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Prawn leukasmus rhabdovirus WP486 gene and coded polypeptide thereof

A WP486, encoding technology, applied in the direction of viral peptides, genetic engineering, plant genetic improvement, etc., can solve the problem of less virus molecules

Inactive Publication Date: 2005-02-09
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is still difficult to prevent and control WSBV. On the one hand, WSBV can survive in the natural environment for a long time. More importantly, people still have little understanding of the molecular level of the virus.

Method used

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  • Prawn leukasmus rhabdovirus WP486 gene and coded polypeptide thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Cloning and sequencing of WP486 gene fragment

[0027] Using WSBV genomic DNA as a template, the WP486 gene was amplified with primers P1 and P2.

[0028] P1: 5′-CGT GGATCC GACACAGACGATTTTGAGCC-3' (SEQ ID No. 3);

[0029] P2: 5′-GA GCGGCCGC A GCTAGC TTGGCTGGACAATAAAT-3' (SEQ ID No. 4);

[0030] underlined part GGATCCis the BamH I restriction site, GCGGCCGC is the NotI restriction site, GCTAGC It is the NheI restriction site.

[0031] The PCR reaction conditions are:

[0032] 94°C 60 seconds

[0033] 55℃ for 60 seconds

[0034] 72°C for 1.5 minutes (30 cycles)

[0035] After the amplified fragment was purified by agarose gel electrophoresis, it was cloned into the prokaryotic expression plasmid vector pGEX4T-1 to obtain the recombinant expression plasmid pGEX-WP486, and then sequenced. See SEQ ID No.1 for the WP486 gene sequence obtained by sequencing.

[0036] The amino acid sequence of WP486 deduced according to the obtai...

Embodiment 2

[0037] Example 2 Expression and Purification of Recombinant WP486 Fragment

[0038] The recombinant expression plasmid pGEX-WP486 containing the WP486 gene was transformed into Escherichia coli BL21, and the positive clones were selected and cultured in LB medium containing 100mg / L ampicillin at 37°C until A 600 When = 0.6, add IPTG to a final concentration of 0.1 mmol / L, and collect the bacteria after induction at 37°C for 6 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; mix the supernatant with Glutathione Sepharose 4B equilibrated with lysis buffer in advance, react at 4°C for 10min, and pack the column; use 5-10 Column bed volume of washing buffer (1×PBS) to wash away impurity proteins; elution buffer (50mM Tris-HCl, 10mM reduced glutathione, pH8.0) to elute target protein. The molecular weight of the purified fusion...

Embodiment 3

[0041] Example 3 Antibody Preparation of Recombinant WP486 Fragment

[0042] 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 7 days, at least twice. The titer and specificity of the obtained antisera were analyzed.

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Abstract

The present invention utilizes prawn white spot baculovirus (WSBV) geome microarray technology to obtain genetic transcription map of 6 hr after viral infection and genetic transcription map after deep infection. One virus gene control related gene WP486 is found in the WSBV genome, and the gene has nucleotide sequence encoding one kind of polypeptide with gene controlling funtion. The present invention also relates to the application of gene WP486 and the encoded polypeptide and the production process of the said active polypeptide. The present invention identifies the function of WP486 gene of WSBV virus in biological method. WP486 gene is one of the WSBV early controlling key gene, and the inhibition on the gene and its expression product may be the effective way of preventing and treating prawn white spot disease and is expected to be used in the prevention and treatment of prawn white spot disease.

Description

technical field [0001] The invention relates to a nucleotide sequence of a prawn white spot baculovirus gene. Specifically, the present invention relates to the nucleotide sequence of the very early regulatory gene WP486 of the white spot baculovirus of prawns, and also relates to a polypeptide encoded by the nucleotide sequence with gene regulation activity. The application of these polynucleotides and polypeptides of the present invention, and the production method of said polynucleotides and active polypeptides. Background technique [0002] Shrimp white spot bacilliform virus (WSBV), also known as white spot syndrome virus (WSSV), is one of the main viral pathogens that have harmed artificially cultured prawns in my country and the Asia-Pacific region in recent years. It can It infects most types of prawns with a high lethality rate. In addition, it can also infect a variety of crustaceans such as crabs, lobsters, amphipods, and water flies in freshwater and marine ecosy...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K14/01C07K16/40C12N5/10C12N15/34C12N15/63C12P21/02
Inventor 杨丰兰永胜
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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