Human urate oxidase protein and preparation method and polyethylene glycol composite thereof

A technology of uric acid oxidase protein and polyethylene glycol, applied in the field of protease, can solve the problems of unsuitability for long-term use, allergy, immunogenicity and the like

Active Publication Date: 2012-10-31
HANGZHOU JUNFENG BIOENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent application 99811738.2 has announced the uric acid oxidase chimeric urate oxidase sequence of pig and baboon, and Chinese patent application 03109150.4 is a kind of preparation method of recombinant Candida uric acid oxidase, and Rasburicase (rasburicase) is gene recombination Aspergillus flavus urate oxidase, still has immunogenicity and possibility of allergies, not suitable for long-term use
Although the curative effect has been achieved, the product pegloticase developed by the company still has the risk of allergic reactions

Method used

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  • Human urate oxidase protein and preparation method and polyethylene glycol composite thereof
  • Human urate oxidase protein and preparation method and polyethylene glycol composite thereof
  • Human urate oxidase protein and preparation method and polyethylene glycol composite thereof

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Experimental program
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Effect test

Embodiment 1

[0038] Embodiment one, the construction of human urate oxidase gene and expression vector

[0039] Acquisition of human urate oxidase gene

[0040] The pseudogene sequence of human urate oxidase spliced ​​according to the pseudogene sequence of human urate oxidase published by Genbank (NCBI Reference Sequence: NR 003927.1), GenBank: S94095.1 and GenBank: AH003594.1 is shown in SEQ ID NO.1. Change the "A" at the 99th position and the "A" at the 561st position of the human urate oxidase pseudogene of SEQ ID NO.1 to "G" to obtain a human backmutated human urate oxidase gene, see SEQ ID NO.2. The human uric acid oxidase gene is synthesized by chemical synthesis method, and its coded amino acid sequence is shown in SEQ ID NO.3. Synthesize 25 complementary oligonucleotide fragments first, add the Nde I restriction site to the 5' end of the gene, introduce the BamH I restriction site at the 3' end, treat with T4 bacteriophage polynucleotide kinase at 37°C for 30 min, and phosphoric ...

Embodiment 2

[0046] Embodiment two, the acquisition of high expression host bacteria

[0047] The recombinant expression plasmid pET-22b-rhUOX was transformed into Escherichia coli host bacteria. Prepare bacterial cells for transformation, including picking a BL21(DE3) colony and inoculating it into 3ml of LB medium, culturing overnight at 37°C with 250rpm shaking; pipetting 2ml into 20ml LB for expansion culture, shaking at 37°C and 250rpm until the OD600 is 0.4-0.6; Under aseptic conditions, transfer the bacteria to an ice-precooled sterile 50ml test tube, place on ice for 10 minutes to cool the culture to 0°C; centrifuge at 4000rpm at 4°C for 10 minutes, and recover the bacteria; pour out the culture solution, and put the test tube Invert for 1 minute to drain the remaining medium; 10ml 0.1mol / LCaCl 2 Resuspend the bacterial pellet and let it stand on ice for 10 minutes; centrifuge at 4000rpm at 4°C for 10 minutes to recover the bacteria; pour out the culture medium and invert the test...

Embodiment 3

[0050] Embodiment three, recombinant human urate oxidase

[0051] Cultivation and induced expression of host bacteria

[0052] Preparation medium: Trypton 10g / L, Yeast extract 5g / L, NaCl 5g / L, pH7.0. After the medium is prepared, it is sterilized by moist heat at 121°C for 20 minutes. After the sterilization, add Amp to the ultra-clean bench to make the final concentration of 50 μg / mL when the culture medium is cooled and not hot, and put it in the refrigerator at 4 °C after cooling for later use.

[0053] Seed culture: Pick a single colony in an ultra-clean bench under aseptic operation and inoculate it in 20ml LB medium (containing Amp), then place it at 37°C and culture overnight on a shaker at 180rpm.

[0054] Scale-up culture: Take 1ml inoculum from the overnight cultured seed culture and transfer it to 20ml LB medium, and store the rest of the seed culture in a refrigerator at 4°C. The amplified culture transfer was placed at 37°C and cultured at 180rpm for 3 hours, a...

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Abstract

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.

Description

field of invention [0001] The invention relates to a protease with uric acid decomposition activity prepared by genetic engineering technology, more specifically, the invention relates to a novel urate oxidase protein and a preparation method thereof. Background technique [0002] Uric acid oxidase (urate oxidase, uricase, uricase, E.C.1.7.3.3) is an enzyme in the degradation and metabolism pathway in organisms, which catalyzes the oxidation of uric acid to allantoin with higher solubility, and the solubility of allantoin is higher than that of uric acid About 10 times higher, is the more easily excreted purine metabolite. Uric acid oxidase is found in many species, but it is lacking in higher mammalian primates and humans, so uric acid is the end product of purine metabolism. The solubility of uric acid and its salts in water is very low. Excessive accumulation in the blood will lead to hyperuricemia. If uric acid forms crystals and deposits in soft tissues, joints, organs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/63C12N1/19C12N1/21C12N5/10A61K38/44A61K47/48A61P19/06
Inventor 徐辉张明劼
Owner HANGZHOU JUNFENG BIOENG
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