FIGURE 2.

Identification of recombinant baculovirus containing target gene. Sf9 insect cells were cotransfected with A. californica multiple nuclear polyhedrosis virus linear DNA and baculovirus transfer vectors pBACgus-70. A, cotransfected Sf9 insect cells were overlaid with SeaPlaque agarose and grown for 5 days. 5-bromo-4-chloro-3-indolyl-b-d-glucuronide (50 μl) was spread on each plate. After 3–24 h, blue plaque identified the cells containing potential recombinant virus. The recombinant virus was purified by three rounds of plaque assay. B, recombinant viral genome was extracted and verified by PCR using EcoRV forward and DOWN1629 reverse primers as described under “Experimental Procedures.” Lane 1, 1-kb DNA ladder; lane 2, pBACgus-2cp transfer vector as negative control; lane 3, pBACgus-70 transfer vector as positive control; lanes 4-18, different viral DNA extracted from independent recombinant viral isolates. The arrow indicated the recombinant baculovirus containing the hsp72 gene within its genome. The results were representative experiments from at least 10 independently performed experiments with similar results.