Generation of a recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus expressing a foreign gene under the control of a very late promoter

A recombinant Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNP V) expressing beta -galactosidase under the control of the polyhedrin promo ter was generated in our laboratory. To this end, we cloned the AgMNPV-2D g enomic DNA fragment containing the polh gene and subcloned and sequenced th e polyhedrin gene and its flanking regions. Based on this sequence informat ion, sets of primers were designed to amplify the flanking regions by PCR, including appropriate restriction sites. The transfer vector (pAgPHZ) was c onstructed by the consecutive cloning of these PCR fragments flanking the E scherichia coli LacZ gene, in place of the polh gene. pAgPHZ was used for c otransfection of UFL-AG-286 insect cells with AgMNPV-2D DNA and the require d recombinant, generated by homologous recombination with the polh locus, w as identified by its polh(-)/LacZ(+) plaque phenotype. Its genome structure was confirmed by PCR, restriction digestion and Southern blot analyses. Th e kinetics and levels of expression of beta -galactosidase in UFL-AG-286 ce lls infected with the recombinant were tested by SDS-PAGE and enzymatic act ivity assays.