Prokaryotic expression, polyclonal antibody preparation and tissue-tropism analysis of cyprinid herpesvirus II non-structural protein ORF4

Abstract:

[Objective] Acquire the polyclonal antibody against non-structural protein ORF4 of cyprinid herpesvirus 2, and reveal the tissue distribution pattern of ORF4 protein during the virus infection. [Methods] The ORF4 structure was analyzed by SMART and BLASTx. The ORF of ORF4 was amplified from viral genomic DNA and cloned into plasmid PGEX-4T-3. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) for recombinant protein production after induction by IPTG. The expressed recombinant protein was then purified by affinity chromatography and the purified protein was used to immunize New Zealand rabbits to obtain the anti-ORF4 polyclonal antibody. The specificity of the anti-ORF4 polyclonal antibody was confirmed by Western blot. Furthermore, real time PCR analysis was employed to monitor the genomic replication level in various tissues of virus-infected Carassius auratus gibelio including muscle, brain, gill, spleen, liver pancreas, heart, kidney. And the tissue distribution pattern of ORF4 in infected Carassius auratus gibelio was subsequently analyzed by Western blot. [Results] The expressed recombinant ORF4 protein in bacteria was detected at 65 kD as expected. Immunoassays suggested that the home-made antibody can specially recognize either the recombinant protein or endogenous ORF4. It proved that the ORF4 was mainly distributed in the kidney, spleen and gill in vivo, which was in consistence with the high level of virus replication in these tissues revealed by real time PCR assay. [Conclusion] Non-structural viral protein ORF4 might involve in efficient viral replication, which could serve as a marker protein for active virus infection. This study paved the way for studying the mechanism of ORF4 in the process of cyprinid herpesvirus 2 infection.