HUMAN TESTIS CDNAS IDENTIFIED BY INFERTILITY SERA

不育症血清鉴定的人类睾丸 CDNAS

• 批准号: 6108642
• 负责人: ERWIN GOLDBERG
• 金额: $ 27万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6108642
• 关键词: Escherichia coli; Macaca fascicularis; active immunization; calpain; chimeric proteins; complementary DNA; contraceptives; enzyme inhibitors; female antifertility drug; fertility immunology; gene targeting; genetically modified animals; human tissue; laboratory mouse; laboratory rabbit; northern blottings; protein purification; surface antigens; testis; transfection; vaccine development; vector vaccine; western blottings

The long-term objective of this research plan is to develop a safe and effective immuocontraceptive for use by women. In previous studies, a completely synthetic chimeric peptide based on the sequence of sperm- specific lactate dehydrogenase (LDH-C4 epitope HC-5-19-TT) reversibly suppressed fertility in female baboons by 70 percent. In the current project period, we used sera from immunologically infertile patients to identify three new sperm-specific antigens which can be used alone or in combination with HC5-19-TT to increase the protection from pregnancy to greater than 90 percent. The immediate goal of this project is to test the safety and efficacy of a B-cell epitope from one of these antigens, the sperm-specific isoform of calpastatin (t-CAST), alone and in combination with the LDH-C4 epitope. Calpastatin is the peptide inhibitor of calpain, a cysteine protease that plays an important preteolytic role in vesicle exocytosis and membrane fusion events. Antibodies to t-CAST in the serum of an infertile patient (YM) agglutinated human sperm and inhibited sperm penetration of cervical mucus. A synthetic chimeric B-cell epitope of the protein (HTC25-41-TT) was immunogenic in rabbits and female macaques, and the anti-peptide sera agglutinated and immobilized human sperm. Therefore, t-CAST is present on the surface of acrosome intact sperm, and is a primary candidate for development of an immunocontraceptive. Four Specific Aims are proposed: 1) to establish the functional role of t-CAST during fertilization by targeted gene disruption in mice; 2) to establish contraceptive efficacy and reversibility of HTC25-41-TT in female cynomologus macaques; 3) to test a subunit vaccine composed of both HTC25-41-TT and HC5-19-TT in female macaques; and 4) to engineer E. coli to express large amounts of a recombinant form of the subunit vaccine for large-scale production under Good Laboratory Practices guidelines. This technology would provide a safe and effective alternative to presently available methods of contraception.

该研究计划的长期目标是开发一种安全且 供女性使用的有效免疫避孕药。 在之前的研究中，一个 基于精子序列完全合成的嵌合肽 可逆地特异性乳酸脱氢酶（LDH-C4 表位 HC-5-19-TT） 雌性狒狒的生育能力下降了 70%。 在当前 项目期间，我们使用免疫不孕患者的血清 鉴定出三种新的精子特异性抗原，可以单独使用或 与 HC5-19-TT 结合使用可增强妊娠保护 达到百分之九十以上。 该项目的近期目标是 测试其中一种 B 细胞表位的安全性和有效性 抗原，即精子特异性钙蛋白酶抑制剂 (t-CAST) 单独和在 与 LDH-C4 表位结合。 Calpastatin是一种肽 钙蛋白酶抑制剂，一种半胱氨酸蛋白酶，在 囊泡胞吐作用和膜融合事件中的预水解作用。 不孕患者血清中的 t-CAST 抗体 (YM) 凝集人类精子并抑制精子穿透宫颈 粘液。 该蛋白的合成嵌合 B 细胞表位 (HTC25-41-TT) 在兔子和雌性猕猴中具有免疫原性，并且抗肽 血清凝集并固定人类精子。 因此，t-CAST 是 存在于完整精子顶体的表面，是主要的 开发免疫避孕药的候选者。 四具体 建议的目标是： 1) 确定 t-CAST 在 通过靶向基因破坏小鼠受精； 2）建立 HTC25-41-TT 对女性的避孕效果和可逆性 食蟹猴； 3）测试由两者组成的亚单位疫苗 雌性猕猴中的 HTC25-41-TT 和 HC5-19-TT； 4) 设计E。 大肠杆菌表达大量重组形式的亚基 根据良好实验室规范大规模生产疫苗 指导方针。 该技术将提供安全有效的 目前可用的避孕方法的替代方法。