Production of recombinant human zona pellucida protein (r-hZPA) and its application to contraceptive vaccine

哺乳动物卵母细胞周围的透明带是由三种主要糖蛋白（ZPA、ZPB、ZPC）组成的细胞外基质。由于这些成分在受精、组织特异性表达和强免疫原性方面的重要作用，它们是避孕疫苗的有希望的候选成分。然而，从天然来源制备具有一致质量的疫苗材料是不可能的。本研究在大肠杆菌中大量制备了一种重组蛋白，并对其抗原性、生物学性质和受精阻断作用进行了检测。(1)制备人透明带蛋白nh2末端区域的DNA片段，生成重组蛋白（rhZPA）。r-hZPA在包裹体的6M尿素中分离后，用Ni^树脂柱纯化。(2)利用特异性单克隆抗体证实重组蛋白与原蛋白具有相同的抗原性。它还显示出与人类精子的结合能力。(3)兔免疫产生的抗血清不仅对人有反应，而且对猪和兔透明带也有反应。结果表明，免疫区含有多种交叉反应性抗原。抗血清在体外抑制人精子与透明带的结合。(4)将rhZPA免疫于非人灵长类动物（Macaca Radiata），产生的抗血清抑制了人体外受精。总的来说，r-hZPA蛋白是开发人类使用的避孕疫苗以及宠物和野生动物的人口控制的一个有吸引力的目标。

The mammalian zona pellucida surrounding oocytes is an extra-cellular matrix composed of three major glycoproteins (ZPA, ZPB, ZPC). These components are promising candidates for a contraceptive vaccine because of their important roles at fertilization, tissue-specific expression and strong immunogenicity. However, it is impossible to prepare the vaccine material from natural source with consistent quality. In this study, a recombinant protein was mass-produced in E. coli and examined for its antigenicity, biological nature and fertilization-blocking effects..(1) The DNA fragment coding for the NH2-terminal region of human zona pellucida protein ZPA was prepared to produce the recombinant protein (rhZPA). r-hZPA was purified by Ni^ resin column after resolution in 6M urea of inclusion bodies.(2) The recombinant protein was confirmed to possess the same antigenicity as the native protein using a specific monoclonal antibody. It also showed binding abilityto human spermatozoa.(3) The antiserum produced by immunization in rabbit reacted not only to human but also to pig and rabbit zona pellucida. It was found that the region used for immunization contained species cross-reactive antigen(s). The antiserum inhibited human sperm binding to the zona pellucida in vitro.(4) When rhZPA was immunized in a non-human primate (Macaca Radiata), the produced antiserum inhibited the human fertilization in vitro. Collectively, r-hZPA protein is an attractive target for developing a contraceptive vaccine for human use as well as population control of pets and wildlife.