CN116200355B

CN116200355B - Mutant uricase, uric acid specific conjugate, and preparation method and application thereof

Info

Publication number: CN116200355B
Application number: CN202210883014.7A
Authority: CN
Inventors: 顾凌巍; 陆雪峰; 王蕾
Original assignee: Jiangsu Qirui Biotechnology Co ltd
Current assignee: Jiangsu Qirui Biotechnology Co ltd
Priority date: 2022-07-26
Filing date: 2022-07-26
Publication date: 2023-09-19
Anticipated expiration: 2042-07-26
Also published as: CN116200355A

Abstract

The invention provides mutant uricase, a uric acid specific conjugate and a preparation method and application thereof, belonging to the technical field of bioengineering. The amino acid sequence of the mutant uricase is shown as SEQ ID NO.1, and the mutant uricase is obtained by mutating the 22 nd lysine of the uricase into alanine and mutating the 67 th lysine into threonine. According to the invention, mutation is carried out according to the structure of uricase, and key catalytic sites are subjected to mutation inactivation, so that the uricase loses catalytic activity, thus a conjugate which only specifically binds uric acid can be constructed, the conjugate has no catalytic activity, the uricase can be specifically combined with uric acid molecules and brought out of the body, thereby playing the role of efficiently reducing uric acid, simultaneously, hydrogen peroxide, carbon dioxide and allantoin can not be generated by enzymolysis of uric acid, oxidation reaction can not be caused by the generated hydrogen peroxide, immune reaction can not be also caused, and adverse symptoms can be reduced.

Description

Mutant uricase, uric acid specific conjugate, and preparation method and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to mutant uricase, uric acid specific conjugate, and a preparation method and application thereof.

Background

At present, the incidence rate of patients suffering from hyperuricemia in China is about 1.2 hundred million, and the patients have a trend of increasing, and in recent years, the patients have a trend of younger. How to specifically remove uric acid in blood has not been studied at present. Uric acid is beneficial to bacteria that grow on uric acid as the sole carbon and nitrogen source. Uricase is a key enzyme in the nitrogen assimilation pathway in leguminous plants. Some mammals utilize uricase to treat excess nitrogen. However, during evolution, the gene encoding uricase in primates is pseudogenoized, resulting in a lack of functional uricase in humans themselves, and the inability to convert uric acid, which is the end product of human purine metabolism. Normal uric acid concentration is harmless to human body, but the high-energy and high-purine eating habit of modern society easily breaks the uric acid balance in vivo, so that more and more people have hyperuricemia due to exceeding uric acid concentration. Hyperuricemia can result in saturation of poorly water soluble urate salt and precipitation as monosodium urate crystals, which occasionally occur in the renal tubules resulting in kidney stones, with gout being the most common type of arthritis in adults, which is caused by accumulation in joint synovial fluid. In addition, hyperuricemia has a close relationship with many metabolic diseases such as cardiovascular diseases, type 2 diabetes, chronic kidney disease, etc. The genetic rate of hyperuricemia is high, about 70%, which predicts the genetic risk of hyperuricemia and gout. In related studies, hyperuricemia increased the incidence of hypertension, and asymptomatic hyperuricemia increased the risk of hypertension, chronic kidney disease, and obesity by a factor of 2-3.

Uric acid, also known as 2,6, 8-trishydroxy purine, is a heterocyclic carbonyl compound having a relative molecular weight of 168. Uric acid is a weak acid, the acidity coefficient pKa in blood is about 5.8, the pKa in urine is about 5.35, and uric acid anions exist in physiological blood with a pH value of 7.4, and the uric acid is a powerful scavenger of active oxygen and peroxynitrite and an antioxidant. Uric acid is synthesized primarily in the liver, intestinal tract and other tissues such as muscle, kidney and vascular intima, and is the end product of exogenous purines derived from animals. Living cells can also degrade their nucleic acids, adenine and guanine to uric acid. The synthesis flow is as follows: first, deamination and dephosphorylation convert adenine and guanine to inosine and guanosine; then, purine nucleoside phosphorylase converts inosine and guanosine to hypoxanthine and guanine, respectively; then, both hypoxanthine and guanine are converted to xanthine by xanthine oxidase; finally, xanthine oxidase in turn catalyzes the further oxidation of xanthine to uric acid.

Under normal conditions, uric acid synthesis and excretion are in dynamic equilibrium, and most daily uric acid treatments are performed by the kidneys. Uric acid is metabolized by intestinal bacteria after entering the intestine, a process called intestinal uric acid, which accounts for about 25% of uric acid elimination, and the remaining 75% is mainly excreted by the kidneys. Most of the urate in the circulation is free, the protein binding rate is less than 5%, so most of the urate is easily filtered by the glomeruli, however up to 90% of urate may be subsequently reabsorbed. Many urate transporters play a role in renal tubule reabsorption and urate secretion, helping to regulate uric acid balance in the body and maintain its levels within a range. Currently, the major uric acid transporters identified are glucose transporter 9, uric acid transporter 1, organic anion transporter, ABC transporter G family member 2. Uricase can metabolize uric acid into highly soluble 5-hydroxyurate, and further degrade into allantoin and ammonia, which are easy to discharge. However, some primates, including humans, have lost uricase synthesis, and the mRNA for the synthetic uricase is detected in the human liver, but the stop codon is present in advance, and thus the coding gene is a pseudogene.

Uricase is a key drug for treating gout, and particularly for those patients who cannot use conventional therapies, uricase may be said to be their good news. However, the human body cannot produce uricase itself, so exogenous uricase must be introduced, which causes serious rejection, anaphylaxis, etc. in human beings, resulting in reduced efficacy of uricase.

Disclosure of Invention

The invention aims to provide mutant uricase, a uric acid specific conjugate, a preparation method and application thereof, wherein key catalytic sites are subjected to mutation deactivation to ensure that the uricase loses catalytic activity, so that the conjugate with specific binding uric acid can be constructed, has no catalytic activity, can combine uricase specificity with uric acid molecules and take the uricase out of the body, thereby having the effect of efficiently reducing uric acid, and simultaneously, the uricase can not be subjected to enzymolysis to produce hydrogen peroxide, carbon dioxide and allantoin, can not cause oxidation reaction due to the produced hydrogen peroxide, can not cause immune reaction, and reduces adverse symptoms.

The technical scheme of the invention is realized as follows:

the invention provides a mutant uricase, the amino acid sequence of which is shown as SEQ ID NO.1, which is obtained by mutating the 22 nd lysine of uricase into alanine and mutating the 67 th lysine into threonine.

The invention further protects a gene for encoding the mutant uricase, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The invention further protects host cells expressing the urease mutants, which are fungi or bacteria.

The invention further protects an expression vector carrying the gene.

The invention further protects a uric acid specific conjugate which is PGA-PEG modified mutant uricase, wherein the mutant uricase is the mutant uricase.

The invention further provides a preparation method of the uric acid specific conjugate, which comprises the following steps:

s1, designing a nucleotide sequence of mutant uricase and performing total gene synthesis;

s2, constructing a synthesized mutant uricase nucleotide sequence into a vector, and performing sequencing verification;

s3, transforming the vector with the constructed target genes into engineering bacteria for expression;

s4, extracting and purifying mutant uricase protein;

s5 synthesis of PGA-PEG modified mutant uricase.

As a further improvement of the invention, the vector is pET28a plasmid, and the engineering bacteria is escherichia coli.

As a further improvement of the invention, the method specifically comprises the following steps:

s1, designing a nucleotide sequence of mutant uricase and carrying out total gene synthesis: mutant uricase obtained by mutating lysine 22 into alanine and mutating threonine 67 into alanine is designed to obtain a gene sequence, and the nucleotide sequence of the mutant uricase is synthesized by utilizing a gene synthesis technology;

s2, constructing a synthesized mutant uricase nucleotide sequence into a vector, and verifying by sequencing: the synthesized mutant uricase nucleotide sequence is connected to pET28a plasmid by an enzyme cutting and inserting method to construct a prokaryotic cell expression vector, and the prokaryotic cell expression vector is subjected to sequencing verification and is transformed into E.coli DH5 alpha competent cells, and a gene recombinant plasmid is extracted;

s3, transforming the vector with the constructed target genes into engineering bacteria for expression: transforming the pET28a plasmid with the constructed target gene into escherichia coli BL21 for expression, and adopting kanamycin resistance gene on the pET28a plasmid as a transformant identification mark to obtain a colony with correct verification;

s4, extracting and purifying mutant uricase protein: collecting and crushing correct colonies, separating nonspecifically bound hybrid proteins by using a nickel column, and eluting target proteins to obtain mutant uricase proteins with purity of more than 95 percent by secondary column hanging;

s5. Synthesis of PGA-PEG modified mutant uricase: and (3) regulating the mutant uricase protein prepared in the step (S4) to a proper concentration by using Tris-HCl buffer solution, adding PGA-PEG-NHS into a system, oscillating a reaction mixture, loading the mixture into a column, eluting by using the Tris-HCl buffer solution, and collecting the PGA-PEG modified mutant uricase part to obtain the PGA-PEG modified mutant uricase.

As a further improvement of the present invention, the nickel column in the step S4 is Ni ²⁺ -NTA column, step S5The pH value of the Tris-HCl buffer solution is 8-8.5, the concentration is 0.05-0.1mol/L, and the mutant uricase protein is regulated to 3-5mg/mL; the mass ratio of the PGA-PEG-NHS to the mutant uricase protein is 10:1, a step of; the column model of the upper column is HiPrep 16/60Sephacryl S-200HR, the diameter is 1.6cm, and the length is 60cm.

The invention further provides application of the mutant uricase or the uric acid specific conjugate in preparing medicines for reducing uric acid.

The invention has the following beneficial effects: the active center of the enzyme consists of a binding site and a catalytic site of enzyme protein, and through genetic engineering, the binding site of uricase is reserved, the catalytic site is mutated, so that the catalytic activity is lost, and the polar amino acids lysine and threonine of the two catalytic active sites are mutated into alanine.

According to the invention, mutation is carried out according to the structure of uricase, and key catalytic sites are subjected to mutation inactivation, so that the uricase loses catalytic activity, thus a conjugate which only specifically binds uric acid can be constructed, the conjugate has no catalytic activity, the uricase can be specifically combined with uric acid molecules and brought out of the body, thereby playing the role of efficiently reducing uric acid, simultaneously, hydrogen peroxide, carbon dioxide and allantoin can not be generated by enzymolysis of uric acid, oxidation reaction can not be caused by the generated hydrogen peroxide, immune reaction can not be also caused, and adverse symptoms can be reduced.

Meanwhile, the prepared modified mutant uricase is further subjected to polyglutamic acid-polyethylene glycol, so that immunogenicity and long-term treatment inefficiency can be effectively reduced, immunogenicity of foreign proteins is obviously reduced, protein solubility and stability are improved, and biocompatibility of the composition is obviously improved, so that the mutant uricase can be specifically combined with uric acid and excreted to the outside of the body, and hyperuricemia and chronic gout patients can be effectively treated, and the composition is good in biocompatibility and high in safety.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.

FIG. 1 is a schematic structural diagram of uricase;

FIG. 2 is a schematic representation of the interaction of uricase with uric acid;

FIG. 3 is an electrophoretogram of mutant uricase expression and purification;

FIG. 4 is a graph showing the comparative antitrypsin hydrolysis ability.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

PGA-PEG-NHS is an active ester-polyethylene glycol-polyglutamic acid, a linear amphiphilic block copolymer with poly (glutamic acid) as the hydrophobic moiety and azide-functionalized PEG as the hydrophilic moiety, with a purity of >95% and a PEG molecular weight of 400-600, supplied by Guangzhou, inc., of carbohydrate technology.

LB medium: mixing 10g/L peptone, 5g/L yeast powder and 10g/L sodium chloride uniformly, and sterilizing at 121deg.C for 20 min.

According to the structure that has been resolved, uricase is a tetrameric structure composed of four single strands, each of which binds to a uric acid molecule (uricase is a tetrameric structure composed of four single strands, as shown in FIG. 1, red, blue, yellow and green each represent a chain; each chain is a uric acid molecule (PDB References: agUOX, complex with uric acid,2 yzb)), and structural resolution of the substrate complex reveals that uric acid is mutated to amino acid residues K22, T67, D68, F163, R180, L222, Q223, N249, H251 on adjacent chains by hydrogen bonding and Van der Waals forces (K22, T67 and D68 with asterisks are amino acid residues on adjacent chains (PDB References: agUOX, complex with uric acid,2 yzb)), where K22 and T67 are catalytically active sites, the invention mutates these two amino acid residues to K22A, T67, i.e., the amino acid residues on adjacent chains.

EXAMPLE 1 preparation of mutant uricase cloning plasmid and expression bacterium

1. Preparation of recombinant plasmid the nucleotide sequence shown as SEQ ID No.2 was chemically synthesized, and ligated with linearized pET28a plasmid to obtain a recombinant plasmid, which was transformed into E.coli DH 5. Alpha. And extracting the pET28a plasmid by using the positive transformant, converting the pET28a plasmid into escherichia coli BL21 by a heat shock conversion method, and transferring a conversion product to an LB culture medium to obtain the transformant. The method comprises the following steps:

synthesizing a gene of which the nucleotide sequence is shown as SEQ ID NO.2 and codes fusion protein uricase; performing PCR amplification by using the synthesized genes as templates by using the upstream primer and the downstream primer, and recovering amplification products by agarose gel electrophoresis; respectively carrying out enzyme digestion on the amplified product and pET28a plasmid by using restriction enzyme NdeI/XhoI (purchased from NEB company), and connecting the digested product after recovering the digested product gel to obtain a connecting product; the ligation product was transformed into E.coli DH 5. Alpha. From Escherichia coli, inc., of Peking's Optimaceae, to give a transformed product; coating the transformation product on LB solid medium containing 100 mug/mL kanamycin, and inversely culturing 12 h in a constant temperature incubator at 37 ℃ to obtain a transformant; picking up the transformant, inoculating the transformant into 5mL of LB liquid medium containing 100 mug/mL kanamycin, shaking and culturing the transformant in a flask at 37 ℃ and 180rpm for 12 h, extracting plasmids, performing enzyme digestion verification, comparing the plasmids with a target gene after sequencing, and obtaining the recombinant plasmids after verification.

TABLE 1

2. Recombinant plasmid transformed BL21

50. Mu.L of E.coli BL21 is taken, subjected to ice bath, 2. Mu.L of recombinant plasmid is added, subjected to ice bath for 15min, subjected to heat shock at 42 ℃ for 90s, subjected to ice bath for 3min, added with 600. Mu.L of LB, and subjected to shaking at 220rpm for 1h at 37 ℃. 100. Mu.L of the mixture was pipetted and spread on LB plates containing the Canada resistance, the plates were first placed in an incubator at 37℃for 10min, and then the solid plates were placed in the incubator at 37℃overnight for culturing to obtain transformants. Identification of transformants: the kanamycin resistance gene on the pET28a plasmid served as a transformant identification marker.

EXAMPLE 2 expression and purification of mutant uricase

Picking up single colony of the transformant which is verified to be correct, inoculating the single colony into 50mL of LB liquid medium containing 100 mug/mL kanamycin, and carrying out shaking culture at 37 ℃ and 180rpm for 12 h to obtain a culture solution; the culture broth was transferred to 1000 mL LB liquid medium containing 100. Mu.g/mL kanamycin at 1:50, and when the culture broth was subjected to shaking culture at 37℃and 220rpm for 4 hours until OD600 = 0.8, the temperature was lowered to 16℃and shaking culture was continued at 180rpm for 1 hour, and after 1 hour, IPTG (final concentration of 1 mM) was added and 12 h was cultured at 16℃and 180rpm to obtain an expression strain.

The purification steps are as follows: precooling the centrifuge to 4 ℃, collecting thalli at 4000 rpm for 20min, and discarding the supernatant; adding 25 mL nickel column balancing solution (20 mM Tris-HCl pH 8.0, 150 mM NaCl,20 mM imidazole) into each liter of the bacterial cells collected by the culture solution, fully suspending the bacterial cells, adding 1 per mill of beta-mercaptoethanol, adding protease inhibitor PMSF to a final concentration of 0.5 mM, and simultaneously adding other protease inhibitors Aprotin and Leupeptin to a final concentration of 1 mug/mL respectively; the high-pressure cell homogenizer is precooled to 4 ℃, the pressure is set to 1200 Bar, the thalli are crushed for three times under high pressure, and the thalli can be obviously seen to be thin from turbid to sticky after the thalli are crushed for three times. Pre-cooling the centrifugal machine to 4 ℃ and centrifuging for 50min under the condition of 14000 rpm; simultaneous pretreatment nickel column (Ni) ² ⁺ -NTA column), washing the three column volumes with water, then washing the three column volumes with nickel column balancing solution, and plugging the column for standby; collecting supernatant, retaining precipitate (sample preparation, running gel), passing supernatant through nickel column, washing column with 90mL nickel column balance solution to remove non-specifically bound impurity proteins, eluting with gradient imidazole (40, 60, 80, 100, 200. 300, 400 mM imidazole) each 5mL, the eluted protein was immediately added to a final concentration of 1mM TCEP and the protein was inserted into ice; collecting supernatant, passing through column effluent, eluting with 10 μL of eluate, adding 10 μL of 5 XSDS Loading Buffer, removing precipitate, placing into centrifuge tube, adding 10 μL of ddH ₂ O and 10. Mu.L of 5 XSDS Loading Buffer. Then, the mixture was inserted into a metal bath at 100℃and heated for 5 minutes, and then taken out at 12000rpm and centrifuged for 5 minutes. The supernatant and the column-passing effluent were subjected to 5. Mu.L of the sample, 20. Mu.L of the eluate, and the protein purification result was detected by running 15% SDS-PAGE gel. The purified protein was relatively heterogeneous, the protein was concentrated and diluted to 20mM, the column was then hung twice, the column was then washed with 90mL of nickel column equilibration, the elution was repeated with an imidazole gradient, and the eluate was run again on a 15% SDS-PAGE gel to see a much improved protein purity, see FIG. 3. Concentrating the eluted protein, and further desalting and purifying to obtain the mutant uricase with purity of more than 95%.

EXAMPLE 3 Synthesis of PGA-PEG modified mutant uricase

The mutant uricase protein prepared in the step S4 is regulated to 4mg/mL by Tris-HCl buffer solution, and PGA-PEG-NHS is added into the system, wherein the mass ratio of the PGA-PEG-NHS to the mutant uricase protein is 10:1, shaking the reaction mixture, loading on a HiPrep 16/60Sephacryl S-200HR column (diameter 1.6cm, length 60 cm) and eluting with Tris-HCl buffer solution with concentration of 0.07mol/L, pH and value of 8.2, collecting PGA-PEG modified mutant uricase part, and detecting purity by HPLC, wherein the content is 97%.

EXAMPLE 4 test of uric acid binding ability of mutant uricase and PGA-PEG modified mutant uricase

The effect of specific binders was detected by immunoprecipitation techniques: adding 100 mug of mutant uricase or PGA-PEG modified mutant uricase into 10 plasma containing 300 mug mol/L uric acid of mL, uniformly mixing and incubating for 10min, adding the mutant uricase or PGA-PEG modified mutant uricase antibody (100 mug) into the plasma, continuously uniformly mixing and incubating for 30min, finally incubating 10 mug protein A agarose beads with shaking for 2h at 50rpm, centrifuging at 3000 rpm for 3min at 4 ℃, and centrifuging the agarose beads to the bottom of a tube; the supernatant was aspirated and its uric acid concentration and the concentration change rate of other components were measured.

The detection of uric acid is detected by a Baowei BW-901 full-automatic uric acid analyzer.

The results are shown in Table 2.

TABLE 2

As can be seen from the above table, after the mutant uricase or the PGA-PEG modified mutant uricase is treated, the uric acid concentration in serum is obviously reduced, and the concentration of other components is hardly changed, so that the mutant uricase or the PGA-PEG modified mutant uricase prepared by the invention can specifically bind uric acid and has no binding to other components.

EXAMPLE 5 determination of enzyme Activity of mutant uricase and PGA-PEG modified mutant uricase

Uric acid solutions (containing 0.001% uric acid) prepared from buffers with pH=7, 7.5 and 8 were incubated at 25 ℃ for 5min, uricase diluted with water, mutant uricase or mutant uricase modified by PGA-PEG were added, accurate reaction was performed for 5min, 20wt% KOH was added for termination, decrease in light absorption value was measured at 290nm, and relative enzyme activities were measured, and the results are shown in Table 3.

The method for calculating the relative enzyme activity comprises the following steps:

relative enzyme activity (%) = (a) ₀ -A ₁ ）/A ₀ ×100%；

A ₀ Light absorbance was measured at 290nm before the reaction; a is that ₁ The light absorption was measured at 290nm after the reaction.

TABLE 3 Table 3

As can be seen from the above table, the mutant uricase and the PGA-PEG modified mutant uricase prepared by the invention have no enzymatic activity on uric acid in a proper pH value range, and the obtained protein completely loses the catalytic activity on uric acid after the mutant uricase and the PGA-PEG modified mutant uricase are subjected to mutation treatment.

EXAMPLE 6 uricase, mutant uricase and PGA-PEG modified mutant uricase antitrypsin hydrolysis Capacity

And (3) regulating uricase, mutant uricase and PGA-PEG modified mutant uricase solution to the same protein concentration, respectively adding trypsin with the same volume of 0.05mg/ml, mixing, carrying out heat preservation reaction at 37 ℃, sampling at different time points in the reaction process, measuring the residual amounts of uricase, mutant uricase and PGA-PEG modified mutant uricase, and calculating the relative residual rate. And (3) plotting by taking the reaction time as an abscissa and the relative residual rate of uricase, mutant uricase and PGA-PEG modified mutant uricase as an ordinate, and comparing the change of the hydrolytic capacity of antitrypsin of the three.

The results are shown in FIG. 4. As shown in FIG. 4, the modified mutant uricase has significantly higher antitrypsin hydrolysis capability than the unmodified mutant uricase and the common uricase, and after the action of trypsin is performed for 150min, the PGA-PEG modified mutant uricase still has 44% of residual quantity, while the unmodified mutant uricase has only 10% of residual quantity and the common uricase has 4% of residual quantity, so that the modified mutant uricase prepared by the invention is further subjected to polyglutamic acid-PEGylation, thereby effectively improving the biocompatibility and stability of the protein in vivo and being not easy to be subjected to enzymolysis in vivo.

Example 7 rat test

The oxazinate potassium salt is selected as a uricase inhibitor, and is injected intraperitoneally to inhibit uricase in liver peroxisome and raise blood uric acid to form hyperuricemia. 40 healthy male Wistar rats are selected, the age is 10 weeks, the weight is 200+/-20 g, the rats are randomly divided into 5 groups, 8 rats are selected from a blank control group, a model group, a mutant uricase group, a PGA-PEG modified mutant uricase group and a positive medicine group. In addition to the blank control group, the other groups of mice were given 25g/L of potassium oxazinate 10mL/kg, and were given by gavage after 1h, and the control group and the model group were given 10mL/kg each time, 2 times/d of sodium carboxymethyl cellulose emulsion (prepared by distilled water); the mutant uricase group and the PGA-PEG modified mutant uricase group are respectively given 10mg/kg and 2 times/d of mutant uricase and PGA-PEG modified mutant uricase; the benzbromarone group was given 5mL/kg of benzbromarone emulsion suspension 2 times/d. After 1h of gastric lavage, the tail of the rat is broken off to obtain blood, the blood sample is placed in a 1.5mL EP tube, incubated for 30min at 37 ℃, the supernatant is centrifugally taken, and a biochemical analyzer is used for measuring blood uric acid by adopting a uricase-EHSPT method.

The results are shown in Table 4.

TABLE 4 Table 4

Annotation: * P <0.05 for comparison to the placebo group; # is P <0.05 compared to model group.

From the above table, the mutant uricase and the PGA-PEG modified mutant uricase provided by the invention can obviously reduce the content of rat haematuria, so that the mutant uricase and the PGA-PEG modified mutant uricase prepared by the invention can be proved to have a good uric acid reducing effect.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. A mutant uricase is characterized in that the amino acid sequence of the mutant uricase is shown as SEQ ID NO.1, and is obtained by mutating lysine at position 22 of the uricase into alanine and mutating threonine at position 67 into alanine.

2. A gene encoding the mutant uricase of claim 1, wherein the nucleotide sequence is set forth in SEQ ID No. 2.

3. A host cell expressing the mutant uricase of claim 1, wherein the host cell is a fungus or a bacterium.

4. An expression vector carrying the gene of claim 2.

5. A uric acid specific conjugate, characterized by being a PGA-PEG modified mutant uricase, said mutant uricase being the mutant uricase of claim 1.

6. A method of preparing a uric acid specific conjugate as defined in claim 5 comprising the steps of:

s3, transforming the vector with the constructed target gene into engineering bacteria for expression;

s4, extracting and purifying mutant uricase protein;

s5 synthesis of PGA-PEG modified mutant uricase.

7. The method according to claim 6, wherein the vector is pET28a plasmid and the engineering bacterium is E.coli.

8. The preparation method according to claim 6, comprising the following steps:

s1, designing a nucleotide sequence of mutant uricase and carrying out total gene synthesis: mutant uricase obtained by mutating lysine at position 22 of uricase into alanine and mutating threonine at position 67 into alanine is designed to obtain a gene sequence thereof, and the nucleotide sequence of the mutant uricase is synthesized by utilizing a gene synthesis technology;

s3, transforming the vector with the constructed target gene into engineering bacteria for expression: transforming the pET28a plasmid with the constructed target gene into escherichia coli BL21 for expression, and adopting kanamycin resistance gene on the pET28a plasmid as a transformant identification mark to obtain a colony with correct verification;

9. The method according to claim 8, wherein the nickel column in step S4 is Ni ²⁺ -NTA column, pH of Tris-HCl buffer in step S5 between 8 and 8.5, concentration between 0.05 and 0.1mol/L, said mutant uricase protein adjusted to 3-5mg/mL; the mass ratio of the PGA-PEG-NHS to the mutant uricase protein is 10:1, a step of; the column model of the upper column is HiPrep 16/60SephacrylS-200HR, the diameter is 1.6cm, and the length is 60cm.

10. Use of a mutant uricase according to claim 1 or a uric acid specific binding as defined in claim 5 for the manufacture of a medicament for lowering uric acid.