Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses

A technology of recombinant protein and sequence, applied in nervous system diseases, peptide/protein components, recombinant DNA technology, etc.

Inactive Publication Date: 2005-01-26
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no effective drug for this viral disease

Method used

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  • Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses
  • Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses
  • Recombinant nerve putrescence virus protein vaccine and its coding sequence and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Acquisition of the main capsid protein gene cDNA of grouper neuronecrosis virus

[0027] According to the determined genome sequence of the grouper neuronecrosis virus, the inventors designed a pair of specific primers for amplifying the main capsid protein gene of the virus. Using the RNA of the grouper neuronecrosis virus as a template, it was reverse-transcribed into the first-strand cDNA, and the double-strand cDNA was amplified with specific primers.

[0028] 1. Synthesis of first-strand cDNA

[0029] Grouper neuronecrosis virus RNA (GenBank accession number: AF534998) was used as a template. Denature the RNA template in a 95°C water bath for 5 minutes, take it out, and put it on ice for later use. Take a 0.2ml centrifuge tube and add the following reagents in sequence:

[0030] 5×buffer 2μl

[0031] DTT 1μl

[0032] 10mmol / l dNTP 1μl

[0033] 20pmol / l 6bp random primer 1μl

[0034] RNase...

Embodiment 2

[0046] Example 2: Construction of the recombinant expression plasmid pET32a-MCP for the main capsid protein gene of the grouper neuronecrosis virus

[0047] 1. Ligation of cDNA and expression vector

[0048] The cDNA obtained in Example 1 and the prokaryotic expression plasmid pET32a(+) were double-digested with Sac I and Hind III, respectively, and then recovered with agarose gel. The recovered product was subjected to a ligation reaction to obtain a recombinant expression vector containing the coding sequence of the main capsid protein of neuronecrosis virus (the nucleotide sequence of Sequence Listing 1), which was named pET32a-MCP. The plasmid pET32a-MCP actually contains the nucleotide sequence of sequence 4 in the sequence listing.

[0049] The connection reaction system is as follows:

[0050] cDNA 2μl

[0051] pET32a 6μl

[0052] 10× ligation buffer 1μl

[0053] T 4 DNA ligase 1 μl

[0054] ...

Embodiment 3

[0063] Example 3: Expression of recombinant expression vector pET32a-MCP in Escherichia coli

[0064] Transform Escherichia coli BL21 competent cells with the plasmid DNA of the recombinant expression vector pET32a-MCP, and pick a single colony grown on the transformation plate the next day, which is the prokaryotic recombinant bacteria containing pET32a-MCP plasmid DNA, named BL21 / pET32a -MCP. Pick a single colony grown on the transformation plate and activate it overnight at 37°C in LA liquid medium, transfer it to fresh LA liquid medium at a ratio of 1% the next day, and cultivate to OD 600 When it is about 0.8, add 100mmol / L IPTG to a final concentration of 1mmol / L, and continue culturing for 2-3 hours. Bacteria were collected and analyzed by SDS-PAGE ( Figure 4 ), the recombinant bacteria had one more band with a molecular weight of about 55.3kD than the empty vector bacteria, and the content was more. Further identified by Western-blot ( Figure 4 ), this band can s...

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Abstract

The invention relates to a recombinant nerve putrescence virus protein vaccine, the nucleic acid sequence coding the recombinant protein, and the use of the recombinant protein in preparing fish use vaccine product for nerve putrescence viruses resistance. Wherein the cDNA of the capsid nucleocapsid for the grouper nerve putrescence viruses is utilized to obtain the polypeptide recombinant proteins through DNA recombination process and pronucleus expression method.

Description

technical field [0001] The invention relates to a recombinant protein vaccine of neuronecrosis virus, a nucleotide sequence encoding the recombinant protein, and the application of the recombinant protein in preparing fish anti-necrosis virus vaccine products. Background technique [0002] Grouper is one of the famous and precious marine fish in the world, with a stable sales market and high prices. In recent years, grouper breeding density has increased, and diseases have become increasingly serious. Among them, viral nervous necrosis (viral nervous necrosis, VNN) is one of the major diseases that seriously harm fish fry, which can cause significant economic losses and even stop the production of nursery. Daya Bay Aquatic Products Experimental Center of Guangdong Province began to conduct artificial breeding experiments of grouper in 1999. In 2000, viral neuronecrosis broke out during the breeding period. Only in the production of the first batch of seedlings in May, 600 g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/16A61K39/00A61P25/00C07K19/00C12N15/62C12N15/63
Inventor 陈晓艳翁少萍殷志新黄剑南何建国
Owner SUN YAT SEN UNIV
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