Clonal Expression Of Mouse Epididymis-specific Colipase And Primary Study Of Its Funtion

Aim:To express mouse epididymis-specific colipase (meClps) with prokaryotic system and prepare of anti-meClps antibody, detect expression specific of meClps in male mouse, and study the possibility of meClps as a immunocontraceptive target.Methods:The characteristics of meClps gene and protein were analysed by bioinformation, and primers were designed with Oligo 6. The full-length of meClps was amplified from mouse epididymal cDNA and cloned into pMD19-T vector. The DNA fragment of mature peptide was obtained by PCR from pMD19-T vector with meClps. Then the product was inserted into the prokaryotic expression vector pET-21 b. The recombinant plasmid was identified by PCR and.sequencing before transformation into E.coli BL21 (DE3). After induced by IPTG, the recombinant meClps protein was expressed and separated by Tricine-SDS-PAGE, then identified by Western blotting. The inclusion body was washed with 2 mol/L urea, then separated by Tricine-SDS-PAGE. After staining and destaining, the gel containing meClps protein was cut, equilibrated with physiological saline solution, rubbed, and emulsified with Freund adjuvant, then used as antigen to immunize rabbit and prepare antibody. The anti-meClps antiserum response was identified by Western blotting. The specific expression of meClps was identified with anti-meClps antiserum by Western blotting. Male mice were immunized passively by muscle injection with anti-meClps antiserum. The antifertility effect was evaluated by mating test and sperm motility analysis.Results:The full-length of meClps was cloned into pMD19-T vector, and the in vitro recombinant expression vector pET21b-meClps was constructed. The meClps protein was successfully expressed in prokaryotic system, but mainly in inclusion body. Anti-meClps antibody response was induced by immunization with meClps in rabbit, preparing high titre rabbit anti-meClps antiserum. The rabbit anti-meClps antiserum reacted with meClps, exhibiting specific imprinting by Western blotting with 1:50000 dilution antiserum. The anti-meClps antiserum could bind to the protein of epididymis, but not to testis, pancreas, intestine, liver and lung. The antifertility study showed that there were no significant difference in sperm mobility and the numbers of fetus borned by female mouse after mating with immunized male mouse among experiment group,negative control group and blank control group.Conclusion:The prokaryotic expression system of meClps protein was established and the recombinant meClps was expressed. Anti-meClps antiserum with high titre was prepared, and the specific expression of meClps in epididymis was identified. But in this study, the fertility of male mouse was not affected by passive immunization with anti-meClps antiserum. The antifertility effect of immunization with meClps need further evaluate.