Filtering medium capable of degrading human blood uric acid, and preparation method thereof

A technology for filtering media and blood uric acid, which is applied in biochemical equipment and methods, chemical instruments and methods, enzymes, etc., and can solve problems such as serious side effects

Inactive Publication Date: 2020-08-07
北京翼方生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] At present, the drugs for hyperuricemia all have their advantages and disadvantages without exception. After entering the human body, the drugs will not only exert their medicinal effects, but also produce a series of side effects, and some of the side effects are quite serious.

Method used

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  • Filtering medium capable of degrading human blood uric acid, and preparation method thereof
  • Filtering medium capable of degrading human blood uric acid, and preparation method thereof
  • Filtering medium capable of degrading human blood uric acid, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1. Preparation of Aspergillus flavus urate oxidase

[0041] 1) Gene synthesis:

[0042] The gene encoding Aspergillus flavus uric acid oxidase was synthesized in a fully synthetic way (sequence shown in SEQ ID NO.1); for the convenience of constructing the vector, an EcoRI restriction site was added to the front of the start codon at the 5' end of the gene , a BamHI restriction site was added to the back end of the stop codon at the 3' end, and cloned into the pBV220 vector, the structure is as follows figure 2 ; Both are entrusted to outsourcing service companies to complete.

[0043] 2) Preparation of strains:

[0044] The expression vector carrying the fully synthetic gene encoding Aspergillus flavus uric acid oxidase was transformed into CaCl 2 Treated E.coli DH5α. The transformed bacteria were plated on Amp+LB plates, and the transformants with Amp resistance were screened out, and expanded to 200ml shake flasks with ordinary LB medium, 37 degrees C...

Embodiment 2

[0050] Example 2. Agarose gel electrophoresis

[0051] Electrophoresis instrument DYY-6C, non-reducing electrophoresis, stacking gel 5%, separating gel 12%;

[0052] Sample: marker, broken bacteria supernatant, the pure product of Aspergillus flavus uricase prepared in Example 1;

[0053] Loading volume: 10 μl each;

[0054] Voltage setting: 80V, 30min; 180V, 60min

[0055] Test results: Aspergillus flavus uricase was expressed soluble in bacteria. Analysis by gel imaging system showed that the molecular weight / purity of the pure product was 34.2 / 95.0%, the molecular weight of the main band was 34.05kDa, and the theoretical molecular weight was 34.11kDa, and the detected value was consistent with the theoretical value. Such as image 3 .

Embodiment 3

[0056] Example 3. Activity assay

[0057] Using uric acid degradation method, the reaction equation is as follows:

[0058]

[0059] The reduction of uric acid was measured by a spectrophotometer at an ultraviolet wavelength of 290nm.

[0060] Definition of activity: One unit of activity is defined as the amount of urate oxidase required to oxidize 1 μmol of uric acid at 25 degrees Celsius.

[0061] Reagent:

[0062] A. 20% KOH: 20g KOH is dissolved in 100ml distilled water.

[0063] B. 0.001% uric acid solution: Dilute 0.01% uric acid 10 times with reagent C;

[0064] 0.01% stock solution: 10mg of uric acid was dissolved in 100ml of reagent C.

[0065] C. Enzyme diluent: 3.09g boric acid, 0.37g EDTA-Na 2 2H 2 O, 0.01g Triton X-100 was dissolved in 800ml distilled water, the pH value was adjusted to 8.5 with 2N NaOH, and distilled water was added to 1000ml.

[0066] Sample: Dissolve the sample with reagent C to 0.01-0.02U / ml, and test the activity immediately.

[00...

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Abstract

The invention relates to a filtering medium capable of degrading human blood uric acid, and a preparation method thereof. Specifically, recombinant urate oxidase is immobilized on a substrate such asagarose, polyacrylate, cellulose or polyacrylamide through a chemical reaction to prepare the plasma filtering medium; and when blood plasma containing high-concentration uric acid flows through the surface of the medium, uric acid oxidase oxidizes uric acid into allantoin, so that the level of uric acid in the blood plasma is reduced. The allantoin has higher solubility in an aqueous solution andis easy to discharge out of the body. The medium has wide application value in preparation of a plasma filtering device for reducing blood uric acid of the human body.

Description

technical field [0001] The invention relates to a filter medium for removing human blood uric acid and a preparation method thereof, and has clinical practical value in the field of treating hyperuricemia caused by diseases such as gout. Background technique [0002] 1. About purine metabolism hyperuricemia [0003] Uric acid is an end product of purine metabolism in some birds, reptiles, primates, and humans. It is produced by the oxidation of xanthine and hypoxanthine in the liver. In most other mammals, uric acid is finally oxidized by urate oxidase to the allantois. white. However, humans lack this enzyme, so uric acid becomes the end product of purine metabolism. If the production of uric acid is increased (excessive intake of high-purine foods from exogenous sources, accelerated catabolism of endogenous nucleic acid) and / or decreased excretion (under normal circumstances, about 2 / 3 of uric acid is excreted by the kidneys, and about 1 / 3 is excreted by the intestines) ...

Claims

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Application Information

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IPC IPC(8): C12N9/06C12P17/10B01D69/08B01D61/00
CPCB01D61/00B01D69/08C12N9/0048C12P17/10C12Y107/03003
Inventor 吴彦卓徐霞陈怡凝
Owner 北京翼方生物科技有限责任公司
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