Strong and regulated expression of Escherichia coli beta-galactosidase in insect cells with a baculovirus vector.
The N-terminal region of the gene encoding polyhedrin, the major occlusion protein of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), has been fused to DNA encoding Escherichia coli beta-galactosidase. The fused gene was inserted into the AcNPV DNA genome by cotransfection of insect cells with recombinant plasmid DNA and wild-type AcNPV genomic DNA. Recombinant viruses were selected as blue plaques in the presence of a beta-galactosidase indicator, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Studies of one such virus, L1GP-gal3, indicated that the synthesis of beta-galactosidase is temporally controlled beginning late (20 h) in infection after the release of infectious virus particles from the cell. By 48 h postinfection, a remarkably high level of expression is achieved. On the basis of these results, AcNPV should be a useful vector for the stable propagation and expression of passenger genes in a lepidopteran cell background. A generalized transplacement vector that facilitates the construction and selection of recombinant viruses carrying passenger genes under their own promoter control has also been developed.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007304:Insect Viruses / Q000235:genetics*; D007915:Lepidoptera; D010957:Plasmids; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase