Construction of a recombinant eukaryotic vector for a grass carp reovirus VP6 gene and its expression invitro and invivo.

Abstract

A recombinant plasmid expressing the VP6 inner capsid coding gene of grass carp reovirus (GCRV) was constructed and expressed in a Ctenopharyngodonidellus kidney (CIK) cell line and grass carps. The VP6 gene was amplified by RT-PCR, cloned into a pEGFP-N1 eukaryotic expression vector and transfected into CIK cells. Results from enhanced green fluorescent protein (EGFP) experiments and flow cytometry showed highest protein expression at 48h. The immunoreactivity of fusion protein was confirmed using an indirect immunofluorescent assay. The specific binding between the fusion protein and polyclonal mouse GCRV VP6-specific antiserum indicated that the fusion protein was translated in vitro and had good immunogenicity. An antiviral activity assay showed that the virus titer was 100-fold lower in the GCRV VP6 expressed cells than in the pEGFP-N1 transfected cells. The expression levels of three immune genes in the head kidney of grass carps injected with the recombinant plasmid were used. Mx, TLR3 and IgM mRNA expression increased sharply at the 1st and 15thdays post-injection (dpi). Specific antibodies were detected 30days after vaccination. Neutralizing titers of the antibodies in vaccinated fish detected ranged from 160 to 320. Intramuscular injection of grass carps with 1μg of pEGFP-N1-VP6 was found to provide strong protection against GCRV. These results suggested that the VP6 gene was a good candidate for the design of GCRV-DNA vaccines and to investigate the use of cytokines as co-stimulatory molecules.