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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% agarosegel 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 enzymedigestion and sequencing. The identification confirmed that the construction was correct, and the positive recombinant plasmid pFastBac HTA-...
[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 expressionsystem, 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 expressionsystem constructed in the prior art is all insoluble protein. For related research applied to molecular virology, such ...
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