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Engineered protein TAT-VP28 against white spot syndrome virus of shrimp and its preparation and purpose

A technology of TAT-VP28 and fusion protein, which is applied in the direction of antiviral agents, peptide/protein components, biochemical equipment and methods, etc., can solve the problems that the molecular mechanism is not fully studied, so as to improve immunity and disease resistance, The effect of low cost and simple operation

Active Publication Date: 2011-10-05
HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although there are many studies on the transmembrane function of the TAT protein transduction domain, so far, the molecular mechanism of the TAT protein transduction domain into the cell has not been fully studied

Method used

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  • Engineered protein TAT-VP28 against white spot syndrome virus of shrimp and its preparation and purpose
  • Engineered protein TAT-VP28 against white spot syndrome virus of shrimp and its preparation and purpose
  • Engineered protein TAT-VP28 against white spot syndrome virus of shrimp and its preparation and purpose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Preparation of tat-vp28 gene.

[0061] (1) Preparation of WSSV virus genome: take gills, stomach and heart of diseased prawns on ice, homogenate in ice bath, then add proteinase K (100ug / ml), boil in boiling water for 15 minutes, and immediately ice bath for 5 minutes , centrifuged at 12,000 rpm for 10 minutes, and the supernatant was taken and stored at 4°C.

[0062] (2) PCR amplification of VP28 gene: PCR primers were designed according to the sequence of WSSV (GeneBank: EU414753) published in GeneBank, and the above supernatant was used as template DNA to amplify the target gene VP28 by PCR, and the PCR product was detected by DNA gel electrophoresis , recover the PCR product and clone it into the pGEM-T vector (purchased from Promega Company), pick a single colony and use alkaline lysis to extract a small amount of plasmid for enzyme digestion identification, and the positive clone obtained by sequencing is the one containing VP28 coli, named pGEM-T-vp28...

Embodiment 2

[0064] Example 2: Recovery, purification and subcloning of PCR products.

[0065] The PCR product of TAT-VP28 obtained in Example 1 was electrophoresed on an agarose gel, and the band to be recovered was rapidly excised under ultraviolet light, purified with a TIANGEN agarose gel DNA recovery kit, and the single target Put the DNA band into a clean Eppendorf tube and weigh it. Add three times the volume of sol solution PN 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 50°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. Take 750uL of the resulting solution and add it to an adsorption column (the adsorption column is placed in a collection tube), place it at room temperature (the same below 20-25°C) for 2 minutes, centrifuge at 12000rpm for 1 minute, pour off the waste liquid in the collection tube, and remove the adsor...

Embodiment 3

[0070] Example 3: Construction of plasmid pGEM-tat-vp28.

[0071] Calcium chloride method prepares Escherichia coli competent cell: its steps are:

[0072] (1) Use an inoculation loop to pick a single colony of newly activated Escherichia coli JM109 on a solid LB plate, and inoculate it into 20ml liquid LB medium (dissolve 1g peptone, 0.5g yeast powder, 1g NaCl in 75ml ddH 2 O, adjust the pH to 7.0, and finally add ddH 2 O was fixed to 100ml, distributed in five Erlenmeyer flasks, sterilized at 115 lbs for 20 minutes and then used for standby), 37°C, 250rpm shaker activation overnight.

[0073] (2) Under sterile conditions, take 60ul of the above-mentioned activated E. coli in fresh 20ml liquid LB medium, culture at 37°C, 250rpm shaker for about 3 hours to OD 600 The value is 0.4-0.6. (All the following steps require aseptic operation)

[0074](3) Under aseptic conditions, take 1.5 ml of the above bacterial solution into a sterile Eppendorf tube, place it on ice for 10 min...

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Abstract

The invention discloses an engineered protein TAT-VP28 against white spot syndrome virus of shrimp and its preparation and purpose. The sequences of the engineered protein TAT-VP28 provided by the invention are nucleotide sequence as shown in SEQIDNO:1 and amino acid sequence as shown in SEQIDNO:2, and the bacterial strain is a recombinant gene engineering bacterial strain Escherichiacoli BL21(Pet-28a-TAT-VP28). WSSV is extracted from shrimp bodies and the VP28 gene is obtained through PCR; TAT-VP28 sequences are amplified by the PCR method and then the recombinant plasmid pET-28a(+)-TAT-VP28is constructed, followed by the transformation of the recombinant plasmid pET-28a(+)-TAT-VP28 into the Escherichia coli BL21(DE3). The gene engineering fusion protein can be applied to preparing medicaments for treating or preventing the white spot syndrome virus of shrimp, has large expression amount and soluble performance, is easy for industrial production at low cost with good security, and has a better lasting protective effect of preventing the white spot syndrome of shrimp.

Description

technical field [0001] The invention relates to the technical field of genetic bioengineering. More specifically, it relates to a recombinant fusion protein TAT-VP28 subunit vaccine, and also relates to a method for preparing a genetically engineered Escherichia coli strain, specifically relating to the construction and high-efficiency expression of a soluble protein transduction domain polypeptide (TAT-PTD) The invention relates to a genetically engineered strain of the fusion protein of the envelope protein VP28 of shrimp white spot syndrome virus (WSSV); it also relates to the use of the fusion protein expressed by the above strain in anti-prawn white spot syndrome virus (WSSV) medicines. Background technique [0002] Since the 1980s, the world's shrimp farming industry has developed rapidly. With the increasing destruction of the environment, disease problems have become more serious, which has become an important factor hindering the development of the shrimp farming in...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N1/21A61K38/16A61P31/12C12R1/19
Inventor 孟小林徐进平王健张毅宁建芳
Owner HUBEI TAIYANGHONG BIOLOGICAL TECH CO LTD
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