Designing of a Recombinant Agip Bacmid Construct with Infectious Properties Against Black Cutworm Agrotis ipsilon Larvae

Abstract

In this study, Agrotis ipsilon nucleopolyhedrovirus bacmid has been constructed as an infectious bacmid in an attempt to allow genome recombination and generation of virus mutants. Since the FseI, a unique restriction site, is located in a viral coding region (ORF_119), PCR was performed to partially amplify the ORF_119 fragment containing the FseI site to facilitate the bacmid construction in a proper way without interrupting the ORF expression. Construction with repeated fragments at the end of the cloned viral was carried out in an attempt to facilitate circulation during infection in insect cells. The amplified gp_119 fragment was cloned into the BAC_Bsu361 plasmid derived from the AcMNPV Bac-to-Bac® system. Recombinant plasmid was used to subclone the Agrotis ipsilon nucleopolyhedrovirus (AgipNPV)-linearized genome using the FseI unique site. The Agip bacmid DNA extracted from Escherichia coli was used to transfect A. ipsilon third instar larvae by injection into the hemolymph. The produced occlusion bodies were purified from infected larvae and used to feed healthy larvae for amplifying the virus, and infectivity was recorded. Using bacmid technology will facilitate manipulation of the AgipNPV genome and help in determining the genetic factors involved in virus virulence and biology.