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Prawn leukasmus rhabdovirus dUTPase gene and coded polypeptide thereof

A technology for encoding and active polypeptides, which is applied in the fields of viral peptides, genetic engineering, and plant genetic improvement, and can solve the problem of fewer viral 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 WSS. 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 this virus.

Method used

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  • Prawn leukasmus rhabdovirus dUTPase gene and coded polypeptide thereof
  • Prawn leukasmus rhabdovirus dUTPase gene and coded polypeptide thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Cloning and sequencing of embodiment 1 W-DUT gene

[0028] Using WSBV genomic DNA as a template, the W-DUT gene was amplified with primers P1 and P2.

[0029] P1: 5′-GAGA GGATCC GACTCATCTGCATCTGTCGTG-3' (SEQ ID No. 3);

[0030] P2: 5′-GAGC GAGCTC TTCAGTAAAATTTGGGTT-3' (SEQ ID No. 4);

[0031] The underlined part GGATCC is the BamH I restriction site, and GAGCTC is the Sac I restriction site.

[0032] The PCR reaction conditions are:

[0033] 94°C for 30 seconds

[0034] 55°C 30 seconds

[0035] 5 minutes at 68°C (30 cycles)

[0036] After the amplified fragment was purified by agarose gel electrophoresis, it was cloned into the prokaryotic expression plasmid vector pQE30 to obtain the recombinant expression plasmid pQE30-dut, and then sequenced. The W-DUT gene sequence obtained by sequencing is shown in SEQ ID No.1.

[0037] The amino acid sequence of W-DUT deduced according to the obtained nucleotide sequence has a total of 176 amino acid ...

Embodiment 2

[0038] Example 2 Expression and purification of recombinant W-DUT

[0039] The recombinant expression plasmid pQE30-dut containing the W-DUT gene was transformed into Escherichia coli BL21, and positive clones were selected and cultured in LB medium containing 100 mg / L ampicillin at 37°C until A 600 =0.6, add IPTG to a final concentration of 0.5mmol / L, and collect the bacteria after induction at 37°C for 6h. Add ice-cold lysis buffer (50mM NaH 2 PO 4 , 300mM NaCl, 15mM imidazole, pH8.0), ultrasonically lyse bacteria (300W×10s×10 times), centrifuge at 15,000rpm at 4°C for 20min; mix the supernatant with Ni-NTAAgarose pre-balanced with lysis buffer, and react at 4°C 10min, pack the column; wash buffer (50mM NaH 2 PO 4 , 300mM NaCl, 30mM imidazole, pH8.0) to wash away impurities; elution buffer (50mMNaH 2 PO 4 , 300mM NaCl, 250mM imidazole, pH8.0) to elute the target protein. The molecular weight of the purified protein was identified by SDS-PAGE to be about 23kD, ...

Embodiment 3

[0041] Example 3 Antibody Preparation of Recombinant W-DUT

[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. Ten days 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 clones one uracil deoxyribonucleoside triphosphatase (dUTPase) gene from prawn white spot baculovirus (WSBV) genome. The gene has nucleotide sequence encoding one kind of polypeptide with dUTPase activity. The present invention also relates to the application of dUTPase gene and the encoded polypeptide and the production process of the said active polypeptide. The present invention identifies the function of dUTPase gene of WSBV virus in biological method. Because dUTPase gene is one of the key enzyme for nucleotide synthesis and metabolism, the fast copying and proliferation of WSBV and the inhibition on the enzyme may be one of 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 gene sequence of the prawn white spot baculovirus W-DUT polypeptide, and the polypeptide protein is a protein with dUTPase activity. The present invention also relates to the polypeptide encoded by the nucleotide sequence, the application of these polynucleotides and polypeptides, and the production method of the 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 ecosystem...

Claims

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Application Information

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IPC IPC(8): C07K14/01C07K16/40C12N5/10C12N9/16C12N15/33C12N15/55C12N15/63C12P21/02
Inventor 杨丰刘小青
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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