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

与人透明带交叉反应的猪带状抗原的基因克隆

基本信息

批准号：
01570944
负责人：
KOYAMA Koji
金额：
$ 1.34万
依托单位：
Hyogo College of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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.

从猪透明带（s-PZP）糖蛋白家族（ZP4）中分离得到5个单克隆抗体（Mads），分别为2A1、2G3、4A2、4E12和5H4。每个单抗不仅能与s-PZP反应，还能与经三氟甲烷磺酸（TFMS）处理的去糖基化s-PZP反应。他们还与猪和人卵母细胞的完整ZP反应。5个单抗中有3个（4A4、4E12和5H4）对人类精子结合和穿透人类ZP具有显著的阻断作用。5H4与ZP4和ZP1糖蛋白家族反应强烈，而其他4个单抗与ZP3的反应较ZP4强烈。5H4与s-PZP的反应性被胰凝乳酶消化破坏，但抗原表位对胰蛋白酶和赖氨酸肽酶的蛋白水解具有抗性。用反相高效液相色谱法分离了与5H4反应的ZP4糖蛋白片段，SDS-PAGE测定其分子量为7kd。这些结果表明，5H4抗原表位是开发避孕疫苗的一个很好的候选抗原。为了分离ZP4基因，我们构建了猪卵巢cDNA文库，并利用5H4进行抗体筛选。分离出与5H4反应的3个克隆并测定其核苷酸序列。其中一个克隆与胶原蛋白的基因极度同源，另外两个克隆与已知序列无同源性。但经氨基酸测序均不含ZP4的核苷酸序列。我们筛选了人类卵巢的cDNA文库，分离出2个与5H4反应的克隆，但推导出的氨基酸序列与猪ZP4没有同源性。