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A codon-optimized fad-glucose dehydrogenase gene and its application

A technology of glucose dehydrogenase and codon optimization, which is applied in the fields of bioenzyme genetic engineering and biosensing, can solve problems such as difficult recombination and expression, and achieve the effect of avoiding inverted repeat sequences

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, at present, most of GDH-FAD is mainly isolated and purified from wild strains, and it is not easy to carry out recombinant expression, which also leads to less research on GDH-FAD as an enzyme electrode biosensing element, especially in China, there are only a few studies on GDH-FAD recombination Therefore, it is of great significance to study the high-efficiency recombinant expression of GDH-FAD and its application in the field of sensors

Method used

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  • A codon-optimized fad-glucose dehydrogenase gene and its application
  • A codon-optimized fad-glucose dehydrogenase gene and its application
  • A codon-optimized fad-glucose dehydrogenase gene and its application

Examples

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Effect test

Embodiment 1

[0023] Example 1, FAD-glucose dehydrogenase gene codon optimization and cloning

[0024] In this embodiment, the FAD-glucose dehydrogenase g5086.t1 gene in the genome of Aspergillus niger An76 is used as the original gene, and the original gene sequence is shown in SEQ ID No.1. According to the difference in frequency of use of different codons encoding the same amino acid in Pichia pastoris, the rare codons in the original gene of FAD-glucose dehydrogenase g5086.t1 were removed to avoid the occurrence of inverted repeat sequences and ensure the stability of RNA According to different strategies, two sequences were optimized, as shown in SEQ ID No.2 and SEQ ID No.3 respectively.

[0025] The optimized FAD-glucose dehydrogenase gene was synthesized, and Nanjing Nuoweizan was used according to the principle of homologous recombination The Ultra One Step Cloning Kit product homologously recombines the codon-optimized FAD-glucose dehydrogenase gene into the pPIC9K plasmid vector...

Embodiment 2

[0026] Embodiment 2, FAD-glucose dehydrogenase gene transformation Escherichia coli enrichment plasmid

[0027] The original FAD-glucose dehydrogenase gene (SEQ ID No.1) and the codon-optimized FAD-glucose dehydrogenase gene (SEQ ID No.2, SEQ ID No.3) and the pPIC9K homologous recombination product were combined with E.coli DH5α was mixed, heat-shocked for 90s, spread on 100ug / mL ampicillin-resistant LB agar culture plate, and cultivated 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, FAD-glucose dehydrogenase 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...

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Abstract

The present invention provides a codon-optimized FAD-glucose dehydrogenase gene derived from Aspergillus niger An76, the sequence of which is shown in SEQ ID No.2. The original FAD-glucose dehydrogenase gene cannot be successfully expressed in Pichia pastoris, but 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 FAD - Glucose dehydrogenase 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 FAD-glucose dehydrogenase gene and its application in preparing biosensing elements. Background technique [0002] Accurate and rapid monitoring of glucose is of great significance in medicine, food and other industries. For this reason, biosensors have become an ideal method for glucose monitoring because of their high specificity, short analysis time, in-situ monitoring, and low manufacturing cost. Despite these advantages, biosensors still need to overcome the problems caused by the enzymatic molecular sensing elements used for detection. [0003] In biosensors with enzymes as sensing elements, oxidoreductases are the main biorecognition elements, among which glucose oxidase (GOX) is the most commonly used enzyme, and GOX can selectively oxidize glucose into Glucolactone, while producing H 2 o 2 . However, the respons...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/81C12N1/21C12N1/19C12N9/04C12Q1/00G01N27/48G01N27/327G01N27/30C12R1/84C12R1/19
CPCC12N9/0006C12N15/815C12N2800/22C12Q1/001C12Y101/9901G01N27/308G01N27/3272G01N27/3277G01N27/48
Inventor 公维丽马耀宏孟庆军王丙莲史建国蔡雷刘庆艾杨艳
Owner BIOLOGY INST OF SHANDONG ACAD OF SCI
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