Recombinant oral protein TAT-GH of tilapia, preparation method for recombinant oral protein TAT-GH and application of recombinant oral protein TAT-GH

A TAT-GH, tilapia technology, applied in the field of genetic bioengineering, can solve problems to be studied, etc., and achieve the effects of simple operation, ensuring accuracy and integrity, and low cost

Active Publication Date: 2012-10-03
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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In conclusion, the specific mechanism of

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  • Recombinant oral protein TAT-GH of tilapia, preparation method for recombinant oral protein TAT-GH and application of recombinant oral protein TAT-GH
  • Recombinant oral protein TAT-GH of tilapia, preparation method for recombinant oral protein TAT-GH and application of recombinant oral protein TAT-GH
  • Recombinant oral protein TAT-GH of tilapia, preparation method for recombinant oral protein TAT-GH and application of recombinant oral protein TAT-GH

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The preparation method of tilapia TAT-GH growth hormone gene, its steps are:

[0057] (1) Extraction of total RNA from tilapia by Trizol method

[0058] The pituitary tissue of the killed tilapia was directly put into the research body, and an appropriate amount (about 3-5 times the volume of the pituitary tissue) was added to grind it with liquid nitrogen. Add Trizol at 50-100 mg / mL, and fully homogenize with a homogenizer for about 1-2 minutes. Place at room temperature (20-25°C, the same below) for 5 minutes to fully lyse. Centrifuge at 12000rpm for 10min, and take the supernatant. Add chloroform at 200 μL chloroform / mL Trizol, shake for 5 minutes, and place at room temperature for 5 minutes. Centrifuge at 12000g at 4°C for 15 minutes, absorb the upper aqueous phase into another centrifuge tube, add an equal volume of isopropanol to mix, and place at room temperature for 20-30 minutes. Centrifuge at 12000g for 10min at 4°C, discard the supernatant, and sink the R...

Embodiment 2

[0074] The construction method of TAT-GH recombinant vector, its steps are:

[0075] The PCR product of TAT-GH obtained in Example 1 was mixed with 10×Loading Buffer, and electrophoresed on a 1% (mass volume ratio, the same below) agarose gel. When the target DNA band is completely separated, quickly excise the agarose gel containing the target DNA band under the ultraviolet light, and transfer it to a 1.5ml EP tube. The target band was purified and recovered with the BIOMIGA Agarose Gel DNA Recovery Kit (purchased from Biomega). Add 1 times the volume of Buffer GC, heat in a water bath at 55-60°C for 10 minutes, and invert and mix 6-8 times until the gel block is completely dissolved. Cool the EP tube to room temperature, and transfer the above mixture (no more than 700 μl each time) to an adsorption column with a collection tube. Centrifuge at 13000×g for 1 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column back into the...

Embodiment 3

[0083] A preparation method for Escherichia coli genetically engineered strains, the steps of which are:

[0084] (1) Preparation of Escherichia coli competent cells by calcium chloride method:

[0085] A single colony of Escherichia coli BL21 (DE3) (purchased from Novagen and maintained in our laboratory) was picked from a fresh solid LB plate, inoculated in 20ml LB liquid medium, and cultured overnight at 37°C on a shaker at 300rpm. Take 200 μl of the overnight activated bacterial solution and transfer it to fresh 20ml LB liquid medium, culture at 37°C and 300rpm for about 2-3h, until OD 600 About 0.4-0.6. Draw the bacterial solution into a pre-cooled 1.5ml EP tube in the ultra-clean workbench, and place it on ice for 10 minutes. Centrifuge at 4000rpm for 10min at 4°C and discard the supernatant to collect the cells. If there are few bacteria, repeat the collection of bacteria. Use 200 μl ice-cold 0.1M CaCl per tube 2 The solution was resuspended. Centrifuge at 4000rpm...

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Abstract

The invention discloses recombinant oral protein TAT-GH of tilapia, a preparation method for the recombinant oral protein TAT-GH and application of the recombinant oral protein TAT-GH. The method comprises the following steps of: extracting total messenger ribonucleic acid (mRNA) from a pituitary gland of male tilapia, performing reverse transcription-polymerase chain reaction (RT-PCR) to obtain complementary deoxyribonucleic acid (cDNA), performing PCR amplification to obtain a growth hormone (GH) gene of the tilapia, and amplifying a TAT-GH gene by a PCR overlap extension method; constructing a recombinant plasmid pET-32a(+)-TAT-GH; and transforming Escherichia coli BL21 (DE3) by using the recombinant plasmid pET-32a(+)-TAT-GH, efficiently expressing fusion protein TAT-GH in the form of an inclusion body under the induction of isopropyl thiogalactoside (IPTG), and purifying and renaturing to obtain active protein. The protein can effectively promote the growth and development of fries of the tilapia after being fed to the fries of the tilapia.

Description

technical field [0001] The present invention relates to the technical field of genetic bioengineering, in particular to a recombinant oral auxin protein TAT-GH, a preparation method for a recombinant oral auxin protein TAT-GH, and a recombinant oral auxin protein TAT capable of expressing - a genetically engineered bacterial strain of GH; it also relates to the application of a recombinant oral auxin protein TAT-GH in promoting the growth of tilapia. technical background [0002] Fish growth hormone (GH) is a kind of single-chain polypeptide secreted and synthesized by eosinophilic cells in the distal pituitary gland of fish. It plays an important regulatory role in fish growth, energy fixation, and gonad development, and it is also an area that has attracted much attention from researchers in aquaculture. [0003] The separation of fish GH began in the 1970s and became a hot spot in the mid-to-late 1980s. Its initial separation technique is similar to the separation of ma...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N1/21C12N15/70C07K19/00A23K1/16C12R1/19
Inventor 孟小林徐进平王健曾辉
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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