IDENTIFICATION OF RECOMBINANT BACULOVIRUSES USING GREEN FLUORESCENT PROTEIN AS A SELECTABLE MARKER

A rapid procedure for the production and identification of recombinant baculoviruses is described that uses the autofluorescent properties o f the Aquorea victoria green fluorescent protein (GFP). Expression of the GFP cDNA (without signal peptide sequence) in Spodoptera frugiperd a cells resulted in the synthesis of a 30-kDa protein, which was confi rmed as GFP by Western blotting and by the emission of green fluoresce nce when illuminated with longwave UV light (495 or 365 nm). To use GF P as a marker for the selection of recombinant baculoviruses, we prepa red a virus, BacGFP1, in which the GFP cDNA was inserted in lieu of la cZ in BacPAK6. Before the use of BacPAK6 or BacGFP1 in a cotransfectio n to prepare recombinant bac uloviruses, the virus DNA was linearized with Bsu36I to improve the recovery of nonparental virus plaques. The use of BacGFP1 DNA resulted in the recovery of 79%-91% plaques with th e non-parental phenotype. Plaques were rapidly identified by simply ex posing them briefly to longwave UV light (365 nm) without the need for exogenous substrates or biological stains.