Characterization of hepatitis A virus capsid proteins with antisera raised to recombinant antigens.
The capsid proteins of hepatitis A virus (HAV) were expressed as fusion proteins of beta-galactosidase in E. coli using the expression vector lambda gt11. Four fusion proteins were stably expressed and used to immunize rabbits to obtain mono-specific antisera. The antisera were unable to neutralize viral infectivity or react with HAV by radioimmunoassay. Three of the antisera were able to recognize HAV antigens in infected BS-C-1 cells by immunofluorescence and denatured capsid proteins by immunoblot analysis. The antisera were used to investigate the migration of the capsid proteins in gels by immunoblot analysis using standard SDS-PAGE conditions and in gels containing urea. The migration of VP1 and VP3 correlated with their molecular weights predicted from the nucleotide sequence and was consistent in either the presence or absence of urea. However, VP2 migrated with an apparent molecular weight significantly higher than the predicted value and, in gels containing urea, migrated as a doublet. It is proposed that the upper band of this doublet represents VP0, the proteolytic precursor of VP2 and VP4. The relative molecular mass (Mr) of VP4 was estimated to be less than 1 kDa, which is substantially lower than the 2.5 kDa predicted from the nucleotide sequence.
Ross
B C
BC
Department of Clinical Pathology, Fairfield Hospital, Victoria, Australia.
Anderson
D A
DA
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology*; D002213:Capsid / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006507:Hepatovirus / Q000254:growth & development / Q000276:immunology*; D007106:Immune Sera; D015151:Immunoblotting; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D001616:beta-Galactosidase / Q000235:genetics
D000914:Antibodies, Viral; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase