CLONING AND EXPRESSION OF THE GENE hZ3.3 ENCODING FRAGMENT OF HUMAN ZONA PELLUCIDA 3 IN PICHIA PASTORS

In order to explore the molecular mechanism of zona pellucida-sperm interaction, a DNA fragment (387bp) corresponding to hZP3 peptide fragment 191~319 was amplified by PCR from hZP3 cDNA and then subcloned into the MCS of expression vector pPICZαA. The recombinant plasmid pPICZαA-hZ3.3 was transformed into Pichia pastoris yeast X-33 via electroporation, and then high-copy transformants were screened out on the YPDS plates with high concentration Zoecin. After induction of 0.5% methanol, a specific peptide fragment of 37 kD or so was expressed and secreted out of the cells. Western-blot result showed that the peptide could bind anti-human zona antibody specifically. These suggested a recombinant human zona pellucida fragment hZ3.3 gene expression vector had been successfully constructed and hZ3.3 peptide had been expressed in the engineered yeasts.