Preparation Of Recombinant Mouse Sperm Protein Izumo And Investigation Of Its Immunocontraceptive Potential

【BACKGROUND & AIM】Among the numerous candidate antigens for development of immunocontraception, sperm antigens currently constitute the most promising and exciting targets for contraceptive vaccines. Izumo, a novel sperm-spectific protein which was identified in 2005 and plays an essential role in sperm-egg fusion, seems to provide a new approach for development of contraceptive vaccines. The aim of this study is to determine if the recombinant mouse Izumo (mIzumo) could be used as a potential immunocontraceptive antigen.【MATERIALS & METHODS】The prokaryotic expression plasmid pET28a(+)-mIzumo was constructed and the recombinant mlzumo fused with 6His tag (6His-mIzumo) was purified by immobilized Ni²⁺ affinity chromatography and refolded by dislysis and quantitated by Bradford assay. Female and male C57BL/6 mice were immunized with 6His-mlzumo by intraperitoneal injection. Western blot was used to detect anti-6His-mIzumo activities of serum from the mice immunized with 6His-mIzumo and ELISA was performed to examine the time course of mean titre of serumal anti-6His-mlzumo IgG antibodies in immunized mice. Effect of the immune serum on mouse sperm-egg fusion in vitro was detected using sperm-egg fusion assay. Dualfluorescent stain was used to locate the recognition site of anti-6His-mlzumo IgG antibodies on sperm surface. Fertility of the 6His-mIzumo immunized male and female mice was evaluated compareing with PBS-control animals.【RESULTS】The prokaryotic expression plasmid pET28a(+)-mIzumo was successfully constructed and the recombinant protein 6His-mlzumo was successfully produced. Female and male mice inoculated with 6His-mIzumo developed the specific serum IgG antibodies that cross-reacted to the native mIzumo. The IgG response of immunized mice started to raise two weeks after the first boost and the antibody titers reached the maximum value two weeks after the final boost and lasted for four weeks. The immune sera against 6His-mIzumo almost completely blocked mouse sperm-egg fusion in vitro. The anti-6His-mIzumo IgG antibodies in the immune sera could specifically recognize the acrosomal region of the acrosome-reacted mouse sperm. However, there was no significant reduction of fertility of both male and female mice immunized with 6His-mlzumo compared with PBS-control mice.【CONCLUSION】The recombinant protein 6His-mIzumo which fused with 6His purification tag was successfully prepared by pET prokaryotic expression system. The immunogenicity of the recombinant 6His-mIzumo was strong in female and male mice. Inoculation with recombinant 6His-mIzumo was able to stimulate the immunized mice to develope the circulated anti-mIzumo antibody which could recognize and bind the acrosomal region of the acrosome-reacted mouse sperm and could block mouse sperm-egg fusion in vitro but not to reduce the fertility of both female and male immunized mice. Thus, there was probably no contraceptive effect of immunization with recombinant mouse Izumo prepared by prokaryotic expression system on mouse model. It seems that application of Izumo as a candidate antigen in development of contraceptive vaccine needs further investigation and exploration.