Purification and Partial Characterization of the 38 kDa Glycolipoprotein Antigen from the Culture Filtrate of Mycobacterium tuberculosis H37Rv

ABSTRACT

Mycobacterium tuberculosis infected macrophages can become ineffective at activating CD4+ T cells through presentation of peptide antigens by MHC class II, possibly contributing to the ability of M tuberculosis to persist despite the presence of an intact immune system. Presentation of lipid antigens may help to overcome this problem. CD1 represents the key component of an MHC independent pathway for presentation nonpeptide lipid antigens to T cells. The 38 kDa glycolipoprotein antigen of M. tuberculosis is actively secreted. The antigen induces strong antibody and T-cell responses and provided partial protection against M. tuberculosis infection in mice when it is administered either entrapped in biodegradable microparticles or in the form of a DNA vaccine. But an selective anergy to stimulation with peptide of the 38 kDa was observed in the majority of tuberculosis patients. An 38 kDa antigen has been isolated by affinity chromatography using a monoclonal antibody. This antigen contains some immunosuppressive cell wall associated antigens such as lipoarabinomannan. Therefore, we purified the 38 kDa glycolipoprotein from the culture filtrate of M tuberculosis H37Rv by ammonium sulfate precipitation (55~80%), hydroxylapatite and DEAE-Sephacel column. The purified antigen showed three major bands on isoelectric focusing gel, and two-dimensional electrophoresis (2-DE) analysis of this antigen revealed five distinct spots of the 38 kDa molecular mass. One of five spots had a N-terminal sequence identical to that of the 38 kDa glycolipoprotein (pstS-1). Other protein spots could not determine sequences. An antiserum against the recombinant 38 kDa antigen of M tuberculosis reacted strongly with the purified the 38 kDa antigen.