Thesis (Ph.D.) - Cairo University - Faculty of Veterinary Medicine - Department of Virology

The glycoprotein D (gD) gene of bovine herpesvirus-1.1 Egyptian strain 2Abu-Hammad3 was cloned and expressed in spodopetra Frugiperda (Sf9) insect cells using baculovirus expression system. Full length gD encoding sequence was amplified by polymerase chain reaction (PCR) and cloned into the baculovirus shuttle vector; pMelBac B. The cloned gene was inserted in the genome of autographa california nuclear polyhydrosis virus (AcMNPV) under control of the polyhedrin promoter, through homologous recombination between the recombinant pMel/gD and a linearized triple cut baculovirus DNA (Bac-N-Blue) using liposome mediated transfection on to Sf9 cells. Recombinant baculovirus was selectively purified by plaque assay and verified for integrity of gD gene of BoHV-1.1 using PCR. The recombinant gD protein maintained their antigenic properties as determined by its reactivity with anti BoHV-1 positive serum using situ immunofluorescent and Western blot assays. The secreted recombinant gD in culture medium of infected insect cells was used as a coating antigen in an indirect enzyme linked immunosorbent assay (ELISA) to test its utility for detection of antibody against gD of BoHV-1. This ELISA was compared to standard virus neutralization test for detection of anti-BoHV-1antibody in a panal of bovine sera demonstrating closely correlated antibody titers in both. The developed indirect gD-ELISA was a reliable candidate for detection and validation of BoHV-1seropositive animals with high speci{uFB01}city

Issued also as CD