PRODUCTION AND APPLICATION OF MONOCLONAL ANTIBODIES AGAINST VP7 CAPSID PROTEIN OF GRASS CARP REOVIRUS

Grass carp reovirus (GCRV) poses great threat to the grass carp aquaculture industry and causes huge economic losses. Grass carp reovirus is the pathogen of this disease, which consists of three types, type Ⅰ, Ⅱ and Ⅲ. GCRV-873 is the most well studied stain of type I GCRV, with its complete genome sequenced in the 1980s. It has a segmented double stranded RNA genome with 11 fragments, encoding 7 structural proteins. VP5 and VP7 proteins form virus capsid. In this study, the capsid protein VP7 (GenBank: AF403396) of GCRV-873 strain was expressed in the E. coli cells. The cDNA fragment encoding the extracellular fragment of VP7 was cloned into pRSET-A vector and the resultant plasmid transformed into the E. coli BL21 (DE3) cells for prokaryotic expression. The recombinant protein was purified by size exclusion chromatography and used to immunize BALB/c mice for generation of monoclonal antibodies. It has been shown that the VP7 protein was highly expressed as inclusion bodies and the size was confirmed to be approximately 40 kD by SDS-PAGE analysis. After immunization, five IgG positive hybridoma cell lines were obtained, among which three were IgG1 subtype and two were IgG2a subtype. Western blotting and direct immunofluorescence analysis showed that the antibody could specifically recognize GCRV-I in the infected CIK cells, and the titer was high, reaching an affinity constant of 4.04×10⁹. The present study provides a practical approach for the development of diagnostic tools for detecting type I GCRV virus and in-depth investigation on the GCRV infection mechanism.