Prokaryotic expression and immunoassay of grass carp reovirus capsid VP6 protein

Abstract

Grass carp reovirus (GCRV) of the genus Aquareovirus and the family Reoviridae causes a severe hemorrhagic disease in grass carp fingerlings in China. GCRV genome is composed of 11 double-stranded RNA segments, of which segment 8 encodes the major core capsid protein VP6. In this study, the VP6 gene following an RT-PCR-amplification from the GCRV 104 strain was cloned into an expression vector pET-32a to obtain pET-32(a)-VP6. The VP6 protein was expressed in Escherichia coli BL21 as a fusion protein of 64 kDa. After purification with the HisLink Spin Protein Purification System the VP6 protein was used to raise a specific polyclonal antibody in Balb/c mice. Presence of VP6 protein was proved in bacterial lysates containing VP6 fusion protein by Western blot analysis and in GCRV-infected CIK cells by immunofluorescent staining using polyclonal antibody. These results may be helpful in further studies of interactions between GCRV and cells and in preparation of an engineered vaccine against GCRV.