Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene

We have constructed a transfer vector (pAgGal) containing the beta -galacto sidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Ant icarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UF L-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The beta -galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were in fected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the p roduction of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. beta -galactosidase was expressed at high levels late on infection as expe cted for a gene under the control of the polh promoter. The highly expresse d beta -galactosidase protein was also shown to be biologically active by a beta -galactosidase assay.