Studies Of The Biological Activities Of The Mutant Recombinant Human Zona Pellucida Protein 3(mrhZP3)

In the present study, the Pichia pastoris strains which protease cleavage sites of hZP3 hadbeen replaced for producing the mutant recombinant human zona pellucida protein 3(mrhZP3)were adopted. Induced by methanol, mrhZP3 were expressed in shake flasks. High-efficiencystrains were chose to produce mrhZP3 in high-density fermentation. The fermentativesupernatants were separated and concentrated. Under the optimized conditions, mrhZP3 werepurified by Ni²⁺ metal chelating chromatography. The purified mrhZP3 were identified bySDS-PAGE and Western blotting. Then mrhZP3 were compared with rhZP3 in molecularweight and yield. Human sperm acrosome reaction was induced by mrhZP3. Female mice wereimmunized with mrhZP3. The mice were divided into four groups at random, each group 10mice. Group A were immunized with mrhZP3 suppository by vagina, the suppository withoutmrhZP3 were administered to group B. Group C were immunized with mrhZP3 by muscleinjection, and group D were injected with adjuvant only. All the groups were administered onweek 0, 1, 2, and 3. Serum samples of the immunized mice were collected on week 1, 2, 3, and5. The antibodies against ZP3 were detected by ELISA. The female mice were mated on week 5and killed on week 8 for checking pregnancy. Ovarian frozen sections were prepared formicroscopical pathological observation. The antigenic activity of mrhZP3 was confirmed byWestern blotting. The human sperm acrosome reaction was induced with mrhZP3. WithmrhZP3 and pZP3 as coating antigens respectively, the antibodies could be detected in group C,while none were detected in the sera of group A, B and D. Pregnancy rate of group A was 0, Bwas 9/10, C was 0 and D was 8/10. There were significant statistic differences between group Aand B, between group C and D (P＜0.01). No obvious pathological changes were observed inthe mice ovarian frozen sections. The results implied mrhZP3 are similar to natural human ZPin inducing sperm acrosome reaction and mrhZP3 possess antigenic activity, mrhZP3 has thepotential to develop diagnostic kits for AR and effective contraceptive vaccine.