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Infection and Immunity, January 2006, p. 213-224, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.213-224.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

A. Jeevan,1* C. T. McFarland,1 T. Yoshimura,2 T. Skwor,1,{dagger} H. Cho,1 T. Lasco,1,{ddagger} and D. N. McMurray1

Texas A&M University SHSC, College Station, Texas 77843-1114,1 NCI-FCRDC, Frederick, Maryland 217022

Received 27 June 2005/ Returned for modification 31 August 2005/ Accepted 14 October 2005

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

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, Texas A&M University SHSC, 407 Reynolds Medical Building, College Station, TX 77843-1114. Phone: (979) 862-2436. Fax: (979) 845-3479. E-mail: ajeevan{at}medicine.tamhsc.edu.

Editor: J. L. Flynn

{dagger} Present address: Children's Hospital and Oakland Research Institute, Oakland, CA 94609.

{ddagger} Present address: MYCOS Research, LLC, Loveland, CO 80538.

Infection and Immunity, January 2006, p. 213-224, Vol. 74, No. 1
0019-9567/06/$08.00+0     doi:10.1128/IAI.74.1.213-224.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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