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A codon-optimized glucose oxidase gene and its application

A technology for glucose oxidase and codon optimization, which is applied in the fields of biological enzyme genetic engineering and biosensing, can solve problems such as failure to express successfully, and achieve the effect of avoiding inverted repeats.

Active Publication Date: 2021-06-22
BIOLOGY INST OF SHANDONG ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when glucose oxidase from different sources is expressed in Pichia pastoris, it usually fails to express successfully, and the gene sequence of glucose oxidase needs to be optimized; there are various ways to optimize the gene sequence, How to adjust the gene sequence and make it express smoothly in Pichia pastoris is difficult to predict

Method used

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  • A codon-optimized glucose oxidase gene and its application
  • A codon-optimized glucose oxidase gene and its application
  • A codon-optimized glucose oxidase gene and its application

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Experimental program
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Embodiment 1

[0023] Embodiment 1, glucose oxidase gene codon optimization and cloning

[0024] In this embodiment, the glucose oxidase g9669.t1 gene in the AspergillusnigerAn76 genome is used as the original gene, and the original gene sequence is shown in SEQ ID No.1. According to the differences in the frequency of use of different codons encoding the same amino acid in Pichia pastoris, the rare codons in the original gene of glucose oxidase g9669.t1 were removed to avoid the occurrence of inverted repeat sequences and ensure the stability of the RNA secondary structure. The splicing site of intronic attribute was removed, the glucose oxidase gene was optimized, and two sequences were optimized according to different strategies, as shown in SEQ ID No.2 and SEQ ID No.3 respectively.

[0025] The optimized glucose oxidase gene was synthesized, and Nanjing Nuoweizan was used according to the principle of homologous recombination The Ultra One Step Cloning Kit product homologously recombin...

Embodiment 2

[0026] Embodiment 2, glucose oxidase gene transformation Escherichia coli enrichment plasmid

[0027] The original glucose oxidase gene (SEQ ID No.1) and the codon-optimized glucose oxidase gene (SEQ ID No.2, SEQ ID No.3) were mixed with the pPIC9K homologous recombination product and E.coli DH5α respectively, After heat shock for 90s, spread on 100ug / mL ampicillin-resistant LB agar culture plate and culture overnight at 37°C. Pick a single colony, then extract the plasmid for electrophoresis detection, and store the plasmid at -20°C. Then use EcoRI and NotI enzyme digestion to detect the target fragment, and then send the bacterial suspension to the company for sequencing, and transform the correctly sequenced plasmid into Escherichia coli in the same way to achieve plasmid enrichment.

Embodiment 3

[0028] Embodiment 3, glucose oxidase gene transformation Pichia host bacterium

[0029] Inoculate a single colony of Pichia pastoris GS115 into a test tube containing 5mL of LYPD liquid medium, and culture overnight at 30°C. Transfer 1% of the inoculum to a Erlenmeyer flask containing 50mL of YPD liquid medium, culture overnight at 30°C until OD600=1.3-1.5; centrifuge the culture medium at 1500g at 4°C for 5min, discard the supernatant, and use a 50mL ice bath Resuspend the cells in double-distilled water; centrifuge the culture medium at 1500g at 4°C for 5 minutes, discard the supernatant, and resuspend the cells in 25 mL ice-bathed double-distilled water; centrifuge the culture medium at 1500g at 4°C for 5 minutes, discard the supernatant, Resuspend the cells with 2 mL of 1 M sorbitol solution in ice bath; centrifuge the culture medium at 1500 g for 5 min at 4 °C, discard the supernatant, and resuspend the cells with 1 mL of 1 M sorbitol solution in ice bath to make the bact...

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Abstract

The present invention provides a codon-optimized glucose oxidase gene, which is derived from AspergillusnigerAn76, and its sequence is shown in SEQ ID No.2. The original glucose oxidase gene cannot be successfully expressed in Pichia pastoris. After sequence optimization, it can be successfully expressed in Pichia pastoris, and after separation and purification, a purified target protein can be obtained. The recombinant glucose oxidase can be used in glucose biosensors.

Description

technical field [0001] The invention relates to the technical fields of bioenzyme genetic engineering and biosensing, in particular to a codon-optimized glucose oxidase gene and its application in preparing biosensing elements. Background technique [0002] At present, the total amount of bio-fermentation products in my country ranks first in the world, and glucose is an important quality control indicator of fermented products. Rapid and accurate detection of glucose content in fermented products is of great significance to ensure the quality of fermented products. Traditional detection methods for glucose in fermented products mainly include high-performance liquid chromatography (HPLC), gas chromatography (GC), optical rotation and thin-layer chromatography, but these detection methods face various problems such as high analysis cost and cumbersome operation. The biosensor method has the characteristics of high sensitivity, good reproducibility, and simple operation, and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/81C12N1/19C12N9/04C12Q1/00C12R1/84
CPCC12N9/0006C12N15/815C12N2800/22C12Q1/006C12Y101/03004G01N2333/904
Inventor 公维丽马耀宏史建国韩庆晔王丙莲刘庆艾郑岚杨俊慧蔡雷
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI
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