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A large-scale preparation process and application of gcrv II S9 and S10 recombinant proteins

A recombinant protein and protein engineering technology, applied in the field of protein purification, can solve the problems of difficult application and high antigen cost, and achieve the effect of reducing work intensity

Active Publication Date: 2020-10-13
GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that the use of inactivated GCRV whole virus vaccines or subunit vaccines prepared from GCRV S9 and S10 encoded proteins has good effects on the prevention and control of GCRV. Or the cost of preparing antigens by affinity chromatography is high and difficult to apply

Method used

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  • A large-scale preparation process and application of gcrv II S9 and S10 recombinant proteins
  • A large-scale preparation process and application of gcrv II S9 and S10 recombinant proteins
  • A large-scale preparation process and application of gcrv II S9 and S10 recombinant proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 A large-scale preparation process of grass carp reovirus type II S9 recombinant protein, comprising:

[0064] 1. Steps for constructing recombinant marker protein engineering bacteria: construct and express GCRV II S9 recombinant protein, which is GV2B9;

[0065] The specific construction method is as follows: Bioinformatics analysis was performed on the S9 encoded protein in the grass carp enterovirus type II virus genome, and the S9 amino acid sequence of the GCRV type II virus was used as a reference sequence (Genebank accession number ADT79740), and the codons of the encoded protein were respectively analyzed. Optimize and artificially synthesize the gene sequence GCRV II opti-S9, and insert the synthesized gene fragments into the prokaryotic expression vector PET21b through NdeI / Xho I and BamH I / XhoI endonucleases respectively; GCRV S9 expression sequence ORF 3' end is fused with 6 histidines Coding sequence; the synthetic sequence was verified by sequ...

Embodiment 2

[0086] Example 2 A large-scale preparation process of grass carp reovirus type II S10 recombinant protein, comprising:

[0087] 1. Steps for constructing recombinant marker protein engineering bacteria: constructing genetically engineered bacteria expressing GCRV II S10 recombinant protein, which is GV2B10;

[0088] The specific construction method is as follows: Bioinformatics analysis was performed on the S10 encoded protein in the grass carp enterovirus type II virus genome, and the S10 amino acid sequence of the GCRV type II virus was used as a reference sequence (Genebank accession number is ADT79741), and the codes encoding the two proteins were respectively analyzed. The gene sequence GCRV II opti-S10 was optimized and artificially synthesized. The two synthetic gene fragments were inserted into the prokaryotic expression vector PET21b through NdeI / Xho I and BamH I / XhoI endonucleases respectively; the 5' end of the GCRV S10 expression sequence ORF was fused with T7 S...

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Abstract

The invention discloses a scale preparation process and application of GCRV II recombinant proteins S9 and S10. The preparation process comprises the steps of constructing and fermenting a recombinantlabelled protein engineering bacterium, recovering a protein inclusion body by means of tangential flow microfiltration, degenerating proteins rGCRV2 S9 and rGCRV2 S10, and continuously renaturing. Asupernatant and a precipitate in various steps are separated by mainly using tangential flow microfiltration and ultrafiltration, and a large-volume sample does not need to be centrifuged; the endotoxin and the mycoprotein are removed through the degeneration and the continuous denaturation of the recombinant proteins S9 and S10 in the form of the inclusion body expressed by escherichia coli, andthe biological activity of the renatured recombinant protein is excellent. The key is that the preparation process is simple to operate, the large-volume sample does not need to be centrifuged in thewhole process, the working strength is reduced, and the preparation process is suitable for large-scale industrial production.

Description

technical field [0001] The invention relates to a protein purification technology, in particular to a large-scale preparation process and application of GCRV II S9 and S10 recombinant proteins. Background technique [0002] Grass carp (Ctenopharyngodon idellus) is the main species of freshwater fish farming in my country. Grass carp hemorrhagic disease is a common disease that plagues grass carp farming in my country. Grass carp hemorrhagic disease is mainly caused by type II grass carp reovirus (grasscarp reovirus, GCRV II) attacking grass carp epithelial tissue Therefore, early prevention of GCRV can effectively prevent and control the loss caused by grass carp hemorrhagic disease to grass carp breeding. Studies have shown that the use of inactivated GCRV whole virus vaccines or subunit vaccines prepared from GCRV S9 and S10 encoded proteins has good effects on the prevention and control of GCRV. Or the cost of preparing antigen by affinity chromatography is relatively hig...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12P21/02C07K14/14C07K1/34C07K1/113G01N33/68G01N33/569A61K39/15A61P31/14
CPCA61K31/12A61K2039/552C07K14/005C12N15/70C12N2720/12022C12N2720/12034G01N33/56983G01N33/6854G01N2333/14G01N2469/20
Inventor 李中圣王凤求伍建敏候月娥张杰赵玉林
Owner GUANGDONG HAID ANIMAL HUSBANDRY & VETERINARY RES INST