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Urate oxidase gene of bacillus subtilis BS04 and application thereof

A technology of Bacillus subtilis and uric acid oxidase, which is applied in oxidoreductase, genetic engineering, plant genetic improvement, etc., can solve the problems of poor thermal stability, low enzyme activity, and limited application, and achieve good heat resistance and efficient degradation Effect

Inactive Publication Date: 2017-09-19
YANGTZE UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In recent years, related studies on urate oxidase have been reported, but the low enzyme activity and poor thermal stability in the actual production process limit its application in the treatment of hyperuricemia and gout-related diseases

Method used

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  • Urate oxidase gene of bacillus subtilis BS04 and application thereof
  • Urate oxidase gene of bacillus subtilis BS04 and application thereof
  • Urate oxidase gene of bacillus subtilis BS04 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Cloning of Example 1 Bacillus subtilis BS04 urate oxidase gene

[0030] Genomic DNA of Bacillus subtilis BS04 was extracted by CTAB method, and a pair of specific primers Uri-F (5'-ATGTTCACAATGGATGACCTG-3') and Uri-R (5' -GGCTTTCAGGCTC-3'), with genomic DNA as template, Uri-F and Uri-R as primers, PCR amplification is carried out to obtain the uric acid oxidase gene of Bacillus subtilis BS04.

[0031] The PCR reaction program is PCR reaction system: ddH 2O (16.2 μL), 10×buffer (2.5 μL), dNTP (2 μL), Uri-F (1 μL), Uri-R (1 μL), DNA (2 μL), TaqE (0.3 μL), total volume 25 μL. PCR reaction program: 94°C 4min; 94°C 30s, 62°C 30s, 72°C 2min 15s, a total of 35 cycles; finally 72°C extension 7min. The PCR amplified product was recovered and purified with Tiangen gel extraction kit, then ligated with pEASY-T1 vector (Takara) and transformed into E. coli DH5α competent cells, and positive clones were selected to extract plasmids for sequencing. Sequencing results show that the...

Embodiment 2

[0032] Example 2 Construction of Bacillus subtilis BS04 urate oxidase gene prokaryotic expression vector

[0033] According to the gene sequence of uric acid oxidase of Bacillus subtilis BS04 and the multiple cloning site of the prokaryotic expression vector PET-28a (+), a pair of specific primers Uri-P-F (5'-CCC GAATTC ATGTTCACAATGGATGACCTG-3') and Uri-P-R (5'-TT GCGGCCGC GGCTTTCAGGCTC-3'), EcoRI and NotI restriction sites were added to the upstream and downstream primers, respectively. Using Bacillus subtilis BS04 genomic DNA as template and Uri-P-F and Uri-P-R as primers, PCR amplification was carried out.

[0034] PCR reaction system: ddH 2 O (16.2 μL), 10×buffer (2.5 μL), dNTP (2 μL), Uri-P-F (1 μL), Uri-P-R (1 μL), DNA (2 μL), TaqE (0.3 μL), total volume 25 μL. PCR reaction program: 94°C 4min; 94°C 30s, 62°C 30s, 72°C 2min 15s, a total of 35 cycles; finally 72°C extension 7min. The PCR product and the prokaryotic expression vector PET-28a(+) were digested with EcoRI...

Embodiment 3

[0035] Induced expression and purification of embodiment 3 recombinant protein

[0036] Take 1 ng of the recombinant plasmid to transform Escherichia coli BL21(DE3), spread it on an ampicillin plate containing 50 μg / mL for overnight culture at 37°C. Positive transformants were picked and cultured in liquid LB medium containing 50 μg / mL ampicillin at 37 °C and 200 r / min until the exponential growth phase, and then IPTG was added to make the final concentration 0.1 mmol / L and cultured for 4 h. The cells were collected by centrifugation and ultrasonically broken, and then 5× loading buffer was added for 12% SDS-PAGE electrophoresis to detect the expression of the recombinant protein. SDS-PAGE electrophoresis was used to detect the expression of the recombinant protein, and the molecular weight of the recombinant protein was about 60kDa.

[0037] Expand the positive recombinant bacteria at a ratio of 1%, induce recombinant expression with IPTG, collect the bacterial cells by cent...

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Abstract

The invention discloses a urate oxidase gene from bacillus subtilis BS04 and application thereof and belongs to the technical field of biology. A nucleotide sequence of the urate oxidase gene of bacillus subtilis BS04 disclosed by the invention is SEQ ID NO:1, an encoding amino acid sequence is SEQ ID NO:2, a full length of the urate oxidase gene is 1485bp, 494 amino acids are encoded, and a serial number for applying GenBank is KX388349. Furthermore, recombinant protein of the urate oxidase gene of bacillus subtilis BS04 disclosed by the invention can degrade uric acid efficiently, enzyme activity is much higher than that of urate oxidase from other sources, heat resistance is better, and a theoretical basis is provided for commercial production.

Description

technical field [0001] The invention belongs to the field of biotechnology. Specifically, a urate oxidase gene is cloned from bacillus subtilis BS04, and the gene can be stably expressed in vitro and produce urate oxidase with higher activity. Background technique [0002] Uricase, also known as uric acid oxidase (Uricase or Urate oxidase), is an important enzyme in the process of purine metabolism in animals. It uses molecular oxygen as the receptor and plays an important role in purine metabolism. It can catalyze the oxidation of uric acid and generate Allantoin, CO 2 and H 2 o 2 . Uric acid oxidase exists widely in the biological world. Except for humans and some higher primates, it is present in most organisms (including archaea, bacteria, fungi, actinomycetes, plants, fish and most non-primate mammals). Can produce biologically active uric acid oxidase. In the process of human evolution, due to the nonsense mutation of the urate oxidase gene, the urate oxidase gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/06C12N15/70
CPCC12N9/0048C12N15/70C12Y107/03003
Inventor 孙文秀李伟陈洪娜
Owner YANGTZE UNIVERSITY
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