IMMUNOSUPPRESSION OF FERTILITY BY HUMAN LDH-C4

这项研究计划继续并完善了长期目标， 开发基于睾丸特异性 乳酸脱氢酶同工酶（LDH-C4; LDH-X）。 以前的研究 已经鉴定了小鼠LDH-C4的几个表位并定位它们 在分子的晶体结构中。 我们提出 将这些发现外推到研究免疫避孕 基于cDNA序列的人同工酶的性质， 我们最近决定。 基因工程将被用来生产大量的 用于生物化学和免疫学研究。 Ldh-c cDNA的5'和3'末端将被工程化，使得 编码区的侧翼是限制酶位点。 这 1）PKK 223 -3用于转化 E. 2）用于与杆状病毒重组的pAc 373， 随后感染Sf 9昆虫细胞。 人LDH-C4将 进行纯化和鉴定。 小鼠和人LDH-C4的结构信息将用于 设计模拟地形决定簇的合成肽 的分子。 将特别强调稳定 将两亲肽转化为预定的超二级结构 代表它们在天然分子中的构型。 这些 将研究肽的化学和免疫学性质， 特性. 作为肽抗原的替代品，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制造疫苗。 这将 通过胸苷激酶 野生型牛痘和pGS 20 +Ldh-c的序列。 重组体 (tk-)在人143 B细胞（tk-）中生长的细胞将通过 pGS 20 +Ldh-c。 在人143 B细胞（tk-）中生长的促凝剂（tk-） 将通过对5'溴脱氧尿苷的抗性来选择。 将测试产生的每种肽和病毒载体的 它在实验动物模型中激发抗体的能力。 推定的T细胞表位将用于刺激次级T细胞免疫应答。 体外增殖。 抗体对精子的影响 在体外使用人精子和仓鼠卵进行评估 受精测定 候选避孕抗原将是 在体内测试受精前和受精后的效果。

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.