A HUMAN SPERM ANTIGEN AS AN IMMUNOCONTRACEPTIVE

人类精子抗原作为免疫避孕药

• 批准号: 3735611
• 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/3735611
• 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.

该应用程序解决了预受精的问题 从特定哺乳动物精子的角度进行免疫避孕 表面成分及其在抗原避孕中的作用 干扰精子带结合的特异性抗体。这项研究将 利用定义的人类精子抗原(Hspa18)，其功能是结合 人类精子到透明带。这项建议被组织成 六个具体的目标是解决抗原的分子生物学和 预防受精的免疫学方面。具体目标是： I.人类精子抗原hspa18将通过对 人的cDNAs及经部分测序证实的序列 分子。Ii.hspa18的组织特异性将由 人和灵长类不同器官RNA的Northern印迹分析 将该基因作为探针。此外，hspa18的融合蛋白将是 在大肠杆菌中生产。三、Hspa18的单克隆性 抑制精子穿透去透明带仓鼠的抗体MA-51 卵母细胞。因此，hspa18序列中的MA-51表位将是 下定决心。Hspa18上MA-51表位的合成肽将是 制备并偶联KLH免疫雌性小鼠。IV. 用多克隆方法在小鼠中鉴定出类似的hspa18抗原 抗血清。它将从小鼠睾丸cDNA文库中进行测序，以提供 一个比较序列，其融合蛋白可以作为 免疫避孕药。小鼠模型将允许任何免疫病理 待评估的效果。V.雌性小鼠将积极接种 将AIM 2的融合蛋白与AIM 3的合成肽(S)进行融合， 并与小鼠抗原融合蛋白在AIM 4中测序。 受精，他们的生育能力将被确定。对照组小鼠将被 用不含该融合蛋白的融合蛋白主动免疫 Hspa18或小鼠抗原插入分子(仅限B-半乳糖苷酶)或仅限KLH。 雌性产生的抗血清将被用来确定小鼠 体外受精受到抑制。