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

从热溶猪透明质酸（s-PZP）中分离到一个糖蛋白家族（ZP 4），制备了2A 1、2G 3、4A 2、4 E12和5 H4五个单克隆抗体（Mads）。每种单抗不仅与s-PZP反应，而且通过三氟甲磺酸（TFMS）处理与去糖基化的s-PZP反应。它们还与猪和人卵母细胞的完整ZP反应。5种单抗中有3种（4A 4、4 E12和5 H4）对人精子与人ZP的结合和穿透显示出显著的阻断作用。5 H4与ZP 4和ZP 1糖蛋白家族表现出较强的反应，而其他4种单克隆抗体与ZP 3的反应比ZP 4更强。5 H4与s-PZP的反应性被糜蛋白酶消化破坏，但抗原表位对胰蛋白酶和赖氨酰内肽酶的蛋白水解具有抗性。用反相高效液相色谱法分离到一个能与5 H4反应的肽段，经SDS-PAGE测得其分子量为7 kd。本研究构建了猪卵巢组织cDNA文库，并利用5 H4进行抗体筛选。分离与5 H4反应的三个克隆并测定其核苷酸序列。其中一个克隆与胶原蛋白基因具有高度同源性，另外两个克隆与已知序列没有同源性。然而，它们中没有一个含有通过氨基酸测序确定的ZP 4的核苷酸序列。我们从人卵巢cDNA文库中筛选出2个与5 H4反应的克隆，但推导的氨基酸序列与猪ZP 4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.