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Nucleotide sequence and expression vector of encoded recombinant urate oxidase protein

A urate oxidase protein and nucleic acid sequence technology, applied in the field of protein engineering, can solve problems such as unfavorable treatment of leukemia, increase the burden on the kidney, reduce the curative effect, etc., and achieve the effects of easy quality control, small batch-to-batch variation, and cost reduction.

Inactive Publication Date: 2018-06-01
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the drug commonly used to treat hyperuricemia is mainly allopurinol. This drug acts on the intermediate link of purine metabolism, inhibits the formation of uric acid, and increases the intermediate products xanthine and hypoxanthine. The disadvantage is that it increases the burden on the kidneys.
The drug can also interact with the leukemia treatment drug 6-mercaptopurine, reducing the curative effect, which is not conducive to the treatment of leukemia

Method used

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  • Nucleotide sequence and expression vector of encoded recombinant urate oxidase protein
  • Nucleotide sequence and expression vector of encoded recombinant urate oxidase protein
  • Nucleotide sequence and expression vector of encoded recombinant urate oxidase protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment l

[0029] Construction of engineering bacteria expressing recombinant urate oxidase

[0030] 1. Target gene synthesis

[0031] According to the known natural amino acid sequence of recombinant urate oxidase, a specific recombinant urate oxidase nucleic acid sequence fragment was obtained through screening, according to the codon preference of Escherichia coli and considering the elimination of hairpin structures and other secondary structures that are not conducive to expression, Under the condition of not changing the amino acid sequence, the coding sequence primer of recombinant urate oxidase is designed, and its sequence is as follows.

[0032] Recombinant urate oxidase upstream primer:

[0033] 5'GATGAT GCTAGC ATG TCA ACA ACG CTC TCA 3'NheI (SEQ ID NO.2)

[0034] Downstream primers for recombinant urate oxidase:

[0035] 5'GATGAT GGATCC TTA CAA CTT GGT CTT CTC C 3'BamH I (SEQ ID NO.3)

[0036] The full-length sequence of recombinant urate oxidase was obtained by bridging ...

Embodiment 2

[0046] Study on High Density Fermentation of Engineering Bacteria

[0047] Configure low phosphorus medium: low phosphate medium contains 10g of peptone, 2.5g of yeast extract, MgSO in every 1000ml medium 4 ·7H 2O 0.5g, 1mol / L Tris-Cl (pH 8.0) 50ml, phenol red 2ml, glucose 10g.

[0048] Pick a single clone from the positive plate into a 250ml Erlenmeyer flask containing 50mI LB medium and culture overnight. The next day, inoculate the overnight cultured bacterial solution in the low-phosphorus medium at a ratio of 1:100.

[0049] Appropriately add carbon source (such as glycerol) and nitrogen source (such as ammonia water) to adjust pH to 7.2-7.4, DO>50%, temperature at 30-32°C, and end of induction for 12 hours. The samples were detected by SDS-PAGE (and scanned) to determine the protein content. results, such as figure 2 As shown, the expression level of recombinant urate oxidase protein reached more than 20%.

[0050] After fermenting and expressing the recombinant u...

Embodiment 3

[0053] Purification of recombinant urate oxidase

[0054] Prepare TSE solution: 20mM Tris-Hcl, pH 7.0, 20% glucose, 1mM EDTA.

[0055] The fermentation broth was centrifuged at 5000 rpm for 10 min at 4°C to obtain bacterial cells. The bacterial cells were resuspended in pre-cooled TSE solution and thoroughly stirred evenly. Centrifuge at 12000rpm for 10min to obtain a precipitate. Add 20Mm, pH7.0 Tris-CL to resuspend, and stir well. Centrifuge at 12000rpm for 10min, and collect the supernatant. The result is as figure 2 Lane 3 shows.

[0056] Chromatography was performed three times.

[0057] 1. Ion exchange chromatography

[0058] Chromatography medium: DEAE chromatography column

[0059] Buffer: Solution A: PBS (pH7.4, 20mM)

[0060] Solution B: PBS (pH7.4, 20mM) + 1M NaCl

[0061] Loading: Load the TSE extraction solution.

[0062] Cleaning: Wash the column with solution A after loading the sample.

[0063] Gradient: Solution B was raised from 0% to 100% after w...

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Abstract

The invention discloses encoded recombinant urate oxidase protein. A nucleotide sequence of the encoded recombinant urate oxidase protein is shown as SEQ ID NO:1. The nucleotide sequence of the encoded recombinant urate oxidase protein is connected with a phoA promoter and a periplasmic secretion signal peptide StII sequence and is transferred into escherichia coli to construct UOX escherichia coli engineering bacteria capable of being efficiently and stably secreted and expressed; then a low-phosphate culture medium is used for culturing; after fermentation is finished, centrifuging is carried out to remove supernatant of fermentation liquid, so as to obtain thalli; a TSE solution is used for extracting to obtain the recombinant urate oxidase protein. After purification is carried out, the urate oxidase protein is detected and has relatively good biological activity.

Description

technical field [0001] The invention belongs to the field of protein engineering, and specifically relates to a nucleic acid sequence encoding recombinant urate oxidase protein, a plasmid vector expressing the recombinant urate oxidase protein, genetic engineering bacteria, and a method for fermenting and producing the recombinant urate oxidase protein. Background technique [0002] Uric acid oxidase (urate oxidase, Uricase, EC.1.7.3.3) is a protease that can oxidize uric acid to allantoin. Uric acid is widely present in intracellular fluid and body fluids. In most mammals, uric acid is rapidly oxidized into allantoin under the action of urate oxidase in the liver, so the content in the blood is very low. Hyperuricemia mainly occurs after gout and organ transplantation, and is also a common complication of leukemia and lymphoma treatment. Due to the great harm of hyperuricemia and the large number of patients, the drug market for the treatment of hyperuricemia is broad. A...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/70C12N1/21C12R1/19
CPCC12N9/0048C12N15/70C12Y107/03003
Inventor 王明珠蔡祥胜李静静李东升朱建斌李昊佘妙琴王进辉于莉
Owner GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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