CN111269899B - Human uric acid oxidase with catalytic activity and application thereof - Google Patents

Abstract

The invention belongs to the technical field of protein engineering, and specifically relates to a human urate oxidase with catalytic activity, a gene sequence encoding the human urate oxidase, a preparation method of the human urate oxidase, and its use in reducing uric acid. Application in pharmaceutical preparation. The sequence of the enzyme is a recombinant amino acid sequence obtained by substituting amino acids or amino acid residues at several positions in the sequence shown in SED ID NO: 1, and the substitutions include at least 8 amino acid or amino acid residue substitutions. The human urate oxidase of the present invention has low immunogenicity, and can effectively reduce the uric acid value in the body through zoological verification, thereby laying the foundation for the preparation of pharmaceutical preparations for reducing uric acid with low or even no immunogenicity.

Description

Human uric acid oxidase with catalytic activity and application thereof

technical field

The invention belongs to the technical field of protein engineering, and in particular relates to human urate oxidase with catalytic activity and application thereof.

Background technique

In the catabolic pathway of purine in organisms, the process of degrading purine to uric acid under the action of xanthine oxidase is highly conserved from prokaryotes to eukaryotes. Due to the low solubility of uric acid and its salts in water, if the concentration is too high, crystallization will occur. Therefore, urate oxidase exists in most organisms, and urate oxidase can use molecular oxygen to oxidize uric acid to produce allantoin, which is more water-soluble and easy to excrete. However, during the long evolutionary process, the urate oxidase gene of higher primates such as humans, chimpanzees, orangutans, gorillas, and gibbons has undergone nonsense mutations and inactivated them, becoming pseudogenes. Therefore, the human body lacks urate oxidase, and uric acid becomes the final product of purine metabolism in the human body and is excreted through urine and intestines.

The daily production and excretion of uric acid in healthy people are approximately equal and in a state of balance. When the production of uric acid increases or the excretion decreases, it can cause the concentration of uric acid in the blood to increase. When the concentration of blood uric acid and its salts exceeds its solubility, hyperuricemia can be caused. Hyperuricemia can cause or exacerbate many diseases: for example, hyperuricemia is significantly positively correlated with obesity, hypertension, diabetes, cardiovascular disease, and chronic kidney disease; hyperuricemia is also a direct cause of gout, about 5%-12% of patients with hyperuricemia will develop gout. In recent years, the incidence of hyperuricemia and gout has been on the rise, and the age of onset is getting younger. With the improvement of living standards, the incidence of patients with hyperuricemia in my country is also increasing year by year. It is conservatively estimated that there are 120 million patients with hyperuricemia in my country. At present, the prevention of hyperuricemia and gout is mainly to limit high-purine foods and control alcohol consumption. The main way to treat hyperuricemia is to balance uric acid production and excretion. The commonly used drugs for the prevention and treatment of hyperuricemia are mainly divided into three categories: xanthine oxidase inhibitors, urate anion transporter 1 (URAT1) inhibitors, and urate oxidase.

Xanthine oxidase can catalyze the oxidation of hypoxanthine to xanthine, and can continue to catalyze the conversion of xanthine into uric acid. It is the key enzyme in the production of uric acid in the body. The xanthine oxidase inhibitors that have been marketed mainly include allopurinol, new febuxostat, and topiroxostat. URAT1 is a membrane transporter encoded by the SLC22A12 gene of the organic anion-encoding family SLC22A, and is mainly located in the brush border of epithelial cells in the proximal convoluted tubule of the renal cortex. Studies have shown that URAT1 is an important renal uric acid transporter, mainly involved in the reabsorption of uric acid in the renal proximal tubule, and is a key protein for maintaining the physiological balance of human uric acid metabolism. Benzbromarone, probenecid, and sulfazone are the typical uricosuric drugs targeting URAT1 in the treatment of hyperuricemia that have been marketed. Reabsorption thereby rapidly lowers the concentration of uric acid in the blood.

The above chemical drugs all have a certain degree of side effects. For example, probenecid is contraindicated for patients with impaired renal function and sulfonamide allergies, and benzbromarone is used with caution for patients with kidney stones; moreover, none of the above drugs can dissolve uric acid stones deposited in the body . Supplementing uric acid oxidase can restore the body's purine metabolism pathway to normal, and decompose uric acid into allantoin. Studies have shown that urate oxidase not only reduces the blood uric acid concentration far better than other drugs, but also promotes the dissolution of tophi. Therefore, urate oxidase is the most ideal drug candidate for the treatment of gout and hyperuricemia.

Although the amino acid residues at the active site of urate oxidase from different sources are very conserved, the catalytic activities of urate oxidase from different sources are different. The activity of urate oxidase from microorganisms is the highest, while the activity of urate oxidase from plants and animals is lower than microbial source. Among all urate oxidases that have been discovered, the activity of urate oxidase derived from Aspergillus flavus is the highest. Therefore, Uricozyme ^TM, the first-generation urate oxidase product on the market, is directly extracted and purified from Aspergillus flavus. However, the extracted natural uric acid oxidase is limited in yield, expensive and immunogenic, which greatly limits its wide application. In 2001, the recombinant Aspergillus flavus-derived uric acid oxidase (Rasburicase) was approved for clinical use. However, clinical studies have shown that the half-life of Rasburicase in patients is short, requiring multiple medications, and the cost is high. At the same time, repeated use of the body is prone to produce antibodies, causing allergic reactions. In 2010, the pig-baboon-derived PEGylated urate oxidase chimera Pegloticase was approved for marketing by the FDA. Pegloticase combines the high catalytic activity of porcine urate oxidase and the high homology between baboon urate oxidase and human urate oxidase, and further PEG modification prolongs the half-life of pig-baboon chimeric urate oxidase and reduces its immunogen sex. However, clinical studies have shown that the activity of PEG-modified chimeric urate oxidase is reduced and the immunogenicity has not been completely eliminated. FDA still gives a black box warning for allergic reactions in the instructions.

According to the theory of immunology, the close relationship between the antigen source and humans is related to the immunogenicity of the antigen: the farther the antigen source is from humans, the more homologous the antigen components (such as proteins or polypeptides, etc.) are to humans. The lower the immunogenicity, the stronger the immunogenicity, and the easier it is recognized by human lymphocyte clones as an antigenic foreign body to generate a specific immune response; on the contrary, the closer the antigen source is to humans, the weaker the immunogenicity. Based on this, if the pseudogene of human urate oxidase can be modified to "resurrect" it and express human urate oxidase with catalytic activity, its immunogenicity in human applications will be reduced, and even it will not produce Immunogenicity.

Contents of the invention

In order to solve the above-mentioned technical problems, the object of the present invention is to propose a series of human urate oxidases with catalytic activity and their applications, so as to solve the immunogenicity problem of non-human oxidases. Based on a general inventive concept, the present invention includes the gene encoding the above-mentioned urate oxidase, the vector and host cell containing the above-mentioned gene, and the preparation method of the above-mentioned human source urate oxidase, so as to prepare a low-immunogenic or even non-immunogenic The pharmaceutical preparations for lowering uric acid lay the foundation.

In order to achieve the above technical purpose, the present invention adopts the following technical solutions:

Human urate oxidase with catalytic activity, the sequence of the enzyme is a recombinant amino acid sequence obtained by substituting amino acids or amino acid residues at several positions in the sequence shown in SED ID NO: 1, and the amino acid or amino acid residue substitutions at least include the following Amino acid or amino acid residue replacement at 8 positions:

Replacement of 112th methionine with valine or isoleucine; 119th histidine with arginine; 121st glycine with glutamic acid or aspartic acid; 208th lysine Acid is replaced by glutamic acid or aspartic acid; 219th methionine is replaced by leucine; 222th serine is replaced by phenylalanine; 232nd leucine is replaced by serine; 240th half Cystine was replaced with tyrosine.

Preferably, the amino acid or amino acid residue substitution also includes the substitution of any one or two or more of the following amino acid or amino acid residues:

The 3rd histidine was replaced with aspartic acid; the 14th valine was replaced with leucine; the 24th glutamic acid was replaced with aspartic acid; the 89th alanine was replaced with threonine; Glycine at position 91 is replaced with alanine; histidine at position 97 is replaced with tyrosine; glutamine at position 151 is replaced with proline or isoleucine; aspartic acid at position 212 is replaced with alanine acid or glycine; threonine at position 233 is replaced by alanine or proline; leucine at position 245 is replaced by histidine; arginine at position 249 is replaced by glutamine; alanine at position 252 Substitute with glutamine, leucine or glutamic acid.

The above human urate oxidase is urate oxidase H1-UOX, H2-UOX, H3-UOX, H4-UOX, H5-UOX, H6-UOX, H7-UOX, H8-UOX, H9-UOX, H10-UOX, Any one of H11-UOX, H12-UOX, H13-UOX, H14-UOX, H15-UOX, the specific amino acid sequence is shown in SEQ ID NO:2 to SEQ ID NO:16.

Based on a general inventive concept, the present invention also includes the gene sequence encoding the above-mentioned human urate oxidase.

Based on a general inventive concept, the present invention also includes a pair of primers for synthesizing the above-mentioned human urate oxidase gene sequence, specifically as follows:

UOX T For: GAGGGAAGGATTTCACATATGATGGCTCATTACCGTAATGACTA

UOX TRev: ACCTGCAGGGAATTCGGATCCTTACAGCCTTGAGCTCAGCTTCTCTT.

Based on a general inventive concept, the present invention also includes an expression vector containing the above-mentioned human urate oxidase gene sequence, and a host cell containing the expression vector.

A method for preparing the human urate oxidase, specifically comprising the following steps: transferring the human urate oxidase gene sequence into an expression vector; then transferring the recombined expression vector into a competent cell; using the competent cell Express the human urate oxidase for the host cells; finally break the host cells, separate and purify the human urate oxidase, and obtain it.

Based on a general inventive concept, the present invention also includes the application of the above-mentioned human urate oxidase in the preparation of a uric acid-lowering medicament.

The present invention utilizes the technical means of bioinformatics, takes human urate oxidase as the main body, and obtains the gene sequence of urate oxidase with potential catalytic activity through gene sequence comparison; The mutation site of human urate oxidase, and a series of human urate oxidase were synthesized based on this.

After verification, the highest enzyme activity value of a series of human urate oxidase obtained in the present invention can reach 32.83U/mL, and the maximum specific activity value is 20.02U/mg, which is much higher than the commercialized Pegloticase enzyme; and the series of human urate oxidase immune It has low originality and has been verified by animal science, and can effectively reduce the uric acid value in the body, thereby laying the foundation for the preparation of pharmaceutical preparations for reducing uric acid with low or even no immunogenicity.

Description of drawings

Figure 1 PCR amplification electrophoresis of different urate oxidase genes;

Figure 2 Screening results of positive clones by bacterial liquid PCR;

Figure 3 The results of SDS-PAGE electrophoresis analysis of samples after protein expression and purification; 1, Maker (Blue Plus ^® II Protein Marker (14-120 kDa)); 2, the supernatant after breaking the bacteria; 3, the sample passed through the column effluent; 4, purified uric acid oxidase H8-UOX obtained after elution;

Fig. 4 Determination results and comparison of specific activity values of different urate oxidases;

Figure 5 SDS-PAGE electrophoresis of purified urate oxidase after removing the MBP tag; where M is marker; lane 2 is H1-UOX; lane 3 is H15-UOX;

Fig. 6 The immunogenicity assay results and comparison of different uric acid oxidases.

Detailed ways

The present invention will be further described in detail below in conjunction with specific examples.

The reagents and equipment used in the present invention are common commercially available products, or can be obtained by those skilled in the art through open channels.

Example 1 Construction of the amino acid sequence of human urate oxidase with catalytic activity

According to the urate oxidase gene sequence determined from different biological sources, such as dogs, pigs, mice, and baboons, the inventors found that human urate oxidase had nonsense mutations at codons 33 and 187, and its second exon The exon is inserted with a 12bp harmful base; after removing the nonsense mutation and the harmful base, the human urate oxidase gene sequence is obtained; according to the codon encoded by the gene, the amino acid sequence of the human urate oxidase is determined, referred to as H-UOX, the specific sequence is shown in SEQ ID NO: 1:

SEQ ID NO: 1:

MAHYHNNYKKNDEVEFVRTGYGKEMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFGVNICEHFLSSFNHVIRAQVYMEEIPWKHLGKNGVKHVHAFIHTPTGTHFCEVEQLRSGPQVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQCRDVDFKATWDTIRDLVMEKSAGPYDKGEYLTSVQKTLCDIQVLSLSRVPAIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL。

Sequence comparison of H-UOX with other urate oxidase amino acid sequences with catalytic activity, specifically including urate oxidase from Aspergillus flavus, pig, dog and baboon, and use pymol or WinCoot software to generate the above amino acid sequence into a cartoon shape, and then use Align or superpose for protein structure superimposition comparison, the results found that there may be more than 16 mutation sites in the amino acid sequence of human urate oxidase, resulting in the loss of human urate oxidase activity.

In order to further identify the mutation site, the inventors used the crystal structure of urate oxidase (PDB code: 4MB8), the closest mammalian ancestor (An19/22) urate oxidase to human urate oxidase, to construct human urate oxidase H using SWISSMODEL software. - UOX three-dimensional structure. Then use PyMOL or WinCoot software to superimpose the H-UOX structure of human urate oxidase and 4MB8, combined with sequence alignment and literature reports, it is determined that mutation of the following amino acid residues can theoretically obtain human urate oxidase with catalytic activity:

The 3rd histidine was replaced with aspartic acid; the 14th valine was replaced with leucine; the 24th glutamic acid was replaced with aspartic acid; the 89th alanine was replaced with threonine; Glycine at position 91 is replaced with alanine; histidine at position 97 is replaced with tyrosine; methionine at position 112 is replaced with valine or isoleucine; histidine at position 119 is replaced with arginine acid; glycine at position 121 is replaced by glutamic acid or aspartic acid; glutamine at position 151 is replaced by proline or isoleucine; lysine at position 208 is replaced by glutamic acid or aspartic acid ;The 212th aspartic acid is replaced by alanine or glycine; the 219th methionine is replaced by leucine; the 222nd serine is replaced by phenylalanine; the 232nd leucine is replaced by serine; Threonine at position 233 is replaced with alanine or proline; cysteine at position 240 is replaced with tyrosine; leucine at position 245 is replaced with histidine; arginine at position 249 is replaced with gluten Aminoamide; alanine at position 252 was replaced by glutamine, leucine, or glutamic acid.

According to the above mutation sites and rules, the inventors obtained the amino acid sequences of partial human urate oxidase, which were named H1-UOX, H2-UOX, H3-UOX, H4-UOX, H5-UOX, H6-UOX, H7- UOX, H8-UOX, H9-UOX, H10-UOX, H11-UOX, H12-UOX, H13-UOX, H14-UOX, H15-UOX, the specific amino acid sequences are shown in SEQ ID NO:2~NO:16.

Example 2 Cell expression and purification

In order to verify whether the human urate oxidase obtained in Example 1 has catalytic activity, the inventors performed cell expression and protein purification for the above urate oxidase.

1. Replication and amplification of urate oxidase gene

Determine the gene sequence of the above H1-UOX~H15-UOX according to the codon principle, and then chemically synthesize the above gene sequence; the primer pair designed for PCR amplification of the human urate oxidase gene is as follows:

UOX T For: GAGGGAAGGATTTCA CATATG ATGGCTCATTACCGTAATGACTA

UOX TRev: ACCTGCAGGGAATTC GGATCC TTACAGCCTTGAGCTCAGCTTCCTCTT;

The underline in the above primer pair is the restriction endonuclease recognition sequence; the synthetic human urate oxidase gene is used as a template, and the above primers are used for PCR amplification. The specific PCR conditions are: 94°C denaturation for 1 minute, 60°C for 1 minute Extend at 72°C for 3 minutes, after 30 cycles, extend at 72°C for 5 minutes; the amplification system (50 µL) is as follows:

The size of the chemically synthesized UOX series gene sequence is 954bp. After the PCR amplification is completed, a 1.3% agarose gel is prepared, and the PCR product is analyzed by gel electrophoresis. The PCR amplification result is shown in Figure 1;

Lane M in Figure 1 is the Takara DL2,000 molecular marker, and lanes H1, H2, H3, and H15 are the gene sequences of H1-UOX, H2-UOX, H3-UOX, and H15-UOX amplified by PCR respectively; the results show that the above amplification The sizes of the products were all in line with the target; the gene amplification results of other human urate oxidase H4-UOX~H14-UOX were consistent with the above results.

2. Construction and identification of recombinant plasmids

The vector pMBL-c5X (NEB Company) was double-digested with Nde I and Bam HI. The enzyme digestion reaction system (50 µL) was: 25 µL of pMBL-c5X plasmid, 2.5 µL of Nde I, 2.5 µL of Bam HI, 5 µL of 10×Curtsmart Buffer , ddH ₂ O 15µL;

Prepare 1.0% agarose gel, and run the gel to recover the carrier fragment;

The vector digestion product and the amplified gene sequence were connected by homologous recombination. The homologous recombination reaction system (20 µL) was: 4 µL of 5´ reaction buffer, 1 µL of recombinase, 5 µL of pMBL-c5X vector, and 6 µL of UOX series gene fragments , ddH ₂ O 4 µL. The obtained recombinant vector was transferred into the Escherichia coli TG1 competent cells made by our laboratory for overnight culture;

Positive clones were screened by bacterial liquid PCR, and the primer pairs used to screen positive clones were as follows:

malE For: GGTCGTCAGACTGTCGATGAAGCC

UOX TRev: ACCTGCAGGGAATTCGGATCCTTACAGCCTTGAGCTCAGCTTCCTCTT

The electrophoresis analysis diagram of the PCR results of the obtained bacterial solution is shown in Figure 2, and the swimming lanes H1, H2, H3, H4, H5, and H15 in the figure are H1-UOX, H2-UOX, H3-UOX, H4-UOX, H5-UOX and The positive clone product of H15-UOX gene, M represents TakaraDL2,000 molecule marker. It can be seen from the figure that the positive clone products of the above gene sequences all conform to the target fragment size. The electrophoresis results of the positive clone products of urate oxidase H6-UOX~H14-UOX genes were consistent with the electrophoresis results of the above sequences. Gene identification was carried out on the positive clone products by gene sequencing, and the recombinant plasmid pMBL-c5X-UOX with correct sequence was obtained.

3. Gene expression and purification

Transform the correctly identified recombinant plasmid pMBL-c5X-UOX into expression bacteria BL21(DE3), pick positive clones and inoculate them in 5ml LB medium, and culture them overnight at 37°C and 180rpm; then transfer all the culture medium to 200ml In LB medium, carry out scale-up culture; culture at 37°C, rotate at 180rpm until OD600 is 1.0, then add IPTG to a final concentration of 1.0 mmol/L; continue to cultivate at 25°C, 180rpm for 24h, then centrifuge at 10,000rpm for 5min to collect the bacteria ;

After the cells were suspended in PBS, ultrasonically disrupted, and then centrifuged at 10,000 rpm for 5 min; the supernatant and precipitate were collected respectively. After 10% SDS-PAGE electrophoresis analysis, the results showed that the target protein was mainly stored in the supernatant. The supernatant was filtered with a 0.02 μm filter membrane, and then urate oxidase was purified by MBPTrap HP affinity column, and the eluent used was 10 mM maltose buffer prepared with PBS;

The obtained purified protein was identified by 10% SDS-PAGE electrophoresis; taking urate oxidase H8-UOX as an example (shown in Figure 3), the protein band appeared at the target band position after induced expression, and passed through the MBP affinity column Impurity bands disappear after purification, and the obtained recombinant protein has high purity. The expression and purification results of the other 14 human urate oxidases were consistent with those of H8-UOX, and then the enzyme activity and antigenicity of these purified human urate oxidases were analyzed.

Example 3 Determination of Enzyme Activity

The activity of the purified human uric acid oxidase obtained above was measured, and the enzyme activity was determined by enzyme reaction-ultraviolet spectrophotometry. The principle is that uric acid oxidase specifically oxidizes uric acid to allantoin, and uric acid has The characteristic absorption peak, allantoin has no absorption peak in this wavelength range, and the 293nm absorption value of uric acid is proportional to its concentration within a certain range, and the decrease of the absorption value of uric acid under the action of urate oxidase can reflect the level of enzyme activity.

The urate oxidase activity assay reaction system is a 50mM boric acid-borax buffer solution with a pH value of 8.6; the specific assay steps are:

After the blank is zeroed, add 3ml substrate and 10ul uric acid oxidase in turn, and time immediately; measure the A340nm value every 0.5min, and measure continuously for 5min; plot the absorbance value against time, take the initial linear part, and calculate the DA _293nm/min decrease value; according to the following calculation formula (1) and formula (2), the activity value and specific activity value of urate oxidase can be obtained;

公式（1）Enzyme activity unit (U/ml) =

Formula 1)

Enzyme specific activity (U/mg) = (enzyme activity unit (U/ml)´total volume of enzyme)/enzyme mass (mg) formula (2)

After calculation, the obtained purified human urate oxidase H-UOX, H1-UOX, H2-UOX, H3-UOX, H4-UOX, H5-UOX, H6-UOX, H7-UOX, H8-UOX, H9-UOX , H10-UOX, H11-UOX, H12-UOX, H13-UOX, H14-UOX, H15-UOX comparison results of specific activity values are shown in Figure 4. In order to demonstrate the activity of the above urate oxidase, the inventors used Pegloticase (trade name Krystexxa) from LGMPharma as a control.

As can be seen from Figure 4, urate oxidase H-UOX without amino acid residue replacement has no catalytic activity; while H1-UOX~H15-UOX urate oxidase with amino acid residue site replacement according to the replacement principle of the present invention The specific activities of uric acid oxidase H15-UOX were all higher than those of Pegloticase; among them, the specific activity of urate oxidase H15-UOX was the highest, reaching 20.02U/mg, and its enzyme activity was 32.83U/mL. Among the above-mentioned human uric acid oxidases, H1-UOX has the fewest amino acid residue replacement sites, only 8 amino acid residues, its specific activity value is 7.62U/mg, enzyme activity value is 11.05U/mL, and it is located in 15 kinds of uric acid The specific activity value of oxidase is near the median value, so the inventors carried out immunological and pharmaceutical analysis with urate oxidase H1-UOX and H15-UOX as the research objects.

Example 4 Immunogenicity assay

The recombinant human urate oxidase expressed by the pMBL-c5X system has a maltose-binding protein (MBP) tag, so after purification, it is necessary to use an enzyme to remove the MBP tag protein. The commonly used endonuclease is Factor Xa;

In view of the fact that the isoelectric points of H1-UOX and H15-UOX are around pH 8.5, the inventors chose 50mM Tris-HCl (pH 8.5) as the enzyme digestion buffer; after enzyme digestion, MBP was removed by affinity chromatography to obtain purified The purified urate oxidase was identified by 10% SDS-PAGE electrophoresis, and the specific results are shown in Figure 5, where M is marker, lane 2 is urate oxidase H1-UOX, and lane 3 is urate oxidase H15-UOX ; As can be seen from Figure 5, the urate oxidase that removes the MBP tag appears at the position of the target band.

Next, the inventors performed an immunological analysis on the urate oxidase that removed the MBP tag, and the specific process was as follows:

The blood donated by volunteers is separated by TBD lymphocyte separation medium to obtain PBMC cells, including B cells, T cells, NK cells, etc.; MBP, H1-UOX, H15-UOX and Pegloticase enzymes are added to cell culture plates (24 wells) respectively 5 μg, and then add 500 μL per well of the PBMC cell suspension obtained from the above separation, the PBMC cell concentration is 1×10 ⁶ cells/mL; place the cell culture plate in a 37°C, 5% CO ₂ incubator for 7 days, half the amount Change the solution once. After the culture, the supernatant was collected, and the immunogenicity of MBP, H1-UOX, H15-UOX and Pegloticase enzymes were analyzed by indirect ELISA. The specific determination results are shown in FIG. 6 .

It can be seen from the figure that compared with Pegloticase, urate oxidase H1-UOX with the fewest amino acid residue substitution sites has lower immunogenicity, while urate oxidase H15-UOX with the highest enzyme activity has lower immunogenicity. high.

Example 5 Pharmacological analysis experiment

The above results show that the human urate oxidase of the present invention has high enzyme activity and low immunogenicity, and then the inventors carried out animal experiments on the above-mentioned urate oxidase, so as to carry out the pharmacological analysis of the above-mentioned urate oxidase; The following animal experiments were completed in the Experimental Animal Center of Zhengzhou University Academy of Sciences;

Select Bailaihang SPF chickens as experimental animals, inject 0.1mg, 0.5mg, 2mg and 4mg of human urate oxidase H1-UOX, H15-UOX and Pegloticase into 5 kg of chickens through the chicken wing root vein respectively; At 1h, 2h, 4h, and 24h, blood was drawn through the chicken wing root vein, centrifuged to prepare plasma, and the concentration of uric acid in the blood was detected. Three parallel experiments were carried out for each condition, and the results were averaged. The measurement results of uric acid concentration in the blood of Bailaihang SPF chickens under different conditions are shown in Table 1;

Table 1 Comparison of determination results of uric acid concentration (μmol/L) in blood of Bailaihang SPF chickens under different conditions

。

.

It can be seen from Table 1 that under the condition of the same injection volume, after the injection of urate oxidase H15-UOX, the uric acid concentration in the blood measured at different times was lower than that of the other two urate oxidase injections, that is to say, H15-UOX UOX has the most obvious degradative effect on uric acid in the blood; and after injecting H1-UOX, the uric acid value in the blood is lower than the situation of injecting Pegloticase under the condition of equal injection volume; This is consistent with the urate oxidase activity value trend measured in embodiment 3, It further shows that the urate oxidase obtained in the present invention has catalytic activity and can effectively degrade the uric acid value in the body.

The present invention directly targets human urate oxidase, through the replacement of amino acid residues at specific sites, "resurrects" urate oxidase, which originally had no catalytic activity, so as to obtain a series of urate oxidases with catalytic activity that can effectively reduce the concentration of uric acid in the body. Human urate oxidase; this series of human urate oxidase has low immunogenicity, thus laying the foundation for the preparation of pharmaceutical preparations for reducing uric acid with low immunogenicity or even no immunogenicity.

SEQUENCE LISTING

<110> Zhengzhou University

<120> Human urate oxidase with catalytic activity and its application

<130> None

<160> 16

<170> PatentIn version 3.5

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<213> Synthetic

<400> 3

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Lys

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 4

<211> 304

<212> PRT

<213> Synthetic

<400> 4

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 5

<211> 304

<212> PRT

<213> Synthetic

<400> 5

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Ala Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 6

<211> 304

<212> PRT

<213> Synthetic

<400> 6

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Gly Val Asn Ile Cys Glu

85 90 95

Arg Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Ala Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 7

<211> 304

<212> PRT

<213> Synthetic

<400> 7

Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Gly Val Asn Ile Cys Glu

85 90 95

Arg Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Ala Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 8

<211> 304

<212> PRT

<213> Synthetic

<400> 8

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Gly Val Asn Ile Cys Glu

85 90 95

Arg Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Leu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 9

<211> 304

<212> PRT

<213> Synthetic

<400> 9

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Asp Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Asp

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Ala Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Glu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 10

<211> 304

<212> PRT

<213> Synthetic

<400> 10

Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Gly Val Asn Ile Cys Glu

85 90 95

Arg Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Gln Val Pro Gln Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 11

<211> 304

<212> PRT

<213> Synthetic

<400> 11

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Ile

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Asp Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Ile Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Asp

195 200 205

Ala Thr Trp Ala Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Ala Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val Leu Ser Leu Ser Arg Val Pro Leu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 12

<211> 304

<212> PRT

<213> Synthetic

<400> 12

Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Leu Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Gly Val Asn Ile Cys Glu

85 90 95

Arg Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val His Ser Leu Ser Gln Val Pro Ala Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 13

<211> 304

<212> PRT

<213> Synthetic

<400> 13

Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Ala Phe Gly Val Asn Ile Cys Glu

85 90 95

Arg Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Thr Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val His Ser Leu Ser Gln Val Pro Leu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 14

<211> 304

<212> PRT

<213> Synthetic

<400> 14

Met Ala Asp Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Asp Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Asp Val Asn Ile Cys Glu

85 90 95

Tyr Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Glu Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Gln Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Glu

195 200 205

Ala Thr Trp Gly Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Pro Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val His Ser Leu Ser Gln Val Pro Leu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 15

<211> 304

<212> PRT

<213> Synthetic

<400> 15

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Asp Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Asp

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Ala Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val His Ser Leu Ser Gln Val Pro Glu Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

<210> 16

<211> 304

<212> PRT

<213> Synthetic

<400> 16

Met Ala His Tyr His Asn Asn Tyr Lys Lys Asn Asp Glu Val Glu Phe

1 5 10 15

Val Arg Thr Gly Tyr Gly Lys Glu Met Val Lys Val Leu His Ile Gln

20 25 30

Arg Asp Gly Lys Tyr His Ser Ile Lys Glu Val Ala Thr Ser Val Gln

35 40 45

Leu Thr Leu Ser Ser Lys Lys Asp Tyr Leu His Gly Asp Asn Ser Asp

50 55 60

Ile Ile Pro Thr Asp Thr Ile Lys Asn Thr Val His Val Leu Ala Lys

65 70 75 80

Phe Lys Gly Ile Lys Ser Ile Glu Thr Phe Ala Val Asn Ile Cys Glu

85 90 95

His Phe Leu Ser Ser Phe Asn His Val Ile Arg Ala Gln Val Tyr Val

100 105 110

Glu Glu Ile Pro Trp Lys Arg Leu Asp Lys Asn Gly Val Lys His Val

115 120 125

His Ala Phe Ile His Thr Pro Thr Gly Thr His Phe Cys Glu Val Glu

130 135 140

Gln Leu Arg Ser Gly Pro Pro Val Ile His Ser Gly Ile Lys Asp Leu

145 150 155 160

Lys Val Leu Lys Thr Thr Gln Ser Gly Phe Glu Gly Phe Ile Lys Asp

165 170 175

Gln Phe Thr Thr Leu Pro Glu Val Lys Asp Arg Cys Phe Ala Thr Gln

180 185 190

Val Tyr Cys Lys Trp Arg Tyr His Gln Cys Arg Asp Val Asp Phe Asp

195 200 205

Ala Thr Trp Asp Thr Ile Arg Asp Leu Val Leu Glu Lys Phe Ala Gly

210 215 220

Pro Tyr Asp Lys Gly Glu Tyr Ser Ala Ser Val Gln Lys Thr Leu Tyr

225 230 235 240

Asp Ile Gln Val His Ser Leu Ser Gln Val Pro Gln Ile Glu Asp Met

245 250 255

Glu Ile Ser Leu Pro Asn Ile His Tyr Phe Asn Ile Asp Met Ser Lys

260 265 270

Met Gly Leu Ile Asn Lys Glu Glu Val Leu Leu Pro Leu Asp Asn Pro

275 280 285

Tyr Gly Lys Ile Thr Gly Thr Val Lys Arg Lys Leu Ser Ser Arg Leu

290 295 300

Claims (8)

1. Human uric acid oxidase with catalytic activity, characterized in that: the sequence of the enzyme is a recombinant amino acid sequence obtained by replacing amino acids or amino acid residues at several positions in the sequence shown in SED ID NO: 1, and the amino acid or amino acid Residue replacement is specifically the replacement of the following 8 positions: The 112th methionine was replaced by valine; The 119th histidine was replaced by arginine; Glycine at position 121 is replaced by glutamic acid or aspartic acid; The 208th lysine is replaced by glutamic acid or aspartic acid; The 219th methionine was replaced by leucine; The 222nd serine was replaced by phenylalanine; Leucine at position 232 was replaced with serine; The 240th cysteine is replaced by tyrosine; After amino acid or amino acid residue substitution, the obtained human urate oxidase is urate oxidase H1-UOX, and the specific amino acid sequence is shown in SEQ ID NO:2. 2. The human urate oxidase as claimed in claim 1, characterized in that: the amino acid or amino acid residue replacement also includes the replacement of the following positions in addition to the 8 positions described in claim 1: The 89th alanine was replaced by threonine; The 91st glycine was replaced by alanine; Glutamine at position 151 was replaced by proline; Threonine at position 233 was replaced with alanine; Leucine at position 245 was replaced by histidine; The 249th arginine was replaced by glutamine; Alanine at position 252 was replaced with glutamine; After amino acid or amino acid residue substitution, the obtained human urate oxidase is urate oxidase H15-UOX, and the specific amino acid sequence is shown in SEQ ID NO:16. 3. The human urate oxidase as claimed in claim 1, characterized in that: the amino acid or amino acid residue replacement includes the replacement of the following positions in addition to the 8 positions described in claim 1: The 89th alanine was replaced by threonine; The 91st glycine was replaced by alanine; Glutamine at position 151 was replaced by proline; Threonine at position 233 was replaced with alanine; Leucine at position 245 was replaced by histidine; Alanine at position 252 was replaced with glutamine; After amino acid or amino acid residue replacement, the obtained human urate oxidase is urate oxidase H8-UOX, and the specific amino acid sequence is shown in SEQ ID NO:9. 4. The gene sequence encoding human urate oxidase according to any one of claims 1-3. 5. An expression vector containing the gene sequence of claim 4. 6. A host cell containing the expression vector of claim 5. 7. The method for preparing the human urate oxidase described in any one of claims 1-3, characterized in that it specifically comprises the following steps: transferring the human urate oxidase gene sequence into an expression vector; then expressing the recombinant urate oxidase The vector is transformed into competent cells; the competent cells are used as host cells to express human urate oxidase; finally, the cells are broken, and the human urate oxidase protein is separated and purified to obtain the product. 8. The application of human urate oxidase described in claim 1 in the preparation of pharmaceutical preparations for reducing uric acid.

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