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Prawn white spot syndrome virus VP37p polypeptide fragment and application thereof

A technology for shrimp and active peptides, applied in the direction of viral peptides, applications, antiviral agents, etc., can solve the problem of less understanding of the molecular level of the virus

Inactive Publication Date: 2010-04-28
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is still difficult to prevent and control WSSV. On the one hand, because WSSV can survive for a long time in the natural environment, and more importantly, people still have little understanding of the molecular level of the virus.

Method used

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  • Prawn white spot syndrome virus VP37p polypeptide fragment and application thereof
  • Prawn white spot syndrome virus VP37p polypeptide fragment and application thereof
  • Prawn white spot syndrome virus VP37p polypeptide fragment and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Cloning and sequencing of WSV254 gene fragments

[0047] Using WSSV genomic DNA as a template, a partial fragment (133bp-299bp) of WSV254 gene was amplified with primers P1 and P2.

[0048] P1: 5'-AGA CTCGAG ATGAGACGTCAAGTTG-3';

[0049] P2: 5'-GAG AAGCTT GTCAATGAGGGAGTTA-3';

[0050] The underlined part CTCGAG is the Xhol restriction site, AAGCTT is the Hind restriction site, and the PCR reaction conditions are:

[0051] 94°C 10 minutes

[0052] 94°C for 30 seconds

[0053] 44.2°C for 30 seconds

[0054] 72°C for 1 minute (8 cycles)

[0055] 94°C for 30 seconds

[0056] 51.7°C for 30 seconds

[0057] 72°C for 1 minute (30 cycles)

[0058] 72°C 10 minutes

[0059] After the amplified fragment was purified by agarose gel electrophoresis, it was cloned into the prokaryotic expression plasmid vector pBADgA to obtain the recombinant expression plasmid pBADgA-rVP37p, and then sequenced. The partial gene sequence of WSV254 obtained by sequencing is show...

Embodiment 2

[0062] Example 2 Expression and Purification of Recombinant VP37p Fragment

[0063] The recombinant expression plasmid pBADgA-rVP37p containing the WSV254 gene was transformed into E. coli TOP 10, and the positive clones were selected and cultured in LB medium containing 100 mg / L ampicillin at 37°C until OD 600 When = 0.6, L-arabinose was added to a final concentration of 0.2%, and the cells were collected after induction at 37°C for 5 hours. Add ice-cold lysis buffer (1×PBS, 10mM NaHPO 4 , 140mM NaCl, 2.7mM KCL, 1.8mM KH 2 HPO 4 ), ultrasonically lyse the bacteria (300W×10s×10 times), centrifuge at 15,000rpm at 4°C for 20min, install a tweezer column (Ni-nitriloacetic acid resins), and wash off with 5-10 column bed volumes of washing buffer (1×PBS). Miscellaneous proteins; elution buffer (50mM Tris-HCL, 100mM, imidazole, pH 8.0) to elute the target protein. The molecular weight of the purified protein was identified by SDS-PAGE to be about 12.09kD, and the purity was over...

Embodiment 3

[0066] Example 3 Binding activity of recombinant VP37p fragments to shrimp cells

[0067] Select well-adhered cells and place them in pre-cooling at 4°C for 1 hour, discard the medium, carefully wash twice with cold PBS buffer, add fluorescein-labeled VP37p, combine at 4°C for 2 hours, and then carefully wash with PBS buffer After washing twice, DAPI stained the nuclei, and observed the results directly under a fluorescent microscope (Olympus). At the same time, unlabeled VP37p and FITC-labeled bovine serum albumin (BSA) were used as blank controls.

[0068] After FITC-labeled VP37p interacts with adherent cells, green fluorescence can be seen under a fluorescent microscope, and DAPI-stained blue nuclei can be observed in the same field of view. After the two fields of view are superimposed, it can be seen that the green fluorescence is just superimposed with the blue fluorescence, while the control only The blue fluorescence of cell nucleus staining was observed, but the gre...

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Abstract

The invention utilizes prawn white spot syndrome virus (WSSV) gene expression technology to obtain a segment which is coded by a prawn white spot bacilliform virus WSV254 gene and is named as VP37p. The invention also relates to a method for manufacturing the VP37p, application of polynucleotide and polypeptide of the VP37p, and an antibody specifically bound with the polypeptide of the VP37p. The invention identifies the function of the VP37p through a biological method for the first time; the VP37p is one of WSSV attachment protein fragments; and the inhibition on the gene and expression products thereof may be one of effective ways of preventing and controlling prawn white spot disease and expects to be applied to the prevention and control of the prawn white spot disease.

Description

technical field [0001] The invention relates to a nucleotide sequence of a prawn white spot syndrome virus (WSSV) gene. Specifically, the present invention relates to a nucleotide sequence fragment (VP37p) of prawn white spot syndrome virus VP37, and also relates to a polypeptide with adhesion activity encoded by the nucleotide partial sequence. The present invention also relates to the use of these polynucleotides and polypeptides, and the production methods of said polynucleotides and polypeptides. Background technique [0002] Shrimp white spot baculovirus (WSSV) is one of the main virus sources that have harmed artificially cultured prawns in my country and the Asia-Pacific region in recent years. A variety of crustaceans such as crabs, lobsters, amphipods, and water flies in the ecosystem have a wide range of hosts, so they not only seriously endanger shrimp farming, but also have an impact on marine ecology. WSSV is a double-stranded DNA virus with a full-length genom...

Claims

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Application Information

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IPC IPC(8): C07K14/01C07K16/08C12N15/34C12N15/63A61K39/00A61K39/395A61K31/7088A61P31/20
Inventor 刘庆慧陈文博黄捷梁艳
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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