Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.
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.
Jeevan
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A
Department of Medical Microbiology and Immunology, Texas A&M University SHSC, 407 Reynolds Medical Building, College Station, TX 77843-1114, USA. ajeevan@medicine.tamhsc.edu
McFarland
C T
CT
Yoshimura
T
T
Skwor
T
T
Cho
H
H
Lasco
T
T
McMurray
D N
DN
D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D000995:Antitubercular Agents / Q000138:chemical synthesis / Q000494:pharmacology; D000998:Antiviral Agents / Q000138:chemical synthesis / Q000494:pharmacology; D002460:Cell Line; D006168:Guinea Pigs; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000737:chemistry* / Q000494:pharmacology; D008262:Macrophage Activation / Q000276:immunology*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000254:growth & development / Q000276:immunology; D009573:Nitrites / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins; D014397:Tuberculosis, Pulmonary / Q000276:immunology
D000995:Antitubercular Agents; D000998:Antiviral Agents; D000949:Histocompatibility Antigens Class II; D009573:Nitrites; D011994:Recombinant Proteins; D007371:Interferon-gamma; D006861:Hydrogen Peroxide