117 results about "Urate oxidase" patented technology

The present invention relates, in general, to urate oxidase (uricase) proteins and nucleic acid molecules encoding same. In particular, the invention relates to uricase proteins which are particularly useful as, for example, intermediates for making improved modified uricase proteins with reduced immunogenicity and increased bioavailability.

A naturally occurring or recombinant protein, especially a mutein of porcine urate oxidase (uricase), that is essentially free of large aggregates can be rendered substantially non-immunogenic by conjugation with a sufficiently small number of strands of polymer such that the bioactivity of the protein is essentially retained in the conjugate. Such conjugates are unusually well suited for treatment of chronic conditions because they are less likely to induce the formation of antibodies and / or accelerated clearance than are similar conjugates prepared from protein preparations containing traces of large aggregates.

Genetically modified proteins with uricolytic activity are described. Proteins comprising truncated urate oxidases and methods for producing them, including PEGylated proteins comprising truncated urate oxidase are described.

The invention discloses a polyethylene glycol (PEG) dog-source urate oxidase analogue, wherein the dog-source urate oxidase analogue is natural dog-source urate oxidase and chimeric protein and mutant protein containing part of dog-source and human-source urate oxidase amino acid sequences, the average molecular weight of polyethylene glycol is about 1kDa-40kDa, and each urate oxidase monomer is coupled with 2-15 polyethylene glycol molecules in average. According to the method for preparing mammal-source urate oxidase subjected to covalent modification by the polyethylene glycol, the enzyme activity retention rate is higher than 85.0%, and the purification is higher than 95.0%. According to the method, the dog-source urate oxidase analogue subjected to covalent modification by the polyethylene glycol and drug compositions thereof can be used for preventing and / or treating acute hyperuricemia caused by hematologic tumor chemotherapy and hyperuricemia and chronic gout caused by metabolic disorders.

The invention belongs to the technical field of biosensing, and relates to a dry chemical test strip used in quantitative test of uric acid and a preparation method thereof. The uric acid quantitative-test dry chemical test strip provided by the invention comprises an insulating substrate, electrodes and an anti-interference enzyme layer printed on the insulating substrate with a screen printing technology, and a siphonic reaction cell composed of a double-sided adhesive, a hydrophilic membrane, and the enzyme layer. According to the invention, with an electrochemical method, uric acid oxidation is catalyzed, and urate oxidase is not needed to participate in the reaction. Therefore, testing precision can be improved, cost can be reduced, and uric acid content can be tested in a short time.

The invention relates to a PEGylation urate oxidase compound as well as the preparation method thereof and the preparation of PEGylation urate oxidase compound as well as the application thereof, particularly relates to a PEGylation urate oxidase compound which has extremely low immunogenicity and anaphylaxis, can be applied repetitively and has a long acting as well as the preparation method thereof and the preparation of PEGylationurate oxidasee compound as well as the application thereof. The invention adopts a modification method which has high selectivity to a N-terminal of a urate oxidase, which means that PEG active ester is connected with the N-terminal of the urate oxidase by covalence bond, the uniformity of products is greatly improved, the products are easy to be purified, the activity of the purified products can be better preserved and the immunogenicity of prototype protein of the urate oxidase can be reduced obviously. The PEGylation urate oxidase compound can prevent systemic anaphylaxis when applied, can be applied repetitively and has the efficacy of treating and preventing gout, reducing the level of serum uric acid, preventing tumor lysis syndrome and improving the renal function.

The invention belongs to the technical field of analytical chemistry and relates to a method for non-enzymatic colorimetric detection of uric acid. First an integrated CoP / NF entire mimetic peroxidase is prepared, and then a color-development substance TMBox is generated by redox reaction of catalyzing TMB and a hydrogen peroxide solution with CoP / NF serving as mimetic peroxidase. Since TMBox can be selectively reduced into colorless TMB by uric acid, the absorbance generated by the original chromogenic reaction reduces with increase of the uric acid concentration, a curve with the absorbance reducing with the increase of the uric acid concentration is obtained by measuring the absorbance of the finally obtained color-development substance, and the uric acid content of a measured object is determined according to the standard curve. According to the analytical method, neither peroxidase nor uric acid oxidase is needed to achieve non-enzymatic detection of uric acid, the detection cost is low, and the operation is simple; the detection range of the uric acid is 1 to 200 [mu]m, and the detection limit is as low as 1 [mu]m; the response to the uric acid is sensitive; the method has good specificity, and accurate analysis of uric acid in serum and urine can be achieved.

The invention discloses a detection reagent and test paper for uric acid. The detection reagent comprises 12 to 18 KU / L of urate oxidase, 5 to 10 KU / L of horseradish peroxidase, 15 to 20 KU / L of ascorbic acid oxidase, 0.20 to 0.35 g / L of 4-aminoantipyrine and 0.20 to 0.30 g / L of a chromogenic substance. The detection reagent provided by the invention contains a plurality of specific enzymes in a specific ratio and can achieve the purpose of rapid and accurate detection of the content of uric acid. The test paper for uric acid comprises a reaction substrate layer, a reaction layer, a hemofiltration layer and a sample suction layer. The above-mentioned layers are superposed to form a siphon system, so the test paper has better anti-interference capability, effectively eliminates interference by endogenous substances and further guarantees rapid and accurate detection of the content of uric acid.

The invention relates to multi parameter whole blood integrated test bar. It combines the testing instrument and could measure the blood sugar, uric acid or cholesterol parameters in time-sharing or multiple steps. The invention uses the base compounded by polymer as main body, and conductive silver layer, conductive carbon layer, insulating layer, undrying layer and covering film layer are over the base. The invention set glucose oxidase, urate oxidase or cholesterol oxidase are set on the same base. The test bar side would take sample, and the reacting pools are separated, and would not disturb with each other while taking electrochemical reaction.

The invention provides a recombinant adeno-associated virus mediated medicament for treating hyperuricemia. A recombinant adeno-associated virus vector carries a gene expression cassette of artificialurate oxidase (AUO for short) containing a human miR-142-3p target sequence. An internal experiment shows that the recombinant adeno-associated virus vector can be efficiently introduced into body, in order to continuously and stably express urate oxidase, reduce content of uric acid, and effectively alleviate gout symptom due to excessive uric acid in blood. A result shows that the recombinant adeno-associated virus vector is hopeful to be developed into a novel medicament for treating hyperuricemia.

The invention discloses a urate oxidase gene recombinant plasmid and a genetic engineering bacterium which produces urate oxidase. The urate oxidase gene recombinant plasmid pRNJ800-U is obtained by inserting a NisA+MCS fragment and a urate oxidase gene in a plasmid vector pRAF800. The recombinant plasmid pRNJ800-U is transformed into Lactococus lactis to obtain the genetic engineering bacterium which produces the urate oxidase. The genetic engineering bacterium can be prepared into a biological preparation for oral administration to ensure that the genetic engineering bacterium is colonized on intestinal mucosa, thereby persistently and effectively decomposing uric acid in an intestinal canal to prevent and treat various diseases related to high uric acid; thus the side effect of drugs can be avoided, and the frequent drug use is also not needed. At the same time, the Lactococus lactis is a probiotic bacterium of the intestinal canal of a human body, is colonized on an inner layer of the intestinal mucosa, has functions of strengthening immunosuppresive pathogen, synthesizing various vitamins, reducing cholesterol and so on, and has a plurality of health care functions.

The invention discloses a human urate oxidase protein which is formed by SEQ ID NO.3 amino acid sequences. By the human urate oxidase protein, mutation codes in human urate oxidase gene can be reserved, and the human urate oxidase gene subjected to reserve mutation can be introduced into engineering bacteria to realize expression of human urate oxidase gene so as to generate urate oxidase protein with enzyme activity. The polyethylene glycol human urate oxidase protein composite and drug composition have oxygenolysis uric acid activity and can be used for treatment of hyperuricemia and diseases (such as gout) caused by high uric acid.

The invention provides a preparation method of PEG recombinant pig-human urate oxidase fusion protein, a method for preparing injecta and lyophilized injection by PEG recombinant pig-human urate oxidase fusion protein, and application for reducing content of blood uric acid. The invention further provides a nucleotide sequence of pig-human urate oxidase, which comprises vectors of nucleotide sequence and host cells containing the vectors. The PEG recombinant pig-human urate oxidase fusion protein provided with the invention has no immunogenicity substantially, maintains activity of urate oxidase before PEG and has good treatment effect for reducing the content of blood uric acid.

Genetically modified proteins with uricolytic activity are described. Proteins comprising truncated urate oxidases and methods for producing them, including PEGylated proteins comprising truncated urate oxidase are described.

The invention discloses an efficient secretory expression and purification method of a recombinant urate oxygen oxidoreductase, and belongs to the field of medical and biological engineering. The technical scheme comprises the following steps of: expressing an escherichia coli operon of the recombinant urate oxygen oxidoreductase using the encoding sequence of a recombinant urate oxygen oxidoreductase which is artificially synthesized from the encoding sequence of an escherichia coli alkaline phosphatase promoter and the encoding sequence of an escherichia coli LamB signal peptide; cloning the operon to a plasmid vector pBR322 carrying the escherichia coli to obtain an expression plasmid; transforming a W3110 escherichia coli empty host using the expression plasmid containing the escherichia coli operon to obtain an engineering strain for the secretory expression of the recombinant urate oxygen oxidoreductase; and carrying out purification technologies of cation-exchange chromatography, hydrophobic chromatography, and anion-exchange chromatography on the recombinant urate oxygen oxidoreductase. The method disclosed by the invention has the beneficial effects that a good secretory expression effect can be achieved through the regulation and control on protein expression by the escherichia coli alkaline phosphatase promoter and induction of the secretory expression of the urate oxygen oxidoreductase by the optimized escherichia coli LamB signal peptide, and the purification method is simple and easy in implementation, short in protein purification period and high in protein yield.

The invention belongs to the technical field of biosensing, and relates to an enzyme detection reagent for detecting uric acid content for an electrochemical test strip. The enzyme detection reagent disclosed by the invention comprises the following components in parts by weight: 20-30 parts of urate oxidase, 5-10 parts of an electronic media body, 5.4-9 parts of a binder, and 60-70 parts of pH buffer solution. The test strip of the enzyme detection reagent disclosed by the invention has any known structure in the technical field. The enzyme detection reagent disclosed by the invention can improve the detection accuracy, is matched with a reagent strip with an optimized structure and corresponding data treatment method, and can detect the content of uric acid by a little of blood within short time.

The invention discloses a fusion protein, coded genes thereof and an application thereof. The fusion protein comprises urate oxidase and albumin. The urate oxidase is non-human urate oxidase and is preferably selected as urate oxidase from microorganisms; the albumin is the protein having more than 90 percent of homology with human serum albumin and is preferably selected as human serum albumin. The fusion protein has the following advantages as: (1) the immunogenicity is extremely low in respect of a human body; (2) the half-life of the urate oxidase in the human body can be prolonged; (3) medicaments can be supplied repeatedly to improve the effect of treating diseases; (4) the original activity of the urate oxidase for decomposing uric acid can be maintained; and (5) potential side effect or toxicity accompanying with the use of urate oxidase can be reduced, which mainly reduces the generation of antibodies and anaphylactic reaction.

The invention discloses a method for extracting recombinant aspergillus flavus uricase from an efficiently-expressed genetic engineering bacterium. The bacterium is bacillus coli rosetta (DE3) carrying a recombinant plasmid, and the recombinant plasmid is a plasmid pET-30c(+) containing an aspergillus flavus uricase gene. A method for producing the aspergillus flavus uricase by using the bacterium comprises the following steps of: A, fermenting the engineering bacterium and accumulating intracellular uricase; B, ultrasonically performing cell disruption and centrifugally collecting supernatant (containing soluble uricase); C, performing ion exchange chromatography, hydrophobic chromatography and gel permeation chromatography in sequence and collecting an activity peak; D, performing freeze drying on the activity peak to obtain the high-purity recombinant aspergillus flavus uricase. The method has the advantages of low production cost of the recombinant aspergillus flavus uricase, high enzyme expression level, simple and convenient purifying steps, high yield, high purity, easiness for industrial mass production and high practical application value.

The invention discloses a polyethylene glycol modifier of aspergillus flavus urate oxidase, wherein polyethylene glycol and the aspergillus flavus urate oxidase are connected through a covalent bond, the subunit of each aspergillus flavus urate oxidase molecule is averagely combined with 2-10 polyethylene glycol molecules, the average molecular weight of the polyethylene glycol molecule is 4-9kd, and the enzymatic specific activity of the polyethylene glycol modified aspergillus flavus urate oxidase is 5.0-10.0U / mg. A preparation method thereof is realized by adopting the following technical scheme: recombinant aspergillus flavus urate oxidase protein with the purity of larger than 95 percent is modified by adopting the activated polyethylene glycol molecule with the average molecular weight of 4-9kd for 0.1-10 hours at the pH value of 5-10 and the temperature of 4-37 DEG C, and the mass ratio of the polyethylene glycol molecule to the aspergillus flavus urate oxidase is 3:1-20:1. The polyethylene glycol modified aspergillus flavus urate oxidase realizes the balance in the aspects of high specific activity, long half life, low immunogenicity, high stability, low cost and the like and has industrialization value.

The invention relates to a stable preparation of polyethylene glycol uricase conjugate, in particular to polyethylene glycol uricase freeze drying powder and a preparation method thereof. The polyethylene glycol uricase freeze drying powder is prepared from solid ingredients of polyethylene glycol uric acid oxidase, a protection agent and a freeze drying shaping agent, wherein the protection agentuses saccharose, the freeze drying shaping agent uses arginine; through being metered in parts by mass, the polyethylene glycol uric acid oxidase accounts for 0.5 to 5.0 parts; the saccharose accounts for 15 to 35 parts; the arginine accounts for 5.0 to 25 parts. The freeze drying powder improves the stability of the polyethylene glycol uricase freeze drying powder; in addition, the shaping performance is good. The invention also provides the preparation method of the freeze drying powder.

The present invention discloses an engineering probiotic capable of degrading uric acid in intestinal tract, a preparation method and an application of the engineering probiotic, and belongs to the technical field of biology. Uric acid permease and uric acid oxidase are respectively constructed into different intestinal probiotic strains, abilities of the intestinal probiotic strains to reduce uric acid are compared, and compared with original probiotics, the probiotic recombining and integrating the uric acid permease can significantly increase intake of exogenous uric acid and thus reduces extracellular uric acid concentration. The uric acid-lowering probiotic has significantly higher uric acid-lowering ability than the previously reported uric acid-lowering probiotics, besides, animal uric acid-lowering effect tests show that the engineering probiotic has the significant uric acid-lowering effect. As a new method to reduce blood uric acid and treat gout, the engineering probiotic has broad application prospects.

The invention discloses a urate oxidase gene from bacillus subtilis BS04 and application thereof and belongs to the technical field of biology. A nucleotide sequence of the urate oxidase gene of bacillus subtilis BS04 disclosed by the invention is SEQ ID NO:1, an encoding amino acid sequence is SEQ ID NO:2, a full length of the urate oxidase gene is 1485bp, 494 amino acids are encoded, and a serial number for applying GenBank is KX388349. Furthermore, recombinant protein of the urate oxidase gene of bacillus subtilis BS04 disclosed by the invention can degrade uric acid efficiently, enzyme activity is much higher than that of urate oxidase from other sources, heat resistance is better, and a theoretical basis is provided for commercial production.

The invention discloses a high-efficiency expression and purification method of aspergillus flavus uricase in Pichia pastoris. The method comprises the steps of: on the basis of the nucleotide composition of an uricase gene, splicing the double-stranded DNA (DeoxyriboNucleic Acid) of the uricase and the double-stranded DNA of alpha-factor signal peptide via SOE-PCR (Splicing Overlap Extension-Polymerase Chain Reaction) to obtain a fusion gene alpha-factor-uricase; and inserting the fusion gene into plasmid pPIC3.5k to obtain recombinant plasmid pPIC3.5k-alpha-factor-uricase and carrying out electrotransformation on the Pichia pastoris to obtain high-expression clone strain Pichia pastoris SMD1168. The expressed recombined aspergillus flavus uricase has the advantages of completely natural N terminal, no any unnecessary amino acid, low production cost, high enzyme activity, simpleness and convenience in the purification step, high yield, high purity, easiness in industrial scale production and greater practical application value.

The invention discloses a recombinant multi shape hansenula yeast, specific recombinant carrier and application thereof. The disclosed recombinant multi shape hansenula yeast is recombinant strain capable of secreting expressive uricoxidase obtained by introducing uricoxidase gene of candida producing prion into multi shape hansenula yeast CGMCC2.2498; encoding gene of the uricoxidase is inserted between MOX promoter and AOX1 terminator in pMOXZ-alpha-A to obtain recombinant expression vector, then uricoxidase expression cassette is integrated onto chromosome of multi shape hansenula yeast by transformation. The recombinant multi shape hansenula yeast can produce uricoxidase on high level by high density fermentation, wherein yield of uricoxidase is 8.5 times of reported yield. Uricoxidase produced by constructed recombinant multi shape hansenula in the invention is high in yield, low in immunogenicity, highly benefit for wide application of uricoxidase in the medical and pharmaceutical field.