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Catalytically active human urate oxidase

A technology of uric acid oxidase and catalytic activity, applied in the field of human uric acid oxidase, can solve the problems of inconclusive immunogenicity, prolong the half-life of protein drugs, limit the effectiveness of patient treatment, etc., and achieve the effect of low immunogenicity

Active Publication Date: 2017-11-07
CHINA PHARM UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

The more successful modification is to covalently modify urate oxidase with PEG (polyethylene glycol); in 2010, the US FDA approved the marketing of Krystexxa, a chimera of PEG-modified pig-baboon urate oxidase, and Krystexxa is used to treat traditional treatments. For gout patients who are ineffective or intolerable to the method, a total of 212 patients with gout symptoms participated in the one-year clinical phase I and II trials. The test proved that Krystexxa can effectively reduce the level of uric acid in the blood and reduce the concentration of uric acid crystals in the blood. Deposition in joints and soft tissues; but in clinical trials, a quarter of patients experienced severe allergic reactions, as well as other adverse reactions: such as gout attacks, nausea, bruising at the injection site, constipation, chest pain and vomiting, etc. ( Corinne SY, William H, Michelle AB et al. J. Clin. Pharmacol. 2008.48:708–718)
For a long time, researchers have believed that PEG has no immunogenicity to the human body. Covalently linking PEG to protein drugs can not only prolong the half-life of protein drugs, but also reduce the immunogenicity of protein drugs. However, Ganson et al. In the phase I clinical trial of urate oxidase, it was found that some patients produced anti-PEG antibodies in their serum, which limited the effectiveness of treatment for some patients (Ganson N, Kelly S, Scarlett E et al.Arthritis Research&Therapy2005. 8:R12)
Therefore, the immunogenicity of PEG to the human body has not yet been determined, and further investigation and research are needed to ensure the safety and effectiveness of clinical use of PEG-modified protein drugs

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0087] Example 1 Human-pig urate oxidase chimeric protein H 1-2 P 3-8 Construction of DNA and its recombinant expression in Escherichia coli BL21

[0088] Obtain the plasmid containing the template: inoculate glycerol tubes containing Escherichia coli engineering bacteria containing human urate oxidase gene and porcine urate oxidase gene respectively, the inoculation amount is 1%, culture overnight, and extract the plasmid.

[0089] H 1-2 ,P 3-8 Fragments, and then use the method of overlap extension PCR to obtain H 1-2 P 3-8 DNA.

[0090] get H 1-2 P 3-8 DNA: The first stage of PCR: the template sequence is the above plasmid containing the human uric acid oxidase gene, the primers are primer 1 and primer 6, and the PCR conditions are: 95°C for 30s, 55°C for 30s, 72°C for 35s, a total of 30 cycles, the first One cycle of denaturation at 95°C for 5 minutes, the last cycle of extension at 72°C for 10 minutes, and finally the product H 1-2 Fragment; the second stage of P...

Embodiment 2

[0096] Example 2 Human-pig urate oxidase chimeric protein P 1-2 h 3 P 4-8 Construction of DNA and its recombinant expression in Escherichia coli BL21

[0097] The same method as in Example 1 was used to obtain plasmids containing human and porcine urate oxidase gene sequence templates.

[0098] get P 1-2 H3P 4-8 DNA: The first stage of PCR: the template sequence is pET-22b(+)-PU, the primers are primer 8 and primer 3, the PCR conditions are: 95°C for 30s, 55°C for 30s, 72°C for 35s, a total of 30 cycles, the first The first cycle was denatured at 95°C for 5 minutes, and the last cycle was extended at 72°C for 10 minutes to obtain the product P 1-2Fragment; the second stage of PCR: the template sequence is pET-22b(+)-HU, the primers are primer 7 and primer 9, the PCR conditions are: 95°C for 30s, 55°C for 30s, 72°C for 20s, a total of 30 cycles, the first The first cycle was denatured at 95°C for 5 minutes, the last cycle was extended at 72°C for 10 minutes, and finally t...

Embodiment 3

[0100] Example 3 Human-pig urate oxidase chimeric protein P 1-3 h 4 P 5-8 Construction of DNA and its recombinant expression in Escherichia coli BL21

[0101] The same method as in Example 1 was used to obtain plasmids containing human and porcine urate oxidase gene sequence templates.

[0102] get P 1-3 h 4 P 5-8 DNA: The first stage of PCR: the template sequence is pET-22b(+)-PU, the primers are primer 11 and primer 3, the PCR conditions are: 95°C for 30s, 55°C for 30s, 72°C for 45s, a total of 30 cycles, the first The first cycle was denatured at 95°C for 5 minutes, and the last cycle was extended at 72°C for 10 minutes to obtain the product P 1-3 Fragment; the second stage of PCR: the template sequence is pET-22b(+)-HU, the primers are primer 12 and primer 13, the PCR conditions are: 95°C for 30s, 55°C for 30s, 72°C for 15s, a total of 30 cycles, the first The first cycle is denatured at 95°C for 5 minutes, and the last cycle is extended at 72°C for 10 minutes to ob...

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Abstract

The invention relates to a series of human-derived urate oxidases with catalytic activity, DNA sequences encoding them, expression vectors containing the DNA sequences, as well as host cells containing the expression vectors. The human-derived urate oxidases have recombinant amino acid residue sequences obtained by specific locus amino acid residue replacement or exon locus amino acid residue replacement on the basis of SEQ ID NO:17. According to the invention, human urate oxidase pseudogene is successfully revived, and human-derived urate oxidases with catalytic activity can be expressed. Thus, the human-derived urate oxidases with catalytic activity have good prospects in preparation of low immunogenicity and even low immunogenicity uric acid lowering drugs.

Description

technical field [0001] The invention relates to a series of human uric acid oxidases with catalytic activity, DNA sequences encoding them, expression vectors containing the DNA sequences, and host cells containing the expression vectors, belonging to the field of biotechnology. Background technique [0002] As far as the applicant knows, uric acid oxidase (Uricase, Urate oxidase, EC1.7.3.3) participates in the purine metabolic pathway of organisms, and it can combine molecular oxygen and water molecules to catalyze the degradation of uric acid into allantoin, CO 2 and H 2 o 2 (Ramazzina I, Folli C, Secchi A et al. Nat. Chem Biol. 2006. 2:144–148). Since urate oxidase was found to contain a copper ion-binding domain by domain search, researchers once believed that urate oxidase was a copper ion-binding enzyme. However, subsequent studies have shown that urate oxidase does not require copper ions to participate in its catalytic function (Chu RY, Lin YL, Rao MS et al. Annals...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/63A61K38/44A61P19/06
CPCA61K38/00C12N9/0048C12Y107/03003
Inventor 陈建华谢光蓉赵百学
Owner CHINA PHARM UNIV
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