Production and Characterization of Guinea Pig Recombinant Gamma Interferon and Its Effect on Macrophage Activation

Abstract

ABSTRACT Gamma interferon (IFN-γ) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-γ (gpIFN-γ) and reported that BCG vaccination induced a significant increase in the IFN-γ mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-γ mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-γ with a histidine tag at the N terminus (His-tagged rgpIFN-γ) in Escherichia coli . rgpIFN-γ was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-γ. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-γ was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-γ did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-γ induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-γ has been successfully produced and characterized in our laboratory. The study of rgpIFN-γ will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.