Recombinant Mouse Sperm Antigen Vaccine Strain And Its Contraceptive Effect

Development of effective contraceptive vaccine probably is an ideal approach to control the overgrowing population. It's mechanism is using the important molecule(s) involved in the fertilization as target antigen(s) employed for immunizing the populations the specific immunological reaction induced by the target antigen(s) will interfere with the fertilization and result in infertility. The researches in recent years indicate that the development of a vaccine based on sperm antigens represent a promising approach for contraception. The key steps inducing fertilization are the interactions of sperm- ZP and sperm- egg plasma membrane, which become the main focus in researching contraceptive vaccine. Fertilin ~ subunit, one of sperm membrane proteins, localized in the equatorial region and the inner acrosomal membrane which regions participated in the early step of sperm-egg membrane fusion. It functions as a cell adhesion molecule involved in the sperm-egg membrane binding and fusion and the sperm-ZP binding. Cyritestin is a transmembrane protein associated with the sperm acrosome, and also localized in the equatorial region and the inner acrosomal membrane. The peptides mimetic from the predicted binding site in the disintegrin domain of cyritestin and the monoclonal antibody against the disintegrin domain strongly inhibited sperm-egg adhesion and fusion in vitro fertilization. Male mice deficient for germ-cell cyritestin are infertile. Sperm without cyritestin protein does not bind with ZP. So both the fertilin P subunit and cyritestin have important roles in the sperm-egg binding and fusion, and are potential sperm-specific target antigens for contraceptive vaccine. As we know, this study is for the first time using the Fertilin P subunit and V cyritestin protein as target antigen and the salmonella typhimurium as vehicle for development the contraceptive vaccine. The genes expressing mouse extracelluiar domain of sperm fertilin P subunit and mature cyritestin protein were inserted into an expressing vector pYA3 149 respectively, two recombinant plasmids pFEC and pCFL were constructed using the vector-host balanced-lethal system. The pFEC and pCFL were transformed into two kinds of attenuated salmonella typhimurium strains respectively and got four recombinant vaccine strains. Kunming mice were immunized with these strains by oral administration. Female mice serum and vaginal secretion antibodies and their fertility were determined. Meanwhile the male mice testicular pathological changes were also observed. On the other hand, the effects of the monoclonal antibodies and anti-serum against fertilin P subunit and Cyritestin on the sperm-egg interaction during in vitro fertilization were tested. The main results and conclusions were as follows: 1. The gene encoding the extracellular domain of mouse Fertilin P subunit was prepared by PCR amplification from mouse Fertilin P plasmid DNA and inserted into the expressing vector pYA3 149, so got the recombinant plasmid pFEC. The gene encoding the mature Cyritestin protein was prepared by directly digest the mouse Cyritestin plasmid DNA using restriction enzyme and inserted into the expressing vector pYA3 149, so got the recombinant plasmid pCFL. 2. The recombinant plasmids pFEC and pCFL were transformed into two kind of attenuated salmonella typhimurium strains X4632 and X4550 respectively. Four kinds of recombinant...