IMMUNOSUPPRESSION OF FERTILITY BY HUMAN LDH-C4

这项研究计划继续并细化了以下长期目标 基于睾丸特异性的免疫避孕药研究进展 乳酸脱氢酶同工酶(LDH-C4；LDH-X)。以前的研究 已经确定了小鼠LDH-C4的几个表位并对它们进行了定位 在分子的晶体结构中。我们建议 来推断这些发现来研究免疫避孕药 基于人类同工酶的基因序列的性质 我们最近确定了。 基因工程将被用来大量生产 用于生化和免疫学研究的人类蛋白质。 LDH-c基因的5‘和3’末端将被设计成 编码区两侧有限制性内切酶位点。这 将片段插入：1)用于转化的PKK223-3 2)pAc373与杆状病毒重组； 随后感染Sf9昆虫细胞。人类的乳酸脱氢酶-C4将 进行提纯和鉴定。 小鼠和人类LDH-C4的结构信息将用于 设计模拟地形决定因素的合成肽 分子的分子。将特别强调稳定 两亲性多肽进入预定的超二级结构 代表它们在天然分子中的构型。这些 将对多肽进行化学和免疫学研究。 属性。 作为多肽抗原的替代品，我们还将开发一种病毒 通过将人类基因克隆到牛痘病毒中来研制疫苗。这将是 是通过胸苷激酶之间的重组来实现的 野生型痘苗病毒和pGS20+LDH-c的序列分析。重组体 在人143B细胞(tk-)中生长的(tk-)将通过 PGS20+LDH-c。重组体(tk-)在人143B细胞中生长(tk-) 将通过对5‘-溴脱氧尿苷的抗性进行选择。 生产的每一种多肽和病毒载体都将进行测试 它在实验动物模型中激发抗体的能力。 假定的T细胞表位将被用来刺激次级T细胞 体外增殖。抗体对精子意志的影响 用人类精子和仓鼠卵子在体外进行评估 受精试验。候选避孕抗原将是 在体内测试受精前后的效果。

This research plan continues and refines the long-term goal of developing immunocontraceptives based on the testis-specific lactate dehydrogenase isozyme (LDH-C4; LDH-X). Previous studies have identified several epitopes of mouse LDH-C4 and located them within the crystallographic structure of the molecule. We propose to extrapolate these findings to study the immunocontraceptive properties of the human isozyme based on the cDNA sequence which we have recently determined. Genetic engineering will be employed to produce large quantities of the human protein for biochemical and immunological studies. The 5' and 3' ends of the Ldh-c cDNA will be engineered such that the coding region is flanked by restriction enzyme sites. This fragment will be inserted into: 1) PKK223-3 for transformation of E. coli, and 2) pAc373 for recombination with Baculovirus and subsequent infection of Sf9 insect cells. The human LDH-C4 will be purified and characterized. Structural information on mouse and human LDH-C4 will be used to design synthetic peptides which mimic the topographic determinants of the molecule. Particular emphasis will be placed on stabilizing amphipathic peptides into predetermined supersecondary structures that represent their configuration in the native molecule. These peptides will be studied for their chemical and immunological properties. As an alternative to peptide antigens, we will also develop a viral vaccine by cloning the human gene into vaccinia virus. This will be accomplished by recombination between the thymidine kinase sequences of wild-type vaccinia and pGS20+Ldh-c. Recombinants (tk-) grown in human 143B cells (tk-) will be selected by pGS20+Ldh-c. Recombinants (tk-) grown in human 143B cells (tk-) will be selected by resistance to 5' bromodeoxyuridine. Each of the peptides and viral vectors produced will be tested for its ability to evoke antibodies in an experimental animal model. Putative T-cell epitopes will be used to stimulate secondary T-cell proliferation in vitro. The effect of the antibodies on sperm will be assessed using human sperm and hamster ova in an in vitro fertilization assay. Candidate contraceptive antigens will be tested for pre- and post-fertilization effects in vivo.