The Expression Of Recombinant Human Zona Pellucida-3(rhZP3) In Pichia Pastoris In High-density Fermentation And Immunization Of Macaca Fascicularis With RhZP3

Zona pellucida (ZP) is the extracellular semitransparent matrix surrounding mammalian oocytes to play the role in protecting the oocytes, it's also the location at which gamete recognization and egg-sperm interactions occur. ZP has the worthness to develop the immunocontraceptive vaccines. However, ZP is a protein of tissue specificity, it's seldom existence in the natural surroundings. For achieving mass high-purity zona pellucida protein for the study of controceptive vaccine and diagnosis of sterility, we recombined the mature peptide of human ZP3 and expressed in vitro.In the present study, the gene fragment of the mature peptide(23-383aa) of human ZP3 were cloned, and incorporated into expression vector shuttle plasmid pPICZαA. When the correct recombinants were selected, the recombinant plasmid was linearized with Sac Ⅰ after enlarged in Escherichia coli, then transformed into Pichia pastoris by electroporation. Pichia transfomants including multicopy inserts were screened with high concentration of Zeocin. SDS-PAGE and Western Blotting were used to appraise the recombination protein which was induced by methanol. The transformant that had the highest yield of rhZP3 protein was choosed for high-density fermentation. The recombination protein, which has a polyhistidine tag designed in the pPICZαA, was purified with Ni-chelating affinity chromatography.Pure rhZP3 as an antigen was used to induce immune response of Macaca fascicularis with the adjuvant of muramyldipeptide(MDP). The antiserum of the monkeys were monitored by ELISA with the pig ZP3 as coating antigen, and it's also detected by immunohistochemistry with frozen section of mouse ovaries and paraffin section of human ovaries. Sperm-egg interaction of mouse was used to evaluate the effect of the antiserum.The results showed that the amplified hZP3 fragment of the mature peptide had been inserted into vector plasmid successfully. Pichia transfomants which can secrete interesting protein steadily were screened. The recombination protein had...