Expression Of Human Sperm-specific Lactatedehy Drogenase In E.coli, Construction Of Its DNA Vaccine And Prelimibary Characterization Of Genetic Immunization

Lactate dehydrogenase C₄ ( LDH-C4 ) is a testis specific lactate dehydrogenase isozyme which is expressed on sperm plasma membrane through out spermatogenesis as well as in the mature sperm type. LDH-C4 plays a importante role in the process of spermatogenesis, sperminogenesis, sperm capacitation and egg fertilization. LDH-C4 is also involved in human involuntary infertility in male and female. Effects on fertility of animals after immunization with LDH-C4 shows that the LDH-C4 induced infertility is reversible. Therefore, LDH-C4 is a known candidate antigen for a contraceptive vaccine.The coding sequence of human LDH-C4 was amplified from human testis λ TripEx cDNA library with specific primers , and cloned into pET-28a(+) after the restriction enzyme digestion. The recombinant vector was introdued into E. coli BL21(DE3 plysS+) .His-tagged fusion protein was expressed at a high level after induction with IPTG. .and analyzed by 12%SDS-PAGE, activity determination of lactate dehydrogenase, LDH isozyme profiling and Western blot. The fusion protein was purified throgh Ni-NTA columns , which was a prerequisite for preparation of anti-hLDH-C4 antibody and DNA immunization experiments in animals.A eukaryotic expression vector pVAXl-hLDHC4 eas also construced.The T7 Quick coupled transcription/translation systems was uesed to detect its expression in vitro translation. BALB/c mice were immunized with pVAXl-hLDHC4 by muscular injection(i.m) .Control mice were administered with pVAX1. Serom were collected for evaluating anti hLDH-C4 antibodyresponse by Western-blot and ELISA. Anti hLDH-C4 IgG response was induced in immunized mice,but no specific IgG response was deteced in control mice.Although LDH-C4 is an autoantigen for male animals, Orchitis was no found in immunized male mice,Possibility because of blood-testis barrier . Further studies are required for clear and detailed understanding of its role in anti fertility.