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Recombinant baculoviruses which express grass carp reovirus spike protein VP56 and application

A technology of recombinant baculovirus and reovirus, applied in the biological field, can solve difficult problems such as molecular virology

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 VP56 protein expressed by the prokaryotic expression system constructed by the existing technology is an insoluble protein, which is difficult to apply to the related research of molecular virology, such as pulling down, co-ip and other experimental operations.

Method used

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  • Recombinant baculoviruses which express grass carp reovirus spike protein VP56 and application
  • Recombinant baculoviruses which express grass carp reovirus spike protein VP56 and application
  • Recombinant baculoviruses which express grass carp reovirus spike protein VP56 and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. PCR amplification:

[0019] Using the genotype II grass carp reovirus spike protein (VP56) gene preserved in our laboratory as a template to amplify the VP56 gene fragment, the amplification conditions are:

[0020] 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.

[0021] 2. Construction of recombinant transfer plasmid pFastBac HT A-VP56

[0022] The PCR positive product was recovered, and primers were designed through the SalI and XhoI restriction sites. The specific primers were as shown in the table below. The amplified VP56 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 recombina...

Embodiment 2

[0060] IFA analysis of SF9 insect cells infected by recombinant baculovirus

[0061] Infection of SF9 Insect Cells with Recombinant Baculovirus

[0062] 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%;

[0063] 2. Remove the supernatant, add 1mL PBS to wash once;

[0064] 3. Remove PBS, add purified baculovirus MOI=10 and incubate with SF9 cells at 4°C for 1 hr;

[0065] 4. Remove virus, add 1mL PBS to wash away residual virus particles, repeat 3 times;

[0066] 5. Set up the normal group with the same conditions but adding baculovirus;

[0067] 6. Observe, compare and record the results after culturing for 5 days.

[0068] The result shows, as figure 1 As shown, the sf9 cells infected with the recombinant baculovirus shrink and lyse, and have specific green fluorescence after incubation and labeling with His monoclonal antibody, indicating that the recombinant virus can efficiently infect s...

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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 VP65 and application. The recombinant baculovirusesare generated when recombinant plasmids of pFast-HTA-VP56 are built and converted into recombinant baculoviruses. According to the recombinant baculoviruses which express the grass carp reovirus spikeprotein VP56 and application, by building the pFast-HTA-VP56 plasmids and using a baculovirus expression system, the baculoviruses with his tag protein of soluble protein of the grass carp reovirus spike protein VP56 are recombined and expressed, by means of the baculoviruses, a large amount of expressed soluble VP56 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 VP56 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. Existing studies have shown that the detection rate of genotype Ⅱ grass carp reovirus in Chinese grass carp breeding areas is as high as 50%. Type grass carp reovirus does not have this protein. The 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 VP56 protein has practical significance for grass carp reovirus scientific research and vaccine development. However, due to its gene structure characteristics, the VP56 protein expressed by the prokaryotic expression system constructed by the ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/866C12N15/31C12R1/93
CPCC07K14/28C12N7/00C12N15/86C12N2710/14021C12N2710/14043C12N2800/105
Inventor 吕利群王浩喻飞李婉娟孙皓盛佳璐
Owner SHANGHAI OCEAN UNIV