Construction of E. coli expression plasmid libraries: localization of a pseudorabies virus glycoprotein gene

Abstract

We describe the use of a combined cloning/expression protocol to identify a gene encoding a Pseudorabies virus (PRV) glycoprotein. Prior to this study, the genome locations of PRV glycoproteins had not been described. We first identified PRV glycoproteins using antibodies directed against PRV virions. Using affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the PRV glycoproteins were separated and antibodies were made against them in rabbits. Using DNase digestion, PRV genomic DNA was fragmented into approximately 500-base-pair regions. These random fragments were inserted in an expression vector and the PRV DNA sequences were expressed as proteins fused to beta-galactosidase. The antibodies made in rabbits were then used as probes in a Western blot analysis to screen for the presence of PRV-specific glycoprotein sequences in these PRV-beta-galactosidase fusion proteins. Two expression plasmids were isolated that specified fusion proteins that reacted in the Western blot analysis with rabbit antibodies directed against a 74,000 molecular weight PRV glycoprotein. The PRV DNA sequences represented in the expression plasmids were mapped within a single BamHI fragment of PRV genomic DNA, but were not overlapping. PRV-beta-galactosidase fusion proteins produced by these two expression plasmids were used to inoculate rabbits. Antibodies produced against both fusion proteins recognized PRV-specific glycoproteins of 74,000 and 92,000 molecular weight. This protocol should have general application in localizing genes within large DNA virus genomes.