Abstract: Abstract Objective: To construct suitable vectors for the secretory expression of hirudin in Pichia pastoris. Methods: The α-facor- Hirudin gene was amplified from pPIC9- Hirudin by PCR and subcloned into PAO815. and then construct multicopy recombinant plasmid pAO815-(α-Hirudin)n .The recombinant was transformed into P. pastoris strain GS115 for induction expression and then the activity of secreted products were identified. Result: A new multicopy vector pAO815-(α-Hirudin)n was successfully constructed and was capable of secreting recombinant hirudin efficiently, confirmed respectively by PCR, SDS-PAGE. The products possess the activity of thrombin inhibitor. Conclusion: This result offers efficient P. pastoris strains for mass production of biologically active hirudin.

Key words: Pichia pastoris, Secrete expression, Hirudin, Multicopy