The Secretory Expression Of Recombinant Porcine Zona Pellucida Glycoprotein-3α(rpZP3α) In Pichia Pastoris And The Determination Of Its Activity

The zona pellucida (ZP) is a transparent extracellular matrix that surrounds the mammalian oocyte. It plays important roles in the species-specific gamete recognition and blockage of polyspermy during fertilization. Porcine ZP (pZP) has been used in the study for developing human contraceptive vaccine because its antibody cross-reacts with human ZP. The porcine zona pellucida 3 is composed of pZP3α and pZP3β, and the former has been implicated as the primary sperm receptor in porcine fertilization. pZP3α was considered a potential antigen candidate for the contraceptive vaccine in early study. Pichia pastoris is an efficient eukaryotic expression system with great potentials, and has unexampled advantages in expressing the heterologous proteins and more wide application. To obtain the recombinant pZP3α (rpZP3α) protein and anti- rpZP3α antibody for the further study of the contraceptive vaccine, the DNA sequence(446-1423) encoding purified pZP3α was inserted into avector-pPICZαA. The recombinant plasmid pPICZαA-pZP3α was linearized andthen transformed into Pichia pastoris GS115 by electroporation. After screening with Zeocin, rpZP3α expression of the engineering strains was optimized for pH and methanol concentration in shake flasks, and for inducing time in fermentation. Under these optimized conditions, the engineering strains were induced to produce rpZP3α in high-density fermentation. The fermentative supematants were separated and concentrated, and then rpZP3α was purified by Cu²⁺ or Ni²⁺ metal affinity column chromatography. The purified rpZP3α was identified by SDS-PAGE and Western blot, and the quantity, purity and rate of recovery of the rpZP3α were analyzed by Quantity One software. One male rabbit was immunized with the Cu-NTA-purified rpZP3α. The antibody responses against rpZP3α and porcine ZP were detected by ELISA and the indirect immunofluorescent technique (IIF). The effect of the anti-rpZP3α antibody on sperm-egg binding was assayed in of the anti-rpZP3α antibody in the mouse and porcine in vitro fertilization. Thirteen weeks after immunization with rpZP3α, the mice were killed and the paraffin slices of ovarian serial section was prepared and stained by H.E. Changes of ovarian morphology were observed.