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White spot syndrome virus VP292 polypeptide and application thereof

A technology for prawns and recombinant proteins, applied in the field of genetic bioengineering, can solve problems such as difficult to eliminate

Inactive Publication Date: 2013-06-26
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 generally believed that WSSV has two transmission routes: horizontal transmission (oral infection or immersion infection) and vertical transmission, which can independently circulate in animals, so it is difficult to completely eliminate it in the short term

Method used

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  • White spot syndrome virus VP292 polypeptide and application thereof
  • White spot syndrome virus VP292 polypeptide and application thereof
  • White spot syndrome virus VP292 polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Cloning and sequencing of embodiment 1WSV254 gene fragment

[0045] Using WSSV genomic DNA as a template, primers P1 and P2 were used to amplify partial fragments of WSV237 gene.

[0046] P1: 5'-TTA CTCGAG ATGTTGTTTGATTTCT-3;

[0047] P2: 5'GAC AAGCTT TAATACGGGACCT-3';

[0048] The underlined part CTCGAG is the Xhol restriction site, AAGCTT is the HindIII restriction site,

[0049] The PCR reaction conditions are:

[0050] 94°C 10 minutes

[0051] 94°C for 30 seconds

[0052] 44.2°C for 30 seconds

[0053] 72°C for 1 minute (8 cycles)

[0054] 94°C for 30 seconds

[0055] 51.7°C for 30 seconds

[0056] 72°C for 1 minute (30 cycles)

[0057] 72°C 10 minutes

[0058] After the amplified fragment was purified by agarose gel electrophoresis, it was cloned into the prokaryotic expression plasmid vector pBAD / gIIIA to obtain the recombinant expression plasmid pBAD / gIIIA-rVP292, and then sequenced. The partial gene sequence of WSV237 obtained by sequencing is show...

Embodiment 2

[0060] Example 2 Expression and Purification of Recombinant VP292 Fragment

[0061] Transform the recombinant expression plasmid pBAD / gIIIA-VP292 containing the WSV237 gene into Escherichia coli TOP10, select positive clones and culture them 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 on a Ni-nitriloacetic acid resin, wash with 5-10 column bed volumes of washing buffer (1×PBS) Remove impurities; elution buffer (50mM Tris-HCL, 100mM, imidazole, pH 8.0) elutes 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 90%. see results figure 1.

Embodiment 3

[0062] Example 3 Antibody Preparation of Recombinant VP292 Fragment

[0063] The purified recombinant protein obtained in Example 2 was emulsified with an equal volume of complete Freund's adjuvant, and the mice were subcutaneously injected with 0.25-0.5 mg / mL emulsified protein, each 0.2 mL. Two weeks later, the same dose of the same antigen emulsified with incomplete Freund's adjuvant was re-injected to boost immunization to produce antibodies, and then boosted immunization every 10 days, at least twice. The titer and specificity of the obtained antisera were analyzed.

[0064] After reading the above content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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Abstract

The invention utilizes white spot syndrome virus (WSSV) gene expression technology to obtain a protein coded by white spot caculovirus WSV237 gene, and the protein is named VP292. The invention also relates to a method for producing VP292, application of VP292 polynucleotide and polypeptide as well as antibody specifically combined with VP292 polypeptide. Inhibition on the gene and expression product thereof can be one effective way for preventing and treating white spot disease, thus being hopeful to be applied to preventing and treating white spot disease.

Description

technical field [0001] The invention relates to the technical field of genetic bioengineering. Specifically, the present invention relates to the nucleotide sequence of prawn white spot syndrome virus VP292, and also relates to a polypeptide encoded by the nucleotide 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 genome of 305Kb, and is the largest animal virus discovered so far (J Virol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/34C12R1/19
Inventor 刘庆慧黄捷陈文博梁艳
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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