Secreted Expression Of Porcine Interferon-gamma Gene In Pichia Pastoris And Studies On Anti-HCV Activity Of Recombinant Protein

Porcine interferon-gamma (PoIFN-γ), produced by activated cytotoxic T cells and natural killer cells, plays important roles on anti-infectious and immunomodulatory effects. In order to develop the application of recombinant PoIFN-y, a recombinant expression vector was constructed in this study. The porcine interferon-gamma gene, in which the sequence encoding signal peptide was replaced by that of the a -factor of Saccharomyces cerevisiae, was cloned into Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K- α -PoIFN- γ was then transformed into Pichia pastoris GS115 cells by electroporation and stable multicopy recombinant Pichia pastoris strains were selected by G418 resistance. Two recombinants of multiple inserts were obtained. PCR analysis of recombinants showed that the porcine interferon-gamma gene had successfully integrated into the Pichia pastoris GS115 genome. SDS-PAGE and Western blot assays of culture broth from this two methanol-induced expression strains demonstrated that recombinant PoIFN-γ 17kD and 23kD proteins, were secreted into the culture medium. Target proteins, about 60% of total proteins, were obtained in the culture medium at the concentration of 105mg/L and 108mg/L respectively under shake cultivation. The recombinant PoIFN-y can well protect PK-15 cells against virulent hog cholera virus (HCV) infection in vitro.This is the first secreted expression of porcine interferon-gamma gene in Pichia pastoris, which establish a good foundation for large-scale production of the recombinant PoIFN-γ.