PROTEIN IMMUNOGENS OF THE HUMAN SPERM SURFACE

这项研究计划的目标是克隆和鉴定蛋白质 它们位于人类精子表面，用来评估 它们的免疫避孕潜力。我们将把注意力集中到 可被精子识别的自身或同种抗原的蛋白质 凝集抗体。一种基于计算机的二维数据库 人精子蛋白在二维凝胶中的迁移 发展起来的。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可获得的蛋白质 已确认身份。这些表面蛋白在二维中的位置 《精子蛋白百科全书》对凝胶进行了分类。的子集 这些精子表面蛋白将被微测序，并完全开放 阅读框将通过分子克隆获得。独特的精子 蛋白质将被鉴定和评估为潜在的避孕药 符合包括睾丸特异性在内的严格标准的免疫原 以及参与精子表面的调节功能。 具体目标是：1)对人类精子表面进行微序列 通过Edman降解和/或串联相结合的蛋白质 2)对完整的开放阅读片段进行克隆和测序 大约20个代表独特精子表面的cDNA的框架 蛋白质的分子组合方法；3)确定 Northern印迹、RT-PCR和免疫印迹法检测克隆的组织特异性 人与非人类器官库的免疫组织化学分析 人类灵长类mRNAs和组织；4)确认精子表面 候选免疫原的定位及其避孕效果检测 通过体外功能分析的潜力；以及5)进行 几种新型精子表面的免疫原性和生育力试验 老鼠和猕猴体内的蛋白质。 这些目标的实现将增加我们对 人体体表暴露蛋白质的分子特征 精子表面和辅助选择合适的候选抗原 通过永生和操纵cDNA实现人类免疫避孕 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.