Penaeus monodon nucleopolyhedrovirus detection using monoclonal antibodies specific to recombinant polyhedrin protein

Two segments of the gene encoding the polyhedrin protein of Penaeus monodon nucleopolyhedrovirus (PemoNPV) were cloned into the pTYB1 (759 bp) and pGEX-6P-1 (614 bp) expression vectors and then transformed into the BL21 Escherichia coli strain. After induction, a fusion of the OB-N-intein (OB-N-intein; 83.2 kDa) and OB-C glutathione-S-transferase (GST-OB-C; 48.4 kDa) proteins were produced. They were purified by SDS-PAGE, electroeluted and injected into Swiss mice for monoclonal antibody (MAb) production. Two MAbs specific to OB-N and three MAbs specific to OB-C were isolated. They can be used to detect natural PemoNPV infection in Penaeus monodon by dot blotting, western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses, including Taura syndrome virus (TSV), yellow head virus (YHV), white spot syndrome virus (WSSV) and Penaeus monodon densovirus (PmDNV). Dot-blotting a combination of the four different MAbs specific to OB-N and OB-C, which were obtained from this study and from previous studies, was approximately 100 times less sensitive than performing 1-step PCR. The combination of MAbs is expected to be useful for the future development of a simple, immunochromatographic strip test for the rapid, pond-side detection of PemoNPV.

► MAbs specific to two fragments of recombinant polyhedrin of PemoNPV were produced. ► They can be used to detect natural PemoNPV infection by various immuno-assays. ► These included dot blotting, Western blotting and immunohistochemistry. ► The detection limit by dot blotting was about 100 times less than that of 1-step PCR. ► MAbs targeted to different epitopes can be combined for producing of a strip test.