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Oral recombinant protein TAT-GH for promoting ricefield eel growth, and preparation method and application thereof

A technology of TAT-GH and eel, applied in the application of oral recombinant protein in promoting the growth of eel, the genetic engineering strain of Escherichia coli expressing oral recombinant protein, and the preparation of oral recombinant protein, which can solve the problem that the detailed mechanism is not very clear, etc.

Active Publication Date: 2013-06-05
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This domain has been proven to be able to transduce foreign fusion proteins into eukaryotic cells in a fast and efficient manner, which is independent of cell membranes and transporters, but the detailed mechanism is still not very clear

Method used

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  • Oral recombinant protein TAT-GH for promoting ricefield eel growth, and preparation method and application thereof
  • Oral recombinant protein TAT-GH for promoting ricefield eel growth, and preparation method and application thereof
  • Oral recombinant protein TAT-GH for promoting ricefield eel growth, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Preparation of rice field eel GH gene

[0069] Artificially synthesized rice field eel GH gene: artificially synthesized rice field eel GH gene according to the sequence of GH (GeneBank: AY265351.1) published in GeneBank, and designed PCR primers. GH upstream primer P1: 5'-GGC GGATCC ATGGACAAAGTCGTCCTCCTG-3' (the underline is the BamHI site), which is the downstream primer P2 of SEQ ID NO.3, GH: 5'-GCC GAATT CCTACAGAGTGCAGTTAGCTTCTGG-3' (the underline is the EcoRI site), which is SEQ ID NO.4. The target gene GH was amplified by PCR using the artificially synthesized eel GH gene as a template. The PCR reaction conditions for the GH gene were: ① 94°C for 2 minutes, ② (94°C for 30 seconds → 56°C for 40 seconds → 68°C for 40 seconds) for a total of 32 cycles, and ③ 68°C for 5 minutes. Store the reacted product at -20°C for future use.

[0070] PCR reaction system:

[0071]

Embodiment 2

[0072] Embodiment 2: Preparation of fusion gene TAT-GH

[0073] PCR amplification of TAT-GH gene: TAT-GH was synthesized by PCR overlapping extension technique in 3 PCRs. That is, the TAT sequence is added to the front end of the GH sequence by a bridge method. Using the artificially synthesized eel GH gene as a template, the upstream primer P3: 5'-CAGCGTCGTCGTGGATCCATGGACAAAGTCGTCC-3; that is, SEQ ID NO.5, and the downstream primer P4: 5'-GCC GAATT CCTACAGAGTGCAGTTAGCTTCTGG-3' (the underline is the EcoRI site), which is SEQ ID NO.6, the annealing temperature is 55°C, the target fragment is amplified by PCR, electrophoresis and recovered; the product recovered in the previous step is used as a template, and the upstream primer P5 : 5'-CGTAAAAAACGTCGTCAGCGTCGTCGTGGATCC-3' is SEQ ID NO.7, downstream primer P4: 5'-GCCGAATTCCTACAGAGTGCAGTTAGCTTCTGG-3; is SEQ ID NO.8, annealing temperature 55°C for PCR, electrophoresis and recovery; recovery in the previous step The product is u...

Embodiment 3

[0074] Example 3: Recovery and purification of PCR products.

[0075] 1) Mix the above PCR product with 6×Loading Buffer, and electrophoresis on 1% (W / V) agarose gel.

[0076] 2) When the target DNA band is completely separated from others, quickly cut off the agarose gel containing the target DNA band under ultraviolet light (cut as small as possible), transfer it to a 1.5ml EP tube, and weigh it. Purify and recover the target band with TIANGEN Agarose Gel DNA Recovery Kit.

[0077] 3) Add 3 times the volume of sol solution to the gel block (the weight of the gel is 0.1g, its volume can be regarded as 100μL, and so on), cover the tube cap, and put it in a water bath at 55°C for 10 minutes, during which time it is constantly inverted and mixed until the gel is completely dissolved.

[0078] 4) Leave it for 2-3 minutes, wait for room temperature (20-25°C, the same below), add to the adsorption column, centrifuge at 12000rpm for 60s, and discard the waste liquid in the collect...

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Abstract

The invention discloses an oral recombinant protein TAT-GH for promoting ricefield eel growth, and a preparation method and application thereof. The preparation method comprises steps of: A. synthesizing a GH gene of ricefield eel according to a known sequence; B. amplifying a protein transduction domain polypeptide TAT through a PCR method, and fusing with the GH gene; C. fusing the gene and a plasmid by a double digestion TAT-GH, and carrying out gel extraction on fragments containing the TAT-GH gene and open-loop fragments to obtain a TAT-GH plasmid; D. transforming the plasmid to a competence BL21 (DE3) to obtain a positive clone which is an objective strain; and E. separating and purifying protein expressed by the objective strains. The purified TAT-GH recombinant protein can effectively promote the growth of ricefield eel by oral administration, and provides a basis for the preparation of TAT mediated oral recombinant protein.

Description

technical field [0001] The present invention relates to the technical field of genetic bioengineering, in particular to an oral recombinant protein that promotes the growth of rice field eel, a preparation method for an oral recombinant protein that promotes the growth of rice field eel, and a genetic engineering strain of Escherichia coli expressing oral recombinant protein , and also relates to the application of an oral recombinant protein in promoting the growth of rice field eel. technical background [0002] Yellow eel (Monopterus albus), commonly known as eel, field eel and long fish, is a freshwater bony fish widely distributed in China, India, Malaysia and Indonesia, and has been cultivated in a large area in central and southern China. China's current total aquaculture output has reached 180,000 tons. Due to its fast growth, high survival rate and good adaptability to cage culture conditions, it is regarded as one of the preferred species for aquaculture in China. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12R1/19
Inventor 孟小林徐进平王健周莉
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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