HUMAN TESTIS CDNAS IDENTIFIED BY INFERTILITY SERA

这项研究计划的长期目标是开发一种安全， 妇女使用的有效免疫避孕药。 在以前的研究中， 基于精子序列的完全合成的嵌合肽- 特异性乳酸脱氢酶（LDH-C4表位HC-5-19-TT） 抑制了雌性狒狒70%的生育能力。 在当前 在项目期间，我们使用免疫不育患者的血清， 鉴定了三种新的精子特异性抗原， 与HC 5 -19-TT联合使用，以增加妊娠保护 超过90%。 该项目的近期目标是 测试来自其中之一的B细胞表位的安全性和有效性， 抗原，精子特异性钙蛋白酶抑制蛋白（t-CAST）亚型，单独和 与LDH-C4表位组合。 钙蛋白酶抑制素是一种肽 钙蛋白酶的抑制剂，一种半胱氨酸蛋白酶， 在囊泡胞吐和膜融合事件中的蛋白溶解作用。 不孕症患者血清中的t-CAST抗体（YM） 凝集人精子并抑制精子穿透宫颈 粘液 蛋白质的合成嵌合B细胞表位（HTC 25 -41-TT） 在家兔和雌性猕猴中具有免疫原性， 血清凝集并固定人精子。 因此，t-CAST是 存在于顶体完整精子的表面，并且是初级的 用于开发免疫避孕药的候选物。 四个具体 目的是：1）建立t-CAST的功能作用， 在小鼠中通过靶向基因破坏进行受精; 2）建立 HTC 25 -41-TT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 HTC 25 -41-TT和HC 5 -19-TT; 大肠杆菌表达大量重组形式的亚基 根据药物非临床研究质量管理规范进行大规模生产的疫苗 指南 这项技术将提供一种安全有效的 替代目前可用的避孕方法。

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.