) Transformation and culture: The recombinant expression plasmids were transformed into Escherichia coli BL21(DE3) competent cells and plated on LB solid medium containing ampicillin, and cultured overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing ampicillin, and cultured overnight at 37°C with shaking at 220 rpm. The colonies were then transferred to fresh LB medium at a ratio of 1:100 and cultured at 37°C until the OD600 reached 0.6-0.8.

[0045] (3) Induction of expression: Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was continued at 16℃ for 20 h.

[0046] (4) Collection of bacterial cells: Centrifuge the induced bacterial solution at 4℃ and 8000rpm for 10min and collect the bacterial cell precipitate.

[0047] (5) Cell lysis: The bacterial cells were resuspended in 50mM HEPES buffer (pH 7.5) containing 50mM NaCl and sonicated under ice bath conditions. The lysate was centrifuged at 12,000 rpm for 20 min at 4°C and the supernatant was collected.

[0048] (6) Affinity purification: Load the supernatant into a pre-equilibrated nickel ion chelating resin (Ni-NTA) column, wash the impurities with buffer containing 20 mM imidazole, and then elute the target protein with buffer containing 50 mM to 250 mM imidazole. Collect the eluent.

[0049] (7) Dialysis and freeze-drying: The eluent was placed in a dialysis bag and dialyzed in deionized water at 4°C for 24 hours, with the dialysis solution being replaced every 6 hours. The dialyzed protein solution was freeze-dried to obtain TT, TCT and ULDC fusion protein powders, the amino acid sequences of which are shown in SEQ ID NO: 3, SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0050] Take the prepared TT fusion protein powder, TCT fusion protein powder and ULDC fusion protein powder, dissolve them in phosphate buffer (pH 7.2~7.4) respectively, and prepare solutions with a mass concentration of 8wt% to obtain reaction solution D (containing TT), reaction solution E (containing TCT) and reaction solution F (containing ULDC).

[0051] Mix reaction solution D or reaction solution E with reaction solution F at a molar ratio of TT:ULDC of 2:1 or TCT:ULDC of 2:1, and vortex to ensure thorough mixing. Place the mixture in a 37°C water bath for 1 hour and observe whether gel formation occurs. Results are as follows... Figure 1 As shown, Figure 1Image 'a' in the image is a macroscopic picture of the mixture of TT and ULDC after the reaction. It can be seen that the mixture failed to form an inverted, non-flowing gel state, indicating that the two cannot form a stable three-dimensional network structure. Figure 1 Image b in the image shows a macroscopic picture of the reaction between TCT and ULDC, demonstrating its ability to form a gel that does not flow when inverted. The gel formed after the reaction of TCT and ULDC was loaded into a 2 ml syringe and then injected into a culture dish, as shown. Figure 2 As shown in the figure, the gel has good injectability.

[0052] Therefore, in subsequent experiments, the gel resulting from the mixing and reaction of TCT fusion protein powder and ULDC fusion protein powder was selected as the carrier for delivering the antioxidant enzyme complex.

[0053] Example 2 This embodiment provides a method for preparing a fusion protein gel complex loaded with SOD and CAT. A schematic flowchart of the preparation process is shown below. Figure 3 As shown.

[0054] (1) Preparation of stock solution: Weigh 0.2 mg of superoxide dismutase (SOD) and 0.2 mg of catalase (CAT) powder, dissolve them together in 1 mL of phosphate buffer (1×PBS, pH 7.4), mix thoroughly to obtain stock solution.

[0055] (2) Preparation of fusion protein solution: Take the TCT fusion protein powder and ULDC fusion protein powder prepared in Example 1 respectively, dissolve them in the mother liquor, and prepare solutions with a mass concentration of 8wt% respectively to obtain the first reaction solution (containing TCT) and the second reaction solution (containing ULDC).

[0056] (3) Formation of gel complex: The first reaction solution and the second reaction solution are mixed at a molar ratio of TCT:ULDC of 2:1, and the mixture is vortexed to ensure that the components are fully mixed. The mixture is placed in a water bath at 37°C and reacted at a constant temperature for 1 hour to form a fusion protein gel complex carrying SOD and CAT.

[0057] Macroscopic images of the SOD and CAT-loaded fusion protein gel complex prepared in this embodiment are shown below. Figure 4 As shown in b, the fusion protein gel without SOD and CAT is as follows: Figure 4 As shown in a, after encapsulating SOD and CAT, the gel remains in an inverted, non-flowing state, and no antioxidant enzyme complex liquid is present, indicating that both SOD and CAT are encapsulated within the gel.

[0058] The cross-sectional structure of the SOD and CAT-loaded fusion protein gel complex prepared in this embodiment was observed using a scanning electron microscope (SEM) after freeze-drying. Figure 5As shown, the gel complex has a porous structure and a smooth surface.

[0059] Test case 1. Release characteristics test of fusion protein gel complex (1) Test method: Superoxide dismutase and catalase were labeled with Cy5 fluorescent dye, and fusion protein gel complexes loaded with Cy5-labeled enzymes were prepared according to the method in Example 2. 200 μL of the gel complex (containing 40 μg each of SOD and CAT) was placed in 1 mL of different release media: phosphate-buffered saline (PBS, pH 7.5), simulated gastric juice (SGF, pH 1.0, containing pepsin), simulated intestinal juice (SIF, pH 8.5, containing pancreatin), and PBS solution containing 1 mM hydrogen peroxide (pH 7.5). The sample was placed in a shaker and shaken at 37°C and 100 rpm. 100 μL of supernatant was collected by centrifugation at preset time points (0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 10 h, 20 h), and an equal volume of fresh media was added simultaneously. After all samples were collected, the fluorescence intensity was measured using an ELISA reader at an excitation wavelength of 630 nm and an emission wavelength of 675 nm. The cumulative release rate of the Cy5-labeled enzyme was calculated, and the release curve was plotted.

[0060] (2) Test results and analysis: Figure 6 The test results showed that in PBS, the release rate of the gel complex was slow, with a cumulative release rate of about 49% after 20 hours; in SIF, the cumulative release rate was about 57% after 20 hours; in SGF, the release rate increased significantly due to the low pH environment and the presence of protease, with a cumulative release rate of about 78% after 20 hours; and in PBS containing 1 mM hydrogen peroxide, the cumulative release rate was about 93% after 20 hours.

[0061] The above results indicate that the fusion protein gel complex prepared in this invention has good sustained-release properties, and the release rate is significantly accelerated in a simulated highly reactive oxygen species environment (containing hydrogen peroxide) at the site of inflammation. This indicates that the gel complex has reactive oxygen species responsive release capability, which is beneficial for achieving on-demand drug release at the site of intestinal inflammation.

[0062] 2. Enzyme activity protection efficacy test of the fusion protein gel complex (1) Test method: To determine the enzyme activity protection efficacy of the fusion protein gel complex, the amounts of superoxide dismutase (SOD) and catalase added to the fusion protein gel complex in Example 2 were adjusted. Specifically, the amounts of SOD and catalase were adjusted to 0.06 mg, 0.08 mg, 0.1 mg, 0.12 mg, 0.14 mg, 0.16 mg, 0.18 mg, and 0.2 mg, respectively, so that the final concentrations of SOD or CAT in the system were 6, 8, 10, 12, 14, 16, or 20 μg / mL. Blank gels and equal concentrations of free SOD and catalase were used as controls, and enzyme activities were measured. SOD activity was determined using a total SOD kit: 20 μL of sample, 160 μL of working solution, and 20 μL of reaction initiation reagent were mixed and incubated at 37°C for 30 min. The absorbance at 450 nm was measured using a microplate reader. Catalase activity assay: 20 μL of sample was added to 180 μL of HEPES buffer (0.1 M, pH 7.4) containing 20 mM hydrogen peroxide, and the decomposition rate of hydrogen peroxide was measured at 240 nm wavelength within 1 min.

[0063] (2) Test results: like Figure 7 As shown in Figure A, at a relatively low concentration (6 μg / mL), 25% of superoxide anions can be removed. The removal rate of superoxide anions increases with increasing concentration, reaching over 80% when the superoxide dismutase concentration is 20 µg / mL. Figure 7 As shown in B, compared with free superoxide dismutase (SOD), the gel complex experimental group (TUC@SC) with a final SOD concentration of 20 µg / mL maintained comparable superoxide dismutase activity. Figure 7 B in the text). CAT activity also shows a similar trend ( Figure 7 The scavenging rate of the gel complex experimental group (TUC@SC) with a final CAT concentration of 20 µg / mL (C and D in the sample) was approximately 60%. This increase in enzyme activity compared to free catalase is likely due to the hydrogel effectively buffering the local hydrogen peroxide concentration and preventing instantaneous enzyme inactivation, thus increasing enzyme stability.

[0064] The above results indicate that the fusion protein gel complex of the present invention can effectively protect the activity of superoxide dismutase and catalase during the encapsulation process, with a high activity retention rate, laying the foundation for subsequent in vivo antioxidant effects.

[0065] 3. Intestinal retention performance test of fusion protein gel complex (1) Test method: Superoxide dismutase and catalase were labeled with Cy5 fluorescent dye, and fusion protein gel complexes loaded with Cy5-labeled enzymes were prepared according to the method in Example 2. Six- to eight-week-old male C57BL / 6J mice were randomly divided into two groups, receiving Cy5-labeled free enzyme (control group, FSC) and Cy5-labeled enzyme-loaded gel complexes (experimental group, TUC@SC) by gavage, respectively. The distribution of fluorescence signals in the mice was observed using a small animal in vivo imaging system at time points of 2, 4, 6, and 8 hours after administration, and the fluorescence intensity in the intestinal region was quantitatively analyzed.

[0066] (2) Test results and analysis: Figure 8 The results showed that at 2 and 4 hours after drug administration, the fluorescence intensity in the intestines of the experimental group mice was significantly higher than that in the control group. Although the fluorescence intensity of both groups gradually decreased over time, the fluorescence intensity of the experimental group remained higher than that of the control group for up to 4 hours.

[0067] The above results indicate that the fusion protein gel complex of the present invention can significantly prolong the retention time of superoxide dismutase and catalase in the intestine, which is beneficial to the enrichment and sustained action of enzymes at sites of intestinal inflammation.

[0068] 4. Evaluation of the preventive and therapeutic effects of fusion protein gel complex on colitis (1) Test method: Male C57BL / 6J mice aged 6-8 weeks were randomly divided into 5 groups of 5 mice each after one week of acclimatization feeding: Group G1 (normal control group): Free access to normal water, and PBS administered by gavage; Group G2 (model control group): Free access to 2.5% sodium dextran sulfate (DSS) solution, and PBS administered by gavage; Group G3 (Free Enzyme Group): Free drinking of 2.5% DSS solution, and administration of free superoxide dismutase and catalase by gavage (total enzyme dose 2 mg / kg). Group G4 (blank gel group): Free drinking of 2.5% DSS solution, and gavage administration of unloaded fusion protein hydrogel (prepared by the same method as the gel after TCT and ULDC mixing reaction in Example 1). Group G5 (drug-loaded gel group): Free access to 2.5% DSS solution, and administered the drug-loaded gel complex prepared in Example 2 (total enzyme dose 2 mg / kg) by gavage.

[0069] The experiment lasted for 7 days, with each group receiving the drug once daily via gavage starting from day 2. Daily changes in mouse body weight, fecal characteristics, and blood in the stool were recorded, and the Disease Activity Index (DAI) was calculated. On day 9, mice were euthanized, and the colon was dissected and its length measured. Colon, heart, liver, spleen, lung, and kidney tissues were collected for hematoxylin-eosin (H&E) staining to observe histopathological changes. Real-time quantitative PCR was used to detect the mRNA expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the colon tissue. Dihydroethidium ether (DHE) staining was used to detect reactive oxygen species (ROS) levels in the colon tissue.

[0070] (2) Test results and analysis: Weight changes such as Figure 9 As shown in A, it can be seen that the body weight of mice in group G2 continued to decrease, the body weight decrease trend in groups G3 and G4 was somewhat alleviated, and the body weight decrease in group G5 was slow, which was significantly improved compared with group G2.

[0071] DAI score as follows Figure 9 As shown in B, it can be seen that the DAI score of group G5 was significantly lower than that of groups G2, G3 and G4.

[0072] Colon length as Figure 9 As shown in C, it can be seen that the colon in group G2 is significantly shortened, the colon length in groups G3 and G4 recovers to some extent, and the colon length in group G5 is closest to that in group G1.

[0073] H&E stained pathological sections as follows Figure 10 As shown, the colon structure in group G5 was more intact, with reduced inflammatory cell infiltration and better recovery of crypts and mucosa, while group G2 showed obvious tissue damage and inflammatory infiltration.

[0074] like Figure 11 As shown in the pathological sections after H&E staining, no obvious damage was observed in the heart, liver, lungs, and kidneys, indicating that the constructed gel complex has good biocompatibility. The spleen in group G2 showed significant damage, while groups G3 and G4 showed some recovery, and group G5 showed the most significant recovery.

[0075] The relative expression levels of inflammatory factors in the mouse colon are as follows: Figure 11 As shown, the mRNA expression levels of TNF-α, IL-1β, and IL-6 in the colon tissue of group G5 were significantly lower than those in groups G2, G3, and G4.

[0076] Results of reactive oxygen species level test as follows Figure 12 As shown, the fluorescence intensity of DHE staining in the colon tissue of group G5 was significantly lower than that of group G2, indicating a decrease in reactive oxygen species levels.

[0077] The above results indicate that the fusion protein gel complex containing superoxide dismutase and catalase prepared in this invention can effectively alleviate DSS-induced colitis symptoms in mice, reduce weight loss, decrease disease activity index, restore colon length, improve histopathological damage, reduce inflammatory factor expression, and scavenge reactive oxygen species. Its therapeutic effect is significantly better than that of free enzymes and blank gels, indicating that the enzymes and carriers in the gel complex play a synergistic role.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0079] The sequence listing information involved in this invention is as follows: SEQ ID NO:1 MAHIVMVDAYKPTKLDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSAPQMLRELQETNAALQDVRELLR QQVKEITFLKNTVMESDASKLNTSVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGGLLDAHIVMVDAYKPTKLEHHHHHH* SEQ ID NO:2 ARGDGTMLPVFCVVEHYENAIEYDCKEEHAEFVLVRKDMLFNQLIEMALLSLGYSHSSAAQAKGLIQVGKWNPVPLSYVTDAPDATVADMLQDVYHVVTLKIQLHSLDGHGVGVPGVGVPGVGVPGEGVPGVGVPGVGVPGVGVPGVGVPGEGVPGVGVPGV GVPGVGVPGVGVPGEGVPGVGVPGVGELGSGLGSGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIREREREHHHHHH* SEQ ID NO: 3 DIMAHIVMVDAYKPTKLDGHGVGVPGVGVPGVPGVPGVPGVGVPGVPGVPGVPGVPGVPGVPGEGVPGVPGVGVPGVGVPGVPGVPGVPGVGVPGVGELGSGLGSTSVPGVGVPGVGVPGEGVPGVPGVPGVPGVPGVGVPGEGVPGVPGVPGVPGVPGVPGVGVPGVPGVPGVPGGLLDAHIVMVDAYKPTKLEHHHHHH* SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6

Claims

1. A fusion protein gel complex loaded with SOD and CAT, characterized in that, It includes superoxide dismutase, catalase, and a fusion protein scaffold; the fusion protein scaffold includes a first fusion protein and a second fusion protein; The amino acid sequence of the first fusion protein is shown in SEQ ID NO: 1; the amino acid sequence of the second fusion protein is shown in SEQ ID NO:

2.

2. The fusion protein gel complex carrying SOD and CAT as described in claim 1, characterized in that, The mass ratio of superoxide dismutase to catalase is 1:(0.5~2).

3. The fusion protein gel complex carrying SOD and CAT as described in claim 1, characterized in that, The molar ratio of the first fusion protein to the second fusion protein is (1.5~2.5):

1.

4. The method for preparing the fusion protein gel complex loaded with SOD and CAT as described in any one of claims 1 to 3, characterized in that, Includes the following steps: Superoxide dismutase and catalase were mixed and dissolved in a solvent to obtain a mother liquor; The mother liquor was mixed with the first fusion protein and the second fusion protein respectively to obtain the first reaction solution and the second reaction solution; The first and second reaction solutions are mixed and reacted to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The solvent is at least one of phosphate buffer, HEPES buffer, and tris(hydroxymethyl)aminomethane hydrochloride buffer.

6. The preparation method according to claim 4, characterized in that, The concentration of superoxide dismutase in the mother liquor is 0.06~0.2 mg / mL, and the concentration of catalase is 0.06~0.2 mg / mL.

7. The preparation method according to claim 4, characterized in that, The mixing reaction is carried out at a temperature of 35-40°C for 0.5-2 hours.

8. The preparation method according to claim 4, characterized in that, In the first reaction solution, the concentration of the first fusion protein is 4-12 wt%; in the second reaction solution, the concentration of the second fusion protein is 4-12 wt%.

9. The preparation method according to claim 4, characterized in that, The preparation method of the first fusion protein or the second fusion protein includes: Construct recombinant expression vectors that express the first or second fusion protein; The recombinant expression vectors were introduced into host cells for induced expression. The first or second fusion protein is obtained by isolation and purification.

10. The use of the SOD and CAT-loaded fusion protein gel complex as described in any one of claims 1 to 3, or the SOD and CAT-loaded fusion protein gel complex prepared by the preparation method as described in any one of claims 4 to 9, in the preparation of a medicament for treating inflammatory bowel disease.

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