Human urate oxidase mutant with weakened immunogenicity and high activity

Abstract

The invention belongs to the technical field of biology, and relates to a human urate oxidase mutant with weakened immunogenicity and high activity. The human urate oxidase mutant with weakened immunogenicity and high activity is a human urate oxidase mutant obtained by mutation of inactive human urate oxidase with an amino acid sequence of SEQ ID NO.1; the amino acid sequence of the amino acid sequence is shown as SEQ ID NO.1. The amino acid sequence has 83rd, 85th, 112th, 119th, 121th, 151th, 208th, 219th, 222nd, 232, 233rd, 240th and 252th amino acid substitutions. The human uricase mutant keeps high homology with the theoretical amino acid sequence of human uricase, has high activity while reducing immunogenicity, is suitable for long-term use in clinical treatment of hyperuricemia and gout, and can be used for preparing drugs for treating hyperuricemia and gout.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to urate oxidase, and particularly relates to a human urate oxidase mutant with weakened immunogenicity and high activity. Background Art

[0002] The clinical treatment drugs for hyperuricemia and gout mainly include allopurinol, xanthine oxidase inhibitors, benzbromarone, probenecid, colchicine, etc. These drugs generally have the risk of multi-organ toxicity, and their applications are severely restricted. As a key enzyme in the terminal link of purine metabolism, urate oxidase is responsible for catalyzing the conversion of uric acid into allantoin with higher solubility, showing a more ideal uric acid-lowering effect. Currently, the recombinant urate oxidase approved for the treatment of hyperuricemia in clinical practice mainly comes from heterologous species, such as Aspergillus flavus and porcine-baboon chimeras, and the heterology often causes severe allergic reactions in patients. Although the urate oxidase modified with polyethylene glycol (PEG) has improved in terms of half-life and stability, patients may still produce anti-drug antibodies (ADAs) and infusion reactions, thus limiting its wide application in clinical practice. Therefore, it is mainly used for the treatment of acute, severe, life-threatening refractory hyperuricemia. The existence of immunogenicity has become the main limitation in the application of urate oxidase. Developing urate oxidase with low immunogenicity has become a hot research field in the current transformation of recombinant urate oxidase.

[0003] During the process of evolution, the human urate oxidase gene has become a pseudogene due to complex missense mutations and their accumulation. Although short peptides of urate oxidase have been found in human sweat glands, there is no activity of urate oxidase. JIANG et al. reported obtaining a low-immunogenic urate oxidase for therapeutic use by resurrecting the human urate oxidase pseudogene (Literature 1: JIANG N, XU C, ZHANG L, et al. “Resurrected” human-source urate oxidase with high uricolytic activity and stability [J]. Enzyme and Microbial Technology, 2021, 149: 109852.). This literature shows that through multiple sequence alignment analysis of the inactive human urate oxidase gene and mutating 15 highly conserved sites, a “resurrected” urate oxidase rHU15 with 95.06% identity to the theoretical amino acid sequence of human urate oxidase was obtained, but the specific enzyme activity was only 2.30 U / mg. By further expanding the mutation of conserved sites, a mutant rHU19 with 19 sites mutated (the identity to the theoretical amino acid sequence of human urate oxidase decreased to 93.75%) was obtained, and the specific enzyme activity reached 8.29 U / mg. This literature did not test the immunogenicity of the mutants. Another literature reported that even baboon urate oxidase with up to 93.75% identity to the theoretical amino acid sequence of human urate oxidase may cause significant immunogenicity (Literature 2: XIONG Runsong. Cloning and Molecular Modification of Baboon Urate Oxidase Gene [D]. Beijing University of Chemical Technology, 2012.). Compared with baboon urate oxidase, rHU19 has a lower sequence identity, so it can be speculated that rHU19 is more similar to heterologous urate oxidase rather than human origin and may still have significant immunogenicity. Since resurrecting the human uricase pseudogene through back mutations at all highly conserved sites is essentially non-humanized, increasing the heterology of the resurrected enzyme and thus increasing immunogenicity.

[0004] As a protein that has never existed in the body in the form of a complete protein molecule during human development, there are dual problems with the resurrection strategy of human urate oxidase: one is that by analyzing the conservation of amino acid sequences of isozymes from different species sources and mutating to restore the activity of human urate oxidase, it is actually non-humanized in terms of protein amino acid sequence. While restoring the enzyme activity by repairing conserved sites, the immunogenicity is enhanced; the other is that when the humanization rate of the amino acid sequence reaches a threshold (such as 95%), further increasing the humanization rate will instead lead to a significant decrease in enzyme activity and even loss of activity.

[0005] Therefore, the current strategy of functional resurrection of human urate oxidase pseudogenes through homologous alignment and conservative amino acid mutations faces challenges and dilemmas. Therefore, it is necessary to find new strategies to obtain human urate oxidase mutants with weakened immunogenicity and high activity. Summary of the Invention

[0006] The primary object of the present invention is to provide a human urate oxidase mutant with weakened immunogenicity and high activity, which has a high homology with the theoretical amino acid sequence of human urate oxidase and has high enzyme activity while reducing immunogenicity.

[0007] The human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention is a human urate oxidase mutant obtained by mutating the inactive human urate oxidase with the amino acid sequence of SEQ ID NO.1; it has amino acid substitutions at positions 83, 85, 112, 119, 121, 151, 208, 219, 222, 232, 233, 240, and 252.

[0008] According to a further feature of the human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention, the amino acid substitution at position 83 is the replacement of glutamic acid (E) with glycine (G), the amino acid substitution at position 85 is the replacement of lysine (K) with cysteine (C), the amino acid substitution at position 112 is the replacement of methionine (M) with valine (V), the amino acid substitution at position 119 is the replacement of histidine (H) with arginine (R), the amino acid substitution at position 121 is the replacement of glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is the replacement of glutamine (Q) with proline (P), the amino acid substitution at position 208 is the replacement of lysine (K) with glutamic acid (E), the amino acid substitution at position 219 is the replacement of methionine (M) with leucine (L), the amino acid substitution at position 222 is the replacement of serine (S) with phenylalanine (F), the amino acid substitution at position 232 is the replacement of leucine (L) with serine (S), the amino acid substitution at position 233 is the replacement of threonine (T) with proline (P), the amino acid substitution at position 240 is the replacement of cysteine (C) with tyrosine (Y), and the amino acid substitution at position 252 is the replacement of alanine (A) with glutamic acid (E); the amino acid sequence of the site-directed mutagenesis-modified human urate oxidase mutant is SEQ ID NO. 2.

[0009] The second object of the present invention is to provide a DNA molecule encoding the human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention. According to the DNA molecule described in the present invention, its nucleotide sequence is SEQ ID NO. 3. The third object of the present invention is to provide a vector containing the DNA molecule described in the present invention.

[0010] The fourth object of the present invention is to provide a host cell containing the DNA molecule described in the present invention, or containing the vector described in the present invention.

[0011] Both the above-mentioned vector and host cell can be prepared by techniques well-known in the art.

[0012] The fifth object of the present invention is to provide a method for producing an immunogenicity-attenuated and highly active human urate oxidase mutant.

[0013] The method for producing an immunogenicity-attenuated and highly active human urate oxidase mutant described in the present invention includes: culturing the host cell described in the present invention under conditions suitable for the expression of the urate oxidase mutant, and isolating the immunogenicity-attenuated and highly active human urate oxidase mutant from the culture medium.

[0014] When the DNA molecule described in the present invention is inserted into the vector in a suitable orientation and correct reading frame, or transferred into the host cell, the DNA molecule can be expressed in any eukaryotic or prokaryotic expression system. Many host-vector systems can be used to express protein-coding sequences. Host-vector systems include, but are not limited to: bacteria transformed with phage, plasmid or cosmid; microorganisms containing yeast vectors, such as yeast; mammalian cell systems infected with viruses; insect cell systems infected with viruses; plant cell systems infected with bacteria. Preferred vectors of the present invention include viral vectors, plasmids, cosmids or oligonucleotides.

[0015] A preferred host of the present invention is a prokaryotic system such as Escherichia coli; a preferred method for protein expression of the present invention is the expression of bacilli.

[0016] The sixth object of the present invention is to provide the application of the immunogenicity-attenuated and highly active human urate oxidase mutant in the preparation of a drug for treating hyperuricemia and gout.

[0017] The present invention performs site-directed mutagenesis on the amino acid sequence of inactive human urate oxidase of the human urate oxidase pseudogene (referred to as the hUOX gene). The GENBANK accession number of the human urate oxidase pseudogene sequence is NR_003927.2. The terminators at positions 3 and 187 in the sequence are changed to R to form the amino acid sequence of inactive human urate oxidase (SEQ ID NO.1).

[0018] Based on SEQ ID NO.1, the inventor obtained a urate oxidase mutant rhUOX with good activity and high humanization degree after immunogenicity attenuation design through site-directed mutagenesismut3 / K85C (SEQ ID NO. 2). The homology of this mutant with the theoretical amino acid sequence of human urate oxidase is 95.06%, and the specific enzyme activity is 7.825 U / mg. While breaking through the 95% homology threshold reported in the literature, it shows relatively high enzyme activity. Experiments show that this mutant has low immunogenicity, similar to that of human serum albumin (HSA), because the hot spot amino acids of the antigenic peptide on its protein surface are further mutated and thus cannot be recognized, thereby reducing immunogenicity. Therefore, the human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention better meets the clinical requirements of low-immunogenic urate oxidase, is suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the SDS-PAGE protein electrophoresis map of the human urate oxidase mutant described in the present invention, wherein, 1: rhUOX wt ; 2: rhUOX mut3 / K85C ; M is Marker.

[0020] Figure 2 Showing the uric acid degradation activity of rhUOX wt , rhUOX mut3 / K85C described in the present invention.

[0021] Figure 3 Showing the human blood monocyte immunogenicity test results of rhUOX mut3 / K85C described in the present invention, as well as urate oxidase from Aspergillus flavus ( Af UOX) and human serum albumin (HAS).

[0022] Figure 4 Showing the in vivo pharmacodynamic results of human urate oxidase, wherein, A: the pharmacodynamic results in male mice; B: the pharmacodynamic results in female mice. DETAILED DESCRIPTION OF THE INVENTION

[0023] The terms used herein, unless otherwise specified, have the meanings commonly understood by those skilled in the art. The following provides the definitions of some special terms used in the present invention.

[0024] "rhUOX wt " refers to the inactive human urate oxidase in which the missense (terminator) codons at positions 33 and 187 of the human urate oxidase pseudogene are restored to arginine (R), and its gene is represented by italic " rhuox wt ". The amino acid sequence of rhUOX wt is SEQ ID NO.1.

[0025] “rhUOX mut3 / K85C ” represents a hypoimmunogenic hUOX mutant, the gene of which is in italics “ rhuox mut3 / K85C ”. The amino acid sequences of rhUOX mut3 / K85C mutants are SEQ ID NO.2 respectively.

[0026] Example 1: rhuox wt 、rhuox mut3 / K85C Synthesis of gene The human urate oxidase pseudogene sequence rhuox wt (GenBank accession number: NR_003927.2) was used in this invention, and after modification, a human urate oxidase mutant rhuox mut3 / K85C was obtained. The above gene was synthesized by BGI and cloned between NcoI and XhoI of the pET28a plasmid (Invitrogen). The recombinant plasmids were named pET28a-huox wt and pET28a-huox mut3 / K85C .

[0027] Example 2: rhuox wt 、rhuox mut3 / K85C Transformation of genes into Escherichia coli Rosetta (DE3) and induction expression of recombinant proteins The recipient bacterium in this experiment was Escherichia coli Rosetta (DE3). After transformation with the recombinant plasmids pET28a-huox wt and pET28a-huox mut3 / K85C , preliminary screening was carried out using a kanamycin (Kan) resistant plate. Then, single colonies on the Kan plate were picked and cultured in 5 mL of LB liquid medium (containing 50 mg / mL Kan) for 12 - 16 h, and then plasmids were extracted for PCR verification to further screen positive clone recombinants.

[0028] The positive recombinants were inoculated into 5 mL of LB medium (50 mg / mL Kan) and cultured overnight at 37 °C and 200 rpm. The revived bacterial liquid was inoculated into 500 mL of LB medium (50 mg / mL Kan) at 1% and cultured at 37 °C and 200 rpm until OD 600It was 0.6, 60 μM lactose was added, and induced expression was carried out at 30 °C under the condition of 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended with carbonate buffer at pH 10.0 (mass-to-volume ratio of 1:20), the bacterial cells were disrupted using an ultrasonic cell disruptor, centrifuged at 6000 g for 10 min, and the supernatant was collected to obtain the recombinant protein.

[0029] Example 3: rhUOX wt and rhUOX mut3 / K85C SDS-PAGE electrophoresis detection of mutant recombinant protein The SDS-PAGE electrophoresis detection in this example includes the following steps: (1) Prepare 10 mL of 10% separating gel, mix well, and use a micropipette to pour the gel into the glass plate until it stops 2 - 3 cm from the upper edge of the short glass plate. Then seal the gel surface with distilled water. You can gently lift one end of the gel maker and then put it down to make the gel surface flat. After polymerization for 40 min, discard the distilled water and use filter paper to absorb the excess water.

[0030] (2) Prepare 4 mL of 5% stacking gel, pour it evenly on top of the separating gel, insert the corresponding comb while avoiding generating bubbles, and wait for 30 min for the gel to solidify.

[0031] (3) Assemble the electrophoresis tank, fill the tank with electrophoresis buffer, and the volume should be greater than half of the volume of the electrophoresis tank. Transfer the prepared gel into the electrophoresis tank and carefully remove the comb.

[0032] (4) Load the samples in sequence. The sample loading volume should not be too much, and 15 μL per well is appropriate.

[0033] (5) At the beginning of electrophoresis, set the voltage to 90 V to run the gel. When the indicator reaches the stacking gel part, change the voltage to 120 V and continue electrophoresis. Stop electrophoresis when the target band runs to the middle position (the target band corresponds to the corresponding band of the Maker, which can be known in advance).

[0034] (6) Carefully peel off the gel, stain it with Coomassie Brilliant Blue R-250 for 30 min, and then decolorize it with the decolorizing solution until the background is lighter and the protein bands are clear.

[0035] (7) Image the gel and observe the results. The SDS-PAGE protein electrophoresis results are as Figure 1 shown.

[0036] The SDS-PAGE protein electrophoresis results are as Figure 1 shown. Lane 1 is the denatured rhUOX wt protein sample, and lane 2 is the mutant rhUOX mut3 / K85CThe target protein sample. The size of the target protein is about 34 kDa. This result indicates that the target gene is successfully expressed in Escherichia coli Rosetta and can be used for subsequent experiments.

[0037] Example 4: Characterization of the activity of mutants At 37 °C, take 20 μL of the purified protein rhUOX mut3 / K85C In 600 μL of uric acid solution (pH 8.0) containing 0.1 M, after reacting for 10 min, an equal volume of pure methanol was added to terminate the reaction. The enzyme activity was calculated by measuring the decrease in ultraviolet absorption at 293 nm by ultraviolet spectrophotometry. The control group was inactivated urate oxidase. The experimental results are as Figure 2 shown. rhUOX mut3 / K85C The specific enzyme activity was 7.825 U / mg, and the mutant had high activity.

[0038] Definition of enzyme activity unit: The amount of enzyme required to catalyze 1 μmol of uric acid oxidation per minute is defined as 1 unit.

[0039] E (U / mg) = (△OD 293 × V1 × D) / (12.3 × V2 × T × C) In the formula, V1 is the total reaction volume; D is the dilution factor; 12.3 is the molar extinction coefficient of uric acid; T is the reaction time; C is the protein concentration.

[0040] Example 5: Immunogenicity analysis of human PBMC cells Using human serum albumin (HAS) and Aspergillus flavus-derived urate oxidase ( Af UOX) as the control group and rhUOX mut3 / K85C as the experimental group. Add 50 μg / mL of the above proteins and PWM (20 μg / mL) in an amount of 100 μL per well (5 μg) to a 24-well cell culture plate, and then add the PBMC cell suspension (1×10 6 / mL), 500 μL per well (the dosing dose is 10 μg / mL). Incubate in an incubator at 37 °C and 5% CO 2 for 7 days. After 2 - 3 days, change half of the culture medium once. After the culture is completed, centrifuge at 3000 rpm for 15 min to collect the cells. Add the cells to the cell culture plate coated with the corresponding above proteins, 500 μL per well. Incubate in the same incubator at 37 °C and 5% CO 2 for 7 days. After 2 - 3 days, change half of the culture medium once. After the culture is completed, collect the culture supernatant. Use indirect ELISA to detect anti-urate oxidase IgG and analyze the immunogenicity.

[0041] Specific steps of indirect ELISA: (1) Antibody coating: Add protein samples (HSA, Af UOX, rhUOX mut3 and rhUOX mut3 / K85C ) to a 96-well plate at a volume of 100 μL per well (5 μg), and prepare two replicates for each sample. After incubating overnight at 4 °C, wash three times with PBST and pat dry. (2) Blocking: Block with Elisa blocking solution for 1 h. (3) Washing: Wash three times with PBST and pat dry. (4) Incubation: Add 100 μL of the supernatant obtained from the above cell culture (HSA, Af UOX, rhUOX mut3 and rhUOX mut3 / K85C ) to each well. Use PBS as the blank control, and incubate at 37 °C for 1.5 h. (5) Washing: Wash three times with the washing solution and pat dry. (6) Incubation with HRP-goat anti-human IgG: Add 100 μL of diluted HRP-goat anti-human IgG (1:3000, PBST, pH 7.4) to each well, and incubate at 37 °C for 1 h. (7) Washing: Wash three times with the washing solution and pat dry. (8) Reaction and color development: Add 100 μL of freshly prepared TMB working solution to each well, and react in the dark at room temperature for 15 min. Add 50 μL of stop solution (2 M H 2 SO 4 ) to each well. Read the OD 450 value with an enzyme-linked immunosorbent assay reader.

[0042] Consider the test well with A450 greater than or equal to 2.1 times that of the negative control well as positive: P / N = OD 450 test serum / OD 450 negative control serum (i.e., P / N ≥ 2.1 is positive).

[0043] The results are as Figure 3 shown. The mutant rhUOX mut3 / K85C has low immunogenicity, comparable to that of HSA.

[0044] Example 7: In Vivo Efficacy Verification of Uricase Gene KO Mice The experiment used 48 C57BL / 6J mice (UOX- / -), with 24 males and 24 females, aged 5 - 8 weeks, weighing 25 - 30 g, of SPF grade. They were purchased from Cyagen Biosciences Inc. and housed in the SPF animal laboratory of the Experimental Animal Management Center of Jinan University. The room temperature was 20 - 25°C, and the experimental animal use license was SYXK (Guangdong) 2022 - 0174. The C57BL / 6J mice had access to clean drinking water and were fed freely before the experiment. After being raised for 3 days, they were used for the experiment.

[0045] A normal control group (with normal serum levels), a hyperuricemia control group, and an experimental group (rhUOX mut3 and rhUOX mut3 / K85C ) were set up respectively. The normal control group consisted of wild-type C57BL / 6J mice, while the other groups used knockout (KO) mice with uricase knockout for the experiment. Each group had 12 mice (6 males and 6 females). The normal control group and the hyperuricemia control group were given an equal volume of normal saline, while the experimental group was administered according to the dosing dose of rasburicase (2 U / kg). Administration was by intraperitoneal injection. Blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 h after dosing. Serum processing was the same as above, and the uric acid content in the collected serum was detected by HPLC. The results were as Figure 4 shown. In the pharmacodynamic experiment of hyperuricemic KO mice, under the experimental conditions, the rhUOX mut3 / K85C with weakened immunogenicity could maintain the serum uric acid level of KO mice at a normal level for at least 3 hours and still had the effect of reducing uric acid after 6 hours.

[0046] HPLC detection conditions Chromatographic column: C18 chromatographic column; length 150 mm, inner diameter 3 mm (PartNO: 70105 - 154330); flow rate: 1 mL / min; ultraviolet detector: ultraviolet wavelengths 225 and 293 nm; column temperature: 30°C; injection volume: 20 μL; mobile phase: A 0.1% formic acid - water; B pure methanol.

[0047] Regarding the number and location of point mutations, homology, specific enzyme activity, immunogenicity, etc., the comparison between the uricase mutants described in the present invention and those in References 1 and 2 is shown in Table 1 below.

[0048] Table 1:

[0049] Document 1: JIANG N, XU C, ZHANG L, et al. “Resurrected” human-source urateoxidase with high uricolytic activity and stability [J]. Enzyme and MicrobialTechnology, 2021, 149: 109852。

[0050] Document 2: XIONG Runsong. Cloning and molecular modification of baboon urate oxidase gene [D]. Beijing University of Chemical Technology, 2012。

[0051] As can be seen from the above table, the present invention first discloses a human urate oxidase mutant rhUOX with weakened immunogenicity and high activity mut3 / K85C , the homology of this mutant with the theoretical amino acid sequence of human urate oxidase is 95.06% (the same as rHU15 reported in Document 1), the specific enzyme activity is 7.825 U / mg (significantly higher than rHU15 reported in Document 1), while maintaining high activity with fewer mutation sites and maintaining a relatively high degree of humanization. Experiments show that they have low immunogenicity, similar to that of HSA. The reason is that the hot-spot amino acids of the antigenic peptides on their protein surface are further mutated and thus cannot be recognized, thereby reducing immunogenicity. Therefore, the human urate oxidase mutant with weakened immunogenicity and high activity described in the present invention better meets the clinical needs of low-immunogenic urate oxidase, is suitable for long-term use in the clinical treatment of hyperuricemia and gout, and can be used to prepare drugs for the treatment of hyperuricemia and gout.

Claims

1. A human urate oxidase mutant with weakened immunogenicity and high activity, characterized in that: The mutant is a human uricase mutant obtained by mutation of an inactive human uricase with an amino acid sequence of SEQ ID NO.1; the human uricase mutant with an amino acid sequence of SEQ ID NO. 2 has amino acid substitutions at positions 83, 85, 112, 119, 121, 151, 208, 219, 222, 232, 233, 240 and 252.

2. The human urate oxidase mutant with weakened immunogenicity and high activity according to claim 1, characterized in that: The amino acid substitution at position 83 is to replace glutamic acid (E) with glycine (G), the amino acid substitution at position 85 is to replace lysine (K) with cysteine ​​(C), the amino acid substitution at position 112 is to replace methionine (M) with valine (V), the amino acid substitution at position 119 is to replace histidine (H) with arginine (R), the amino acid substitution at position 121 is to replace glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is to replace glutamine (Q) with proline (P), the amino acid substitution at position 208 is to replace lysine (K) with glutamic acid (E), and the amino acid substitution at position 210 is to replace lysine (K) with glutamic acid (E), and the amino acid substitution at position 211 is to replace glutamine (Q) with proline (P). The amino acid substitution at position 219 is to replace methionine (M) with leucine (L), the amino acid substitution at position 222 is to replace serine (S) with phenylalanine (F), the amino acid substitution at position 232 is to replace leucine (L) with serine (S), the amino acid substitution at position 233 is to replace threonine (T) with proline (P), the amino acid substitution at position 240 is to replace cysteine ​​(C) with tyrosine (Y), and the amino acid substitution at position 252 is to replace alanine (A) with glutamic acid (E); the amino acid sequence of the human urate oxidase mutant modified by site-directed mutagenesis is SEQ ID NO.

2.

3. A DNA molecule, characterized in that: It encodes the highly active human urate oxidase mutant with weakened immunogenicity as described in claim 1.

4. The DNA molecule according to claim 3, characterized in that: Its nucleotide sequence is SEQ ID NO.

4.

5. A carrier, characterized in that: It contains the DNA molecule according to claim 3 or 4.

6. A host cell, characterized in that: It contains the DNA molecule according to claim 3 or 4, or contains the vector according to claim 5.

7. A method for producing a human urate oxidase mutant with weakened immunogenicity and high activity according to claim 1, characterized in that: The method comprises: culturing the host cell according to claim 6 under conditions suitable for the expression of uricase, and isolating the human uricase mutant from the culture medium.

8. Use of the human urate oxidase mutant with weakened immunogenicity and high activity as claimed in claim 1 for preparing a drug for treating hyperuricemia and gout.

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