We have constructed a transfer vector (pAgGal) containing the β-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin ( polh ) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A . gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The β-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 infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. β-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed β-galactosidase protein was also shown to be biologically active by a β-galactosidase assay.
Archives of Virology – Springer Journals
Published: Jul 1, 2001
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