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

针对S-PZP的一个糖蛋白家族(ZP4)，制备了5株MADs，分别为2A1、2G3、4A2、4E12和5H4。各单抗不仅可与S-PZP反应，还可与经三氟甲磺酸(TFMS)处理的脱糖S-PZP反应。它们还与猪和人卵母细胞的完整ZP反应。5株单抗中有3株(4A4、4E12和5H4)对人精子与人ZP的结合和穿透有明显的阻断作用。5H4与ZP4和ZP1糖蛋白家族有较强的反应，其余4株单抗与ZP3的反应强于ZP4。5H4与S-PZP的反应性被胰凝乳酶消化破坏，但其抗原表位对胰酶和赖氨酸内肽酶的蛋白水解酶有抵抗力。赖氨酸内切酶处理的ZP4糖蛋白经反相高效液相色谱分离得到一段与5H4反应的多肽片段，经SDS-PAGE测定其相对分子质量为7kd。这些结果表明，5H4的抗原表位可能是开发避孕疫苗的一个很好的候选者。为了分离ZP4的cDNA，我们构建了猪卵巢的cDNA文库，并用5H4进行了抗体筛选。分离了与5H4反应的3个克隆，并测定了它们的核苷酸序列。其中一个克隆与胶原蛋白基因高度同源，另外两个克隆与已知序列没有同源性。但经氨基酸序列测定，没有一株含有ZP4的核苷酸序列。我们筛选了一个人卵巢的cDNA文库，分离到了2个与5H4反应的克隆，但推导的氨基酸序列与猪ZP4没有任何同源性。

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.