Georgia
Veterinary Scholar
Program

GLYCOSYLATION OF RECOMBINANT PORCINE ZONA PELLUCIDA.

The zona pellucida surrounding the mammalian oocyte is a nutritive and protective paracrystalline matrix. Glycosylation of the zona pellucida is involved with sperm-egg interaction by providing sperm binding sites. The porcine zonae is comprised of three major glycoproteins: ZP1; ZP3?? and ZP3?. ZP3? and ZP3? are involved with sperm binding and stabilization of the sperm-egg interaction. An antigen that initiates an immune response against these carbohydrate moieties could mechanically disrupt the sperm-egg interaction. Recombinant proteins modeled after pZP3? and pZP3? are, therefore, produced as a potential immunocontraceptive. The recombinant porcine zona pellucida vaccine is produced using Trichoplusia ni- High FiveTM insect cell lines. While the genetic information for the protein component of the ZP is encoded in the transfected plasmid, carbohydrate moieties are added in the ER and modified in the secretory pathway. This experiment will determine if recombinant protein undergoes the same post-translational glycosylation in insect cells as in porcine cells. We established cell cultures that synthesize recombinant protein. To conduct carbohydrate composition analysis, we produced ~600mg of each pZP3? and pZP3?. The harvested protein was purified and analyzed using Roche DIG Glycan Detection Kit, an immunoassay. Once we affirmed the presence of glycoconjugates on the recombinant protein, the samples were electrophoresed, transferred to PVDF membranes and stained. The recombinant bands were then cut from the membrane and analyzed by Dr. Roberta Merkle at CCRC to determine: (i) molar ratio of individual sugars to protein; (ii) class of the oligosaccharides present. Knowing the glycosyl composition of the recombinant glycoprotein may elucidate carbohydrate roles in the sperm-egg interaction by allowing comparison of recombinant to native porcine ZP glycosylation and comparison of immunogenicity of recombinant with native vaccine.