关键词: Izumo, 亲和层析, 精卵融合, 免疫血清, 免疫避孕

Abstract: BACKGROUND ＆ AIM: The sperm membrane protein Izumo was vital for sperm-egg fusion. The aim of this study was to express and purified recombinant mouse Izumo protein (mIzumo) and to assess the immunogenicity of the recombinant mIzumo and the effect of an antibody against recombinant mIzumo on mouse sperm-egg fusion in vitro. MATERIALS AND METHODS: The coding sequence of mIzumo was subcloned into pET28a(＋). The recombinant fusion protein, 6His-mIzumo, was expressed in E coli. BL21(DE3) strain and purified by Ni2＋ affinity chromatography. Mice of both sexes were immunized with purified 6His-mIzumo combined with Freund's adjuvant. Pre-immunized and post-immunized serum samples were tested for anti-6His-mIzumo activities by Western blot and ELISA. The effect of anti-6His-mIzumo antibodies on sperm-egg fusion was detected by in vitro fertilization (IVF) and sperm-egg fusion assay. RESULTS: The purified 6His-mIzumo appeared as a single ≈60 kD band on SDS-PAGE. Both immunized male and female mice developed serum antibodies, which, on Western blot, cross-reacted with a single ≈60 kD protein band corresponding to mIzumo in membrane protein extracts of mouse testis, epididymis and sperm. According to ELISA, the titre of anti-6His-mIzumo antibodies remained at the highest level for 6 weeks after final immunization. The ability of post-immunized sera treated sperm to fuse with zona-free oocytes was significantly inferior to that of the sperm treated with pre-immune sera (P<0.01). CONCLUSION: The purified 6His-mIzumo could stimulate immune response in mice of both sexes and induce specific antibodies which could inhibit sperm-egg fusion in vitro. Therefore, as an allogeneic antigen, 6His-mIzumo could be as a candidate for development of immunocontraceptive vaccine.

Key words: Izumo, affinity chromatography, sperm-egg fusion, immune serum, immunocontraception