Urate oxidase with catalytic activity

A technology of urate oxidase and urate oxidase protein, which is applied in the biological field and can solve the problems of low immunogenicity and in vivo stability of urate oxidase protein

Pending Publication Date: 2021-04-16
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Thus, the higher the amino acid sequence homology deduced from the pseudogene of human urate oxidase, the lower the immunogenicity and the improved stability in vivo of the urate oxidase protein used for therapy may be

Method used

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  • Urate oxidase with catalytic activity
  • Urate oxidase with catalytic activity
  • Urate oxidase with catalytic activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of rhUOX-N mutants (resurrected human-source uricase with mutants, on the basis of human uricase pseudogene, an active uricase protein obtained by mutation of amino acid sites, N in rhUOX-N refers to The number of amino acid positions where site-directed mutagenesis is completed).

[0039] It should be noted that the construction process of the mutant rhUOX-N in this experiment is based on the gene of the previous mutant product rhUOX-(N-1) as a template, using overlap extension PCR technology, and site-directed mutation of 17 amino acid sites that have not yet been identified. A certain site of the mutation was verified by positive screening, and then the mutant rhUOX-N was obtained. And the mutant rhUOX-N is also the site-directed mutation template for the next mutant rhUOX-(N+1).

[0040] Obtain the plasmid template: inoculate the strain of plasmid pET-22b(+) / rhUOX-(N-1) containing the previous successfully mutated amino acid site in LB liqui...

Embodiment 2

[0049] Example 2: Expression, separation and purification of rhUOX-17Mutant (resurrected human-source urate oxidase with 19 mutant of amino acid site, active uricase protein obtained by mutation of 17 amino acid sites on the basis of human uricase pseudogene)

[0050] Induced expression of rhUOX-17Mutant engineered strain: Inoculate the preserved rhUOX-17Mutant engineered strain into liquid LB medium containing 100 μg / mL, culture on a shaker at 200 rpm at 37°C for 11 hours, then transfer to fresh fermentation medium to continue After culturing for 4 hours, 0.2 mM IPTG was added, continued culturing for 5 hours, and the fermentation broth was collected.

[0051] Bacterial collection by centrifugation: Collect the fermented rhUOX-17Mutant engineering strain by centrifugation at 4°C at 8000g for 20 minutes, resuspend the collected bacteria with purified water, and centrifuge under the same conditions to wash the collected bacteria.

[0052] Ultrasonic disruption of bacteria: Add ...

Embodiment 3

[0058] Example 3: Analysis of specific activity and enzymatic properties of rhUOX-17Mutant

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Abstract

The invention relates to a urate oxidase with catalytic activity. A 'resurrection' human urate oxidase amino acid sequence with catalytic activity, a nucleotide sequence for encoding the protein, a vector containing the nucleotide sequence and a host cell containing the vector are obtained by adopting genetic engineering technology modification. The invention also discloses a method for preparing the resurrection protein by using a genetic engineering technology, and a product obtained by modifying the protein. The urate oxidase has good enzymatic activity and stability, the coding sequence of the urate oxidase maintains high homology with human urate oxidase pseudogenes, and the urate oxidase has good prospects in preparation of low-immunogenicity or even immunogenicity-free uric acid lowering drugs or pharmaceutical compositions.

Description

technical field [0001] The invention relates to a uric acid oxidase with catalytic activity, which is a "resurrected" human source uric acid oxidase with catalytic activity obtained through the transformation of genetic engineering technology, and belongs to the field of biotechnology. Background technique [0002] Uric acid (Uric Acid) is the end product of purine metabolism in the human body. When its metabolism is disturbed or its excretion is reduced, long-term oversaturated monosodium urate (MSU) crystals are deposited in the joint cavity or soft tissue, inducing local inflammatory reactions and Tissue destruction, forming gout (gout), the most common arthritis attack, its symptoms include acute onset arthritis, tophi formation, tophi chronic arthritis, urate nephropathy and uric acid urolithiasis, in severe cases Can cause joint disability and renal insufficiency. In addition, gout is associated with clinical diseases such as hypertension, hyperlipidemia, type 2 diabe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/70C12N1/21A61K38/44A61P19/06C12R1/19
Inventor 陈建华蒋楠许春琴
Owner CHINA PHARM UNIV
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