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Epinephelus coioides insulin gene, coded protein and application thereof

A pet22b-insulin and oblique grouper technology, applied in the field of genetic engineering, can solve the problems of lower production cost and high cost of animal and plant proteins, and achieve the effect of reducing usage and production cost

Active Publication Date: 2018-04-20
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the feed for grouper still uses animal and plant protein as the main additive. However, the cost of animal and plant protein is relatively high. If the utilization of glycolipids by grouper can be improved, the production cost will be greatly reduced.
However, there is no report on the use of insulin protein as a bait additive or growth-promoting agent to effectively promote the utilization of glycolipids by the grouper in artificial culture of grouper.

Method used

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  • Epinephelus coioides insulin gene, coded protein and application thereof
  • Epinephelus coioides insulin gene, coded protein and application thereof
  • Epinephelus coioides insulin gene, coded protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] banded grouper insulin The cloning of gene comprises the following steps:

[0035] 1, insulin Cloning of mid-gene fragments

[0036] For the reported Nile tilapia ( Oreochromis niloticus ),carp( Cyprinus carpio ), redfin puffer ( takifugu rubripes ), medaka ( oryzias latipes ) obtained in insulin Sequence comparison, finding relatively conserved regions, and designing and cloning the oblique-banded grouper insulin A pair of degenerate primers for the middle fragment of the sequence, the upstream primer (SEQ ID NO:3) is 5'-GGHTTCTTCTAYAVCCCCARGAG-3', the downstream primer (SEQ ID NO:4) is 5'-GTTRCAGTAGTTTCTSSAGSTCRA-3'; The cDNA after reverse transcription of the grouper brucella RNA was used as a template for PCR. The PCR conditions were: 94°C for 3min; 35 cycles of 94°C for 30s, 56°C for 30s, and 72°C for 30s; 72°C for 10min; 4°C ∞. The gel electrophoresis image after the PCR reaction is shown in figure 1 shown.

[0037] 2, insulin Cloning of genes 5'R...

Embodiment 2

[0052] Plaque-banded grouper insulin Escherichia coli expression vector pET22b -insulin The construction steps are as follows:

[0053] 1. Grouper oblique banded with enzyme cleavage sites insulin gene synthesis

[0054] According to the above cloned grouper insulin The sequence of the gene, the recognition sequence of the restriction endonucleases EcoRI and NotI and the protection bases were designed and synthesized. A pair of specific primers were designed and synthesized. insulin EcoRI recognition site and 6×HIS tag were added before the mature peptide sequence of the gene; the downstream primer R (SEQ ID NO:14) was 5’-ATAGTTTAGCGGCCGCTCAGTTGCAGTAGTTCTG-3’, which was in insulin A stop codon and a NotI digestion recognition site were added after the mature peptide sequence of the gene. Using the PCR2.1 constructed in Example 1- insulin A PCR reaction was performed for the template, and the PCR reaction conditions were: 94°C for 3 minutes; 35 cycles of 94°C for 30s, 56...

Embodiment 3

[0057] Escherichia coli recombinant strain pET22b- insulin -The construction of BL21, the steps are as follows:

[0058] The prepared recombinant plasmid pET22b- insulin Transform Escherichia coli BL21 competent cells into Escherichia coli BL21 competent cells by standard calcium chloride transformation method, and culture them on LB plates containing Ampicillin at 37°C. After overnight, a single clone was grown, and the single clone was picked, induced, cultured and identified to screen out the Escherichia coli recombinant strain pET22b- insulin -BL21.

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular discloses an epinephelus coioides insulin gene, coded protein and an application thereof. The nucleotide sequenceof the insulin protein is shown as SEQ ID NO:1, and the amino acid sequence of the insulin protein coded by the gene is shown as SEQ ID NO.2. The epinephelus coioides insulin gene provided by the invention is obtained for the first time through cloning, and the gene is applied to construction of an expression vector and a recombinant escherichia coli strain; the recombinant epinephelus coioides insulin protein, which is high in expression, is obtained on the basis of the recombinant escherichia coli strain in vitro; and the protein, which can promote glycolipid utilization and growth of suchmarine fishes as epinephelus coioides and the like, can be used for preparing growth promoters or bait additives applicable to the marine fishes; therefore, the protein has a relatively broad application prospect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a kind of insulin Genes, encoded proteins and their applications. Background technique [0002] Insulin plays an important regulatory role in the metabolic regulation of the body. The pancreas has always been considered as an important insulin-producing organ, but recent studies have shown that other organs besides the pancreas can also secrete insulin. As an important hormone, insulin plays an important role in the growth, cell differentiation and metabolism of the body. As one of the earliest discovered insulins, fish insulin plays an important role in many aspects such as fish feeding, growth, development and regulation of glucose and lipid metabolism. After insulin binds to the receptor, it can trigger a variety of downstream biological functions through PI3K / Akt, ERK and STAT / Jak–STAT signaling pathways. [0003] Grouper (grouper) Serranidae ), Grouper ( E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/17C07K14/62C12N15/70C12N1/21A61K38/28A61P3/02A23K20/184A23K50/80C12R1/19
CPCA61K38/00A23K20/184A23K50/80C07K14/62C12N15/70
Inventor 李文笙杨国坤
Owner SUN YAT SEN UNIV
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