Uricase mutant, nucleic acid molecule, recombinant bacterium and application of uricase mutant, nucleic acid molecule and recombinant bacterium in reducing uric acid

Abstract

The invention discloses a uricase mutant, a nucleic acid molecule, a recombinant bacterium and application of the uricase mutant to reduction of uric acid, and relates to the technical field of genetically engineered bacteria. It is found that alanine of the 87th amino acid of wild uricase is mutated into glycine, and the degradation effect of uric acid degrading enzyme on uric acid can be improved. Furthermore, the gene for coding the uricase mutant is integrated into chassis bacteria or cells through a technical means of genetic engineering, and the obtained recombinant bacteria can efficiently degrade uric acid, so that the nucleic acid molecule for coding the uricase mutant, the recombinant vector and the recombinant bacteria provided by the invention have a good uric acid reducing effect and can be used for preparing a high-efficiency uric acid degrading agent. The compound is expected to be used for treating hyperuricemia as well as gout, gout complications or kidney injury caused by hyperuricemia.

Description

Technical Field

[0001] The present invention relates to the technical field of genetically engineered bacteria, and in particular, to a uricase mutant, a nucleic acid molecule, a recombinant bacterium, and their applications in reducing uric acid. Background Art

[0002] Uric acid is a heterocyclic compound composed of an imidazole ring and a pyrimidine ring, and is an intermediate product of purine metabolism. Humans and primates lack uricase for metabolizing uric acid, so uric acid can only be excreted from the body as the final metabolite of purine metabolism. If the uric acid metabolism in the body is disordered, it will cause gout. Gout is a relatively common joint inflammation. When the blood uric acid concentration exceeds the solubility saturation of urate, urate precipitates at the joints, which will then trigger inflammation. Clinical studies have found that kidney diseases, cardiovascular diseases, and diabetes are all closely related to gout. With the continuous improvement of living standards, the incidence of gout has increased significantly compared with before, and the number of patients with hyperuricemia, the early symptom of gout, has reached 13.3%. At present, there are mainly two treatments for gout and hyperuricemia: one is a low-purine diet; the other is to control the blood uric acid concentration through drugs. For example, allopurinol can inhibit the production of uric acid by inhibiting the activity of xanthine dehydrogenase. However, both current methods have their own drawbacks, and at the same time, the treatment effect for gout is relatively limited.

[0003] Currently, some people have detected a certain degree of reduction in blood uric acid concentration by orally administering probiotics such as Lactobacillus and Bifidobacterium, but the degree of reduction is limited. And the method of modifying the intestinal flora through synthetic biology has gradually become an important field of medical research. Escherichia coli Nissle 1917 is also a probiotic and has been widely used by humans to treat gastrointestinal problems such as gastroenteritis since 1917. Since Nissle 1917 does not damage the structure of the intestinal flora and can also quickly colonize in the intestine, it is currently regarded as an important strain for engineering treatment. Some people have constructed an engineered bacterium through Escherichia coli Nissle 1917 to effectively metabolize phenylalanine and fed it to mice and monkeys to relieve phenylketonuria. Among them, the activity of an important enzyme, LAAD, requires oxygen and is also successfully expressed in the intestine. It shows that it is promising to use the constructed engineered probiotics to maintain the blood uric acid concentration. However, the inventor found that the current recombinant probiotics for degrading uric acid cannot efficiently degrade uric acid.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The object of the present invention is to provide a uricase mutant, a nucleic acid molecule, a recombinant bacterium and their application in reducing uric acid to efficiently degrade uric acid, providing a new idea for developing products helpful for degrading uric acid.

[0006] The present invention is implemented as follows:

[0007] In the first aspect, the present invention provides a uricase mutant, which has the following mutations compared with the wild-type uricase: A87G, and the amino acid sequence of the wild-type uricase is as shown in SEQ ID NO: 1.

[0008] In the second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned uricase mutant.

[0009] In the third aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0010] In the fourth aspect, the present invention provides a recombinant bacterium comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0011] In the fifth aspect, the present invention provides the application of the nucleic acid molecule, the recombinant vector and the recombinant bacterium in reducing uric acid, and this application is not for the purpose of treating diseases.

[0012] In the sixth aspect, the present invention provides the application of the nucleic acid molecule, the recombinant vector and the recombinant bacterium in preparing drugs for reducing uric acid.

[0013] In the seventh aspect, the present invention provides a drug, and the active ingredient of the drug comprises the above-mentioned recombinant bacterium.

[0014] The present invention has the following beneficial effects:

[0015] By analyzing and performing saturation mutagenesis on the amino acid sequence of the wild-type uricase, the present invention unexpectedly finds that mutating the 87th amino acid alanine of the wild-type uricase to glycine can improve the degradation effect of the uric acid-degrading enzyme on uric acid. Further, through genetic engineering technical means, integrating the gene encoding the uricase mutant into chassis bacteria or cells, the obtained recombinant bacterium can efficiently degrade uric acid. Therefore, the nucleic acid molecule, the recombinant vector and the recombinant bacterium encoding the uricase mutant provided by the present invention have good uric acid-reducing effects and are expected to be used in drugs for hyperuricemia, gout, gout complications or kidney injury caused by hyperuricemia. Description of the Drawings

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0017] Figure 1 It is a primer design position diagram for the A87G mutation site;

[0018] Figure 2 It is a statistical result diagram of the uric acid residue amounts of 9 strains;

[0019] Figure 3 It is a diagram of the YMG-BfUOX-pTargetF editing plasmid vector;

[0020] Figure 4 It is a diagram of the YMG-uricase-A87G gene editing vector;

[0021] Figure 5 It is a diagram of the YKG-BFUOX-ptargetF gene editing vector;

[0022] Figure 6 It is a diagram of the YKG-uricase-A87G gene editing vector;

[0023] Figure 7 It is a diagram of the YJI-BFUOX-ptargetF gene editing vector;

[0024] Figure 8 It is a diagram of the YJI-uricase-A87G-ptargetF gene editing vector;

[0025] Figure 9 It is a statistical result diagram of the blood uric acid concentrations of mice in each treatment group 2 hours after intraperitoneal injection of potassium oxonate;

[0026] Figure 10 It is a statistical result diagram of the blood uric acid concentrations of mice in each treatment group after continuous high-purine diet modeling for two weeks. Detailed Embodiments

[0027] References to embodiments of the present invention will now be provided in detail, with one or more examples described below. Each example is provided by way of explanation and not limitation of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features described or illustrated as part of one embodiment can be used in another embodiment to yield a still further embodiment.

[0028] By performing bioinformatics analysis and saturation mutagenesis on the amino acid sequence of wild-type uricase, the present invention unexpectedly discovers that mutating the 87th amino acid alanine of wild-type uricase to glycine can improve the degradation effect of uric acid-degrading enzyme on uric acid. Compared with amino acid mutations at other positions of uricase, the A87G mutation has a more significant uric acid degradation effect, degrading uric acid more thoroughly and efficiently.

[0029] In a first aspect, the present invention provides a uricase mutant, which has the following mutation compared with wild-type uricase: A87G, and the amino acid sequence of wild-type uricase is as shown in SEQ ID NO: 1.

[0030] MSTTLSSSTYGKDNVKFLKVKKDPQNPKKQEVMEATVTCLLEGGFDTSYTEADNSSIVPTDTVKNTILVLAKTTEIWPIERFAAKLATHFVEKYSHVSGVSVKIVQDRWVKYAVDGKPHDHSFIHEGGEKRITDLYYKRSGDYKLSSAIKDLTVLKSTGSMFYGYNQCDFTTLQPTTDRILSTDVDATWVWDNKKIGSVYDIAKAADKGIFDNVYNQAREITLTTFALENSPSVQATMFNMATQILEKACSVYSVSYALPNKHYFLIDLKWKGLENDNELFYPSPHPNGLIKCTVVRKEKTKL

[0031] After the A87G mutation, the nucleotide sequence is mutated from GCT to GGT.

[0032] In a second aspect, the present invention provides a nucleic acid molecule encoding the above-mentioned uricase mutant.

[0033] As used herein, the term "nucleic acid molecule" refers to a sequence of nucleoside or nucleotide monomers composed of natural bases, sugars, and sugar-sugar (backbone) linkages. The term also includes modified or substituted sequences containing non-naturally occurring monomers or portions thereof. The nucleic acid molecule of the present invention can be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and can contain natural bases, including adenine, guanine, cytosine, thymine, and uracil. Modified bases can also be included. Examples of these modified bases include nitrogen-containing and de-nitrogenated adenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine.

[0034] In a third aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule.

[0035] The term "vector" is used herein in its most common sense and includes any intermediate agent for nucleic acids that can enable the nucleic acids to be introduced into, for example, prokaryotic and / or eukaryotic cells and, where appropriate, integrated into the genome. Vectors of this type preferably replicate and / or express in cells. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually circular double-stranded DNA, which can replicate independently of chromosomal DNA. Any plasmid and vector can be used as long as it can replicate and be stable in the host.

[0036] In an alternative embodiment, the above-mentioned vector is an expression vector. An important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene and translation control elements.

[0037] For example, the recombinant vector is a gene editing plasmid, such as pTargetF plasmid, pkd4 plasmid, pKD20 plasmid.

[0038] In a fourth aspect, the present invention provides a recombinant bacterium comprising the above-mentioned nucleic acid molecule or the above-mentioned recombinant vector.

[0039] In a preferred embodiment of the application of the present invention, the recombinant bacterium further comprises at least one of the following genes: BfUOX gene, ymgF gene, ycgH gene, ykgH gene, betA gene, yjiP gene and yjiR gene. The nucleotide sequence of the BfUOX gene has at least 90% homology with the sequence shown in SEQ ID NO: 2. For example, it has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology.

[0040] SEQ ID NO: 2:

[0041] atgCGTACCATGTTCTACGGTAAAGGTGACGTTTACGTTTTCCGTACCTACGCTAACCCGCTGAAAGGTCTGAAACAGATCCCGGAATCTAACTTCACCGAAAAACACAACACCATCTTCGGTATGAACGCTAAAGTTGCTCTGAAAGGTGAACAGCTGCTGACCTCTTTCACCGAAGGTGACAACTCTCTGGTTGTTGCTACCGACTCTATGAAAAACTTCATCCAGCGTCACGCTGCTTCTTACGAAGGTGCTACCCTGGAAGGTTTCCTGCAGTACGTTTGCGAAGCTTTCCTGGCTAAATACTCTCACCTGGACGCTGTTCGTCTGGAAGCTAAAGAATACGCTTTCGACGACATCCAGGTTGGTACCGACAAAGGTGTTGTTACCTCTGACCTGGTTTTCCGTAAATCTCGTAACGAATACGCTACCGCTACCGTTGAAGTTGCTCGTACCGCTTCTGGTACCGAAGTTGTTGAACAGGCTTCTGGTATCGCTGACATCCAGCTGATCAAAGTTTCTGGTTCTTCTTTCTACGGTTACATCATCGACGAATACACCACCCTGGCTGAAGCTACCGACCGTCCGCTGTACATCTTCCTGAACATCGGTTGGGCTTACGAAAACCAGGACGACGCTAAAGGTGACAACCCGGCTAACTACGTTGCTGCTGAACAGGTTCGTGACATCGCTGCTTCTGTTTTCCACACCCTGGACAACAAATCTATCCAGCACCTGATCTACCACATCGGTCTGACCATCCTGGACCGTTTCCCGCAGCTGACCGAAGTTAACTTCGGTACCAACAACCGTACCTGGGACACCGTTGTTGAAGGTACCGACGGTTTCAAAGGTGCTGTTTTCACCGAACCGCGTCCGCCGTTCGGTTTCCAGGGTTTCTCTGTTCACCAGGAAGACCTGGCTCGTGAAAAAGCTTCTGCTAACTCTGAATACGTTGCTCTGTAA。

[0042] The functions of the BfUOX gene, ymgF gene, ycgH gene, ykgH gene, betA gene, yjiP gene, and yjiR gene are as follows:

[0043] BfUOX gene: It can degrade uric acid and decompose uric acid.

[0044] ymgF gene: YmgF is an inner membrane protein of 72 residues and is related to the cell division mechanism of Escherichia coli.

[0045] ycgH gene: It encodes one of eight glutathione (GSH) transferase homologs.

[0046] betA gene: It encodes choline dehydrogenase.

[0047] yjiP gene: It encodes an Rpn family recombination-promoting nuclease / transposase.

[0048] yjiR gene: It encodes a protein of the plp-dependent aminotransferase family.

[0049] In a preferred embodiment of the application of the present invention, the nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is single-copy, double-copy, or triple-copy.

[0050] In a preferred embodiment of the application of the present invention, the nucleic acid molecule encoding the uricase mutant is located in at least one of the following blank coding regions: the blank coding region between the ymgF gene and the ycgH gene, the blank coding region between the ykgH gene and the betA gene, and the blank coding region between the yjiP gene and the yjiR gene.

[0051] Integrating genes into the blank coding region will not affect the normal growth and expression of the strain. The blank coding region in the present invention is the non-coding region.

[0052] In a preferred embodiment of the application of the present invention, the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene, and / or, the blank coding region between the yjiP gene and the yjiR gene.

[0053] In a preferred embodiment of the application of the present invention, the genome of the recombinant bacterium is integrated with any one of the following sequences:

[0054] (1) The nucleic acid molecule in the genome of the recombinant bacterium is single-copy, and the nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene;

[0055] (2) The nucleic acid molecule in the genome of the recombinant bacterium is a single copy, the nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene, and the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene;

[0056] (3) The nucleic acid molecule in the genome of the recombinant bacterium is a single copy, the BfUOX gene is a double copy, the nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene, one copy of the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene, and the other copy of the BfUOX gene is located in the blank coding region between the yjiP gene and the yjiR gene;

[0057] (4) The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is a double copy, one copy of the nucleic acid molecule is located in the blank coding region between the ymgF gene and the ycgH gene, and the other copy of the nucleic acid molecule is located in the blank coding region between the ykgH gene and the betA gene;

[0058] (5) The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is a triple copy, the first copy of the nucleic acid molecule is located in the blank coding region between the ymgF gene and the ycgH gene, the second copy of the nucleic acid molecule is located in the blank coding region between the ykgH gene and the betA gene; the third copy of the nucleic acid molecule is located in the blank coding region between the yjiP gene and the yjiR gene;

[0059] (6) The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is a double copy, the first copy of the nucleic acid molecule is located in the blank coding region between the ymgF gene and the ycgH gene, the second copy of the nucleic acid molecule is located in the blank coding region between the ykgH gene and the betA gene; the BfUOX gene is located in the blank coding region between the yjiP gene and the yjiR gene.

[0060] The integration mode (3) of the recombinant bacterium has a better uric acid lowering effect.

[0061] The basic bacterium of the recombinant bacterium is Escherichia coli.

[0062] In a preferred embodiment of the application of the present invention, the basic bacterium of the recombinant bacterium is 4502. It is purchased from Shanghai Taoyusheng Biotechnology Co., Ltd., product number: TYS4502.

[0063] Fifthly, the present invention provides the application of the nucleic acid molecule, the recombinant vector and the recombinant bacterium in lowering uric acid, and this application is not for the purpose of treating diseases.

[0064] In a sixth aspect, the present invention provides the use of a nucleic acid molecule, a recombinant vector, and a recombinant bacterium in the preparation of a drug for reducing uric acid.

[0065] This drug can be used to treat related diseases caused by excessive uric acid (such as gout and hyperuricemia, etc.). Compared with the prior art, it has a stronger ability to degrade uric acid and better treatment effects.

[0066] In a seventh aspect, the present invention provides a drug, the active ingredient of which includes the above-mentioned recombinant bacterium. The drug is, for example, a probiotic agent.

[0067] In a preferred embodiment of the application of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0068] In a preferred embodiment of the application of the present invention, the pharmaceutically acceptable excipients are selected from at least one of fillers, disintegrants, lubricants, flavoring agents, binders, suspending agents, and fragrances.

[0069] Pharmaceutically acceptable excipients include, but are not limited to: pharmaceutically acceptable carriers, auxiliary substances, or solvents. Pharmaceutically acceptable excipients include various organic or inorganic carriers and / or auxiliary materials because they are usually used for pharmaceutical purposes, especially for solid pharmaceutical preparations. Examples include: excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch; disintegrants such as starch, hydrolyzed starch, carboxymethylcellulose, calcium carboxymethylcellulose, hydroxypropyl starch, sodium glycol starch, sodium bicarbonate, calcium phosphate, calcium citrate; lubricants such as magnesium stearate, talc, sodium lauryl sulfate; fragrances such as citric acid, menthol, glycine, orange powder; preservatives such as sodium benzoate, sodium bisulfite, parabens (such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben); stabilizers such as citric acid, sodium citrate, acetic acid, and polycarboxylic acids from the titriplex series such as diethylenetriaminepentaacetic acid (DTPA); suspending agents such as methylcellulose, polyvinylpyrrolidone, aluminum stearate; dispersants; diluents such as water, organic solvents; waxes, fats, and oils such as beeswax, cocoa butter; polyethylene glycol; white petrolatum, etc.

[0070] In a preferred embodiment of the application of the present invention, the dosage form of the drug is tablets, pills, powders, suspensions, gels, emulsions, creams, granules, nanoparticles, capsules, suppositories, injections, or sprays.

[0071] In an alternative embodiment, the above-mentioned drug is a liquid pharmaceutical preparation (such as an injection), for example, solutions, suspensions, and gels usually contain a liquid carrier, such as water and / or pharmaceutically acceptable organic solvents. In addition, such liquid preparations may also contain pH regulators, emulsifiers or dispersants, buffers, preservatives, wetting agents, gelling agents (such as methylcellulose), dyes, and / or flavoring agents, as defined above. The drugs can be isotonic, that is, they can have the same osmotic pressure as blood. The isotonicity of the drugs can be adjusted by using sodium chloride and other pharmaceutically acceptable reagents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol, and other inorganic or organic soluble substances. The viscosity of the liquid composition can be adjusted by a pharmaceutically acceptable thickening agent, such as methylcellulose. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickening agent depends on the reagent selected.

[0072] In an alternative embodiment, the above-mentioned drug is a solid pharmaceutical preparation, such as freeze-dried bacterial powder, particulate preparation, etc.

[0073] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not indicated by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0074] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0075] Experimental materials:

[0076] The primers used in the editing process were all synthesized by Genewiz;

[0077] The editing systems pCasM plasmid and pTargetF plasmid were purchased from Genewiz;

[0078] The amplification enzyme used was 2xPhanta Flash Master Mix purchased from Genewiz, with the product number P520-02;

[0079] The ligation solution used was 2xClonExpress Mix purchased from Genewiz, with the product number C115-02;

[0080] The plasmid extraction kit used was FastPure Plasmid Mini Kit-BOX 2 purchased from Genewiz, with the product number DC201-01;

[0081] The product purification kit used was the FastPure Gel DNA Extraction MiniKit purchased from Genewiz, with the product number DC301-01;

[0082] The DNA Marker used was Trans2K Plus DNA Marker;

[0083] The PCR instrument used was the gene amplification thermal cycler produced by Xi'an Tianlong Technology Co., Ltd., with the model Genesy96T.

[0084] Example 1

[0085] This example provides a method for constructing a genetically engineered bacterium:

[0086] 1. Construction of the pET28a(+)-uricase-A87G mutant expression plasmid

[0087] (1) Site-directed mutagenesis of the gene:

[0088] The original gene used for uricase was the CfUOX gene, and the sequence of this gene is as follows:

[0089] atgTCTACCACCCTGTCTTCTTCTACCTACGGTAAAGACAACGTTAAATTCCTGAAAGTTAAAAAA

[0090] GACCCGCAGAACCCGAAAAAACAGGAAGTTATGGAAGCTACCGTTACCTGCCTGCTGGAAGGTGGT

[0091] TTCGACACCTCTTACACCGAAGCTGACAACTCTTCTATCGTTCCGACCGACACCGTTAAAAACACCAT

[0092] CCTGGTTCTGGCTAAAACCACCGAAATCTGGCCGATCGAACGTTTCGCTGCTAAACTGGCTACCCACT

[0093] TCGTTGAAAAATACTCTCACGTTTCTGGTGTTTCTGTTAAAATCGTTCAGGACCGTTGGGTTAAATACG

[0094] CTGTTGACGGTAAACCGCACGACCACTCTTTCATCCACGAAGGTGGTGAAAAACGTATCACCGACCT

[0095] GTACTACAAACGTTCTGGTGACTACAAACTGTCTTCTGCTATCAAAGACCTGACCGTTCTGAAATCTA

[0096] CCGGTTCTATGTTCTACGGTTACAACCAGTGCGACTTCACCACCCTGCAGCCGACCACCGACCGTATC

[0097] CTGTCTACCGACGTTGACGCTACCTGGGTTTGGGACAACAAAAAAATCGGTTCTGTTTACGACATCGC

[0098] TAAAGCTGCTGACAAAGGTATCTTCGACAACGTTTACAACCAGGCTCGTGAAATCACCCTGACCACC

[0099] TTCGCTCTGGAAAACTCTCCGTCTGTTCAGGCTACCATGTTCAACATGGCTACCCAGATCCTGGAAAA

[0100] AGCTTGCTCTGTTTACTCTGTTTCTTACGCTCTGCCGAACAAACACTACTTCCTGATCGACCTGAAATG

[0101] GAAAGGTCTGGAAAACGACAACGAACTGTTCTACCCGTCTCCGCACCCGAACGGTCTGATCAAATGC

[0102] ACCGTTGTTCGTAAAGAAAAAACCAAACTGTAA。

[0103] After bioinformatics analysis, the 87th amino acid alanine of this gene was mutated to glycine, and the original nucleotide sequence GCT was changed to GGT. The specific mutation process is as follows:

[0104] Figure 1 Design a pair of primers at the positions shown:

[0105] A87G-F: GGGTACCCAGTTTAGCAGCG;

[0106] A87G-R: TGCTAAACTGGGTACCCACT.

[0107] After amplifying the wild-type uricase-encoding gene using this primer pair, a plasmid fragment with a mutant gene was obtained. The template used in this amplification system was the purchased pET28a(+)-uricase expression plasmid (Sangon Biotech (Shanghai) Co., Ltd., product number: B540183). The reaction system was as follows:

[0108] Substance Volume Primer A87G-F 1 μL Primer A87G-R 1 μL Amplification enzyme 25 μL Template plasmid 1 μL Water 22 μL Total volume 50 μL

[0109] After preparing the reaction system as above, use a PCR instrument to perform the amplification reaction. The reaction program was as follows:

[0110]

[0111] Electrophorese the amplified fragment in a 1% agarose gel. The electrophoresis conditions were 250V for 25 minutes;

[0112] A band with a size of 6614bp will appear under the gel imager. Cut this band and recover it.

[0113] (2) Transformation:

[0114] Ligate the recovered fragment using the ligation solution. After reacting in a 50°C water bath for half an hour, perform transformation:

[0115] The ligation system was as follows:

[0116]

[0117]

[0118] The transformation process was as follows: Take the purchased DH5α competent cells and place them on ice for dissolution; add the ligation solution to the dissolved competent cells and place them on ice for an ice bath for 30 minutes; heat shock the ice-bathed competent cell mixture in a 42°C water bath for 90 seconds; return the heat-shocked mixture to ice for an ice bath for 5 minutes; add 1 mL of sterilized liquid LB to the ice-bathed mixture and resuscitate it at 220 rpm and 37°C for 1 hour; take 200 μL and 500 μL of the resuscitated mixture respectively and spread them evenly on the LB solid medium containing 50 mg / L kanamycin, and culture them overnight in a 37°C incubator; select single colonies the next day and place them in a 1.5 mL EP tube containing 1 mL of LB and send them for sequencing; subsequently, select the strain with the correctly sequenced gene for shaking culture;

[0119] Extract the plasmid from the shaken bacterial liquid to obtain the expression plasmid after the mutation is completed, named pET28a(+)-uricase-A87G.

[0120] The pET28a(+)-uricase-A87G plasmid and the wild-type pET28a(+)-uricase plasmid were respectively transferred into the 4502 expression strain to test the uric acid-lowering effect. The 4502 strain was purchased from Shanghai Taoyusheng Biotechnology Co., Ltd., product number: TYS4502.

[0121] (1) Shake the original expression strain 4502, transfer it into 25 mL of medium the next day, and culture it at 220 rpm and 37 °C for 4 h; place the cultured bacterial solution on ice for 30 min; centrifuge the ice-bathed strain at 4 °C and 4000 rpm for 5 min; pour out the centrifuged bacterial solution completely, add 20 ml of pre-cooled sterile pure water, pipette and mix well, and continue to centrifuge at 4 °C and 4000 rpm for 5 min; repeat the above operations 2 times and then take the centrifuged precipitate; add pre-cooled sterile 10% glycerol aqueous solution, pipette and mix well, and continue to centrifuge at 4 °C and 4000 rpm for 5 min; repeat the above operations 2 times and then take the centrifuged precipitate, add 1 ml of pre-cooled sterile 10% glycerol aqueous solution, pipette and mix well, then aliquot, 200 μL per tube, and store in a -80 °C refrigerator; take two of them and dissolve them on ice, then add the extracted pET28a(+)-uricase-A87G plasmid to one, and add the original pET28a(+)-uricase plasmid to the other, gently mix and then perform electrotransformation using a BioRad electroporator; use a 2 mM electroporation cuvette, and the electrotransformation conditions are 3 kV, 200 ohms, and 25 μF; after electrotransformation, add 800 μL of LB liquid medium containing 2,6-diaminopimelic acid (0.1 g / L), aspirate it into a clean 1.5 mL EP tube, and resuscitate at 220 rpm and 37 °C for 1 h; take 200 μL of the resuscitated bacterial solution and evenly spread it on an LB solid medium containing 50 mg / L kanamycin and 2,6-diaminopimelic acid (0.1 g / L), and culture it overnight in a 37 °C incubator; the next day, take the cultured bacterial plate, pick monoclonal colonies into an LB liquid medium containing 50 mg / L kanamycin and 2,6-diaminopimelic acid (0.1 g / L) and culture it overnight; mix the cultured bacterial solution and glycerol in a 1:1 ratio, and store it in a -80 °C refrigerator.

[0122] Take 2 ml of each of the fresh above-mentioned bacterial solutions, centrifuge at a speed of 2000 rpm for 5 min, discard the supernatant, add 5 ml of a reaction solution with a final concentration of 1000 mmol uric acid, and take the reaction solution without bacteria as a control. After reacting at 220 rpm and 37 °C for 1 h, use a detector to detect the concentration. The final detected concentrations are as follows:

[0123] Name Detect uric acid concentration Reaction solution control group 1000 mmol 4502-pET28a(+)-uricase strain 581 mmol 4502-pET28a(+)-uricase-A87G strain 417 mmol

[0124] The final detection results showed that the mutant strain had relatively less residual uric acid. After repeating the experiment multiple times, the conclusion was the same. Therefore, it was concluded that mutating at this locus would indeed improve the uric acid degradation effect of uricase.

[0125] Example 2

[0126] I. Construct gene editing plasmids separately

[0127] In this example, the uricase-A87G mutant editing plasmid and the BfUOX uric acid degradation editing plasmid were constructed separately. Specifically, at the YMG locus (this position is in the blank coding region between the ymgF gene and the ycgH gene, named the YMG locus, and editing at this locus will not affect the normal growth and expression of the strain), the YKG locus (between the ykgH gene and the betA gene, with the same selection result as the YMG locus), and the YJI locus (between the yjiP gene and the yjiR gene), 6 uricase-A87G mutant editing plasmids and BfUOX uric acid degradation editing plasmids were constructed, namely:

[0128] YMG-uricase-A87G-pTargetF, YKG-uricase-A87G-pTargetF, YJI-uricase-A87G-pTargetF, and YMG-BfUOX-pTargetF, YKG-BfUOX-pTargetF, YJI-BfUOX-pTargetF.

[0129] The following takes the construction of the BfUOX editing plasmid (YMG-BfUOX-pTargetF) at the YMG locus as an example:

[0130] (1) Synthesize the following primers:

[0131]

[0132] (2) Use the primers J23119-R and ymgF-N20-F to amplify the purchased pTargetF editing quality. The amplification system is:

[0133] Substance Volume Primer J23119-R 1 μL Primer ymgF-N20-F 1 μL Template pTargetF plasmid 1 μL Amplification enzyme 25 μL Water 22 μL Total volume 50 μL

[0134] The amplification conditions are:

[0135]

[0136]

[0137] (3) Electrophorese the PCR liquid after amplification in 1% agarose gel, add a DNA Marker with a size of 5000bp, and the electrophoresis conditions are 250V for 25 minutes;

[0138] Cut the band with a size of 2117bp after electrophoresis, and use the gel extraction kit from Novizan to extract it;

[0139] (4) Ligate the recovered band with ligation solution. The ligation system is as follows:

[0140] Substance Volume Recycled pTarget fragment containing N20 sequence 2.5 μL Ligation solution 2.5 μL Total volume 5 μL

[0141] After mixing the ligation solution, place it in a 50°C water bath for 30 minutes for ligation; Transform the ligated product into the purchased DH5α competent cells: Take the purchased DH5α competent cells and dissolve them on ice; Add the ligation solution to the dissolved competent cells and place them on ice for 30 minutes; Incubate the ice-bathed competent cell mixture in a 42°C water bath for 90 seconds; Return the heat-shocked mixture to ice for 5 minutes; Add 1 mL of sterile liquid LB to the ice-bathed mixture and resuscitate it at 220 rpm and 37°C for 1 hour; Take 200 μL and 500 μL of the resuscitated mixture respectively and spread them evenly on the LB solid medium containing 50 mg / L spectinomycin, and culture them overnight in a 37°C incubator; The next day, pick monoclonal colonies and place them in a 1.5 mL EP tube containing 1 mL of LB for sequencing; Subsequently, select the monoclonal colonies with correct sequencing for shaking culture; Take the monoclonal colonies with correct sequencing and add them to 5 mL of LB liquid medium containing 50 mg / L spectinomycin, and culture them overnight at 220 rpm and 37°C.

[0142] (5) The next day, extract the plasmid from the shaken bacterial liquid according to the plasmid extraction kit from Novizan, and name the extracted plasmid YMG-pTargetF;

[0143] (6) Amplify the extracted plasmid with primers N20-CPF and N20-CPR, and prepare the amplification system:

[0144] Substance Volume Primer N20-CPF 1 μL Primer N20-CPR 1 μL Template YMG-pTargetF plasmid 1 μL Amplification enzyme 25 μL Water 22 μL Total volume 50 μL

[0145] The reaction program for amplification is as follows:

[0146]

[0147] Electrophorese the amplified fragment in 1% agarose gel, and the electrophoresis conditions are 250V for 25 minutes;

[0148] (7) Cut the band with a size of 2117bp after electrophoresis, and use the gel extraction kit from Novizan to extract it;

[0149] (8) Amplify the genome of strain 4502 using primers YMG-HL-F and YMG-HL-R to obtain the upstream homologous arm; use YMG-HR-F and YMG-HR-R to amplify the downstream homologous arm;

[0150] The reaction system for amplification is as follows:

[0151] Substance Volume Primer YMG-HL-F / YMG-HR-F 1 μL Primer YMG-HL-R / YMG-HR-R 1 μL Template 4502 strain genome 1 μL Amplification enzyme 25 μL Water 22 μL Total volume 50 μL

[0152] The reaction program for amplification is as follows:

[0153]

[0154] Electrophorese the amplified fragment in a 1% agarose gel under the electrophoresis conditions of 250V for 25 min;

[0155] Cut the bands with sizes of 535 bp and 560 bp after electrophoresis, and recover them using the gel extraction kit from Novoprotein;

[0156] (9) Amplify the synthesized BfUOX fragment using primers YMG-BfUOX-F and YMG-BfUOX-R. The reaction system prepared is as follows:

[0157] Substance Volume Primer YMG-BfUOX-F 1 μL Primer YMG-BfUOX-R 1 μL Template BfUOX synthetic fragment 1 μL Amplification enzyme 25 μL Water 22 μL Total volume 50 μL

[0158] The reaction program for amplification is as follows:

[0159]

[0160] Electrophorese the amplified fragment in a 1% agarose gel under the electrophoresis conditions of 250V for 25 min;

[0161] Cut the band with a size of 1225 bp after electrophoresis, and recover it using the gel extraction kit from Novoprotein;

[0162] (10) Ligate all the recovered fragments. The ligation system used is as follows:

[0163]

[0164]

[0165] Mix the ligation solution and place it in a water bath at 50 °C for 45 min for ligation; Transform the ligated product into the purchased DH5α competent cells: Take the purchased DH5α competent cells and place them on ice for dissolution; Add the ligation solution to the dissolved competent cells and place them on ice for 30 min of ice bath; Heat shock the ice-bathed competent cell mixture in a water bath at 42 °C for 90 s; Return the heat-shocked mixture to ice for 5 min of ice bath; Add 1 mL of sterilized liquid LB to the ice-bathed mixture and resuscitate it at 220 rpm and 37 °C for 1 h; Take 200 μL and 500 μL of the resuscitated mixture respectively and spread them evenly on the LB solid medium containing 50 mg / L spectinomycin, and culture them overnight in a 37 °C incubator; Select monoclonal colonies the next day and place them in a 1.5 mL EP tube containing 1 mL of LB for sequencing; The finally correctly sequenced plasmid is the required YMG-BfUOX-pTargetF editing plasmid( Figure 3 as the vector map), and the construction methods of other plasmids are similar. The site selection is different, and the required amplification primers and editing sites are different, while others are the same.

[0166] When constructing the YKG-uricase-A87G gene editing plasmid, the primer sequences used are as follows:

[0167]

[0168] The YKG-uricase-A87G gene editing vector is referred to Figure 6 as shown.

[0169] When constructing the YJI-BFUOX-ptargetF gene editing plasmid, the primer sequences used are as follows:

[0170]

[0171]

[0172] The YJI-BFUOX-ptargetF gene editing vector is referred to Figure 7 as shown.

[0173] II. Construction of recombinant 4502 bacteria: Transfer the above gene editing vector into 4502 bacteria.

[0174] Edit three sites (YKG, YMG, and YJI) respectively in the host expression bacterium 4502, and finally obtain the following 8 strains:

[0175] 4502-YMG-uricase-A87G (No. 4502-C);

[0176] 4502-YMG-BfUOX (No. 4502-B);

[0177] 4502 - YMG - uricase - A87G - YKG - uricase - A87G (No. 4502 - CC);

[0178] 4502 - YMG - uricase - A87G - YKG - BfUOX (No. 4502 - CB);

[0179] 4502 - YMG - BfUOX - YKG - BfUOX (No. 4502 - BB);

[0180] 4502 - YMG - uricase - A87G - YKG - uricase - A87G - YJI - uricase - A87G (No. 4502 - CCC);

[0181] 4502 - YMG - uricase - A87G - YKG - uricase - A87G - YJI - BfUOX (No. 4502 - CCB);

[0182] 4502 - YMG - uricase - A87G - YKG - BfUOX - YJI - BfUOX (No. 4502 - CBB).

[0183] Taking the construction method of the 4502 - YMG - BfUOX strain as an example: Based on the previously constructed YMG - BfUOX - pTargetF vector, gene editing was performed on the 4502 bacteria:

[0184] A. Preparation of 4502 competent bacteria

[0185] (1) Pick a single colony of 4502 and culture it overnight. The next day, transfer the bacterial liquid to 100 mL of LB liquid medium at a ratio of 1:50 and culture it at 37 °C and 200 rpm until OD600 is about 0.5.

[0186] (2) Transfer the bacterial liquid from step (1) to a 50 mL centrifuge tube, place it on ice for 10 min, centrifuge at 4000 rmp for 10 min at 4 °C, and discard the supernatant.

[0187] (3) Gently suspend the bacterial cells with 10 mL of pre - cooled 10% glycerol solution, centrifuge at 4000 rmp for 10 min at 4 °C, and discard the supernatant and repeat once.

[0188] (4) Gently suspend the bacterial cells with 10 mL of pre - cooled deionized water solution, centrifuge at 4000 rmp for 10 min at 4 °C, and discard the supernatant and repeat once.

[0189] (5) Discard the supernatant, add 4 mL of pre - cooled 10% glycerol solution, gently suspend the cells, and place them on ice for 5 min to obtain competent cells.

[0190] (6) Divide the competent cells into small aliquots of 200 μL and quickly store them at -80 °C in the refrigerator.

[0191] Transformation of B.pEcCas plasmid into competent 4502 cells:

[0192] (1) Take out the competent 4502 cells from the -80 °C refrigerator and place them on ice.

[0193] (2) Add the pEcCas plasmid to the competent 4502 cells. After placing them on ice for 30 min, perform heat shock at 42 °C for 90 s in a metal bath, and then place them on ice for 3 min.

[0194] (3) Add 1 mL of LB medium without resistance, culture at 37 °C and 200 rpm in a shaker for 1 h. Then, take 100 μL of the bacterial solution and spread it on an LB plate containing kanamycin sulfate, and culture overnight at 37 °C.

[0195] (4) The colonies grown after culture are the 4502-pEcCas strains. Pick a single colony on the plate and inoculate it into 5 mL of LB medium containing kanamycin sulfate for overnight culture, and then preserve the bacteria after overnight culture.

[0196] C. Preparation of competent cells containing the pEcCas plasmid

[0197] (1) Inoculate at an inoculation amount of 1% into 50 mL of LB liquid medium containing 50 μg / mL kanamycin resistance.

[0198] (2) Culture at 37 °C and 200 rpm until the OD600 is about 0.2. Add 30 mM L-arabinose (induced by λ-RED) and continue to culture until the OD600 reaches 0.6. Then transfer the culture to a sterilized 50 mL centrifuge tube and place it on ice for pre-cooling for 20 min.

[0199] (3) Centrifuge the bacterial solution in step (2) at 4000 rpm for 10 min at 4 °C, discard the supernatant, resuspend the cells with 40 mL of pre-cooled sterile water, centrifuge at 4000 rpm for 10 min at 4 °C, and repeat step 3.

[0200] (4) Resuspend the bacteria in step (3) with 20 mL of pre-cooled 10% (V / V) glycerol and centrifuge at 4000 rpm

[0201] for 10 min at 4 °C.

[0202] (5) Discard the supernatant, resuspend the cells gently with pre-cooled 10% (V / V) glycerol at 3% of the initial volume of the bacterial solution, and finally aliquot them into 1.5 mL centrifuge tubes at 100 μL per tube and store them at -80 °C in the refrigerator.

[0203] D. Transformation and Editing: Transfer the pTargetF-BfUOX-pTargetF editing plasmid into the 4502-pEcCas competent cells.

[0204] (1) Add 5 μL of the pTargetF-BfUOX-pTargetF editing plasmid to the 4502-pEcCas competent cells on ice and place on ice for 1 min.

[0205] (2) Transfer the sample in (1) to a 2 mm electroporation cuvette and place on ice for 5 min.

[0206] (3) Set the electroporator conditions to a voltage of 2500 V and a pulse time of 3 ms. Transfer the sample in (2) to the electroporator for electroporation. After electroporation, quickly add 1 mL of antibiotic-free LB medium in a laminar flow hood and incubate at 37°C and 200 rpm for 1 h.

[0207] (4) Centrifuge the culture at 4000 rpm for 4 min. Discard part of the supernatant and spread the culture on solid LB medium containing 50 μg / mL kanamycin sulfate and 40 μg / mL streptomycin sulfate. Incubate at 37°C until single colonies grow.

[0208] Pick single colonies for colony PCR verification. Keep the bacteria with correct sequencing results.

[0209] E. Plasmid Loss

[0210] 1) Pick a single clone of the 4502-BfUOX-pTargetF-pEcCas positive strain and culture it overnight in 2 ml of LB liquid medium containing 10 mM rhamnose and kanamycin.

[0211] 2) Dilute the overnight culture and spread it on a kanamycin LB plate. Incubate at 37°C overnight.

[0212] 3) Randomly pick single clones and spot them on LB plates containing kanamycin and spectinomycin and on LB plates containing only kanamycin. Incubate at 37°C. Strains that do not grow on the spectinomycin plate are pTargetF plasmid-cured strains and are used as subsequent positive strain screening.

[0213] 4) Take the positive strains obtained from the screening in 3) and culture them overnight in LB liquid medium containing 5 g / l glucose.

[0214] 5) Take 10 μl of the bacterial solution and spread it on solid LB medium containing 5 g / l glucose and 10 g / l sucrose. Incubate overnight.

[0215] 6) Randomly pick single clones and spot them on LB antibiotic-free medium and LB medium containing kanamycin for continued culture. Strains that do not grow on the kanamycin plate are 4502-YMG-BfUOX strains without plasmids.

[0216] The editing methods of other genes are similar, but the selection of sites and genes is different, and the construction of the required editing plasmid pTargetF is different, while the others are the same.

[0217] III. To more clearly demonstrate the construction method of the recombinant strain of the present invention, the present invention also provides the construction method of 4502 - YMG - uricase - A87G - YKG - BfUOX - YJI - BfUOX (No. 4502 - CBB) below.

[0218] 1. Construct gene editing plasmids YMG - uricase - A87G - pTargetF, YKG - BfUOX - pTargetF, and YJI - BfUOX - pTargetF. When constructing the YMG - uricase - A87G gene editing plasmid, the primer sequences used are as follows:

[0219]

[0220] The YMG - uricase - A87G gene editing vector is referred to Figure 4 as shown.

[0221] When constructing the YKG - BFUOX - ptargetF gene editing plasmid, the primer sequences used are as follows:

[0222]

[0223] The YKG - BFUOX - ptargetF gene editing vector is referred to Figure 5 as shown.

[0224] When constructing the YJI - uricase - A87G - ptargetF gene editing plasmid, the primer sequences used are as follows:

[0225]

[0226] The YJI - uricase - A87G - ptargetF gene editing vector is referred to Figure 8 as shown.

[0227] 2. Construct recombinant 4502 bacteria based on the aforementioned vectors

[0228] Transfer the above - mentioned YMG - uricase - A87G - pTargetF, YKG - BfUOX - pTargetF, and YJI - BfUOX - pTargetF into 4502 bacteria.

[0229] First, prepare the 4502 competent cells, then transfer the pEcCas plasmid into the 4502 competent cells. Next, prepare the competent cells containing the pEcCas plasmid, and then transfer YMG-uricase-A87G-pTargetF into the competent cells containing the pEcCas plasmid to obtain the 4502-YMG-uricase-A87G-pTargetF-pEcCas positive strain. Further plasmid loss is carried out to obtain the 4502-YMG-uricase-A87G strain. Then, prepare the competent cells of the 4502-YMG-uricase-A87G strain, transfer the pEcCas plasmid into the 4502 competent cells, prepare the 4502-YMG-uricase-A87G competent cells containing the pEcCas plasmid, and then transfer YKG-BfUOX-pTargetF into the 4502-YMG-uricase-A87G competent cells containing the pEcCas plasmid to obtain the 4502-YMG-uricase-A87G-YKG-BfUOX-pTargetF-pEcCas positive strain. After plasmid loss, the 4502-YMG-uricase-A87G-YKG-BfUOX strain is prepared. Prepare the competent cells of the 4502-YMG-uricase-A87G-YKG-BfUOX strain, transform the pEcCas plasmid into the competent cells to obtain the 4502-YMG-uricase-A87G-YKG-BfUOX-pEcCas positive strain, and then transfer YJI-BfUOX-pTargetF into the 4502-YMG-uricase-A87G-YKG-BfUOX-pEcCas competent cells to obtain the 4502-YMG-uricase-A87G-YKG-BfUOX-YJI-BfUOX-pEcCas-pTargetF positive strain. After plasmid loss, the 4502-YMG-uricase-A87G-YKG-BfUOX-YJI-BfUOX (designated as 4502-CBB) strain is obtained.

[0230] Experimental Example 1

[0231] Cultivate the above 9 strains overnight. The next day, take 2 mL of fresh bacterial liquid, centrifuge at 2000 rpm for 5 min, then add the uric acid reaction solution with a final concentration of 1000 mmol, and set the uric acid reaction solution without bacteria as a blank control.

[0232] After reacting at 37 °C and 220 rpm for 1 h, detect the residual uric acid. The detection results are as follows:

[0233]

[0234]

[0235] From the above detection results and Figure 2 the statistical results graph of uric acid residue shown, it can be seen that the strain numbered 4502-CBB has the best uric acid degradation effect. This strain is 4502-YMG-uricase-A87G-YKG-BfUOX-YJI-BfUOX. The nucleic acid molecule encoding the uricase mutant of the strain numbered 4502-CBB is single-copy, the BfUOX gene is double-copy. The nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene (i.e., at the YMG locus). One copy of the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene (i.e., at the YKG locus), and the other copy of the BfUOX gene is located in the blank coding region between the yjiP gene and the yjiR gene (i.e., at the YJI locus). The uric acid degradation ability of the triple-copy (uricase-A87G) strain is higher than that of the double-copy strain, and the uric acid degradation ability of the double-copy strain is higher than that of the single-copy strain. After combining the uricase-A87G and the BfUOX gene, it has a better uric acid degradation effect.

[0236] Control example

[0237] Four uricase mutant strains at other mutation positions (P59A, P232A) were cultured overnight. The next day, 2 mL of fresh bacterial liquid was taken, centrifuged at 2000 rpm for 5 min, and then a uric acid reaction solution with a final concentration of 1000 mmol was added. A uric acid reaction solution without bacteria was set as a blank control.

[0238] After reacting at 37 °C and 220 rpm for 1 h, the uric acid residue was detected. The detection results are as follows:

[0239]

[0240] From the statistical results of the uric acid residue shown in the above detection results, it can be seen that the strain numbered 4502-YMG-uricase-A87G has the best uric acid degradation effect.

[0241] Experimental example 2

[0242] A mouse hyperuricemia model was induced using 10% yeast powder + 0.15% adenine (XT19008, Synergistic Biology). On the first day of the experiment, an acute hyperuricemia model was induced using adenine + potassium oxonate, and the test strain was administered by oral gavage to evaluate its therapeutic effect.

[0243] Test design:

[0244] The animal grouping information and the test article administration information are shown in Table 1 for details.

[0245] Table 1. Experimental grouping design

[0246]

[0247]

[0248] Experimental method:

[0249] Fifty 7- to 8-week-old Balb / c mice were evenly grouped according to body weight. There were 8 mice in the model group (the second group), and 7 mice in each of the other groups. Before the start of the experimental operation, all mice were allowed to acclimatize to the environment for 7 days.

[0250] On the 1st day after the environmental acclimatization ended, except for Group-1, mice in the other groups were given adenine 75 mg / kg p.o. + potassium oxonate 250 mg / kg i.p. The test strain was administered by gavage together with adenine. Blood samples were taken 2 hours after dosing to detect the uric acid content. The mice were fasted for 16 hours before blood sampling. After blood sampling on the 1st day, except for the 1st group, mice in the other groups were treated with 10% yeast powder + 0.15% adenine (XT19008, Synergistic Biology) for 16 consecutive days. On the 8th and 14th days of modeling, blood samples were taken after fasting for 3 hours for detection. On the 17th day of modeling, detection was performed after fasting for 16 hours. The test strain was administered by gavage once a day.

[0251] Data collection:

[0252] ① Weigh the mice twice a week.

[0253] ② Fast the mice for 16 h before blood sampling and allow normal drinking water. On the 1st day, the probiotic, adenine, and potassium oxonate were administered together. Plasma was taken 2 hours after the injection of potassium oxonate, and about 100 μL of blood was collected from the orbital sinus. Immediately after plasma separation, 10 times the volume of protein precipitant was added. After blood sampling, the mice were fed the modeling diet.

[0254] ④ Fast the mice for 3 h before blood sampling and allow normal drinking water. On the 15th day, plasma was taken, and about 100 μL of blood was collected from the orbital sinus. Immediately after plasma separation, 10 times the volume of protein precipitant was added.

[0255] Statistical analysis:

[0256] Independent samples T-test was used for statistical analysis of the inter-group differences in the experimental data. All data were analyzed using Graphpad prism 8. The P value was rounded to three decimal places, and only the original P value less than 0.001 was expressed as P < 0.001. All tests were two-sided. P < 0.05 was considered statistically significant.

[0257] Experimental results:

[0258] On the 1st day of modeling: Figure 9The results showed that 2 hours after intraperitoneal injection of potassium oxonate, the blood uric acid concentration of the mice in the model group was significantly higher than that in the control group, and the blood uric acid concentration of the basic strain 4502 in each treatment group (4502-CCB, 4502-CBB, 4502-BBB) was significantly lower than that in the model group.

[0259] On the 14th day of model establishment: Figure 10 The results showed that after continuous high-purine diet for two weeks to establish the model, on the 14th day of model establishment, blood samples were taken 3 hours after drug administration after fasting. The blood uric acid concentration of the mice in the model group was significantly higher than that in the control group, and the uric acid concentration in the 4502-CCB and 4502-BBB treatment groups was significantly lower than that in the basic strain 4502 group.

[0260] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A urate oxidase mutant, characterized in that, Compared with wild-type uricase, it has the following mutations: A87G, and the amino acid sequence of the wild-type uricase is as shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that, It encodes the uricase mutant described in claim 1.

3. A recombinant vector, characterized in that, It comprises the nucleic acid molecule described in claim 2.

4. A recombinant bacterium, characterized in that, It comprises the nucleic acid molecule described in claim 2 or the recombinant vector described in claim 3.

5. The recombinant bacterium according to claim 4, characterized in that, The recombinant bacterium further comprises at least one of the following genes: BfUOX gene, ymgF gene, ycgH gene, ykgH gene, betA gene, yjiP gene, and yjiR gene, and the nucleotide sequence of the BfUOX gene has at least 90% homology with the sequence shown in SEQ ID NO:

2.

6. The recombinant bacterium according to claim 5, characterized in that, The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is single-copy, double-copy, or triple-copy; Preferably, the nucleic acid molecule encoding the uricase mutant is located in at least one of the following blank coding regions: the blank coding region between the ymgF gene and the ycgH gene, the blank coding region between the ykgH gene and the betA gene, and the blank coding region between the yjiP gene and the yjiR gene; Preferably, the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene, and / or, the blank coding region between the yjiP gene and the yjiR gene; Preferably, the genome of the recombinant bacterium is integrated with any one of the following sequences: (1) The nucleic acid molecule in the genome of the recombinant bacterium is single-copy, and the nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene; (2) The nucleic acid molecule in the genome of the recombinant bacterium is single-copy, the nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene, and the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene; (3) The nucleic acid molecule in the genome of the recombinant bacterium is single-copy, the BfUOX gene is double-copy, the nucleic acid molecule encoding the uricase mutant is located in the blank coding region between the ymgF gene and the ycgH gene, one copy of the BfUOX gene is located in the blank coding region between the ykgH gene and the betA gene, and the other copy of the BfUOX gene is located in the blank coding region between the yjiP gene and the yjiR gene; (4) The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is double-copy, one copy of the nucleic acid molecule is located in the blank coding region between the ymgF gene and the ycgH gene, and the other copy of the nucleic acid molecule is located in the blank coding region between the ykgH gene and the betA gene; (5) The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is in three copies, wherein the first copy of the nucleic acid molecule is located in the blank coding region between the ymgF gene and the ycgH gene, the second copy of the nucleic acid molecule is located in the blank coding region between the ykgH gene and the betA gene; the third copy of the nucleic acid molecule is located in the blank coding region between the yjiP gene and the yjiR gene; (6) The nucleic acid molecule encoding the uricase mutant in the genome of the recombinant bacterium is in two copies, wherein the first copy of the nucleic acid molecule is located in the blank coding region between the ymgF gene and the ycgH gene, the second copy of the nucleic acid molecule is located in the blank coding region between the ykgH gene and the betA gene; the BfUOX gene is located in the blank coding region between the yjiP gene and the yjiR gene.

7. The recombinant bacterium according to claim 6, characterized in that, The basic bacterium of the recombinant bacterium is Escherichia coli; Preferably, the basic bacterium of the recombinant bacterium is 4502.

8. Use of the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, and the recombinant bacterium according to any one of claims 4-7 in reducing uric acid, and the use is not for the purpose of treating diseases.

9. Use of the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3, and the recombinant bacterium according to any one of claims 4-7 in preparing a drug for reducing uric acid.

10. A drug, characterized in that, The active ingredient of the drug comprises the recombinant bacterium according to any one of claims 4-7.

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