Preliminary Studies For Constructing Sperm-specific CATSPER1 DNA Contraceptive Vaccine

Aim: CATSPER1 is exclusively expressed in testis and located specifically in the principal piece of the sperm tail. Targeted disruption of the gene results in markedly decreased sperm motility, and catsperl^-/- sperm fail to fertilize intact egg. Therefore, CATSPER1 might be an excellent potential target for contraceptive vaccine. This study aims to construct sperm-specific CATSPER1 DNA contraceptive vaccine, test the antibody response induced by immunizing with it and explore the efficacy of C3d molecular as adjuvant, thus accumulate data for futher studies. Methods: After analyzing the topological structure of CATSPER1, a pair of primer was designed to amplify the trans-membrane domain gene by RT-PCR, and then the segment was inserted into the mammalian expression vector pVAX1 to construct recombinant plasmid pVAX1-Catsper1_s1-s6. In order to test the efficacy of C3d3 as molecular adjuvant, the transitional vector pVAX1-Catsper1_s1-s6-C3_link was constructed. Then the C3d3 gene was cutted from pSG5.C3d3.YL and fused into the plasmid pVAX1-Catsper1_s1-s6-C3_link to construct plasmid pVAX1-Catsper1_s1-S6-C3d3. pVAX1-Catsper1_s1-s6 and pVAX1-Catsper1_s1-s6-C3d3 vectors were prepared and administered into each experimental male rabbit and mouse on weeks 0, 2, 4, 6 and 8 by intramuscular injection, whereas Alum-PBS solution without DNA was given to control group. Serum collected at intervals of two week was used to determine antisperm antibody response by ELISA. IIF (indirect immunofluorescence) was used to locate the position where anti-sperm antibodies react with the sperm. Results: Electrophoresis, restricting enzyme cutting and sequencing shows that the fragments of CATSPER1 and C3d3 DNA are correctly inserted into pVAX1 and pVAX1-Catsper1_s1-s6-C3_link respectively. Antisperm antibody in the serum of rabbits was detected by ELISA though the intensity of immune response was low relatively. IIF shows that the antibodies in rabbits induced by the vaccines recognized the whole sperm including the principle piece. In the mice, antisperm antibody also was detected by ELISA, the intensity of antibody response immunized by pVAX1-Catsper1_s1-s6-C3d3 is a litthle higer than that of pVAX1-Catsper1_s1-s6. Conclusion: Fragments of Catsperl and C3d3 DNA are correctly inserted into the plasmids, thus pVAX1-Catsper1_s1-s6 and pVAX1-Catsper1_s1-s6-C3d3 are constructed successfully. pVAX1-Catsper1_s1-s6and pVAX1-Catsper1_s1-s6-C3d3 vectors can induce the rabbits and mice to produce antibodies against sperm, and C3d3 molecular can increase the immune response in mice to some degree in this study. The antibodies induced by Plasmids pVAX1-Catsper1_s1-s6 and pVAX1-Catsper1_s1-s6-C3d3 are not exclusively react with sperm principle piece but the whole sperm.