Construction of Pichia pastoris expression vector of chicken IL-18 and optimization of expression conditions

Chicken IL-18 mature peptide gene was amplified from the recombinant plasmid pMD18-T-ChIL-18 by PCR,and subcloned into Pichia pastoris expression vector pPICZαA,thus constructing the recombinant plasmid pPICZαA-ChIL-18.After identified by restriction enzymes digestion,PCR and DNA sequencing,the recombinant plasmid was transformed into P.pastoris X-33.Then the multi-copy recombinant strains were induced for expression.The expression conditions were further optimized by analyzing the influences of pH value,inducer concentration,inducing time,and inducing temperature on the yields of target protein.The results showed that chicken IL-18 could be secreted by the recombinant P.pastoris and the expression level of recombinant protein was highest under pH 6.0,1.5% of methanol,temperature 26 ℃,and 96 hours of inducing time.This study laid a foundation for the large-scale fermentation of chicken IL-18.