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The application of orexin‑a in the grouper orexin-a in the regulation of glucose metabolism in the grouper

A technology of slanted grouper and grouper candy, which is applied in the field of genetic engineering and can solve problems such as lack of grouper

Active Publication Date: 2017-02-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved by the present invention is to overcome the lack of bait additives that effectively promote the growth of grouper seedlings in the prior art, and to provide the application of the orexin-A gene of the grouper in regulating the sugar metabolism of the grouper

Method used

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  • The application of orexin‑a in the grouper orexin-a in the regulation of glucose metabolism in the grouper
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  • The application of orexin‑a in the grouper orexin-a in the regulation of glucose metabolism in the grouper

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Embodiment 1

[0032] Example 1: Synthesis of the orexin-A gene of the oblique-banded grouper and construction of an expression vector

[0033] According to the published cDNA sequence of grouper precursor orexin, using the complete cDNA sequence of grouper hypothalamus as a template for primer design, a pair of specific primers were designed and synthesized and protective bases were added. The upstream primer F The sequence is: 5'-CGGGATCCGCACAGCGTGTCTGAGTGC-3', and the sequence of the downstream primer R is: 5'-CCCAAGCTTCAGGGTGAGAATGCCAGC-3'. The PCR reaction is based on the cDNA obtained by reverse transcription of the total RNA of the hypothalamus of the oblique-banded grouper as a template, and the orexin-A sequence is amplified by PCR. The reaction conditions are: 95°C pre-denaturation for 5 minutes; Denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 15 seconds, a total of 40 cycles; see the electrophoresis identification diagram of PCR amplifi...

Embodiment 2

[0036] Construction of Escherichia coli Recombinant Strain pET22b-orexin-A-Origami Highly Expressing Grouper orexin-A

[0037] CaCl 2 Transform pET22b-orexin-A into Origami 2(DE3) by using the method, screen the transformants on LB plates containing ampicillin, streptomycin and tetracycline, and obtain recombinant transformants containing pET22b-orexin-A through plasmid detection and enzyme digestion analysis pET22b-orexin-A-Origami.

Embodiment 3

[0039] Utilize Escherichia coli genetically engineered bacteria pET22b-orexin-A-Origami to produce recombinant grouper orexin-A: Pick a single colony of Escherichia coli genetically engineered bacteria pET22b-orexin-A-Origami and inoculate it in a mixture containing 100ug / ml ampicillin, 50ug / ml streptomycin, 25ug / ml tetracycline LB liquid medium, 37 ℃, 200 rev / min culture overnight, inoculate in the same medium of appropriate volume according to 1:50 inoculum the next day, 37 ℃ culture to A 600 0.5-0.6, add IPTG (final concentration: 0.8mmol / l) and 20% glucose (final concentration: 0.2%), induce culture at 37°C for 6 hours, and harvest the bacteria. Synthetic orexin-A in the embryonic stage of grouper has been highly expressed in Escherichia coli genetically engineered strain pET22b-orexin-A-Origami.

[0040] Take a small amount of cells and add 2× electrophoresis loading buffer, boil for 5 minutes and run SDS-PAGE gel electrophoresis according to the standard method. see re...

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Abstract

The invention discloses an application of epinephelus daemelii orexin-A in regulation of glycometabolism of groupers. A research result proves that an orexin-A gene has a regulating function on the glycometabolism of the groupers. On the basis of finding the glycometabolism function, a large amount of recombinant epinephelus daemelii orexin-A in a crude extract grade is obtained through an Escherichia coli prokaryotic expression system and is added to a feed of the groupers for carrying out a physiological experiment to research an influence of the orexin-A on growth of the groupers. By means of the application, epinephelus daemelii orexin-A recombinant proteins, which are stable in sources and low in cost, can be obtained and further a fish growth promoting agent or an additive, which are suitable for marine fishes, can be prepared.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the application of orexin-A of the clotted grouper in regulating the sugar metabolism of the grouper. The specific application is that the grouper orexin-A is used to prepare a growth-promoting agent for fish fry or a feed additive. Background technique [0002] orexin-A (also known as orexin) is a protein composed of 43 amino acids. Orexin is a new type of hypothalamic neuropeptide discovered in mammals in 1998. It is divided into orexin-A and orexin-B, which are derived from the same orexin precursor messenger RNA after different splicing and translation. Orexin-A of the oblique-banded grouper contains a pair of disulfide bonds, composed of cysteines at the 7th and 14th positions, and the protein size is about 4.7Kda. Its sequence and structure are similar to mammalian orexin-A, and mammalian Animal orexin-A is composed of 33 amino acids and two pairs ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/46A23K20/147A23K50/80
CPCA61K38/22
Inventor 李文笙张聪
Owner SUN YAT SEN UNIV
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