A kind of preparation method of polyclonal antibody of rice field eel aldehyde and ketone reductase

A technology of polyclonal antibody, rice field eel aldehyde and ketone, applied in the field of genetic engineering

Active Publication Date: 2019-06-28
YANGTZE UNIVERSITY
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  • Abstract
  • Description
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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.

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  • A kind of preparation method of polyclonal antibody of rice field eel aldehyde and ketone reductase
  • A kind of preparation method of polyclonal antibody of rice field eel aldehyde and ketone reductase
  • A kind of preparation method of polyclonal antibody of rice field eel aldehyde and ketone reductase

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Embodiment Construction

[0037] 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.

[0038] The embodiment of the present invention provides a method for preparing a rice field eel Eakr polyclonal antibody, comprising the following steps:

[0039] The cloning of step 1 field eel Eakr gene:

[0040]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 homology 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. Using the first strand of the eel c...

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Abstract

The invention discloses a preparation method of a ricefield eel aldo-keto reductase polyclonal antibody. The preparation method comprises the following steps of: (1) cloning of ricefield eel Eakr gene; (2) establishment of a recombinant expression vector of the ricefield eel Eakr gene; (3) inducible expression and purification of recombinant protein of ricefield eel Eakr; and (4) preparation and Western blot analysis of polyclonal antibody. In the invention, a recombinant expression vector of ricefield eel Eakr gene is established by molecular biology and genetic engineering to induce expression, and purified recombinant expression protein is obtained through affinity chromatography. The ricefield eel Eakr polyclonal antibody prepared by the method disclosed by the invention can meet the experimental requirements of immunohistochemistry and Western blot, realizes in-vitro immunoassay, studies the functions of ricefield eel Eakr as well as ricefield eel immunofluorescent staining laser confocal microscopic observation and also can be applied to the immunodetection of aldo-keto reductase of grass carp, pseudobagrus fulvidraco, megalobrama amblycephala, snakehead and crucian.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for preparing polyclonal antibody of rice field eel aldehyde and ketone reductase. 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 between 34kDa and 37kDa depending on NAD(P)H. [0003] The 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 plays ...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/40C07K16/06C12N9/02C12N15/53C12N15/70
CPCC07K16/40C07K2317/10C12N9/0008
Inventor 李伟孙文秀
Owner YANGTZE UNIVERSITY
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