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 expression system to obtain nonfusion glycosylated recombinant ZP protein (BV-55). SDS-PAGE and immunoblot analysis demonstrated that the recombinant protein is expressed as two forms having relative molecular masses of 70 kDa and 80 kDa. Because cells treated with tunicamycin produce predominantly the 70-kDa form, this heterogeneity is presumed to be due to differential glycosylation. Further studies using lectin blot and immunoblot analyses showed that the BV-55 protein has both N-linked and O-linked oligosaccharides. However, this glycosylation is distinct from that of the native 55-kDa ZP protein, since it was not recognized by a monoclonal antibody associated with lactosaminoglycan-type carbohydrate epitopes in native ZP proteins. Immunogenicity studies demonstrated that antibodies against the BV-55 protein are developed by female rabbits and guinea pigs and that these antibodies recognize epitopes associated with native, enzyme-deglycosylated as well as nonglycosylated 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 expression of the 55-kDa recombinant protein in the baculovirus expression system enhances its immunogenicity.
Prasad
S V
SV
Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mujtaba
S
S
Lee
V H
VH
Dunbar
B S
BS
D000818:Animals; D000906:Antibodies / Q000097:blood / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D004527:Egg Proteins / Q000235:genetics / Q000276:immunology*; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D006168:Guinea Pigs; D007114:Immunization; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D018411:Spodoptera / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D015044:Zona Pellucida / Q000737:chemistry*
D000906:Antibodies; D000941:Antigens; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; C094916:zona pellucida protein, 55-kDa, Oryctolagus cuniculus; D014415:Tunicamycin