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Monopterus albus AKR (aldo-keto reductase) gene and in-vitro expression method thereof

A technique for expressing and reducing enzymes in vitro, applied in the field of genetic engineering

Active Publication Date: 2017-03-08
YANGTZE UNIVERSITY
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  • Abstract
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Problems solved by technology

In addition, there is no report on the AKR gene and its function in other fishes at home and abroad.

Method used

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  • Monopterus albus AKR (aldo-keto reductase) gene and in-vitro expression method thereof
  • Monopterus albus AKR (aldo-keto reductase) gene and in-vitro expression method thereof
  • Monopterus albus AKR (aldo-keto reductase) gene and in-vitro expression method thereof

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Embodiment Construction

[0024] Embodiments of the present invention are described in detail below. The embodiments described below by referring to the figures are exemplary only for explaining the present invention and should not be construed as limiting the present invention.

[0025] For the embodiment of the present invention, an in vitro expression method of aldehyde and ketone reductase gene of rice field eel is provided, which comprises the following steps:

[0026] The cloning of step 1 field eel Eakr gene:

[0027] Total RNA was extracted from rice field eel according to the instructions of Trizol reagent, cDNA was synthesized by M-MLV reverse transcriptase, the first strand of cDNA was synthesized according to the instructions of Smart RACE kit, and stored at -80°C. According to the homologous comparison of the AKR gene sequence reported in the GenBank database, the specific primer AKR-F (5'-ACATCCAGACCAGCGACCTGCA-3') and the primer oligod (T) (5'-TGCCGAATCTTTTTTTTTTTTAGC-3') were designed....

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Abstract

The invention relates to the field of genetic engineering, in particular to a monopterus albus AKR (aldo-keto reductase) gene with nucleotide and amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2. A recombinant expression vector of a monopterus albus Eakr gene is constructed with methods in molecular biology and genetic engineering, induction expression and affinity chromatography are performed, and purified recombinant expression protein is obtained. Monopterus albus Eakr recombinant protein can catalyze different substrates such as methylglyoxal, acetone, progesterone, methyltestosterone and the like and has theoretical guidance significance and application reference value for industrial application of AKR. Besides, the invention further provides an in-vitro expression method of the monopterus albus AKR gene.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a rice field eel aldehyde and ketone reductase gene and an in vitro expression method thereof. Background technique [0002] Aldo-ketone reductase (Aldo-keto reductase, AKR) widely exists in the biological world, and is a kind of monomeric reductase protein with a molecular weight of 34kDa-37kDa that depends on NAD(P)H. [0003] The currently reported AKR superfamily members have 16 subfamilies with a total of more than 190 members, including aldose reductase, ketoreductase, hydroxylated steroid dehydrogenase, 2,5-diketo-D-gluconate reductase, Polyol dehydrogenase, dihydrodiol dehydrogenase and many other enzymes that can metabolize carbonyl compounds in the body. [0004] Structurally, the AKR superfamily contains a (α / β) 8 The barrel structure, similar coenzyme binding domain and a highly conserved Asp, Tyr, Lys, His catalytic tetrad. Functionally, AKR not only ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/70
CPCC12N9/0008
Inventor 李伟孙文秀
Owner YANGTZE UNIVERSITY
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