Polyclonal antibody preparation and application of grass carp reovirus genotype III nonstructural protein NS66

Abstract:

[Background] Grass carp reovirus (GCRV) 104 strain can cause typical grass carp hemorrhagic disease, the analysis of its coding fragments is helpful to provide a basis for clinical immunological detection. [Objective] To study the possible function of the NS66 protein encoded by the s6 gene segment of GCRV 104 strain, the highly specific polyclonal antibody against NS66 protein was prepared, and its specificity was identified. [Methods] s6 gene segment amplified by PCR was cloned into the expression vector pGEX-4T-3, transformed into E. coli BL21 and induced by IPTG. After analysis and identification by SDS-PAGE, the target protein is obtained through purification. Mice were then immunized with purified recombinant protein to obtain polyclonal antibody. Titers were determined by Western blot, and antibody specificity was identified by Western blot and IFA (indirect immunofluorescence assay). [Results] The recombinant protein analysed by SDS-PAGE was about 66 kD, the same size as expected. The target protein was mainly present in inclusion bodies. The polyclonal antibody titer prepared by Western blot was more than 1:50 000. Western blot and IFA showed that the prepared polyclonal antibody recognizes the 104 strain, and the polyclonal antibody recognizes a single band with high specificity. [Conclusion] The study showed that the polyclonal antibody against NS66 protein can specifically recognize GCRV104 virus, which lays a foundation for the establishment of GCRV104 immunodiagnostic method and the study of GCRV-encoded NS66 protein.

Key words:Grass carp reovirus, 104 strain, NS66, polyclonal antibody