Recombinant protein based on fugu rubripes FoxO1 gene

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The invention discloses a recombinant protein based on a fugu rubripes FoxO1 gene as well as a preparation method and an application thereof, belonging to the biochemical technical field. According to a cDNA coding sequence of fugu rubripes FoxO1 protein, a primer pair for the coding sequence of the fugu rubripes FoxO1 protein is designed; and RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification is carried out after extracting total RNA (Ribonucleic Acid) of the liver by an ISOGEN reagent. An RT-PCR product is connected with a p GEM-T vector to obtain a recombinant plasmid; and a target gene after dual enzyme digestion is connected with an expression vector p ET-32a (+) to obtain a recombinant expression plasmid which is transferred into escherichia coli Rosetta (DE3); and soluble expression of the recombinant protein is realized by IPGT (Isopropyl-beta-d-Thiogalactoside) induction. The fugu rubripes FoxO1 recombinant protein disclosed by the invention can restrain expression of a peroxysome peroxisome proliferator-activated receptor PPAR gamma gene, and can regulate metabolic balance of lipids in the fugu rubripes. The purified recombinant product caries out living body injection or feed addition onto fishes, and can improve growth of bred fishes and can lower lipid content of fishes, so that the fishes are delicious and tasty in meat quality.

The object of the present invention is to provide a ravioli FoxOl based rubripes Recombinant protein.

Also, the present invention also provides a method for preparing a recombinant protein based rubripes ravioli FoxOl gene.

Finally, the present invention also provides a method based on recombinant protein rubripes ravioli FoxOl gene for the preparation of adjustment rubripes ravioli adipose tissue expression of PPAR Y amount of a pharmaceutical composition or feed.

To achieve the above objective, technical solution adopted by the invention are:

A recombinant protein-based rubripes ravioli FoxOl gene, which contains as N0 SEQ ID: the amino acid sequence shown.

wherein the amino acid sequence as SEQ ID NO: 3 encoding nucleotide sequence shown. [0011] A method for preparing a recombinant protein based rubripes ravioli FoxOl gene, comprising the steps of:

(I) by reverse transcription PCR (RT-PCR) amplification of total RNA rubripes ravioli adipose tissue, amplified product pGEM_T vector to obtain the recombinant plasmid, the target gene by double digestion and then with the pET-32a ( +) expression vector to obtain a recombinant plasmid;

(2) The recombinant plasmids were transformed into host bacteria, IPTG was added to induce the expression of recombinant proteins FoxOl.

The reverse transcription PCR amplification primer pair P is:

h tour primer Pl: 5 '-CGGAATTCATGGCGGAAGCAGCCCA-3' (see SEQ ID NO: 1),

downstream primer P2: 5 '-CCGCTCGAGCCCTGACACCCAGCTGTGC-3' (see SEQ ID NO: 2).

The step (1), reverse transcription PCR amplification reaction conditions: 94 ° C denaturation 40s carried out after 94 ° C denaturation for 5min, 61 ° C annealing 50s, 72 ° C extended 2min of 35 amplification cycles, 72 ° C after extending IOmin stored at 4 ° C.

A recombinant protein-based rubripes ravioli FoxOl genes regulate PPARy in the preparation of the expression of adipose tissue rubripes ravioli pharmaceutical composition or feed.

The present invention beneficial effects:

According to the present invention, cDNA coding sequence rubripes ravioli FoxOl protein designed for primer rubripes ravioli FoxOl protein-coding sequences is to extract ISOGEN Reagent red fins liver after oriental ravioli total RNA RT-PCR amplification. RT-PCR products and pGEM-T vector to obtain the recombinant plasmid by double digestion and then with the target gene expression vector pET-32a (+) connection, eligible recombinant plasmid was transformed into E. coli Rosetta (DE3); and by IPTG Induction in soluble recombinant protein expression. The fusion protein expression plasmid with a histidine tag to facilitate the identification and purification of the sample, avoiding denaturation and refolding samples, not only easy to operate, reduce costs, more important is the protein structure from destruction, restructuring Protein purity, high yield and resistant to degradation, adapted to industrial production, lay the foundation for further research rubripes ravioli FoxOl protein.

The present invention rubripes ravioli FoxOl recombinant protein can inhibit the peroxisome proliferator-activated receptor PPARy gene expression, regulation of rubripes ravioli on lipid metabolism in balance. Specifically, there are two advantages: (1) The main part of the fish lipid deposition in the liver and muscles, but also has a very distinct difference with these two organizations proportions of lipid species , FoxOl recombinant protein can inhibit peroxisome proliferator-activated receptor PPAR Y gene expression, in order to maintain the balance of farmed fish fat metabolism; (2) the regulation of the present invention rubripes ravioli FoxOl fish lipid metabolism regulating gene expression of recombinant protein can be applied in fish farming, fish reduce fat deposition, improve meat quality, with significant economic value.