Fusion protein, encoding gene and uses thereof

A technology of fusion protein and coding gene, which is applied in the field of fusion protein and its coding gene and application, to achieve the effect of reducing immunogenicity, reducing allergic reaction and maintaining activity

Inactive Publication Date: 2009-04-08
JOINN LAB (SUZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because urate oxidase is an exogenous human protein, repeated injections of recombinant UOX will produce anti-UOX antibodies and inhibit the activity of UOX

Method used

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  • Fusion protein, encoding gene and uses thereof
  • Fusion protein, encoding gene and uses thereof
  • Fusion protein, encoding gene and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1, Preparation of Albumin and Urate Oxidase Fusion Protein

[0041] 1. Acquisition of human serum albumin coding gene

[0042] Using the human fetal liver cDNA library (purchased from Clontech, product number 637230) as a template, primers were designed to amplify the human serum albumin gene (HSA) without the signal peptide coding sequence. The specific primers are as follows:

[0043] HSA5:5′-TCG CTCGAG AAAAGAGATGCACACAAGAGTGAGG-3' (Xho I),

[0044] HSA3:5′-TT GGATCC ACCTCCACCGGAACCTCCACCTCCTAAGCCTAAGGCAGCTTGACTTG-3' (BamHI).

[0045] PCR amplification system: human fetal liver cDNA 1.0 μL, HSA5 primer and HSA3 primer 0.5 μL each, dNTP 2 μL, KOD DNA Polymerase 0.1 μL, 10×KOD buffer 2 μL, MgSO 4 2 μL, ddH 2 O 35 μL, total volume 50 μL. The PCR reaction conditions were: 94°C for 30s, 53°C for 30s, 72°C for 2min, a total of 30 cycles.

[0046] The PCR amplification product was detected by agarose gel electrophoresis, and the results showed that a band o...

Embodiment 2

[0071] Embodiment 2, the determination of the immunogenicity of fusion protein in rat body

[0072] Male SD rats were divided into two groups, HSAUOA fusion protein group and rUO protein group (for the preparation process of rUO protein, see: Wu Weili, Fang Hongqing, etc. Expression, purification and active form identification of uric acid oxidase. Journal of the Academy of Military Medical Sciences, 2005, 29(2): 124-126.), five rats in each group were intravenously injected with the HSAUOA fusion protein prepared in Example 1 above and the rUO protein prepared according to the above literature method. The dosage of HSAUOA fusion protein group was 0.9 mg / kg body weight, and the dosage of rUO group was 0.3 mg / kg body weight. Administered once a week for four weeks. Blood was collected before the first administration and 14, 21, 28, and 35 days after the first administration, and the serum was separated, put in a 4°C refrigerator for testing, and observed continuously for 2 mon...

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Abstract

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.

Description

technical field [0001] The present invention relates to a fusion protein and its encoding gene and application. Background technique [0002] Uric acid oxidase (Uricase or Urate Oxidase, UOX EC 1.7.3.3) is a key enzyme in the purine metabolic pathway, which can catalyze the formation of allantoin from uric acid. Uric acid and allantoin are the final products of purine metabolism in different organisms. The solubility of the two is very different. The solubility of uric acid in the body is very low, about 11mg / dl water, while allantoin is 13.5 times that, about 147mg / dl water. The accumulation of uric acid in the body can cause gout and urate nephropathy (Duk-HeeKang, Takahiko Nakagawa, Lili Feng et al.A Role for Uric Acid in the Progression of Renal Disease[J].J.Am.Soc.Nephrol.,2002 , 13: 2888-2897). Studies have shown that the accumulation of uric acid in the body is related to the dysfunction of tissues and organs such as the heart and kidney. The mouse model of hyperur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A61K38/44A61K38/38A61P19/06
Inventor 方宏清李招发戴红梅李树龙陈惠鹏
Owner JOINN LAB (SUZHOU) INC
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