Human urate oxidase mutant with immunogenicity weakening and high activity

Abstract

The invention belongs to the technical field of biology, and relates to a human urate oxidase mutant with immunogenicity weakening and high activity. The human urate oxidase mutant with immunogenicity weakening 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 human urate oxidase mutant has 83rd, 84th, 119th, 121st, 151st, 222nd, 232, 233rd, 240th and 252nd amino acid substitutions. The human urate oxidase mutant with immunogenicity weakening and high activity has the advantages that the human urate oxidase mutant with immunogenicity weakening and high activity is obtained by mutation of inactive human urate oxidase with an amino acid sequence of SEQ ID NO.1, and the human urate oxidase mutant with immunogenicity weakening and high activity is obtained by mutation of inactive human urate oxidase with an amino acid sequence of SEQ ID NO.1; the human uricase mutant keeps high homology with a theoretical amino acid sequence of human uricase, has high enzyme activity while reducing immunogenicity, and is suitable for long-term use in clinical treatment of hyperuricemia and gout. The traditional Chinese medicine composition can be used for preparing medicines for treating hyperuricemia and gout.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and relates to a modified urate oxidase, and particularly 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 oxidases approved for the treatment of hyperuricemia in clinics mainly come 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 half-life and stability, patients may still produce anti-drug antibodies (ADAs) and infusion reactions, thus limiting its wide application in clinics. Therefore, it is mainly used to treat 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 for the modification 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 urate oxidase activity. JIANG et al. reported obtaining a low-immunogenic therapeutic urate oxidase 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, the “resurrected” urate oxidase rHU15 with 95.06% identity to the theoretical amino acid sequence of human urate oxidase was obtained, but the specific activity of the enzyme 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 activity of the enzyme 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, and there will still be significant immunogenicity. Since resurrecting the human urate oxidase 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 two problems with the resurrection strategy for human urate oxidase: First, by analyzing the conservation of the amino acid sequences of isozymes from different species and mutating to restore the activity of human urate oxidase, it is actually non-humanized in terms of the protein amino acid sequence. Repairing conserved sites enhances immunogenicity while restoring enzyme activity; Second, when the humanization rate of the amino acid sequence reaches a threshold (such as 95%), further increasing the humanization rate instead leads to a significant decrease in enzyme activity or 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 objective of the present invention is to provide a human urate oxidase mutant with weakened immunogenicity and high activity. This human urate oxidase mutant 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, 84, 119, 121, 151, 222, 232, 233, 240, and 252.

[0008] According to further characteristics 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 replacing glutamic acid (E) with glycine (G), the amino acid substitution at position 84 is replacing isoleucine (I) with cysteine (C) or histidine (H) or asparagine (N) or arginine (R), the amino acid substitution at position 119 is replacing histidine (H) with arginine (R), the amino acid substitution at position 121 is replacing glycine (G) with glutamic acid (E), the amino acid substitution at position 151 is replacing glutamine (Q) with proline (P), the amino acid substitution at position 222 is replacing serine (S) with phenylalanine (F), the amino acid substitution at position 232 is replacing leucine (L) with serine (S), the amino acid substitution at position 233 is replacing threonine (T) with proline (P), the amino acid substitution at position 240 is replacing cysteine (C) with tyrosine (Y), and the amino acid substitution at position 252 is replacing alanine (A) with glutamic acid (E); the amino acid sequences of the site-directed mutagenesis-modified human urate oxidase mutants are SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5 respectively.

[0009] When the amino acid substitution at position 84 is replacing isoleucine (I) with cysteine (C), the amino acid sequence of the site-directed mutagenesis-modified human urate oxidase mutant is SEQ ID NO. 2.

[0010] When the 84th amino acid substitution is the replacement of isoleucine (I) with histidine (H), the amino acid sequence of the site-directed mutagenesis-modified human urate oxidase mutant is SEQ ID NO. 3.

[0011] When the 84th amino acid substitution is the replacement of isoleucine (I) with asparagine (N), the amino acid sequence of the site-directed mutagenesis-modified human urate oxidase mutant is SEQ ID NO. 4.

[0012] When the 84th amino acid substitution is the replacement of isoleucine (I) with arginine (R), the amino acid sequence of the site-directed mutagenesis-modified human urate oxidase mutant is SEQ ID NO. 5. The second object of the present invention is to provide a DNA molecule encoding the immunogenicity-attenuated and highly active human urate oxidase mutant of the present invention. When the amino acid sequence of the human urate oxidase mutant of the present invention is SEQ ID NO. 2, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 6.

[0013] When the amino acid sequence of the human urate oxidase mutant of the present invention is SEQ ID NO.3, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 7.

[0014] When the amino acid sequence of the human urate oxidase mutant of the present invention is SEQ ID NO. 4, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 8.

[0015] When the amino acid sequence of the human urate oxidase mutant of the present invention is SEQ ID NO. 5, the nucleotide sequence of the human urate oxidase mutant is SEQ ID NO. 9.

[0016] The third object of the present invention is to provide a vector containing the DNA molecule of the present invention.

[0017] The fourth object of the present invention is to provide a host cell containing the DNA molecule of the present invention, or containing the vector of the present invention.

[0018] The above-mentioned vector and host cell can both be prepared by means of techniques well known in the art.

[0019] The fifth object of the present invention is to provide a production method for the immunogenicity-attenuated and highly active human urate oxidase mutant.

[0020] The production method of the human urate oxidase mutant with weakened immunogenicity and high activity according to the present invention includes: culturing the host cell according to the present invention under conditions suitable for the expression of the urate oxidase mutant, and separating the human urate oxidase mutant with weakened immunogenicity and high activity from the culture medium.

[0021] When the DNA molecule according to 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 virus; insect cell systems infected with virus; plant cell systems infected with bacteria. Preferred vectors of the present invention include viral vectors, plasmids, cosmids or oligonucleotides.

[0022] A preferred host of the present invention is a prokaryotic system such as Escherichia coli; a preferred protein expression method of the present invention is the expression of bacilli.

[0023] The sixth object of the present invention is to provide the application of the human urate oxidase mutant with weakened immunogenicity and high activity in the preparation of drugs for the treatment of hyperuricemia and gout.

[0024] The present invention performs site-directed mutagenesis on the amino acid sequence of the 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 the inactive human urate oxidase (SEQ ID NO.1).

[0025] Based on SEQ ID NO.1, the inventor obtained a urate oxidase mutant rhUOX with good activity and high humanization degree after immunological weakening design through site-directed mutagenesis mut1 / I84C (SEQ ID NO. 2), rhUOX mut1 / I84H (SEQ ID NO. 3), rhUOX mut1 / I84N (SEQ ID NO. 4) and rhUOX mut1 / I84R(SEQ ID NO.5). They have a homology of 96.05% with the theoretical amino acid sequence of human urate oxidase, showing a higher degree of humanization than the previously reported urate oxidase. Their specific enzyme activities are 7.998 U / mg, 7.254 U / mg, 7.439 U / mg, and 7.922 U / mg respectively, showing high enzyme activity while breaking through the 95% homology threshold reported in the literature. Experiments show that they have low immunogenicity, similar to that of human serum albumin (HSA), because 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 mutants with weakened immunogenicity and high activity described in the present invention better meet the clinical requirements of low-immunogenic urate oxidase, are 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

[0026] Figure 1 It is the SDS-PAGE protein electrophoresis diagram of the human urate oxidase mutant described in the present invention, where 1: rhUOX wt ; 2: rhUOX mut1 / I84C mutant; 3: rhUOX mut1 / I84H mutant; 4: rhUOX mut1 / I84N mutant; 5: rhUOX mut1 / I84R mutant; M is Marker.

[0027] Figure 2 Showing the uric acid degradation activity of rhUOX wt , rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N , rhUOX mut1 / I84R described in the present invention.

[0028] Figure 3 Showing the human blood monocyte immunogenicity test results of rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R as well as urate oxidase from Aspergillus flavus ( Af UOX) and human serum albumin (HAS).

[0029] Figure 4 Showing the in vivo efficacy results of human urate oxidase, where A: the in vivo efficacy results in male mice; B: the in vivo efficacy results in female mice. DETAILED DESCRIPTION OF THE INVENTION

[0030] Unless otherwise defined, the terms used in this document have the meanings commonly understood by those skilled in the art. Definitions of some special terms used in the present invention are provided below.

[0031] “rhUOX wt ” refers to 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 italicized “ rhuox wt ”. The amino acid sequence of rhUOX wt is SEQ ID NO.1.

[0032] “rhUOX mut1 / I84C 、rhUOX mut1 / I84H 、rhUOX mut1 / I84N and rhUOX mut1 / I84R ” refer to the hUOX mutants described in the present invention, and their genes are represented by italicized “ rhuox mut1 / I84C 、 rhuox mut1 / I84H 、 rhuox mut1 / I84N 、 rhuox mut1 / I84R ”. The amino acid sequences of rhUOX mut1 / I84C 、rhUOX mut1 / I84H 、rhUOX mut1 / I84N and rhUOX mut1 / I84R mutants are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively.

[0033] Example 1: rhuox wt 、rhuox mut1 、rhuox mut1 / I84C 、rhuox mut1 / I84H 、rhuox mut1 / I84N 、rhuox mut1 / I84R Synthesis of gene The present invention uses the human urate oxidase pseudogene sequence rhuox wt (GenBank accession number: NR_003927.2), and through modification, the human urate oxidase mutant rhuox mut1 / I84C 、rhuox mut1 / I84H 、rhuox mut1 / I84N 、rhuoxmut1 / I84R The above-mentioned gene was synthesized by BGI and cloned between NcoI and XhoI of the pET28a plasmid (Invitrogen). The recombinant plasmids were named pET28a-huox wt 、pET28a-huox mut1 / I84C 、pET28a-huox mut1 / I84H 、pET28a-huox mut1 / I84N and pET28a-huox mut1 / I84R respectively.

[0034] Example 2: rhuox wt 、rhuox mut1 、rhuox mut1 / I84C 、rhuox mut1 / I84H 、rhuox mut1 / I84N 、rhuox mut1 / I84R Transformation of the genes into Escherichia coli Rosetta (DE3) and induction of expression of recombinant proteins The recipient bacterium in this experiment was Escherichia coli Rosetta (DE3). After transformation with the recombinant plasmids pET28a-huox wt 、pET28a-huox mut1 / I84C 、pET28a-huox mut1 / I84H 、pET28a-huox mut1 / I84N and pET28a-huox mut1 / I84R , preliminary screening was carried out using a kanamycin (Kan) resistance 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.

[0035] The positive recombinants were inoculated into 5 mL of LB medium (50 mg / mL Kan) and cultured overnight at 37°C with 200 rpm. The revived bacterial solution was inoculated into 500 mL of LB medium (50 mg / mL Kan) at a ratio of 1%, and cultured at 37°C with 200 rpm until the OD 600 reached 0.6. Then, 60 μM lactose was added, and induction of expression was carried out at 30°C with 200 rpm for 12 h. After induction, the bacterial cells were collected, resuspended in a carbonate buffer solution with pH 10.0 (mass - volume ratio of 1:20), and the bacterial cells were disrupted using an ultrasonic cell disruptor. After centrifugation at 6000g for 10 min, the supernatant was collected to obtain the recombinant protein.

[0036] Example 3: rhUOX wt , rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R SDS-PAGE electrophoresis detection of mutant recombinant proteins The SDS-PAGE electrophoresis detection in this example includes the following steps: (1) Prepare 10 mL of 10% separating gel. After mixing, 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.

[0037] (2) Prepare 4 mL of 5% stacking gel and evenly pour it on top of the separating gel. Insert the corresponding comb while avoiding air bubbles. After polymerization for 30 min, wait for the gel to solidify.

[0038] (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.

[0039] (4) Load the samples in sequence. The sample loading volume should not be too much, and 15 μL per well is appropriate.

[0040] (5) At the beginning of electrophoresis, set the voltage to 90 V for running 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).

[0041] (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.

[0042] (7) Image the gel and observe the results. The SDS-PAGE protein electrophoresis results are as Figure 1 shown.

[0043] The SDS-PAGE protein electrophoresis results are as Figure 1 shown. Lane 1 is the denatured rhUOX wt protein sample, and lanes 2 - 5 are the mutant rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R target protein samples. 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.

[0044] Example 4: Characterization of the bioactivity of mutants At 37 °C, 20 μL of purified protein (rhUOX mut1 / I84C 、rhUOX mut1 / I84H 、rhUOX mut1 / I84N and rhUOX mut1 / I84R ) was taken and added to 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 decreased UV absorption value at 293 nm using ultraviolet spectrophotometry. The control group was inactivated uricase. The experimental results are as Figure 2 shown. The specific enzyme activities of rhUOX mut1 / I84C 、rhUOX mut1 / I84H 、rhUOX mut1 / I84N and rhUOX mut1 / I84R were 7.998 U / mg, 7.254 U / mg, 7.439 U / mg and 7.922 U / mg respectively, and these mutants all had high activities.

[0045] Definition of enzyme activity unit: The amount of enzyme required to catalyze the oxidation of 1 μmol of uric acid per minute is defined as 1 unit.

[0046] 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.

[0047] Example 5: Immunogenicity analysis of human PBMC cells Using human serum albumin (HAS) and uricase from Aspergillus flavus ( Af UOX) as the control group, and rhUOX mut1 / I84C 、rhUOX mut1 / I84H 、rhUOX mut1 / I84N and rhUOX mut1 / I84R as the experimental group. 50 μg / mL of the above-mentioned protein and PWM (20 μg / mL) were added to a 24-well cell culture plate at a volume of 100 μL per well (5 μg), and then PBMC cell suspension (1×10 6 / mL) was added, 500 μL per well (the dosing dose was 10 μg / mL). Incubated at 37 °C, 5% CO 2Culture in an incubator 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 and collect the cells. Add the cells into the cell culture plates coated with the corresponding proteins above, 500 μL per well. Incubate at 37 °C with 5% CO 2 Culture in an incubator for 7 days. After 2 - 3 days, change half of the culture medium once. After the culture is completed, collect the culture supernatant. Detect anti - uricase IgG by indirect ELISA and analyze the immunogenicity.

[0048] Specific steps of indirect ELISA: (1) Antibody coating: Add protein samples (HSA, Af UOX, rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R ) into the 96 - well plate at a volume of 100 μL per well (5 μg). Make two replicates for each sample. Incubate overnight at 4 °C and then wash 3 times with PBST. Pat dry. (2) Blocking: Block with Elisa blocking solution for 1 h. (3) Washing: Wash 3 times with PBST. Pat dry. (4) Incubation: Add 100 μL of the supernatant obtained from the above cell culture (HSA, Af UOX, rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R ) into each well. Use PBS as the blank control. Incubate at 37 °C for 1.5 h. (5) Washing: Wash three times with the washing solution. 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) into each well. Incubate at 37 °C for 1 h. (7) Washing: Wash three times with the washing solution. Pat dry. (8) Reaction and color development: Add 100 μL of freshly prepared TMB working solution into each well. React at room temperature in the dark for 15 min. Add 50 μL of the termination solution (2 M H 2 SO 4 ). Read the OD 450 value with an enzyme - linked immunosorbent assay reader.

[0049] The test well is considered positive when the A450 value is greater than or equal to 2.1 times that of the negative control well: P / N = OD450 Serum to be tested / OD 450 Negative control serum (i.e., P / N ≥ 2.1 is positive).

[0050] The results are as Figure 3 shown, the mutant rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R have low immunogenicity, which is similar to that of HSA.

[0051] Example 7: Verification of the Pharmacodynamic Effect of Uricase Gene KO Mice in Vivo A total of 48 C57BL / 6J mice (UOX- / -), half male and half female, 5 - 8 weeks old, weighing 25 - 30 g, SPF grade, were purchased from Cyagen Biosciences Inc. and housed in the SPF experimental animal room 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 were provided with clean drinking water and free diet before the experiment and were used for the experiment after 3 days of feeding.

[0052] A normal control group (normal serum level), a hyperuricemia control group, and an experimental group (rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R ) were set up respectively. The normal control group was wild-type C57BL / 6J mice, while the other groups used KO mice with uricase knockout for the experiment, with 12 mice in each group (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). Intraperitoneal injection was used for administration, and blood was collected from the orbital vein at 0, 0.5, 1, 3, and 6 h after administration. The serum was processed as above, and the uric acid content in the collected serum was detected by HPLC. The results are as Figure 4 shown, in the pharmacodynamic experiment of hyperuricemic KO mice, under the experimental conditions, the rhUOX mut1 / I84C , rhUOX mut1 / I84H , rhUOX mut1 / I84N and rhUOX mut1 / I84R with weakened immunogenicity can maintain the serum uric acid level of KO mice at a normal level for at least 3 hours and still play a role in reducing uric acid after 6 hours.

[0053] HPLC Detection Conditions Chromatographic column: C18 chromatographic column; length 150 mm, inner diameter 3 mm (PartNO: 70105 - 154330); flow rate: 1 mL / min; UV detector: UV wavelengths 225, 293 nm; column temperature: 30 °C; injection volume: 20 μL; mobile phase: A 0.1% formic acid - water; B pure methanol.

[0054] Regarding the number and position of point mutations, homology, specific enzyme activity, immunogenicity, etc., the comparison between the urate oxidase mutants of the present invention and Literature 1 and 2 is shown in Table 1 below.

[0055] Table 1:

[0056] 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.

[0057] Literature 2: XIONG Runsong. Cloning and Molecular Modification of Baboon Urate Oxidase Gene [D]. Beijing University of Chemical Technology, 2012.

[0058] As can be seen from the above table, the present invention first discloses human - source urate oxidase mutants rhUOX mut1 / I84C 、rhUOX mut1 / I84H 、rhUOX mut1 / I84N and rhUOX mut1 / I84R with weakened immunogenicity and high activity. The homology of these mutants with the theoretical amino acid sequence of human - source urate oxidase is 96.05%. The specific enzyme activities are 8.098 U / mg, 8.254 U / mg, 7.439 U / mg, and 7.922 U / mg respectively. While reducing the mutation sites, high activity is retained, and a relatively high degree of humanization is maintained. This mutant is higher than rHU15 (Literature 1) in terms of humanization rate and activity. 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 surfaces are further mutated and thus cannot be recognized, thereby reducing immunogenicity. Therefore, the human - source urate oxidase mutants with weakened immunogenicity and high activity described in the present invention better meet the clinical requirements of low - immunogenic urate oxidase, are 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, and has amino acid substitutions at positions 83, 84, 119, 121, 151, 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 84 is to replace isoleucine (I) with cysteine ​​(C) or histidine (H) or asparagine (N) or arginine (R), 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 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 sequences of the human uricase mutants modified by site-directed mutagenesis are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.

5.

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

4. The DNA molecule according to claim 3, characterized in that: Its nucleotide sequence is SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO.

9.

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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