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Preparation method and application of recombinant baculovirus co-expressing grass carp reovirus capsid proteins VP4 and VP35

A technology for recombining baculovirus and reovirus disease, applied in chemical instruments and methods, botany equipment and methods, biochemical equipment and methods, etc., can solve inconsistent shear efficiency, change load tRNA abundance, shear Incomplete and other problems, to achieve the effect of improving the immune protection rate, high production practice significance, and important immunogenicity

Active Publication Date: 2019-08-20
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cleavage efficiency of different 2A peptides is inconsistent, and there may be incomplete cleavage
2A Peptide Upstream Sequence Affects Cleavage Efficiency
However, host cells do not use every codon equally to encode amino acids. Host cells tend to use one or several synonymous codons. In different host cells, the usage of codons is very different. , even between high-expression proteins and low-expression proteins in the same host, and even between different proteins under the same operon, there are large differences in codon usage
Studies have shown that although high-frequency codons can increase the elongation speed of ribosomes, overexpression will lead to amino acid "starvation", thereby changing the abundance of loaded tRNA
Since codon changes will affect the co-translation process of proteins, optimized codons can accelerate the movement of ribosomes on mRNA, but may affect the activity of proteins and cause proteins with conformational changes to be hydrolyzed

Method used

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  • Preparation method and application of recombinant baculovirus co-expressing grass carp reovirus capsid proteins VP4 and VP35
  • Preparation method and application of recombinant baculovirus co-expressing grass carp reovirus capsid proteins VP4 and VP35
  • Preparation method and application of recombinant baculovirus co-expressing grass carp reovirus capsid proteins VP4 and VP35

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] A method for preparing a recombinant baculovirus co-expressing grass carp reovirus outer capsid proteins VP4 and VP35, comprising the steps of:

[0035] 1) PCR amplification of outer capsid proteins VP35 and VP4 and construction of T-A cloning vector

[0036] The total RNA of the grass carp ovary cell line (GCO) infected with grass carp reovirus 106 strains was extracted by the TRIzol method, reverse-transcribed into cDNA with a reverse transcription kit, and used as a template for S6 (shown in SEQ ID NO.2 , encoding VP4), S11 (shown in SEQID NO.1, encoding VP35) genes were amplified by PCR, and the PCR product was detected by 1% agarose gel electrophoresis, and the results showed that, as figure 2 As shown, VP4 of 1827bp and VP35 of 945bp can be obtained respectively. Use the PCR product purification and recovery kit to recover and purify the target fragments respectively.

[0037] The primer sequence used to amplify VP35 is: Upstream primer VP35-F: 5'-G GGATCCATG...

Embodiment 2

[0123] Recombinant baculovirus highly expresses VP4 and VP5 proteins:

[0124] SF9 cells (approximately 9×10 5 1) to be subcultured to T25 cell flasks for culture, when the SF9 cell confluence reached 70%, insert the recombinant baculovirus prepared in Example 1 of the P3 generation into each hole with the inoculation MOI=1, extract the total cell protein, After purification by affinity chromatography, the concentration of the recombinant target protein was determined to be 1.6 μg / mL, and the purity was 90.5%. In this application, through large-scale expression and purification of recombinant proteins, it was found that SF9 cells cultured in 400-600 mL of medium could purify 225-338 μg of protein; ) The unoptimized baculovirus expression system can purify 200-300 μg of protein from SF9 cells cultured in 400-600 mL of medium. Compared with it, the recombinant baculovirus provided by the invention significantly improves the protein expression .

Embodiment 3

[0126] The cleavage effect of different optimized codons for the cleavage peptide:

[0127] According to the method of Example 1, recombinant baculoviruses with different optimized codons for cleavage peptides were constructed, and the expression of proteins was investigated. The specific results are as follows:

[0128] Codon and protein expression results of different optimized cleaved peptides

[0129]

[0130]

[0131] Note: After experimental verification, the optimized T2A(4) sequence is the optimal sequence, and this patent uses this sequence

[0132] The recombinant protein was purified by affinity chromatography, and the immune protection experiment of fish was carried out. One week before the experiment, the same batch of healthy and disease-free grass carp fingerlings with a weight of 25-30 g were selected and raised in a water temperature of 28°C-30°C with oxygenation. The experiment was divided into 3 groups, the injection dose (protein amount / body weight ...

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Abstract

The invention belongs to the technical field of biology, and particularly discloses a preparation method and application of a recombinant baculovirus co-expressing grass carp reovirus capsid proteinsVP4 and VP35. Sequences as shown in SEQ ID NO.1, SEQ ID NO.3 and SEQ ID NO.2 are sequentially inserted among restriction enzyme cutting sites of BamHIand EcoRI pFastBacTMIto obtain recombinant plasmids pFastBac-S11-T2A-S6, and finally obtain the recombinant baculovirus co-expressing grass carp reovirus capsid proteins VP4 and VP35. The recombinant virus has high expression quantity, can be used for preparing proteins applicable to production of grass carp reovirus vaccines and have a wide application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of a recombinant baculovirus co-expressing grass carp reovirus outer capsid proteins VP4 and VP35. [0002] technical background [0003] Grass carp hemorrhagic disease is a viral infectious disease caused by grass carp reovirus that seriously harms grass carp farming. It mainly causes various degrees of bleeding in various organs and tissues of diseased grass carp, necrosis of visceral tissues, and extremely high mortality. It is harmful to aquaculture in my country. Bring huge harm and economic loss. At present, the domestic research is mainly on the genotype Ⅰ grass carp reovirus, but the research on the genotype Ⅱ grass carp reovirus is less. According to the epidemiological survey results of the main grass carp breeding areas in the country, type Ⅱ grass carp reovirus is the main epidemic strain in my country at present. Grass car...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866A61K39/15A61P31/14
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C12N7/00C12N15/86C12N2710/14043C12N2720/12022C12N2720/12034
Inventor 母昌勇范玉顶周勇许晨江南刘文枝曾令兵
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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