Expression And Biological Activity Detection Of Recombinant Chicken Interferon Gama In Pichia Pastoris

Interferon is a pleiotropic cytokine with immunomodulatory effects on a variety of immune cells. Pichia Pastoris expression system could provides an alternative for scale-up production of recombinant proteins, and the bioactivities of products close to natural proteins, so we choose it to express chicken interferon-gamma (ChIFN-γ), to lay theoretical foundations for the industrial production and clinical application.The nucleotide sequence of ChIFN-γ mature protein was cloned into the expression vector pPICZaA to construct recombinant plasmid pPICZaA-IFN-γ. After linearized, the recombinant plasmid was then transformed into the P. pastoris GS115by electroporation. After screening by YPDS-ZeocinTM, two recombinant strains (His+Mut+) were isolated and identified by PCR and MMH/MDH plates. Similarly to the natural ChIFN-γ, the molecular weight of the expressed recombinant ChIFN-γ (rchIFN-γ) was approximate17kD. Western blotting analysis showed that the rchIFN-γ could react with the polyclonal antibody against ChIFN-γ we prepared.The bioinformatic analysis showed that isoelectric point of rchIFN-γ was approximate pH5.2, so we choose anion exchange chromatography to purify it. After membrane ultrafiltration and dialysis, the rchIFN-γ was purified by DEAE anion exchange chromatography. The purity of rchIFN-γ was approximate70%. The bioactive test of the purified protein showed that the antiviral activity of rchIFN-γ could reach10,240IU/mLTo evaluate the adjuvant effect of rchIFN-γ, SPF chickens were immunized with combination of rchIFN-γ and H9subtype avian influenza virus (AI) inactivated vaccine. The percent of CD4+and CD8+T lymphocytes in periphery blood were detected by flow cytometry post immunization. The results demonstrated that the percent of CD4+T lymphocytes as well as ratios of CD4+/CD8+of chickens immunized vaccine plus rchIFN-γ were significantly higher than chickens immunized vaccine (p<0.05). The serum antibody titers were analyzed by hemagglutinin inhibition (HI) test post immunization. All chickens were challenged with H9N2AIV35days post immunization, then virus shedding was detected through SPF chicken embryo inoculation. The results showed that HI antibody titers of chickens immunized with vaccine plus rchIFN-γ reached the peak at14days post-immunization, earlier than chickens immunized vaccine. And HI antibody levels of the former were also higher than the later (p<0.05). After challenged with H9N2AIV, the virus shedding rates of chickens immunization with vaccine plus rchIFN-γ were significantly lower than chickens immunized vaccine (p<0.05). These results suggested that rchIFN-γ had high biological activity in vitro and vivo, and could be used as an adjuvant.