| Ideal contraceptive agents should be effective, cheap, safe and reversible. The immune contraceptive research based on sperm antigens is a good approach with great potential matching the request. Now, it's possible to develop the vaccine with the progress of knowledge in the field. The nature models of immune infertility in human have provided us research objects and evidence. The development of the vaccine based on sperm antigens represents a promising approach in the 21st century.The crucial steps inducing fertilization are the interactions of sperm-ZP and sperm-egg plasma membrane. The sperm antigens that can block the two steps are the candidates for contraceptive vaccine. Many sperm-specific antigens have been identified. Fertilin beta, one of the ADAM protein family, localizes in the posterior region of sperm head after acrosomal reaction, where the interactions of sperm-egg take place definitively. It mediates the interactions of mammalian gametes membrane as a cell adhesion molecule. The molecule is constituted of several conservative domains. In IVF assay, the recombinant forms of fertilin beta including the disintegrin domain(βDCE) is able to inhibit sperm-egg binding, and the recombinant forms of fertilin beta truncated the disintegrin domains (βCE) is relatively ineffective, indicating that the disintegrin domain of fertilin beta is required for the interactions of sperm-egg and is the most important domain. The studies of fertilin beta still focus on the gene-function relations, and as we know, there is no report about construction of attenuated Salmonella strains expressing the disintegrin domain of human fertilin beta.It's to vaccinate directly intact antigen into individuals in conventional method by injection. But the method is difficult to induce the antigen-specific secretory antibody in reproductive tract. So some researchers are devoting to develop oral immunocontraceptive vaccine. They have found that Salmonella typhimurium was able to enhance immune response, which elicited sperm-specific mucosal, cellular and humoral immune responses at high titer. It's a promising delivery system to develop an oral contraceptive vaccine, which ensures the recombinant bacterium to express the target antigen stably by balanced-lethalsystem of Salmonella. This study used human fertilin beta as target antigen and Salmonella typhimurium as vehicle for construction of the vaccine. The cDNA sequences encoding the disintegrin domain of human fertilin beta were inserted into an expressing vector pYA3339 (high copy number plasmid) by genetic recombination techniques. Then the recombinant plasmid pYA3339/hf279 was constructed through the balanced lethal asd selection system of Salmonella. At last the recombinant plasmid was transformed into the attenuated Salmonella typhimurium strain X4550 to construct the recombinant vaccine X4550 (pYA3339/hf279) successfully. The main results and conclusions are as follows:1. The cDNA sequences encoding the disintegrin domain of human fertilin beta was obtained by PCR amplification from the plasmid pT7-7/hf including human fertilin beta gene. The cDNA sequences were inserted into the expressing vector pYA3339, and then the recombinant plasmid pYA3339/hf279 was constructed;2. The recombinant plasmid was transformed into the attenuated mouse Salmonella typhimurium strain X4550, and the recombinant vaccine strain X4550 (pYA3339/hf279) was constructed successfully;3. Western blot assay indicated that the protein HF93 expressed in the recombinant bacterium was recognized by anti-human fertilin beta polyclonal antibody. The recombinant protein HF93 took 6 percents of total bacterial protein. Its molecule weight was about 16KDa;4. This study provided some important basis for the selection of target antigens and delivery systems for the development of oral immuocontraceptive vaccine for human. It also laid the foundation for futher protein functions identification and animals experiments.
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