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

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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.