A HUMAN SPERM ANTIGEN AS AN IMMUNOCONTRACEPTIVE

人类精子抗原作为免疫避孕药

• 批准号: 3757360
• 负责人: MICHAEL G O'RAND
• 金额: --
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3757360
• 关键词: Escherichia coli; active immunization; antiserum; biological models; chimeric proteins; complementary DNA; contraceptives; egg /ovum; epitope mapping; female; fertility immunology; human genetic material tag; human tissue; immunopathology; in vitro fertilization; laboratory mouse; laboratory rabbit; northern blottings; nucleic acid sequence; protein sequence; sperm; surface antigens; synthetic peptide; synthetic vaccines

This application addresses the problem of prefertilization immunocontraception from the perspective of a defined mammalian sperm surface component and its role in achieving contraception by the antigen's specific antibody interfering with sperm-zona binding. This study will utilize a defined human sperm antigen (hspa18) whose function is to bind human spermatozoa to the zona pellucida. This proposal is organized into six specific aims to address the molecular biology of the antigen and the immunological aspects of preventing fertilization. The specific aims are: I. The human sperm antigen hspa18 will be characterized by sequencing the human cDNA and the sequence confirmed by partial sequencing of the native molecule. II. The tissue specificity of hspa18 will be determined by Northern blot analysis of RNA from various human and primate organs using the cDNA as a probe. Additionally, a fusion protein to hspa18 will be produced in E. coli. III. Hspa18 has been characterized by the monoclonal antibody MA-51 which inhibits sperm penetration into zona-free hamster oocytes. Therefore, the MA-51 epitope in the hspa18 sequence will be determined. A synthetic peptide of the MA-51 epitope on hspa18 will be made and coupled to KLH for immunization of female mice. IV. The analogous hspa18 antigen in mice has been identified by polyclonal antisera. It will be sequenced from a mouse testis cDNA library to provide a comparative sequence whose fusion protein can be tested as an immunocontraceptive. The mouse model will allow any immunopathological effects to be evaluated. V. Female mice will be actively immunized with the fusion protein from AIM 2, with the synthetic peptide(s) from AIM 3, and with the fusion protein of the mouse antigen sequenced in AIM 4. After insemination, their fertility will be determined. Control mice will be actively immunized with the fusion protein which does not contain the hspa18 or mouse antigen insert molecule (B-galactosidase only) or KLH only. VI. Antisera produced by the females will be used to determine if mouse fertilization in vitro is inhibited.

本申请解决了优选化的问题， 从确定的哺乳动物精子的角度来看免疫避孕 表面组分及其在通过抗原的 干扰精子结合的特异性抗体。 本研究将 利用确定的人精子抗原（hspa 18），其功能是结合 人类精子与透明质酸的关系。 本提案分为 六个具体的目标，以解决抗原的分子生物学和 防止受精的免疫学方面。 具体目标是： I.人类精子抗原hspa 18将通过测序来表征 人cDNA和通过天然cDNA的部分测序证实的序列。 分子。 二. hspa 18的组织特异性将通过 使用北方印迹分析来自各种人类和灵长类动物器官的RNA cDNA作为探针。 另外，将制备与hspa 18的融合蛋白。 在E.杆菌 三. Hspa 18的特点是单克隆抗体， 抗体MA-51，其抑制精子穿透无透明带仓鼠 卵母细胞 因此，hspa 18序列中的MA-51表位将是 测定 将合成hspa 18上MA-51表位的合成肽。 制备并与KLH偶联用于雌性小鼠的免疫。 四. 的 小鼠中类似的hspa 18抗原已经通过多克隆抗体鉴定， 抗血清 它将从小鼠睾丸cDNA文库中测序， 其融合蛋白可以作为比较序列进行测试， 免疫避孕。 小鼠模型将允许任何免疫病理学 效果有待评估。 V.雌性小鼠将被主动免疫， 来自AIM 2的融合蛋白，与来自AIM 3的合成肽， 并与AIM 4中测序的小鼠抗原的融合蛋白进行比较。 后 受精，他们的生育能力将被确定。 对照小鼠将 用不含所述抗体的融合蛋白主动免疫， hspa 18或小鼠抗原插入分子（仅B-半乳糖苷酶）或仅KLH。 六.雌性产生的抗血清将用于确定小鼠是否 体外受精受到抑制。