Silurus asotus growth hormone (saGH)-cell penetrating peptide trans-activating transcriptional activator (TAT) fusion protein and its preparation method and use

A fusion protein, Escherichia coli technology, applied in biochemical equipment and methods, microorganism-based methods, applications, etc., to achieve the effect of large expression, low cost, and promoting fish growth

Active Publication Date: 2013-01-30
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the application of TAT membrane-penetrating peptide has been very extensive, there is no report at home and abroad on the use of TAT to carry fish auxin across the intestinal membrane and significantly increase fish production

Method used

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  • Silurus asotus growth hormone (saGH)-cell penetrating peptide trans-activating transcriptional activator (TAT) fusion protein and its preparation method and use
  • Silurus asotus growth hormone (saGH)-cell penetrating peptide trans-activating transcriptional activator (TAT) fusion protein and its preparation method and use
  • Silurus asotus growth hormone (saGH)-cell penetrating peptide trans-activating transcriptional activator (TAT) fusion protein and its preparation method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Embodiment 1: prepare catfish growth hormone gene, its steps are:

[0080] 1.1 Extraction of RNA from catfish pituitary gland

[0081] Follow the operation of the TIANGEN RNA extraction kit with minor changes, the specific steps are as follows:

[0082] Sampling: Cut open the fish skull, take out the pituitary gland with tweezers, weighing about 100mg;

[0083] Put the pituitary gland into an Eppendorf tube with 1ml of TRNzol, and mash it with a homogenizer;

[0084] Place the homogenized sample at 20°C for 5 minutes to completely separate the nucleic acid-protein complex;

[0085] Centrifuge at 12000rpm at 4°C for 10min, and take the supernatant;

[0086] Add 0.2ml of chloroform, cover the tube cap, shake vigorously for 15 seconds, and place at room temperature (20-25°C, the same below) for 3 minutes;

[0087] Centrifuge at 12000rpm at 4°C for 15min, the sample will be divided into three layers: yellow organic phase, middle layer and upper colorless aqueous phase, ...

Embodiment 2

[0171] Example 2: PCR amplification of saGH / saGH-TAT gene fragment and construction of expression vector

[0172] Primers were designed according to the sequence of saGH (GenBank: AY157496.1):

[0173] Upstream primer: 5'GGACTG CATATG TTCGAGAACCAGCGTCTCT 3' (the underline is the Nde I restriction site,)

[0174] Downstream primer: 5′CAT CTCGAG CAGGGTGCAGTTGGAATCC 3' (the underline is the Xho I restriction site)

[0175] In addition, according to the base sequence of 11 amino acids in TAT PTD, it was connected to the saGH fragment by PCR extension method, and the primers were designed as follows:

[0176] Upstream primer: 5'-GGACTGCATATGTTCGAGAACCAGCGTCTCT-3'

[0177] Downstream primer 1: 5'-TTTCTTACGGCCATACAGGGTGCAG-3'

[0178] Downstream primer 2: 5'-ACGACGCTGACGACGTTTCTTACGG-3'

[0179] Downstream primer 3: 5'-CATCTCGAGACGACGACGCTGACGACGT-3'

[0180] The above primers were synthesized by Sunny Company.

[0181] Using the extracted pMD18T-saGH as a template, a 50 μl...

Embodiment 3

[0195] Example 3: Construction of genetically engineered bacteria and induced expression and purification of fusion proteins

[0196] The transformation method is the same as the JM109 transformation method.

[0197] 1) Pick a single colony transformed with the plasmid, inoculate it in 5 ml of selective LB liquid medium, and cultivate overnight at 37° C. with shaking at 250 rpm / min.

[0198] 2) The next day, re-inoculate 200 μl of the cultured bacteria solution overnight into 20ml (1:100) selective 2×YT liquid medium (17g peptone, 10g yeast extract, 5g NaCl, add water to 1000ml), 37°C, 250rpm / min shaking culture to optical density (OD 600 =0.6), take a 1ml sample as the pre-induction sample, centrifuge at 10000g for 1min to collect the bacterial pellet, and freeze it at -20°C for later use.

[0199] 3) Add 1mol / L IPTG to the bacterial solution to make the final concentration of IPTG 1mM, 37°C, 250rpm / min

[0200] 4) Culture with shaking for 4 hours. Take a 1ml sample as t...

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Abstract

The invention discloses a silurus asotus growth hormone (saGH)-cell penetrating peptide trans-activating transcriptional activator (TAT) fusion protein and its preparation method and use. The saGH-TAT fusion protein can be used for promoting bony fish growth and resisting osmotic pressure. The preparation method of the saGH-TAT fusion protein comprises the following steps of extracting RNA from the pituitary gland of a live silurus asotusm, carrying out a reverse transcription-polymerase chain reaction (RT-PCR) process to obtain a cDNA fragment containing a GH gene mature peptide encoding segment, connecting GH and TAT, introducing the complex of GH and TAT into an expression vector pET22b (+), and carrying out expression in an escherichia coli expression strain BL21(DE3) to obtain the purified saGH-TAT fusion protein. Through a feeding, injection or immersion method, the saGH-TAT fusion protein can improve a fish growth rate to different degrees and improve strong saline water tolerance. Compared with a single GH expression protein used for fishes, the saGH-TAT fusion protein has better effects.

Description

technical field [0001] The present invention relates to the technical field of genetic bioengineering, in particular to a saGH-TAT recombinant fusion protein, a method for preparing the saGH-TAT recombinant fusion protein, and a genetically engineered bacterium expressing the saGH-TAT recombinant fusion protein. It also relates to the application of a saGH-TAT recombinant fusion protein in promoting fish growth. Background technique [0002] Growth hormone (Growth Hormone, GH) is a class of single-chain peptide hormones secreted by pituitary cells, and its essence is protein. GH widely exists in vertebrates (Mammalia, Aves, Reptiles, Fishes, etc.), and plays an important role in many important endocrine regulation activities, especially in promoting the rapid growth of fish. Therefore, it has been a research hotspot since its discovery. [0003] A large amount of growth hormone can be obtained by fermentation of recombinant growth hormone genetically engineered bacteria. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N1/21C12N15/70A23K1/165C12R1/19
Inventor 孟小林徐进平王健于京佑
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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