Expression of the Epstein-Barr virus envelope fusion glycoprotein gp85 gene by a recombinant baculovirus.
The gp85 envelope glycoprotein of Epstein-Barr virus (EBV) has a role in the molecular mechanism of infection, enabling fusion between the viral and host cell envelopes, a role in common with the homologous gH glycoproteins in other herpesviruses. A glutathione S-transferase bacterial fusion protein (GST85N-S) was generated, containing 178 amino acids from the C terminus of gp85 and including a known gp85 linear epitope. A panel of EBV-positive human antisera contained no antibodies to linear epitopes presented on the purified GST85N-S protein, indicating that primary protein structure in this region of gp85 is not a B cell target. This bacterial fusion protein was used to raise a rabbit monospecific polyclonal antiserum capable of detecting gp85 in a Western blot. The majority of recombinant baculovirus-expressed gp85 obtained from cell extracts prepared with SDS appeared on Western blots as heterogeneous high M(r) protein aggregates and consistently included 84K, 81K and 70K bands. Recombinant gp85 aggregation was increased by boiling the sample prior to gel electrophoresis. The 84K and 81K proteins were completely sensitive to endoglycosidase H treatment, indicating that these glycosylated species did not undergo further post-translational processing. Immunofluorescence studies revealed that recombinant gp85 was not transported to the insect cell surface. It reacted only with antibodies recognizing denatured gp85 and not with antibody to native gp85. Therefore expression of the gene encoding gp85, BXLF2, alone in the baculovirus expression system is insufficient for the synthesis of a correctly transported, processed, folded and antigenically native form of recombinant gp85.
Pulford
D
D
Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, U.K.
Lowrey
P
P
Morgan
A J
AJ
D000818:Animals; D000906:Antibodies; D016367:Baculoviridae; D001483:Base Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D005260:Female; D005814:Genes, Viral; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000096:biosynthesis; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007106:Immune Sera; D007313:Insecta; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014759:Viral Envelope Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D000906:Antibodies; D017931:DNA Primers; D000939:Epitopes; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C090195:glycoprotein 85, Epstein-Barr virus; D005982:Glutathione Transferase