Preparation and immunogenicity of polyclonal antibodies against VP4, VP35 protein of type Ⅱ grass carp reovirus

To develop serological methods for detecting type Ⅱ grass carp reovirus (GCRV), the genes encoding outer capsid proteins VP4 and VP35 were cloned into the prokaryotic expression vector PGEX-4T-3 respectively. Polyclonal antibodies were generated by immunization of mices with the purified recombinant VP4 and VP35 proteins respectively and titered by ELISA.The specificity of antibodies was determined by Western blot. SDSPAGE results showed that the size of the recombinant VP4 and VP35 proteins was about 98ku and 61ku, respectively. The recombinant proteins mainly existed in the form of inclusion bodies. The titer of antibody against VP4 was up to 1:4×10⁵, the titer of antibody against VP35 was up to 1:10⁶. The results of Western blot showed that antibodies were able to identify both recombinant proteins and corresponding capsid proteins in virus particles. Furthermore, serum antibodies of grass carp infected by JX02 were able to recognize VP4 and VP35 proteins specifically. In this paper, the polyclonal antibodies against VP4 and VP35 were successfully prepared, which laid a foundation for developing a serological diagnostic method for major epidemic strains of GCRV and functional studies of these two viral capsid proteins.