Characterization of immune response in mice to plasmid DNA encoding dog zona pellucida glycoprotein-3

To investigate the immunogenicity of plasmid DNA encoding dog zona pellucida glycoprotein-3 (dZP3), the cDNA corresponding to dZP3, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRdZP3). In vitro transfection of COS-1 mammalian cells with VRdZP3 plasmid DNA led to its cytosolic expression. The expressed dZP3 has an apparent molecular weight of 45 kDa as compared to calculated molecular weight of 38.4 kDa, suggesting possible glycosylation. Immunization of male BALB/cJ mice with VRdZP3 plasmid DNA in saline, by electroporation or adsorbed onto gold microcarriers (delivered by gene gun) generated antibody response against Escherichia coli expressed recombinant dZP3 (r-dZP3). Administration of r-dZP3 in saline following immunization with plasmid DNA led to boosting of the antibody response. Although mice immunized with gene gun exhibited highest antibody titres, the differences in the antibody titres seen by the three modes of plasmid DNA delivery were not statistically significant ( P>0.05). Interestingly, female mice immunized with VRdZP3 plasmid DNA using gene gun also generated antibodies against r-dZP3. A dominant IgG1 isotype response was observed in mice immunized with VRdZP3 plasmid DNA using gene gun as compared to a mixed IgG1–IgG2a isotype response when delivered in saline or by electroporation. Immunization with VRdZP3 plasmid DNA also generated cell mediated immune response. The antibodies generated by VRdZP3 plasmid DNA recognized dog native zona pellucida. These studies for the first time, demonstrate the feasibility of generating an immune response to ZP3 by DNA vaccine and that the antibodies thus generated recognize native zona pellucida.