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Recombinant baculoviruses which express grass carp reovirus spike protein VP55 and application

A technology for recombining baculoviruses and reoviruses, applied in the fields of applications, viruses, and viral peptides

Inactive Publication Date: 2018-04-06
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to its gene structure characteristics, the VP55 protein expressed by the prokaryotic expression system constructed by the prior art is an insoluble protein

Method used

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  • Recombinant baculoviruses which express grass carp reovirus spike protein VP55 and application
  • Recombinant baculoviruses which express grass carp reovirus spike protein VP55 and application
  • Recombinant baculoviruses which express grass carp reovirus spike protein VP55 and application

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Experimental program
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Effect test

Embodiment 1

[0017] 1. PCR amplification:

[0018] Using the gene type III grass carp reovirus spike protein (VP55) gene preserved in our laboratory as a template to amplify the VP55 gene fragment, the amplification conditions are:

[0019] After denaturation at 94°C for 1 min, it enters the cycle; cycle parameters are: 98°C for 10 sec, 55°C for 15 sec, 68°C for 30 sec; after 30 cycles, extend at 72°C for 10 min. After the reaction, the amplification results were detected by 0.6% agarose gel electrophoresis.

[0020] 2. Construction of recombinant transfer plasmid pFastBac HT A-VP55

[0021] The PCR positive product was recovered, and primers were designed through the EcoRI and HindIII restriction sites, as shown in Table 1, and the amplified VP55 gene was cloned into the donor plasmid pFastBacTM HTA vector (Invitrogen, USA), and subjected to enzyme digestion and sequencing. The identification confirmed that the construction was correct, and the positive recombinant plasmid pFastBac HTA-...

Embodiment 2

[0058] IFA analysis of SF9 insect cells infected by recombinant baculovirus

[0059] (1) Put the slides into a 24-well plate, spread the sf9 cells to the well plate, and plate for 3 hours until the cells grow to 80%;

[0060] (2) Remove the supernatant, add 1mL PBS to wash once;

[0061] (3) Remove PBS, add purified baculovirus MOI=10 and incubate with SF9 cells at 4°C for 1 hr;

[0062] (4) Remove virus, add 1mL PBS to wash away residual virus particles, repeat 3 times;

[0063] (5) Add anti-his monoclonal antibody (1:500) and incubate at 4°C for 1 hr;

[0064] (6) Remove the antibody and wash with PBS 3 times;

[0065] (7) Add FITC-conjugated anti-mouse antibody (green) (1:500) to remove the supernatant, add PBS to wash 3 times;

[0066] (8) Fix with 4% paraformaldehyde for 10 minutes, add PBS and wash 3 times at room temperature;

[0067] (9) Add mounting medium and observe with laser confocal microscope.

[0068] (10) Another control group was set without adding viru...

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Abstract

The invention relates to the technical field of biology, in particular to recombinant baculoviruses which express grass carp reovirus spike protein VP55 and application. The recombinant baculovirusesare generated when recombinant plasmids of pFast-HTA-VP55 are built and converted into recombinant baculoviruses. According to the recombinant baculoviruses which express the grass carp reovirus spikeprotein VP55 and application, by building the pFast-HTA-VP55 plasmids and using a baculovirus expression system, the baculoviruses with his tag protein of soluble protein of the grass carp reovirus spike protein VP55 are recombined and expressed, by means of the baculoviruses, a large amount of expressed soluble VP55 protein in SF9 (insect cells) can be infected, and the baculoviruses are appliedto relative operation of molecular virology.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant baculovirus expressing grass carp reovirus spike protein VP55 and its application. Background technique [0002] Grass carp reovirus is a non-enveloped virus with a double-stranded RNA structure that can cause grass carp viral hemorrhagic disease. Bioinformatics data showed that the VP55 protein was the spike protein of genotype III grass carp reovirus. Spike protein plays an important role in the process of virus adsorption and host invasion. Therefore, the establishment of an expression system capable of efficiently expressing soluble VP55 protein has practical significance for grass carp reovirus scientific research and vaccine development. However, due to its gene structure characteristics, the VP55 protein expressed by the prokaryotic expression system constructed in the prior art is all insoluble protein. For related research applied to molecular virology, such ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866C12N15/46C07K14/14C12R1/93
CPCC07K14/005C12N7/00C12N15/86C12N2710/14021C12N2710/14043C12N2720/12022
Inventor 王浩吕利群喻飞刘玮莎王龙龙
Owner SHANGHAI OCEAN UNIV
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