IMMUNOLOCALIZATION OF THE PSEUDORABIES VIRUS IMMEDIATE-EARLY PROTEIN IE180 BY IMMUNOPEROXIDASE STAINING

The immediate-early (IE) gene of pseudorabies virus (PRV) expresses im mediately upon infection, a phosphorylated protein (immediate-early pr otein, IE180) that can transactivate viral other genes and plays an es sential role in regulating viral gene expression. In order to detect a nd localize IE180 in infected cells early on, this gene was cloned for overexpression, and the expressed products were applied to generate s pecific antibodies against IE180 protein. Two recombinant expression p lasmids pN and pNB were constructed by cloning the IE gene onto pET 30 a(+) expression vector via NcoI and BamHI sites. Plasmid pN contains t he 1.8-kb NcoI-NcoI fragment of IE gene coding for the N-terminus of 6 16 amino acid residues, while pNB contains the 2.8-kb NcoI-BamHI fragm ent coding for the rest of the IE180 protein. Both pN and pNB were tra nsformed, respectively, into E. coli cells and produced large amounts of IE protein products during induction with 1 mM IPTG. The expressed IE proteins for pN and pNB were 60 kDa and 100 kDa in size, respective ly. These expression products were purified and then used as antigens to immunize mice for preparing specific antibodies against PRV IE180 p rotein. The specificities of the mice immune sera were confirmed by th eir abilities to react with IE180 protein present in the PRV infected cells in the Western immunoblotting assay. Furthermore, immunoperoxida se staining of PRV infected cells undertaken with these antisera revea led the subcellular distribution of the IE proteins in the infected ce lls and also demonstrated their transportation from the cytoplasm to t he nucleus during infection. (C) 1997 Elsevier Science B.V.