PROTEIN IMMUNOGENS OF THE HUMAN SPERM SURFACE

该研究计划的目标是克隆和表征蛋白质 它们位于人类精子的表面， 免疫避孕的潜力。 请注意， 作为自身抗原或同种抗原并被精子识别的蛋白质 凝集抗体 计算机化的二维数据库， 人类精子蛋白在二维凝胶中的迁移已经被 开发 在这个数据库中，98个人类精子表面的子集 表面碘化和生物素化的蛋白质已经被 鉴定 这些表面蛋白在二维空间中的位置 凝胶已经被编入精子蛋白百科全书。 的子集 这些精子表面蛋白将被微测序， 通过分子克隆获得阅读框。 独特的精子 蛋白质将被表征和评估为潜在的避孕药 根据严格的标准，包括睾丸特异性 并参与精子表面介导的功能。 具体目的是：1）对人类精子表面进行微序列分析 通过Edman降解和/或串联降解的组合， 2）克隆和测序完整的开放阅读 估计有20个代表独特精子表面的cDNA的框架 使用分子方法的组合; 3）为了确定 通过北方印迹、RT-PCR和 人和非人器官库免疫组化分析 人类灵长类动物mRNA和组织; 4）确认精子表面 候选免疫原的定位和测试其避孕 通过体外功能测定的潜力;和5）进行 几种新型精子表面免疫原性和生育力试验 小鼠和猕猴体内的蛋白质。 这些目标的实现将增进我们对 暴露在人体外部的蛋白质的分子特征 并有助于选择适合于 通过永生化和操纵cDNA的人免疫避孕 编码这些蛋白质。

The goal of this research program is to clone and characterize proteins which are located on the surface of the human sperm and to evaluate their immunocontraceptive potential. Attention will be directed to proteins which are auto- or iso-antigens and are recognized by sperm agglutinating antibodies. A computerized 2-D database based on migration of human sperm proteins in two dimensional gels has been developed. Within this database a subset of 98 human sperm surface proteins accessible to surface iodination and biotinylation has been identified. The locations of these surface proteins in two dimensional gels have been catalogued in a sperm protein encyclopedia. A subset of these sperm surface proteins will be microsequenced, and complete open reading frames will be obtained by molecular cloning. Unique sperm proteins will be characterized and evaluated as potential contraceptive immunogens according to strict criteria which include testis specificity and involvement in functions mediated at the sperm surface. The specific aims are: 1) To microsequence human sperm surface proteins through a combination of Edman degradation and/or tandem mass spectrometry; 2) To clone and sequence the complete open reading frames for an estimated 20 cDNAs representing unique sperm surface proteins using a combination of molecular approaches; 3) To determine tissue specificity of the cloned cDNAs by Northern blot, RT-PCR, and immunohistochemical analyses on an organ bank of human and non- human primate mRNAs and tissues; 4) To confirm the sperm surface localization of candidate immunogens and test their contraceptive potential through in vitro functional assays; and 5) To conduct immunogenicity and fertility trials of several novel sperm surface proteins in mice and macaques. Achievement of these aims will increase our understanding of the molecular character of proteins exposed on the exterior of the human sperm surface and aid in selection of candidate antigens appropriate for human immunocontraception by immortalizing and manipulating the cDNAs encoding these proteins.