Gene Cloning of a Porcine Zona Antigen Which Cross-Reacts With Human Zona Pellucida

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Five monoclonal antibodies (Mads), 2A1, 2G3, 4A2, 4E12 and 5H4, were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zona pellucida (s-PZP). Each Mab reacted not only with s-PZP, but also reacted with deglycosylated s-PZP by trifluoromethanesulfonic acid (TFMS) treatment. They also reacted with intact ZP of porcine and human oocytes. Three (4A4, 4E12 and 5H4) of the 5 Mabs showed a significant blocking effect on human sperm binding and penetration to human ZP. The 5H4 showed a strong reaction with ZP4 and ZP1 glycoprotein families, while other 4 Mabs reacted more strongly with ZP3 than ZP4. The reactivity of 5H4 with s-PZP was destroyed by chymotrypsin digestion but the antigen epitope was resistant to proteolyses by trypsin and lysylendopeptidase. A peptide fragment reactive to 5H4 was isolated by a reverse-phase HPLC of lysylendoproteinase-treated ZP4 glycoproteins and the molecular weight was determined to be 7kd by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 would be a good candidate for development of contraceptive vaccine.To isolate a cDNA for ZP4, we constructed a cDNA library of a porcine ovary, and antibody screening was done by using 5H4. The three clones that reacted with 5H4 were isolated and their nucleotide sequence determined. One of them showed extreme homology with a gene for collagen and the other two clones showed no homology with known sequences. However none of them contained the nucleotide sequence for ZP4 determined by the amino acid sequencing. We screened the cDNA library of a human ovary and isolated 2 clones that reacted with 5H4, but the deduced amino acid sequence did not show any homology with porcine ZP4.