Construction and expression of a site-specific mutant of chicken interleukin-18 gene in Pichia pastoris

In order to express highly mature chicken interleukin-18(MChIL-18) in Pichia pastoris,it is necessary to reconstruct MChIL-18*,using the technique of site-specific matagenesis which three low-usuage codons have been successfully converted into P.pastoris-preferred codons.The constructed plasmid,pPICZαA-MChIL18*,was linearized by SacⅠ and transformed into Pichia pastoris X-33 by Pichia EasyComIM Transformation Kit.Through selection of X-33 resistant transformation and expression clones,a high expression transformant was obtained.The espressed products were analyzed by SDS-PAGE and Western-blot.SDS-PAGE showed that Pichia pastoris yeast cells integrating the plasmid pPICZαA-MChIL18* produced a high level of MChIL-18*,a relative molecular weight of expressed protein of about 23 000,about 45% of the total yeast body protein.The results of Western-blot immunogenicity showed that MChIL-18* had strong antigen immune reactions with the rabbit sera to chicken IL-18.MChIL-18* protein was successfully expressed in Pichia.pastoris.