Recombinant uricase, coding gene and application of recombinant uricase in preparation of uricase degrading medicine

Abstract

The invention discloses recombinant uricase, a coding gene and application of the recombinant uricase in preparation of uricase degradation drugs, and relates to the technical field of gene engineering. According to the recombinant engineering bacteria, pichia pastoris serves as host bacteria, after uricase genes are introduced, efficient expression of the recombinant uricase in the pichia pastoris is achieved by optimizing codons and improving fermentation conditions, and after the fermentation conditions are optimized, the highest culture yield of a fermentation tank can reach 10.2 g / L. The recombinant uricase is good in catalytic activity and high in expression quantity, and the recombinant uricase can be used for preparing the recombinant uricase. Through microbial fermentation preparation, the method is suitable for large-scale industrial production.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and protein engineering technology, specifically relating to a recombinant uricase, its encoding gene, and its application in the preparation of drugs that degrade uricase. Background Technology

[0002] Urate oxidase (UOX, EC 1.7.3.3), also known as uricase, is a redox enzyme involved in the metabolic pathway of purine degradation in organisms. Under the influence of oxygen, it oxidizes uric acid to 5-hydroxyisouric acid, releasing hydrogen peroxide in the process. 5-Hydroxyisouric acid is extremely unstable and degrades into allantoin through various chemical and enzymatic reactions. Uric acid has low solubility and readily crystallizes, while allantoin, catalyzed by uricase, is more soluble in water and can be excreted through the kidneys. Uricase exhibits wide species diversity, found in animals, plants, fungi, yeast, and bacteria. Active uricase has been found in the livers of mammals, root nodules of legumes, and microorganisms. However, in humans and other apes, due to the evolutionary loss of uricase function, active uricase is absent, preventing further degradation of uric acid, which then becomes the final product of purine catabolism.

[0003] Excessive uric acid accumulation in the body can trigger gout and increase the risk of cardiovascular and metabolic diseases. Furthermore, during chemotherapy for hematologic malignancies such as leukemia, lymphoma, and myeloma, massive lysis of tumor cells leads to significant degradation of intracellular nucleic acids, causing a sharp rise in plasma uric acid levels. This results in metabolic disorders primarily characterized by hyperuricemia and can lead to kidney failure and death, a phenomenon known as tumor lysis syndrome. Therefore, serum uric acid levels are of significant diagnostic value for diseases such as kidney dysfunction, gout, hyperuricemia, and tumor lysis syndrome, and it is necessary to take measures to lower plasma uric acid levels to treat these diseases.

[0004] Uricase can be used as a protein drug to treat hyperuricemia and gout. In addition, it can be used as a diagnostic reagent to detect uric acid levels in human blood and urine. Besides traditional uric acid detection methods, uric acid levels can also be detected using uricase biosensors. Measuring uric acid concentration in blood and urine is a necessary condition for gout diagnosis. Uricase oxidizes slightly soluble uric acid into easily soluble allantoin, which is then excreted from the body. Studies have shown that uricase therapy is more effective than allopurinol therapy in reducing uric acid concentration in the blood, thus dissolving tophi. Furthermore, with the development of genetic engineering and molecular biology techniques, uricase-encoding genes from various organisms have been obtained. Comparison of genes from different sources and prediction of amino acid sequences from nucleotide sequences indicate that uricase is relatively conserved during evolution. The uricase structure contains highly conserved regions that may be involved in maintaining the enzyme's active site, providing a foundation for further research into the higher-order structure of uricase.

[0005] Uricase, a key enzyme in the diagnosis of hyperuricemia, boasts advantages such as high reliability, sensitivity, specificity, and minimal interference, making it the optimal method for detecting blood uric acid. Researchers both domestically and internationally have used various expression systems to prepare recombinant uricases, with significant differences in physicochemical properties among uricases from different sources. Uricases from mammalian and plant sources are difficult to dissolve under physiological pH conditions, exhibiting relatively low enzyme activity; recombinant expression typically requires eukaryotic host cells, resulting in high preparation costs. Fungal uricases have high solubility in human plasma, high specific activity, a low Michaelis constant (Km), and a strong affinity for uric acid. Studies have found that uricase produced through recombinant expression in *E. coli* exhibits relatively good stability, and the *E. coli* expression system is mature and easy to implement. One of the earliest marketed uricase drugs was uricozyme, a non-intestinal uricase extracted from *Aspergillus flavus* cultures, which was launched in Europe in 1967 for the treatment of hyperuricemia associated with leukemia chemotherapy. Currently, uricase drugs marketed internationally include recombinant aflatoxin-derived uricase (Rasburicase), approved by the U.S. Food and Drug Administration (FDA) in 2002. Rasburicase, due to its high immunogenicity and short half-life, is used to treat and prevent acute hyperuricemia in patients with hematological malignancies, particularly chemotherapy-induced hyperuricemia. Pegloticase, a PEG-modified recombinant porcine uricase approved in the U.S. in 2010, is a long-acting protein drug used to treat chronic gout in adults unresponsive to conventional treatments. It has the unique effect of rapidly dissolving tophi and is hailed as a breakthrough treatment for refractory gout; however, due to the action of Pegloticase antibodies, it may lose its uric acid-lowering efficacy. Therefore, exploring uricases with high enzyme activity is of great significance for the future prevention of diseases caused by uric acid. Summary of the Invention

[0006] The existing commercially available lysozyme resources suffer from limited and high-cost natural extraction raw materials, difficulties in large-scale chemical synthesis, and low expression levels and insufficient enzyme activity in the genetic engineering expression of wild-type lysozyme, making it difficult to meet industrial needs. This invention aims to provide a recombinant uricase, its encoding gene, and its application in the preparation of uricase-degrading drugs.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: On one hand, this application provides a recombinant uricase, which is obtained by amino acid mutation of porcine uricase with a natural full-length amino acid sequence as shown in SEQ ID NO.1. The recombinant uricase contains three amino acid mutations, and the mutated amino acid residues are L48F, D68L and V204A.

[0008] The nucleotide sequence of the porcine uricase encoding the natural full-length amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.2.

[0009] The amino acid sequence of the recombinant uricase is shown in SEQ ID NO.3.

[0010] A nucleotide encoding the aforementioned recombinant uricase.

[0011] The nucleotide sequence is shown in SEQ ID NO.4.

[0012] The nucleotide sequence also includes the sequence shown in SEQ ID NO.4 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.2.

[0013] A recombinant expression vector containing the encoding gene for the recombinant uricase, for expressing the recombinant uricase.

[0014] The plasmid vector for the recombinant expression vector is pET22b.

[0015] A recombinant engineered bacterium, wherein the recombinant engineered bacterium contains the recombinant expression vector.

[0016] The method for preparing recombinant uricase involves transforming a recombinant vector into a host bacterium, culturing the transformed host bacterium, inducing expression, centrifuging, and sonicating to obtain recombinant uricase.

[0017] Preferably, IPTG at a concentration of 0.05–0.15 mM is used, and expression is induced at 37°C for 3–5 h.

[0018] Preferably, the centrifugation temperature is 0℃~8℃, the centrifugation speed is 7000 rpm~7800 rpm, and the centrifugation time is 20 min~30 min; the ultrasonic crushing temperature is 0℃~4℃, and the ultrasonic crushing process is performed with intermittent ultrasonic treatment, with an ultrasonic interval of 2 seconds after 1 second of ultrasonic treatment; the total ultrasonic crushing time is 15 min~25 min.

[0019] Furthermore, the recombinant microorganism is one or more of Pichia pastoris, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

[0020] The host bacteria are one or more of Escherichia coli, Pichia pastoris, Bacillus subtilis, and Saccharomyces cerevisiae.

[0021] Preferably, the host bacterium is Escherichia coli. E. coli BL21(DE3).

[0022] This application also provides the use of the aforementioned recombinant uricase, or the encoding gene of the aforementioned recombinant uricase, or the aforementioned recombinant expression vector, or the aforementioned recombinant engineered bacteria in the preparation of drugs that degrade uricase.

[0023] Compared with the prior art, the technical solution of the present invention achieves the following beneficial technical effects: The recombinant uricase provided by this invention is obtained by mutating the amino acids L48F, D68L and V204A of natural porcine uricase, thereby changing the structure and properties of the enzyme and affecting its activity, stability and other properties, providing uricase materials with new characteristics for subsequent research and application.

[0024] This invention yields the nucleotide sequence of a recombinant uricase gene, which is inexpensive, simple to manipulate at the molecular level, easy to clone and express, and has good reproducibility, making it a potential application in industrial production. This invention constructs a prokaryotic expression vector for uricase, successfully obtaining an enzyme protein with a molecular weight of approximately 54 kDa. The protein expression effect is good, providing a vector tool for achieving efficient expression of recombinant uricase in host cells. It is a key step in the genetic engineering production of recombinant uricase, with high enzyme activity and a simple preparation process.

[0025] Furthermore, pET22b is a commonly used and highly efficient expression vector. Choosing this vector helps to improve the expression efficiency of recombinant uricase in host cells, simplify the expression process, and increase yield.

[0026] Recombinant engineered bacteria can express recombinant uricase using their metabolic system. These engineered bacteria have advantages such as rapid growth and ease of cultivation, providing a feasible biological system for large-scale production of recombinant uricase.

[0027] The method for preparing recombinant uricase provided by this invention involves transforming a recombinant vector into a host bacterium, followed by culturing, inducing expression, centrifugation, and ultrasonic disruption to obtain recombinant uricase. This method is relatively simple to operate and provides a feasible process flow for the industrial production of recombinant uricase.

[0028] The application provided by this invention, recombinant uricase, has high degradation activity for uric acid and is a protein that specifically degrades uric acid. It has good prospects for industrial production of uricase and its efficient application in the human body to treat gout and hyperuricemia. Attached Figure Description

[0029] Figure 1 The image shows the recombinant plasmid pET22b-Uricase in the example.

[0030] Figure 2 The image shown is an SDS-PAGE gel electrophoresis image of the recombinant uricase protein in the example. Detailed Implementation

[0031] The following embodiments are provided to further illustrate the present invention, but these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following. Unless otherwise specified, all test methods used are conventional methods, and all raw materials used are commercially available products.

[0032] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0033] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0034] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0035] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0036] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0037] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0038] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0039] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0040] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0041] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0042] Recombinant engineered bacteria refer to engineered microorganisms that, through genetic engineering technology, introduce exogenous target genes (such as genes encoding valuable proteins or enzymes) into suitable host bacteria (commonly Escherichia coli, Bacillus subtilis, etc.), so that the target gene exists stably in the host bacteria and expresses the corresponding product.

[0043] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0044] To more clearly illustrate the overall concept of this application, a detailed description is provided below by way of embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0045] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0046] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0047] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions. Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0048] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0049] In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage.

[0050] In this specification, the amino acids at the corresponding sites are represented by the recognized IUPAC single-letter abbreviations, where each amino acid and its abbreviation are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamine (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0051] The pET22b vector used in this invention was purchased from Shanghai Lianmai Biotechnology Co., Ltd., LMAI Bio, 5ug plasmid, catalog number: LM1200.

[0052] The present invention also provides a recombinant uricase with catalytic activity expressed by the cloned uricase gene, the amino acid sequence of which is shown in SEQ ID NO.3; The recombinant uricase gene (SEQ ID NO. 3) of this invention is obtained by amino acid mutation of porcine uricase with the natural full-length amino acid sequence as shown in SEQ ID NO. 1; The nucleotide sequence encoding the natural full-length amino acid sequence of the porcine uricase, as shown in SEQ ID NO.1, is shown in SEQ ID NO.2.

[0053] This invention provides a nucleotide encoding recombinant uricase, the nucleotide sequence of which is shown in SEQ ID NO.4: The original amino acid sequence of the recombinant uricase (SEQ ID NO.3) described in this invention is shown in SEQ ID NO.1: The present invention also provides a recombinant vector for expressing a recombinant uricase gene, comprising an initial vector and the uricase gene.

[0054] The present invention also provides a recombinant strain expressing a recombinant uricase gene, wherein the recombinant strain is transformed with the recombinant vector described above. The present invention also provides a method for cloning and expressing the recombinant uricase gene, comprising the following steps: (1) The uricase gene and pET22b vector were digested with enzymes and ligated to obtain the recombinant plasmid pET22b-Uricase (see Appendix). Figure 1 ); (2) The recombinant plasmid pET22b-Uricase was transformed into the host bacteria to obtain the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL; (3) Culture steps (2) Recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plusRIL, and then induce expression; (4) Centrifugation to collect step (3) Induced expression cells, sonicated and collected supernatant to obtain uricase solution. In the cloning and expression method of the recombinant uricase gene of the present invention, in step (1), the uricase gene and the pET22b vector are digested with enzymes respectively, and after ligation, the recombinant plasmid pET22b-Uricase is obtained; the uricase gene is preferably amplified by PCR before the treatment in step (1); the uricase gene in step (1) is preferably the nucleotide sequence shown in SEQ ID NO. 3; the enzymes used for digestion are preferably restriction endonucleases Nco I and Xho I, and double digestion is performed; the ligase used for ligation is preferably 2X ClonExpress Mix ligase. In the cloning and expression method of the uricase gene of the present invention, step (2) involves transforming the recombinant plasmid pET22b-Uricase obtained in step (1) into the host bacteria to obtain the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL; after transformation, preferably, PCR screening is performed to obtain positive transformants, and the target cloning strain is determined after sequencing; the host bacteria is preferably E.coli DH5a; then, the plasmid pET22b-Uricase extracted from the cloning strain pET22b-Uricase-E.coli DH5a is transformed into E.coli BL21(DE3)codon plus RIL, and the positive transformants are extracted and directly cultured to obtain the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL. In the cloning and expression method of the recombinant uricase gene of the present invention, step (3) involves culturing the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL from step (2), and then inducing expression; preferably, the specific method for culturing the recombinant expression strain includes the following steps: inoculating the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL into LB medium containing ampicillin, culturing overnight at 37°C for 12-18 h, and then inoculating it into LB medium containing ampicillin at an inoculation rate of 0.5-1.5%; the ampicillin concentration in the LB medium containing ampicillin is preferably 40-60 mg / mL, more preferably 45-55 mg / mL, and even more preferably 48-52 mg / mL; the culture time is preferably 12-18 h, more preferably 13-17 h, and even more preferably 14-16 h; the inoculation amount is preferably 0.5-1 0.5%, more preferably 0.6% to 1.4%, and even more preferably 0.8% to 1.2%. In the cloning and expression method of the recombinant uricase gene of the present invention, step (3) involves culturing the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL and then inducing expression. Preferably, the specific steps for inducing expression in step (3) include: OD of the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL. 600 When the OD value is 0.6–0.8, IPTG at a concentration of 0.05–0.15 mM is added, and expression is induced at 37°C for 3–5 h; the OD value is... 600The value is further preferably 0.7; the concentration of IPTG is 0.05-0.15 mM, further preferably 0.08-0.12 mM; the induction time is 3-5 h, further preferably 3.5-4.5 h. In the cloning and expression method of the recombinant uricase gene of the present invention, step (4) involves centrifuging to collect the cells induced in step (3), ultrasonically disrupting them, and collecting the supernatant to obtain the uricase solution; the centrifugation temperature is preferably 0-8℃, more preferably 2-6℃, and even more preferably 3-5℃; the centrifugation speed is preferably 7000-7800 rpm, more preferably 7200-7600 rpm, and even more preferably 7300-7500 rpm; the centrifugation time is preferably 20-30 min, more preferably 22-28 min, and even more preferably 24-26 min; the ultrasonic disruption temperature is preferably 0-4℃, more preferably 1-3℃, and even more preferably 2℃; the ultrasonic disruption... The preferred method for intermittent ultrasonic treatment during the crushing process is 1 second of ultrasonic treatment followed by a 2-second interval; the total ultrasonic crushing time is preferably 15-25 min, more preferably 16-24 min, and even more preferably 18-22 min; the supernatant is collected by centrifugation; the temperature of the collected supernatant is preferably 0-8℃, more preferably 2-6℃, and even more preferably 4℃; the centrifugation speed for collecting the supernatant is preferably 11000-13000 rpm, more preferably 11500-12500 rpm, and even more preferably 12000 rpm; the centrifugation time for collecting the supernatant is preferably 20-30 min, more preferably 22-28 min, and even more preferably 24-26 min. Unless otherwise specified, all materials and reagents used in the examples are commercially available. Example 1 The target gene SEQ ID No. 3 was designed and synthesized. The vector pET22b was digested with restriction endonucleases NcoI and XhoI (digestion conditions: 37℃ water bath for 2 hours, 65℃ inactivation for 20 minutes). The purified product (5746bp) was recovered. The digestion system of the vector pET22b is shown in Table 1. Table 1: pET22b digestion system

[0055] The amplified product of gene SEQ ID No. 3 and the enzyme digestion and purification product were ligated using 2X ClonExpress Mix for homologous recombination to obtain the recombinant plasmid pET22b-Uricase. The homologous recombination system of the amplified product and the enzyme digestion and purification product is shown in Table 2; Table 2: Homologous recombination system of amplification products and enzyme digestion purified products

[0056] The recombinant plasmid pET22b-Uricase was transformed into E. coli DH5a. The specific transformation procedure was as follows: E. coli DH5a was stored on ice. 2.5 μL of the homologous recombinant plasmid was added to an EP tube containing 100 μL of E. coli DH5a and incubated on ice for 30 min. After the ice incubation, the tube was incubated in a 42℃ water bath for 90 s, followed by rapid cooling on ice for 3 min. Shaking of the centrifuge tube was prohibited. After cooling, 700 μL of sterile LB medium was added, and the tube was incubated at 37℃ and 220 rpm for 1 h on a shaker. The bacterial culture was then spread onto LB agar plates containing antibiotic resistance (Amp) and incubated overnight at 37℃. The plasmid pET22b-Uricase extracted from the cloned strain pET22b-Uricase-E.coli DH5a was screened by enzyme digestion to obtain positive transformants, which were then sequenced to confirm them as the target strain. The enzyme digestion verification and screening system for positive transformants is shown in Table 3. Table 3: Enzyme digestion verification system

[0057] The plasmid pET22b-Uricase extracted from the cloned strain pET22b-Uricase-E.coli DH5a was transformed into E.coli BL21(DE3)codon plus RIL, and the transformation method was as described above. Positive transformants were extracted and directly cultured to obtain the recombinant expression strain pET22b-Uricase-E.coli BL21(DE3)codon plus RIL. The recombinant expression strain pET22b-Uricase- E. coli BL21(DE3)codon plus RIL was inoculated into 10 mL of LB medium containing ampicillin (50 mg / mL) and cultured overnight at 37 °C for 15 h. Then, 1% of the culture was inoculated into 500 mL of LB medium containing ampicillin (50 mg / mL). When OD... 600 =0.7, add IPTG to a final concentration of 0.1 mM, and induce at 37 ℃ and 190 rpm for 4 h; the LB medium formulation is shown in Table 4: Table 4: Luria-Bertani (LB) Culture Medium Formulation

[0058] After in vitro induction of expression, bacterial cells were collected by centrifugation at 7400 rpm for 25 min at 4°C. The cells were resuspended in Tris-buffered saline and sonicated on ice for a total duration of 30 min, with sonication intervals of 1 s and 2 s. The supernatant was then collected by centrifugation at 12000 rpm for 25 min at 4°C, yielding the crude uricase extract. The collected supernatant was purified using a Ni-NTA Beads 6FF column. The purified recombinant protein solution was subjected to SDS-PAGE gel electrophoresis, yielding a 34 kDa protein band. Figure 2 As shown.

[0059] From the appendix Figure 2 The expected molecular weight of the expressed uricase protein is 34 kDa, and the presence of a 34 kDa band in the electrophoresis results indicates successful expression of the target protein. This demonstrates that the entire process, from plasmid construction, transformation, induction of expression, cell disruption to protein purification, was effective, the target gene was correctly expressed in the host bacteria, and the purification process yielded a relatively pure target protein. The clear and single bands indicate good purification efficiency and high purity of the recombinant protein.

[0060] Experimental Example 2: Determination of Recombinant Uricase Activity 1. Plot the uric acid standard curve 3.36 g of uric acid was weighed and dissolved in 100 mM sodium tetraborate solution to prepare a standard reagent with a final concentration of 200 mM. Uric acid standard reagent and 100 mM sodium tetraborate solution were added sequentially according to Table 2, mixed well, and three replicates were performed for each group to obtain the average value. The results are shown in Table 5. 200 μL of liquid from each gradient was added to a 96-well plate, and the absorbance at 293 nm was measured using a microplate reader. Using 100 mM sodium tetraborate as a blank control, and plotting different concentrations of uric acid solution as the x-axis, OD... 293 The absorbance value is used as the ordinate. The correlation coefficient of the linear fitting of the uric acid standard curve is R2 = 0.9933, and the linear regression equation is y = 0.0076x - 0.8778. The results are shown in Table 5. Table 5: Correlation between uric acid content and absorbance value

[0061] Since uric acid has a characteristic absorption peak at 293 nm, while the product has no absorption peak in this wavelength range, and different uric acid concentrations correspond to different absorbance values, exhibiting a linear change, the amount of decrease in absorbance at 293 nm is periodically measured as uric acid is degraded by uricase to calculate enzyme activity.

[0062] Uricase activity assay: Using 100 mM uric acid solution as substrate, the uricase activity was determined. 800 μL of substrate (pH 8.0) was added to 200 μL of crude enzyme solution, and the reaction was incubated in a 37℃ water bath for 1 h. 200 μL of each solution was then transferred to a 96-well plate, and the absorbance at 293 nm was measured using a microplate reader. An equal volume of inactivated enzyme solution served as a blank control; the supernatant was used to measure its absorbance at 293 nm.

[0063] The enzyme activity unit (U) is defined as the amount of enzyme required to consume 1 μmol of uric acid per minute under the above conditions. The specific activity of uricase in different samples (this invention, RhUOX-17Mutant, and porcine uricase) was compared, and the results are shown in Table 5. The reduction in uric acid was calculated based on the standard curve, resulting in a crude enzyme specific activity of 10.81 U / mg and a pure enzyme activity of 10.45 U / mg. The L48F / D68L / V204A (recombinant uricase of this invention) mutant showed better specific activity compared to the wild type.

[0064]

[0065] As can be seen from the above embodiments, the present invention provides a recombinant uricase protein that specifically degrades uric acid, which has good prospects for industrial production of uricase and efficient application in the human body to treat gout and hyperuricemia.

[0066] The above embodiments are merely exemplary models of the present invention and are not intended to limit the present invention. The scope of protection of the present invention is defined by the claims. The above content is only used as an example and illustration of the concept of the present invention. Various modifications or additions made by those skilled in the art to the described specific embodiments, or substitutions made in a similar manner, as long as they do not deviate from the inventive concept or exceed the scope defined by the claims, shall fall within the scope of protection of the present invention.

Claims

1. A recombinant uricase, characterized in that, The recombinant uricase was obtained by mutating the natural full-length amino acid sequence of porcine uricase as shown in SEQ ID NO.

1. The recombinant uricase contains three amino acid mutations, with the mutated amino acid residues being L48F, D68L, and V204A.

2. The recombinant uricase according to claim 1, characterized in that, The amino acid sequence of the recombinant uricase is shown in SEQ ID NO.

3.

3. A nucleotide, characterized in that, Encoding a recombinant uricase as described in claim 1 or 2.

4. The nucleotide according to claim 3, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

4.

5. The nucleotide according to claim 3 or 4, characterized in that, The nucleotide sequence also includes the sequence shown in SEQ ID NO.4 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO.

2.

6. A recombinant expression vector, characterized in that, The recombinant expression vector contains the encoding gene for recombinant uricase as described in any one of claims 3 to 5, and expresses the recombinant uricase as described in claim 1 or 2.

7. The recombinant expression vector as described in claim 6, characterized in that, The plasmid vector for the recombinant expression vector is pET22b.

8. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria contain the recombinant expression vector as described in claim 5 or 6.

9. The method for preparing recombinant uricase according to claim 1 or 2, characterized in that, The recombinant vector of claim 6 or 7 is transformed into a host bacterium, the transformed host bacterium is cultured, induced to express, centrifuged, and sonicated to obtain recombinant uricase.

10. The use of a recombinant uricase according to claim 1 or 2, or a nucleotide according to any one of claims 3 to 5, or a recombinant expression vector according to claim 6 or 7, or a recombinant engineered bacterium according to claim 8, in the preparation of a uricase-degrading drug.

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