Purification and molecular analysis of BCG antigen 60

ABSTRACT

Tuberculosis remains as an important socioeconomical and medical problem throughout the world and especially in Iran. Early and timely diagnosis of pulmonary and extrapulmonary tuberculosis is vital to initiate prompt treatment. Current diagnostic methods are either slow or lack enough sensitivity or specificity. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. Their identification is important for both diagnosis and protection against mycobacteria. Antigen 60 [A60] is a thermostable antigen found in the cytosol of M. bovis and M. tuberculosis. An ELISA test using A60 is designed for diagnosis of tuberculosis with satisfactory results. In previous studies, A60 has also showed a protective effect against experimental infections and useful immunotherapeutic effects in promotion of cancer development. In the present work we tried to purify A60 from the cytoplasm of BCG. A60 was purified by exclusion gel chromatography using sepharose 4B. A60 was recognized by bidimensional immunoelectrophoresis with anti-BCG and anti-A60 antiserum, where it appears as the less mobile component. In agarose electrophoresis, A60 showed only one band but in immunodiffusion it showed two immunoprecipitinogen lines with anti-BCG anti-serum. In analyzing with dot blotting, both cytoplasm and cell wall of BCG showed positive reaction with anti-A60 anti-serum. When A60 was fractionated by SDS-PAGE and analyzed by western blot using anti-A60 antibody, 65,46,40, 38 and 35 KDa protein fractions were identified. It is concluded that A60 is a macromolecular antigen of BCG with a molecular weight of 10[6]-10[7] Da and is a lipoprotein-polysaccharide complex which contains several proteins. A60 is present in both cytoplasm and cell wall of BCG and can easily be purified from BCG vaccine using exclusion chromatography by sepharose 4B, to be used for designing diagnostic tests for TB