Fusion protein, pharmaceutical composition and application

Abstract

The invention discloses a fusion protein, a pharmaceutical composition and application. The fusion protein is formed by fusing uricase and polypeptide with mucoprotein binding property. The original uricase activity of the fusion protein is not affected, the binding property of the fusion protein and II-type mucoprotein covering the intestinal epithelium mucous layer is remarkably improved, and the fusion protein can be orally taken to treat diseases or disorders related to increase of uric acid amount, such as hyperuricemia, hyperuricemia and gout.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a fusion protein, in particular to a uricase fusion protein and a pharmaceutical composition and use thereof. Background Art

[0002] During the evolution process, humans and higher primates have undergone pseudogenesis of urate oxidase, lacking functional urate oxidase and unable to degrade uric acid into allantoin, so uric acid becomes the final product of purine metabolism in the human body. The blood uric acid concentration depends on the balance between uric acid production and excretion. When purine metabolism is disordered, uric acid is produced excessively or excreted less, the blood uric acid level will continue to exceed the standard and suffer from hyperuricemia (HUA). Long-term HUA can cause weakly water-soluble urate to saturate and precipitate in the form of crystals and deposit in soft tissues or joints, which in turn causes inflammation and induces gout. In addition, hyperuricemia can lead to a variety of complications, including hypertension, chronic kidney disease, obesity, metabolic disorders, type 2 diabetes, etc.

[0003] There are three types of drugs for uric acid lowering therapy. The first type of drug is xanthine oxidase inhibitors, such as allopurinol and febuxostat. The treatment strategy aims to reduce the synthesis of uric acid, but its effectiveness in lowering uric acid is limited, and only 30%-60% of patients treated achieve plasma uric acid concentrations <6mg / dL. In addition, allopurinol is excreted by the kidneys, and patients with chronic kidney disease (CKD) may face a higher risk of toxicity. The second type of drug is uricosuric drugs, which can inhibit the reabsorption of uric acid into the blood in the renal tubules and promote the excretion of uric acid, thereby reducing the level of uric acid in the serum, but may cause the formation of urate kidney stones and cause acute kidney disease. The third type is recombinant uricase drugs, which can efficiently catalyze the conversion of uric acid into allantoin. Rasburicase, which is approved for tumor lysis syndrome, has high immunogenicity and a short half-life. Pegloticase, which is marketed in the United States for the treatment of refractory gout, has been clinically detected with anti-polyethylene glycol (PEG) antibodies and allergic reactions. In addition, the adverse reactions and side effects of intravenous infusion of these urate oxidase preparations limit their widespread clinical application.

[0004] In a human body with normal uric acid metabolism, more than 70% of uric acid is excreted by the kidneys every day, and the remaining 30% is secreted through the intestines and then decomposed by the flora. In patients with chronic gouty nephropathy, the kidney's function of excreting uric acid is impaired, and the proportion of uric acid excreted by the intestines increases to 50% to 70%, becoming the main route of uric acid excretion. Current therapies still have many limitations. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of poor efficacy and large side effects in existing drug therapies, and to provide a fusion protein and its application. The present invention connects uricase oxidase with gastrointestinal mucin binding peptide (Mucus-binding Protein, MUB) to obtain a uricase fusion protein, which not only retains the original uricase activity through oral administration, but also increases the retention time of uricase oxidase in the intestine, significantly improves the binding properties with type II mucin covering the intestinal epithelial mucus layer, degrades urate secreted or formed in the intestine (formed by a large amount of uric acid accumulation), increases the efficacy, and is safe and reliable.

[0006] The first aspect of the present invention provides a fusion protein, which is formed by the fusion of uricase and a polypeptide having mucin binding properties.

[0007] In some embodiments, the uricase is recombinant porcine-baboon uricase or Candida utilis uricase.

[0008] In some embodiments, the sequence of the polypeptide having mucin binding properties is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25.

[0009] In some embodiments, the sequence of the recombinant pig-baboon uricase is SEQ ID NO:2, and the sequence of the polypeptide having mucin binding properties is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:25,

[0010] Preferably, when the C-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, preferably SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, more preferably SEQ ID NO:24; when the N-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:23, SEQ ID NO:25, preferably SEQ ID NO:23.

[0011] In some embodiments, the sequence of the Candida utilis uricase is SEQ ID NO:3, and the sequence of the polypeptide having mucin binding properties is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25,

[0012] Preferably, when the C-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, preferably SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, more preferably SEQ ID NO:24; when the N-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, preferably SEQ ID NO:23.

[0013] In some embodiments, the uricase is fused directly or via a linker to the polypeptide having mucin binding properties, and / or the uricase is at the N-terminus or C-terminus of the polypeptide having mucin binding properties.

[0014] In some embodiments, the linker sequence is SEQ ID NO:19.

[0015] In some embodiments, the sequence of the fusion protein is as follows: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

[0016] The second aspect of the present invention provides a pharmaceutical composition comprising the fusion protein and a pharmaceutically acceptable excipient.

[0017] The third aspect of the present invention provides the use of the fusion protein or the pharmaceutical composition in the preparation of an oral drug for treating diseases or disorders associated with elevated uric acid levels, preferably hyperuricemia, hyperuricosuria and gout.

[0018] the term:

[0019] In order to make the present invention more easily understood, some technical terms are specifically defined below. Unless otherwise specified herein, the technical terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0020] Numerical ranges include the numbers within the range. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were expressly written herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as if such lower (or higher) numerical limits were expressly written herein.

[0021] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively.

[0022] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (eg, glycosylation or phosphorylation).

[0023] As used herein, the term "amino acid" is referred to herein by either its commonly known three letter symbols or the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may also be referred to by their commonly accepted single-letter codes.

[0024] As used herein, the terms "engineered," "recombinant," when applied to cells, polynucleotides or polypeptides, refer to a material or material corresponding to the native or native form of a material that has been modified in a manner not found or identical to it in nature but is produced or derived by synthetic material and / or manipulation using recombinant technology.

[0025] The terms "mucin" and "mucin" used herein have the same meaning and refer to a cell surface or secreted glycoprotein characterized by the presence of a large filamentous domain rich in complex O-linked oligosaccharides. It exists in the mucosa of animals and humans, and the mucus layer covering the intestinal epithelium is mainly composed of type II mucin.

[0026] The term "fusion protein" as used herein refers to a protein produced by connecting at least two genes (or portions of genes) that were originally encoded as separate proteins. In some embodiments, a portion of the fusion protein is a mucin binding protein or a fragment thereof. In some additional embodiments, a portion of the fusion protein is an enzyme.

[0027] The term "vector" as used herein is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence that can achieve expression of a polypeptide encoded in the DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgenic) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0028] The term "expression" as used herein includes any step involved in the production of the polypeptide, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also includes secretion of the polypeptide from the cell.

[0029] The term "production" as used herein refers to the production of proteins and / or other compounds by cells. The term includes any step involved in the production of polypeptides, including but not limited to transcription, post-transcriptional modification, translation, and post-translational modification.

[0030] As used herein, the terms "host cell" and "host strain" refer to suitable hosts for expression vectors (e.g., polynucleotides encoding mucin binding fusion protein variants) combined with DNA provided herein. In some embodiments, the host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0031] As used herein, the term "culturing" refers to growing a population of microbial cells under any suitable conditions (eg, using liquid, gel, or solid culture media).

[0032] As used herein, the terms "isolated" and "purified" are used to refer to molecules (e.g., isolated nucleic acids, polypeptides, etc.) or other components that are removed from at least one other component with which they are naturally associated. The term "purified" does not require absolute purity, but is used as a relative definition.

[0033] The term "homogenization" as used herein refers to the process of lysing or mechanically lysing host cells to release intracellular substances from the host cells. The system containing lysed host cells and intracellular substances obtained after the cells are broken by homogenization is called a "homogenate".

[0034] The term "enzyme activity unit (U)" used herein is defined as the amount of enzyme required to consume 1 μmol of uric acid per minute under the conditions of 37°C and 50 mmol / L potassium dihydrogen phosphate buffer system (pH 6.8).

[0035] The term "specific activity" refers to the number of enzyme activity units per milligram (mg) of protein.

[0036] The terms "uricase" and "uricase oxidase" used in this article have the same meaning. It is a tetrameric enzyme composed of four uricase oxidase subunits. Each monomer can bind to a uric acid molecule and can decompose uric acid into allantoin with better water solubility.

[0037] The term "hyperuricemia" as used herein refers to a blood uric acid concentration above the upper limit of normal, usually above 420 μmol / L for men and above 360 ​​μmol / L for women.

[0038] The term "gout" used in this article refers to a disease characterized by abnormal purine metabolism and increased uric acid, which results in the formation of urate deposits in the joint cavity and is accompanied by tissue damage, and is mainly manifested as acute joint pain.

[0039] The term "biotinylation" used in this article refers to the covalent attachment of biotin to proteins, peptides and other biological molecules. The interaction between biotin and streptavidin and biotin labeling is widely used in enzyme-linked reaction, biomembrane interferometry and other interaction detection.

[0040] The positive and progressive effects of the present invention are:

[0041] 1. The present invention provides a new fusion protein, the enzyme activity of which is not affected or even improved. At the same time, in oral therapy, the retention time in the intestine is significantly prolonged, and the binding property with type II mucin covering the intestinal epithelial mucus layer is significantly improved, thereby improving the efficacy.

[0042] 2. The fusion protein of the present invention is suitable for oral treatment of diseases or disorders associated with elevated uric acid levels, including, for example, hyperuricemia, hyperuricosuria and gout, and lays a new foundation for the development of a series of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the SDS-PAGE image of the pig-baboon uricase purification;

[0044] Figure 2 This is the SDS-PAGE image of the purification of uricase from Candida utilis;

[0045] Figure 3 This is a graph showing the results of the uricase fusion protein binding activity experiment with mucin;

[0046] Figure 4 This is a graph showing the results of an experiment on the binding activity of uricase from Candida utilis to mucin. DETAILED DESCRIPTION

[0047] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0048] Unless otherwise specified, all reagents and raw materials used in the examples are commercially available.

[0049] Example 1: Preparation of uricase fusion protein

[0050] The amino acid sequence of the mucin binding polypeptide (MUB) is publicly available and can be found in the UniProt database by accession number Q9RGN5, as shown in SEQ ID NO: 1. The amino acid sequence of the recombinant porcine-baboon uricase (PBC) is publicly available and is shown in SEQ ID NO: 2. The amino acid sequence of the Candida utilis uricase (CuUOX) is publicly available and is shown in SEQ ID NO: 3. CuUOX is reported to have improved stability to proteolytic digestion by pancreatic enzymes (a collection of enzymes secreted by the pancreas) compared to the naturally occurring enzyme. The fusion protein (SEQ ID NO: 4-18) was prepared as follows.

[0051] (1) Uricase fusion protein is expressed in the form of fusion protein. An expression cassette for uricase fusion protein is constructed and inserted into the pET30 plasmid. Then, the expression cassette is transformed into competent E. coli BL21 (DE3) bacteria, and an engineered strain for expressing uricase fusion protein (SEQ ID NO: 4-18) is obtained by screening. After shake flask fermentation of the engineered bacteria, the uricase fusion protein is expressed at a high level in the cytoplasm in a soluble form. According to the protein electrophoresis results (such as Figure 1 , Figure 2 The strains selected after transformation were confirmed to express the target protein, and the sequence of the designed recombinant uricase was confirmed by DNA sequencing. The constructed strains were frozen and stored with 50% glycerol.

[0052] (2) The strain was fermented by shake flask fermentation. The culture medium formula is as follows: LB liquid culture medium: 5 g / L yeast extract, 10 g / L soy peptone, 10 g / L sodium chloride; fermentation medium: 2 g / L glucose, 20 g / L glycerol, 15 g / L yeast extract, 3 g / L ammonium sulfate, 17.2 g / L disodium hydrogen phosphate dodecahydrate, 3 g / L potassium dihydrogen phosphate, 1 g / L potassium chloride, 0.5 g / L sodium chloride. Under sterile conditions, 10 μl was taken from the glycerol tube containing the recombinant strain and inoculated into a shake flask containing 30 ml LB liquid culture medium containing 25 μg / ml kanamycin for seed liquid culture. After culturing at 37°C and 220 rpm for 12 hours, the inoculation amount was transferred to a shake flask containing 200 ml ULP1 fermentation medium under sterile conditions for shake flask fermentation at 2% (v / v). The culture was cultured at 37°C and 220 rpm until the bacterial OD 600 After the pH value was 0.6-0.8, IPTG was added to a final concentration of 0.1 mmol / L, and the culture was continued at 30°C and 220 rpm to induce the expression of urate oxidase. After 6 h of culture, the shake flask fermentation was terminated and the bacteria were collected by centrifugation at 10,000×g for 10 min.

[0053] (3) Use a cell disruptor JN-02C (Ju Neng Biotech) to homogenize and disrupt the recombinant E. coli cells. After centrifugation, the collected bacteria were resuspended in homogenization buffer PBS (ratio of 1 g bacteria / 10 ml buffer), filtered through a mesh, set the homogenization pressure to 1000 bar, and the cooling circulating water temperature to 5°C. Repeat 3 times to disrupt the recombinant E. coli cells. Collect the cell lysis solution and collect the supernatant of the cell lysis solution by centrifugation. Pre-equilibrate a chromatography column filled with xanthine-agarose (Sigma) with mobile phase A: 50 mmol / L disodium hydrogen phosphate (pH 8.5). The supernatant of the cell lysis solution was filtered with a 0.22 μm aqueous filter membrane and then loaded. After loading, the column was rinsed with mobile phase A, and the protein bound to the filler was isocratically eluted with mobile phase B (50 mmol / L triethanolamine, 50 mmol / L sodium chloride, 0.5 mmol / L xanthine). The eluted sample containing the target protein was detected and collected, and PBS (pH 7.4) was added to an ultrafiltration centrifuge tube, and the xanthine was removed by centrifugation at 3000×g for 15 min for 3 times to obtain a purified solution of urate oxidase fusion protein.

[0054] Example 2: Determination of uricase activity

[0055] The enzyme activity of the uricase fusion protein was determined by ultraviolet spectrophotometry. Uric acid, the substrate of uricase oxidase, has a maximum absorption at 293 nm, so the decrease in absorbance at 293 nm per minute detected by the kinetic continuous monitoring method can represent the enzyme activity.

[0056] Using a 50mmol / L potassium dihydrogen phosphate buffer system (pH6.8), add 50μl of appropriately diluted enzyme solution to a quartz 96-well plate, preheat in a 37℃ microplate incubator for 5min, add 200μl of 20μg / ml uric acid solution and mix quickly, put it into a multifunctional microplate reader controlled at 37℃, and continuously measure the decrease in absorbance at 293nm within 3min every 30s. Enzyme activity, that is, the initial rate of enzyme reaction, is reflected by the decrease rate of absorbance measured at the detection wavelength of 293nm at the 2nd to 5th time points. At the same time, the absorbance values ​​of borate buffers containing different molar concentrations of uric acid at the detection wavelength of 293nm were measured, and the molar concentration OD293nm standard curve was drawn. The enzyme activity was calculated in combination with the decrease rate of absorbance measured above.

[0057] The results are shown in Table 1. The specific activity of the original recombinant pig-baboon uricase SEQ ID NO: 2 was 1.20 U / mg, and the specific activity of the recombinant pig-baboon uricase fusion protein SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, which was connected to SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24 at the C-terminus, was increased to 1.26 U / mg, 2.26 U / mg, 1.89 U / mg, and 1.87 U / mg. The specific activity of the recombinant pig-baboon uricase fusion protein SEQ ID NO: 7, which was connected to SEQ ID NO: 23 at the C-terminus, was reduced to 0.67 U / mg, indicating that the recombinant pig-baboon uricase C-terminus connected to SEQ ID NO: 23 had an effect on the activity of the enzyme. The specific activity of the recombinant porcine-baboon uricase, which was connected to the N-terminus of SEQ ID NO:23 and SEQ ID NO:25 by fusion proteins SEQ ID NO:8 and SEQ ID NO:9, was increased to 2.25 U / mg and 1.67 U / mg, respectively. The specific activity of the original Candida utilis uricase SEQ ID NO:3 was 1.07 U / mg, and the specific activity of the Candida utilis uricase, which was connected to the C-terminus of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 by fusion proteins SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, was increased to 1.28 U / mg, 1.20 U / mg, 1.13 U / mg, 1.15 U / mg, and 1.21 U / mg, respectively. The enzyme specific activities of the fusion proteins SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18 of the Candida utilis uricase with the N-terminus connected to SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25 were increased to 1.71 U / mg and 1.26 U / mg, respectively, indicating that the enzyme activity of the fusion protein of uricase connected to the mucin binding peptide was maintained and was partially superior to the original sequence.

[0058] Table 1

[0059]

[0060] Example 3: Evaluation of mucin binding capacity

[0061] (1) Biotinylation of urate oxidase

[0062] Biotin was covalently coupled to the amino group of uricase using the Genemore G-MM-IGT biotinylation kit. The buffer should not contain any other substances with amino groups except the target. If any, they must be removed by desalting or dialysis. Biotinylation reagent: target = 3:1 (molar ratio MCR), reaction volume is 400μl. The required volume of 10mM biotinylation reagent (μl) = target concentration (mg / ml) target molecular weight (kDa) × MCR × target volume (μl) × 0.1. Add the required reaction volume of biotinylation reagent and uricase fusion protein and mix evenly, react at room temperature for 30min. After the reaction is completed, centrifuge at 5000×g for 2min, aspirate the supernatant and remove free biotin by gravity desalting or ultrafiltration.

[0063] (2) Indirect ELISA test

[0064] Type II porcine gastric mucin (Sigma-Aldrich) was dissolved in pH 7.4 phosphate buffer (PBS) at about 4 mg / ml, 100 μL per well, incubated at 37°C for 1 h on a shaker, and coated on a 96-well ELISA plate (NUNC TM [Thermo Scientific]). After coating, pour off the solution in the wells, add 200 μL of 0.05% Tween20-PBS (PBST) as the washing solution to each well, and wash three times. Add 200 μL of 1% (w / v) bovine serum albumin (BSA) as the blocking solution to each well, and incubate at 37°C for 1 hour. After incubation, remove the blocking solution, add 200 μL of PBST to each well and wash three times. Dilute the biotinylated uricase fusion protein with PBS to different concentrations, add 100 μL to each well of the ELISA plate, set up 2 replicate wells for each sample, and incubate at 37°C for 1 hour. After incubation, wash each well with 200 μL of PBST three times. Add 100 μL of diluted HRP-streptomycin to each well, and incubate at 37°C for 30 minutes. After incubation, wash each well with 200 μL of PBST three times. Add 100 μL TMB reaction solution to each well and incubate at 37°C for 20 min. Then add 50 μL stop solution (2 mol / L H2SO4 solution) to stop the reaction. After adding the stop solution, detect the OD on the microplate reader in time. 450nm Finally, the half-maximal effect concentration (EC 50 )value.

[0065] The results are shown in Table 2. Figure 3 , Figure 4As shown, the EC50 value of the original recombinant pig-baboon uricase was 207.90 nM, and the EC50 value of the fusion proteins of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24 was 207.90 nM. 50 The EC50 values ​​of the recombinant pig-baboon uricase were reduced to 74.28nM, 31.45nM, 32.18nM, 9.45nM, and 3.08nM, respectively. The EC50 values ​​of the fusion proteins SEQ ID NO:8 and SEQ ID NO:9, whose N-termini were connected to SEQ ID NO:23 and SEQ ID NO:25, respectively, were reduced to 3.27nM and 5.15nM. The EC50 value of the original Candida utilis uricase was 1220.00nM, and the EC50 value of the fusion proteins SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, whose C-termini were connected to SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, were reduced to 74.28nM, 31.45nM, 32.18nM, 9.45nM, and 3.08nM, respectively. The EC50 values ​​of the recombinant pig-baboon uricase were reduced to 3.27nM and 5.15nM, respectively. 50 The values ​​were reduced to 0.89nM, 1.40nM, 0.91nM, 1.99nM and 0.70nM, respectively. The EC50 of the fusion proteins SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18 of the Candida utilis uricase linked to SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:25 at the N-terminus were reduced to 6.93nM, 1.15nM and 3.96nM, respectively.

[0066] It can be seen that the uricase fusion protein connected to the MUB fragment can enhance the binding ability of uricase to mucin. Enhancing the binding ability of uricase to mucin can prolong the retention time of uricase in the intestine after oral administration, and have a longer active window in the intestine, thereby enhancing its therapeutic effect.

[0067] Table 2

[0068]

[0069]

[0070] The sequences involved in the embodiment are:

[0071] SEQ ID NO:1

[0072] >MUB

[0073]

[0074] SEQ ID NO:2

[0075] >PBC

[0076] MTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL

[0077] SEQ ID NO:3

[0078] >CuUOX

[0079] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL

[0080] SEQ ID NO:4

[0081] >PBC-MUB2

[0082] MTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRLGGGGSGGGSGGGSGGGGSSDVQHAVINYIDGESDEILHTDKVNGHSDEKINYSTADMIKQLEAKGYELFKDNFPAGEKFDNDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNDAEQGPTLTVIYTAD

[0083] SEQ ID NO:5

[0084] >PBC-MUB4

[0085] MTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRLGGGGSGGGSGGGSQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDTTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNDAEQGPTLTVIYTAD

[0086] SEQ ID NO:6

[0087] >PBC-MUB6

[0088] MTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRLGGGGSGGGSGGGSQEAAINFYDETGHKLLDNQTIHLTGKTGEKVDRTQADQTLADLVKQGYVLDKENTAKAFPADAVYDNNDQTPQEFTIYLKHGTTHDATSSKADQKTVSETIHYVYKDGVNANKPVADDANTTVTFKRGYTTDKVTGKIVSYDPWTVDGKQADSKTFDAVKSPVIAGYTADQAEVAAQTVTPDSQNINKTVYYTAD

[0089] SEQ ID NO:7

[0090] >PBC-MUB8

[0091] MTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRLGGGGSGGGSGGGSQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKYTADTNEIPAITYHAGDSDVTYVVKYN

[0092] SEQ ID NO:8

[0093] >MUB8-PBC

[0094] MNAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKYTADTNEIPAITYHAGDSDVTYVVKYNGGGGSGGGSGGGSGGGGSTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL

[0095] SEQ ID NO:9

[0096] >MUB84-PBC

[0097] MNAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYNAQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPT IAGYAPSEAVVKRSSNSDAEQGPTLTVIYTADGGGGSGGGSGGGSTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL

[0098] SEQ ID NO:10

[0099] >PBC-MUB48

[0100] MTYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRLGGGGSGGGSGGGSQKVHVQYIDGETDQMLRQDDLDGYTDETI PYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNDAEQGPTLTVIYTADAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYN

[0101] SEQ ID NO:11

[0102] >CuUOX-MUB2

[0103] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKLGGGGSGGGGSGGGGSDVQHAVINYIDGESDEILHTDKVNGHSDEKINYSTADMIKQLEAKGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTAD

[0104] SEQ ID NO:12

[0105] >CuUOX-MUB4

[0106] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKLGGGGSGGGGSGGGGSQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTAD

[0107] SEQ ID NO:13

[0108] >CuUOX-MUB6

[0109] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKLGGGGSGGGGSGGGGSQEAAINFYDETGHKLLDNQTIHLTGKTGEKVDRTQADQTLADLVKQGYVLDKENTAKAFPADAVYDNNDQTPQEFTIYLKHGTTHTDATSSKADQKTVSETIHYVYKDGVNANKPVADDANTTVTFKRGYTTDKVTGKIVSYDPWTVDGKQADSKTFDAVKSPVIAGYTADQAEVAAQTVTPDSQNINKTVYYTAD

[0110] SEQ ID NO:14

[0111] >CuUOX-MUB8

[0112] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKLGGGGSGGGGSGGGGSQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYN

[0113] SEQ ID NO:15

[0114] >CuUOX-MUB48

[0115] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKLGGGGSGGGGSGGGGSQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTADAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYN

[0116] SEQ ID NO:16

[0117] >MUB4-CuUOX

[0118] MQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTADGGGGSGGGGGSGGGGSSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL

[0119] SEQ ID NO:17

[0120] >MUB8-CuUOX

[0121] MNAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYNGGGGSGGGGSGGGGSSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL

[0122] SEQ ID NO:18

[0123] >MUB84-CuUOX

[0124] MNAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYNAQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTADGGGGSGGGGSGGGGSSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGAFDTSYTKADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRRTDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGFNKCDFTTLQPTTDRVLSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL

[0125] SEQ ID NO:19

[0126] GGGGSGGGGSGGGGS

[0127] SEQ ID NO:20

[0128] >MUB2

[0129] DVQHAVINYIDGESDEILHTDKVNGHSDEKINYSTADMIKQLEAKGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTAD

[0130] SEQ ID NO:21

[0131] >MUB4

[0132] QKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTAD

[0133] SEQ ID NO:22

[0134] >MUB6

[0135] QEAAINFYDETGHKLLDNQTIHLTGKTGEKVDRTQADQTLADLVKQGYVLDKENTAKAFPADAVYDNNDQTPQEFTIYLKHGTTHTDATSSKADQKTVSETIHYVYKDGVNANKPVADDANTTVTFKRGYTTDKVTGKIVSYDPWTVDGKQADSKTFDAVKSPVIAGYTADQAEVAAQTVTPDSQNINKTVYYTAD

[0136] SEQ ID NO:23

[0137] >MUB8

[0138] QEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYN

[0139] SEQ ID NO:24

[0140] >MUB48

[0141] QKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNSDAEQGPTLTVIYTADAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYN

[0142] SEQ ID NO:25

[0143] >MUB84

[0144] MNAQEAQAIFYDETTGKEISGTREIATGKTDETISFTKDPNEVVKELEKQGYVFDKDNAKNNVFVAGTAYDKNSEVHQYFKYYLKHGHATVTPDQDPQKGQKTVTQTIKYEYADGTATGLADNVQTLTFKRTGDKDLVTHEVTWPDWSTVAGQQTSVVTSPALKGYTADTNEIPAITYHAGDSDVTYVVKYNAQKVHVQYIDGETDQMLRQDDLDGYTDETIPYSTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIFKHHRENVDPNHSSADGTKGTKTLTETVHYKYANGTKAAEDQTAQVTFTRNGVLDDVTGIVAWGKWNEASQSYKALTSPTIAGYAPSEAVVKRSSNDAEQGPTLTVIYTAD.

Claims

1. A fusion protein, characterized in that It is composed of a fusion of uricase and a polypeptide with mucin-binding properties.

2. The fusion protein according to claim 1, characterized in that The uricase is recombinant porcine-baboon uricase or Candida utilis uricase.

3. The fusion protein according to claim 1, characterized in that The sequence of the polypeptide having mucin binding properties is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 or SEQ ID NO:

25.

4. The fusion protein according to claim 2 or 3, characterized in that The sequence of the recombinant pig-baboon uricase is SEQ ID NO: 2, and the sequence of the polypeptide having mucin binding properties is SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, Preferably, when the C-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, preferably SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, more preferably SEQ ID NO:24; when the N-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:23, SEQ ID NO:25, preferably SEQ ID NO:

23.

5. The fusion protein according to claim 2 or 3, characterized in that The sequence of the Candida utilis uricase is SEQ ID NO: 3, and the sequence of the polypeptide having mucin binding properties is SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 or SEQ ID NO: 25, Preferably, when the C-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, preferably SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, more preferably SEQ ID NO:24; when the N-terminus is connected to a polypeptide having mucin binding properties, the sequence is SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, preferably SEQ ID NO:

23.

6. The fusion protein according to claim 1, characterized in that The uricase is directly fused to the polypeptide having mucin binding properties or is fused with a linker, and / or the uricase is at the N-terminus or C-terminus of the polypeptide having mucin binding properties.

7. The fusion protein according to claim 6, characterized in that The linker sequence is SEQ ID NO:

19.

8. The fusion protein according to claim 1, characterized in that The sequence of the fusion protein is as follows: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:

18.

9. A pharmaceutical composition, characterized in that The composition comprises the fusion protein according to any one of claims 1 to 8 and a pharmaceutically acceptable excipient.

10. Use of the fusion protein according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of an oral drug for treating diseases or disorders associated with elevated uric acid levels, preferably hyperuricemia, hyperuricosuria and gout.

Images