Grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as preparation method and application thereof

A technology of artificial preparation and protein, applied in the direction of antiviral agents, biochemical equipment and methods, virus antigen components, etc., can solve the problem that the molecular mechanism is not fully studied, and achieve the prevention of grass carp hemorrhagic disease, rapid immune response, membrane penetration The effect of activity enhancement

Active Publication Date: 2015-07-01
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although there are many studies on the transmembrane function of the TAT protein transduction domain, the molecular mechanism of the TAT protein transduction domain entering the cell has not been fully studied so far.

Method used

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  • Grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as preparation method and application thereof
  • Grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as preparation method and application thereof
  • Grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The preparation of fusion gene TAT-VP7-TAT comprises the following steps:

[0044] The amino acid sequence of the mature GCRV VP7 protein was obtained from the gene bank of the National Center for Biotechnology (NCBI), and the sequence of the VP7 gene was converted into a nucleotide sequence containing E. The gene encoded by TAT and the foreign protein gene can be fused and expressed. The 5' and 3' ends of the optimized VP7 gene sequence are respectively connected with the nucleotide sequence of TAT, named: TAT-VP7-TAT, the sequence It is the nucleotide sequence shown in SEQ ID NO:1.

[0045] The TAT-VP7-TAT gene was artificially synthesized and connected to the pGEM-T vector, that is, the plasmid pGEM-T-TAT-VP7-TAT was synthesized.

[0046] The plasmid pGEM-T-TAT-VP7-TAT was transformed into Escherichia coli E.coli DH5α (purchased from Novagen and kept in our laboratory) to obtain strain E.coli DH5α (pGEM-T-TAT-VP7-TAT), For the value increase and preservation of gen...

Embodiment 2

[0056] Construction of expression plasmids.

[0057] In Example 1, the plasmid pGEM-T-TAT-VP7-TAT was digested by BamHI and HindIII, electrophoresed on agarose gel, and the band to be recovered was cut out quickly under ultraviolet light, and gelatinized with Kangwei Century agarose. Gel DNA recovery kit for purification, put a single target DNA band into a clean Eppendorf tube, and weigh it. Add three times the volume of sol solution PG to the gel block (the weight of the gel is 0.1g, its volume can be regarded as 100uL, and so on). Water bath at 60°C for 10 minutes, during which time the Eppendorf tube was gently turned up and down every 2 minutes to ensure that the gel was fully dissolved. Add 250 μl of Buffer PS to the adsorption column, let it stand for 2 minutes, then centrifuge at 12000 rpm for 2 minutes, and pour off the liquid in the collection tube. Take 750ul of the obtained solution and add it to an adsorption column (the adsorption column is placed in a collecti...

Embodiment 3

[0071] Construction of pET32a-TAT-VP7-TAT engineering bacteria.

[0072] Take 1 ul plasmid pET32a-TAT-VP7-TAT to transform Escherichia coli BL21(DE3) competent cells. Positive transformants were screened out through PCR identification, and the resulting positive clone was the recombinant genetically engineered strain Escherichia coli BL21pET32a-TAT-VP7-TAT capable of expressing TAT-VP7-TAT.

[0073] The strain was sent to the China Type Culture Collection Center for preservation on March 13, 2015, with a classification name: Escherichia coli BL21(DE3) / pET-32a-TAT-VP7-TAT, and a preservation number: CCTCC NO : M2015111, Address: Wuhan University, Wuhan, China.

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Abstract

The invention discloses a grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as a preparation method and application thereof. The amino acid sequence of the TAT-VP7-TAT protein is shown as SEQ ID NO:2, and a corresponding nucleotide sequence is shown as SEQ ID NO:1. By using Eschierichia coli BL21(DE3) / pET-32a-TAT-VP7-TAT to perform inducible expression for 4 to 5 hours, a TAT-VP7-TAT fusion protein can be efficiently expressed; the N end and the C end of the protein are respectively provided with protein transduction domain polypeptide TAT. The genetic engineering fusion protein provided by the invention can be used for preventing grass carp haemorrhagic virus diseases. The fusion protein has the advantages of high expression, easiness for industrial production, low cost and good safety; grass carp orally takes the fusion protein, so that better immune protection is realized.

Description

technical field [0001] The invention belongs to the technical field of genetic bioengineering, and specifically relates to an anti-grass carp haemorrhagic disease virus engineering protein TAT-VP7-T AT and its preparation method and application, and more specifically relates to a 5' and 3' terminal respectively linking protein transduction domain polypeptide The fusion protein gene TAT-VP7-TAT subunit vaccine of the gene sequence of (TAT-PTD) also involves a kind of E. The invention relates to a genetically engineered bacterial strain of hemorrhagic disease virus (GCRV) capsid protein VP7 fusion protein; it also relates to the use of the fusion protein expressed by the above bacterial strain in anti-grass carp hemorrhagic disease virus medicine. Background technique [0002] Grass carp (Ctenopharyngodon idellus) is the main freshwater cultured species in China and one of the "four major family carps". The feeding cost of grass carp is low, and the breeding range is wide. Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N1/21A61K39/15A61K47/48A61P31/14C12R1/19
Inventor 孟小林徐进平孟明翔王健杨莉莉
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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