Expression of bovine leukemia virus ENV glycoprotein in insect cells by recombinant baculovirus

Abstract 

The gp51-p30 glycoprotein constituting BLV envelope was expressed in Sf-21 insect cells by means of recombinant baculoviruses. Post-infection cell lysates were analyzed, in order to define the immunologic reactivity of recombinant products. Oligosaccharide chains, containing N-acetylglucosamine, mannose, galactose and sialic acid were found on recombinant gp51-p30. In order to investigate the timing of transcription and translation of the glycoprotein, kinetic assays were carried out on cell lysates and directly in situ on Sf-21 cells during the course of baculovirus infection. The use of different solubilizing reagents was also evaluated in order to rescue recombinant glycoprotein from its subcellular location.

Keywords:  Baculovirus, Biotechnology, Bovine leukemia virus, Glycosylation, Recombinant glycoprotein

Abbreviations:  AcMNPV, Autographa californica multiple nuclear polyhedrosis virus, BCA, bichinchoninic acid protein quantitative assay, BEVS, baculovirus expression vector system, BLV, bovine leukemia virus, bp, base pair, BSA, bovine serum albumin, DEPC, diethyl-pyrocarbonate, FCS, fetal calf serum, FLK, fetal lamb kidney cell line, FLK-gp51, gp51 purified from persistently infected FLK cell, gp51-p30, glycoproteins constituting BLV envelope, kbp, 10³ base pairs, kD, 10³ daltons, MAb, monoclonal antibody, MOI, multiplicity of infection, occ⁻, AcMNPV plaques showing polh⁻ phenotype, PBS, phosphate buffered saline, PCR, polymerase chain reaction, p.i., post infection, polh, polyhedrin coding gene of AcMNPV, PRINS, primed in situ labelling of nucleic acids, SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis