IMMUNOGENICITY ENHANCEMENT OF RECOMBINANT RABBIT 55-KILODALTON ZONA-PELLUCIDA PROTEIN EXPRESSED USING THE BACULOVIRUS EXPRESSION SYSTEM

In the present study we have used a molecular approach to evaluate the immunogenicity and antigenicity of glycosylated and non-glycosylated recombinant rabbit 55-kDa zona pellucida (ZP) protein. The 55-kDa cDNA was expressed in insect (Sf9) cells through use of a baculovirus expr ession system to obtain nonfusion glycosylated recombinant ZP protein (BV-55). SDS-PAGE and immunoblot analysis demonstrated that the recomb inant protein is expressed as two forms having relative molecular mass es of 70 kDa and 80 kDa. Because cells treated with tunicamycin produc e predominantly the 70-kDa form, this heterogeneity is presumed to be due to differential glycosylation. Further studies using lectin blot a nd immunoblot analyses showed that the BV-55 protein has both N-linked and O-linked oligosaccharides. However, this glycosylation is distinc t from that of the native 55-kDa ZP protein, since it was not recogniz ed by a monoclonal antibody associated with lactosaminoglycan-type car bohydrate epitopes in native ZP proteins. Immunogenicity studies demon strated that antibodies against the BV-55 protein are developed by fem ale rabbits and guinea pigs and that these antibodies recognize epitop es associated with native, enzyme-deglycosylated as well as nonglycosy lated recombinant forms of the rabbit 55-kDa ZP protein. In contrast, recombinant protein expressed in bacteria did not elicit antibodies in either rabbits or guinea pigs. These results demonstrate that express ion of the 55-kDa recombinant protein in the baculovirus expression sy stem enhances its immunogenicity.