Preparation of Polyclonal Antibody Against the Capsid Protein of Grouper Nervous Necrosis Virus

Grouper nervous necrosis virus (GNNV) infects the nervous system of juvenile fish, with the mortality rates as high as 90 % -100 %, which was a bottleneck restricting the development of grouper aquaculture. The preparation of polyclonal antibodies against GNNV is essential for the in-depth study of viral protein functions and the development of related detection methods. In this study, an expression vector containing the protruding domain (P-domain) of the GNNV capsid protein, designated GNNV-P-pET-15b, was constructed and transformed into Escherichia coli BL21 (DE3) for the prokaryotic expression. The purified GNNV-P protein was used as an antigen to immunize New Zealand white rabbits, generating the rabbit-derived polyclonal antiserum. The antiserum titer was determined by indirect ELISA, and its specificity was analyzed by Western blot. The results showed that: the 15 kDa soluble recombinant GNNV-P protein was successfully obtained by prokaryotic expression of GNNV-P-pET-15 b vector, and then further used as an antigen to immunize New Zealand white rabbits to prepare the rabbit-derived polyclonal antiserum against GNNV-P, with the titer of antiserum exceeding 1∶150 000 by using the method of indirect ELISA. The Westernblot showed that GNNV-P polyclonal antibody had high specificity. The results provided key biological materials for the subsequent study of GNNV infection mechanism and the development of related immunological detection methods.