Molecular cloning and prokaryotic expression of vp5 gene of grass carp reovirus strain GCRV096.

VP5 is an outer capsid protein of grass carp reovirus (GCRV). It is predicted to involve in helping GCRV enter the host cells. In this study, the full-length vp5 gene (accession number in GenBank: JN206664.1) was cloned from GCRV strain GCRV096, which was isolated from diseased grass carp (Ctenopharyngodon idella) in southern China by RT-PCR technique using the primers designed from the known vp5 gene sequences of other strains of GCRV published in GenBank. The ORF sequence of vp5 is composed of 1,947 nucleotides encoding a 648-residues protein with a calculated molecular mass of 68.6 kDa and an estimated isoelectric point of 6.1. Sequence analysis results showed that VP5 might serve as a penetration protein and play an important role in GCRV penetration into the host cells. A full length of vp5 gene was subcloned into the prokaryotic expression vector pET-28a (+) and the recombinant plasmid (pET/GCRV-VP5) was then transduced into Escherichia coli BL21 (DE3) cells to express VP5 in vitro. SDS-PAGE and western blotting analysis indicated that the protein expressed successfully. Results also showed that the fusion protein expressed in the form of inclusion body, and it expressed in the highest level when induced with 0.2-mM IPTG at 28 °C for 4 h. These results are important for the future study on the molecular structure, function, and immunogenicity of GCRV capsid protein.