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、组织特异性表达和较强的免疫原性方面具有重要作用，因此有望成为避孕疫苗的候选成分。然而，从天然来源制备质量稳定的疫苗材料是不可能的。本研究在大肠杆菌中大量表达重组蛋白，并对其抗原性、生物学特性和受精阻断作用进行了检测。(1)制备了编码人透明带蛋白ZPA NH2末端的DNA片段，用于表达重组蛋白(RhZPA)。R-hZPA经6M尿素包涵体溶解后，经镍树脂柱层析纯化。(2)用特异性单抗证实重组蛋白具有与天然蛋白相同的抗原性。免疫兔产生的抗血清不仅能与人反应，还能与猪和兔透明带反应。发现用于免疫的区域含有物种交叉反应抗原(S)。该抗血清在体外能抑制人精子与透明带的结合。(4)用重组人ZPA免疫猕猴后，产生的抗血清对人体外受精有抑制作用。总的来说，r-hZPA蛋白是开发人类使用的避孕疫苗以及控制宠物和野生动物种群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.